P1616L-2-hydroxyglutarate dehydrogenase (L2HGDH) is a novel target for heart failure due to pressure overload

Abstract

Abstract Background We have found L-2-hydroxyglutarate dehydrogenase (L2HGDH) to be downregulated in the myocardium of mice subjected to transverse aortic constriction (TAC). L2HGDH is an important regulator of mitochondrial bioenergetics by catalyzing the conversion of L-2-hydroxyglutarate (L2-HG) to α-ketoglutarate. However, the connection between L2-HG accumulation and heart failure is not yet understood. Purpose Purpose of our study was to investigate the role of increased L2-HG levels in heart failure and the potential role of L2HGDH overexpression as therapeutic strategy. Methods For in vitro studies, primary rat neonatal cardiomyocytes (NRVCMs) were incubated with L2-HG. L2HGDH was overexpressed using adeno-associated virus (AAV) 6 vectors. Mitochondrial membrane potential was measured using TMRE (tetramethylrhodamine ethyl ester) dye. Mitochondrial reactive oxygen species production was monitored using MitoSOX. We further determined activation of fetal gene program by real time qPCR and macrophage migration using RAW 264.7 cells and transwell inserts. mTOR activation was analyzed by Western blot with antibodies against phosphorylated mTOR and ribosomal protein S6. AAV9 expressing L2HGDH or luciferase was injected in C57BL/6N mice two weeks prior to TAC and heart function was monitored by echocardiography for 6 weeks. Results L2-HG acts as a pro-hypertrophic stimulus in NRVCMs as shown by upregulation of a fetal gene expression pattern and an increase in cardiomyocyte cross-sectional area upon L2-HG treatment. Furthermore, mRNA levels of macrophage chemoattractant protein 1 were increased in L2-HG treated cells, which correlated with enhanced macrophage migration towards supernatant of L2-HG treated NRVCMs. Furthermore, we could confirm that L2-HG augmented mTOR signaling by affecting the phosphorylation status of ribosomal protein S6. AAV-mediated L2HGDH overexpression in NRVCMs led to a significant 2.1-fold decrease in the accumulation of ROS production. Moreover, we found an inhibition of endothelin-1 induced mitochondrial membrane depolarization in AAV6-L2HGDH transduced cells. Pretreatment of mice with AAV9-L2HGDH prior to TAC resulted in significantly reduced heart weight to tibia length ratios (HW/TL) and cardiomyocyte area. Importantly, heart function was notably improved in mice receiving gene therapy (ejection fraction, EF: 36.18±6.63%, fractional shortening, FS: 16.72±4.01%) whereas control animals showed marked decline in myocardial contractility (EF: 20.14±8.24%, FS: 12.66±6.66%). Conclusion L2-HG causes cardiomyocyte dysfunction by activating mTOR signaling pathway, a well-characterized critical inducer of myocyte hypertrophy, and enhancing macrophage migration, leading to establishment of a pro-inflammatory environment in the myocardium. Moreover, our results point out towards a novel preventive approach for cardiac hypertrophy and heart failure by cardiomyocyte-specific L2HGDH overexpression. Acknowledgement/Funding DZHK (Deutsches Zentrum für Herz-Kreislaufforschung)