|Year : 2017 | Volume
| Issue : 4 | Page : 17-39
|Date of Web Publication||6-Apr-2017|
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
. Poster/Oral Presentations. J Pract Cardiovasc Sci 2017;3, Suppl S1:17-39
Gene Polymorphism in Essential Hypertension and Its Relation to Environmental Factors in North Indians
Qulsoom Naz, Narsingh Verma, M. Serajuddin, A. A. Mahdi, M. L. Patel
King George's Medical University, Lucknow, Uttar Pradesh, India
Aim: We aimed to determine genotype and allele frequencies of angiotensinogen (AGT) and alpha adducing (ADD1) polymorphisms in patients with essential hypertension. Methods: We recruited total 205 subjects. In which 105 were essential hypertensive and 100 were Healthy controls. DNA samples for each individual were isolated from peripheral blood by standard phenol/chloroform method analyzed by polymerase chain reaction & enzymatic digestion. Lipid profile was analyzed by VITROS ° 250 Dry Biochemistry Fully auto-analyzer (Gonson & Gonson Company). Electrolytes (in serum & urine) were measured by ion-selective electrodes (Roche Hitachi MODULAR, Hitachi Ltd) in Clinical lab of Biochemistry. Results: The distribution for each ADD1 genotypes were 61.96% for GG (69), 33.51% for GT (21) and 4.53% for TT (11) in the essential hypertensive group; 82.72% for GG (91), 16.46% for GT (9) and 0.82% for TT in the control group. The distribution of AGT genotypes was found significantly different between groups (x2 = 10.00: df = 2; P = 0. 006). The frequencies for each of the AGT genotypes were found as 44.66% for MM (43), 44.33% for GT (49), and 11% for TT (9) in essential hypertensive group; 64% for MM (66), 32% for GT (36), and 4% for TT (3) in healthy control group. The distribution of AGT genotypes did not highly significant as compared to AGT between the groups. We suggest that AGT and ADD1 gene polymorphism play a role for development of essential hypertension (x2 = 9.767: df = 2; P = 0.007). Conclusion: Patients with essential hypertension exhibited higher levels of Serum cholesterol, LDL Cholesterol & TG than in control subjects. Taken together the genotype and biochemical parameters & considering the restrictive selection criteria used, the present results suggest a relationship between these gene polymorphism and essential hypertension in North Indians.
Endothelial Nitric Oxide Synthase Gene Expression of Endothelial Progenitor Cell in Premature Coronary Artery Disease Patients in Indian Population: A Determinant of Circulatory
Introduction: A significant number of Ex-vivo and animal studies has already established that the reduced eNOS gene expression in EPC may leads to impaired mobilization of cells from bone marrow and that in turn results a reduced number of circulatory EPC in CAD patients. However in vivo eNOS gene expression of EPC from PCAD patients is yet to be known. Since in developing countries like India, the occurrence of cardiovascular disease at young age is markedly increasing, our present study aimed to investigate in vivo eNOS gene expression of EPC in PCAD patients and age matched healthy controls. Method: Endothelial progenitor cells were isolated from peripheral blood on the basis of cell surface antigens CD34+/KDR+ by Magnetic Activated Cell sorting (MACS) method from 50 PCAD patients and 50 healthy controls. The intracellular eNOS gene expression was assessed by RT PCR method by using constitutive gene GAPDH as a reference gene. Result: A reduced eNOS gene expression (mean eNOS/GAPDH integrated density ratio) in EPC from PCAD patients compared to healthy controls were found (0.998 ± 0.096/1.063 ± 0.107) with a p-value of 0.002 and this difference was persisted even after adjustment for confounding factors like age, sex, BMI, smoking and statin therapy (p = 0.002). Conclusion: Our previous study had shown a significantly reduced number of CD34+/KDR+ cells in PCAD patients in comparison with healthy controls (.01868 ±.0176975/.039972 ±.0299683; p < 0.0001), which when supported by the result of the present study may delineate a possible cause of reduced circulatory level of EPC. Also as the study determined the eNOS gene expression directly in EPC isolated from study subjects, the result more realistically reflect the in vivo condition.
Post-transcriptional Regulation of 3-Hydroxy-3-Methyl Glutaryl-Coenzyme A Reductase: Crucial Role of the MicroRNA miR-27a
Abrar A. Khan, Vinayak Gupta, Kalyani Ananthamohan, Vikas Arige, Anil Kiran Chokkalla, Nitish R. Mahapatra
Department of Biotechnology, Bhupat and Jyoti Mehta School of Bioscience, Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Dyslipidemia is a strong predictor of cardiovascular diseases including Essential Hypertension (EH). 3-Hydroxy-3-Methyl Glutaryl-Coenzyme A reductase (Hmgcr), coding for the rate-limiting enzyme in the cholesterol biosynthesis, is an important candidate gene for EH. However, the regulation of Hmgcr, especially at the post-transcriptional level, is incompletely understood. We set out to explore the possible roles of miRNAs in the regulation of Hmgcr. In silico predictions coupled with systematic functional analysis revealed specific interaction of miR-27a with mouse Hmgcr 3'-UTR in mouse liver fibroblasts AML-12. Furthermore, miR-27a showed a significant negative correlation with Hmgcr expression in cultured AML-12 cells as well as rat liver, kidney and skeletal muscle tissues. Ribonucleoprotein Immunoprecipitation (RIP) assays using antibodies against Ago2 in human hepatocellular HuH-7 cells over-expressing miR-27a revealed enrichment of HMGCR in the Ago2-IP fraction. Next, we quantified the expression of miR-27a and Hmgcr protein levels in liver tissues of genetically hypertensive blood pressure high (BPH) and genetically hypotensive blood pressure low (BPL) mice. Liver tissues from BPH showed elevated Hmgcr protein levels and diminished miR-27a levels as compared to BPL. Interestingly, cholesterol depletion in AML-12 cells resulted in down-regulation of endogenous miR-27a while augmenting the Hmgcr protein levels. In corroboration, exogenous cholesterol treatment diminished Hmgcr protein levels while enhancing the miR-27a levels. In addition, miR-27a expression and Egr1 protein levels were elevated in AML-12 cells under hypoxic conditions. Interestingly, computational analysis of the 1 kb promoter region of mmu-miR-27a revealed multiple putative Egr1 binding sites. Moreover, over-expression/down-regulation of Egr1 augmented/diminished the miR-27a promoter activity in both AML-12 and HuH-7 cells respectively. This study highlights the role of miR-27a in the post-transcriptional regulation of Hmgcr, thereby implicating its plausible role in cholesterol homeostasis and hypoxia-like pathological conditions.
Inhibitors of Dopamine-β-Hydroxylase Identified by Structure Based Lead Discovery Methods Exhibited Anti-hypertensive Effect in L-NAME Induced Hypertensive Rats
Sanjay Kumar Dey1, Pankaj Prabhakar2, Manisha Saini1, B. K. Thelma1, Subir Kumar Maulik2, Suman Kundu1
1University of Delhi,2 All India Institute of Medical Science, New Delhi, India
Hypertension is the leading cause of mortality worldwide (AHA report, 2014). While classical anti-hypertensives like ACE inhibitors, ARBs, calcium channel blockers, beta blockers and diuretics are widely used, they have their own limitations and no new type of drugs emerged in the last few years that use body's own blood pressure lowering mechanism. Dopamine β-hydroxylase (hDBH) expressed in noradrenergic nerve terminals as well as in adrenal medullary chromaffin cells, converts dopamine into norepinephrine, is a novel target whose inhibition has been shown to help the treatment of hypertension with several advantages over the classical anti-hypertensives. However, hDBH inhibitors are few in number, often result in side-effects and are frequently non-responsive to specific population, probably since the known inhibitors so far have been designed on ligand based homologues. To address this lacuna, we have employed structure based drug discovery approach and used an experimentally validated computational model for hDBH, built in our lab, for virtual-screening against small molecule databases from NCI, USA and elsewhere. Virtual screening of NCI libraries revealed 69 hits showing better efficacies compared to known inhibitors nepicastat and disulfiram. These hits were then assessed in vitro using a repertoire of biochemical and biophysical methods. UDSC171, UDSC180 and UDSC142 were discovered to be potent inhibitors of DBH with IC50s of 1 μM, 5.5 μM and 18 μM, respectively. The inhibitors displayed KD values against DBH in the range of 100 nm to 1 μM. In silico pharmacokinetic analysis indicated that the molecules are unable to cross the BBB. High doses (up to 50 μM) of the lead compounds showed acceptable cellular tolerance against HEK293 cell line and insignificant hemo-toxicities against RBCs. UDSC171, UDSC180 and UDSC142 leads were successful in preventing elevated systolic blood pressure in L-NAME induced hypertensive rats.
Arjunolic Acid Regresses Cardiac Fibrosis by Inhibiting Non-canonical Transforming Growth Factor- β Signaling
Trisha Bansal1, Arjun Ray2, Shantanu Sengupta2, Sagartirtha Sarkar1
1 Department of Zoology, University of Calcutta, Kolkata, West Bengal,2 CSIR-Institute of Genomics & and Integrative Biology, New Delhi, India
Left ventricular cardiac hypertrophy with excess accumulation of fibrillar collagen causes myocardial stiffness and eventual ventricular dysfunction. Therefore, preventing or inhibiting the excess accumulation of fibrotic tissue in the ventricular myocardium is a major therapeutic goal. Use of plant derived biomolecules has been considered very promising for developing such therapeutics. Our study was aimed to delineate the role of a phytochemical, Arjunolic Acid (AA), a triterpenoid extracted from the bark of Terminalia arjuna plant in amelioration of pathological level of collagen synthesis and associated cardiac hypertrophic remodelling. Administration of AA during hypertrophy significantly regressed collagen transcription with concomitant inhibition of hypertrophy marker genes along with significant betterment in cardiac function. Previous studies although indicated an affinity of triterpenoids to interact with PPAR family proteins, specific molecular targets of Arjunolic Acid remained unidentified. Bioinformatic analyses suggested AA to be an agonistic ligand of PPAR-α which was further confirmed by biophysical characterization. Knock down of PPAR-α in AA treated hypertrophy samples reversed the beneficial effects of Arjunolic acid establishing that its antifibrotic role is principally contributed by increased expression of PPAR-α during cardiac hypertrophy with overall regression of TGF-β pathway. Arjunolic acid treatment in TGF-β treated fibroblasts also suggested that AA principally affects the noncanonical branch of TGF-β signalling possibly via modulation of TAK-1 activity. Further, TAK-1 showed pronounced interaction with the N terminal transactivation domain (AF-1) of PPAR-α that can be correlated with significantly higher attenuation of collagen gene expression. Our study thus proposes a novel signalling mechanism showing PPAR-α as the molecular target of AA and subsequently deciphers mechanism of PPAR-α driven regression of cardiac fibrosis via inhibition of non-canonical TGF-β axis.
Association of mirSNP rs11174811 in AVPR1A Gene with Arterial Blood Pressure in Coronary Artery Disease Patients from South India
Linda Koshy1, S. V. Vijayalekshmi, S. Harikrishnan2, V. Raman Kutty3, V. T. Jissa3, A. Jayakumaran Nair1, A. Gangaprasad4, G. M. Nair1, P. R. Sudhakaran1
1Inter-University Centre for Genomics and Gene Technology, University of Kerala,2 Sree Chitra Tirunal Institute for Medical Sciences and Technology,3 Achutha Menon Centre for Health Science Studies, Sree Chitra Tirunal Institute for Medical Sciences and Technology,4 Department of Botany, University of Kerala, Thiruvananthapuram, Kerala, India
Persistently elevated blood pressure is a risk factor for cardiovascular disease and death. Recent post-GWAS studies have identified genetic mutations in non-coding regions that have the capacity to regulate gene-expression through mechanisms that interfere with miRNA recruitment. MirSNPs, a novel class of human polymorphisms located within the miRNA recognition sites in 3'UTR region of target genes can create, destroy or modulate miRNA-mRNA binding, interfere with target protein translation, and thereby serve as functional candidates for eQTL analysis. Supporting the mechanistic role of mirSNPs in influencing arterial blood pressure, a mutation in the AVPR1A gene was associated with increased blood pressure in a Caucasian population. Individuals homozygous for the T-allele of the mirSNP rs11174811 in the AVPR1A gene showed an increase of ~16 mm Hg in systolic and ~7 mm Hg in diastolic pressure compared to those homozygous for the G-allele (Nossent et al., 2011). The AVPR1A gene encodes a G-protein coupled receptor that binds to arginine vasopressin, a nonapeptide hormone secreted by the hypothalamus in response to hypotension, and regulates fluid balance and blood pressure in vascular smooth muscles. Elevated circulating levels of arginine vasopressin have been associated with chronic heart failure in humans and animal models. Despite experimental evidences implicating the AVPR1A gene as a plausible candidate gene in the regulating blood pressure, few studies have attempted to replicate the association of mirSNP rs11174811 with elevated BP in a different ethnic population. Therefore, to examine the frequency of this polymorphism among individuals of south Indian ethnicity and to investigate its association with blood pressure variation we undertook this study using a hospital based case-control design consisting of 415 known cases of hypertension (HTN) and 416 control subjects. We found that the minor allele frequency (MAF) of rs11174811 was 14.5% among controls in the south Indian population, which was similar to that reported in the SMILE study by Nossent et al. (14.6%). Analyses of the coefficients of the censored normal regression was done after adjustment for anti-hypertensive treatment therapy and other risk factors such as age, BMI, dyslipidemia, family history of HTN and hypothyroidism. Our results indicated that among female patients the presence of the AVPR1 mirSNP was significantly associated with an increase of ~17 mm Hg in systolic (p = 0.026) and ~8 mm Hg in diastolic blood pressure (p = 0.027). This implies that common mirSNPs in RAAS-pathway genes can influence blood pressure variation, albeit in a gender-specific manner.
Cytochrome b5 Reductase 3 Can be Targeted in Modulating Nitric Oxide-hemoglobin Interaction to Counter Hypertension
Gaurav Kumar1, Sanjay Kumar Dey1, Priti Shenoy2, Suman Kundu1
1University of Delhi, New Delhi,2 Ramnarain Ruia College, Mumbai, Maharashtra, India
Hypertension or high blood pressure is attributed as a critical health problem. Currently available therapeutics are indeed effective in combating hypertension, but are not always bereft of complications. In addition, new and novel drugs are sparse and all the available drugs target the renin-angiotensin pathway. Therefore, there is scope for alternate strategies to deal with hypertension. As a step forward, we targeted hsCYB5R3 enzyme as a therapeutic protein, since this enzyme is involved in modulating the bioavailability of nitric oxide, which in turn act as a potent vasodilator, and hence is involved in governing the blood vessel tone which is an important aspect for treating hypertension.1] In the present investigation, small molecule databases were virtually screened against the crystal structure of the enzyme to identify potential inhibitors as well as to obtain binding energies and inhibitory constants. The enzyme was purified to homogeneity from recombinant sources and the inhibitors so obtained were tested in vitro using potassium ferricyanide assay in order to calculate IC50 as a measure of inhibitor efficacy of these compounds against hsCYB5R3. Further, the effect of these inhibitors was tested using various biophysical techniques including fluorescence and CD spectroscopy so as to obtain binding affinity, number of binding sites and effect on secondary structure, respectively. Fluorescence analysis has revealed promising binding constants (in micro molar ranges) and stoichiometric ratios (≈1:1) for some of the inhibitors. CD analysis revealed that these inhibitors do not alter the secondary structure of the FAD containing enzyme. The encouraging findings have poised us to screen the inhibitors in in vivo studies.
- Straub AC, Lohman AW, Billaud M, Johnstone SR, Dwyer ST, Lee MY, et al. Endothelial cell expression of haemoglobin α regulates nitric oxide signalling. Nature 2012;491:473-7.
All-trans Retinoic Acid and Valporic Acid Suppresses the Isoproterenol Induced Cardiac Hypertrophic Growth by Modulating the NPR-A Mediated Anti-hypertrophic Signaling in H9c2 Cells In vitro
Senthamizharasi Manivasagam1, Tamilselvi Velusamy1, Divya Kumar1, Elangovan Vellaichamy1
1 University of Madras, Chennai, Tamil Nadu, India
Background: ANP/NPR-A system acts as an intrinsic negative regulator of abnormal cardiac remodeling. NPR-A mediated anti-hypertrophic signaling mechanism is down-regulated in diseased hearts. All-trans retinoic acid (ATRA) and Valporic acid (VA) have shown to induce the transcriptional activation of Npr1 gene (coding for NPR-A) in the heart. Hence, the present study was sought to determine whether the stimulation of Npr1 gene by ATRA and VA suppresses the isoproterenol (ISO) induced cardiac hypertrophy in H9c2 cells in vitro. Methods: H9c2 cells were treated with ISO (10-5 M) and co-treated with ATRA (10-5 M) and VA (10-5 M) in the presence and absence of ANP (10-8 M), respectively for 48 hours. The mRNA expression of hypertrophic markers (ANP, BNP, α-sk and β-MyHC), proto-oncogenes (c-fos, c-jun and c-myc), Npr1 and PKG-I genes were determined by RT- PCR. The protein profiling of NPR-A, PKG-I and cGMP was evaluated by Western blot, immunofluorescence and ELISA analysis. Results: ISO-treated H9c2 cells showed an increased expression of hypertrophic marker genes, and proto-oncogenes (p < 0.001), and reduced mRNA expression of Npr1 and PKG-I (p < 0.001) genes as compared with control cells. Interestingly, ATRA and VA co-treatment significantly increased the mRNA and protein expression of Npr1 (p < 0.01) and intracellular accumulation of cGMP in ISO-treated cells. Moreover, ATRA and VA co-treated H9c2 cells exhibited significant reduction in the expression of ANP, BNP, c-fos, c-jun, c-myc, α-sk, β-MyHC (p < 0.01) genes. Conclusion: Together, the present results demonstrate that ATRA and VA co-treatment normalizes the ISO-induced down-regulation of Npr1 levels thereby repressing the hypertrophic growth probably by NPR-A mediated anti-hypertrophic signaling.
Diet with High Content of Dietary Advanced Glycation End Products Induces Systemic Inflammation in Experimental Mice: Protective Role of Natural Anti-oxidants Gallic Acid and Curcumin
Boopathi Sowndhar Rajan1, Kalaiselvi Krishnan1, Elangovan Vellaichamy1
1 University of Madras, Chennai, Tamil Nadu, India
Introduction: Advanced glycation end products (AGEs)/glycotoxins are involved in the pathogenesis of various cardiovascular diseases. High intake of dietary AGEs has been linked with elevated inflammatory and pro-atherogenic responses in humans. Aim: The present study was aimed to investigate the impact of food derived AGEs on the circulation inflammatory markers levels and on the inflammatory marker gene expression in internal organs specifically cardiac tissue. In addition, the efficacy of natural polyphenols gallic acid and curcumin were tested against the dietary AGEs - induced inflammation in experimental swiss albino mice. Methodology: AIN-76 rodent diet was exposed to elevated temperature (90°C) for the production of dietary AGEs. Experimental mice were divided into six groups (n = 6/group). Group I served as control fed with basal AIN-76 diet, group II fed with AIN-76 heat exposed diet, group III fed with N-ɛ CML a known AGE, group IV and group V mice were fed with AIN-76 heat exposed diet and N-ɛ CML and co-treated with gallic acid and curcumin respectively. The circulatory cytokines levels and the mRNA and protein level expression of inflammatory marker genes were analyzed using ELISA, RT-PCR, Western Blot, immunohistochemistry methods. Results: A significant elevation in the serum cytokines and chemokines levels (p < 0.001) were seen in mice fed with AIN-76 heat exposed diet and N-ɛ CML as compared with control mice. Further, Mice fed with AIN-76 heat exposed diet exhibited a marked increase in heart weight (19.7%, p < 0.01) accompanied by a significant increase in the cardiac inflammatory marker genes and protein expression (Cox-2, TNF-α, IL-6, TGF-β and NF-κB, p < 0.001) as compared with control. Interestingly, gallic acid and curcumin co-treatment significantly reverted back the altered levels to near normal respectively. Conclusion: In conclusion, the result of the present study clearly demonstrates a positive association between AGEs and systemic inflammatory response. Increased consumption of natural polyphenols gallic acid and curcumin have the ability to neutralize the AGEs induced systemic inflammation in experimental mice.
C-type Natriuretic Peptide, Exhibits Anti-fibrotic Activity Against Isoproterenol-induced Hypertrophic Remodeling in Experimental Rats
Navvi Chandrasekar1, Senthamizharasi Manivasagam1, Anusha Tummala1, Elangovan Vellaichamy1
1 University of Madras, Chennai, Tamil Nadu, India
Background: C-type natriuretic peptide (CNP), a 22 amino acid containing cyclic peptide hormone, is a member of the natriuretic peptide family. CNP is a selective ligand for Natriuretic Peptide Receptor B (NPR-B). NPR-B is the predominantly expressed receptor in the heart, while very few studies have carried out on the cardio protective activity of CNP/NPR-B signaling system as compared with ANP/NPR-A. Hence the aim of the present study was to study the anti-fibrotic activity of CNP against ISO (isoproterenol)-induced left ventricular hypertrophy in experimental rats. Method: Left ventricular hypertrophy in swiss albino male rats induced by the chronic infusion of isoproterenol intraperitoneally at a dosage of 10 mg/kg body weight. ISO-induced rats were co-treated with CNP intravenously at 0.1 μg/kg body weight after an initial period of 3rd day of ISO treatment. The anti-fibrotic efficacy of CNP was studied in comparison with the standard drugs atenolol and valsartan. Results: ISO-induced rats exhibited left ventricular hypertrophy on 7th day, which was confirmed by macroscopic, microscopic and marker gene analysis. ISO-treated rats exhibited marked increase in HW/BW ratio (p < 0.01), as a measure of hypertrophy induction. A marked increased expression of fibrotic genes MMP-2, MMP-9, Collagen I and Collagen III were also observed in ISO-treated rats. In parallel, hypertrophic marker gene expression (α-sk, β-Myhc and ANP) were elevated by 2.5, 4.2 and 2.1 folds, respectively. The circulatory ANP level was also found to be significantly decreased in ISO treated rats. Upon CNP co-treatment circulatory ANP level was increased to near normal. A significant decrease in HW/BW ratio (p < 0.01) and fibrotic marker genes expression was observed. Conclusion: The results of present study suggests that CNP has a potential anti-fibrotic activity. CNP/NPR-B system can be considered as a potential therapeutic target for the treatment of cardiac hypertrophy and heart failure.
Identification of Natural Inhibitors of Dopamine-β-Hydroxylase to Combat Hypertension
Manisha Saini1, Sanjay Kumar Dey1, Pankaj Prabhakar1, Suman Kundu1
1 University of Delhi, New Delhi, India
With the escalation in deaths by hypertension novel drugs with lesser side effects is required; hence healing by naturopathy is indispensable. The aim is to uncover the bioactive components in plant extracts against peripheral dopamine beta hydroxylase (DBH) which is a pivotal enzyme involved in the conversion of dopamine to norepinephrine which leads to vasoconstriction and causes hypertension or cardiac hypertrophy. High throughput screening was applied to speed up the search of a novel inhibitor. Diverse natural compound libraries from available databases were docked against the active site of DBH. In addition, various plant extracts were assessed via colorimetric assay against DBH. The ones which exhibited higher or equivalent inhibitory constant as compared to the known inhibitors like Nepicastat and Disulfiram were taken forward. Furthermore reversed phase HPLC based assay was performed to determine the IC50. To validate the binding of leads and the enzyme, spectroscopic techniques like fluorescence and CD were used. Fluorescence measurements were used to determine the KD and number of binding sites in the enzyme. Then ex-vivo (HEK293 cell lines) experiments were done to investigate about toxic properties of inhibitor, if any. In the near future, we aspire to check the lead candidates in vivo most likely in SHR and DOCA-salt induced rat models to evaluate their therapeutic efficacies. Kidney, liver and brain tissues of the rats will also be examined to evaluate the dose dependent repercussions on these organs.
Inhibition of Endoplasmic Reticulum Stress - Stimulated Mitochondrial Damage by Taurourso Deoxycholic Acid in Isoproterenol Induced Myocardial Injury
Ashok Kumar Rathinavel1, Jamuna Sankar1, S. Niranjali Devaraj1
1 University of Madras, Chennai, Tamil Nadu, India
Cardiac remodeling after myocardial infarction is the instrumental cause of left ventricle enlargement which leads to progression of congestive heart failure. Persistent ischemia leads to loss of myocytes. Endoplasmic reticulum (ER) is a major regulator to induce apoptotic machinery in myocytes. In this study, we have focused on ER stress - mediated modulation of mitochondrial homeostasis through activation of apoptosis. Taurourso deoxycholic acid (TUDCA) is a chemical chaperone which inhibits ER stress. We have examined the effect of TUDCA on expression of ER stress proteins GRP-78, Eif2α and CHOP. TUDCA significantly down regulates the expression of GRP-78, Eif2α and CHOP. In our study, we found that phosphorylation of Bcl2 was inhibited by ER stress during myocardial ischemia, because phosphorylation and translocation of Bcl2 to mitochondria is necessary for suppression of pro-apoptotic machinery in the myocyte. TUDCA increases the phosphorylation of Bcl2 remarkably when compared to isoproterenol (ISO) induced myocardial injury. Furthermore, TUDCA significantly reduced the expression of Caspase-9, Caspase-3 and cleaved caspase-3. In addition, we found that significantly increased expression of Bnip3 in ISO induced group when compared to control group. These results suggest that chronic induction of cardiac injury causes severe oxidative stress leading to accumulation of misfolded or unfolded proteins in the ER lumen, which executes ER stress in the myocardium. ER permeabilization through ER stress inhibits the phosphorylation of Bcl2 and increases mitochondrial outer membrane permeability leading to myocyte death.
Keywords: Apoptosis, Bcl2, endoplasmic reticulum stress, isoproterenol, taurourso deoxycholic acid
SIRT6 Inhibits Cardiac Fibrosis by Repressing SMAD3 Transcription Factor
Jaseer Muhamed1, Faiz Ahamed1, Mahesh P. Gupta2, Nagalingam R. Sundaresan1
1 Indian Institute of Science, Bengaluru, Karnataka, India,2 Biological Sciences Division, Cardiothoracic Surgery Research Program, Pritzker School of Medicine, University of Chicago, Chicago, USA
Objective: Cardiac fibrosis is a major complication associated with aging and several cardiovascular diseases. Till now, there is no definitive therapy available for treatment of cardiac fibrosis. Our previous study identified the class-III histone deacetylase SIRT6 as a key regulator of cardiomyocyte hypertrophy. Objective of this study is to understand the role of SIRT6 in cardiac fibrosis. Methods: Cardiac fibrosis and TGF-β1 signaling was evaluated by histology and western blotting in SIRT6 deficient mice hearts. Gene expression was studied by real time RT-PCR. Neonatal cardiac fibroblasts were used for in vitro genetic experiments. TGF-β/SMAD3 signaling and α-SMA promoter activity was evaluated by luciferase assays. SIRT6 and SMAD3 interaction was probed by co-immunoprecipitation. SMAD3 occupancy in TGF-β1 promoter was investigated by chromatin immunoprecipitation (ChIP) assay. Results: Histology and or immunoblot analysis of SIRT6-/- mice heart, and SIRT6-/- fibroblasts showed increased expression of fibrotic markers like fibronectin 1, collagen 1a, and alpha smooth muscle actin (SMA). Interestingly, TGF-β/SMAD3 signaling was found to be spontaneously activated in SIRT6-/- heart and fibroblasts, which is associated with transcriptional up-regulation of TGF-β/SMAD3 signaling genes. Moreover, SIRT6 deficient cells express increased membrane associated TGF-β1 under basal conditions. On the other hand, over expression of SIRT6 in cardiac fibroblasts decreased the expression of fibrotic markers and TGF-β/SMAD3 signaling. Immunoprecipitation and Chromatin immunoprecipitation results suggested that SIRT6 interact with SMAD3 transcription factor and reduces the occupancy of SMAD3 to TGF-β1 promoter resulting in the repression of TGF-β1 promoter. Finally, inhibition of SMAD3 with specific inhibitor rescued the fibrotic phenotype of SIRT6 deficient cardiac fibroblasts.
Screening and Characterization of Indigenous Medicinal Plant Extracts Against Dopamine Beta-Hydroxylase to Combat Hypertension and Cardiac Hypertrophy
Pankaj Prabhakar1, Sanjay Kumar Dey1, Manisha Saini1, Subir Kumar Maulik2, Suman Kundu1
1 University of Delhi,2 All India Institute of Medical Science, New Delhi, India
Dopamine beta-hydroxylase (DBH) catalyzes the oxidative hydroxylation of dopamine to norepinephrine (NE). NE is the essential transmitter in the sympathetic nervous system (SNS). It has been reported that excessive sympathoadrenal activity is implicated in the pathogenesis of hypertension (HTN). Moreover, chronic hypertension can lead to cardiac hypertrophy (CH). As such, if the concentration of NE is reduced, HTN and CH can be prevented. It is reported that inhibition of DBH can reduce the level of NE and thus can be targeted to prevent HTN and CH. Presently, most of the inhibitors being investigated against this target are mainly synthetic compounds, which are often seen to exhibit higher risk of in vivo toxicity. Thus, the present study was designed to screen and characterize indigenous medicinal plant extracts against dopamine beta-hydroxylase to identify naturally occurring inhibitors of the enzyme target. Different plant extracts like aqueous extract of Terminalia arjuna, hydroalcoholic extract of Punica granatum and Emblica officinalis were screened against purified DBH from native sources and IC50 were evaluated. Interaction of extracts with DBH was further evaluated using an array of biochemical and biophysical methods (like fluorescence and CD spectroscopy). We observed that some of the plant extracts inhibited DBH activity and IC50 were calculated to be in low nanogram range. These extracts are being evaluated for their interaction with DBH to calculate binding affinity against DBH.
Role of Sodium-dependant Glucose Transporter 1, a Novel Glucose Transporter, in Ischemic Preconditioning-induced Cardioprotection
Hina Lateef Nizami1, Abhinav Kanwal1, Sanjay K. Banerjee
1Translational Health Science and Technology Institute, Faridabad, Haryana, India
Ischemic-preconditioning has been established as a protective approach against subsequent ischemia-reperfusion injury. Since impaired glucose metabolism has been observed in hearts during ischemia-reperfusion injury, and sodium-dependant glucose transporter 1 (SGLT1) has been shown to be a novel glucose transporter involved in glucose metabolism in rat heart, we speculated that it might play an important role in ischemic preconditioning-induced cardioprotection. Also, among the possible mechanisms responsible for ischemic-preconditioning induced cardioprotection, PKC activation has gained attention. In this study, we utilised ex-vivo model of myocardial ischemia-reperfusion injury (I/R), or ischemic preconditioning (IPC) followed by I/R, in presence and absence of the SGLT1 inhibitor phlorizin, to find the role of SGLT1, and its possible relationship with PKC in preconditioning-induced cardioprotection. IPC increased membrane SGLT1 expression in rat heart as observed by immunoblotting and immunohistochemistry. Significant improvement in oxidative stress, apoptosis parameters and cardiac injury was observed in I/R hearts when subjected to IPC. However, all beneficial effects of preconditioning were lost when hearts were pre-treated with phlorizin. Further, we observed increased PKC activation enhanced glucose uptake through SGLT1 and protected against subsequent I/R injury as observed by reduction of cardiac oxidative stress and apoptosis. However, the protective effects of PKC activation were blunted by SGLT1 inhibition, indicating a possible cross-talk between PKC and SGLT1 in ischemic preconditioning-induced cardioprotection. Thus, the present study provides first-reported evidence that SGLT1 plays a crucial role in ischemic preconditioning-induced cardioprotection, through activation of PKC.
Role of Mitochondrial Genes in Inflammation and Essential Hypertension
Bhargavi Natarajan1, Nitish R. Mahapatra1
1Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Mitochondria are the site of oxidative phosphorylation, generating energy in the form of ATP. Incapacity to produce the required amount of ATP (mitochondrial dysfunction) affects cardiac output and may lead to heart failure. The rate of ATP synthesis in liver and brain of SHR (Spontaneously Hypertensive Rat) is observed to be lesser than WKY (Wistar-Kyoto Normotensive rat) counterparts. Recent studies indicate that more than 50 mitochondrial genes are differentially expressed between SHR and WKY. Mitochondria, being ancestral proteobacteria, contain a 16 kb circular genome coding for 13 proteins of the electron transport chain, tRNAs and rRNAs. Mitochondrial gene expression is enabled through nuclear-encoded genes. These include genes for DNA replication (POLG – DNA polymerase, TWINKLE - helicase), transcription (POLRMT – RNA polymerase, TFAM, TFBM1 and TFBM2 – transcription factors) and tRNA processing (ELAC2 and RNAseP). We hypothesize that mitochondrial dysfunction in SHR might be, in part, due to differential expression of these genes. Therefore, we sought to obtain an expression profile of these genes in various tissues of SHR and WKY and explain the differences. We found that the expression all the aforementioned genes are differentially expressed in the liver, brain and kidney tissues of SHR. Noteably, the expression profile in polymorphonuclear cells (PMNs) revealed interesting clues about inflammation in SHR. The PMNs from blood of SHR showed high expression of DNA replication related genes (POLG, TFAM and TWINKLE) and lesser expression of transcription related genes (TFB1M, TFB2M and POLRMT) than the PMNs from WKY. This observation prompts us to hypothesize that SHR might have higher mtDNA in blood in comparison to WKY. Since high mtDNA content in blood is a trigger and a recent marker for inflammation, differential/deregulated expression of mitochondrial genes could be one of the possible causes for inflammation in SHR.”
Sulfur Metabolites from Garlic Activate SIRT-3 to Prevent Mitochondrial Dysfunction in Diabetic Heart
Parameshwar B. Katare1, Mohamed Razia Sultana2, Pankaj K. Bagul1, Raju Padiya2, Tarak Nath Khatua1, Sanjay K. Banerjee1
1 Drug Discovery Research Center, Translational Health Science and Technology Institute, Faridabad, Haryana,2 CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana, India
Energy homeostasis is crucial regulator for the cardiac complications in diabetic cardiomyopathy. Therefore, to mitigate these complications, we hypothesized whether garlic can prevent mitochondrial dysfunction in these animals. Rats were injected single dose of streptozotocin to develop hyperglycemia. Hyperglycemic rats were divided in two groups as diabetic (Dia) and diabetic + garlic (Dia + Garl). Raw garlic was administered orally at a dose of 250 mg/kg/day. Control group rats were maintained along with these groups. Altered functioning of cardiac mitochondrial enzymes involved in ETC and metabolism were altered in diabetic rats. We have observed increased levels of ROS and decreased activity of catalase and SOD in diabetic rats. TFAM, PGC-1α, MnSOD and CO1 mRNA expression were altered in diabetes. This indicates increased oxidative stress with mitochondrial dysfunctioning in diabetic heart. Raw garlic administration improved the mitochondrial enzyme activity and oxidative stress with normalizing the gene expression. SIRT-3 activity was decreased in diabetic heart. This reduced SIRT-3 activity leads to increased acetylation of MnSOD. However, administration of garlic normalized the SIRT-3 activity and reduced acetylation of MnSOD. MnSOD deacetylation increases its activity and reduced the levels of ROS in garlic administered rats. Collectively our results provide an insight into garlic's protection in T1DM cardiomyopathy through activation of SIRT3-MnSOD pathway.
GDF-11 and GDF-15 Levels in Diabetes and Diabetes with Hypertensive Patients in South India
Ramu Adela1, Soheb Anwar Mohammed1, Abhinav Kanwal1, P. Naveen Chander Reddy2, Sanjay K. Banerjee1
1 Translational Health Science and Technology Institute, Faridabad, Haryana,2 Mediciti Institute of Medical Sciences, Hyderabad, Telangana, India
Patients with type 2 diabetes mellitus have an increased risk of cardiovascular diseases. Recently, GDF-11 and GDF-15 are considered as new biomarkers for cardiovascular diseases. This study was designed to find the alteration of GDF-11 and GDF-15 levels in hypertensive patients associated with type 2 diabetes compared to diabetes and healthy group. In this study, we examined plasma GDF-11 and GDF-15 levels in control (CT), type-2 diabetes (T2DM) and type-2 diabetes with hypertension (T2DM_HTN) subjects. There was a decrease (p < 0.05) in GDF-11 levels in T2DM and T2DM_HTN group as compared with control subjects. There was no correlation between GDF-11 and Ang II, HbA1C and FBS levels. Unlike GDF-11, plasma GDF-15 and angiotensin II levels were significantly higher in T2DM (p < 0.01) and T2DM_HTN (p < 0.001) subjects as compared with the control subjects. Significant positive correlation was found between serum Ang II and plasma GDF-15 levels (r = 0.572, p < 0.0001). GDF-15 mRNA expression from blood cells was significantly elevated in T2DM_HTN (p < 0.05) when compared to control subjects but not increased in T2DM subjects. GDF-15 mRNA expression was significantly elevated in Ang II (50 nM) treated THP-1 (p < 0.001) and H9C2 (p < 0.05) cells as compared to controls. However, GDF-15 mRNA expression was not increased in THP-1 and H9C2 cells after high glucose treatment. Collectively, our data suggest that higher level of GDF-15 is associated with increased Ang II levels in diabetic patients with concurrent hypertension. The paradoxical increase of plasma GDF-15 in diabetes with hypertension may indicate compensatory changes in response to Ang II elevation.
Sirtuin 6 Regulates Glucose Homeostasis in the Heart
Danish Khan1, Subhajit Dasgupta1, Mohsen Sarikhani1, Babukrishna Maniyadath2, Faiz Ahamed1, Abhinav Dubey1, Hanudatta S. Atreya1, Ullas Kolthur-Seetharam2, Nagalingam R. Sundaresan1
1 Indian Institute of Science, Bengaluru, Karnataka,2 Tata Institute of Fundamental Research, Mumbai, Maharashtra, India
Objective: Heart relies mostly on fatty acid for its energy supplies while glucose as a minor energy source. Cardiac pathologies like cardiomyopathies, infarction, and arrhythmia often involve a significant skewedness toward glucose uptake and glycolysis accompanied by a decrease in glucose oxidation. The phenomenon is referred to as a metabolic shift. However, the molecular event(s) responsible for metabolic shift are not fully understood. In this study, we identify SIRT6 as a key regulator of metabolic shift in heart. Methods: Cardiac metabolite levels were analyzed by 2D Nuclear Magnetic Resonance (NMR). Glucose uptake was analyzed using 2- NBDG (2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose) both by confocal microscopy and flow cytometry. Confocal microscopy was performed to assess membrane proteins. Gene expression was studied by quantitative Polymerase Chain Reaction (qPCR). FoxO1 and SIRT6 interaction was assessed by co-immunoprecipitation. Chromatin Immunoprecipitation (ChIP) were performed for FoxO1 occupancy in pyruvate dehydrogenase kinase 4 (PDK4) promoter. Sea Horse flux analyzer was used for glucose oxidation analysis. Results: Metabolite analysis revealed higher amounts of glucose and lactate in heart samples of SIRT6 +/- mice compared to wild type. Similarly, SIRT6 deficient cells also showed increased glucose uptake. Biochemical assay for glucose oxidation revealed reduced oxygen consumption rate (OCR) and ATP production in SIRT6 deficient cells. Further qPCR analysis suggested transcriptional upregulation of GLUT4, GLUT1, LDHA1, PDK4 and PFKM genes in SIRT6 deficient heart indicating increased glucose uptake and impaired glucose oxidation. Interestingly, SIRT6+/- mice had increased expression of FoxO1 and its downstream target PDK4, which in turn increased the inhibitory phosphorylation on pyruvate dehydrogenase complex (p-PDH), a marker for impaired glucose oxidation and metabolic shift. On opposite, overexpression of SIRT6 had decreased FoxO1, PDK4, and p-PDH levels, while increasing the OCR and ATP production in cardiomyocytes. Further, molecular analysis revealed that FoxO1 occupancy was increased in the promoter of PDK4 in SIRT6 deficient cells compared to control cells. Finally inhibition of FoxO1 by using dominant negative form of FoxO1 rescued defects in glucose uptake, glucose oxidation and ATP production in SIRT6 deficient cardiomyocytes. Conclusion: SIRT6 deficiency induces enhanced glucose uptake and meanwhile inhibiting glucose oxidation in cardiomyocytes, thus causing the metabolic shift, a hallmark of failing heart. SIRT6 suppresses metabolic shift/remodeling by transcriptional regulation of FoxO1 transcription factor. Results suggest that activators of SIRT6 might have potential benefits in heart failure patients.
Dysregulated miR-25, -99, -155, -451 and Renin Angiotensin System in High Salt Diet – Induced Cardiac Dysfunction of Uninephrectomized Rats
Venkateswara Rao Amara1, Sunil Kumar Surapaneni1, Kulbhushan Tikoo1
1 National Institute of Pharmaceutical Education and Research, Mohali, Punjab, India
Background and Rationale: Uninephrectomy is not associated with major adverse cardiovascular or renal events both preclinically and clinically. Due to modernisation, excursion in food habits such as increased intake of salt, fructose, sucrose, carbohydrates, fats, etc. is seen at alarming rate. The deleterious effects of high salt intake are well known in normal (binephric) condition, but that of in the live kidney donors is largely unknown. Hence, the present study was undertaken to get an insight into the development and progression of cardiac dysfunction in uninephrectomized rats fed with high salt diet. Methods: Studies conducted so far started high salt diet immediately after uninephrectomy surgery, which is least likely to occur in clinical scenario. In order to exactly mimic the clinical situation, we fed the uninephrectomized rats initially with normal pellet diet for 12 weeks and then for 20 weeks with high salt diet (10% w/w NaCl). Kidney function tests (albumin, urea and creatinine), cardiovascular functions (SBP, DBP, HR, LVSP, LVEDP, +dP/dt, -dP/dt, baroreflex sensitivity) were measured at the end and histological, molecular studies (western blotting and RT-qPCR) were carried out after sacrifice. Results: Uninephrectomized rats fed with high salt diet exhibited renal and cardiovascular dysfunction manifested in the form of decreased baroreflex sensitivity, increased in vivo cardiovascular reactivity to angiotensin II. Cardiac dysfunction is associated with decreased SERCA, AMPK and increased AKT and corresponding alteration in the miRNA (miR-25, -451, -99b, -155) regulating the expression of these proteins. The pattern of circulating miRNAs is opposite to that of the tissue miRs. Conclusion: This study gives a preliminary idea on the cardiovascular and renal function of live kidney donors consuming high salt diet. Since the levels of tissue and circulating miRs do not match with each other, caution must be exercised in harnessing their role as diagnostic and prognostic markers.
SIRT6 Deacetylase is Required for Cardiac Myofibroblast Cell Death
Veena Subramanian1, J. Faiz Ahamed1, Jaseer Muhamed1, N. Ravi Sundaresan1
1 Indian Institute of Science, Bengaluru, Karnataka, India
Introduction: Fibroblasts play a major role in maintaining cardiac homeostasis by aiding in repair process post any damage to the heart. Under physiological conditions, myofibroblasts undergo programmed cell death following the completion of repair. However, myofibroblasts do not die in several forms of heart failure, but persist and proliferate, leading to fibrosis. However the mechanisms conferring survival advantage to myofibroblasts of cardiac origin are mostly unknown. In our work we studied the role of SIRT6 deacetylase in regulating myofibroblast cell death. Methods: Doxorubicin induced cell death in wild type and SIRT6-deficient cardiac fibroblasts were analysed by PI staining coupled with flow cytometry analysis. While cell viability was assessed using the standard MTT assay and live/dead assay (calcein/EtBr staining), studies on mitochondrial membrane potential were carried using TMRM and JC-1 staining. Myofibroblast markers and the protein levels of AIF and PARP-1 were measured by western blotting. PARP1 expression was analysed by quantitative-PCR. Confocal microscopy was used to assess the translocation of apoptosis inducing factor (AIF) from mitochondria to nucleus. To assess the stability of PARP-1 protein, cells were treated with cycloheximide for different time points and the protein levels were measured by western blotting. Results: We observed increased expression of myofibroblast markers in fibroblasts isolated from heart tissues of SIRT6 KO mice compared to wild type mice, suggesting that SIRT6 deficient hearts have a high proportion of resident myofibroblasts. When SIRT6 deficient fibroblasts were subjected to genotoxic stress, they showed reduced cell death and impaired mitochondrial to nuclear AIF translocation as compared to wild type controls. When we checked the protein levels of PARP-1, a key regulator of AIF translocation, we found their level to be low under SIRT6 deficient conditions. However qPCR analysis revealed that the transcript levels of PARP-1 were unaffected by SIRT6, suggesting that the regulation might not be at the transcriptional level. Interestingly, PARP-1 stability was significantly affected under SIRT6 deficient conditions. Further molecular analysis revealed that SIRT6 is required for maintaining PARP-1 acetylation, a posttranslational modification which competes with ubiquitination. Conclusion: SIRT6 deficiency impairs myofibroblast cell death and favours the persistence of myofibroblasts in heart. SIRT6 is essential for maintaining the PARP-1 acetylation that prevents its degradation; AIF translocation and myofibroblast cell death. Our study explains a plausible mechanism behind the persistence of myofibroblasts in diseased heart.
Multiple Transcriptional Factors Regulate Human Cystathionine Gamma Lyase Gene Expression
Toiba Mushtaq1, Piyushkumar R. Kapopara1, R. Gopikrishna1, Vinayak Gupta1, Abrar A. Khan1, Vikas Arige1, Nitish R. Mahapatra1
1 Indian Institute of Technology Madras, Chennai, Tamil Nadu, India
Enzymatic synthesis of H2S is mediated by three enzymes: cystathionine gamma-lyase (CTH), cystathionine beta-synthase (CBS) and 3-mercaptopyruvate 5 sulfurtransferase (3-MST). H2S is synthesized in the vasculature, heart, liver, kidney, brain, nervous system, lung, upper and lower GI tract, reproductive organs, skeletal muscle, pancreas, synovial joints, connective tissues, cochlea, and adipose tissues. H2S has recently been identified as the third gaseous messenger molecule exhibiting properties similar to nitric oxide (NO) and carbon monoxide (CO). CTH is the key enzyme that catalyzes the formation of H2S in the cardiovascular system. Accumulating evidence suggests that H2S influences a wide range of physiological and pathological processes including blood vessel relaxation, cardioprotection, atherosclerosis and hypertension. Despite the well-known role of CTH in cardiovascular pathophysiology, transcriptional regulation of CTH remains incompletely understood. In the present study, ~1 kb promoter region of human CTH (hCTH) was analyzed for putative transcription factor binding sites by using several in silico tools (viz. Consite, TFSEARCH and PROMO). Based on the predictions, various 5'-promoter deletion constructs were generated in the promoterless luciferase reporter vector pGL3-Basic. The luciferase activities of hCTH-123, hCTH-232, hCTH-275, hCTH-375, hCTH-616 and hCTH-816 constructs were similar while the hCTH-75 construct showed a dramatically diminished (~12-fold) activity as compared to the hCTH-123 construct in HEK-293 and HuH-7 cells suggesting that the-123 bp to -75 bp region harbors crucial cis- elements for basal expression of CTH. Interestingly, computational analysis of the -123 bp/-75 bp domain showed the presence of two Sp1 (-17/-8 bp and -112/-103 bp) two CREB (at -101 bp/-95 bp and -57 bp/-46 bp), one AP2-alpha (-72/-57 bp) and one NF-Y (-72/- 57 bp) binding sites. Co-transfection of the core promoter-reporter construct with Sp1 or CREB dose-dependently enhanced hCTH promoter activity. Co-transfection of the hCTH-123 and hCTH-75 promoter constructs with Sp1 revealed that both the predicted Sp1 binding sites are functional; on the other hand, between the two CREB sites only the -101 bp/-95 bp site appears to be functional. Further studies are in progress to confirm/verify the role of predicted transcription factors in regulating hCTH promoter. Taken together, multiple transcription factors seem to regulate hCTH expression. These findings provide new insights into the mechanism of H2S regulation.
Impact of Gene Polymorphisms, Platelet Reactivity, and the SYNTAX Score on 1-year Clinical Outcomes in Patients with Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention
1 Nizam's Institute of Medical Sciences, Hyderabad, Telangana, India
Background: High on-treatment platelet reactivity (HPR) has emerged as risk factor for stent thrombosis and major adverse cardiovascular events (MACE) in patients who receive Clopidogrel after percutaneous coronary intervention (PCI). the SYNTAX score (SS) has been shown to be associated with an increased risk of mortality, myocardial infarction MI, and the stent thrombosis in patients with ST-segment elevation acute coronary syndromes (ACS) undergoing PCI. We studied (1) the association between platelet reactivity and the SS for the risk of MACE in patients with ACS undergoing PCI treated with Clopidogrel; (2) the association between genetic variants involved in Clopidogrel-mediated platelet effects and the risk of MACE; (3) the incremental prognostic value of the platelet reactivity measured at several time points Methods Eighty four in-patients with ACS were enrolled. HPR was determined before PCI, at hospital discharge, and at1 month. The risk of MACE (cardiac mortality, MI, and stent thrombosis) with 1-month HPR and the SS in the period between 1 month and 1 year was investigated. The determination of platelet aggregation was done by using a platelet aggregometer >46 AU and <19 AU HPR and LPR in the setting of PCI have been defined by the (ROC) curve analyses. Genetic analysis of CYP2C19 was done by PCR-RFLP technique. Results: The relative impact of HPR, SS and genetic variants modulating Clopidogrel effects and the risk of MACE in patients with ACS undergoing PCI was studied. CYP2C19*2 was associated with HPR but was not independently associated with the risk of MACE at any point. Patients with HPR and a high SS displayed a higher risk of MACE compared with patients with HPR and a low SS. By cox regression analysis for MACE, it was found that there was 3 fold more hazard of developing MACE though statistically not significant with increase in platelet reactivity at 1 month. Conclusion: HPR is associated with increased rates of ischemic events in patients with high SS suggesting a possibility that platelet function testing could be implemented to optimise antiplatelet therapy.
Histone Deacetylase Inhibition Regulates miR-449a Levels in Skeletal Muscle Cells
Shagun Poddar1, Devesh Kesharwani1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
MicroRNAs (miRNAs) are small non-coding RNAs that regulate cellular processes by fine-tuning the levels of their target mRNAs. However, the regulatory elements determining cellular miRNA levels are not well studied. We show that histone deacetylase (HDAC) protein levels and activity are upregulated in the skeletal muscle of diabetic mice. In C2C12 skeletal muscle cells, HDAC inhibition using suberoylanilide hydroxamic acid (SAHA) altered the levels of 24 miRNAs: 15 were downregulated and 9 were upregulated. miR-449a, an intronic miRNA localized within the Cdc20b gene, while being downregulated in the skeletal muscle of diabetic mice, was the most highly upregulated during HDAC inhibition. The host gene, Cdc20b, was also significantly upregulated during HDAC inhibition. Bioinformatics analyses identified a common promoter for both Cdc20b and miR-449a that harbors major histone acetylation marks, suggesting the possibility of regulation by histone acetylation-deacetylation. These observations suggest an inverse correlation between miR-449a levels and HDAC activity, in both SAHA-treated skeletal muscle cells and db/db mice skeletal muscle. Further, in SAHA-treated C2C12 cells, we observed augmented occupancy of acetylated histones on the Cdc20b/miR- 449a promoter, which possibly promotes their upregulation. Our results provide insights into the potential regulatory role of histone acetylation of the miR-449a promoter that may regulate its expression in the diabetic skeletal muscle.
The Roles of Mitochondria-associated Endoplasmic Reticulum Membrane proteins in Endoplasmic Reticulum-Mitochondria Interaction during Pancreatic β-Cell Death
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Pancreatic β cell apoptosis is a common feature associated with both type 1 and type 2 diabetes. Several contributing factors include oxidative stress, glucotoxicity, free fatty acids and inflammatory cytokines. This programmed cell death is a highly coordinated process which includes a coordinated crosstalk between the endoplasmic reticulum (ER) and the mitochondria. Although ER and mitochondria play distinct cellular roles, these organelles also form physical interactions with each other at sites defined as mitochondria-associated ER-membranes (MAMs) that are made up of specific proteins and are essential for calcium, lipid and metabolite exchange. However, the detailed roles and mechanisms of the MAM proteins during the onset and progression of pancreatic β-cell death remains unknown. Here, we show that MAM proteins are critical during free fatty acid induced pancreatic β-cell death. MIN6 cells were incubated in the absence and presence of palmitate and the transcript levels of MAM proteins, namely Mfn1, Mfn2, Grp75, FACL4, PACS2 and Sigma-1R were evaluated. Among these, only levels of GRP75 (glucose regulated protein 75) and FACL4 were upregulated in palmitate treated cells while the levels of PACS2, Mfn1, Mfn2 and Sigma 1R were unchanged. This was also validated by Western Blot where Grp75 protein levels were significantly elevated. These suggest that Grp75 might be critical in the ER-mitochondria interaction during palmitate induced pancreatic β-cell death.
A Randomized Controlled Trial to Assess the Effectiveness of Nurse Based Heart Failure Management Programme
Mamata Rai1, Kamlesh K. Sharma1, Sandeep Seth1, Pragya Pathak1
1 All India Institute of Medical Science, New Delhi, India
Background: Heart failure (HF) is characterized by an intolerance to activities of daily living, depending on their level of HF which significantly reduces quality of life (QOL). Heart failure management involves various complex management of drugs, devices and interventions, putting everything together needs more than doctors and drugs. so, nurse based HF management program (HFMP) is a possible in improving patients' self-care, drug adherence, making them experts in heart failure self-management and help to recognize deterioration thus leads to fewer unplanned readmissions and improved quality of life. Aim: To assess the effectiveness of nurse based heart failure management programme. Methods: The study was conducted from June 2016 to December 2016 in the Cardio-Neuro-Centre, OPD, AIIMS, New Delhi in 50 HF outpatients (25 experimental and 25 controls) with mean age 39.52 ± 15.7 years NYHA class–I, II & III with 3 months follow-up. Nurse based HFMP is a set of activities which includes formal health teaching, Hriday card (which includes demographic profile, medicine chart, B.P & weight monitoring log, self-management plan and control measures), telemonitoring through a mobile application named Dhadkan app (it is telemonitoring system in android mobile, which helps to transfer subject's vital parameter record i.e. B.P, weight, heart rate) and phone call once a week. Outcome measures were QOL, drug adherence, re-admission and mortality rate. Results: The results showed QOL score among experimental group was (60.00 ± 17.51), (66.66 ± 12.26) at pre and post respectively whereas in Control group it was (51.66 ± 17.0) (53.33 ± 16.13) as measured by kansas city cardiomyopathy questionnaire (KCCQ). The drug adherence score was (17.48 ± 2.32) (15.24 ±1.36) at pre and post in experimental and (17.68 ± 2.23) (16.72 ± 1.76) in control group as measured by adherence to refills and medications scale (ARMS) after 3 months of intervention. No re-hospitalization and mortality were reported in any of the study groups. Conclusion: Nurse based HFMP was found to better in terms of improving QOL and drug adherence in the experimental group as compared to control group.
Keywords: Drug adherence, heart failure, nurse based heart failure management programme, quality of life, re-admission and mortality rate
Risk Prediction of Acute Coronary Syndrome by Circulating Soluble TREM Like Transcript 1 Level in Indian Subpopulation
Apabrita Ayan Das1, Tanima Banerjee1, Prakash Chandra Mandal1, Arun Bandyopadhyay1
1 CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
Soluble TREM like transcript 1 (sTLT1) is reported to be associated with inflammation and major processes related to atherosclerosis. This study aimed to examine the expression of sTLT1 in normal and patient population as well as to evaluate its prognostic significance. Here, ELISA and Western blot analysis were performed to check expression of sTLT1 in study groups. The results showed that sTLT1 expression was significantly higher in ACS patients and asymptomatic population than control population (p < 0.05). Moreover, correlation studies showed that sTLT1 level was not only associated with common ACS risk factors in both patient and asymptomatic group but also correlated with disease severity (LVEF, GRACE and TIMI score). Further correlation studies on sTLT1 level and total atherosclerotic burden (tunica intima-tunica media thickness) indicated a significant relation between these two variables pointing the potential of this protein as a risk predictor. ROC curve analysis for both the groups showed that sTLT1 had two definite cut off values for two separate study populations with better sensitivity and specificity. Lastly, logistic regression analysis against the risk factors showed it can act as an independent risk predictor in both patient and normal population respectively.
Genome-wide Expression Analysis in High Altitude Deep Vein Thrombosis: Insight into Hypoxia Triggered Hypercoagulation
Prabhash Kumar Jha1, Anita Sahu1, Amit Prabhakar1, Babita Kumari1, Tathagat Chatterjee2, Nitin Bajaj3, Velu Nair4, Manish Sharma1, Mohammad Zahid Ashraf1
1 Defence Institute of Physiology and Allied Sciences,2 Army Hospital (Research and Referral), New Delhi,3 Western Command Hospital, Chandimandir, Chandigarh,4 Armed Forces Medical College, Pune, Maharashtra, India
Venous thromboembolism (VTE), a multifactorial disease, is one of the major causes of death worldwide with both inherited and acquired risk factors responsible for its prospensity. High altitude (HA) also poses as an additional risk factor in the individual predisposition to VTE. Despite extensive research, the detailed molecular mechanisms underlying the development of thrombosis at HA still remain unclear. Comprehensive global expression analysis was conducted to identify genes that showed differential expression and their associated pathways in cases of venous thrombosis at high altitude relative to sea level in a representative cohort. Subsequent systematic bioinformatic analysis including enrichment analysis, pathway classification and network analysis provide comprehensive biological insights into the pathogenesis of High Altitude Deep Vein Thrombosis (HA-DVT). Both gene ontology (GO) and pathway analysis showed enrichment of genes involved in hemostasis including fibrin polymerization, platelet function and development. As anticipated, all are closely connected with regulation of blood coagulation and platelet homeostasis at the physiological level. Interestingly, 'response to hypoxia' turned out to be the most distinctive pathway in HA-DVT patients which could be an important determining factor for the pathophysiology of DVT at high altitude. Technical and biological validation of the selected genes was done on the independent set of samples using qRT-PCR which further strengthen the microaaray datasets. Our results suggest that hypoxia at HA induce alterations in the expression of genes involved in hemostasis and blood coagulation pathway, which in turn causes platelet dysfunction and thereby lead to increased susceptibility towards thrombosis. The findings of the study provide early inroads into possible mechanisms of altitude induced thrombosis.”
NLRP3 Inflammasome Complex as Key Regulator for Hypoxia Induced Venous Thrombosis
Neha Gupta1, Manish Sharma1, Tathagata Chatterjee2, Anita Sahu1, Tarun Tyagi1, Amit Prabhakar1, Mohammad Zahid Ashraf1
1 Defence Institute of Physiology and Allied Sciences,2 Army Hospital (Research and Referral), New Delhi, India
Introduction: Venous thrombosis (VT) remains the third most common cause of mortality amongst all cardiovascular conditions. Besides established genetic and acquired risk factors, exposure to hypoxic environments (such as at High Altitude or during air travel) also predispose otherwise healthy individuals to VT. Despite the recently reported role of platelets and novel regulator calpain, the exact mechanism for hypoxia induced VT remains poorly understood. Methods and Results: The inferior vena cava ligation model in combination with simulated hypobaric hypoxia rat models were used to explore the possible early factors that regulate thrombogenesis under hypoxic environments. Preliminary observations demonstrated that acute hypoxic exposures (3 h, 6 h and 24 h) accelerate both coagulation and fibrinolytic pathways & with extended exposures (≥48 h) hypercoagulation is overwhelmed by hyperfibrinolysis. Further, the functional role of hypoxia response pathways investigated using targeted real–time PCR array revealed that thrombus formation under normoxia is a function of hypoxia response pathways and this response is exacerbated under hypoxic environments. The whole transcriptome analysis of thrombus tissue using NGS approach implicated the novel role of NOD like receptors, protein 3 (NLRP3) and inflammasome complex in hypoxia induced VT. Multiple lines of evidence emerging from pharmacological inhibition of caspase-1, in vivo NLRP3 knockdown and IL-1β-neutralizing antibodies, revealed the causal involvement of inflammasome activation in augmenting VT under hypoxic settings. Moreover, the inflammasome complex was likely to be activated as an early response to hypoxia, which precedes the platelet aggregation. Interestingly, the increased expression of NLRP3, caspase-1, and IL-1β was also observed in human patients with clinically established, altitude-induced VT as compared to controls. Conclusion: The study proposes that the pro-inflammatory response generated via inflammasome activation involving 'NLRP3-caspase-1-IL-1β' axis is a key determinant for onset of VT under hypoxic conditions.
MiRNA-145 Administration Attenuates Thrombus Formation in Animal Model of Venous Thrombosis
Anita Sahu1, Prabhash Kumar Jha1, Neha Gupta1, Tarun Tyagi1, Amit Prabhakar1, Tathagata Chatterjee2, Mohammad Zahid Ashraf1
1 Defence Institute of Physiology and Allied Sciences,2 Army Hospital (Research and Referral), New Delhi, India
Introduction: Venous thromboembolism (VTE) remains the third most common cardiovascular disease (CVD) with inexplicit pathogenesis. MiRNAs are small regulatory RNAs; emerge as a promising class of therapeutics in CVDs and malignancies. Targeting differentially expressed miRNAs could represent a new approach for the treatment of VTE also. In our study miR-145 expression decreased in thrombosed IVC of animal model of venous thrombosis (VT) as well as in plasma of VT patients. We assessed the therapeutic potential of miR-145 restoration in animal model. Methods: Animals were subjected to a sham operation (n = 10) or Inferior vena cava (IVC) ligation, n = 14) for 6 h to induce thrombus formation in IVC. Animals were intravenously administered miR-145 mimic or Negative control mimic. Coagulation markers were assessed by various assays post treatment, followed by molecular and histological analyses. Results: Expression of miR-145 decreased in thrombosed IVC subjected to ligation compared to sham operated animals (~2 fold, n = 5, P < 0.01), but was increased in thrombosed IVC of animals administered with miR-145 mimic (~4 fold n = 5, P < 0.05). Restoration of miR-145 attenuated thrombus formation. Histology study of thrombus showed reduced thrombus formation and thrombus weight (35 ± 5%, n = 5, P < 0.01). Furthermore, decrease in coagulation markers - Clotting time, prothrombin time, prothrombin F1+2, (all P < 0.05, n = 4-7/group) confirmed antithrombotic effect of miR-145 treatment. To elucidate the mechanisms underlying the miR-145 mediated antithrombotic effect, we performed bioinformatics analysis, luciferase reporter assay, and western-blotting. Our results showed that miR-145 negatively regulated tissue factor. Tissue Factor mRNA and activity were decreased in thrombosed IVC in treated animals (3 fold and 2 fold respectively, n = 5, P < 0.05). Conclusion: Therapeutic administration of miR-145 attenuates the thrombus formation, decreased thrombogenesis by downregulation of tissue factor. In conclusion this study provides the first evidence that restoration of miR-145 could represent an attractive approach for the treatment of VTE.
Molecular Mechanism behind Toxicity Caused by Excess Cysteine
Ajay Bhat1,2, Rahul Chakraborty1,2, Trayambak Basak1, Shuvadeep Maity1, Ganesh Agam1, Kausik Chakraborty1, Shantanu Sengupta1
1 CSIR-Institute of Genomics and Integrative Biology,2 Academy of Scientific and Innovative Research, New Delhi, India
Background: Elevated levels of cysteine have been shown to be associated with Vitamin B12 deficiency and cardiovascular disease in Indian population (Kumar et al 2009). Apart from cardiovascular disease high levels of cysteine is also reported to be associated with Obesity and Neurological disorders. Excess cysteine is toxic in various model organisms and its toxicity is known from more than 50 years now. However, despite the fact that cysteine can cause toxicity and is associated with many diseases the mechanism of how excess cysteine causes toxicity is not known. Using Saccharomyces cerevisiae as a model system this study is focused on delineating the detailed molecular mechanism of cysteine toxicity. Results and Discussion: Quantitative proteomics approach, iTRAQ and SWATH-MS were used to characterize the cellular response during hyper- cysteinemia. We found that Ribosomal proteins and proteins involved in amino acid biosynthesis were upregulated; however proteins involved in Glycolysis, TCA cycle were downregulated due to high levels of cysteine. Further after an un-biased screen for amino acids and TCA intermediates (including Pyruvate), we found that Leucine and pyruvate supplementation can rescue cysteine toxicity. Genetic screen of around 4800 non-essential genes was performed to identify genes required for survival in high levels of cysteine. Among the cysteine sensitive deletion strains pyruvate was unable to rescue in Δleu3 strain (Leu3 is a transcription factor regulates genes involved in branched chain amino acid biosynthesis) and leucine was not able to rescue Δncl1 deleted strain (Ncl1 is a tRNA m5C-methyltransferase, methylates tRNALeu (CAA) at the anticodon wobble position). It clearly indicates that pyruvate and leucine can alleviate cysteine induced toxicity through Leu3 and Ncl1 respectively. Intercellular amino acid measurement using o-Phtalaldehyde based derivatization revealed that hyper-cysteinemia causes an imbalance of other amino acids. Further by using s35 radiolabelling and polysome profiling we found that high levels of cysteine induces translational arrest. Conclusion: Excess cysteine leads to translational arrest and amino acid imbalance.
Understanding the Effects of Vitamin B12Restriction on Wistar Rats through Global Proteomic Profiling
Swati Varshney1,2, Arunachal Chatterjee1, Ajay Bhat1,2, R. Usha Sree3, Lovejeet Kaur3, Vislavath Jyothi3, Pujitha Kommineni4, Rakesh Mishra3, G. R. Chandak3, M. Raghunath4, Shantanu Sengupta1
1 CSIR-Institute of Genomics and Integrative Biology,2 Academy of Scientific and Innovative Research, New Delhi,3 CSIR-Centre for Cellular and Molecular Biology,4 National Institute of Nutrition, Hyderabad, Telangana, India
Vitamin B12 is a water soluble vitamin and its deficiency is common in Indian population especially in pregnant mothers. Vitamin B12 is known to affect the changes in DNA methylation patterns as it plays an important role in one carbon metabolism. Thus, in-utero vitamin B12 deficiency during early pregnancy may rewire the methylome and hence proteome of the foetus in a way that programs development of complex disorders like cardiovascular diseases and insulin resistance in later stages of life. Through this study we are interested in understanding whether changes caused by vitamin B12 deficiency are transmitted multi-generationally in pups born to vitamin restricted mother's to set the permanent change in physiology. To assess the multigenerational effect of maternal B12 deficiency, we developed the B12 deficient wistar rat female model (F0) and mated them with normal male rats. We measured biochemical and lipid levels for F1 and F2 generation pups born to B12 restricted mothers from 3-12 months. We generated the proteome profile of 3-month old F1 pups using 4-plex iTRAQ experiments performed in triplicates for liver, kidney and skeletal muscle to generate differential proteome of male and female separately. In each 4-plex, tissues from control group, vitamin B12 restricted, rehabilitated at conception, and rehabilitated at parturition were included. We identified a total of 2279, 2212, 2449 and 2199, 2182, 2379 proteins at 1%FDR in three biological replicate experiments of male and female liver respectively. We found 239 (129 up and 110 down-regulated) proteins to be differentially expressed in male liver and 317 proteins (195 up and 122 down-regulated) in female livers. Of these, 18 up-regulated and 11 down-regulated proteins were common. Similar change in protein expression is also seen in kidney and skeletal muscle as well suggesting tissue specific effect of vitamin restriction. We also observe distinct differences in protein expression among males and females suggesting sex-specific effects of maternal vitamin B12 restriction through changes in distinct pathways. This study will provide a comprehensive understanding of the effect of vitamin B12 deficiency on different tissue types.
Evaluating Global Pharmacogenomic Studies towards Personalised Therapy Guidance for Cardiovascular Diseases
Debleena Guin1, Ritushree Kukreti1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality across the globe. Since O' Reilly's breakthrough discovery on a family require high warfarin doses to achieve appropriate anticoagulation, pharmacogenomics (PGx) has anchored cardiovascular clinical research. Genetic variants have implicated their roles in certain classes of treatment used in cardiovascular medicine. These variants affect drug response to 3 classes of cardiovascular drugs: statins, platelet inhibitors and anti-coagulants. They can, therefore, explain the difference in drug response such as lipid-lowering, prevention of CVD, prevention of stroke, adverse effects like musculoskeletal side effects, and bleeding. Minimizing life-threatening adverse effects like statin- induced myopathy, thrombosis events during anti-coagulant therapy and identify patients at increased risk is the purpose of PGx. FDA approved biomarker based clinical recommendations are already available for drugs like warfarin and clopidogrel. However, further investigations addressing genomic predictors of variability in CVD drug response, translating into routine clinical practices are underway. In this regard, we conducted literature searches to provide a field synopsis of the current understanding of the genetic architecture of PGx response in different population. A total of 1420 publications were identified with functionally significant single nucleotide polymorphisms (SNPs) that show strong association with the CVD phenotype. These SNPs are associated with 298 genes involved in several pharmacological functions for 86 drugs belonging from different classes like angiotensin converting enzyme inhibitors, antiarrhythmic agents, beta-adrenergic blocking agents comprising within 132 CVDs (hypertension to heart failure, arrhythmia to stroke) which could impact population variations in drug efficacy and toxicity profiling. Several meta-analyses are warranted from this study for identification of such significantly associated variants. Evidences suggested that CYP2C19*2 and *3 being the most reproducible polymorphism with PGx relevance of clopidogrel on platelet inhibition, followed by CYP2C9*2, *3, rs9923231 (VKORC1) and rs2108622 (CYP4F2) for warfarin dosing. Other variants like I/D polymorphism in ACE1 gene in ACE inhibitor therapy, SLCO1B1*5 in statin induced side effects, 677C>T, 1298A>C in the efficacy of HMG-CoA reductase inhibitors in hypertension have been reported. The difference in drug metabolism and response due to these variants in CVD drugs can be assessed in different populations to achieve optimum therapeutic outcome. These variants and others are responsible for a fraction of the total variation seen in the treatment response to CVD. New pharmacogenomic studies like this ours will advance the ability to tailor the treatment of cardio-vascular disorders, developing ethnicity-based treatment regimen.
Keywords: Adverse effects, cardiovascular disease, drug efficacy, drug response, pharmacogenomics
Association between Toll-like Receptor Genes Polymorphism and Risk of High Altitude Induced Venous Thrombosis: Indian Population Study
Swati Sharma1, Babita Kumari1, Tathagat Chatterjee2, Nitin Bajaj3, Mohammad Z. Ashraf1, Iti Garg1
1 Defence Institute of Physiology and Allied Sciences,2 Army Hospital (Research and Referral), New Delhi,3 Western Command Hospital, Chandimandir, Chandigarh, India
Introduction: Venous thrombo-embolism (VTE) is a multifactorial disease; individual genetic background being one of the factors is potentially responsible for development of VTE along with several acquired risk factors as well as genetic pre-dispositions. The TLRs play a major role in both manifestation and elimination of diseases depending upon the recognition of specific ligand and downstream signalling therefore, the genetic variation in TLRs could be implicated for imparting varying response of individuals to discrete diseases. Methodology: Genotyping of TLR 2, TLR 4 and TLR 9 was done in patients of high altitude induced venous thrombosis (n = 25) compared to normal healthy controls (n = 40) in Indian population. Allelic sizes were determined by comparison of bands with molecular weight markers. Genotypic and Allelic frequencies were determined by gene counting and compared by 2x2 contingency table. The statistical significance of differences between patients group and control group were estimated by chi square test, fisher's exact test and calculation of odd's ratio (with 95% confidence interval) using Graph Pad (Prism). Statistical significance criteria is p < 0.05 for all tests. Results: In this study, we conducted a case-control study to investigate whether polymorphisms in the toll like receptor gene i.e. T399I SNP of TLR4 (rs 4986791), R753G SNP of TLR2 (rs 5743708) and T-1237C SNP of TLR9 (rs 5743836) are associated with high altitude induced thrombosis in Indian population. Our initial results show low frequency of mutation in these genes in Indian population; and no significant difference is seen between the patients and controls which established the fact that these SNPs are not associated with venous thrombosis in Indian population unlike in western population.
Molecular Docking Analysis to Study the Inhibitory Effect of Plant Compound in Heart Diseases
Sreshtha Mondal1, Bharti Rakhecha1, Ankita Sharma1, Sagarika Biswas1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Coronary artery disease (CAD) is a progressive inflammatory disorder of the arterial wall. The disease is characterized by focal- lipid rich deposits of atheroma. Extensive evidence indicates that environmental factors also contribute to CAD risk, incidence and severity. Additionally, CAD risk is also affected by changes in nutritional diet, lifestyle choices and different pathogens. Available drugs (aspirins, statins) exhibit various side effects such as unknown long term effect, flushing and hepatic dysfunction etc. Hence there is an urgent need to study non-toxic plant based medicines which can reduce chronic disease prevalence and mortality either in the general population or subsets of individual at high risk. Presently there are hundreds of plant based medicines for the treatment of CAD which are used as herbal or dietary supplements. The lack of prevalence of their usage has been attributed to less scientific investigation, subsequently correlating to less clinical data of such medication. Earlier, in several studies it has been found that Interleukin-6 (IL-6) a pro- inflammatory cytokine is up-regulated in CAD. IL-6 plays a central role in starting the inflammatory cascade which leads to progression of atherosclerotic lesion in vascular intima. We therefore made an attempt to carry out docking studies of IL-6 with Apigenin. Apigenin is a flavonoid compound which we tested for its toxicity and Quantitative Structure Activity Relationship (QSAR) using Lazar toxicity and Molinspiration servers respectively, along with Lipinski's filter from Super Computing Facility of Bioinformatics and Computational Biology of Indian Institute of Technology, Delhi (SCFBio, IIT-D). Docking analysis indicated that this compound have inhibitory constant 227.75 μM and binding energy -4.97 kcal/mol. The result thus implicates the possibility to become the candidate drug in the near future.
Identification of Differentially Methylated Regions in Peripheral Blood of Dilated, Hypertrophic and Restrictive Cardiomyopathy Patients by MeDIP Sequencing
Subhoshree Ghose1, Sourav Ghosh1, Anju Sharma1, Priya Tolani1, Shantanu Sengupta1, Sandeep Seth2
1 CSIR-Institute of Genomics and Integrative Biology,2 All India Institute of Medical Science, New Delhi, India
Introduction: Cardiomyopathy is a complex broad-spectrum disease of the myocardium with unknown etiology. Cardiomyopathy is majorly classified in three categories i.e. dilated (DCM), hypertrophic (HCM) and restrictive cardiomyopathy (RCM) depending on their pathophysiological phenotype. Recent studies suggest that gene environmental interactions play a major role in manifestation of these complex disorders. Here we have used MeDIP sequencing approach to look for epigenetic markers in peripheral blood of three different forms of cardiomyopathy. Methodology: Patients afflicted with DCM (n = 5), HCM (n = 5), RCM (n = 5), and controls (n = 5) were recruited in this study from AIIMS. Individual PBL DNA samples from each group were considered for Methylated DNA immunoprecipitation sequencing to look at the altered global DNA methylation pattern. Results and Discussions: We obtained more than 4 GB data in each sample. After quality control and trimming we obtained mapped reads which could align up to 97% to the hg38 genome. Further methylation peak calling was done using MACS algorithm and differentially methylated regions were identified using DiffReps. P value cut off was chosen as 0.001. We found several genes related to reverse cholesterol pathway like APOA1, APOC3, APOA4 to be differentially methylated. Moreover several extracellular matrix components like myosin heavy chain 3 gene showed altered methylation. Following KEGG analysis, we observed a change of methylation in different genes related to myocardial infarction, stenosis, hypertrophy etc. which points towards involvement of these pathways with cardiomyopathy pathogenesis. Conclusions: This study is unique because identifying DNA methylation based markers in peripheral blood would circumvent the complications related to identifying markers in tissue biopsies.
Altered Plasma Proteome Promotes Venous Thromboembolism at High Altitude
Amit Prabhakar1, Tarun Tyagi1, Swati Varshney2, Prosenjit Ganguli3, Surinder Pal Singh4, Nitin Bajaj5, Shantanu Sengupta2, Velu Nair3, Mohammad Zahid Ashraf1
1 Defence Institute of Physiology and Allied Sciences,2 CSIR-Institute of Genomics and Integrative Biology,3 Army Hospital (Research and Referral), New Delhi,4 Armed Forces Medical College, Pune, Maharashtra,5 Western Command Hospital, Chandimandir, Chandigarh, India
Venous thromboembolism (VTE) is a broad spectrum disorder arises due to multiple genetic or acquired predisposing factors. In the recent years, high altitude (HA) has been recognized as a probable risk factor for VTE. Despite the several reports suggesting exposure to HA hypoxia contributes to the development of a prothrombotic milieu, the association between high altitude and incidences of VTE have been the subject of continuing debate. Though the traditional risk factors are useful in diagnosis, yet lack the desired predictive precision. Thus, we aimed to identify differentially expressed plasma proteins in the persons who developed the disease at HA in order to identify novel markers for VTE. HA-VTE patients (n = 3) and comparable exposed controls (n = 3) subjected to 8-plex iTRAQ based quantitative proteomics analysis in the set of three biological replicates. Differentially expressed proteins (both up and down regulated), common to all three sets, were validated in more HA-VTE patients and HA-exposed controls. Proteins related to blood coagulation, platelet activation and ECM receptor interaction were highly expressed and common downregulated proteins were related to complement activation and lipoprotein metabolism. Some proteins obtained here have straight or subsidiary impact on cardiovascular system and might be significantly associated with HA-VTE onset. These findings provide proof of concept for altered expression of plasma proteome on HA exposure promotes prothrombotic state.
Pleomorphic Manifestations of Sodium Channelopathy in a Family
Soumya R. Mahapatra1, Praloy Chakraborty1, H. S. Isser1, Sudheer Arava2, Kausik Mandal3
1 Vardhman Mahavir Medical College, Safdarjang Hospital,2 All India Institute of Medical Science, New Delhi,3 Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Introduction: Cardiac Channelopathy affecting sodium channel mutation may have multiple clinical manifestations. Results: A 50 year old female presented with sustained monomorphic ventricular tachycardia requiring DC cardioversion. Post cardioversion she developed bradycardia with irregular ventricular rate and no visible P wave. Echocardiography showed dilatation of right atrium (RA) and right ventricle (RV). Invasive electrophysiological testing showed presence atrial electrogram but despite highest output pacing, right atrium could not be captured. Son of the proband (30 year, male) presented with history of syncope. Surface ECG revealed absent P wave. Echocardiography showed dilatation of RA and RV. Invasive electrophysiological testing was suggestive of atrial standstill. A histological specimen from the right ventricular endocardium was normal. Sister of proband (38 year female) presented with intermittent atrial fibrillation and incomplete RBBB. 24 hours monitoring showed long sinus pauses. Echocardiography showed dilated RA and RV. Invasive electrophysiological testing showed electrically active right atrium. Son of the proband was subjected to genetic analysis by Next Generation sequencing (NGS) technique, which showed that the individual harbours variation in SCN5A (p. Asp1275Asn) and BRCA1 (p. Ser1503Ter) gene. Mutation analysis study showed that proband and her sister harbour the same variation in SCN5A and BRCA1. Apart from LQTS3 and Brugada syndrome, SCN5A mutation can cause inherited progressive cardiac conduction defect (PCCD), sick sinus syndrome, atrial fibrillation, dilated cardiomyopathy and atrial standstill. Overlap syndromes of cardiac sodium Channelopathy consisting of multiple rhythm disturbances within one family, as in our case have also been reported. Concurrent role of BRCA1 mutation is not clear. BRCA1 mutation has been reported to be associated with increased cardiac apoptosis. Whether it is implicated with the disease manifestations (direct or as genetic modifier) or it is an incidental polymorphism, requires further research. Conclusion: We report a family presenting with various arrhythmias associated with SCN5A and BRCA1.
Two Different Spectrums of Arrhythmogenic Cardiomyopathy
Deepak Tewari1, Hermohander S. Isser2, Praloy Chakraborty2, Sudheer Arava3
1 V.V.V. College for Women, Virudhunagar, Tamil Nadu,2 Vardhman Mahavir Medical College, Safdarjang Hospital,3 All India Institute of Medical Science, New Delhi, India
Background: Although classically known as ARVC/ARVD, Arrhythmogenic cardiomyopathy can affect both ventricles, RV only and rarely LV only. We present two cases of different spectrum, one of the common types (biventricular type) and another rare type (Left dominant type). Patients and Methods: Case 1: 64 years female patient presented with VT of RV origin. Echocardiography documented LVEF = 50%. Coronary angiography was normal. Contrast enhanced cardiac MRI showed mild biventricular dysfunction and late gadolinium enhancement (LGE) distributed in both ventricles. Endomyocardial Biopsy (EMB) shoed fibro fatty changes and myocyte loss. Case 2: 45 years male presented with VT from LV. Echocardiography and coronary angiography was normal. CMRI showed normal biventricular function with patchy LGE involving LV and IVS. EMB from IVS showed shoed fibro fatty changes and myocyte loss. A diagnosis of left dominant Arrhythmogenic cardiomyopathy (LDAC) was made. The individual showed a variation in TMEM 43 gene (p. Thr277Ser). Discussion: Arrhythmogenic cardiomyopathy is a rare form of inherited cardiomyopathy characterized by fibro fatty replacement and myocyte loss. It presents with ventricular arrhythmias with or without features of ventricular dysfunction. The commonest form involves the free wall of right ventricle and known as ARVC/D however in later stage in affects the LV also. Isolated involvement of LV is rare and occurs in less than 5% cases. The disease is caused by mutation in desmosomal genes. TMEM 43 is is a highly conserved inner nuclear membrane (INM) protein. It has been hypothesized that mutant TMEM43 protein would disrupt structure and function of desmocollin-2, desmoglein-2, desmoplakin, and junctional plakoglobin, leading to Arrhythmogenic cardiomyopathy.
Clinical and Angiographic Profile of Coronary Artery Disease in Women
Soumya R. Mahapatra1, Sandeep Bansal1, H. S. Isser1, Praloy Chakraborty1, Preeti Gupta1
1 Vardhman Mahavir Medical College, Safdarjang Hospital, New Delhi, India
Background: Coronary artery disease is traditionally considered as disease of “Male”. However there is growing evidence that death from coronary disease is increasing in women. As per Centers for Disease Control (CDC) data mortality from CAD (38%) is higher than cancer death (22%). The data regarding clinical and angiographic profile of CAD in women is limited. Aims and Objectives: To look for the difference of clinical and angiographic profile of CAD in women compared to male. Materials and Methods: 1000 consecutive patients with CAD who underwent coronary angiography included in this observational the study. Data were collected on demographic profile, Clinical presentation, risk factors, socioeconomic status using a structured interview schedule. After detailed clinical evaluation the patients underwent coronary angiography. Results: Among 1000 studied patients 30% were female (n = 300). Mean age of female (57.2 years) was not different from male (53.2 years). Although the peak incidence at around 51-60 years was found in both the groups, CAD was more prevalent in female age >50 yrs. Risk factors like Diabetes (38.3% vs 22.7%, p < 0.001), Hypertension (46.1% vs 27.3%, p < 0.001) and Family history of Premature CAD (2.3% vs 0.6%, p = 0.014) were more prevalent among women, whereas smoking (14.3 vs 67.9, p < 0.001) and obesity (48.7 vs 55.6, p =.044) were significantly more common in men. Chronic stable angina as presenting symptom was more common in women (33.3% vs 22.6%) whereas of acute coronary syndrome was higher in males (77.4% vs 66.7%). STEMI and NSTEMI were commonest form of ACS in males and females respectively. On comparing the angiographic data, more women had normal coronary arteries (21.6% vs 8.6% p < 0.001) whereas prevalence of obstructive CAD was higher among men (78.5% vs 69.4% p < 0.001). LV dysfunction (LVEF <55%) was more prevalent in male (62.3% vs 50.3% p = 0.001). Significantly greater subset of men received thrombolytic therapy than women (21% vs 9.6% p < 0.001). Any intervention in form of PCI or CABG at admission or within 3 months of follow up was significantly greater in men than in women (40% vs 28.3% p < 0.001). Conclusions: CAD is significantly prevalent in women especially in elderly age group. Women with CAD differ in risk factors, clinical and angiographic profile from their male counterpart.
Genetic Polymorphism in Cytochrome P450 and Vitamin K Reductase Enzyme Genes Predict the Risk of Venous Thromboembolism in Male Indian Population
Babita Kumari1, Amit Prabhakar1, Anita Sahu1, Tathagata Chatterjee2, Nitin Bajaj3, Mohammad Zahid Ashraf1
1 Defence Institute of Physiology and Allied Sciences,2 Army Hospital (Research and Referral), New Delhi,3 Western Command Hospital, Chandimandir, Chandigarh, India
Genes related to Cytochrome 450 enzymes function (CYPs) and vitamin K epoxide reductase complex are thought to be responsible for the regulation of vascular functions. Available reports on animal model and a limited human studies suggests the involvement of these genes in different insults to the heart, inhibiting inflammation and blood vessel formation. Thus, to decipher the role of CYP genes polymorphism and association with other polymorphisms to predict the risk for venous thromboembolism (VTE), we studied 40 VTE patients and 40 healthy controls. CYP2C9 *2 allele, CYP2C9 *3 allele, CYP4V2 (rs13146272), CYPV2 (rs3736456), VCoRC1 1639G/A, VCoRC1 1173C/T and VCoRC1 497C/T polymorphisms were genotyped by restriction fragment length polymorphism (RFLP). The genotype and allele frequency between control and VTE patients were significantly different with respect to CYP2C9 *2 allele, CYP2C9 *3 allele, and VCoRC1 1173C/T polymorphisms. Logistic regression model analysis indicated the significant association of these polymorphisms with VTE risk. Significant linkage disequilibrium was observed with p < 0.05. Additionally, different haplotypes were significantly associated with the responses for all the SNPs. These results indicate that CYP and VCoRC1 gene polymorphisms may play a significant role in individual's susceptibility to the VTE and its clinical progression.
Age-wise Identification of Global Proteomic Changes Associated with Ischemia in Rat Model
Trayambak Basak1, Arkadeep Mitra2, Swati Varshney1, Ritwik Datta2, Arunachal Chatterjee1, Khushboo Adlakha1, Sagartirtha Sarkar2, Shantanu Sengupta1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi,2 University of Calcutta, Kolkata, West Bengal, India
Myocardial ischemia has been one of the major threats to human population nowadays. This causes myocardial infarction (MI) and has been critical for optimal recognition and timely management in patients of different age groups since ageing itself contributes as a significant risk factor in this disease. Very limited studies have shown to be implicated in age specific alterations in heart with respect to MI and hence potential age specific MI markers are yet to be recognized. Here in this study we have tried to identify novel age specific proteomic markers in a murine model of MI and elucidate major pathways that are affected exclusively in different age groups of MI model. Male Wister rats of 9 and 70 weeks old were used to develop MI by occlusion of left anterior descending coronary artery (LAD) for 10 days. Age and sex matched rats were sham operated to use as controls. iTRAQ based quantitative LC-MS proteomic workflow was undertaken to identify the differentially expressed proteins. We identified a total of 1160 proteins in four different groups (9 weeks and 70 weeks MI and control) at 1% FDR. A total of 174 and 205 differentially expressed proteins were identified in 9 weeks MI and 70 weeks MI tissue respectively when compared with their respective age and sex matched controls. A total of 78 and 109 proteins were altered exclusively in 9 weeks and 70 weeks MI groups respectively compared to their age-matched controls. ER-stress induced apoptotic mechanism was found to be a significant regulator for age dependent post-MI consequences. Biological pathway prediction revealed enrichment of different pathways in these two different age group of MI model which suggests that recovery from stress due to MI at early age are more conspicuous than late age MI. This study would help us to identify novel age specific markers of MI and discover the specific pathways which are affected exclusively in each age group of MI.
Liquid Chromatography-Mass Spectrometry Based Investigation of Plasma Lipidomics
Mainak Dutta1, Akash Kumar Bhaskar1, Dipankar Malakar2, Shantanu Sengupta1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi,2 Ab Sciex, Gurgaon, Haryana, India
Lipidomics is a field to characterize the lipid compositions of biological samples including biofluids. This approach of making quantitative, comprehensive molecular measurements of lipids provides access to the understanding of various biological processes. Lipid compositions of plasma are complex, reflecting a wide range of diversity and concentration of different lipid classes. Their varying degree of chemical complexity makes their elution from liquid chromatography challenging. Also, several lipid species are present as isobars making their interpretation in mass spectrometry difficult. Here, we performed targeted liquid chromatography-mass spectrometry (LC-MS) with multiple reaction monitoring (MRM) for 212 plasma lipid species in positive mode. We have compared two different LC protocols: (A) Methanol/water and (B) IPA/ACN/water. Mass spectrometry parameters such as declustering potential (DP), entrance potential (EP), collision cell exit potential (CXP) and collision energy (CE) were optimized for each lipid class. High resolution MS/MS was generated to identify the isobaric peaks by fragmenting the two most intense peaks with trap fill time of 10 ms and Q0 trapping turned on. Isobaric peaks were identified based on neutral loss of fatty acyl groups from the parent ion. Technical variation was assessed using triplicate measurements. Our results indicate that though IPA has relatively poorer ionization efficiency than methanol, IPA/ACN/water protocol provides better peak shape. Further, both neutral and phospholipids can be detected in a single LC run. Also, internal standards show relatively poorer linearity with increasing concentration in protocol A than in protocol B indicating that the latter helps in better elution of lipids from the column. Our study with the use of technical replicates highlights the importance of the range of biological and technical variation observed in plasma lipids analysis.
Genomics for Understanding Rare Diseases India Alliance Network
Shamsudheen Karuthedath Vellarikkal1, Ankit Verma1, Rijith Jayarajan1, Vinod Scaria1, Sridhar Sivasubbu1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Rare genetic diseases affect close to 70 million people in India. Accurate diagnosis of rare genetic diseases is challenged by the overlapping phenotypes, non-availability of resources and issues with scalability. Large-scale systematic efforts to address this issue are still in their infancy in India. We have created a unique collaborative programme, GUaRDIAN, a framework in health care planning, implementation and delivery in the area of rare genetic diseases. GUaRDIAN leverages the power of genomics for systematic characterization and diagnosis of rare genetic diseases in India. The genome sequence data analysis pipeline and interpretation engine forms the heart of the programme. This engine heavily relies on open source architectures, datasets, tools and resources, to enable easy replication, scalability and implementation in clinical setups. The GUaRDIAN consortium is also sensitive to ethical, cultural and legal framework, which is strongly anchored on the basic principles of beneficence, reciprocity, justice and professional responsibility. The GUaRDIAN consortia has already established a pilot network presently involving 25 hospitals, mostly tertiary care medical centers and teaching hospitals involving over 100 clinicians across the country. This pilot network has been able to solve several cases of undiagnosed diseases and in many cases, able to identify novel variations associated with rare genetic diseases. GUaRDIAN offers a unique opportunity for clinicians to refer cases of rare genetic diseases and get appropriate high-quality assessment of the genomic variants. The network also provides a motivating forum for interaction and exchange of ideas and knowledge. For more information, visit http://guardian.meragenome.com/.
Whole Exome Sequencing Identifies a Novel Mutation in Sodium Voltage-gated Channel Beta Subunit 4 in a Child with Long QT Syndrome 10 and Congenital Complete Heart Block
Rajnish Juneja1, Ankit Verma2, Neeraj Parakh1, Nitish Naik1, Gautam Sharma1, Shamsudheen Karuthedath Vellarikkal2, Rijith Jayarajan2, Rowmika Ravi2, Vinod Scaria2, Sridhar Sivasubbu2
1 Department of Cardiology, All India Institute of Medical Sciences,2 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Long QT syndrome 10 is among the very rare forms of Long QT syndrome described sparingly. Phenotypically it is supposed to mimic LQTS 3 and is associated with sodium channel dysfunction due to defective Sodium Voltage-Gated Channel Beta Subunit 4 (SCN4B)gene. Here, we present a case of a boy from Punjabi family, presented with recurrent “seizures” on crying and immune mediated congenital complete heart block in 2009. The child had bradycardia related polymorphic Ventricular Tachycardia and a prolonged QT. An ECG of 2008 showed QT of 480 and QTC of 537 ms. His mother apart from being positive for anti Ro and anti La antibodies had history of documented seizures with an MRI showing demyelinating lesions in the brain. She, her husband and paternal grandmother had a normal ECG. The child was given an endocardial VVIR pacemaker and put on beta blockers after which he stopped having “seizures”. We performed whole exome sequencing using trio based approach. Exome data analysis pipeline revealed a heterozygous frameshift deletion (c.512delG; p.G171fs) in exon 4 of SCN4B gene, which is predicted to be deleterious by SIFT Indel tool. Sanger sequencing analysis confirms that the variation is present in affected child and mother but absent in unaffected father and paternal grandmother. The present variation is not present in 1000 genome, ExAC, and internal control database, suggesting that the mutation is novel. The child is now nearly 11 years old and is on extended release Propranolol 480 mg/day.
Integrative Analysis of Whole Exome Sequence Data to Aid Diagnosis of a Case of Autosomal Recessive Dilated Cardiomyopathy
Bibhas Kar1, Shamsudheen Karuthedath Vellarikkal2,3, K. Sivakumar, S. Sivamani, Rajesh Kumar, Ankit Verma2, Anoop Kumar2, Rijith Jayarajan2, Rowmika Ravi2, Ambily Sivadas3,4, Vinod Scaria3,4, Sridhar Sivasubbu2,3
1 Centre for Genetic Studies and Research, The Madras Medical Mission, Chennai, Tamil Nadu,2 Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology,3 Academy of Scientific and Innovative Research,4 GN Ramachandran Knowledge Center for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Dilated cardiomyopathy has known to have genetic basis implying the involvement of number of genes. Majorly, dilated cardiomyopathy is inherited in an autosomal dominant pattern. Familial cases of dilated cardiomyopathy could also inherit in an autosomal recessive, X linked or mitochondrial inheritance, making the accurate genetic diagnosis a tedious, time consuming and costly workup. Recently, the advancement in the area of next generation sequencing has enabled to identify and understand the nature of genetic variations in an affected individuals and thus in turn allow downstream processing. In the present report, we have utilised whole exome sequencing to identify the genetic mutation in an infant from South India, clinically diagnosed with dilated cardiomyopathy. The patient had familial history of multiple siblings death but the parents were phenotypically normal. We have performed whole exome sequencing using trio based approach to prioritise variants. The variant prioritization revealed a novel variant p.Q208* in myosin-binding protein C gene (MYBPC3). The variant was homozygous in the proband and heterozygous in the parents, showing the inheritance in autosomal recessive manner. The mutation introduces a premature stop codon in the C1 domain of cMyBP-C, which possibly truncate the protein. This domain is required for interaction with other proteins and probably in its absence protein will no longer be functional. The variant was further validated using Sanger sequencing in the trio.
Whole Exome Sequencing Resolves Diagnostic Dilemma in a Rare Case of Cardiomyopathy with Ataxia
Sreejith Valappil1, Shamsudheen Karuthedath Vellarikkal2,3, Rowmika Ravi2, Rijith Jayarajan2, Chakanalil Govindan Sajeev1, Gopalan Nair Rajesh1, Ankit Verma2, Anoop Kumar2, Ajay Garg4, Varun Suroliya2, Mohammed Faruq2,3, Srinivasan Ramachandran2,5, Sridhar Sivasubbu2,3, Vinod Scaria2,5
1 Department of Cardiology, Government Medical College, Kozhikode, Kerala,2 Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology,3 Academy of Scientific and Innovative Research,4 Department of Neuroradiology, All India Institute of Medical Science,5 GN Ramachandran Knowledge Center for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Cardiomyopathies with ataxias are rare clinical presentations and often associated with a genetic etiology. Though there is no cure for the disease in many cases, clinical presentations can be managed if accurate diagnosis is made early in life. However the heterogeneity in both etiology and phenotype makes disease diagnosis often challenging. Here we describe a clinically challenging case of an extreme cardiomyopathy with multiple phenotypic manifestations including high fever, extreme cardiomyopathy, progressive ataxia and recurrent seizures. The clinically characterized patient was subjected to a systematic genetic analysis for ataxia including common mutation screening, whole mitochondrial and whole exome sequencing. We identified a novel non-synonymous variation (c.419C>T, p.S140F) in the exon 3 of alpha tocopherol transfer protein gene (TTPA), which enabled the clinician to successfully arrive at a right diagnosis. The variant was further technically validated in the family and in silico modelled in order to determine the effect of the variant. Current study reports the first genetically characterized rare ataxia with vitamin E deficiency from India.
Nuclear–Mitochondrial Cross-talk: Implications in Mitochondrial Function and Disease Pathophysiology
K. V. Shamsudheen1,2, Ankit Sabharwal1,2, Ranjith Kumar3, Ankit Verma1, Nitin Dhochak3, Anushree Mishra3, Neerja Gupta3, Sheffali Gulati3, Vinod Scaria4,2, Madhulika Kabra3, Sridhar Sivasubbu1,2
1 Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology,2 Academy of Scientific and Innovative Research,3 Department of Pediatrics, All India Institute of Medical Sciences,4 GN Ramachandran Knowledge Center for Genome Informatics, CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Mitochondria are cellular organelles involved in a variety of biological functions in the cell, apart from their principal role in generation of ATP. Mitochondrion, in spite of being a compact organelle, is capable of performing complex biological functions largely because of its ability to exchange proteins, RNA, chemical metabolites and other biomolecules between cellular compartments. A close network of biomolecular interactions is known to modulate the crosstalk between the mitochondria and the nuclear genome. Apart from the small repertoire of genes encoded by the mitochondrial genome, it is now known that the functionality of the organelle is highly reliant on a number of proteins encoded by the nuclear genome, which localize to the mitochondria. However, not much is known about the functional role of the nuclear encoded proteins in the pathophysiology of the human mitochondrial disorders. We describe a case study of a 9-month-old female who presented with global developmental delay, visual problems and regression of attained mile stones since 6 months of age. On examination she had microcephaly central hypotonia and nystagmus. MRI of brain showed hyperintense symmetrical lesions in crus cerebri and brain stem tracts. MRS showed lactate peaks in the involved regions. Blood lactate was elevated multiple times. A high Nijmegan score (>9) and other evidences suggested a possible mitochondrial disease. Therefore, we attempted whole mitochondrial sequencing of mother and child. Analysis using our mitomatic pipeline ruled out any mitochondrial disease associated variations. Further, we performed whole exome analysis on the trio (mother, father and child) to identify any disease causing variations. Coverage based analysis revealed a deletion on chromosome 5, only in the proband suggesting a de novo deletion. Whole genome sequencing of the proband accurately mapped the deletion boundary of ~150 kb on chromosome 5, encompassing genes responsible for mitochondrial function and DNA damage repair. The synteny of these genes is conserved in model organisms such as zebrafish and mouse, allowing us to investigate the role of this deletion in pathophysiology of the disease. Our study provides insights into a relatively unexplored layer of biomolecular pathways modulating mitochondrial-nuclear cross-talk and its role in human diseases.
Long Noncoding RNAs Regulating Vascular Development in Zebrafish
Paras Sehgal1,2, Samatha Mathew1,2, Ambily Sivadas1,2, K. V. Shamsudeen1,2, Rijith Jayarajan1,2, Ankit Verma1,2, Archana Vats1, Vinod Scaria1, Sridhar Sivasubbu1
1 Genomics and Molecular Medicine Unit, CSIR-Institute of Genomics and Integrative Biology,2 Academy of Scientific and Innovative Research, New Delhi, India
A functional vascular system is essential for the growth and development of vertebrates. Endothelial cells (ECs), which are key components of vasculature, create a semi- permeable barrier to control the supply of nutrients and oxygen to surrounding tissues. EC organization is modulated by various transcriptional cues. Aberrant regulation of these signals leads to impaired organ development and disease conditions. Inquisitive studies of eukaryotic transcriptomes have led to the discovery of long non-coding RNAs (lncRNAs). LncRNAs exhibit cell specific expression as well as restricted subcellular distribution. Role of lncRNAs in EC development has been of keen interest to us. In this study we identified over 3,000 EC enriched unique and novel lncRNAs using transgenic zebrafish Tg (fli1a:EGFP, gata1a:DsRed) as a template. of these, about 150 lncRNAs displayed significantly high enrichment in the ECs. We confirmed endothelial specific expression of candidate lncRNAs using RT-PCR and WISH studies. The overexpression of a candidate lncRNA induced the sprouting of SIV vessels in zebrafish embryos. TALEN-mediated knockout of the candidate lncRNA resulted in vascular defects in zebrafish embryos. A stable zebrafish mutant of the candidate lncRNA transmitted the genotype and hemorrhage phenotype to subsequent generations. RNA sequencing analysis of lncRNA mutant zebrafish embryo revealed differential expression of genes involved in EC function, suggesting that their function is being attuned by the candidate lncRNA. Cellular fractionation followed by RT-PCR analysis hinted that the candidate lncRNA localizes within the chromatin fraction. In order to further understand the molecular networks of the lncRNA, we adopted a RNA pull down based approach to identify interacting proteins. The characterization of few candidate proteins is ongoing. Our study opens up possibilities of exploring the role of lncRNAs in the regulation of EC development and could potentially provide leads for better understanding of regulatory mechanisms during angiogenesis in humans.
T2DiACoD: A Gene Atlas of Type 2 Diabetes Mellitus Associated Complex Disorders
Namita Singh1, Jyoti Rani1, Inna Mittal1, Atreyi Pramanik1, Namita Dube1, Smriti Sharma1, Bhanwar Lal Puniya1, Muthukurussi Varieth Raghunandanan1, Ahmed Mobeen1, Srinivasan Ramachandran1
1 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Purpose: Type 2 diabetes mellitus (T2DM) is characterized by hyperglycaemia due to insulin resistance and decreased insulin activity. Genetic factors, environmental agents and their interactions have been recognized as contributors to the development of T2DM. Hyperglycaemic conditions in late stages of diabetic individuals include well known microvascular complications (nephropathy, neuropathy, retinopathy) and macrovascular complications (atherosclerosis and cardiovascular). Here our goal is to organize the dispersed information on the genes reportedly associated with complications in T2DM conditions accumulating in the literature. we are identifying the genes implicated in T2DM associated complications that can serve for identifying drug targets, potential biomarkers and clinical applications. Methods: Extensive studies are being conducted world-wide to elucidate the associated genes and their variants and aberrant pathways underlying the disease development. The information available in the voluminous literature requires systematic organization of the data to enable further investigations. For this we developed T2DiACoD database, using the R package pubmed.mineR developed in house for literature mining, we obtained 650 genes for type 2 diabetes and associated complications, classified in Atherosclerosis (115), Nephropathy (403), Retinopathy (161), Neuropathy (130), and Cardiovascular disease (172). Results: Differentially Expressed genes in patients' and normals' tissues including adipose, pancreas, liver and skeletal muscle. Nephropathy genes topped in differentially expressed genes (70.4%) followed by cardiovascular (26.87%), retinopathy (21.5%), atherosclerosis (17.6%) and neuropathy (16.7%) and 34 microRNAs leading with potential causation role in diabetes are also considered and compiled. 25 drug targets are identified for T2DM and its associated complications where 13 drug targets are for T2DM and 12 others are for associated complications. It includes classification of genes studied in different population, Japanese, Chinese, Mexican, Indian, European, American. It also contains various risk factors (obesity, inflammation, stress, diet) that progress to diabetes and its associated complications. Conclusion: T2DiACoD is more comprehensive encompassing highest number of genes up to date, to help users to identify genes playing role in the pathogenesis of diabetes under various risk factors and associated complications.
Impact of Obesity on Clinical and Angiographic Profile of Coronary Artery Disease Patients
Deepak Tewari1, Hermohander S. Isser1, Sandeep Bansal1
1 Vardhman Mahavir Medical College, Safdarjang Hospital, New Delhi, India
Background: Obesity is an independent risk factor for CVD on a par with cigarette smoking, physical inactivity, and high blood cholesterol. Impact of obesity on profile of CAD patients is not known. Clinical, angiographic profile & out come in obese vs non obese CAD patients were compared to study the Impact of obesity on CAD patients. Methods: In the prospective observational study, 778 consecutive patients of CAD who underwent coronary angiography were included. Obesity was defined as a BMI ? 25 kg/m2. Clinical presentation, risk factor profile, and angiographic profile was compared between obese and non-obese groups. The patients were followed up for six months for major adverse cardiovascular events (MACE), and six month out comes were also compared between two groups. Results: 54.6% patients were obese and 45.4% non-obese. Prevalence of smoking was Obese 56.9% & non obese 50.1%, DM was 24% & 31.2% (p = 0.026), HT was 36.9% & 31.7%, dyslipidemia 53.2% & 48.7% respectively. Total cholesterol and LDL cholesterol was higher in obese vs non obese 184.11 vs 177.86 (P- 0.007), and 116.24 vs 111.28 (P- 0.02). EF was 49.72% vs 48.07% (P-0.026) in obese vs non obese CAD pts. Clinical presentation in obese and non-obese was STEMI 46% vs 47.5%, NSTMI/UA 27%, CSA 26.4% vs 25.5% respectively. CAG profile was SVD 37.4% vs 43.9%, DVD 21.9% vs 18.7%, TVD 15.1% vs 15% in obese vs non obese CAD patients. Six month MACE was.09% &.08% in obese vs non obese. Conclusion: Although obesity is a major risk factor for CAD, clinical and angiographic profile as well as 6 months MACE are similar in obese and non-obese CAD patients, except for diabetes mellitus which was more common in non obese. Total cholesterol and LDL cholesterol was higher in obese. EF was higher in obese as compared to non obese CAD patients.
Genome-wide Association Study of Glycemic Traits in Indians
Khushdeep Bandesh1, Dwaipayan Bharadwaj1,2
1 CSIR-Institute of Genomics and Integrative Biology,2 Systems Genomics Laboratory, School of Biotechnology, Jawaharlal Nehru University, New Delhi, India
Diverse heritability of metabolic traits accounts for mechanistic variability of complex disorders, especially in a genetically distinct population such as Indians. The present study pioneers genome-wide search for variants perturbing metabolic glycemic traits – plasma Fasting Glucose (FG), Fasting Insulin (FI) and C-peptide levels in Indians. A genome wide scan was done in 1,157 healthy people to identify associated variants. After stringent quality check and association analysis, variants with p < 10-4 were selected for validation in an independent sample set for each trait (FG- 3847 subjects, FI- 2814 people, C-peptide- 1829 individuals). The results of two phases were combined through meta-analysis. Publicly available regulatory data was mined to secure a biological rationale for observed association. None of the variants reached genome-wide significance. Known variants for FG GWAS in G6PC2 and MTNR1B genes were nominally significant in Indians (p = 0.002; p = 0.008 respectively). In FG GWAS, lead signal was obtained with a p-value of 3.66 x 10?? while some signals in FI GWAS (Meta-analysis p = 8.38 x 10??) and C-peptide (Meta-analysis p = 7.93 x 10??) were also obtained. Biological relevance of these lead variants was deciphered by integrating gene regulatory and expression quantitative trait locus (eQTL) data.
Cardiovascular Disease and Heart Harmony
Ankush Bhardwaj, Kriti
Amity Institute of Biotechnology, Noida, Uttar Pradesh, India
Researchers doctors and scientists around the globe are making progress in the battle against heart disease. From printing the heart tissue to novel medications for heart failure. In the past year, progress in the fight against heart disease and stroke came in many forms, from novel drugs and procedures to improvement and newfound benefits from existing treatments. As the population ages, cardiovascular disease will have an ever-greater human and monetary impact. Statins are the cornerstone of treatment to help regulate cholesterol production. They repress an enzyme involved in the making of cholesterol in the liver and boost the number of low density lipoprotein receptors (LDL-R) to help clear the body of LDL (“”bad cholesterol). The PCSK9 inhibitors (PSK9i) are a newer class of injectable drugs that have been appeared to significantly bring down LDL cholesterol levels, by up to 60% when combined with a statin. PCSK9 inhibitors are monoclonal antibodies (MABs), a type of biologic drug. In individuals with chronic heart failure, a new drug known as LCZ696 lowered the risk of being hospitalized with heart failure or dying from heart disease by 20%. Entresto is at present undergoing review by Health Authorities around the world. Entresto is a first in class pharmaceutical (an ARNI, Angiotensin Receptor Neprilysin Inhibitor) that decreases the strain on the failing heart. Keeping systolic pressure under 120, instead 140 lowered the risk of death among blood-pressure patients by more than 25%. Numerous diabetic patients get cardiovascular disease, and of patients who have cardiovascular problems, those with diabetes do worse. A new diabetes drug EMPA-REG (empagliflozin) appears to be in reducing deaths from cardiac diseases as well as maintaining sound blood sugars. Vorapaxar (Zontivity), the first in class of drugs called PAR-1 antagonists, prevents platelets from clumping and forming clots. Vorapaxar is utilised for persons with a history of myocardial infarction (heart attack) or persons with peripheral arterial disease. Transcatheter aortic valve replacement (TAVR) offers a non-surgical way to fix a stiff, narrowed aortic valve. TAVR delivers the new collapsible valve to heart through a catheter threaded into an artery in the groin. Nanostim - are Tiny new pacemakers, about the span of your pinky tip, can be embedded via catheter directly into the heart. Normally human heart cells stop dividing a week after birth. But the heart muscle tissue of salamanders and fish regenerates itself. Wouldn't it be great if the same were for humans? Damaged heart could recuperate more rapidly and completely after a heart attack. By manipulating the protein Neuregulin – a protein essential for normal heart development – in mice, scientists were able to stimulate the regeneration of heart cells. The strategy worked in adult mice as well as adolescents and made damaged heart cells as good as new, the study found. The complexity of cardiovascular function is an overall expression of its component from myocardium to cardiomyocyte with subcellular compartments interacting for synchronized muscle contraction. Advancement in cardiologic molecular research, as in other fields are aimed to reach the final goal of improving current clinical practice. However, complex biological networks are right now in an early stage of clinical application. Blood pressure, the immune system, and the biology of the vessel walls, are regulated by multiple genes. As these genes are defined (by human genome project), individuals at risk can be identified and focused for specific preventive interventions directed at factors contributing to atherosclerosis, such as blood levels of homocysteine and lipoprotein (a). It will be some time before all genes and gene polymorphism involved in the development of cardiovascular diseases are identified. Ultimately, it will be possible to predict disease long before it occurs and to develop therapies that cure and prevent these diseases. A National Institute of Health supported study provide some of the first clues about the impact of sustained calorie restriction in adults. Although the expected metabolic effects were not found, calorie limitations dramatically lowered several predictors of cardiovascular disease compared to the control group, decreasing average blood pressure by 4% and total cholesterol by 6%. Cardiovascular disease is the greatest threat to human life and health. The past decade has seen remarkable progress in clinical and basic cardiovascular research, and many areas of opportunity are promising. The pace of current progress in clinical and basic research is such that noteworthy improvement in the quality and length of life for those at risk for cardiovascular disease is likely.
Disease Related Biomarker in Patients with Obstructive Sleep Apnea
Surendra K. Sharma1, Mikashmi Kohli1, Swati Varshney2, Shantanu Sengupta2
1 Department of Internal Medicine, All India Institute of Medical Science,2 CSIR-Institute of Genomics and Integrative Biology, New Delhi, India
Introduction: Obstructive sleep apnea (OSA) is an emerging major public health problem worldwide and is characterized by habitual snoring, partial or complete upper airway obstruction. It is associated with several adverse health consequences. Besides road traffic accidents, OSA patients have neurocognitive impairments, cerebrovascular and cardiovascular (CVD) co-morbidities.1,2,3,4 The prevalence of OSA in patients with coronary artery disease (CAD) is about 30%.5 Cross-sectional data from the Sleep Heart Health Study showed the presence of OSA with an AHI >11 events/hr to be associated with a 2.38 relative increase in the likelihood of having heart failure, independently of confounding factors.6 The aim of the present study was to identify any differentially expressed proteins in plasma and urine. Methods: The study was carried out in three phases; (i) identification; (ii) verification. Isobaric tagging for relative and absolute quantification (iTRAQ), which is now considered as a robust proteomics technique was used in the present study to identify differentially expressed proteins, which were subsequently verified and validated using ELISA technique in OSA patients Patients with BMI ≥25 kg/m 2 were screened for OSA to avoid obesity being a confounding factor. Subjects included in identification and verification phase did not have any co-morbidities like hypertension, diabetes, dyslipidemia, cardiovascular disorder, chronic renal disorder, urinary tract infection, impaired neurocognitive function and were naïve to continuous positive air pressure (CPAP) therapy. Patients who were on drugs like non-steroidal anti-inflammatory drugs (NSAIDs) and angiotensin converting enzyme (ACE) inhibitors were also excluded. Results: For the identification phase, 15 subjects were enrolled as 'OSA subjects' [mild: 3; moderate: 5; severe: 7] and 6 were enrolled as non-OSA subjects in the study. From the iTRAQ list of proteins for plasma samples, a total of 233, 250, 255, 254 proteins at 1% FDR were identified in four sets of iTRAQ experiment respectively. Similarly in urine samples, a total of 496, 505, 441 proteins were identified at 1% FDR in three sets of iTRAQ experiment respectively. Seventeen differentially expressed proteins were selected from iTRAQ data based on consistently being up-regulated or down-regulated across different sets for verification (non-OSA = 10; OSA = 30). Receiver operating characteristics curve analysis for all OSA vs. non-OSA subjects ensured optimal diagnostic utility one urinary protein: dermcidin (AUC= 74%) with a sensitivity of 75% and specificity of 71% respectively. Additionally, urinary dermcidin levels represented a dose-response relationship with disease severity. This protein has recently been studied in cardiovascular disease and is known to be a double-edged sword causing hypertension and diabetes. Conclusion: Dermcidin appears to be a promising marker for screening patients with OSA. Additionally, the data in the present study acquiesces with the fact that there is an association of OSA and CVD; however, future studies are required to validate these findings.
- Briançon-Marjollet A, Weiszenstein M, Henri M, Thomas A, Godin-Ribuot D, Polak J. The impact of sleep disorders on glucose metabolism: endocrine and molecular mechanisms. Diabetol Metab Syndr 2015;7:25.
- Yaggi HK, Concato J, Kernan WN, Lichtman JH, Brass LM, Mohsenin V. Obstructive sleep apnea as a risk factor for stroke and death. N Engl J Med 2005;353:2034-41.
- Dopp JM, Reichmuth KJ, Morgan BJ. Obstructive sleep apnea and hypertension: mechanisms, evaluation, and management. Curr Hypertens Rep 2007;9:529-34.
- Somers VK, White DP, Amin R, Abraham WT, Costa F, Culebras A, et al. Sleep apnea and cardiovascular disease: an American Heart Association/American College of Cardiology Foundation Scientific Statement from the American Heart Association Council for High Blood Pressure Research Professional Education Committee, Council on Clinical Cardiology, Stroke Council, and Council On Cardiovascular Nursing. In collaboration with the National Heart, Lung, and Blood Institute National Center on Sleep Disorders Research (National Institutes of Health). Circulation 2008;118:1080-111.
- Peker Y, Kraiczi H, Hedner J, Löth S, Johansson A, Bende M. An independent association between obstructive sleep apnoea and coronary artery disease. Eur Respir J 1999;14:179-84.
- Shahar E, Whitney CW, Redline S, Lee ET, Newman AB, Nieto FJ, et al. Sleep-disordered breathing and cardiovascular disease: cross-sectional results of the Sleep Heart Health Study. Am J Respir Crit Care Med 2001;163:19-25.
Transcriptional Regulation of Hyperglycemia Induced Endothelial to Mesenchymal Transition: Role of Myocardin Related Transcription Factor-A and Activating Transcription Factor 3
Vibhuti Sharma, Nilambra Dogra1, Uma Saikia, Madhu Khullar2
Departments of Histopathology and 1Experimental Medicine and Biotechnology, PGIMER, 2National Centre for Human Genome Studies and Research, Punjab University, Chandigarh, India
Constant exposure of vascular endothelial cells to high blood glucose levels in a diabetic condition may result in diabetes associated microvascular complications. Under pathological conditions endothelial cells are known to contribute to fibrosis by transitioning into myofibroblasts by a process termed as endothelial to mesenchymal transition (EndMT). Molecular mechanisms and the transcriptional mediators controlling EndMT in heart during development or disease remain relatively undefined. Myocardin related transcription factor-A (MRTF-A) is known to facilitate serum response factor (SRF) in the transcription of contractile and cytoskeletal genes during fibrosis, therefore its specific role in cardiac EndMT might be of importance. Activating transcription factor 3 (ATF3) activation during cardiac EndMT is speculated, since it is known to immediately respond to various stress signals or stimuli such as TGF-β and directly control the expression of primary EMT marker genes snail, slug and twist. Although the role of TGF-β has been established in inducing EndMT mediated cardiac fibrosis, the contribution of hyperglycemia (HG) to this transition is not clearly defined. In the present study, the effect of hyperglycemia on the mesenchymal transitioning of cardiac endothelial cells and potential involvement of fibrosis associated transcription factors MRTF-A and ATF3 during that transition was explored. EndMT changes were examined in type-II diabetic rat heart sections, as well as cardiac autopsy tissues of diabetic subjects. Protein expression and localization of transcription factors MRTF-A and ATF3 was also observed in these tissue sections. Nuclear localization of transcription factor MRTF-A was observed in the vascular endothelium of diabetic hearts, parallel with an increase in interstitial collagen deposition. Transcription factor ATF3 was induced and specifically expressed in the small cardiac vessels of diabetic hearts. However, ATF3 expression was not observed in large cardiac vessels. In vitro results showed HG mediated EndMT response in cell specific manner. Prominent EndMT features were observed in cardiac microvascular endothelial cells (CMVECs) upon HG treatment, as compared to the aortic endothelial cells (AECs). ATF3 induction in response to HG treatment was specifically observed in CMVECs. HG stimulated EndMT marker gene slug expression through ATF3 was also observed. Our study suggests HG as a potent stimulus of cardiac EndMT and an important contributor to diabetes associated cardiac fibrosis. In addition, differential effects of HG treatment on macrovascular and microvascular endothelial cells were observed. Results suggest that HG induced EndMT more significantly in CMVECs as compared to AECs. Further, role of transcription factor ATF3 in HG mediated EndMT in CMVECs is indicated but needs further investigation in order to determine its exact role in the transition.
ACE2, CALM3 and TNNI3K polymorphisms as disease modifiers in HCM and DCM
Amit Kumar, Bindu Rani, Rajni Sharma, Ajay Bahl1, Madhu Khullar
Departments of Experimental Medicine and Biotechnology and 1Cardiology, PGIMER, Chandigarh, India
The marked clinical and genetic heterogeneity suggests involvement of disease modifiers and environmental factors in the pathophysiology of the disease. In our present study, we examined the association SNPs of ACE 2 (rs6632677), TNNI3K (rs49812611) and CALM3 (rs13477425) genes in our patients with clinical phenotypes. Prevalence of CG and GG genotypes and G allele was significantly higher in DCM patients as compared to controls. No significant difference in clinical characteristics in HCM or DCM patients was observed on the basis of their genotypes. Prevalence of TNNI3K 3784TT genotype was significantly higher in HCM and DCM patients as compared to controls. DCM patients with CT genotype showed significant decrease in LVEF as compared to CC genotype (p < 0.03). Prevalence of CALM3 (-34T>A) AA genotype was significantly higher in HCM and DCM patients as compared to controls. Also the frequency of A allele was significantly higher in HCM patients as compared to controls. We also observed Gene-gene interaction among ACE 2 C>G, TNN13K C>T polymorphisms and their association with risk of HCM and DCM. Our results suggest ACE2, TNNI3K and CALM3 polymorphisms as disease modifiers in HCM and DCM and associated with increased risk of these diseases.
Keywords: ACE2, CALM3, cardiomyopathy, modifier gene, TNNI3K
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