Galanin Regulates Myocardial Mitochondrial ROS Homeostasis and Hypertrophic Remodeling Through GalR2.

The regulatory peptide galanin is broadly distributed in the central nervous systems and peripheral tissues where it modulates numerous physiological and pathological processes through binding to its three G-protein-coupled receptors, GalR1-3. However, the function and identity of the galaninergic system in the heart remain unclear. Therefore, we investigated the expression of the galanin receptors in cardiac cells and tissues and found that GalR2 is the dominant receptor subtype in adult mouse hearts, cardiomyocytes and H9C2 cardiomyoblasts. In vivo, genetic suppression of GalR2 promotes cardiac hypertrophy, fibrosis and mitochondrial oxidative stress in the heart. In vitro, GalR2 silencing by siRNA abolished the beneficial effects of galanin on cell hypertrophy and mitochondrial reactive oxygen species (ROS) production. These findings unravel new insights into the role of galaninergic system in the heart and suggest novel therapeutic strategies in heart disease.

Apilimod alters TGFß signaling pathway and prevents cardiac fibrotic remodeling.

Rationale: TGFß signaling pathway controls tissue fibrotic remodeling, a hallmark in many diseases leading to organ injury and failure. In this study, we address the role of Apilimod, a pharmacological inhibitor of the lipid kinase PIKfyve, in the regulation of cardiac pathological fibrotic remodeling and TGFß signaling pathway. Methods: The effects of Apilimod treatment on myocardial fibrosis, hypertrophy and cardiac function were assessed in vivo in a mouse model of pressure overload-induced heart failure. Primary cardiac fibroblasts and HeLa cells treated with Apilimod as well as genetic mutation of PIKfyve in mouse embryonic fibroblasts were used as cell models. Results: When administered in vivo, Apilimod reduced myocardial interstitial fibrosis development and prevented left ventricular dysfunction. In vitro, Apilimod controlled TGFß-dependent activation of primary murine cardiac fibroblasts. Mechanistically, both Apilimod and genetic mutation of PIKfyve induced TGFß receptor blockade in intracellular vesicles, negatively modulating its downstream signaling pathway and ultimately dampening TGFß response. Conclusions: Altogether, our findings propose a novel function for PIKfyve in the control of myocardial fibrotic remodeling and the TGFß signaling pathway, therefore opening the way to new therapeutic perspectives to prevent adverse fibrotic remodeling using Apilimod treatment.

Galanin promotes autophagy and alleviates apoptosis in the hypertrophied heart through FoxO1 pathway.

Autophagy and apoptosis are powerful regulators of multiple facets of cellular metabolism and homeostasis. Here, we uncover that galanin, a pleiotropic peptide, regulates cardiac autophagy and deactivates apoptotic cell death through the Forkhead box protein O1 (FoxO1) pathway. In hypertrophied heart, galanin promotes autophagy and metabolic shift from fatty acid (FA) to glucose oxidation and preserves mitochondrial integrity. In cardiomyoblasts, galanin triggers autophagosome formation and alleviates hypertrophy, apoptotic cell death, and mitochondrial stress. Mechanistically, galanin dictates cell autophagic and anti-apoptotic phenotypes through FoxO1 pathway. Together, these findings uncover a previously unknown role for galanin in the regulation of cardiac autophagy and provide new insights into the molecular mechanisms supporting cell survival in the hypertrophic reprogramming of the heart.

Differential protein profiling as a potential multi-marker approach for obese patients with heart failure: A retrospective study.

Identification of novel circulating biomarkers predicting death and major cardio-metabolic events in obese patients with heart failure (HF) remains a research priority. In this study, we compared multi-marker profile of non-obese (NOB) and obese (OB) HF patients in relation to mortality outcome. The new multiplex proximity extension assay technology was used to analyze the levels of 92 proteins in plasma samples from HF patients according to body mass index (BMI) categories. At 2-year follow-up, all-cause mortality rates were significantly greater in NOB patients (BMI < 30 kg/m2) compared to the OB patients (BMI > 30 kg/m2) with HF (odds ratio 26; 95% CI: 1.14-624, p < 0,04). Quantitative proteomic analysis revealed thirteen distinct proteins expression profiles of OB and NOB HF patients. Among these proteins, RAGE, CXCL6, CXCL1, CD40, NEMO, VEGF-A, KLK6, PECAM1, PAR1, MMP1, BNP and NTproBNP were down-regulated, whereas leptin was up-regulated in OB HF patients. In addition, an inverse correlation between plasma BNP levels and leptin in OB HF patients was observed (r = -0.58 p = 0.02). This study identifies specific plasma protein signature in OB and NOB patients with HF in relation to mortality outcome.

Cardioprotective properties of N-terminal galanin fragment (2-15) in experimental ischemia/reperfusion injury.

BACKGROUND AND PURPOSE: Galanin is an endogenous peptide involved in diverse physiological functions in the central nervous system including central cardiovascular regulation. The present study was designed to evaluate the potential effects of the short N-terminal galanin fragment 2-15 (G) on cardiac ischemia/reperfusion (I/R) injury. EXPERIMENTAL APPROACH: Peptide G was synthesized by the automatic solid phase method and identified by 1H-NMR spectroscopy and mass spectrometry. Experiments were performed on cultured rat cardiomyoblast (H9C2) cells, isolated perfused working rat hearts and anaesthetized open-chest rats. KEY RESULTS: Cell viability increased significantly after treatment with 10 and 50 nM of G peptide. In hypoxia and reoxygenation conditions, exposure of H9C2 cells to G peptide decreased cell apoptosis and mitochondrial reactive oxygen species (ROS) production. Postischemic infusion of G peptide reduced cell membrane damage and improved functional recovery in isolated hearts during reperfusion. These effects were accompanied by enhanced restoration of myocardial metabolic state. Treatment with G peptide at the onset of reperfusion induced minor changes in hemodynamic variables but significantly reduced infarct size and plasma levels of necrosis markers. CONCLUSION AND IMPLICATIONS: These findings suggest that G peptide is effective in mitigating cardiac I/R injury, thereby providing a rationale for promising tool for the treatment of cardiovascular diseases.

Inhibition of PIKfyve prevents myocardial apoptosis and hypertrophy through activation of SIRT3 in obese mice.

PIKfyve is an evolutionarily conserved lipid kinase that regulates pleiotropic cellular functions. Here, we identify PIKfyve as a key regulator of cardiometabolic status and mitochondrial integrity in chronic diet-induced obesity. In vitro, we show that PIKfyve is critical for the control of mitochondrial fragmentation and hypertrophic and apoptotic responses to stress. We also provide evidence that inactivation of PIKfyve by the selective inhibitor STA suppresses excessive mitochondrial ROS production and apoptosis through a SIRT3-dependent pathway in cardiomyoblasts. In addition, we report that chronic STA treatment improves cardiometabolic profile in a mouse model of cardiomyopathy linked to obesity. We provide evidence that PIKfyve inhibition reverses obesity-induced cardiac mitochondrial damage and apoptosis by activating SIRT3. Furthermore, treatment of obese mice with STA improves left ventricular function and attenuates cardiac hypertrophy. In contrast, STA is not able to reduce isoproterenol-induced cardiac hypertrophy in SIRT3.KO mice. Altogether, these results unravel a novel role for PIKfyve in obesity-associated cardiomyopathy and provide a promising therapeutic strategy to combat cardiometabolic complications in obesity.

Myocardial protection from ischemia/reperfusion injury by exogenous galanin fragment.

BACKGROUND AND PURPOSE: Galanin is a multifunctional neuropeptide with pleiotropic roles. The present study was designed to evaluate the potential effects of galanin (2-11) (G1) on functional and metabolic abnormalities in response to myocardial ischemia-reperfusion (I/R) injury. EXPERIMENTAL APPROACH: Peptide G1 was synthesized by the 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase method. The chemical structure was identified by 1H-NMR spectroscopy and mass spectrometry. Experiments were conducted using a rat model of I/R injury in vivo, isolated perfused rat hearts ex vivo and cultured rat cardiomyoblast H9C2 cells in vitro. Cardiac function, infarct size, myocardial energy metabolism, hemodynamic parameters, plasma levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) were measured in order to evaluate the effects of G1 on myocardial I/R injury. KEY RESULTS: Treatment with G1 increased cell viability in a dose-dependent manner, inhibited cell apoptosis and excessive mitochondrial reactive oxygen species (ROS) production in response to oxidative stress in H9C2 cells. Pre- or postischemic infusion of G1 enhanced functional and metabolic recovery during reperfusion of the ischemic isolated rat heart. Administration of G1 at the onset of reperfusion significantly reduced infarct size and plasma levels of CK-MB and LDH in rats subjected to myocardial I/R injury. CONCLUSIONS AND IMPLICATIONS: These data provide the first evidence for cardioprotective activity of galanin G1 against myocardial I/R injury. Therefore, peptide G1 may represent a promising treatment strategy for ischemic heart disease.

Apelin-13 administration protects against ischaemia/reperfusion-mediated apoptosis through the FoxO1 pathway in high-fat diet-induced obesity.

BACKGROUND AND PURPOSE: Apelin-13, an endogenous ligand for the apelin (APJ) receptor, behaves as a potent modulator of metabolic and cardiovascular disorders. Here, we examined the effects of apelin-13 on myocardial injury in a mouse model combining ischaemia/reperfusion (I/R) and obesity and explored their underlying mechanisms. EXPERIMENTAL APPROACH: Adult male C57BL/6J mice were fed a normal diet (ND) or high-fat diet (HFD) for 6 months and then subjected to cardiac I/R. The effects of apelin-13 post-treatment on myocardial injury were evaluated in HFD-fed mice after 24 h I/R. Changes in protein abundance, phosphorylation, subcellular localization and mRNA expression were determined in cardiomyoblast cell line H9C2, primary cardiomyocytes and cardiac tissue from ND- and HFD-fed mice. Apoptosis was evaluated by TUNEL staining and caspase-3 activity. Mitochondrial ultrastructure was analysed by electron microscopy. KEY RESULTS: In HFD-fed mice subjected to cardiac I/R, i.v. administration of apelin-13 significantly reduced infarct size, myocardial apoptosis and mitochondrial damage compared with vehicle-treated animals. In H9C2 cells and primary cardiomyocytes, apelin-13 induced FoxO1 phosphorylation and nuclear exclusion. FoxO1 silencing by siRNA abolished the protective effects of apelin-13 against hypoxia-induced apoptosis and mitochondrial ROS generation. Finally, apelin deficiency in mice fed a HFD resulted in reduced myocardial FoxO1 expression and impaired FoxO1 distribution. CONCLUSIONS AND IMPLICATIONS: These data reveal apelin as a novel regulator of FoxO1 in cardiac cells and provide evidence for the potential of apelin-13 in prevention of apoptosis and mitochondrial damage in conditions combining I/R injury and obesity.

Apelin regulates FoxO3 translocation to mediate cardioprotective responses to myocardial injury and obesity.

The increasing incidence of obesity accentuates the importance of identifying mechanisms and optimal therapeutic strategies for patients with heart failure (HF) in relation to obesity status. Here, we investigated the association between plasma level of apelin, an adipocyte-derived factor, and clinicopathological features of obese and non-obese patients with HF. We further explored potential regulatory mechanisms of cardiac cell fate responses in conditions combining myocardial injury and obesity. In a prospective, cross-sectional study involving patients with HF we show that obese patients (BMI ≥ 30 kg/m(2)) have higher left ventricular ejection fraction (LVEF) and greater levels of plasma apelin (p < 0.005) than non-obese patients (< 30 kg/m(2)), independently of ischemic etiology. In a mouse model combining ischemia-reperfusion (I/R) injury and high-fat diet (HFD)-induced obesity, we identify apelin as a novel regulator of FoxO3 trafficking in cardiomyocytes. Confocal microscopy analysis of cardiac cells revealed that apelin prevents nuclear translocation of FoxO3 in response to oxygen deprivation through a PI3K pathway. These findings uncover apelin as a novel regulator of FoxO3 nucleocytoplasmic trafficking in cardiac cells in response to stress and provide insight into its potential clinical relevance in obese patients with HF.