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Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro.

ABSTRACT: To examine the inhibitory effects of adenosine monophosphate-activated protein kinase (AMPK) activation on cardiac hypertrophy in vitro and to investigate the underlying molecular mechanisms.Cultured neonatal rat cardiomyocytes were treated with the specific AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and the specific AMPK antagonist Compound C, and then stimulated with phenylephrine (PE). The Muscle RING finger 1 (MuRF1)-small interfering RNA (siRNA) was transfected into cardiomyocytes using Lipofectamine 2000. The surface area of cultured cardiomyocytes was measured using planimetry. The protein degradation was determined using high performance liquid chromatography (HPLC). The expression of beta-myosin heavy chain (beta-MHC) and MuRF1, as well as the phosphorylation levels of AMPK and Forkhead box O 1 (FOXO1), were separately measured using Western blot or real-time polymerase chain reaction.Activation of AMPK by AICAR 0.5 mmol/L inhibited PE-induced increase in cardiomyocyte area and beta-MHC protein expression and PE-induced decrease in protein degradation. Furthermore, AMPK activation increased the activity of transcription factor FOXO1 and up-regulated downstream atrogene MuRF1 mRNA and protein expression. Treatment of hypertrophied cardiomyocytes with Compound C 1 micromol/L blunted the effects of AMPK on cardiomyocyte hypertrophy and changes to the FOXO1/MuRF1 pathway. The effects of AICAR on cardiomyocyte hypertrophy were also blocked after MuRF1 was silenced by transfection of cardiomyocytes with MuRF1-siRNA.The present study demonstrates that AMPK activation attenuates cardiomyocyte hypertrophy by modulating the atrophy-related FOXO1/MuRF1 signaling pathway in vitro.

SUBMITTER: Chen BL

PROVIDER: S-EPMC4007721 | biostudies-other | 2010 Jul

REPOSITORIES: biostudies-other

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Project description:Pathological cardiac hypertrophy is an independent risk factor for complications such as arrhythmia, myocardial infarction, sudden mortality and heart failure. Succinate, an intermediate product of the Krebs cycle, is released into the bloodstream by cells; its levels increase with exacerbations of hypertension, myocardial and other tissue damage and metabolic disease. Succinate may also be involved in several metabolic pathways and mediates numerous pathological effects through its receptor, succinate receptor 1 (SUCNR1; previously known as GPR91). Succinate-induced activation of SUCNR1 has been reported to be related to cardiac hypertrophy, making SUCNR1 a potential target for treating cardiac hypertrophy. Traditional Chinese medicine (TCM) and its active ingredients have served important roles in improving cardiac functions and treating heart failure. The present study investigated whether 4'-O-methylbavachadone (MeBavaC), an active ingredient of the herbal remedy Fructus Psoraleae, which is often used in TCM and has protective effect on myocardial injury and hypertrophy induced by adriamycin, ischemia-reperfusion and sepsis, could ameliorate succinate-induced cardiomyocyte hypertrophy by inhibiting the NFATc4 pathway. Using immunofluorescence staining, reverse transcription-quantitative PCR, western blotting and molecular docking analysis, it was determined that succinate activated the calcineurin/NFATc4 and ERK1/2 pathways to promote cardiomyocyte hypertrophy. MeBavaC inhibited cardiomyocyte hypertrophy, nuclear translocation of NFATc4 and ERK1/2 signaling activation in succinate-induced cardiomyocytes. Molecular docking analysis revealed that MeBavaC interacts with SUCNR1 to form a relatively stable binding and inhibits the succinate-SUCNR1 interaction. The results demonstrated that MeBavaC suppressed cardiomyocyte hypertrophy by blocking SUCNR1 receptor activity and inhibiting NFATc4 and ERK1/2 signaling, which will contribute to the preclinical development of this compound.

Project description:BackgroundAMP-activated Protein Kinase (AMPK) is a stress-activated kinase that protects against cardiomyocyte injury during ischemia and reperfusion. c-Jun N-terminal kinase (JNK), a mitogen activated protein kinase, is activated by ischemia and reperfusion. NF-κB is an important transcription factor involved in ischemia and reperfusion injury.Methods and resultsThe intrinsic activation of AMPK attenuates the inflammation which occurred during ischemia/reperfusion through the modulation of the JNK mediated NF-κB signaling pathway. Rat cardiac myoblast H9c2 cells were subjected to hypoxia and/or reoxygenation to investigate the signal transduction that occurred during myocardial ischemia/reperfusion. Mitochondrial function was measured by the Seahorse XF24 V7 PS system. Hypoxia treatment triggered AMPK activation in H9c2 cells in a time dependent manner. The inhibition of hypoxic AMPK activation through a pharmacological approach (Compound C) or siRNA knockdown of AMPK α catalytic subunits caused dramatic augmentation in JNK activation, inflammatory NF-κB phosphorylation, and apoptosis during hypoxia and reoxygenation. Inhibition of AMPK activation significantly impaired mitochondrial function and increased the generation of reactive oxygen species (ROS) during hypoxia and reoxygenation. In contrast, pharmacological activation of AMPK by metformin significantly inhibited mitochondrial permeability transition pore (mPTP) opening and ROS generation. Moreover, AMPK activation significantly attenuated the JNK-NF-κB signaling cascade and inhibited mRNA and protein levels of pro-inflammatory cytokines, such as TNF-α and IL-6, during hyopoxia/reoxygenation in H9c2 cells. Intriguingly, both pharmacologic inhibition of JNK by JNK-IN-8 and siRNA knockdown of JNK signaling pathway attenuated NF-κB phosphorylation and apoptosis but did not affect AMPK activation in response to hypoxia and reoxygenation.ConclusionsAMPK activation modulates JNK-NF-κB signaling cascade during hypoxia and reoxygenation stress conditions. Cardiac AMPK activation plays a critical role in maintaining mitochondrial function and inhibiting the inflammatory response caused by ischemic insults.

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Activation of AMPK inhibits cardiomyocyte hypertrophy by modulating of the FOXO1/MuRF1 signaling pathway in vitro.

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