CURRICULUM IN CARDIOLOGY - JOURNAL CLUB

Year : 2016  |  Volume : 2  |  Issue : 2  |  Page : 110-113

RNA sequencing analysis identifying new human collagen genes involved in cardiac remodeling

Pooja Singh¹, Suganthi Ramchandran¹, Sudip Sen^2
1 Department of Pharmacology, AIIMS, New Delhi, India
² Department of Biochemistry, AIIMS, New Delhi, India

Correspondence Address:
Pooja Singh
Department of Pharmacology, AIIMS, New Delhi
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/2395-5414.191535

Introduction

• Ventricular remodeling involves changes in cardiomyocytes and extracellular matrix (ECM) [Figure 1]
• Remodeling initially occurs as a compensatory response to preserve the structural integrity of the myocardium, but progressive collagen deposition can lead to cardiac fibrosis and impairment of systolic and diastolic function
• Collagen deposition in ECM consists primarily of collagen Type I and III
• These collagens are constantly synthesized and secreted as procollagens in the interstitial space. Increased collagen turnover has been associated with myocardial fibrosis and plays an important role in determining the functional properties of arterial vasculature and ventricular myocardium ^[1]
• The ratio of collagen Type I: Type III is thought to be responsible for cardiac remodeling
• Myocardial stiffness does not always correlate with the amount of collagen deposition. This has led to the proposal that it is not the amount of collagen that matters; it is the cross-linking ^[2],[3] or the relative proportion of stiffer collagen phenotype (Type I) to that of more elastic type (Type III),^[4] which determines the stiffness.^[5]

Figure 1: Changes of fibrosis in heart failure. Click here to view

• To analyze the changes in expression profile of collagen-related genes in patients with ischemic cardiomyopathy (ICM)
• To examine the relationship of changes in expression profile of collagen-related genes and left ventricular (LV) dysfunction.

• A nonrandomized case–control study
• Twenty-three human LV tissue samples from patients with ICM (13 men; mean age 54 ± 8 years) undergoing heart transplantation and control donors (CNT) (8 men, 2 women; mean age 47 ± 16 years)
• All control donors had normal LV function (ejection fraction >50%) and no history of cardiac disease
• As an established condition for inclusion in the study, all samples displayed a 260/280 nm absorbance ratio >2.0 and RNA integrity number (RIN) ≥9. It is possible to relate the amount of light absorbed to the concentration of the absorbing molecule such as DNA or RNA. It is common for nucleic acid samples to be contaminated with other molecules (i.e., proteins, organic compounds). The ratio of absorbance at 260 and 280 nm (A260/280) is used to assess the purity of nucleic acids. For pure DNA, A260/280 is ~1.8, and for pure RNA, A260/280 is ~2. The RIN software algorithm allows the classification of total RNA based on a numbering system from 1 to 10, with 1 being the most degraded and 10 being the most intact
• The 260 nm/280 nm ratio of purified RNA is believed to be around 2.0, and it is generally accepted that a lower 260 nm/280 nm ratio indicates contamination and a higher one is a result of the purified RNA.

Methods

Results

• ICM and CNT samples were compared and it was found that significantly increased mRNA levels of 11 collagen genes, such as COL9A1, COL11A2, COL14A1, and COL16A1 (P < 0.05 for all), not previously described in cardiac remodeling
• A heat map and hierarchical clustering analysis identified two divergent gene expression profiles, showing a clear separation of ICM and CNT groups [Figure 2].

Figure 2: Heat map and hierarchical clustering analysis explained. Click here to view

• For validation of novel regulated transcripts, quantitative RT-PCR and Western blot analysis were performed focusing on those related significantly with LV function: COL16A1 and COL4A5, and COL14A1, newly described. These results confirm that patients with ICM showed direction changes in expression identical to what we found in RNA sequencing analysis.

Discussion

• Collagen Types IX, XIV, and XVI belong to the fibril-associated collagens with interrupted triple helices class, highly expressed in tissues that have high mechanical stress, such as heart tissue
• Collagen Type IX is thought to be required for several purposes, including eye and heart development and its overexpression has been observed in pathologies such as pectus excavatum
• Previous studies suggest that collagen Type XIV is required to establish and maintain an organized ECM environment in the developing myocardium and play an important role in regulating cardiomyocyte growth and cardiac fibroblast survival. During replacement fibrosis, cardiomyocytes undergo hypertrophic adaptive changes, whereas myofibroblast remains at the site of injury; this results in collagen deposition and scar formation
• Collagen Type XVI, a minor collagen component of connective tissue, is thought to act as a linker protein, helping to organize the large fibrillar networks that modulate ECM integrity and stability. A recent study proposed that in Crohn's disease, increased collagen Type XVI expression would promote the formation and maturation of focal adhesion contacts on intestinal subepithelial myofibroblasts. This would retain the cells at inflammation site, promoting fibrotic responses in the tissue and prolonging disturbances of cellular and ECM homeostasis
• Overexpression of COL9A1 could lead to increased cardiac tissue stiffness and may be potentially involved in human heart development and remodeling
• It is suggested that dysregulated collagen Type XVI expression could play a similar role in cardiac tissue by keeping myofibroblast at the inflammation site and promoting pathological remodeling
• Collagen Type XI promotes the nucleation of Type I and II fibrils and is required for myocardial morphogenesis. The overexpression of COL11A2 may be related to formation of heterotypic fibrils with collagen Type I, involved in cardiac remodeling.

• Patients with end-stage heart failure are under heavy medical treatment and some therapies might influence mRNA levels.

Conclusions

• The study findings propose that collagen Types IV, XIV, and XVI may have a novel role in:
• 
  
  
  • Remodeling process
  • Regulating size of cardiomyocytes
  • Survival of myofibroblasts at the inflammation site and assisting the organization in fibers other collagen, such as Types I and III
  • All these processes may slightly facilitate the development of cardiac fibrosis and consequently ventricular dysfunction
  • Inhibition of cardiac remodeling can lead to improved cardiac function, demonstrating the relevance of new insights into the compensatory remodeling mechanism
  • Thus, the study findings support new theoretical evidence for treatment of patients with ICM.

References

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4.	Mukherjee D, Sen S. Collagen phenotypes during development and regression of myocardial hypertrophy in spontaneously hypertensive rats. Circ Res 1990;67:1474-80.
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