SPTLC3 Is Essential for Complex I Activity and Contributes to Ischemic Cardiomyopathy.

BACKGROUND: Dysregulated metabolism of bioactive sphingolipids, including ceramides and sphingosine-1-phosphate, has been implicated in cardiovascular disease, although the specific species, disease contexts, and cellular roles are not completely understood. Sphingolipids are produced by the serine palmitoyltransferase enzyme, canonically composed of 2 subunits, SPTLC1 (serine palmitoyltransferase long chain base subunit 1) and SPTLC2 (serine palmitoyltransferase long chain base subunit 2). Noncanonical sphingolipids are produced by a more recently described subunit, SPTLC3 (serine palmitoyltransferase long chain base subunit 3). METHODS: The noncanonical (d16) and canonical (d18) sphingolipidome profiles in cardiac tissues of patients with end-stage ischemic cardiomyopathy and in mice with ischemic cardiomyopathy were analyzed by targeted lipidomics. Regulation of SPTLC3 by HIF1α under ischemic conditions was determined with chromatin immunoprecipitation. Transcriptomics, lipidomics, metabolomics, echocardiography, mitochondrial electron transport chain, mitochondrial membrane fluidity, and mitochondrial membrane potential were assessed in the cSPTLC3KO transgenic mice we generated. Furthermore, morphological and functional studies were performed on cSPTLC3KO mice subjected to permanent nonreperfused myocardial infarction. RESULTS: Herein, we report that SPTLC3 is induced in both human and mouse models of ischemic cardiomyopathy and leads to production of atypical sphingolipids bearing 16-carbon sphingoid bases, resulting in broad changes in cell sphingolipid composition. This induction is in part attributable to transcriptional regulation by HIF1α under ischemic conditions. Furthermore, cardiomyocyte-specific depletion of SPTLC3 in mice attenuates oxidative stress, fibrosis, and hypertrophy in chronic ischemia, and mice demonstrate improved cardiac function and increased survival along with increased ketone and glucose substrate metabolism utilization. Depletion of SPTLC3 mechanistically alters the membrane environment and subunit composition of mitochondrial complex I of the electron transport chain, decreasing its activity. CONCLUSIONS: Our findings suggest a novel essential role for SPTLC3 in electron transport chain function and a contribution to ischemic injury by regulating complex I activity.

Chronic treatment with serelaxin mitigates adverse remodeling in a murine model of ischemic heart failure and modulates bioactive sphingolipid signaling.

Relaxin is a pleiotropic hormone demonstrated to confer cardioprotection in animal models of myocardial infarction and ischemic heart failure by modulating inflammation, fibrosis and arrhythmogenesis. Several of these pathways in the ischemic myocardium are intricately tied with the downstream signaling of bioactive sphingolipids, which play an active role during post-infarction remodeling. In this current study, we examined the effects of relaxin on sphingosine 1-phosphate (S1P), and the potential benefits of relaxin treatment on cardiac health in a rodent model of ischemic heart failure. Acute (30 min) and sub-acute (24 h) treatment of primary cardiomyocytes with serelaxin (recombinant human relaxin-2) increased the cardiomyocyte content of S1P. In the rodent model, treatment with relaxin for 28 days following myocardial ischemia by way of permanent left coronary artery occlusion improved survival and cardiac function, reduced fibrosis and apoptosis, and mitigated the expression of several pro-inflammatory and pro-fibrotic markers. The expression of beclin-1 (autophagy marker) was also reduced. The expression of S1P was significantly higher in cardiac tissue and plasma samples extracted from serelaxin-treated mice at day 28. In conclusion, our studies show a significant protection from relaxin in ischemic heart disease, and demonstrate the association between relaxin signaling and S1P generation.

Review of Mechanisms and Treatment of Cancer-Induced Cardiac Cachexia.

Cancer cachexia is a multifactorial, paraneoplastic syndrome that impacts roughly half of all cancer patients. It can negatively impact patient quality of life and prognosis by causing physical impairment, reducing chemotherapy tolerance, and precluding them as surgical candidates. While there is substantial research on cancer-induced skeletal muscle cachexia, there are comparatively fewer studies and therapies regarding cardiac cachexia in the setting of malignancy. A literature review was performed using the PubMed database to identify original articles pertaining to cancer-induced cardiac cachexia, including its mechanisms and potential therapeutic modalities. Seventy studies were identified by two independent reviewers based on inclusion and exclusion criteria. While there are multiple studies addressing the pathophysiology of cardiac-induced cancer cachexia, there are no studies evaluating therapeutic options in the clinical setting. Many treatment modalities including nutrition, heart failure medication, cancer drugs, exercise, and gene therapy have been explored in in vitro and mice models with varying degrees of success. While these may be beneficial in cancer patients, further prospective studies specifically focusing on the assessment and treatment of the cardiac component of cachexia are needed.

Cardiac Gene Therapy With Relaxin Receptor 1 Overexpression Protects Against Acute Myocardial Infarction.

Relaxin is a pleiotropic hormone shown to confer cardioprotection in several preclinical models of cardiac ischemia-reperfusion injury. In the present study, the effects of up-regulating relaxin family peptide receptor 1 (RXFP1) via adeno-associated virus serotype 9 (AAV9) vectors were investigated in a mouse model of myocardial infarction. AAV9-RXFP1 vectors were generated and injected in adult male CD1 mice. Up-regulation of Rxfp1 was confirmed via quantitative polymerase chain reaction, and overexpressing animals showed increased sensitivity to relaxin-induced ventricular inotropic response. Overexpressing animals also demonstrated reduced infarct size and preserved cardiac function 24 hours after ischemia-reperfusion. Up-regulation of RXFP1 via AAV9 vectors has potential therapeutic utility in preventing adverse remodeling after myocardial infarction.

B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Attenuates Myocardial Infarction-Related Adverse Cardiac Remodeling in Mice.

Background Human relaxin-2 is a peptide hormone capable of pleiotropic effects in several organ systems. Its recombinant formulation (serelaxin) has been demonstrated to reduce infarct size and prevent excessive scar formation in animal models of cardiac ischemia-reperfusion injury. B7-33, a synthetically designed peptide analogous to B-chain of relaxin-2, invokes signaling at relaxin family peptide receptor 1 (cognate receptor for relaxin-2) by preferentially phosphorylating the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2. We sought to investigate the effects of B7-33 treatment post ischemia-reperfusion injury in mice. Methods and Results Adult male CD1 mice were subjected to ischemia-reperfusion via ligation of left anterior descending artery for 30 minutes, followed by 24 hours or 7 days of reperfusion. Echocardiography was performed to assess cardiac function, and cardiac tissue was stained to determine infarct size at 24 hours. B7-33 significantly reduced infarct size (21.99% versus 45.32%; P=0.02) and preserved fractional shortening (29% versus 23%; P=0.02) compared with vehicle. The difference in fractional shortening further increased at 7 days post myocardial infarction (29% versus 20% for B7-33 and vehicle groups, respectively). In vitro, primary cardiomyocytes were isolated from adult hearts and subjected to simulated ischemia-reperfusion injury (simulated ischemia reoxygenation). B7-33 (50 and 100 nmol/L) improved cell survival and reduced the expression of GRP78 (glucose regulated protein), an endoplasmic reticulum stress marker. Subsequently, B7-33 (100 nmol/L) reduced tunicamycin (2.5 µg/mL) induced upregulation of GRP78 in an extracellular signal-regulated kinase 1/2-dependent manner. Conclusions B7-33 confers acute cardioprotection and limits myocardial infarction-related adverse remodeling in mice by attenuating cardiomyocyte death and endoplasmic reticulum stress as well as preserving cardiac function.

Functional analysis of molecular and pharmacological modulators of mitochondrial fatty acid oxidation.

Fatty acid oxidation (FAO) is a key bioenergetic pathway often dysregulated in diseases. The current knowledge on FAO regulators in mammalian cells is limited and sometimes controversial. Previous FAO analyses involve nonphysiological culture conditions or lack adequate quantification. We herein described a convenient and quantitative assay to monitor dynamic FAO activities of mammalian cells in physiologically relevant settings. The method enabled us to assess various molecular and pharmacological modulators of the FAO pathway in established cell lines, primary cells and mice. Surprisingly, many previously proposed FAO inhibitors such as ranolazine and trimetazidine lacked FAO-interfering activity. In comparison, etomoxir at low micromolar concentrations was sufficient to saturate its target proteins and to block cellular FAO function. Oxfenicine, on the other hand, acted as a partial inhibitor of FAO. As another class of FAO inhibitors that transcriptionally repress FAO genes, antagonists of peroxisome proliferator-activated receptors (PPARs), particularly that of PPARα, significantly decreased cellular FAO activity. Our assay also had sufficient sensitivity to monitor upregulation of FAO in response to environmental glucose depletion and other energy-demanding cues. Altogether this study provided a reliable FAO assay and a clear picture of biological properties of potential FAO modulators in the mammalian system.

Remote Ischemic Pre-Conditioning Attenuates Adverse Cardiac Remodeling and Mortality Following Doxorubicin Administration in Mice.

OBJECTIVES: Because of its multifaceted cardioprotective effects, remote ischemic pre-conditioning (RIPC) was examined as a strategy to attenuate doxorubicin (DOX) cardiotoxicity. BACKGROUND: The use of DOX is limited by dose-dependent cardiotoxicity and heart failure. Oxidative stress, mitochondrial dysfunction, inflammation, and autophagy modulation have been proposed as mediators of DOX cardiotoxicity. METHODS: After baseline echocardiography, adult male CD1 mice were randomized to either sham or RIPC protocol (3 cycles of 5 min femoral artery occlusion followed by 5 min reperfusion) 1 h before receiving DOX (20 mg/kg, intraperitoneal). The mice were observed primarily for survival over 85 days (86 mice). An additional cohort of 50 mice was randomized to either sham or RIPC 1 h before DOX treatment and was followed for 25 days, at which time cardiac fibrosis, apoptosis, and mitochondrial oxidative phosphorylation were assessed, as well as the expression profiles of apoptosis and autophagy markers. RESULTS: Survival was significantly improved in the RIPC cohort compared with the sham cohort (p = 0.007). DOX-induced cardiac fibrosis and apoptosis were significantly attenuated with RIPC compared with sham (p < 0.05 and p < 0.001, respectively). Although no mitochondrial dysfunction was detected at 25 days, there was a significant increase in autophagy markers with DOX that was attenuated with RIPC. Moreover, DOX caused a 49% decline in cardiac BCL2/BAX expression, which was restored with RIPC (p < 0.05 vs. DOX). DOX also resulted in a 17% reduction in left ventricular mass at 25 days, which was prevented with RIPC (p < 0.01), despite the lack of significant changes in left ventricular ejection fraction. CONCLUSIONS: Our preclinical results suggested that RIPC before DOX administration might be a promising approach for attenuating DOX cardiotoxicity.

Sacubitril/Valsartan Averts Adverse Post-Infarction Ventricular Remodeling and Preserves Systolic Function in Rabbits.

BACKGROUND: Sacubitril/valsartan (SAC/VAL) is approved by the U.S. Food and Drug Administration for heart failure with reduced ejection fraction (HFrEF). OBJECTIVES: This study investigated the effects of SAC/VAL on acute myocardial infarction (MI) and cardiac remodeling in a translational rabbit model of MI. METHODS: New Zealand White rabbits were sedated and underwent conscious MI (45-min ischemia) by balloon inflation (previously implanted surgically) followed by 72 h (acute protocol) or 10 weeks (chronic protocols) of reperfusion. "Infarct-sparing" protocol: SAC/VAL, VAL, or placebo were randomly allocated and administered at reperfusion. "HFrEF-treatment" protocol: rabbits were randomized, and treatment commenced after echocardiography-confirmed left ventricular ejection fraction (LVEF) ≤40%. "HFrEF-prevention" protocol: treatment started at reperfusion and continued daily throughout the study. RESULTS: Compared with placebo, SAC/VAL and VAL significantly reduced infarct size (TTC staining) and plasma troponin levels; however, only SAC/VAL preserved LVEF at 72 h post-MI. In the HFrEF-treatment protocol, LVEF improvement was observed with SAC/VAL compared with both placebo and VAL starting 2 weeks post-treatment, a benefit that persisted throughout study duration. In the HFrEF-prevention protocol, SAC/VAL and VAL attenuated the decline in LVEF post-MI, although SAC/VAL offered better functional protection. The functional improvement observed in both treatment protocols was paralleled by significant reduction in left ventricular (LV) scar size (Picrosirius red staining) in the SAC/VAL groups. CONCLUSIONS: Reperfusion therapy with SAC/VAL or VAL offers robust acute infarct-sparing benefits; however, SAC/VAL treatment offered superior short-term and long-term benefits in preventing MI-induced LV dysfunction compared with VAL. SAC/VAL also significantly attenuated LV scar size following MI compared with placebo, whereas VAL did not reach statistical significance in scar reduction.

Heart Disease and Relaxin: New Actions for an Old Hormone.

The hormone relaxin has long been recognized for its involvement in maternal adaptation during pregnancy. However, discoveries during the past two decades on the mechanism of action of relaxin, its family of receptors, and newly described roles in attenuating ischemia/reperfusion (I/R) injury, inflammation, and arrhythmias have prompted vast interest in exploring its therapeutic potential in cardiovascular disease. These observations inspired recently concluded clinical trials in patients with acute heart failure. This review discusses our current understanding of the protective signaling pathways elicited by relaxin in the heart, and highlights important new breakthroughs about relaxin signaling that may pave the way to more carefully designed future trials.

Reperfusion therapy with recombinant human relaxin-2 (Serelaxin) attenuates myocardial infarct size and NLRP3 inflammasome following ischemia/reperfusion injury via eNOS-dependent mechanism.

AIMS: The preconditioning-like infarct-sparing and anti-inflammatory effects of the peptide hormone relaxin following ischemic injury have been studied in the heart. Whether reperfusion therapy with recombinant human relaxin-2, serelaxin, reduces myocardial infarct size and attenuates the subsequent NLRP3 inflammasome activation leading to further loss of functional myocardium following ischemia/reperfusion (I/R) injury is unknown. METHODS AND RESULTS: After baseline echocardiography, adult male wild-type C57BL or eNOS knockout mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min followed by 24 h reperfusion. Mice were treated with either serelaxin (10 µg/kg; sc) or saline 1 h prior to ischemia or 5 min before reperfusion. In both pre-treatment and reperfusion therapy arms, serelaxin improved survival at 24 h post MI in wild-type mice (79% and 82%) as compared with controls (46% and 50%, P = 0.01), whereas there was no difference in survival between serelaxin- and saline-treated eNOS knockout mice. Moreover, serelaxin significantly reduced infarct size (64% and 67% reduction, P < 0.05), measured with TTC staining, and preserved LV fractional shortening (FS) and end-systolic diameter (LVESD) in wild-type mice as compared with controls (P < 0.05). Interestingly, caspase-1 activity in the heart tissue, a measure of inflammasome formation, was markedly reduced in serelaxin-treated wild-type mice compared with controls at 24 h post-MI in both treatment modalities (P < 0.05). Genetic deletion of eNOS abolished the infarct-sparing and anti-inflammatory effects of serelaxin as well as functional preservation. Serelaxin plasma levels assessed at 5 min and 1 h after treatment, using ELISA, approximated physiologic relaxin levels during pregnancy in mice and parallels that in humans. CONCLUSION: Serelaxin attenuates myocardial I/R injury and the subsequent caspase-1 activation via eNOS-dependent mechanism.