The Mechanisms Of Neonatal Rat Cardiomyocyte Hypertrophy Induced By Leptin

BackgroundObesity is a major risk factor for the development of cardiovascular diseases (CVD) such as hypertension, atherosclerosis, and heart failure. Leptin, the product of the ob gene, is a plasma protein secreted by adipocytes and is involved in the regulation of appetite and energy metabolism. Several studies have shown that leptin directly induced hypertrophy in neonatal rat cardiomyocytes and vascular smooth muscle cells in vitro. Our previous study has demonstrated that leptin stimulated endothelin-1 (ET-1) production and induced hypertrophy via ET-1-reactive oxygen species (ROS) pathway in cultured neonatal rat cardiomyocytes. However, the blockade of ETA receptors by ABT-627 was not able to completely abolish leptin-induced ROS production and the hypertrophic effect. These results suggest that there may be other pathway(s) by which leptin induces cardiomyocyte hypertrophy.Peroxisome proliferator-activated receptor (PPAR)αis abundant in tissues with high rate of mitochondrial fatty acid (FA) oxidation, such as heart, liver, and skeletal muscle. It regulates the expression of genes involved in every step of FA utilization including FA-uptake/-esterification, mitochondrial transport-, and, mitochondrialβ-oxidation. Recently, PPARαhas been thought as a key regulator of FA-metabolism in the heart, being substantially involved in the determination of substrate utilization during the development of cardiac dysfunction. Animal studies have shown that cardiac PPARαactivity was increased in diabetic rodent models including streptozotocin-induced diabetes and obese db/db mice and cardiac–restricted over expression of PPARα(MHC-PPAR) leaded diabetic cardiomyopathy in mice. A number of studies in humans have also shown that patients with heart failure exhibited increased rates of myocardial FA-uptake, the hearts of patients with end-stage dilated cardiomyopathy (DCM) presented upregulation of cardiac PPARαexpression and higher rate of cardiac FA-metabolism. Exogenous ligands of PPARα-fibrates- have been used for many years as effective hypolipidemic drugs. However, to date, no novel PPARαagent has reached an advanced stage of development. According to the report from US Food and Drug Administration (FDA), more than 50 PPAR agonists have been discontinued due to various types of toxicity including cardiomyopathy. These findings indicate that PPARα-driven reliance on fatty acid utilization and coordinate inhibition of glucose metabolism may be responsible for pathologic remodeling and severe cardiomyopathy.Leptin, as PPARαendogenous activator, regulates energy metabolism through activating PPARαin many cells. Therefore, we hypothesized that leptin induces cardiomyocyte hypertrophy through activating cardiac PPARαpathway. In part one, we investigated the relationship between PPARαoveractivation and cardiomyocyte hypertrophy. In part two, we investigated the role of PPARαoveractivation in leptin-induced cardiomyocyte hypertrophy.Part oneThe relationship between PPARαactivation and cardiomyocyte hypertrophyObjectiveTo investigate the relationship between PPARαactivation and cardiomyocyte hypertrophy.MethodThe study was performed with primary cultured neonatal rat cardiac myocytes. Ad-CMV-PPARαwas constructed to upregulate PPARαexpression. The cultured cardiomyocytes were treatment with 6.25-100μmol/L PPARαagonist fenofibrate. After 72 hours of treatment, total RNA levels, total protein levels, protein synthesis, ANF mRNA expression and cell surface area were measured. Meanwhile, the cardiomyocytes were infected by Ad-CMV-PPARα. After 48 hours of infection, protein synthesis, ANF mRNA expression and cell surface area were measured. After 4 hours of incubation with 6.25-100μmol/L fenofibrate, ROS levels were measured by a ROS-specific probe, 2', 7'-dichlorofluorescein diacetate (DCF-DA).Result1. PPARαagonist fenofibrate (6.25-100μmol/L) directly increased total RNA content, [3H]-leucine incorporation, and cell surface area of cultured neonatal rat cardiomyocytes. Fenofibrate also enhanced ANF mRNA expression in cultured neonatal rat cardiomyocytes (6.25μmol/L group P>0.05,12.5-100μmol/L groups P<0.05).2. Ad-CMV-PPARαdirectly increased PPARαmRNA and protein expression (P<0.05). After 48 hours of infection, [3H]-leucine incorporation and cell surface area of cardiomyocytes were increased, and ANF mRNA expression was enhanced (P<0.05).3. PPARαagonist fenofibrate significantly increased the levels of intracellular ROS in cultured neonatal rat cardiomyocytes in a concentration- dependent manner (6.25μmol/L group P>0.05,12.5-100μmol/L groups P<0.05).Brief summary1. PPARαoveractivation induces cardiomyocyte hypertrophy;2. PPARαoveractivation enhances ROS production of cultured neonatal rat cardiomyocytes. Part twoLeptin induces hypertrophy via activating PPARαpathway in cultured neonatal rat cardiomyocytes Objective1. To observe the role of PPARαdownregulation in leptin-induced neonatal rat cardiomyocyte hypertrophy.2. To investigate the mechanism of PPARαactivation induced by leptin.Method1. The cultured cardiomyoctes were incubation with 100 ng/mL leptin+ PPARαantagonist GW6471 (0.01-10μmol/L). Treatment 72 hours later, total RNA levels, and total protein levels, protein synthesis, ANF mRNA expression and cell surface area were measured. Meanwhile, the cardiomyocytes were transfected by PPARαsiRNA. Treatment with 100 ng/mL leptin+ PPARαsiRNA 48 hours, protein synthesis, ANF mRNA expression and cell surface area of cultured neonatal cardiomyocytes were measured.2. After 4 hours of incubation with 100 ng/mL leptin+ PPARαantagonist GW6471 (0.01-10μmol/L) and leptin+ PPARαsiRNA, ROS levels were measured by a ROS-specific probe, 2', 7'-dichlorofluorescein diacetate (DCF-DA).3. The cardiomyocytes were incubated with 1-1000 ng/mL leptin 24 hours. PPARαmRNA expression was measursed by Real-time PCR; PPARαprotein expression was detected by Western-blot.4. The cardiomyocytes were incubated with 1-1000 ng/mL leptin 24 hours. The PPARαactivity was measured by EMSA and DNA binding-ELISA.5. The cardiomyocytes were treatement with 100 ng/mL leptin 24 hours. M-CPT1,ACO,ACS,FAS,GLUT-4,GLUT-1,PFK,PDK4 mRNA expression were detected by Real-time PCR. Then, the cardiomyocytes were treatement with 100 ng/mL leptin at different time (10 mins,20 mins,30 mins,1 hour,3 hours,6 hours,12 hours,24 hours), the expression of p-AMPKα,p-ACC were detected by Western-blot. 6. The cardiomyocytes were preincubated with 40μmol/L Compond C 40 minutes, and then were treatment with 100 ng/mL leptin 24 hours. The protein expression of PPARαwas detected by Western-blot.Result1. The selective PPARαantagonist GW6471 concentration-dependently decreased ANF mRNA expression, and significantly decreased total RNA levels, total protein, protein synthesis, and cell surface areas (0.01μmol/L group P>0.05,0.1-10μmol/L groups P<0.05), all of which were elevated by 72 hours leptin treatment.2. RNA interference inhibited PPARαexpression. Protein synthesis, ANF mRNA expression, and cell surface areas which were elevated by 48 hours leptin treatment, were decreased by PPARαsiRNA (P<0.05).3. The augmentation of reactive oxygen species levels in leptin treated cardiomyocytes was reversed by 0.1-10μmol/L GW6471 and PPARαsiRNA respectively (0.01μmol/L group P>0.05,other groups P<0.05).4. After 24 hours treatment, leptin concentration-dependently enhanced mRNA expression and protein expression and activity of PPARα.5. 100 ng/mL leptin enhanced M-CPT1, ACO, FAS, PDK4 mRNA expression; Meanwhile, leptin decreased GLUT-1,PFK mRNA expression. Leptin induced phosphorylation of AMPKαand ACC.6. AMPK inhibitor Compond C (40μmol/L) inhibited the PPARαexpression elevated by 100 ng/mL leptin. Brief summary1. Cardiomyocytes hypertrophy induced by leptin was inhibited by PPARαantagonist GW6471 and PPARαsiRNA.2. Leptin induced PPARαmRNA and protein expression, upregulation PPARαactivity in a concentration-dependently manner.3. Leptin activated AMPKαthrough induced phosphorylation of AMPKα. Leptin increased metabolism of fatty acid, decreased metabolism of glucose.4. Leptin regulated PPARαexpression and activity through AMPKαactivation. Conclusion1. Chronic activation of PPARαinduces the cardiomyocytes hypertrophy and ROS production;2. Leptin induces hypertrophy through the PPARα-ROS pathway in cultured neonatal rat cardiomyocytes.3. Leptin maybe regulate cardiac energy metabolism through AMPKαactivation.4. Leptin maybe regulate PPARαexpression through AMPKαactivation.