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ObjectivesDetermine the role of apolipoprotein M (ApoM) in anthracycline (Dox) cardiotoxicity.BackgroundApoM binds the cardioprotective sphingolipid sphingosine-1-phosphate (S1P). Circulating ApoM is inversely associated with mortality in human heart failure (HF).MethodsIn the Penn HF Study (PHFS), we tested the relationship between ApoM and mortality in a subset with anthracycline-induced cardiomyopathy. We measured ApoM in humans and mice treated with Dox and utilized hepatic ApoM transgenic (ApomTG), ApoM knockout (ApomKO), ApoM knock-in mice with impaired S1P binding, and S1P receptor 3 (S1PR3) knockout mice in Dox cardiotoxicity. We assayed autophagy in left ventricular tissue from anthracycline-induced HF patients versus donor controls.ResultsApoM was inversely associated with mortality in PHFS, and Dox reduced circulating ApoM in mice and breast cancer patients.ApomTGmice were protected from Dox-induced cardiac dysfunction and loss of left ventricular mass.ApomTGattenuated Dox-induced impairment in autophagic flux in vivo and accumulation of insoluble p62, which was also observed in the myocardium of patients with anthracycline-induced HF. In vehicle-treated mice, ApoM negatively regulated transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. The effect of ApoM on TFEB required both S1P binding and S1PR3. In the presence of Dox, ApoM preserved TFEB and cardiomyocyte lysosomal abundance assessed as lysosomal associated membrane protein 1 positive structures in vivo, while S1P mimetic pretreatment of cardiomyocytes prevented Dox-induced changes in lysosomal pH.ConclusionsApoM attenuates Dox cardiotoxicity via the autophagy-lysosome pathway. The association between ApoM and reduced mortality may be explained by its role in sustaining autophagy.HighlightsCirculating ApoM is inversely associated with survival in human anthracycline-induced cardiomyopathyAnthracycline treatment reduces circulating ApoM in humans and miceIncreasing ApoM attenuates doxorubicin cardiotoxicity, lysosomal injury and preserves myocardial autophagic flux, but does not impact doxorubicin anti-neoplastic efficacyAutophagic impairment is characteristic of human anthracycline cardiomyopathy

Background: Sodium-glucose cotransporter-2 inhibitors are foundational therapy in patients with heart failure with reduced ejection fraction (HFrEF), but underlying mechanisms of benefit are not well defined. We sought to investigate the relationships between sodium-glucose cotransporter-2 inhibitor treatment, changes in metabolic pathways, and outcomes using targeted metabolomics. Methods: DEFINE-HF (Dapagliflozin Effects on Biomarkers, Symptoms and Functional Status in Patients With HF With Reduced Ejection Fraction) was a placebo-controlled trial of dapagliflozin in HFrEF. We performed targeted mass spectrometry profiling of 63 metabolites (45 acylcarnitines [markers of fatty acid oxidation], 15 amino acids, and 3 conventional metabolites) in plasma samples at randomization and 12 weeks. Using mixed models, we identified principal components analysis–defined metabolite clusters that changed differentially with treatment and examined the relationship between change in metabolite clusters and change in Kansas City Cardiomyopathy Questionnaire scores and NT-proBNP (N-terminal probrain natriuretic peptide). Models were adjusted for relevant clinical covariates and nominal P P Results: Among the 234 DEFINE-HF participants with targeted metabolomic data, the mean age was 62.0±11.1 years, 25% were women, 38% were Black, and mean ejection fraction was 27±8%. Dapagliflozin increased ketone-related and short-chain acylcarnitine as well as medium-chain acylcarnitine principal components analysis–defined metabolite clusters compared with placebo (nominal P =0.01, false discovery rate–adjusted P =0.08 for both clusters). However, ketosis (β-hydroxybutyrate levels >500 μmol/L) was achieved infrequently (3 [2.5%] in dapagliflozin arm versus 1 [0.9%] in placebo arm) and supraphysiologic levels were not observed. Increases in long-chain acylcarnitine, long-chain dicarboxylacylcarnitine, and aromatic amino acid metabolite clusters were associated with decreases in Kansas City Cardiomyopathy Questionnaire scores (ie, worse quality of life) and increases in NT-proBNP levels, without interaction by treatment group. Conclusions: In this study of targeted metabolomics in a placebo-controlled trial of sodium-glucose cotransporter-2 inhibitors in HFrEF, we observed effects of dapagliflozin on key metabolic pathways, supporting a role for altered ketone and fatty acid biology with sodium-glucose cotransporter-2 inhibitors in patients with HFrEF. Only physiologic levels of ketosis were observed. In addition, we identified several metabolic biomarkers associated with adverse HFrEF outcomes. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02653482.

Background: Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα–TRAF2–NF-κB signaling in the pathogenesis of DOX cardiotoxicity. Methods: Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg −1 ·wk −1 ) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell–derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. Results: In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T–adeno-associated virus 9–TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. Conclusions: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

Background and objectives: Patients who were hospitalized with coronavirus disease 2019 (COVID-19) infection are at high risk of AKI and KRT, especially in the presence of CKD. The Dapagliflozin in Respiratory Failure in Patients with COVID-19 (DARE-19) trial showed that in patients hospitalized with COVID-19, treatment with dapagliflozin versus placebo resulted in numerically fewer participants who experienced organ failure or death, although these differences were not statistically significant. We performed a secondary analysis of the DARE-19 trial to determine the efficacy and safety of dapagliflozin on kidney outcomes in the overall population and in prespecified subgroups of participants defined by baseline eGFR.Design, setting, participants, & measurements: The DARE-19 trial randomized 1250 patients who were hospitalized (231 [18%] had eGFR 2) with COVID-19 and cardiometabolic risk factors to dapagliflozin or placebo. Dual primary outcomes (time to new or worsened organ dysfunction or death, and a hierarchical composite end point of recovery [change in clinical status by day 30]), and the key secondary kidney outcome (composite of AKI, KRT, or death), and safety were assessed in participants with baseline eGFR 2.Results: The effect of dapagliflozin versus placebo on the primary prevention outcome (hazard ratio, 0.80; 95% confidence interval, 0.58 to 1.10), primary recovery outcome (win ratio, 1.09; 95% confidence interval, 0.97 to 1.22), and the composite kidney outcome (hazard ratio, 0.74; 95% confidence interval, 0.50 to 1.07) were consistent across eGFR subgroups (P for interaction: 0.98, 0.67, and 0.44, respectively). The effects of dapagliflozin on AKI were also similar in participants with eGFR 2 (hazard ratio, 0.71; 95% confidence interval, 0.29 to 1.77) and ≥60 ml/min per 1.73 m2 (hazard ratio, 0.69; 95% confidence interval, 0.37 to 1.29). Dapagliflozin was well tolerated in participants with eGFR 2.Conclusions: The effects of dapagliflozin on primary and secondary outcomes in hospitalized participants with COVID-19 were consistent in those with eGFR below/above 60 ml/min per 1.73 m2. Dapagliflozin was well tolerated and did not increase the risk of AKI in participants with eGFR below or above 60 ml/min per 1.73 m2.

Background:Right heart failure (RHF) is associated with worse clinical outcomes. In addition to hemodynamic perturbations, the syndrome of RHF involves liver congestion and dysfunction. The mechanisms that underlie heart-liver interactions are poorly understood and may involve secreted factors. As a first step to understand the cardiohepatic axis, we sought to elucidate the circulating inflammatory milieu in patients with RHF.Methods:Blood samples were collected from the IVC and hepatic veins during right heart catheterization from 3 groups of patients: 1) controls with normal cardiac function, 2) patients with heart failure (HF) who did not meet all criteria of RHF, and 3) patients who met prespecified criteria for RHF defined by hemodynamic and echocardiographic parameters. We performed multiplex protein assay to survey levels of several circulating markers and analyzed their association with mortality and need for left ventricular assist device or heart transplant. Finally, we leveraged publicly available single cell RNA sequencing (scRNAseq) data and performed tissue imaging to evaluate expression of these factors in the liver.Results:In this study of 43 patients, RHF was associated with elevated levels of a subset of cytokines/chemokines/growth factors compared to controls. In particular, soluble CD163 (sCD163) and CXCL12 were higher in RHF and predicted survival in an independent validation cohort. Furthermore, scRNAseq and immunohistochemistry of human liver biopsies suggest that these factors are expressed by Kupffer cells and may be liver derived.Conclusions:RHF is associated with a distinct circulating inflammatory profile. sCD163 and CXCL12 are novel biomarkers that can prognosticate patient outcomes. Future studies to define how these molecules influence HF phenotypes and disease progression may lead to new approaches to management of patients with RHF.

Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, glucose levels in the brain plummet, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1α transcriptional program that induces the expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) bothin vitroandin vivo. We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by powering the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 ensures the metabolic plasticity of synaptic transmission.HighlightsGlucose deprivation drives transcriptional reprogramming of neuronal metabolism via CREB and PGC1α.Glucose or food deprivation trigger the neuronal expression of mitochondrial deacetylase sirtuin 3 (Sirt3) bothin vitroandin vivo.Sirt3 stimulates oxidative ATP synthesis in nerve terminals.Sirt3 sustains the synaptic vesicle cycle in the absence of glucose.

Summary Background COVID-19 can lead to multiorgan failure. Dapagliflozin, a SGLT2 inhibitor, has significant protective benefits for the heart and kidney. We aimed to see whether this agent might provide organ protection in patients with COVID-19 by affecting processes dysregulated during acute illness. Methods DARE-19 was a randomised, double-blind, placebo-controlled trial of patients hospitalised with COVID-19 and with at least one cardiometabolic risk factor (ie, hypertension, type 2 diabetes, atherosclerotic cardiovascular disease, heart failure, and chronic kidney disease). Patients critically ill at screening were excluded. Patients were randomly assigned 1:1 to dapagliflozin (10 mg daily orally) or matched placebo for 30 days. Dual primary outcomes were assessed in the intention-to-treat population: the outcome of prevention (time to new or worsened organ dysfunction or death), and the hierarchial composite outcome of recovery (change in clinical status by day 30). Safety outcomes, in patients who received at least one study medication dose, included serious adverse events, adverse events leading to discontinuation, and adverse events of interest. This study is registered with ClinicalTrials.gov , NCT04350593 . Findings Between April 22, 2020 and Jan 1, 2021, 1250 patients were randomly assigned with 625 in each group. The primary composite outcome of prevention showed organ dysfunction or death occurred in 70 patients (11·2%) in the dapagliflozin group, and 86 (13·8%) in the placebo group (hazard ratio [HR] 0·80, 95% CI 0·58–1·10; p=0·17). For the primary outcome of recovery, 547 patients (87·5%) in the dapagliflozin group and 532 (85·1%) in the placebo group showed clinical status improvement, although this was not statistically significant (win ratio 1·09, 95% CI 0·97–1·22; p=0·14). There were 41 deaths (6·6%) in the dapagliflozin group, and 54 (8·6%) in the placebo group (HR 0·77, 95% CI 0·52–1·16). Serious adverse events were reported in 65 (10·6%) of 613 patients treated with dapagliflozin and in 82 (13·3%) of 616 patients given the placebo. Interpretation In patients with cardiometabolic risk factors who were hospitalised with COVID-19, treatment with dapagliflozin did not result in a statistically significant risk reduction in organ dysfunction or death, or improvement in clinical recovery, but was well tolerated. Funding AstraZeneca.

Background: The TOPCAT trial (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial) suggested clinical benefits of spironolactone treatment among patients with heart failure with preserved ejection fraction enrolled in the Americas. However, a comprehensive assessment of biologic pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction has not been performed. Methods: We conducted aptamer-based proteomic analysis utilizing 5284 modified aptamers to 4928 unique proteins on plasma samples from TOPCAT participants from the Americas (n=164 subjects with paired samples at baseline and 1 year) to identify proteins and pathways impacted by spironolactone therapy in heart failure with preserved ejection fraction. Mean percentage change from baseline was calculated for each protein. Additionally, we conducted pathway analysis of proteins altered by spironolactone. Results: Spironolactone therapy was associated with proteome-wide significant changes in 7 proteins. Among these, CARD18 (caspase recruitment domain-containing protein 18), PKD2 (polycystin 2), and PSG2 (pregnancy-specific glycoprotein 2) were upregulated, whereas HGF (hepatic growth factor), PLTP (phospholipid transfer protein), IGF2R (insulin growth factor 2 receptor), and SWP70 (switch-associated protein 70) were downregulated. CARD18, a caspase-1 inhibitor, was the most upregulated protein by spironolactone (−0.5% with placebo versus +66.5% with spironolactone, P Conclusions: Proteomic analysis in the TOPCAT trial revealed proteins and pathways altered by spironolactone, including the caspase inhibitor CARD18 and multiple pathways that involved collagens. In addition to effects on fibrosis, our studies suggest potential antiapoptotic effects of spironolactone in heart failure with preserved ejection fraction, a hypothesis that merits further exploration.

Background: Anthracyclines such as doxorubicin (Dox) cause cardiotoxicity and associated autophagic impairment, though controversy exists increasing autophagy ameliorates or exacerbates Dox cardiotoxicity. Because intermittent fasting (IF), a potent stimulus of autophagy, is presently under clinical investigation in multiple human clinical trials of cancer patients receiving chemotherapy, we tested whether IF would ameliorate or exacerbate Dox cardiotoxicity in multiple murine models. Methods: Chow-fed C57BL/6 mice (n=150) were randomized to ad libitum feeding ( adlib ) or IF (every other day) and treated with vehicle or Dox (5 mg/kg IP x 4 doses). Alternatively, to stimulate autophagy and lysosomal biogenesis transcriptionally, we employed adeno-associated virus 9 (AAV9)-driven overexpression of transcription factor EB (TFEB) vs null, followed by vehicle or Dox (n=40). We further analyzed TFEB nuclear content in myocardial tissue from patients with anthracycline cardiomyopathy vs non-failing donors and patients with other non-ischemic cardiomyopathies (n=17). Results: In Dox-treated mice (but not in vehicle controls), IF significantly reduced survival (18% absolute reduction or AR) ( figure A ), left ventricular ejection fraction (LVEF) (8% AR) ( figure B ) and heart weight index (12% relative reduction) ( figure C ) compared to adlib group. Mechanistically, in Dox-treated mice, IF significantly decreased phosphorylation of mammalian target of rapamycin, increased active, nuclear TFEB content, and increased levels of the atrophy factor muscle RING-finger protein-1. In myocardial tissue from patients with anthracycline cardiomyopathy, we observed a 3.5-fold increase (p Conclusions: IF exacerbates Dox-related mortality and cardiomyopathy, likely by stimulating the autophagy-lysosomal transcriptional regulator TFEB. Our data raise concerns about the safety of IF and augmentation of TFEB activity in the context of anthracycline chemotherapy.

AIMS: Coronavirus disease 2019 (COVID-19) is caused by a novel severe acute respiratory syndrome coronavirus 2. It can lead to multiorgan failure, including respiratory and cardiovascular decompensation, and kidney injury, with significant associated morbidity and mortality, particularly in patients with underlying metabolic, cardiovascular, respiratory or kidney disease. Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, has shown significant cardio- and renoprotective benefits in patients with type 2 diabetes (with and without atherosclerotic cardiovascular disease), heart failure and chronic kidney disease, and may provide similar organ protection in high-risk patients with COVID-19. MATERIALS AND METHODS: DARE-19 (NCT04350593) is an investigator-initiated, collaborative, international, multicentre, randomized, double-blind, placebo-controlled study testing the dual hypotheses that dapagliflozin can reduce the incidence of cardiovascular, kidney and/or respiratory complications or all-cause mortality, or improve clinical recovery, in adult patients hospitalized with COVID-19 but not critically ill on admission. Eligible patients will have ≥1 cardiometabolic risk factor for COVID-19 complications. Patients will be randomized 1:1 to dapagliflozin 10 mg or placebo. Primary efficacy endpoints are time to development of new or worsened organ dysfunction during index hospitalization, or all-cause mortality, and the hierarchical composite endpoint of change in clinical status through day 30 of treatment. Safety of dapagliflozin in individuals with COVID-19 will be assessed. CONCLUSIONS: DARE-19 will evaluate whether dapagliflozin can prevent COVID-19-related complications and all-cause mortality, or improve clinical recovery, and assess the safety profile of dapagliflozin in this patient population. Currently, DARE-19 is the first large randomized controlled trial investigating use of sodium-glucose cotransporter 2 inhibitors in patients with COVID-19.

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined. We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, and maintain myocardial homeostasis.

Ceramides are a class of bioactive sphingolipids increasingly recognized in metabolic disease, heart failure (HF), atherosclerosis, and cardiovascular death ([1][1]). Despite accumulating evidence linking ceramides to HF risk, little is known about circulating ceramides and clinical outcomes in HF

Mitochondrial damage triggers cell death signaling with catastrophic consequences in long-lived and irreplaceable cells, such as cardiac myocytes. Sensing of leaked mitochondrial DNA upon mitochondrial damage is also a potent trigger of inflammation. Whether the innate immune response pathways monitor mitochondrial damage in mitochondria-rich cardiac myocytes to prevent inflammation and cell death, remains unknown. TRAF2, an adaptor protein downstream of innate immune receptors, localizes to the mitochondria in the unstressed heart, with increased mitochondrial targeting in cardiomyopathic human hearts and after cardiac ischemia-reperfusion injury in mice. Inducible cardiomyocyte-specific deletion of TRAF2 in young adult mice impairs mitophagy with rapid decline in mitochondrial quality, upregulates TLR9 expression in cardiac myocytes, and results in inflammation and cell death manifesting as a fulminant cardiomyopathy. Preventing TLR9-mediated mitochondrial DNA sensing and resultant inflammation provides a short-term reprieve from cardiomyopathy, but persistence of damaged mitochondria results in long-term recrudescence. Restoration of wild-type TRAF2, but not the E3 ubiquitin ligase deficient mutant, improves mitochondrial quality and rescues cardiomyopathy to restore homeostasis. Thus, the innate immune response acts via TRAF2 as the first line of defense against mitochondrial damage by orchestrating homeostatic mitophagy to dampen myocardial inflammation and prevent cell death.

Visual Abstract, Highlights • DM is a significant risk factor for major adverse cardiovascular events in patients with HFpEF. • Patients with diabetes with HFpEF have a distinct proteome compared with patients without diabetes with HFpEF. • Proteomics analysis identified higher levels of alpha-1-microglobulin/bikunin precursor protein in patients with diabetes with HFpEF and lower levels of CILP2 and Apo M. • Lower Apo M levels mediate most of the association between diabetes and major adverse cardiovascular events in HFpEF., Summary Diabetes mellitus (DM) is associated with a higher risk of heart failure hospitalization and mortality in patients with heart failure with preserved ejection fraction (HFpEF). Using SomaScan assays and proteomics analysis of plasma from participants in the TOPCAT (Treatment of Preserved Cardiac Function Heart Failure with an Aldosterone Antagonist) trial and the Penn Heart Failure Study, this study identified 10 proteins with significantly different expression in patients with HFpEF and DM. Of these, apolipoprotein M was found to mediate 72% (95% CI: 36% to 100%; p

Background: Apolipoprotein M (APOM) is a lipoprotein that binds sphingosine-1-phosphate (S1P) and high-density lipoprotein (HDL). APOM/S1P attenuate myocardial ischemia/reperfusion injury by reducing infarct size. We recently published that reduced APOM plasma protein levels are associated with mortality across the spectrum of human heart failure. With the goal of uncovering mechanisms by which APOM impacts heart failure mortality, we modeled doxorubicin cardiotoxicity in hepatocyte specific APOM overexpression (APOM TG ) and littermate controls. Results: Doxorubicin treatment acutely reduces APOM plasma protein levels in control mice. APOM TG mice exhibit ~3-5 fold increased APOM and ~2.5 fold increased S1P. In acute and chronic doxorubicin cardiotoxicity models, APOM TG mice were protected from mortality and reductions in left-ventricular mass and ejection fraction observed in littermate controls. In murine models of acute leukemia, doxorubicin effectively killed leukemic cells in control and APOM TG mice. In the myocardium, APOM TG mice and controls had similar levels of γ-H2AX foci after doxorubicin treatment, indicating similar levels of DNA damage, and electron microscopy indicated similar mitochondrial morphology. However, APOM TG mice exhibited enhanced autophagic flux, and APOM prevented doxorubicin-induced reductions in autophagic flux observed in littermate control mice (Figure). Mechanistically, treatment of neonatal rat cardiomyocytes with S1P mimetics attenuated doxorubicin-induced loss of lysosomal pH, suggesting that APOM may enhance lysosomal function via S1P. In the murine myocardium, doxorubicin reduced the nuclear protein content of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, in control mice but not APOM TG mice. Furthermore, adeno-associated virus mediated knockdown of TFEB completely reversed the beneficial effects of APOM on the myocardium, leading to cardiomyopathy and mortality in APOM TG mice. Conclusions: Our studies identify APOM and S1P as regulators of TFEB in the myocardium, hence providing a mechanistic link between HDL and the autophagy-lysosome pathway in the murine heart. The role of APOM in myocardial autophagy may explain our observed clinical associations between reduced APOM and mortality.

Background: Apo (apolipoprotein) M mediates the physical interaction between high-density lipoprotein (HDL) particles and sphingosine-1-phosphate (S1P). Apo M exerts anti-inflammatory and cardioprotective effects in animal models. Methods: In a subset of PHFS (Penn Heart Failure Study) participants (n=297), we measured apo M by Enzyme-Linked ImmunoSorbent Assay (ELISA). We also measured total S1P by liquid chromatography–mass spectrometry and isolated HDL particles to test the association between apo M and HDL-associated S1P. We confirmed the relationship between apo M and outcomes using modified aptamer-based apo M measurements among 2170 adults in the PHFS and 2 independent cohorts: the Washington University Heart Failure Registry (n=173) and a subset of TOPCAT (Treatment of Preserved Cardiac Function Heart Failure With an Aldosterone Antagonist Trial; n=218). Last, we examined the relationship between apo M and ≈5000 other proteins (SomaScan assay) to identify biological pathways associated with apo M in heart failure. Results: In the PHFS, apo M was inversely associated with the risk of death (standardized hazard ratio, 0.56 [95% CI, 0.51–0.61]; P P P P =0.001) in models that adjusted for multiple confounders. This association was present in both heart failure with reduced and preserved ejection fraction and was replicated in the Washington University cohort and a cohort with heart failure with preserved ejection fraction only (TOPCAT). The S1P and apo M content of isolated HDL particles strongly correlated ( R =0.81, P Conclusions: Reduced circulating apo M is independently associated with adverse outcomes across the spectrum of human heart failure. Further research is needed to assess whether the apo M/S1P axis is a suitable therapeutic target in heart failure.

Macrophages specialize in removing lipids and debris present in the atherosclerotic plaque. However, plaque progression renders macrophages unable to degrade exogenous atherogenic material and endogenous cargo including dysfunctional proteins and organelles. Here we show that a decline in the autophagy–lysosome system contributes to this as evidenced by a derangement in key autophagy markers in both mouse and human atherosclerotic plaques. By augmenting macrophage TFEB, the master transcriptional regulator of autophagy–lysosomal biogenesis, we can reverse the autophagy dysfunction of plaques, enhance aggrephagy of p62-enriched protein aggregates and blunt macrophage apoptosis and pro-inflammatory IL-1β levels, leading to reduced atherosclerosis. In order to harness this degradative response therapeutically, we also describe a natural sugar called trehalose as an inducer of macrophage autophagy–lysosomal biogenesis and show trehalose's ability to recapitulate the atheroprotective properties of macrophage TFEB overexpression. Our data support this practical method of enhancing the degradative capacity of macrophages as a therapy for atherosclerotic vascular disease., Dysfunction of autophagy in plaque macrophages aggravates atherosclerosis. Here the authors show that induction of macrophage autophagy–lysosomal biogenesis either genetically by overexpression of the master transcriptional regulator of this process, TFEB, or pharmacologically with trehalose is atheroprotective.

Lysosomes are at the epicenter of cellular processes critical for inflammasome activation in macrophages. Inflammasome activation and IL-1β secretion are implicated in myocardial infarction (MI) and resultant heart failure; however, little is known about how macrophage lysosomes regulate these processes. In mice subjected to cardiac ischemia/reperfusion (IR) injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal impairment in macrophages. Inducible macrophage-specific overexpression of transcription factor EB (TFEB), a master regulator of lysosome biogenesis (Mϕ-TFEB), attenuated postinfarction remodeling, decreased abundance of proinflammatory macrophages, and reduced levels of myocardial IL-1β compared with controls. Surprisingly, neither inflammasome suppression nor Mϕ-TFEB-mediated attenuation of postinfarction myocardial dysfunction required intact ATG5-dependent macroautophagy (hereafter termed "autophagy"). RNA-seq of flow-sorted macrophages postinfarction revealed that Mϕ-TFEB upregulated key targets involved in lysosomal lipid metabolism. Specifically, inhibition of the TFEB target, lysosomal acid lipase, in vivo abrogated the beneficial effect of Mϕ-TFEB on postinfarction ventricular function. Thus, TFEB reprograms macrophage lysosomal lipid metabolism to attenuate remodeling after IR, suggesting an alternative paradigm whereby lysosome function affects inflammation.

Background: Autophagy, lipid metabolism, and inflammation are interrelated cellular processes that implicate lysosomes in human disease. After ischemia reperfusion (IR) injury, inflammasome activation and interleukin 1-beta (IL1-beta) secretion promote heart failure progression. Whether macrophage autophagy and lysosomal biogenesis can attenuate post-IR remodeling and inflammation is unknown. We hypothesized that macrophages exhibit lysosome dysfunction and autophagic impairment after IR injury, and that augmentation of macrophage lysosomal biogenesis via macrophage-specific overexpression of transcription factor EB (Mf-TFEB), a master regulator of autophagy and lysosomal biogenesis, would attenuate myocardial remodeling and inflammation in ischemic cardiomyopathy. Methods and Results: In mice subject to IR injury and humans with ischemic cardiomyopathy, we observed evidence of lysosomal and autophagic impairment. To ameliorate post-IR macrophage lysosomal injury, we expressed Mf-TFEB in a closed-chest IR murine model using a tamoxifen-inducible CX3CR1erCre and TFEB overexpression cassette bearing a Cre-excisable STOP codon. Compared to Cre-only controls, Mf-TFEB mice had significantly increased left ventricular (LV) ejection fraction 28-days post-IR (40% relative increase, p=0.002, n=15-17 per group), decreased abundance of pro-inflammatory macrophages, and reduced levels of IL1-beta in myocardial tissue. Surprisingly, neither inflammasome suppression nor TFEB-mediated attenuation of post-IR remodeling required intact macro-autophagy as evidenced by Mf-TFEB-mediated rescue of post-IR LV dysfunction in mice with concomitant inducible ATG5 ablation. RNA sequencing of flow-sorted macrophages from post-IR mice identified lysosomal acid lipase amongst other lipases regulated by TFEB. Mechanistically, pharmacologic inhibition of lysosomal acid lipase specifically abrogated the in vivo effects of TFEB on post-IR remodeling. Conclusions: Our findings suggest that macrophage TFEB regulates lysosomal lipolysis to attenuate inflammasome activity and protect against post-IR LV dysfunction, suggesting an alternative paradigm for how lysosome function may impact acute inflammation in vivo.

Background Mutations in αB‐crystallin result in proteotoxic cardiomyopathy with desmin mislocalization to protein aggregates. Intermittent fasting (IF) is a novel approach to activate transcription factor EB (TFEB), a master regulator of the autophagy‐lysosomal pathway, in the myocardium. We tested whether TFEB activation can be harnessed to treat advanced proteotoxic cardiomyopathy. Methods and Results Mice overexpressing the R120G mutant of αB‐crystallin in cardiomyocytes (Myh6‐CryABR120G) were subjected to IF or ad‐lib feeding, or transduced with adeno‐associated virus–TFEB or adeno‐associated virus—green fluorescent protein after development of advanced proteotoxic cardiomyopathy. Adeno‐associated virus–short hairpin RNA–mediated knockdown of TFEB and HSPB8 was performed simultaneously with IF. Myh6‐CryABR120G mice demonstrated impaired autophagic flux, reduced lysosome abundance, and mammalian target of rapamycin activation in the myocardium. IF resulted in mammalian target of rapamycin inhibition and nuclear translocation of TFEB with restored lysosome abundance and autophagic flux; and reduced aggregates with normalized desmin localization. IF also attenuated left ventricular dilation and myocardial hypertrophy, increased percentage fractional shortening, and increased survival. Adeno‐associated virus–TFEB transduction was sufficient to rescue cardiomyopathic manifestations, and resulted in reduced aggregates and normalized desmin localization in Myh6‐CryABR120G mice. CryABR120G‐expressing hearts demonstrated increased interaction of desmin with αB‐crystallin and reduced interaction with chaperone protein, HSPB8, compared with wild type, which was reversed by both IF and TFEB transduction. TFEB stimulated autophagic flux to remove protein aggregates and transcriptionally upregulated HSPB8, to restore normal desmin localization in CryABR120G‐expressing cardiomyocytes. Short hairpin RNA–mediated knockdown of TFEB and HSPB8 abrogated IF effects, in vivo. Conclusions IF and TFEB activation are clinically relevant therapeutic strategies to rescue advanced R120G αB‐crystallin mutant‐induced cardiomyopathy by normalizing desmin localization via autophagy‐dependent and autophagy‐independent mechanisms., See Editorial by Mukai et al

Endothelial function may be deranged in heart failure with preserved ejection fraction (HFpEF). Serum NO-derived metabolites (NO m ) might provide a biochemical surrogate of endothelial function in patients with heart failure (HF). We measured serum NO m in 415 participants in the Penn HF Study. Participants with HFpEF (n = 82) and those whose EF had recovered (Recovered-HF, n = 125) were matched 1:1 to heart failure with reduced ejection fraction (HFrEF) participants based on age, gender, race, tobacco use, and eGFR. Serum NO m levels were quantified after chemical reduction coupled with gas-phase chemiluminescence detection. After adjustment for matching covariates and BMI, HFpEF (34.5 μM; interquartile range [IQR] 25.0, 51.5) participants had lower NO m levels than HFrEF (41.0 μM; IQR 28.3, 58.0; ratio of HFpEF:HFrEF 0.82, 95% confidence interval [CI] 0.67 to 0.99; p = 0.04), which further decreased when adjusted for covariates that affect endothelial function (ratio 0.79, 95% CI 0.65 to 0.98; p = 0.03). There were no differences between HFrEF (34.0; IQR 25.3, 49.0) and matched Recovered-HF (36.0 μM; IQR 25.0, 55.0) or HFpEF and Recovered-HF. Age (+21%/10-year increase, p m in HFpEF, whereas age (+11%/10-year increase, p = 0.03), current tobacco use (+67%, p = 0.01), and eGFR (p = 0.01) associated with NO m in Recovered-HF. In conclusion, HFpEF participants have reduced NO m compared with HFrEF in this matched cohort. This might suggest either compromised endothelial function or poor dietary intake. Black race was associated with lower NO m in HFpEF.

Background Cardiac allograft vasculopathy (CAV) is a major cause of mortality after cardiac transplantation. High-density lipoprotein (HDL) cholesterol efflux capacity (CEC) is inversely associated with coronary artery disease. In 2 independent studies, we tested the hypothesis that reduced CEC is associated with mortality and disease progression in CAV. Methods We tested the relationship between CEC and survival in a cohort of patients with CAV ( n = 35). To determine whether reduced CEC is associated with CAV progression, we utilized samples from the Clinical Trials in Organ Transplantation 05 (CTOT05) study to determine the association between CEC and CAV progression and status at 1 year ( n = 81), as assessed by average change in maximal intimal thickness (MIT) on intravascular ultrasound. Results Multivariable Cox proportional hazard models demonstrated that higher levels of CEC were associated with improved survival (hazard ratio 0.26, 95% confidence interval 0.11 to 0.63) per standard deviation CEC, p = 0.002). Patients who developed CAV had reduced CEC at baseline and 1-year post-transplant. We observed a significant association between pre-transplant CEC and the average change in MIT, particularly among patients who developed CAV at 1 year (β = −0.59, p = 0.02, R 2 = 0.35). Conclusion Reduced CEC is associated with disease progression and mortality in CAV patients. These findings suggest the hypothesis that interventions to increase CEC may be useful in cardiac transplant patients for prevention or treatment of CAV.