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 (Δψ_m) 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.

MicroRNA and ROS Crosstalk in Cardiac and Pulmonary Diseases

“…Extracellular stimuli, including angiotensin II (AngII), endothelin-1 (ET-1), isoproterenol (ISO), phenylephrine (PE), and tumor necrosis factor alpha (TNFα), can induce myocyte hypertrophy through the activation of signaling pathways downstream transduction factors, e.g., mitogen-activated protein kinases (MAPK), protein kinase C (PKC), NF-κB, calcineurin, and tyrosine kinases that can be activated by ROS [32,33]. Mitochondrial dysfunction and dysregulation can also directly impact morphological and biochemical (mal-)adaptation of cardiomyocytes [34,35]. Several miRNAs have been involved in the regulation of oxidative stress by targeting ROS generators, antioxidant pathways, and antioxidant effectors [36] in cardiac hypertrophy, having either positive or negative effects on the cardiac function [37,38].…”

“…Kar et al found that PE-induced hypertrophy reduced the trans-membrane potential of mitochondria, affecting the voltage-dependent anion channel (VDAC) expression, as well as mitochondrial activity and ATP generation [ 34 ]. Interestingly, the authors demonstrated that miR-28 directly targets VDAC, being responsible for its reduction and leading to an overall mitochondrial dysfunction [ 34 ]. In summary, while miR-25 inhibits it [ 48 ], miR-106 and miR-28 promote mitochondrial-derived oxidative stress in cardiac hypertrophy [ 34 , 50 ].…”

Dual PPARα/γ activation inhibitsSIRT1-PGC1α axis and causes cardiac dysfunction

“…However, constitutive PPARγ expression in cardiomyocytes of Ppara -/mice did not cause cardiac dysfunction, despite increased myocardial lipid content (11), indicating a toxic role for cardiac PPARα when PPARγ is activated as well. Moreover, rosiglitazone-mediated PPARγ activation promotes cardiomyocyte hypertrophy in vitro (46), while fenofibrate-mediated PPARα activation has the opposite effect in isolated cardiomyocytes and rescues mitochondrial function (47). Activation of PPARs relies on availability of FAs that are released either via LpL-mediated hydrolysis of lipoprotein triglycerides (48) or from intracellular triglycerides via ATGL-mediated lipolysis (49).…”

Downregulation of PPARα during Experimental Left Ventricular Hypertrophy is Critically Dependent on Nox2 NADPH Oxidase Signalling

“…Prior to stimulation, cells were seeded in 12-well plates at a density of 12,000 cells/well and incubated in 0.5% serum-containing DMEM for 24 h to induce quiescence. Cell hypertrophy was then stimulated by exposure to phenylephrine (PE; 10 μmol/L) for 24 h in the presence or absence of the pan-NADPH oxidase inhibitor, VAS2870 (10 μmol/L, Vasopharm, Würzburg, Germany), to dissect the specific contribution of Nox-derived ROS [ 61 ], or fenofibrate (10 μmol/L, Sigma-Aldrich), to identify the involvement of PPARα in H9c2 hypertrophy [ 62 ]. VAS2870 treatment of H9c2 cardiomyocytes completely abolished PE-induced increases in NADPH-dependent superoxide production ( Figure 1 F), whilst fenofibrate treatment of H9c2 cardiomyocytes increased protein expression of PPARα protein expression ( Figure S2 ), demonstrating efficacy in our employed culture model.…”