Role of Perilipins in Oxidative Stress-Implications for Cardiovascular Disease.

Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and antioxidants in a cell. In the heart, oxidative stress may deteriorate calcium handling, cause arrhythmia, and enhance maladaptive cardiac remodeling by the induction of hypertrophic and apoptotic signaling pathways. Consequently, dysregulated ROS production and oxidative stress have been implicated in numerous cardiac diseases, including heart failure, cardiac ischemia-reperfusion injury, cardiac hypertrophy, and diabetic cardiomyopathy. Lipid droplets (LDs) are conserved intracellular organelles that enable the safe and stable storage of neutral lipids within the cytosol. LDs are coated with proteins, perilipins (Plins) being one of the most abundant. In this review, we will discuss the interplay between oxidative stress and Plins. Indeed, LDs and Plins are increasingly being recognized for playing a critical role beyond energy metabolism and lipid handling. Numerous reports suggest that an essential purpose of LD biogenesis is to alleviate cellular stress, such as oxidative stress. Given the yet unmet suitability of ROS as targets for the intervention of cardiovascular disease, the endogenous antioxidant capacity of Plins may be beneficial.

Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

AIMS: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. CONCLUSION: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Cardiac Plin5 interacts with SERCA2 and promotes calcium handling and cardiomyocyte contractility.

The adult heart develops hypertrophy to reduce ventricular wall stress and maintain cardiac function in response to an increased workload. Although pathological hypertrophy generally progresses to heart failure, physiological hypertrophy may be cardioprotective. Cardiac-specific overexpression of the lipid-droplet protein perilipin 5 (Plin5) promotes cardiac hypertrophy, but it is unclear whether this response is beneficial. We analyzed RNA-sequencing data from human left ventricle and showed that cardiac PLIN5 expression correlates with up-regulation of cardiac contraction-related processes. To investigate how elevated cardiac Plin5 levels affect cardiac contractility, we generated mice with cardiac-specific overexpression of Plin5 (MHC-Plin5 mice). These mice displayed increased left ventricular mass and cardiomyocyte size but preserved heart function. Quantitative proteomics identified sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) as a Plin5-interacting protein. In situ proximity ligation assay further confirmed the Plin5/SERCA2 interaction. Live imaging showed increases in intracellular Ca2+ release during contraction, Ca2+ removal during relaxation, and SERCA2 function in MHC-Plin5 versus WT cardiomyocytes. These results identify a role of Plin5 in improving cardiac contractility through enhanced Ca2+ signaling.

Cardiac Cell Exposure to Electromagnetic Fields: Focus on Oxdative Stress and Apoptosis.

Exposure to electromagnetic fields (EMFs) is a sensitive research topic. Despite extensive research, to date there is no evidence to conclude that exposure to EMFs influences the cardiovascular system. In the present study, we examined whether 915 MHz EMF exposure affects myocardial antioxidative and apoptotic status in vitro and in vivo. No statistically significant difference in the apoptotic cell profile and antioxidant capacity was observed between controls and short-term EMF-exposed mouse cardiomyocytes and H9C2 cardiomyoblasts. Compared with sham-exposed controls, mice subjected to a 915 MHz EMF for 48 h and 72 h had no significant effect on structural tissue integrity and myocardial expression of apoptosis and antioxidant genes. Therefore, these results indicate that short-term exposure to EMF in cardiac cells and tissues did not translate into a significant effect on the myocardial antioxidant defense system and apoptotic cell death.

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.

Glucosylceramide synthase deficiency in the heart compromises ß1-adrenergic receptor trafficking.

AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to ß-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of ß1-adrenergic receptors. CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain ß-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

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.

In vitro and in vivo cardioprotective and metabolic efficacy of vitamin E TPGS/Apelin.

AIMS: Apelin and vitamin E have been proposed as signaling molecules, but their synergistic role is unknown. The aim of this work was to develop vitamin E TPGS/Apelin system to test their cardioprotective and metabolic efficacy in vitro and in vivo. METHODS: FDA-approved surfactant D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS-1000) and Apelin complex were characterized by physico-chemical methods (CMC determination, dynamic light scattering and circular dichroism). In vitro studies were carried out on H9C2 cardiomyoblasts and isolated murine cardiomyocytes. In vivo studies were performed in isoproterenol- and high-fat diet-induced cardiac remodeling models in mice. RESULTS: We found that vitamin E TPGS/Apelin provide cardioprotective and metabolic efficacy in vitro and in vivo. In vitro studies revealed that vitamin E TPGS/Apelin reduces hypoxia-induced mitochondrial ROS production in cultured cardiomyocytes and H9C2 cardiomyoblasts. In addition, vitamin E TPGS/Apelin confers apoptotic response to hypoxic stress in cells. In a mouse model of isoproterenol-induced cardiac injury, TPGS is not able to affect cardiac remodeling, however combination of vitamin E TPGS and Apelin counteracts myocardial apoptosis, oxidative stress, hypertrophy and fibrosis. Furthermore, combination treatment attenuated obesity-induced cardiometabolic and fibrotic remodeling in mice. CONCLUSION: Together, our data demonstrated the therapeutic benefits of vitamin E TPGS/Apelin complex to combat cardiovascular and metabolic disorders.

Metformin Protects the Heart Against Hypertrophic and Apoptotic Remodeling After Myocardial Infarction.

Cardiovascular complications are the most prevalent cause of morbidity and mortality in diabetic patients. Metformin is currently the first-line blood glucose-lowering agent with potential relevance to cardiovascular diseases. However, the underpinning mechanisms of action remain elusive. Here, we report that metformin represses cardiac apoptosis at least in part through inhibition of Forkhead box O1 (FoxO1) pathway. In a mouse model of ischemia-reperfusion (I/R), treatment with metformin attenuated cardiac and hypertrophic remodeling after 14 days of post-reperfusion. Additionally, cardiac expression of brain-like natriuretic peptide (BNP) was significantly reduced in metformin-treated mice after 14 days of cardiac I/R. In cultured H9C2 cells, metformin counteracted hypertrophic and apoptotic responses to metabolic or hypoxic stress. FoxO1 silencing by siRNA abolished anti-apoptotic effect of metformin under hypoxic stress in H9C2 cells. Taken together, these results suggest that metformin protects the heart against hypertrophic and apoptotic remodeling after myocardial infarction.

Periprostatic Adipose Tissue Favors Prostate Cancer Cell Invasion in an Obesity-Dependent Manner: Role of Oxidative Stress.

Prostate gland is surrounded by periprostatic adipose tissue (PPAT), which is increasingly believed to play a paracrine role in prostate cancer progression. Our previous work demonstrates that adipocytes promote homing of prostate cancer cells to PPAT and that this effect is upregulated by obesity. Here, we show that once tumor cells have invaded PPAT (mimicked by an in vitro model of coculture), they establish a bidirectional crosstalk with adipocytes, which promotes tumor cell invasion. Indeed, tumor cells induce adipocyte lipolysis and the free fatty acids (FFA) released are taken up and stored by tumor cells. Incubation with exogenous lipids also stimulates tumor cell invasion, underlining the importance of lipid transfer in prostate cancer aggressiveness. Transferred FFAs (after coculture or exogenous lipid treatment) stimulate the expression of one isoform of the pro-oxidant enzyme NADPH oxidase, NOX5. NOX5 increases intracellular reactive oxygen species (ROS) that, in turn, activate a HIF1/MMP14 pathway, which is responsible for the increased tumor cell invasion. In obesity, tumor-surrounding adipocytes are more prone to activate the depicted signaling pathway and to induce tumor invasion. Finally, the expression of NOX5 and MMP14 is upregulated at the invasive front of human tumors where cancer cells are in close proximity to adipocytes and this process is amplified in obese patients, underlining the clinical relevance of our results. IMPLICATIONS: Our work emphasizes the key role of adjacent PPAT in prostate cancer dissemination and proposes new molecular targets for the treatment of obese patients exhibiting aggressive diseases.

IL-26 in the induced sputum is associated with the level of systemic inflammation, lung functions and body weight in COPD patients.

Background: Chronic inflammatory process is the main link in COPD pathogenesis, which causes structural changes in the respiratory tract and lungs. In overweight patients, an adipose tissue could contribute to activation of the inflammatory process. Therefore, it is highly important to identify potential biomarkers of inflammation for patients with COPD and obesity. The aim of this study was to investigate the role of interleukin-26 (IL-26) and evaluate the relationship between the level of systemic inflammation, lung function, and body mass index (BMI) in patients with COPD. Patients and methods: Eighty-three patients with COPD in the stable condition (stage 2 according to the 2016 Global Initiative for Chronic Obstructive Lung Disease recommendations), aged 40-70 years, were included in the study. All patients were divided into 2 groups: obese (n=53) (BMI - 30.0-39.9 kg/m2) and non-obese (n=30) (BMI - 18.5-24.9 kg/m2). We conducted patients' examination, spirometry, induced the sputum, determined the level of C-reactive protein (CRP), leptin in serum and IL-26 in sputum. Results: Obese and non-obese COPD patients had a significant increase in IL-26 compared with healthy subjects by 2.3 and 2.6 (P=0.0003). We also observed a higher level of CRP by 1.38 times (P=0.0008), compared with the rate in non-obese COPD patients, and by 1.8 times (P=0.015) higher concentration level of leptin compared with healthy subjects. The sputum IL-26 level had positive correlation with BMI, CRP, and leptin, and a negative - with forced expiratory volume in 1 second (FEV1) and FEV1/forced vital capacity. Leptin level had positive correlation with BMI and CRP, and negative with FEV1, FEV1/forced vital capacity. Conclusion: Obese COPD patients had a higher level of persistent systemic inflammation than non-obese ones, which is confirmed by a significant increase of CRP and leptin in serum. The data confirm that IL-26 can be considered as a perspective marker to detect the inflammation level in lung tissue of COPD patients.

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.