Aims Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization, but the molecular events underlying the metabolic remodelling remain poorly understood. We explored metabolic remodelling and mitochondrial dysfunction in cardiac hypertrophy and investigated the cardioprotective effects of choline. Methods and results The experiments were conducted using a model of ventricular hypertrophy by partially banding the abdominal aorta of Sprague Dawley rats. Cardiomyocyte size and cardiac fibrosis were significantly increased in hypertrophic hearts. In vitro cardiomyocyte hypertrophy was induced by exposing neonatal rat cardiomyocytes to angiotensin II (Ang II) (10−6 M, 24 h). Choline attenuated the mito-nuclear protein imbalance and activated the mitochondrial-unfolded protein response (UPRmt) in the heart, thereby preserving the ultrastructure and function of mitochondria in the context of cardiac hypertrophy. Moreover, choline inhibited myocardial metabolic dysfunction by promoting the expression of proteins involved in ketone body and fatty acid metabolism in response to pressure overload, accompanied by the activation of sirtuin 3/AMP-activated protein kinase (SIRT3-AMPK) signalling. In vitro analyses demonstrated that SIRT3 siRNA diminished choline-mediated activation of ketone body metabolism and UPRmt, as well as inhibition of hypertrophic signals. Intriguingly, serum from choline-treated abdominal aorta banding models (where β-hydroxybutyrate was increased) attenuated Ang II-induced myocyte hypertrophy, which indicates that β-hydroxybutyrate is important for the cardioprotective effects of choline. Conclusion Choline attenuated cardiac dysfunction by modulating the expression of proteins involved in ketone body and fatty acid metabolism, and induction of UPRmt; this was likely mediated by activation of the SIRT3-AMPK pathway. Taken together, these results identify SIRT3-AMPK as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to cardiac stress. Choline treatment may represent a new therapeutic strategy for optimizing myocardial metabolism in the context of hypertrophy and heart failure.

Cardiac ischemia-reperfusion (I/R) injury results mainly from mitochondrial dysfunction and cardiomyocyte death. Mitophagy sustains mitochondrial function and exerts a pro-survival effect on the reperfused heart tissue. Mammalian STE20-like kinase 1 (Mst1) regulates chronic cardiac metabolic damage and autophagic activity, but its role in acute cardiac I/R injury, especially its effect on mitophagy, is unknown. The aim of this study is to explore whether Mst1 is involved in reperfusion-mediated cardiomyocyte death via modulation of FUN14 domain containing 1 (FUNDC1)-related mitophagy. Our data indicated that Mst1 was markedly increased in reperfused hearts. However, genetic ablation of Mst1 in Mst1-knockout (Mst1-KO) mice significantly reduced the expansion of the cardiac infarction area, maintained myocardial function and abolished I/R-mediated cardiomyocyte death. At the molecular level, upregulation of Mst1 promoted ROS production, reduced mitochondrial membrane potential, facilitated the leakage of mitochondrial pro-apoptotic factors into the nucleus, and activated the caspase-9-related apoptotic pathway in reperfused cardiomyocytes. Mechanistically, Mst1 activation repressed FUNDC1 expression and consequently inhibited mitophagy. However, deletion of Mst1 was able to reverse FUNDC1 expression and thus re-activate protective mitophagy, effectively sustaining mitochondrial homeostasis and blocking mitochondrial apoptosis in reperfused cardiomyocytes. Finally, we demonstrated that Mst1 regulated FUNDC1 expression via the MAPK/ERK-CREB pathway. Inhibition of the MAPK/ERK-CREB pathway prevented FUNDC1 activation caused by Mst1 deletion. Altogether, our data confirm that Mst1 deficiency sends a pro-survival signal for the reperfused heart by reversing FUNDC1-related mitophagy and thus reducing cardiomyocyte mitochondrial apoptosis, which identifies Mst1 as a novel regulator for cardiac reperfusion injury via modulation of mitochondrial homeostasis.

Voltage-gated calcium (Ca 2؉ ) channels play a key role in the control of heart contraction and are essential for normal heart development. The Ca v 1.2 L-type calcium channel is the predominant isoform in cardiomyocytes and is essential for excitation-contraction coupling. Although the inactivation of the Ca v 1.2 gene caused embryonic lethality before embryonic day E14.5, hearts were contracting before E14 depending on a dihydropyridine-sensitive calcium influx. We analyzed the consequences of the deletion of the Ca v 1.2 channel on the expression level of other voltage-gated calcium channels in the embryonic mouse heart and isolated car-

Mutations in mitochondrial DNA (mtDNA) have been associated with Leber's hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON.

Vascular dysfunction, characterized by the endothelial-to-mesenchymal transition (EndMT), contributes to the development of cardiac fibrosis induced by pressure overload. Toll-like receptor (TLR)5 is a member of the TLR family that is expressed on not only immune cells but also nonimmune cells including cardiomyocytes and vascular endothelial cells. The level of TLR5 expression on endothelial cells is low under normal circumstances but is increased in response to stimuli such as pressure overload. The aim of this study was to investigate the importance of TLR5 in cardiac endothelial dysfunction during the development of cardiac fibrosis induced by pressure overload. Global TLR5-deficient mice and wild-type littermates underwent aortic banding (AB) for 8weeks to induce cardiac fibrosis, hypertrophy and dysfunction. The deficiency of TLR5 in this model exerted no basal effects but attenuated the cardiac fibrosis, hypertrophy and dysfunction induced by pressure overload. AB-induced endothelial TLR5 activation enhanced the development of cardiac fibrosis independent of cardiomyocyte hypertrophy and triggered left ventricular dysfunction. TLR5-deficient mice also exhibited ameliorated myocardial pro-inflammatory cytokine expression and macrophage infiltration and inhibited the EndMT, all of which contribute to the development of cardiac fibrosis. These findings suggest that TLR5 triggers inflammatory responses and promotes the EndMT, which may be an important mechanism underlying the promotion of cardiac fibrosis and left ventricular dysfunction during pressure overload.