Journal of the Practice of Cardiovascular Sciences

: 2022  |  Volume : 8  |  Issue : 2  |  Page : 117--132

Cardiovascular Research Convergence 2022

Arun Bandhopadhyaya1, Partha Chakrabarti2, Sanjay Banerjee3, Shivani Kumar Vashista4, Saheli Chowdhaury1,  
1 Department of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India
2 Cell Biology and Physiology Division (IICB), National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India
3 Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India
4 All India Institute of Medical Science, New Delhi, India

Correspondence Address:
Shivani Kumar Vashista
All India Institute of Medical Science, New Delhi

How to cite this article:
Bandhopadhyaya A, Chakrabarti P, Banerjee S, Vashista SK, Chowdhaury S. Cardiovascular Research Convergence 2022.J Pract Cardiovasc Sci 2022;8:117-132

How to cite this URL:
Bandhopadhyaya A, Chakrabarti P, Banerjee S, Vashista SK, Chowdhaury S. Cardiovascular Research Convergence 2022. J Pract Cardiovasc Sci [serial online] 2022 [cited 2022 Dec 6 ];8:117-132
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The Cardiovascular Research Convergence meetings came into existence years back when cardiac treatment was established and the need for basic scientists came in the role for better diagnosis to rule the nitty gritty at the molecular level during treatment of cardiac patients. This was the 11th successful convergence organized in CSIR-Indian Institute of Chemical Biology, Kolkata, in collaboration with the Department of Cardiology, AIIMS, New Delhi, on June 25, 2022, Saturday. There was active participation from different institutes around the country, i.e., CSIR Institute of Genomics and Integrative Biology; Department of Cardiology, AIIMS, New Delhi; Indian Institute of Chemical Technology, Hyderabad; Centre for Cellular and Molecular Biology, Hyderabad; and Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati. The Convergence topics were planned according to the various shortcomings in cardiac patients, as we are witnessing a significant rise every year. There were three categories of presentation: (1) Oral Presentation for Prof. S. K. Maulik Award (Basic Science); (2) Oral Presentation for Prof. Balram Bhargava Award (Clinical Science); (3) Poster Presentation. The active participation of students from various institutes was seen during the presentation.

The Convergence maintained the legacy of providing new horizons to both Clinical and Basic scientists, by exchanging various ideas and developing future collaborations. Hoping to continue the standard of Convergence by widening the network all over the country for a better future in cardiac treatment [Figure 1] and [Figure 2].{Figure 1}{Figure 2}

 Winners of Basic Science: Prof. S. K. Maulik Award

1a. Understanding the Pathophysiological Implications of Dysregulated Cholesterol Homeostasis

Aleepta Guha Ray, Kamalika Roy Choudhury, Devasmita Chakravarty, Vivek Chander, Khawer N. Siddiqui1, Aditya Konar, Arun Bandyopadhyay

Department of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, 1Division of Cardiac Research, Ruby General Hospital, Kolkata, West Bengal, India

E-mail: [email protected]

The efflux of excess cholesterol in macrophages, primarily mediated by ABCA1, is essential for alleviating atherosclerosis, but the role of secondary transporters has not been explored in detail. We report, for the first time, the inverse relationship between plasma levels of ABCA1 and ABCA5 in high cholesterol diet-fed mouse models of atherosclerosis (ApoE−/−) and hyperlipidemia (PPARα−/−) and their clinical correlation in human samples. Taking our study further, we have performed a multiomic study to understand the changes in the plaque macrophages subpopulation in response to statin therapy and the resulting cytokines and chemokines profile, which contributes to the healing phenotype. Surprisingly, an increase of inflammatory macrophages is observed in response to a significant lowering of cholesterol build-up which we have characterized further. Our study indicates that upregulation of apoptosis-inducing factors seems to be necessary for efferocytosis in the plaque and increase in reverse cholesterol transport.

Keywords: macrophages, cytokines, apoptosis

1b. Peptidoglycan Recognition Protein 2 Activates NOD2-NFκβ Inflammatory Axis in Atherosclerosis

Pratitusti Basu, Apabrita Ayan Das, Khawer N. Siddiqui1, Prakash C. Mandal2, Arun Bandyopadhyay

Division of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, 1Department of Cardiology, Apollo Gleneagles Hospital, 2Department of Cardiology, Ruby General Hospital, Kolkata, West Bengal, India

E-mail: [email protected]

Inflammation is one of the major drivers of atherogenesis. The inflammation caused by atherosclerosis does not only act locally, but the manifestation can also be found in the entire circulatory system. Hence, the proteins that show altered expression in plasma, in the background of coronary artery disease, can provide an insight into the mediators that may have an interesting role in plaque development and progression. Plasma-soluble peptidoglycan recognition protein 2 (PGLYRP2) is reported to be associated with chronic inflammation by recognizing the peptidoglycan component of bacterial cell membrane and cleaving it with the C-terminal amidase domain. The correlation of elevated levels of plasma PGLYRP2 with the severity of coronary artery disease raises interest to understand the role of the protein in atherosclerosis. In this study, we report, for the first time, that plasma-soluble PGLYRP2 interacts with the receptor NOD2, present on the macrophage cell membrane at the site of the lesion. The N-terminal domain of the protein PGLYRP2 directly binds to NOD2 on the surface of macrophages and activates NOD2-RIP2-NFκβ cascade that ultimately promotes further inflammation. We showed that treatment with human recombinant PGLYRP2 protein to the macrophages in vitro results in increased secretion of proinflammatory cytokines (tumor necrosis factor-α, interleukin 1 beta and interleukin 8). In vivo study using atherosclerosis mice model showed elevated plasma PGLYRP2 along with increased TNF-α level associated with advanced plaque in the coronary artery, indicating the contribution of PGLYRP2-NOD2 interaction promoting inflammation. Here, we demonstrate that increased plasma-soluble PGLYRP2 interacts with NOD2 and activates the downstream inflammatory pathway that accelerates the inflammation toward an unresolved state that might eventually promote persistent chronic inflammation.

Keywords: Peptidoglycan, inflammation, plaque

1c. Uncovering Sex-Specific Differential Molecular Mechanisms in Cerebrovascular Incidents using Mouse Mild-Stroke Model

Mydhili Radhakrishnan1,2, Kalyani Soren1,2, Vincy Vijay1,2, Arvind Kumar2,3, Sumana Chakravarty1,2

1Department of Applied Biology, Indian Institute of Chemical Technology, 3Centre for Cellular and Molecular Biology, Hyderabad, Telangana, 2Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh, India

E-mail: [email protected]

Cerebral stroke is one of the leading causes of death and disability worldwide. Therapeutic interventions to minimize ischemia-induced neural damage are limited due to poor understanding of molecular mechanisms of the complex pathophysiology due to a dearth of appropriate animal model. The most popular rodent cerebral stroke model is middle cerebral artery occlusion model, although 25% of stroke cases are due to the internal carotid artery occlusion (ICAO). Moreover, earlier studies showed that gender plays an important role in regulating pathophysiological changes in cerebral stroke. Sex difference seems to reflect a major influence not only on stroke vulnerability but also on poststroke cellular and molecular event. To address this, we have used recently developed ICAO mouse (CD1) model considerably close to human cerebrovascular incidents, to explore the sequential molecular events, including epigenetic changes during neural damage and repair in both the sexes. Various behavioral paradigms were employed to assess ICAO-induced deficits in motor/locomotor abilities such as NDS, rotarod test, grip strength, and open field tests at different time points up to 7 days of post-ICAO. The results revealed that females recover faster than males. By investigating regulatory molecular mechanisms, as well as triggering recovery mechanisms individually, we could observe interesting sex-specific patterns in the onset of epigenetic, inflammatory, and mitochondrial activity pathways. Interestingly, in females, the post-ICAO recovery mechanism appears to be triggered earlier than in males. Overall results clearly indicated that the differential sex-specific recovery mechanism might play a key role in regulating the susceptibility to cerebrovascular diseases [Figure 3].{Figure 3}

Keywords: Therapeutic, ischemia, cerebovascular

 Winner of Clinical Science Prof. Balram Bhargava Award

2a. Common Promoter Variants in the Matrix Metalloproteinase 8 Gene Contribute to Hypertension in Human Populations

Sakthisree Maghajothi, Lakshmi Subramanian, Preethi Mani, Mrityunjay Singh1, Dhanya R. Iyer, Saurabh Sharma2, Madhu Khullar2, Suma M. Victor3, Shailendra Asthana1, Ajit S. Mullasari3, Nitish R. Mahapatra

Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, 4Institute of Cardiovascular Diseases, Madras Medical Mission, Chennai, 1Translational Health Science and Technology Institute, NCR Biotech Science Cluster, Faridabad, Haryana, 2Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education and Research, Chandigarh, India

E-mail: [email protected]

Altered matrix metalloproteinase 8 (MMP8, collagenase 2) levels are associated with increased risk of cardiovascular diseases (CVDs). MMP8 degrades matrix proteins such as collagen through its proteolytic activity. Disturbed collagen turn-over in the blood vessels leads to hypertension, an independent predictor of CVDs. However, the contribution of MMP8 to hypertension remains poorly understood. Hence, we aimed to investigate the association of MMP8 regulatory single-nucleotide polymorphisms (SNPs) with hypertension in Indian populations. We genotyped 2565 unrelated individuals for the promoter SNPs in MMP8 gene and identified 4 SNPs in the upstream ~1.3 kb promoter region of MMP8: -763A/T (rs35308160), -799C/T (rs11225395), -815G/T (rs17099451), and -1089A/G (rs17099452); these SNPs form three major haplotypes. Hap3 (haplotype comprising variant allele of all 4 SNPs) displayed significant association with hypertension (Chennai: odds ratio [OR] = 1.47, 95% confidence interval [CI] = 1.16–1.86, P = 2 × 10−3; Chandigarh: OR = 1.85, 95% CI = 1.21–2.81, P = 4 × 10−3) and exhibited elevated Systolic blood pressure (SBP), Diastolic blood pressure (DBP), and mean arterial pressure (MAP) compared to wild-type haplotype (Hap1). Hap3 exhibited diminished promoter activity compared to the wild-type haplotype. In line with the in silico predictions and molecular dynamics analysis, co-transfection of the Hap3 with NF-κB expression plasmid displayed a diminished enhancement in the reporter activity compared to wild-type haplotype. Moreover, Hap3 also showed weak response to TNF-α treatment, which could be attributed to the weaker binding of NF-κB to Hap3. Plasma MMP8 level of Hap3 heterozygous genotype individuals was lower, whereas the endothelial dysfunction markers (endothelin-1 and von Willebrand factor (vWF)) were elevated compared to wild-type individuals. Our findings suggest that altered MMP8 expression in individual's harboring these MMP8 promoter variants may cause differential response to inflammatory queues and contribute to the development of hypertension.

Keywords: heterozygous genotype, single-nucleotide polymorphisms, haplotype

2b. Inflammatory Cardiomyopathy: Validating a Diagnostic Algorithm Combining Biomarkers, Magnetic Resonance Imaging, and Somatostatin Receptor-Based Positron Emission Tomography/Computed Tomography

Kavita Kumari, Shivani Vashista, Deepika Jindal, Santoshi Sahani, Sandeep Seth, R Narang

Department of Cardiology, AIIMS, New Delhi, India

E-mail: [email protected]

Objective: Inflammatory cardiomyopathy, majorly caused by viral infections, autoimmune diseases, hypersensitivity, and sarcoidosis, is a primary cause of heart failure in India. It is estimated that about one-third of myocarditis patients develop dialated cardiomyopathy (DCM). With the help of imaging techniques like Magnetic resonance (MRI) and positron emission tomography resulted in better diagnosis of Myocarditis. This study is an attempt to create a combined diagnostic score based on the above information, to diagnose inflammatory cardiomyopathy. Methods: A total of 100 patients with inflammatory cardiomyopathy have been recruited. Investigation includes echo, MRI, nuclear scans, and Endomyocardial biopsy (EMBx). Information from all this was combined to create a diagnostic score, and this was then tested on the recruited patient cohort. Results: The components of the score include viral prodrome, inflammation and cardiac stress, and injury biomarkers such as C reactive Protein (CRP), Brain natriuretic peptide (BNP), and troponins. Electrocardiographic changes and imaging by MRI or Dotanoc were also scored. EMBx was scored. Most parameters if positive were given a score of 2, and a total score of 7 was considered, suggestive of probable myocarditis. This score was applied to the 100 patients for validation and accuracy checking. Conclusion: A diagnostic score has been created for myocarditis and validated on 100 patients. Wider application to more patients will show the robustness of this score.

Keywords: cardiomyopathy, Electrocardiographic, hypersensitivity

2c. Targeted Placental Metabolomic Profiling to Unravel the Disease-Specific Biomarkers in Gestational Hypertension

Bincy Varghese, Soumya Reddy, Aishwarya Jala1, Roshan M. Borkar1, Ramu Adela

Departments of Pharmacy Practice and 1Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India

E-mail: [email protected]

Women with a history of hypertensive disorders during pregnancy have a 2–3 folds greater risk of cardiovascular disease in their later life. Gestational hypertension (GH) accounts for about 3%–10% of maternal-fetal morbidity and mortality, worldwide. Recent advances in the field of high-throughput omic techniques contributed significantly to identifying potential therapeutic targets and biomarkers for various pathological conditions. Identification of placental metabolic adaptations during pregnancy may help in getting a deeper understanding of maternal, placental, and fetal health, especially in pathological pregnancies such as GH. We have determined the placental metabolic alterations of healthy pregnant (n = 20) and GH patients (n = 20) using a targeted metabolomic approach. The signature metabolites were extracted using univariate and multivariate analyses. Statistical analysis showed a total of 98 significantly altered metabolites, with 65 downregulated and 33 upregulated ones. The major pathways affected were the purine, amino acid, and aminoacyl-tRNA biosynthesis. Receiver operator characteristic curve analysis showed five significant metabolites (spermine, inosine, sarcosine, alanine, and eicosatetraenoic acid) that can discriminate GH than the hypertension (HP) with area under the curve, area under the receiver operating characteristics (AUROC) ≥0.90, with higher sensitivity and specificity. Overall, this is the first attempt to unravel tissue metabolomic aberrations in Hypertensive Disorders in Pregnancy (HDP) patients in the Indian population. These results may add to the knowledge of the molecular mechanisms behind the development of these progressive diseases, which might aid in better understanding of disease pathophysiology and monitoring therapeutic response in severe hypertensive patients in the future [Figure 4].{Figure 4}

Keywords: Biomarkers, gestational hypertension, metabolomics, pregnancy

 Poster Presentation Award

3a. Regulation of Peroxiredoxin-3 Gene Expression by Transcription Factors Specificity Protein 1, cAMP Response Element–Binding Protein, and Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells in Diabetic Cardiomyopathy

Silpa Arkat, Rohini Dhat1, Sandhya L. Sitasawad1, Nitish R. Mahapatra

Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai, Tamil Nadu, 2National Centre for Cell Science, Savitribai Phule Pune University Campus, Pune, Maharashtra, India

E-mail: [email protected]

Background: Diabetes contributes to a distinct form of heart muscle disorder known as diabetic cardiomyopathy (DCM), which profoundly increases the risk of heart failure. Mitochondrial oxidative stress is a major contributor to the progression of DCM. Peroxiredoxin-3 (Prx-3), a mitochondrial antioxidant protein, is crucial for regulating hydrogen peroxide levels in most cells. In fact, a reduced level of Prx-3 has been linked to various cardiovascular diseases (viz., myocardial infarction/DCM/dilated cardiomyopathy), and overexpression of Prx-3 prevents the above pathological conditions in various animal models. However, the transcriptional regulation of Prx-3 is poorly understood. In this study, we aim to identify the transcription factors (TFs) governing Prx-3 gene expression under basal and DCM conditions. Methods: Systematic in silico TF prediction tools and experimental analyses (promoter-reporter, co-transfection, and chromatin immunoprecipitation [ChIP] assays) were used to identify potential TFs that regulate Prx-3 gene expression. Results: To identify the core promoter region of Prx-3, varying lengths of rat Prx-3 promoter (−1200, −457, −251, −190, −134 bp to +20 bp) were polymerase chain reaction (PCR)-amplified and cloned into promoter-less pGL4.2 basic vector. Transfection of these deletion constructs into HEK-293, H9c2, N2a, E9, and Huh-7 cells shows the highest promoter activity in the case of −190 bp construct. Systematic in silico analysis of the −190/+20 bp promoter domain revealed putative binding sites for specificity protein 1 (Sp1), cAMP response element–binding protein (CREB), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Consistently, co-transfection of the −190/+20 bp construct with Sp1/CREB diminished and NF-κB augmented the Prx3 promoter-reporter activity, mRNA, and protein levels. Co-transfection of Sp1/CREB/NF-κB shRNA plasmids showed promoter-reporter activity, mRNA, and protein levels, consistent with these results. Furthermore, in vivo interaction of Sp1, CREB, and NF-κB with the rPrx-3 promoter in the context of chromatin was confirmed by ChIP assays. High-glucose treatment of H9c2 cells and streptozotocin (STZ)-treated diabetic rats showed an inverse correlation between rPrx-3 and Sp1/CREB levels. Microarray data from GEO Dataset also demonstrated similar trends between rPrx-3 and Sp1/CREB levels in the hearts of control and STZ-induced diabetic rats. Thus, Sp1/CREB/NF-κB emerges as important regulators of rPrx-3 gene expression in DCM.

Keywords: Co-transfection, streptozotocin, geneexpression

3b. Reduction in PTEN-Mediated Apoptosis Is Counteracted by Calpain-1 Regulated Autophagy in Cardiac Hypertrophy of Peroxisome Proliferator-Activated Receptor alpha−/− Mice

Ritu Kumari, Aleepta Guha Ray, Dibyanti Mukherjee, Dipak Kar, Aditya Konar, Arun Bandyopadhyay1

1Department of Cell Biology and Physiology, CSIR- Indian Institute of Chemical Biology, Kolkata, West Bengal, India

E-mail: [email protected]

Cardiac hypertrophy is characterized by an increase in the size of the cardiomyocytes, which is initially triggered as an adaptive response due to various kinds of stimuli but ultimately becomes maladaptive with chronic exposure. Peroxisome proliferator-activated receptor alpha (PPARα), which is critical for mitochondrial biogenesis and fatty acid oxidation, is known to be downregulated in hypertrophied cardiomyocytes. Yet, the role of PPARα in cardiomyocyte death is largely unknown. To assess the role of PPARα in chronic hypertrophy, isoproterenol, a β-adrenergic receptor agonist, was administered in PPARα knock-out (PPARα−/−) mice for 2 weeks, and hypertrophy-associated changes in cardiac tissues were observed. Proteomic analysis using Orbitrap mass spectrometer revealed an unexpected downregulation of apoptotic markers, Annexin V and p53. The study revealed a significant downregulation of apoptosis regulator, PTEN, along with other apoptosis markers such as p53, caspase 9, and c-PARP Poly (ADP-ribose) polymerase. The autophagy markers were upregulated in PPAR α−/−mice, indicating an upsurge in autophagy. The results were further validated in vitro using NRVMs and H9C2 cell line by blocking PPARα that resulting in enhanced autophagosome formation upon hypertrophic stimulation. The results demonstrate that in the absence of PPARα, apoptotic pathway is inhibited while autophagy is enhanced. The data suggest that PPARα signaling might act as a molecular switch between apoptosis and autophagy, thereby playing a critical role in adaptive process of cardiac hypertrophy.

Keywords: Autophagy, biogenesis, autophagosome

3c. Identification of Adamts4, a Matrix Metalloproteinase as a Novel Cardiac Injury Biomarker with Implications in Patients with Cardiac Injury

Riffat Khanam, Santanu Charaborty

Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

E-mail: [email protected]

Pathological cardiac remodeling as an aftermath of a severe cardiac injury can lead to ventricular dysfunction and subsequent heart failure. Our study focuses on Adamts4, a matrix metalloproteinase (MMP) and extracellular matrix (ECM) marker following cardiac injury. Our studies show that the widespread prevalence of Adamts4 throughout chamber myocardium in the embryonic stages severely wanes and is only restricted to the edge of the IVS but reactivation of Adamts4 in the left ventricle of chamber myocardium post-myocardial infarction (MI) induction in an adult murine model is observed, and interestingly, the expression of Adamts4 co-localized with cardiomyocytes. To further decipher the signaling, Adamts4 induction was induced by hypoxia and reactive oxygen species (ROS) stress treatment in H9c2, a rat cardiomyocyte cell line. In response to both the stress conditions, Admats4 expression along with Tgf-β, α-SMA, Col-III, and periostin was significantly enhanced as validated by Western Blot, IF, and qPCR data. Moreover, Tgf-β inhibition by ALKI treatment shows Adamts4 downregulation and subsequent repression of the above-mentioned ECM and fibrosis markers. However, Adamts4 loss of function by Adamts4-specific siRNA transfection showed no significant change in the expression of Tgf, indicating Tgf-β–dependent Adamts4 functioning. Finally, Adamts4 and α-SMA expression was studied in clinical samples with a history of MI Anterior wall myocardial infarction (AWMI), Inferior wall myocardial infarction (IWMI) and dilated cardiomyopathy where the expression of Adamts4 was significantly elevated as quantified by Western blot for Adamts4 and α-SMA in addition to Adamts4-specific ELISA. Our work highlights the emerging role of Adamts4 as an alternative cardiac injury marker in addition to the routinely assessed cardiac biomarkers.

Keywords: Matrix Metalloproteinase, Adamts4, siRNA

 Various Other Studies Presented in Cardiovascular Research Convergence 2022

4a. Role of Bmp2 in Regulating Cardiac Remodelling Postendoplasmic Reticulum Stress

Shreya Das, Santanu Chakraborty1, Arunima Sengupta

Department of Life Science and Biotechnology, Jadavpur University, 1Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

E-mail: [email protected]

Adult cardiomyocytes in mammals fail to retain the proliferative capacity exhibited by neonatal cardiomyocytes before birth. An insult to endoplasmic reticulum (ER) machinery often results in the development of ER stress-mediated cardiomyopathy. There is a very fine balance between pro-survival and pro-apoptosis during ER stress-induced cardiomyopathy. In this study, we investigate the role of Bmp2 as a potential marker in imparting this pro-survival response in the milieu of ER stress-induced cardiomyopathy. We observed increased expression of Tbx20 and Bmp2 with increased cardiomyocyte proliferation and decreased apoptosis upon ER stress induction. Their expression decreased with increased intensity and duration of ER stress with concomitant increase in cardiomyocyte apoptosis, hypertrophy, and fibrosis. Administration of recombinant Bmp2 protein resulted in restoration of cardiomyocyte proliferation even during chronic ER stress in vitro. Knockdown of Tbx20 and use of Bmp2 inhibitor noggin showed Bmp2 to be downstream of Tbx20. In mice, sustained ER stress resulted in drastic increase in Bmp2 expression, which is attributed to the heterogeneity of heart tissue. Sustained ER stress also results in increased inflammation which in turn increases Bmp2 expression. Increased Bmp2 in turn causes augmentation of pro-inflammatory phenotype in the endothelial cell following injury. Thus, Bmp2 imparts protection post-ER stress by augmenting cardiomyocyte proliferation; however, its drastic increase during chronic ER stress is due to factors such as inflammation and other cell types of the heart, thus making it a suitable candidate for a novel biomarker for early detection of ER stress-mediated cardiomyopathy.

Keywords: endoplasmic reticulum, Tbx20 and Bmp2, heterogeneity

4b. Transcriptional Regulation of Adult Cardiac Fibrosis Process

Ipshit Dey, Santanu Chakraborty

Heart Development and Disease Laboratory, Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

E-mail: [email protected]

Cardiovascular disease is one of the main causes of death worldwide, and nearly all etiologies of cardiovascular disease are associated with activation of cardiac fibroblasts. After adult cardiac injuries such as myocardial infarction, these resident cardiac fibroblasts proliferated and differentiated into activated myofibroblasts with the production of excessive amount of fibrous collagens and subsequent fibrotic scar formation. This pathological myocardial remodeling associated with cardiomyocyte hypertrophy and death leads to overall cardiac dysfunction and progression toward heart failure. Despite the prevalence of cardiac fibrosis downstream of many cardiac injuries, we have a little understanding of its transcriptional regulation in vivo. T-box transcription factor, Tbx20 has a role in heart development and also plays an important role in embryonic, fetal, and neonatal cardiomyocyte proliferation and cardiac chamber maturation, but its role in cardiac fibrosis process is unknown. Here, we have generated a cardiac fibrosis model in adult male rats by isoproterenol treatment. Treatment of isoproterenol induces hypertrophic response along with increased expression of activated myofibroblasts and collagen type III associated with active fibrosis. To our surprise, preliminary experiment also detected an increased number of Tbx20 positive-activated myofibroblasts in isoproterenol-treated adult hearts. Overall, this study, for the first time, explores the Tbx20 function in adult fibroblasts postinjury with possible therapeutic intervention in the near future.

Keywords: cardiac fibrosis, postinjury, isoproterenol

4c. The Study of Arsenic-Induced Cardiotoxicity upon Multiple Adult Cardiac Cell Types Cardiac Lineages and Identification of Affected Downstream Regulators in Adult Murine Heart

Sankha Banerjee, Santanu Chakraborty

Heart Development and Disease Laboratory, Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

E-mail: [email protected]

Chronic arsenic exposure associated with heart failure leads to an increase in mortality worldwide centralizing cardiovascular diseases. However, the detailed molecular insight toward arsenic-mediated cardiotoxicity in adult heart homeostasis and function is largely unknown. Therefore, our current study is solely focused on determining the effects of arsenate upon adult cardiac cell types, including cardiomyocytes and fibroblasts originating from the epicardial cells through epithelial-to-mesenchymal transition (EMT) via epicardial-derived cells in utero. While analyzing the lineage-specific marker expression levels, we have observed a significant increase in the expression of activated fibroblasts in the arsenic-exposed hearts compared to controls. Moreover, the Wheat germ agglutinin staining, commonly used to label the glycoproteins for plasma membrane, has shown us an increase in the cell surface area of arsenate-exposed samples compared to control ones, indicating hypertrophic response. Studies have shown that Yap molecule of the Hippo-Yap signaling pathway is associated with cellular hypertrophy as well as proliferation, leading to proper organ size development in embryonic condition. Hence, our hypothesis, for now, is whether Yap after getting activated upon arsenate exposure is activating the EMT mechanism in the adult heart to generate the activated fibroblasts from the arsenic-exposed epicardial cells. Preliminary data have been obtained to support EMT mechanism in adult heart by checking the expression level of EMT-specific marker Twist1. Overall, we try to explore the arsenate-dependent–affected signaling pathway and its manipulation in the adult heart to reduce the burden of cardiac pathological hypertrophy and fibrosis in vivo with future therapeutic implications.

Keywords: cardiotoxicity, fibroblasts, expression

4d. A Critical Role of FOXM1 in Pathophysiology of Diabetic Cardiomyopathy

Arunima Mondal, Santanu Chakraborty1, Arunima Sengupta

Department of Life Science and Biotechnology, Jadavpur University, 1Department of Life Sciences, Presidency University, West Bengal, Kolkata, India

E-mail: [email protected]

Cardiac diseases are one of the most common complications with higher susceptibility in diabetic patients. Hypertrophy of cardiomyocyte and fibrosis of heart muscle often lead to structural and functional abnormalities, leading to risks of heart failure. The study shown that up-regulation of developmental regulatory molecule FOXM1 plays a important role in pathogenesis of myocardium. As the underlying mechanism, we have further observed that improper glucose metabolism activates YAP1, a key organ size regulatory molecule that further acts to activate AKT-GSK3 signaling. A suppression of GSK3-mediated FOXM1 inactivation results in imbalanced expression and accumulation of FOXM1 in the cardiomyocyte. In our study, under high glucose stress, YAP1 and FOXM1 have been found to overexpress, leading to hypertrophic enlargement and myofibroblast differentiation. Further, YAP1 manipulation has been resulted in a reduced expression of FOXM1, pointing to a possible interaction of YAP1 and FOXM1 in the cardiomyocyte. Modulation of both these molecules also decreases the overexpression of hypertrophic and fibrotic markers of high glucose-stimulated cardiomyocytes. In the control cells, FOXM1 overexpression alone has also been observed to have a profound effect on the induction of hypertrophy of H9c2 and fibrosis, indicating that an upregulated FOXM1 expression in the adult cardiac myocyte cells may be a potential marker of cardiomyocyte pathogenesis. Further observation regarding cardiac FOXM1 level may prove to be an indication of cardiac damage, as well as therapeutic option in diabetic individuals.

Keywords: FOXM1, AKT-GSK3 signaling, myofibroblast

4e. Polyphenol-Enriched Bioactive Fraction of Clerodendrum glandulosum Effectively Ameliorates Oxidative Stress and Atherosclerosis, in vitro

Puspanjali Khound, Rajlakshmi Devi

Division of Life Sciences, Institute of Advanced Study in Science and Technology, Gauhati, Assam, India

E-mail: [email protected]

Excess free fatty acids (FFAs) and glucose are responsible for causing oxidative stress in patients with cardiovascular diseases. Reactive oxygen species (ROS) oxidize and damage DNA, proteins, and lipids, resulting in the activation of several stress-signaling pathways that cause endothelial injury and dysfunction, which results in the development of atherosclerosis and cardiovascular diseases. Reduction in ROS production and enhancement of antioxidant availability are considered important strategies for decreasing cardiovascular complications by utilization of herbal remedies. Clerodendrum glandulosum is endemic to Northeast India and used in traditional medicinal practices as a hypotensive agent. The polyphenol-enriched bioactivity-guided fraction (PECGF) was screened for different antioxidant and enzyme-based assays. FFA (palmitic acid, 0.25 μM)-induced oxidative and mitochondrial stress was effectively ameliorated by PECGF in HepG2 cells. Pretreatment with PECGF effectively ameliorated high levels of proinflammatory cytokines in lipopolysaccharide LPS-induced inflammation human monocytic cell line THP-1 cells. It was observed that pretreatment with PECGF exhibited significant antiadhesion activity in high glucose-induced atherosclerosis in vitro, in endothelial Human Umbilical Vascular Endothelial Cell (HUVEC) and monocyte (THP-1) cells. Therefore, these results indicate that PECGF can be effective in the amelioration of atherosclerosis and related cardiovascular complications.

Keywords: Atherosclerosis, cardiovascular, Clerodendrum glandulosum, free fatty acid, reactive oxygen species

4f. Isoproterenol-Mediated Oxidative Injury Results in the Generation of Early Cardiomyocyte Progenitor Cells in the Adult Heart In vivo

Madhurima Ghosh, Santanu Chakraborty

Heart Development and Disease Laboratory, Department of Life Sciences, Presidency University, Kolkata, West Bengal, India

E-mail: [email protected]

Adult heart failure is a major public health issue, and its prevalence is increasing in recent postpandemic times at an alarming rate. The pathophysiological effect of oxygen depletion during heart failure leads to the massive loss of cardiomyocytes, which are ultimately replaced by permanent scar tissue. Unlike zebrafish or neonatal rodent hearts with limited cardiac regenerative capability, adult mammalian hearts with terminally differentiated cardiomyocytes cannot repair or regenerate damaged cardiac muscles postinjury. Here, in this study, we have used isoproterenol for cardiac injury induction in adult male rat hearts via generation of oxidative stress in vivo. Adult cardiac hypertrophy and fibrosis deposition demonstrated successful injury induction in isoproterenol-injected animals. To our surprise, an increased number of Wt1 and Nkx2.5-positive cells are detected in the injured heart, indicative of epicardial activation and generation of early cardiomyocyte lineage cells, respectively, compared to untreated adult hearts. Interestingly, in the injured adult hearts, the expression of epithelial-to-mesenchymal transition markers such as Twist1 and Snail1 was also seen to be upregulated. Furthermore, using an in vitro avian epicardial cell culture system, the inhibition of Bmp2 signaling represses the generation of early cardiomyocyte lineage differentiation. Overall, knowledge of possible molecular pathway(s) responsible for early cardiomyocyte lineage differentiation in the injured adult heart will open up new avenues toward cardiac repair and regenerative therapeutics.

Keywords: zebrafish, epicardial activation, epicardial activation

4g. Role of Vitronectin in Atheroinflammation

Devasmita Chakravarty1, Aleepta Guha Ray1, Vivek Chander1, Ulaganathan Mabalirajan1, Prakash Chandra Mondal2, Khawer N. Siddiqui3, Bishnu Prasad Sinha4, Aditya Konar5, Arun Bandyopadhyay1,4,5

Departments of 1Cell Biology and Physiology, 4Cancer Biology and Inflammatory Disorder and 5Laboratory Animal Facility, CSIR-Indian Institute of Chemical Biology, 2Department of Cardiology, Apollo Hospitals, 3Cardiac Research Division, Ruby General Hospital, Kolkata, West Bengal, India

E-mail: [email protected]

Cellular spreading and interplay of vascular smooth muscle cells (VSMCs), inflammatory cells, and cell adhesion molecules (CAMs) are essential in regard to atherosclerotic progression and cardiovascular complications. A major cell attachment glycoprotein, whose role in atherosclerotic pathogenesis remains elusive, is vitronectin (VTN). We attempted to examine the pathological role of VTN in maintaining architecture of arteries and progression of plaque. We found that VTN levels are modulated by cholesterol levels in vitro and in vivo by influencing its promoter. Downregulation of VTN in vivo accelerates inflammation in atheroma along with upregulation of proinflammatory cytokines in the plasma. In addition, the expression of matrix metalloproteases was increased in the plaque, in VTN-deficient conditions posing a challenge to plaque integrity. Human subjects with acute coronary syndrome or having risk factors of atherosclerosis were found to have lower levels of VTN compared to healthy controls, suggesting a clinical significance of plasma VTN in the pathophysiology of coronary artery disease. We establish that VTN plays a pivotal role in cholesterol-driven atherosclerosis and aortic inflammation and might be a useful indicator for atherosclerotic plaque burden and stability.

Keywords: pathophysiology, glycoprotein, atherosclerotic

4h. Understanding the Role of PPARα Signaling in Oxido–Reductase Pathways in Dyslipidemic Condition

Trina Roy, Aleepta Guha Ray, Aditya Konar, Arun Bandyopadhyay

Department of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India

E-mail: [email protected]

PPARα, a member of the peroxisome proliferator-activated receptor family, is a ligand-activated transcription factor predominantly expressed in tissues (liver, heart, and adipose tissue) involved in monitoring systemic nutrient homeostasis. Abnormalities in PPARα expression often trigger hepatic steatosis, liver cancer, and increased risk of cardiovascular disorder, mainly because of its uncompromised role in regulating the expression of more than a few genes critical in lipid and lipoprotein metabolism. PPARα ligands, “Fibrates” are frequently used in treating dyslipidemia for their ability to lower circulating triglyceride and elevate plasma high-density lipoprotein cholesterol. Because PPARs are intriguing therapeutic targets for metabolic syndromes and cardiovascular disorders, we have undertaken a multiomic study approach to understand how cholesterol is affecting the major pathways in dyslipidemic PPARα-deficient mice.

Keywords: ligand-activated, triglyceride, cholesterol

4i. Circulating Cell-Free DNA Acts as a Trigger toward Extracellular Matrix Remodeling

Abhi Dutta, Moumita Das, Ankita Ghosh, Santanu Rana

Raja Peary Mohan College Integrated Biological Research Facility, Raja Peary Mohan College, Hooghly, West Bengal, India

E-mail: [email protected]

Pathological cardiovascular conditions are developed due to an increased workload and are largely associated with alterations in gene expression within cardiac-resident cell types. To strive, the myocardium adapts to increased workload by structural and functional remodeling of myocardium – known as hypertrophy. Isoproterenol, a catecholamine, induces cardiac hypertrophy by mimicking the sustained adrenergic stimulation and pathogenesis of maladaptive cardiac hypertrophy. Hypertrophic response further aggravates as an inflammatory response, leading to a reparative phase, and a permanent scar formation due to increased collagen accumulation in the myocardium, leading to extracellular matrix (ECM) remodeling along with myocyte necrosis or apoptosis. Experimental data from our studies demonstrate an elevation of circulating cell-free DNA (cfDNA) in the bloodstream in induced hypertrophied mice groups compared to controls. cfDNA are well-known Damage-associated molecular patterns and are able to upregulate hypertrophic gene expression in myocytes, independently. Isolated cfDNA on being treated to H9C2 myocytes showed upregulation of atrial natriuretic factor and beta-myosin heavy chain gene expression. Interestingly, the cGAS-STING pathway was stimulated during hypertrophy leading to upregulated expression of type 1 IFN and stimulated genes and NF-κB regulated downstream inflammatory genes. Further, fibroblasts when supplemented with media from cfDNA-induced hypertrophied H9C2 culture, in vitro, showed an upregulation in collagen 1 and collagen 3 gene expression, associated with ECM remodeling, thereby projecting a significant role of cfDNA toward ECM remodeling and collagen deposition, leading to myocardial scar formation.

Keywords: hypertrophied, catecholamine, Isoproterenol

4j. Biomarkers and Drug Discovery in Cardiometabolic Diseases

Puja Kundu

Dinabandhu Andrews College, Calcutta University, Kolkata, West Bengal, India

E-mail: [email protected]

For effective drug discovery, several specific biomarkers need to be selected. Free fatty acid (FFA) receptor 4 (FFR4), long noncoding RNAs (lncRNAs) such as Apolipoprotein A1 (APOA1)-AS, human lncRNA metabolic regulator 1 (hlMR1), and AK098656 are acting as biomarkers and targets for drug delivery in several cardiometabolic diseases. FFA causes diabetes mellitus-2 and obesity. However, it is unable to exert its effect while bound with the FFR. FFR4 (also called G-protein coupled receptor-120) is present in endothelial cells, adipocytes, and macrophages and enhances the homeostatic regulations (insulin sensitivity, inflammation, and adipogenesis) by interacting with FFA. Defective FFR4 can lead to obesity and insulin resistance. FFR4 also has a great role in atherosclerosis. Activated FFR4 can increase the efflux of cholesterol from macrophages and decrease the damaged size. lncRNAs have immense roles in regulating cardiometabolic diseases. APOA1, a key component of high-density lipoprotein, is negatively regulated by lncRNAs APOA1-AS epigenetically. Application of short-antisense-oligonucleotides against APOA1-AS amplifies the expression of APOA1 and also helps in the transportation of reverse cholesterol. lncRNA hlMR1 is present in liver tissue and participates in lipid metabolism. Activated hlMR1 can enhance cholesterol biosynthesis whereas knockdown of it can reduce 40% or more low-density lipoprotein. Hypertensive patients have high AK098656 lncRNA in their plasma. It regulates the proliferation of human vascular smooth muscle cells by binding with myosin heavy chain-11 (contractile proteins) and fibronectin-1 (ECM proteins), causing their degradation by 26S proteasome, and leading to hypertension. The use of these biomarkers can help in effective drug discovery in cardiometabolic diseases.

Keywords: proteasome, proliferation, cardiometabolic diseases

4k. Pregestational Diabetes Alters Metabolic Flexibility in Neonatal Heart: Impact on Amino Acid and Fat Metabolism

Uppulapu Shravan Kumar, Vikas Tiwari, Md Jahangir Alam, Parul Kamboj1, Yashwant Kumar, Sanjay K Banerjee

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, 1Non-Communicable Disease Group, Translational Health Science and Technology Institute, Faridabad, Haryana, India

E-mail: [email protected]

Background: Maternal health plays a pivotal role in determining and predicting the health of offspring later in life. Pregestational diabetes mellitus (PGDM) remains a major risk for wide-ranging birth defects, such as congenital heart defects. Chronic pathological condition such as hyperglycemia compromises metabolic flexibility, resulting in modulation of myocardial energetics and contractile function. However, the metabolic reprogramming during the early development of the heart in PGDM is largely undefined. Methods: To assess the causal relationship, we conducted untargeted metabolic profiling using Orbitrap fusion mass spectrometry coupled with Ultra-performance liquid chromatography (UPLC). We draw out the metabolic pathways involved in the perturbation of myocardial energetics and contractile function of 7-day-old rat hearts from neonates of PGDM mothers. We homogenized 50 mg of heart tissue collected from control and diabetic mother-born pups in 100% methanol following standard protocol. Systolic and diastolic functions of the pups were assessed from M-mode ultrasound echocardiography. Results: With the help of metabolomics, we identified a total of 322 metabolites in heart samples, of which 32 were significantly changed. The key downregulated metabolites are kynurenic acid, cADP-ribose, and GMP. The key upregulated metabolites are methionine, oxoproline, asparagine, riboflavin, 4-pyridoxic acid, and taurine. The significantly enriched pathways are “aromatic amino acid (AAA) and branched-chain amino acid (BCAA) biosynthesis,” biosynthesis of unsaturated fatty acids, pyrimidine metabolism, and Vitamin B5 and B6 metabolism. The echocardiography of the pups showed a decrease in stroke volume, cardiac output, left ventricular mass, left ventricular anterior wall, and left ventricular posterior wall. Conclusion: In the present study, PGDM hearts exhibited compromised contractility and energetics compared to control, accompanied by extensive metabolic remodeling as demonstrated by metabolomics combined with echocardiography. The PGDM heart metabolome showed significant differences in the abundance of metabolites in pathways that may lead to reduced heart rate (kynurenic acid), contractility (c-ADP-ribose, taurine), abnormal heart development (methionine), altered energy homeostasis and fat metabolism (pantothenic acid, taurine, linolenate), myocardial dysfunction (4-oxoproline), and diabetic cardiomyopathy (4-pyridoxic acid). Moreover, increased abundance of the AAA, BCAA, and asparagine in the neonates from diabetic mothers predicts risk of future insulin resistance and cardiovascular disease in offspring [Figure 5].{Figure 5}

Keywords: kynurenic acid, methionine, pantothenic acid, taurine, linolenate

4l. Modulating Nrf2/HO-1 and TGF-β1/Smad2/3 Signaling Cascade Ameliorates TAC-induced Cardiac Remodeling in Mice

Abu Mohammad Syed, Sourav Kundu, Bidya Dhar Sahu

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India

E-mail: [email protected]

Cardiac hypertrophy is one of the significant contributors to heart failure. Pathological cardiac hypertrophy is characterized by excessive accumulation of extracellular matrix (ECM) proteins and enhanced expression of hypertrophic genes, ROS and oxidative stress, and apoptosis. Studies demonstrated that the Nrf2 transcription factor plays a crucial role in oxidative damage and cardiac remodeling. Daphnetin is a coumarin isolated from the Daphne genus. It has a broader range of pharmacological activities. In this study, daphnetin reversed the increased cell surface area, ROS production, fibrosis, and apoptosis protein expression stimulated by angiotensin-II in H9c2 rat cardiomyoblasts. We found that Si-Nrf2 transfection eliminated the protective effect of daphnetin in H9c2 cells. However, the treatment of daphnetin increased the protein expression of Nrf2, NQO1, and HO-1. Besides, daphnetin oral administration normalized the cardiac functional parameters against TAC-induced cardiac hypertrophy in mice. Furthermore, daphnetin significantly enhanced the nuclear translocation of Nrf2, activated many antioxidants signaling, and inhibited oxidative damage. In addition, daphnetin downregulated the hypertrophic mRNA markers such as α-SA, ANP, BNP, and β-MHC. The TGFβ1/Smad2/3 fibrosis signaling axis and ECM proteins such as fibronectin, α-SMA, and collagen were downregulated in daphnetin-treated mice. Our study suggested that the daphnetin treatment group significantly downregulated TUNEL-positive nuclei, Bax/Bcl2, and cleaved caspase-3 protein expression compared to TAC group mice. Combined results propose that daphnetin ameliorates cardiac remodeling via modulating Nrf2/HO-1 axis, TGFβ1/Smad2/3, and apoptosis signaling cascade [Figure 6].{Figure 6}

Keywords: fibrosis, daphnetin, pharmacological

4m. Dopamine Beta Hydroxylase: An Optimal Target to Counter-Hypertension

Varnita Anand, Sanjay Kumar Dey1, Manisha Saini2, Suman Kundu

Department of Biochemistry, University of Delhi, South Campus, 1Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, New Delhi, India, 2Department of Chemistry and Chemical Biology, Rutgers University, New Jersey, USA

E-mail: [email protected]

The sympathetic nervous system is an important system involved in the pathophysiology of hypertension. An enhanced sympathetic activity generally results in increased production of norepinephrine (NE) and epinephrine (E), hormones that have receptors on the heart and blood vessels and have vasostimulatory effects that increase blood pressure. Just as α and β blockers block the binding of NE and E on their receptors, another direct approach is to inhibit the biosynthesis of these hormones so as to counter the adverse effects caused due to enhanced sympathetic activity. Dopamine beta-hydroxylase (DBH) is an enzyme involved in the production of these hormones. DBH converts dopamine to NE (which is converted to E) within chromaffin granules of the adrenal medulla. Inhibition of DBH decreases the production of NE and E, resulting in a decreased stimulation of the adrenoreceptors on the heart and blood vessels, thereby reducing cardiac output and total peripheral resistance to reduce blood pressure. In addition, this increases dopamine levels, which has a beneficial effect, as it binds to dopaminergic receptors on renal tubules and causes natriuresis and diuresis, lowering blood pressure. We have found a promising inhibitor of DBH using computer-aided drug designing. The identified hit was validated using in vitro, ex vivo, and in vivo experiments. In this study, we summarize the acute, subacute, and genotoxicity studies, which have regarded the drug as nontoxic. We also present a 1-day pharmacokinetic study in a rat model showing changes in its concentration with time after oral and intravenous administration [Figure 7].{Figure 7}

Keywords: sympathetic nervous system, Dopamine beta-hydroxylase (DBH), genotoxicity

4n. Allyl Methyl Sulfide Attenuating Platelet Activation in Diabetes by Inhibiting the Metabolism of Arachidonic Acid Pathway

Malladi Navya, Ebin Johny1, Aishwarya Jala2, Roshan M. Borkar2, Ramu Adela1, Sanjay Kumar Banerjee

Departments of Biotechnology, 1Pharmacy Practice and 2Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India

E-mail: [email protected]

Background: Platelet activation plays a pivotal role in the pathophysiology of many cardiovascular diseases as well as diabetes. Current antiplatelet drugs have demonstrated reduced efficacy and increased adverse effects in diabetic patients. We proposed that allyl methyl sulfide (AMS), an active metabolite of garlic, could be a safe and alternative antiplatelet therapy for diabetes. Therefore, the goal of this study was to determine the therapeutic effect of AMS in reducing platelet activation in diabetes and the molecular mechanism thereof. Materials and Methods: The streptozotocin-induced diabetic rats were given AMS for 10 weeks. At 3-and 10-week postdiabetes, platelet activation, aggregation, platelet–macrophage interaction, and platelet endogenous ROS production were assessed. In addition, we did Liquid chromatography–mass spectrometry (LCMS)study with the platelets to understand the molecular mechanism through metabolic alterations. Results: The platelet activation was significantly reduced after 10 weeks of AMS treatment, but there was no effect after the first 3 weeks. Furthermore, at 3rd and 10th weeks, AMS significantly reduced both baseline and ADP-induced platelet aggregation. In addition, after 10 weeks of AMS treatment, platelet–macrophage interactions and platelet endogenous ROS concentration decreased. Moreover, a metabolomic study revealed that AMS reduced numerous compounds associated with arachidonic acid such as 15-HETE, 15-keto prostaglandin A1, 13,14-dihydro-15-keto prostaglandin F2 alpha, and also 7-hydroxy docosahexaenoic acid in the platelets of diabetic rats. Conclusion: In the current study, elevated platelet activation was linked to increased metabolites of arachidonic acid pathways in diabetic rats. AMS attenuated platelet activation, aggregation, and platelet–macrophage interaction by lowering arachidonic acid pathway metabolites in the platelets of diabetic rats [Figure 8].{Figure 8}

Keywords: aggregation, allyl methyl sulfide, endogenous, 7-hydroxy docosahexaenoic acid

4o. Terbium Hydroxide Nanoparticle, an Innovative and Alternative Medicine for the Treatment of Myocardial Ischemia-Reperfusion Injury

Papia Basuthakur1,2, Arpita Roy1,2, Chitta Ranjan Patra1,2, Sumana Chakravarty1,2

1Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad, Telangana, 2Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh, India

E-mail: [email protected]

Myocardial ischemia-reperfusion injury (MIRI) is a major and invincible underlying cause of heart failure. Considering the escalating cost and mediocrities of current therapeutic approaches, there is a dire need to conceive safer and more versatile strategies for the treatment of MIRI. In this regard, nanomedicine, with its vast meritorious applications in biomedicine, may offer new breakthroughs in the treatment of MIRI. In the light of this prospect, the present study was initiated to explore the therapeutic potential of proangiogenic terbium hydroxide nanorods (THNs) for the treatment of MIRI. For our study, cellular model of MIRI was developed by chemical (CoCl2) induction of hypoxia reperfusion injury (I/R) in cardiomyocytes (H9C2). Our studies displayed major protective role of THN by scavenging I/R-induced oxidative stress and subsequent lipid peroxidation. In addition, THN shown to restore impaired mitochondrial dynamics by preserving mitochondrial membrane potential and also to restrict the progress of I/R-induced mitochondrial apoptotic pathway by quenching the activation of caspase 3 with simultaneously suppressing the release of mitochondrial cytochrome C into cytosol. Concomitantly, the treatment also compliments the survivability of cells by restoring stress-induced DNA damage. Finally, THN seems to enhance intracellular tolerance for calcium overload and thereby may support normal cardiac rhythm. Overall, the results warrant in-depth molecular investigations and more comprehensive optimization of THN to be translated into clinical research, for the treatment of MIRI [Figure 9].{Figure 9}

Keywords: Apoptosis, calcium overload, cardiomyocyte, CoCl2, ischemia-reperfusion, mitochondria dysfunction, oxidative stress, terbium hydroxide nanorods

4p. Aloin Inhibits Cardiac Hypertrophy by Upregulating Nuclear Factor Erythroid 2–Related Factor-2-Mediated Antioxidant Responses and Alleviating TGF-β/Smad2/3 Signaling Axis

Sourav Kundu, Bidya Dhar Sahu

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Guwahati, Assam, India

E-mail: [email protected]

Cardiac hypertrophy is a serious concern of heart failure, resulting in high mortality and morbidity. It can be detected by excessive oxidative stress and extracellular matrix accumulation. Aloin, an anthraquinone glycoside isolated from Aloe species, has a wide spectrum of cardioprotective activities. As the effect of aloin on cardiac hypertrophy and its mechanism is less explored, hence we investigated. Cardiac hypertrophy was successfully induced in male Sprague–Dawley rats by subcutaneous isoproterenol (5 mg/kg) injection. Meanwhile, the animals were administered aloin (25 and 50 mg/kg/day) orally. Oxidative stress markers, echocardiographic parameters, histopathological alterations, cardiac injury markers, fibrotic markers, and their upstream signaling cascades were evaluated to find the effect of aloin in attenuating cardiac hypertrophy. Our results indicate that aloin treatment resulted in amelioration of oxidative stress by activating master regulator of antioxidant defense system nuclear factor erythroid 2–related factor-2 (Nrf2). Further evaluation showed that aloin increased the translocation of Nrf2 along with the expression of antioxidant genes NQO1 and heme oxygenase-1 (HO-1). Besides, aloin also impeded oxidative stress by decreasing ROS generation and increasing the antioxidant levels including superoxide dismutase and reduced glutathione. In addition, aloin significantly reduced the expression of hypertrophic markers and oxidative stress in H9c2 cells. Moreover, aloin treatment markedly reduced the fibrotic protein expression of collagen I, α-smooth muscle actin, fibronectin, transforming growth factor (TGF-β), and Smad2/3. Thus, aloin has the potential to be a promising therapeutic agent that stimulates Nrf2 signaling and blocks the TGF-β/Smad2/3 pathway and so prevents the cardiac hypertrophy [Figure 10].{Figure 10}

Keywords: aloin, erythroid 2–related factor-2 (Nrf2), anthraquinone glycoside, fibronectin

4q. Efficacy and Cardiovascular Safety of SGLT-2 Inhibitors: A Network Meta-Analysis of Randomized Controlled Trials

Raja Chakraverty

Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India

E-mail: [email protected]

Aim: This meta-analysis of randomized clinical trials intended to evaluate the efficacy and safety of sodium-glucose cotransporter 2 inhibitors (SGLT2is) have been released recently including trials enrolling patients with congestive heart failure (CHF) and chronic kidney disease. Methods: We searched the PubMed, EMBASE, and Cochrane databases for relevant studies published up until December 26, 2021. The study protocol was preregistered in PROSPERO. The effect size (mean difference or risk ratio [RR]) was reported with its 95% confidence interval (CI). Results: A total of 14 trials with 89,930 participants were included in this network meta-analysis. SGLT2is were associated with lower risks of 3P-MACE than placebo (RR 0.69, 95% CI 0.81, 0.94 and RR 0.78, 95% CI 0.81, 0.92, respectively). Conclusion: SGLT2i affords better protection to Dipeptidyl Peptidase-4 (DPP-4) Inhibitors in terms of cardiovascular and renal outcomes. SGLT2i appears to be superior in reducing the incidence of CHF and improving renal outcomes based on our findings.

Keywords: EMBASE, Cochrane databases, PROSPERO

4r. Identification of Prognostic Markers for Acute Heart Failure using SWATH-MS-Based Plasma Proteomics

Praveen Singh1,2, Harshita Singh3, Anurag Raj1,2, Manoj Kushwaha1, Debasis Dash1,2, Sandeep Seth3, Shantanu Sengupta1,2

1Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India, 3Department of Cardiology, All India Institute of Medical Sciences, New Delhi, 2Academy of Scientific and Innovative Research, Ghaziabad, Uttar Pradesh, India

E-mail: [email protected]

Acute heart failure (AHF) is defined as a syndrome with rapid onset or gradual worsening of signs and symptoms of heart failure (HF) primarily as a result of pulmonary congestion. In the presence of preexisting cardiomyopathy or an undiagnosed de novo HF, conditions such as hypertension, coronary heart disease, valvular heart disease, or other factors may precipitate or induce AHF. It is characterized by high mortality and hospital readmission rates. In the first 3 months after hospitalization, the readmission rates are as high as 30% and the overall 1-year mortality rates are up to 23% in India. Current treatment for AHF is mostly symptomatic, based on decongestive drugs. Poor management often leads to worsening of the condition. To identify proteomic prognostic markers, we segregated AHF patients in three categories (recovered, sick, and expired) based on baseline and 3 months' follow-up data of NT-pro BNP, ejection fraction, NYHA functional classification, and physical signs such as climbing a flight of stairs without stopping. “Recovered” showed improvement with symptoms, “sick” presented worsened symptoms on follow-up, while “expired” could not survive till 3 months after their first hospital admission. We recruited 30 patients in each category and performed SWATH-MS-based label-free quantitative plasma proteomics on baseline and follow-up samples. Identified plasma proteins were analyzed using Boruta feature selection algorithm to identify features (proteins) that can segregate between the three categories of patients on baseline data. We could identify 215 common proteins among all samples. The feature selection tool provides 16 proteins which can act as potential prognostic markers among AHF patients.

Keywords: pulmonary congestion, SWATH-MS, Boruta feature selection algorithm

4s. Identification of Potential Lipid Species Associated with Coronary Artery Disease

Salwa Naushin, Akash Kumar Bhaskar, Anurag Raj, Sandeep Seth, Debasis Dash, Shantanu Sengupta

CSIR-Institute of Genomics and Integrative Biology, New Delhi, India

E-mail: [email protected]

Background: Complex diseases such as coronary artery disease (CAD) are controlled by various genetic, environmental, and lifestyle factors. Lipidomic profiling is a newer approach for identifying potential biomarkers. It could also help delineate the mechanisms associated with cardiovascular diseases (CVDs) since perturbation of lipid metabolism is prominent in CVD and is one of the main mechanisms that cause disease progression. In this study, lipid species that might be exploited as possible biomarkers for CAD were identified using multiple reaction monitoring (MRM)-based mass spectrometric approach. Methods: The study was conducted at CSIR-IGIB in collaboration with All India Institute of Medical Sciences, New Delhi. We analyzed 304 samples (153 control and 151 CAD) using our recently developed MRM-based lipidomic approach where more than 1000 lipid species representing different types of lipid classes can be identified. MultiQuant 3.0.2 software is sufficient was used for peak review and data processing. Results: This approach led to the identification of several lipid species that were significantly differentially expressed in CAD samples (adjusted by BH and confirmed/tentative selection using Boruta random forest). We found ceramides species, CER.24.1, CER.18.0 were elevated in CAD and CER.24.0 were down compared to control. Plasma ceramides have been shown to be a promising marker in the identification of patients at risk of adverse cardiovascular events. We also show that triglycerides containing long-chain fatty acids and not short- and medium-chain fatty acids are associated with CAD risk in the Indian population. Conclusions: Our study identified a range of potential lipid biomarkers for the differentiation of CAD. Lipid biomarkers along with clinical predictors might be useful candidates in differentiating CAD from healthy individuals.

Keywords: coronary artery disease, cardiovascular diseases (CVDs), perturbation, disease progression

4t. Identification of Potential Diagnostic Markers for Coronary Artery Disease

Salwa Naushin, Praveen Singh, Rajat Ujjainiya, Anurag Raj, Sandeep Seth, Debasis Dash, Shantanu Sengupta

CSIR-Institute of Genomics and Integrative Biology, New Delhi, India

E-mail: [email protected]

Background: Complex diseases such as coronary artery disease (CAD) are multifactorial in nature. Identification of precise markers indicative of its occurrence and progression is important for improving disease diagnosis. Proteomic profiling is a robust approach for exploring new biomarkers. In this study, we utilized global proteomic approach for the identification of proteins that can act as the potential biomarkers for CAD. Methods: For this study, we employed data independent approach-based SWATH-MS for the identification of differentially expressed proteins between control and CAD in 120 samples (60 control and 60 CAD). Results: We are able to identify a total of 368 proteins, of which 15 were found to be significantly differentially expressed between control and CAD samples. Using a random forest-based feature selection algorithm (BORUTA), we identified a list of five proteins that could significantly differentiate CAD from control with 77.8% confidence. Validation of differentially expressed proteins will be done in large sample size using targeted peptide detection approach. Conclusions: Identification of proteins that can significantly differentiate CAD from controls might improve disease diagnosis and prognosis. This might further help in understanding the disease mechanism.

Keywords: Potential Diagnostic Markers, algorithm, SWATH-MS

4u. Effect of Vitamin D Supplementation in Cardiovascular Disease Prevention in Type 2 Diabetes Patients: Focusing on Platelet-Mediated Inflammation

Ebin Johny, Amir Ali, Aishwarya Jala1, Bishamber Nath, Indra Kuladhipati2, Rupam Das2, Roshan M. Borkar1, Ramu Adela

Departments of Pharmacy Practice and 1Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, 2Department of Cardiology, Down Town Hospital, Guwahati, Assam, India

E-mail: [email protected]

Introduction: Diabetic patients exhibit a two–four-fold higher risk of cardiovascular disease compared to nondiabetic patients. In type 2 diabetes mellitus (T2DM) and coronary artery patients, increased platelet activation was observed and initiated platelet-mediated inflammation. Moreover, severe Vitamin D insufficiency was observed in T2DM and T2DM with coronary artery disease patients. From previous reports, it is known that Vitamin D plays a vital role in platelet hyperactivity and various immune function regulations, but the effect of Vitamin D on platelet-induced inflammation is not well studied. Hence, we hypothesized that Vitamin D supplements might suppress platelet-mediated inflammation and reduce the cardiovascular disease risk in T2DM patients. Methods: We conducted a randomized, double-blind, placebo-controlled trial in which a dose of 60,000 IU/week of Vitamin D3 (cholecalciferol) was given as a management dose for the first 3 months followed by a maintenance dose of 60,000 IU/month for the next 3 months. Serum Vitamin D metabolite levels, platelet activation, platelet-immune cell aggregate formation, and immunome profiling were assessed at baseline and 6 months of the trial. Results: This study has shown improvement in circulatory Vitamin D levels in T2DM patients. Our study highlights that Vitamin D supplementation downregulated platelet activation and platelet-immune cell aggregation (P < 0.05). Moreover, it also downregulated the serum TNF-α, IL-18, IFN-γ, CCL-2, CCL-5, CCL-11, CXCL-10, and CXCL-12 levels compared to the baseline levels (P < 0.05). However, Vitamin D3 supplementation does not improve the glycemic parameters. Conclusion: The outcomes of this study suggest that supportive therapy with Vitamin D may help decrease or minimize the risk of cardiovascular disease development in T2DM patients by attenuating platelet-mediated inflammation [Figure 11].{Figure 11}

Keywords: Inflammation, platelet, Vitamin D, type 2 diabetes

4v. Acute Heart Failure Registry-II: Predictors of Acute Outcome: A Preliminary Report

Shivani Vashista, Kavita Kumari, Deepika Jindal, Santoshi Sahani, Sandeep Seth, Anurag Rathore1, Shantanu Sengupta2, Chandra Bhan Meena3, Deeraj Gandotra4

Department of Cardiology AIIMS, 1Department of Chemical Engineering, IIT, 2CSIR-Institute of Genomics and Integrative Biology, New Delhi, 3Department of Cardiology, SMS Medical College Jaipur, Rajasthan, 4Department of Cardiology, BLK Hospital, Delhi, India

E-mail: [email protected]

Objective: With increasing numbers of heart failure patients, there was a serious need for studying the predictors of adverse outcome in Indian patients with heart failure. We herein report the in-hospital and 6-month outcomes of Indian patients admitted with heart failure. Methods: We enrolled patients with heart failure with systolic dysfunction in the acute failure registry and followed them up for 6 months. We analyzed the data on events (death, hospitalization, and any surgery during the study) in 158 patients over 6 months. Results: A total of 158 patients were enrolled with a mean age of 0.70 ± 14.32 years, with females:male 47:111 and median age 43 years, and the majority were male (77%). The mean left ventricular ejection fraction was 29.2% ± 11.9%. In-hospital mortality was 20.8%. 6-month postdischarge major adverse events occurred (mortality, re-hospitalization) combined rates were 15%. This group had higher eGFR and liver function and higher values of inflammatory biomarkers such as Nt proBNP. Detailed results will be discussed. Conclusions: Indian patients with heart failure have higher morbidity and mortality as compared to the West. The patients who have adverse events have higher levels of BNP and impaired renal and liver function at presentation. These can act as markers of future adverse events.

Keywords: predictors, eGFR and liver function, morbidity and mortality

4w. Efficacy and Safety of an Indian System of Medicine Lifestyle Intervention Program Including Yoga in Heart Failure: The KRYOG Randomized Controlled Trial

Sandeep Seth, Gautam Sharma, S. K. Maulik1, Raj K. Yadav 2, Shivani Vashista, Archana Saini, Karishma Landge, Pooja Bhardawaj, Rabindra Acharya3, Hemant Bhargava 3

Departments of Cardiology and 2 Physiology, AIIMS, Delhi, 3Swami Vivekananda Yoga Anusandhana Samsthana) University, Bengaluru, Karnataka, 2Dr. Subir K Maulik, Emeritus Scientist ICMR, New Delhi, India

E-mail: [email protected]

Background: Cardiac rehabilitation (CR) is one of the established treatment options for heart failure. CR improves exercise capacity, quality of life, morbidity, and mortality. Studies of Yoga in heart failure have shown improved quality of life, decrease in heart rate and inflammatory markers. Objectives: The study compared the clinical outcomes of lifestyle management through an Indian System of Medicine including Yoga in patients with heart failure. Parameters assessed included clinical outcomes, functional capacity (6-min walk test), and quality of life. Methods: This was an open-label, randomized interventional study. The intervention arm included patients with heart failure allocated to standard therapy for heart failure with lifestyle management through the Indian System of Medicine including Yoga. The study period was 6 months. Results: The intervention (KRYOG Study) led to a significant improvement in the functional status of the patients as manifested by the 6-min walk test and the quality of life scores as assessed by the SF36 scales, over 6 months. This was over and above guideline-directed medical therapy. The Yoga intervention was safe and effective. Conclusions: Yoga lifestyle intervention provides improvement in functional capacity in patients on guideline-mandated heart failure medication. DST Reference No.: SR/SATYAM/13/2016(G). AIIMS Ethics Committee: Date (IEC-131/04.03.2016,RP-12/2016), CTRI/2018/05/013605 (Registered on May 2, 2018).

Keywords: 6-min walk test, Yoga intervention, Medicine including Yoga

4x. Heterogeneity among Uncontrolled Type 2 Diabetes Patients

Sujay Krishna Maity, Jit Sarkar, Avishek Paul, Partha Chakrabarti

Department of Cell Biology and Physiology, CSIR-Indian Institute of Chemical Biology, Kolkata, West Bengal, India

E-mail: [email protected]

Type 2 diabetes mellitus (T2DM) is earlier thought to be a homogenous clinical entity but has now been recognized as a heterogeneous disease with varying manifestations including disease progression, drug response, and the risk of cardiometabolic complications. Identification of these various manifestations and disease etiologies would result in customized T2DM management and would open up avenues for personalized medicine. Here, health records of 339 patients with uncontrolled T2DM were followed up for a median period of 14 months and were analyzed using uniform manifold approximation and projection followed by density-based spatial clustering of applications with noise. Oral glucose tolerance test was performed to assess insulin resistance and β-cell dysfunction. We identified three major clusters based on clinical parameters and various drug combinations. Among the clusters identified, the first cluster characterized by recent-onset T2DM had moderately preserved β-cell function. The second cluster with a longer duration of T2DM and associated hypertension showed the best glycemic control with dual antidiabetic therapy. The third cluster with the longest history of T2DM and no history of hypertension had the worst glycemic control in spite of the highest percentage of patients on triple therapy (34.58%) and quadruple therapy (8.41%). Uncontrolled T2DM comprises a heterogeneous population concerning disease duration, presence of hypertension, and β-cell function without significant difference in insulin resistance. Stratifying them based on pathological features is the first step toward personalized management in T2DM.

Keywords: β-cell dysfunction, glycemic, Stratifying

4y. COVID-19 Infection and Outcomes of Heart-Transplant Patients in India

Deepika Jindal, Santoshi Kumari, Shivani Vashista, Kavita Kumari, Sandeep Seth, Manoj Kumar Sahu, Sarvesh Pal Singh, Milind P. Hote

Department of Cardiology AIIMS, New Delhi, India

E-mail: [email protected]

Introduction: Heart transplant (HT) recipients are on chronic immunosuppressants and have an increased burden of comorbidities. Hence, there is increased susceptibility to severe COVID-19 infection which may result in worse prognosis. Objectives: The aim of this study was to investigate the events in postheart-transplant patients during different waves of COVID-19. Events evaluated included clinical presentation, hospitalization, home isolation, and treatment changes. Methods: A retrospective observational study was conducted to analyze frequency of events in 30 heart-transplant recipients in AIIMS, New Delhi. Eleven patients were COVID-19 positive from January 2020 to May 2022 (3 different COVID-19 pandemic waves). Results: Eleven (36.6%) heart-transplant patients got COVID-19 infection in the first, second, and third waves. COVID-19 infection ratio during all three waves was 4:4:3. In the first wave, 4 patients were admitted electively, observed, and discharged with no clinical symptoms. In the second wave, 4 patients were exposed, and due to the scarcity of beds in the hospital, the patients were home isolated and managed. One 65-year-old male patient desaturated, was admitted for 1 week, kept on high-flow oxygen and steroid, and discharged with no complications. In the third wave, three patients who got COVID-19 were kept in home isolation and recovered with no complications. Two patients were exposed twice with COVID-19 in different waves of pandemic and recovered well. Mean age of the patients was 40.1 years with female:male ratio of 3:8. Patients clinically presented with fever, myalgia, cough, and anosmia. Vaccination percentage was 72.7%. Covishield:Covaxin ratio was 4:3. Conclusion: Patients were managed by discontinuing mycophenolate doses and increasing steroid doses, respectively. Their hemogram and total leukocyte count were evaluated for a week and were discharged when values were normal. Severity of infection was mild to moderate. Mortality was none and all patients had good prognosis.

Keywords: Heart transplant, retrospective observational study, COVID-19 infection, Vaccination

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Conflicts of interest

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