Targeting Peroxisome Proliferator Activated Receptor α (PPAR α) for the Prevention of Mitochondrial Impairment and Hypertrophy in Cardiomyocytes

Abstract

Background/Aims: Morphological and biochemical maladaptation of cardiomyocytes are associated with mitochondrial dysfunction and dysregulation in hypertrophic conditions. Peroxisome proliferator activated receptor α (PPARα), a drug target for dyslipidemia, is known to be downregulated in cardiomyocytes in response to hypertrophic stimuli. The current study was undertaken to investigate the role of PPARα signaling in mitochondrial remodeling and thereby dysregulation of cardiomyocytes due to hypertrophy in vitro. Methods: Rat cardiomyocytes H9c2 (2-1) and neonatal rat ventricular myocytes (NRVMs) were cultured and treated with α1-adrenergic agonist phenylephrine (PE, 100 µM, 24 hours) in the presence or absence of 10 µM fenofibrate or bezafibrate. Cellular hypertrophy was observed by atomic force microscopy and immunofluorescence with F-actin antibody. mRNA levels of hypertrophic marker genes and other genes were examined by quantitative real time PCR. Structural as well as functional remodeling of the mitochondria were evaluated by immunofluorescence (F-actin and COX-I), live cell imaging microscopy (JC-I, mitotracker), mitochondrial complex V activity, MPTP activity and ATP assay. Oxidative stress was measured by using sensitive fluorescent indicator probes. Cellular and mitochondrial calcium were measured by using fluorescent indicator probes Rhod-2 AM and X-rhod-1 AM, respectively. Targetscan prediction analysis was performed to find out miRNAs as putative regulators of VDAC. Luciferase assay was conducted to confirm binding of miR28 with VDAC. Results: Co-treatment of H9c2(2-1) cells with PE and fenofibrate restricted increase in cell size and expression of marker genes such as atrial-natriuretic peptide (ANP), brain-natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC) compared to those with PE alone. Fenofibrate prevented PE-induced down regulation of PPARα-target genes like CPT-I and MCAD. Mitochondrial trans-membrane potential (Δψm) and motility were reduced by PE which were significantly checked by fenofibrate. Increased ROS production and calcium level in PE-treated cells were ameliorated by fenofibrate. Mitochondrial activity and ATP generation were reduced by PE which was rescued by fenofibrate. Fenofibrate also prevented PE-induced down regulation of mitochondrial genes like VDAC-I and COX-IV. Expression of several miRNAs was altered in hypertrophic cardiomyocytes which were restored when co-treated with fenofibrate. miR28 was found to target 3’ untranslated region of VDAC-I. Conclusion: Overall, the results demonstrate that PPARα signaling is critically involved in mitochondrial dysfunction in hypertrophic cardiomyocytes in which miR28 plays a pivotal role.