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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: 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.

Although research on high-density lipoprotein (HDL) has historically focused on atherosclerotic coronary disease, there exists untapped potential of HDL biology for the treatment of heart failure. Anti-oxidant, anti-inflammatory, and endothelial protective properties of HDL could impact heart failure pathogenesis. HDL-associated proteins such as apolipoprotein A-I and M may have significant therapeutic effects on the myocardium, in part by modulating signal transduction pathways and sphingosine-1-phosphate biology. Furthermore, because heart failure is a complex syndrome characterized by multiple comorbidities, there are complex interactions between heart failure, its comorbidities, and lipoprotein homeostatic mechanisms. In this review, we will discuss the effects of heart failure and associated comorbidities on HDL, explore potential cardioprotective properties of HDL, and review novel HDL therapeutic targets in heart failure.

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

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.

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.

Background— The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. Methods and Results— We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic β-hydroxybutyryl-CoA, in association with increased myocardial utilization of β-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of β-hydroxybutyrate and acetoacetate. Conclusions— These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart.

Heart transplantation is one of the most definitive therapies for end-stage heart failure. The therapy is unfortunately marred by the devastating complications of cardiac allograft vasculopathy (CAV). Non-invasive screening and assessment for CAV has been greatly limited by both low sensitivity and poor correlation with adverse outcomes. As such, invasive imaging with coronary angiography has emerged as the gold standard for detection of CAV. Although conventional coronary angiography serves well for larger lesions, the modality has been significantly enhanced with adjunct imaging to visualize the intimal hyperplasia that is a hallmark of the disease process. These modalities include intravascular ultrasound (IVUS) and optical coherence tomography (OCT). In the following review, we summarize both the invasive and non-invasive assessments of CAV. We further conclude that the current evidence poorly supports the use of non-invasive testing for early CAV and that a transition should be considered to routine early angiography with adjunctive intravascular imaging.

Background Radiation-treated head and neck cancer (HNC) patients are at high risk for developing radiation vasculopathy, as evidenced by an increased stroke risk. The benefits of screening and assessing the cardiovascular (CV) risk of HNC patients using carotid intima-media thickness (CIMT) ultrasound are not known. Our objective was to determine the prevalence of high CV risk in patients without known CV diseases who received radiation for HNC, determine the percentage of screened patients who had a change in clinical management as a result of an increased CIMT, and to compare this risk-assessment tool to patients' risk classification using the Framingham Risk Score (FRS) and Pooled Cohort Atherosclerotic Cardiovascular Disease (ASCVD) Risk Equation (recommended by American College of Cardiology/American Heart Association Guidelines on the Assessment of Cardiovascular Risk). Hypothesis Risk calculators may not accurately predict risk in this population with a unique risk factor. Carotid IMT may be used to detect radiation vasculopathy in HNC patients. Methods Retrospective medical chart review was conducted on 134 radiation-treated HNC patients. The main outcome measures were CV risk (as determined by CIMT) and clinical management. Also, the FRS and the Pooled Cohort ASCVD Risk Equation were used to compare classification with CIMT. Results Approximately 74% of the cases were at high CV risk using CIMT technique. Approximately half of the HNC patients screened had a change in clinical management characterized by recorded initiation of aspirin and recorded initiation or increase of statin therapies. The FRS and the Pooled Cohort ASCVD Risk Equation failed to detect 40% to 50% of cases found to be at high risk using the CIMT technique. Conclusions Carotid IMT identified a much greater percentage of radiation-treated HNC patients at high CV risk compared with standard CV-risk calculators. By more accurately identifying the patients at high risk, this may lead to more effective prevention, and therefore a reduction in CV events.

Introduction: Animal models and human studies have identified increased intramyocardial lipid accumulation and a lipotoxicity hypothesis has been emerging as a mechanism of myocardial dysfunction in diabetes. We have identified a significant decrease in energetic lipids in chronic advanced non-diabetic heart failure. We hypothesized that intramyocardial lipid species would be increased in diabetic as compared to non-diabetic end-stage heart failure patients. Methods: Left ventricular samples procured at the time of orthotopic heart transplantation from non-diabetic (IDCM n=8) and diabetic (DCMDM n=8) patients as well as organ donors with (NFDM) and without a history of diabetes (Donor) were quantitated for lipids with high-resolution mass spectrometry . Stable isotope labeled essential nutrient in cell culture internal standards for acyl-CoAs were generated using [ 13 C 3 15 N 1 ]-pantotheonate in Hepa1c1c7 cells. Results: The lipidomic signature of end-stage failing myocardium marked by a significant decrease in energetic lipids, a decreased myocardial Succinyl CoA (p-value 0.0079 for failing versus non-failing diabetic subjects) and a decreased ratio of [Succinyl CoA]/[Acetyl Coa] consistent with deficient TCA cycling, is indistinguishable in diabetic and non-diabetic patients (Figure 1). Discussion: Despite the known bioenergetic deficits in insulin-resistant diabetes, we have not identified any significant differences between diabetic and non-diabetic subjects in the lipidomic signature of end-stae failing myocardium. Future studies are needed with focused metabolomics to elucidate differences in the diabetic phenotype of human heart failure.

Background: Coronary allograft vasculopathy (CAV) is an important cause of mortality after cardiac transplantation. High density lipoprotein (HDL) cholesterol efflux capacity has been inversely associated with coronary artery disease and is impaired in cardiac transplant recipients. We performed a single center case-cohort study to test the hypothesis that reduced efflux capacity is a risk factor for mortality and a second, multi-center retrospective study to test if efflux capacity is associated with CAV progression. Methods: We designed a single center case-cohort study in which we identified cardiac transplant patients who died between 2009-2012 (cases, n=34) and controls as cardiac transplant patients who were alive as of the fourth quarter of 2013 (n=57). Efflux capacity was measured by incubating apolipoprotein B-depleted serum with macrophages in a validated ex vivo system. In a second study, we utilized pre-transplant samples from the Clinical Trials in Organ Transplantation 5 (CTOT5) study to determine the association between ATP-binding-cassette (ABC) A1-dependent cholesterol efflux and CAV progression at 1 year. Results: In our single center study, the average time from transplant to study entry was well-matched between cases and controls (7.6±1.0 vs 7.7±0.8 years, respectively, p=0.48). Multivariable Cox proportional hazard ratios demonstrated that higher levels of HDL cholesterol efflux capacity were associated with survival (HR 0.61, 95% CI 0.43-0.85), even after adjustment for HDL cholesterol mass. To determine whether excess mortality observed in subjects with reduced efflux could be attributable to CAV progression, we tested the relationship between intravascular ultrasound (IVUS) progression of CAV and cholesterol efflux capacity using linear regression. ABCA1-dependent efflux and IVUS progression were significantly associated (β = -0.90, 95% CI [-1.73 - -0.07], p = 0.037, R2 = 0.37). Conclusion: Reduced efflux capacity is an important mediator of CAV progression and mortality in cardiac transplant recipients. This finding suggests that interventions to increase HDL cholesterol efflux capacity may provide clinical benefit in cardiac transplant recipients.

Stable plasma nitric oxide metabolites (NOx), predominantly composed of nitrate, are markers of endogenous nitric oxide (NO) production, NO utilization, and dietary intake and may be indicators of vascular health. We enrolled subjects undergoing a clinically-indicated cardiac MRI. Plasma NOx levels

Introduction: Several studies have implicated energetic deficits in chronic heart failure. Recently, our group has described a state of myocardial substrate deficiency across a broad spectrum of lipid species in non-diabetic end-stage heart failure. We hypothesized that lipid substrate depletion in end-stage heart failure would be associated with dysregulation of short chain acyl-CoA intermediates implicated in important metabolic pathways. Methods: Left ventricular samples were obtained at the time of orthotopic heart transplantation for failing cases with idiopathic dilated cardiomyopathy (DCM) n=16, and brain-dead organ donors without a history of heart failure (NF) n=18. Samples were snap frozen at -80°C for further analysis with liquid chromatography in tandem mass spec LC MS/MS. Stable isotope labeled essential nutrient in cell culture (SILEC) internal standards for acyl-CoAs were generated using 13 C 3 15 N 1 pantotheonate in Hepa1c1c7 cells. Results: In the myocardium of failing as compared to NF, we identified a significant decrease in Succinyl-CoA (Avg 10.5 versus 17.7 pmol/mg, p = 0.004 ), Propionyl-CoA (0.9 versus 1.8 pmol/mg, p=0.004) and a concomitant increase in β-hydroxybutyryl -CoA (BHB-CoA Avg 0.57 versus 0.29 pmol/mg, p =0.008 ). The ratio of myocardial Succinyl-CoA to Acetyl-CoA, a potential marker of tricarboxylic acid cycling, is significantly decreased in end-stage heart failure ( 0.84 versus 1.93, p= 0.005 ). The figure depicts the levels of myocardial acyl-CoA species in the non-diabetic cohort. Conclusion: We have identified important differences in myocardial acyl CoA species—an increase in the ketogenic BHB-CoA and decreased Succinyl-CoA and Propionyl-CoA in end-stage human heart failure. These data, along with the recently identified state of myocardial lipodeficiency, are strongly supporting the concept of a bio-energetic deficit in end-stage heart failure.

Despite epidemiological studies showing that increased levels of high-density lipoprotein cholesterol (HDL-C) are associated with a reduced risk of future coronary heart disease, neither monogenic diseases characterized by extreme HDL-C levels nor genetic variants associated with higher HDL-C levels are associated with coronary heart disease risk reduction.1,2 Most importantly, increasing HDL-C levels as a therapeutic approach to reduce cardiovascular risk has been called into question by the recent failure of several randomized trials in which therapies aimed at increasing HDL-C levels, such as niacin and cholesteryl ester transferase protein inhibitors, have failed to improve cardiovascular outcomes.3–5 See accompanying article on page 2106 However, it remains clear that HDL has multiple potential antiatherogenic functions,6 including its well-described role in reverse cholesterol transport (RCT)7 (Figure [A]). Thus, a novel approach toward coronary heart disease prevention and HDL-targeting therapies focuses on enhancing HDL function rather than HDL-C levels. That cholesterol efflux capacity of HDL isolated from human subjects is inversely correlated with coronary heart disease status8 reinforces the concept that atheroprotection is associated with HDL function rather than HDL-C levels. In this issue of Arteriosclerosis Thrombosis and Vascular Biology , Gille et al9 present evidence that in healthy volunteers, infusion of CSL112, a reconstituted HDL (rHDL) containing apolipoprotein AI (apoAI) and phospholipids improves biomarkers of RCT and cholesterol efflux when measured ex vivo. Figure. A , Reverse cholesterol transport. Apolipoprotein AI (apoAI; synthesized in the liver and intestines) acquires phospholipid and a small amount of free cholesterol via the ATP-binding cassette transporter A1 (ABCA1) to …

High-density lipoprotein (HDL) cholesterol is strongly and inversely associated with coronary heart disease. However, recent developments have raised major questions about the causal nature of this relationship. Several randomized controlled clinical trials of HDL-raising interventions have failed to demonstrate reduction in risk of major adverse cardiac events.1–4 Furthermore, in 1 Mendelian randomization study, genetic variants associated with increased HDL cholesterol were not associated with protection from coronary heart disease.5 It has been proposed that HDL can be dysfunctional and that this might cloud the relationship between HDL cholesterol and coronary heart disease. In this issue of Circulation Research , Shao et al6 further our understanding of the concept of HDL dysfunction by showing that oxidative modifications of apoA-I, the main protein constituent of HDL, impair its ability to accept cholesterol from macrophages. Article, see p 1733 HDL may protect against atherogenesis via several potential mechanisms. HDL has been shown to inhibit inflammation,7 regulate nitric oxide production,8 function in innate immunity,9 and, in its most extensively studied property, remove excess cholesterol from macrophages in the process of reverse cholesterol transport. During cholesterol efflux, lipid-poor apoA-I and mature HDL interact with integral membrane proteins ATP-binding cassette transporter A1 and ATP-binding cassette transporter G1, respectively, to accept cholesterol from cells.10 Our group and others have shown that the cholesterol efflux capacity of HDL inversely correlates with atherosclerotic vascular disease.6,11,12 The addition of niacin to statin therapy failed to improve cardiovascular outcomes but also did not improve HDL cholesterol efflux capacity,13 providing a …

Obesity, Sleep Apnea, LV Remodeling and Myocardial Fibrosis Assessed with Cardiac MRI Both obesity and sleep apnea (SA) are predictive of heart failure (HF). Limited data are available regarding the independent effects of SA and obesity on left ventricular (LV) remodeling. We recruited 280

Despite numerous epidemiological studies demonstrating that high-density lipoprotein cholesterol (HDL-C) levels are inversely associated with cardiovascular risk,1–3 several lines of evidence now indicate that targeting HDL-C levels to reduce the risk of cardiovascular events is unlikely to be effective. Studies of pharmacological interventions to raise HDL-C, such as niacin4,5 and 2 cholesteryl ester transfer protein inhibitors,6,7 have shown no benefit in reducing cardiovascular events. In addition, data from a large Mendelian randomization study have shown that some genetic variants associated with HDL-C seem to have little relationship to coronary heart disease.8 As a result, there is currently skepticism about whether interventions specifically to raise HDL-C levels will decrease the risk of cardiovascular events. Article, see p 1345 This failure of the so-called HDL cholesterol hypothesis has been accompanied by a shift toward a more rigorous, basic understanding of HDL as a molecule with multiple functions that can be differentiated from simple measures of HDL cholesterol mass. One of the important functions of HDL is its role in promoting cellular cholesterol efflux and reverse cholesterol transport. Our group and others have shown that the capacity of HDL to promote cholesterol efflux from macrophages ex vivo is inversely related to the risk of coronary heart disease even after controlling for HDL-C levels.9,10 Furthermore, niacin therapy does not augment cholesterol efflux despite raising HDL levels in statin-treated patients,11 which could explain the lack of efficacy of niacin despite increased HDL-C levels. Although more studies are certainly warranted, one hypothesis is that therapies that improve cholesterol efflux capacity and reverse cholesterol transport, such as infusion of a reconstituted HDL12 composed of apolipoprotein A1 and phospholipids, may improve cardiovascular outcomes. Beyond promoting cholesterol efflux, HDL is known to have anti-inflammatory, …

Despite the emergence of sleep apnea (SA) as a significant risk factor for heart failure (HF) mortality, data indicate that SA remains under-diagnosed and under-treated. Less well established, though perhaps more emphasized, is the role of sleep apnea in pulmonary hypertension (PH). SA occurs in approximately 50 % of HF patients, and its consequences include intermittent hypoxemia, arousal, and intra-thoracic pressure swings leading to neurohormonal stimulation, oxidative stress and inflammation. While SA is also considered a cause of PH, severe PH due solely to SA is rare. Combining the results of several studies using Swan-Ganz catheters for diagnosis of PH, approximately 10 % of patients with OSA have PH. Effective treatment of SA in HF is associated with improved survival, while treatment of SA in PH is typically associated with modest hemodynamic improvement.

Radiofrequency epicardial ablation of ventricular tachycardia (VT) is a safe, effective modality for treatment of VT.1 Epicardial VT circuits were originally described in Chagas disease, but the importance of nonendocardial substrates for VT increasingly are recognized in other cardiomyopathies.2 In arrhythmogenic right ventricular cardiomyopathy (ARVC), failure of endocardial VT ablation often is related to epicardial VT origination, with high rates of success seen with epicardial VT ablation.3 While hemopericardium or pericardial inflammation are rare complications of epicardial VT ablation, to our knowledge no case of pericardial constriction and heart failure following epicardial VT ablation has been reported.4 Here we describe a case of pericardial constriction and severe heart failure requiring stripping and “bridge to recovery” right-ventricular assist-device (RVAD) support, which occurred subsequent to recurrent epicardial VT ablations. ### Case Presentation A 46-year-old male who presented with palpitations and episodic syncope underwent cardiac magnetic resonance imaging, which was consistent with ARVC, followed by implantable …