Elucidation of Mitochondrial Regulatory Pathways and Molecular Stress Scavengers in Cardiovascular Diseases

Abstract

Cardiac hypertrophy in which heart muscles enlargement occur and myocardial infarction (MI) due to arteries blockage are disorders considered in current study. Pro free radical oxidative stresses such as Reactive Oxygen Species (ROS) generation due to mitochondrial stress as well as regulating factors are believed to be the important players in diabetes associated heart disorders. The first aim investigates the cardioprotective effect of plant extracts Incarviella emodi (I.E) and its Myoinositol rich fraction (M.I). Quantitative analysis of Incarvillea emodi through HPLC showed presence of active antioxidative potential metabolites. FT-IR analysis showed presence of active functional groups. Cell viability was checked through MTT assay in H9c2 and Neonatal rat cardiomyocytes (NRCMs) at different doses of N-acetyl cysteine (NAC), I.E and M.I. Hypertrophic response analysis was done in Neonatal rat cardiomyocytes (NRCMs) and H9c2 through phalloidin staining. For measurement of oxidative stress, we have performed DCFDA analysis. Sprague Dawley rat in vivo hypertrophic model was confirmed by assessing the morphological parameters, cell surface area and histopathological examination. Biochemical analysis on serum and tissue samples showed significant increases oxidative profile and decreased antioxidative expression of antioxidative markers. Additionally, liver damage was evaluated by toxicity markers, which showed safer dosage administration. At molecular level, increased expression of p53, PUMA (P53 upregulated modulator of apoptosis), Tp53BP2 (tumor protein P53 binding protein 2), Drp-1 (dynamin related protein-1), MDM4 (murine double minute 2), cytochrome c and BID (BH3-interacting domain death agonist), and miR-15a-5p and downregulation of MFN-2 (mitofusin-2) and miR-214-3p were observed in both in vivo and in vitro model. Additionally, increased mRNA levels of PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha), OXPHOS-2 (mitochondrial oxidative phosphorylation system-2), SOD-2 (superoxide oxide dismutase), Gpx-1 (glutathione peroxidase 1), and decreased expression of miR-151-3p, OXPHOS-1 (mitochondrial oxidative phosphorylation system-1) was observed in H9c2 cells. Transfection with miR-151 mimic in H9c2 regulated the expression of mitochondrial linked biomarkers. Increased expression of biomarkers p53, Phosphorylated P53 (P-p53), Drp1, PUMA, cytochrome c was observed at translational level in in vivo and in vitro model. Decreased protein xvii Abstract expression of PGC-1α, OXPHOS-1 and increased expression of OXPHOS-2, OXPHOS-3 and OXPHOS-4 was observed in H9c2 cells. The 2nd aim of study was to investigate the therapeutic potential of antioxidants and molecular player’s microRNAs in alloxan and streptozotocin induced cardiac hypertrophy. In the first part of this objective, we initially established a diabetic rat model by alloxan-administration, which was validated by peripheral glucose measurement. Diabetic rats displayed myocardial stiffness and fibrosis, changes in heart weight/body weight, heart weight/tibia length ratios, increased oxidative stress and decreased antioxidative expression profile, enhanced size of myocytes, which altogether demonstrated the establishment of diabetic cardiac hypertrophy (DCH). Furthermore, we examined the expression of genes associated with mitochondrial signalling impairment. In the 2nd part of diabetic associated cardiac pathologies research, we have investigated the cardio protective role of miRNA-98-5p mimic in diabetes linked cardiac hypertrophy and its mechanistic regulation in the progression of diseases. This part of our study aimed to analyze the role of endothelin-1 in cardiac hypertrophy. Experimental groups of Sprague Dawley rats were divided in normal control, isoproterenol, synthetic immunogenic endothelin-1 peptide, Freund’s Complete Adjuvant (FCA), and peptide + adjuvant groups including 14 and 28 days. Baseline parameters including elevated heart weight/body weight and heart weight/tibia length ratios, increased cellular surface area, and disrupted cellular organization was found and confirmed cardiac hypertrophy. High immunogenic activity of peptide and adjuvant+ peptide groups reduced oxidative stress, upregulated antioxidative enzymes production and lower the levels of liver marker enzymes, triglycerides, and cholesterol. Quantitative real time PCR showed upregulated transcriptional level expression of ET 1, ANP and BNP. Western blot analysis showed elevated levels of endothelin-1 both in serum and heart homogenates in 28-days groups. ELISA results showed elevated total IgG count in 14- days disease groups and upregulated endothelin-1 specific antibodies in 28-days groups. Thus, these results indicated that upregulated circulating levels can help in early-stage diagnosis of disease before progression to heart failure. Collectively, our research provides a plat form for development of therapeutic and diagnostic strategy against cardiovascular disease. Further studies are needed to employ our research in therapeutic and diagnostic applications.