Multi-Omics Profiling of Hypertrophic Cardiomyopathy Reveals Altered Mechanisms in Mitochondrial Dynamics and Excitation-Contraction Coupling.

Hypertrophic cardiomyopathy is one of the most common inherited cardiomyopathies and a leading cause of sudden cardiac death in young adults. Despite profound insights into the genetics, there is imperfect correlation between mutation and clinical prognosis, suggesting complex molecular cascades driving pathogenesis. To investigate this, we performed an integrated quantitative multi-omics (proteomic, phosphoproteomic, and metabolomic) analysis to illuminate the early and direct consequences of mutations in myosin heavy chain in engineered human induced pluripotent stem-cell-derived cardiomyocytes relative to late-stage disease using patient myectomies. We captured hundreds of differential features, which map to distinct molecular mechanisms modulating mitochondrial homeostasis at the earliest stages of pathobiology, as well as stage-specific metabolic and excitation-coupling maladaptation. Collectively, this study fills in gaps from previous studies by expanding knowledge of the initial responses to mutations that protect cells against the early stress prior to contractile dysfunction and overt disease.

Redox-Resistant SERCA [Sarco(endo)plasmic Reticulum Calcium ATPase] Attenuates Oxidant-Stimulated Mitochondrial Calcium and Apoptosis in Cardiac Myocytes and Pressure Overload-Induced Myocardial Failure in Mice.

BACKGROUND: SERCA [sarco(endo)plasmic reticulum calcium ATPase] is regulated by oxidative posttranslational modifications at cysteine 674 (C674). Because sarcoplasmic reticulum (SR) calcium has been shown to play a critical role in mediating mitochondrial dysfunction in response to reactive oxygen species, we hypothesized that SERCA oxidation at C674 would modulate the effects of reactive oxygen species on mitochondrial calcium and mitochondria-dependent apoptosis in cardiac myocytes. METHODS: Adult rat ventricular myocytes expressing wild-type SERCA2b or a redox-insensitive mutant in which C674 is replaced by serine (C674S) were exposed to H2O2 (100 µmol/Lµ). Free mitochondrial calcium concentration was measured in adult rat ventricular myocytes with a genetically targeted fluorescent probe, and SR calcium content was assessed by measuring caffeine-stimulated release. Mice with heterozygous knock-in of the SERCA C674S mutation were subjected to chronic ascending aortic constriction. RESULTS: In adult rat ventricular myocytes expressing wild-type SERCA, H2O2 caused a 25% increase in mitochondrial calcium concentration that was associated with a 50% decrease in SR calcium content, both of which were prevented by the ryanodine receptor inhibitor tetracaine. In cells expressing the C674S mutant, basal SR calcium content was decreased by 31% and the H2O2-stimulated rise in mitochondrial calcium concentration was attenuated by 40%. In wild-type cells, H2O2 caused cytochrome c release and apoptosis, both of which were prevented in C674S-expressing cells. In myocytes from SERCA knock-in mice, basal SERCA activity and SR calcium content were decreased. To test the effect of C674 oxidation on apoptosis in vivo, SERCA knock-in mice were subjected to chronic ascending aortic constriction. In wild-type mice, ascending aortic constriction caused myocyte apoptosis, LV dilation, and systolic failure, all of which were inhibited in SERCA knock-in mice. CONCLUSIONS: Redox activation of SERCA C674 regulates basal SR calcium content, thereby mediating the pathologic reactive oxygen species-stimulated rise in mitochondrial calcium required for myocyte apoptosis and myocardial failure.

Increasing mitochondrial ATP synthesis with butyrate normalizes ADP and contractile function in metabolic heart disease.

Metabolic heart disease (MHD), which is strongly associated with heart failure with preserved ejection fraction, is characterized by reduced mitochondrial energy production and contractile performance. In this study, we tested the hypothesis that an acute increase in ATP synthesis, via short chain fatty acid (butyrate) perfusion, restores contractile function in MHD. Isolated hearts of mice with MHD due to consumption of a high fat high sucrose (HFHS) diet or on a control diet (CD) for 4 months were studied using 31 P NMR spectroscopy to measure high energy phosphates and ATP synthesis rates during increased work demand. At baseline, HFHS hearts had increased ADP and decreased free energy of ATP hydrolysis (ΔG~ATP ), although contractile function was similar between the two groups. At high work demand, the ATP synthesis rate in HFHS hearts was reduced by over 50%. Unlike CD hearts, HFHS hearts did not increase contractile function at high work demand, indicating a lack of contractile reserve. However, acutely supplementing HFHS hearts with 4mM butyrate normalized ATP synthesis, ADP, ΔG~ATP and contractile reserve. Thus, acute reversal of depressed mitochondrial ATP production improves contractile dysfunction in MHD. These findings suggest that energy starvation may be a reversible cause of myocardial dysfunction in MHD, and opens new therapeutic opportunities.

Role of Glutaredoxin-1 and Glutathionylation in Cardiovascular Diseases.

Cardiovascular diseases are the leading cause of death worldwide, and as rates continue to increase, discovering mechanisms and therapeutic targets become increasingly important. An underlying cause of most cardiovascular diseases is believed to be excess reactive oxygen or nitrogen species. Glutathione, the most abundant cellular antioxidant, plays an important role in the body's reaction to oxidative stress by forming reversible disulfide bridges with a variety of proteins, termed glutathionylation (GSylation). GSylation can alter the activity, function, and structure of proteins, making it a major regulator of cellular processes. Glutathione-protein mixed disulfide bonds are regulated by glutaredoxins (Glrxs), thioltransferase members of the thioredoxin family. Glrxs reduce GSylated proteins and make them available for another redox signaling cycle. Glrxs and GSylation play an important role in cardiovascular diseases, such as myocardial ischemia and reperfusion, cardiac hypertrophy, peripheral arterial disease, and atherosclerosis. This review primarily concerns the role of GSylation and Glrxs, particularly glutaredoxin-1 (Glrx), in cardiovascular diseases and the potential of Glrx as therapeutic agents.

Decreased ATP production and myocardial contractile reserve in metabolic heart disease.

Metabolic syndrome is a cluster of obesity-related metabolic abnormalities that lead to metabolic heart disease (MHD) with left ventricular pump dysfunction. Although MHD is thought to be associated with myocardial energetic deficiency, two key questions have not been answered. First, it is not known whether there is a sufficient energy deficit to contribute to pump dysfunction. Second, the basis for the energy deficit is not clear. To address these questions, mice were fed a high fat, high sucrose (HFHS) 'Western' diet to recapitulate the MHD phenotype. In isolated beating hearts, we used 31P NMR spectroscopy with magnetization transfer to determine a) the concentrations of high energy phosphates ([ATP], [ADP], [PCr]), b) the free energy of ATP hydrolysis (∆G~ATP), c) the rate of ATP production and d) flux through the creatine kinase (CK) reaction. At the lowest workload, the diastolic pressure-volume relationship was shifted upward in HFHS hearts, indicative of diastolic dysfunction, whereas systolic function was preserved. At this workload, the rate of ATP synthesis was decreased in HFHS hearts, and was associated with decreases in both [PCr] and ∆G~ATP. Higher work demands unmasked the inability of HFHS hearts to increase systolic function and led to a further decrease in ∆G~ATP to a level that is not sufficient to maintain normal function of sarcoplasmic Ca2+-ATPase (SERCA). While [ATP] was preserved at all work demands in HFHS hearts, the progressive increase in [ADP] led to a decrease in ∆G~ATP with increased work demands. Surprisingly, CK flux, CK activity and total creatine were normal in HFHS hearts. These findings differ from dilated cardiomyopathy, in which the energetic deficiency is associated with decreases in CK flux, CK activity and total creatine. Thus, in HFHS-fed mice with MHD there is a distinct metabolic phenotype of the heart characterized by a decrease in ATP production that leads to a functionally-important energetic deficiency and an elevation of [ADP], with preservation of CK flux.

Glucose transporter 4-deficient hearts develop maladaptive hypertrophy in response to physiological or pathological stresses.

Pathological cardiac hypertrophy may be associated with reduced expression of glucose transporter 4 (GLUT4) in contrast to exercise-induced cardiac hypertrophy, where GLUT4 levels are increased. However, mice with cardiac-specific deletion of GLUT4 (G4H-/-) have normal cardiac function in the unstressed state. This study tested the hypothesis that cardiac GLUT4 is required for myocardial adaptations to hemodynamic demands. G4H-/- and control littermates were subjected to either a pathological model of left ventricular pressure overload [transverse aortic constriction (TAC)] or a physiological model of endurance exercise (swim training). As predicted after TAC, G4H-/- mice developed significantly greater hypertrophy and more severe contractile dysfunction. Somewhat surprisingly, after exercise training, G4H-/- mice developed increased fibrosis and apoptosis that was associated with dephosphorylation of the prosurvival kinase Akt in concert with an increase in protein levels of the upstream phosphatase protein phosphatase 2A (PP2A). Exercise has been shown to decrease levels of ceramide; G4H-/- hearts failed to decrease myocardial ceramide in response to exercise. Furthermore, G4H-/- hearts have reduced levels of the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1, lower carnitine palmitoyl-transferase activity, and reduced hydroxyacyl-CoA dehydrogenase activity. These basal changes may also contribute to the impaired ability of G4H-/- hearts to adapt to hemodynamic stresses. In conclusion, GLUT4 is required for the maintenance of cardiac structure and function in response to physiological or pathological processes that increase energy demands, in part through secondary changes in mitochondrial metabolism and cellular stress survival pathways such as Akt.NEW & NOTEWORTHY Glucose transporter 4 (GLUT4) is required for myocardial adaptations to exercise, and its absence accelerates heart dysfunction after pressure overload. The requirement for GLUT4 may extend beyond glucose uptake to include defects in mitochondrial metabolism and survival signaling pathways that develop in its absence. Therefore, GLUT4 is critical for responses to hemodynamic stresses.

High fat, high sucrose diet causes cardiac mitochondrial dysfunction due in part to oxidative post-translational modification of mitochondrial complex II.

BACKGROUND: Diet-induced obesity leads to metabolic heart disease (MHD) characterized by increased oxidative stress that may cause oxidative post-translational modifications (OPTM) of cardiac mitochondrial proteins. The functional consequences of OPTM of cardiac mitochondrial proteins in MHD are unknown. Our objective was to determine whether cardiac mitochondrial dysfunction in MHD due to diet-induced obesity is associated with cysteine OPTM. METHODS AND RESULTS: Male C57BL/6J mice were fed either a high-fat, high-sucrose (HFHS) or control diet for 8months. Cardiac mitochondria from HFHS-fed mice (vs. control diet) had an increased rate of H2O2 production, a decreased GSH/GSSG ratio, a decreased rate of complex II substrate-driven ATP synthesis and decreased complex II activity. Complex II substrate-driven ATP synthesis and complex II activity were partially restored ex-vivo by reducing conditions. A biotin switch assay showed that HFHS feeding increased cysteine OPTM in complex II subunits A (SDHA) and B (SDHB). Using iodo-TMT multiplex tags we found that HFHS feeding is associated with reversible oxidation of cysteines 89 and 231 in SDHA, and 100, 103 and 115 in SDHB. CONCLUSIONS: MHD due to consumption of a HFHS "Western" diet causes increased H2O2 production and oxidative stress in cardiac mitochondria associated with decreased ATP synthesis and decreased complex II activity. Impaired complex II activity and ATP production are associated with reversible cysteine OPTM of complex II. Possible sites of reversible cysteine OPTM in SDHA and SDHB were identified by iodo-TMT tag labeling. Mitochondrial ROS may contribute to the pathophysiology of MHD by impairing the function of complex II. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".

Mitochondrial remodeling in mice with cardiomyocyte-specific lipid overload.

BACKGROUND: Obesity leads to metabolic heart disease (MHD) that is associated with a pathologic increase in myocardial fatty acid (FA) uptake and impairment of mitochondrial function. The mechanism of mitochondrial dysfunction in MHD, which results in oxidant production and decreased energetics, is poorly understood but may be related to excess FAs. Determining the effects of cardiac FA excess on mitochondria can be hindered by the systemic sequelae of obesity. Mice with cardiomyocyte-specific overexpression of the fatty acid transport protein FATP1 have increased cardiomyocyte FA uptake and develop MHD in the absence of systemic lipotoxicity, obesity or diabetes. We utilized this model to assess 1) the effect of cardiomyocyte lipid accumulation on mitochondrial structure and energetic function and 2) the role of lipid-driven transcriptional regulation, signaling, toxic metabolite accumulation, and mitochondrial oxidative stress in lipid-induced MHD. METHODS: Cardiac lipid species, lipid-dependent signaling, and mitochondrial structure/function were examined from FATP1 mice. Cardiac structure and function were assessed in mice overexpressing both FATP1 and mitochondrial-targeted catalase. RESULTS: FATP1 hearts exhibited a net increase (+12%) in diacylglycerol, with increases in several very long-chain diacylglycerol species (+160-212%, p<0.001) and no change in ceramide, sphingomyelin, or acylcarnitine content. This was associated with an increase in phosphorylation of PKCα and PKCδ, and a decrease in phosphorylation of AKT and expression of CREB, PGC1α, PPARα and the mitochondrial fusion genes MFN1, MFN2 and OPA1. FATP1 overexpression also led to marked decreases in mitochondrial size (-49%, p<0.01), complex II-driven respiration (-28.6%, p<0.05), activity of isolated complex II (-62%, p=0.05), and expression of complex II subunit B (SDHB) (-60% and -31%, p<0.01) in the absence of change in ATP synthesis. Hydrogen peroxide production was not increased in FATP1 mitochondria, and cardiac hypertrophy and diastolic dysfunction were not attenuated by overexpression of catalase in mitochondria in FATP1 mice. CONCLUSIONS: Excessive delivery of FAs to the cardiac myocyte in the absence of systemic disorders leads to activation of lipid-driven signaling and remodeling of mitochondrial structure and function.

The polyphenols resveratrol and S17834 prevent the structural and functional sequelae of diet-induced metabolic heart disease in mice.

BACKGROUND: Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes mellitus and chronic hemodynamic overload. METHODS AND RESULTS: We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or resveratrol for up to 8 months. HFHS diet-fed mice developed progressive left ventricular hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS diet-fed mice, there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol adduction. HFHS diet-fed mice also exhibited increases in plasma fasting glucose, insulin, and homeostasis model assessment of insulin resistance indicative of insulin resistance. Treatment with S17834 or resveratrol prevented left ventricular hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyperinsulinemia and increased plasma adiponectin. CONCLUSIONS: Resveratrol and S17834 administered concurrently with a HFHS diet prevent the development of left ventricular hypertrophy, interstitial fibrosis, and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols, including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance, and increased plasma adiponectin. The polyphenols resveratrol and S17834 may be of value in the prevention of diet-induced metabolic heart disease.

Hemodynamic testing to guide vasodilator therapy in cardiac amyloidosis.

Cardiac amyloidosis is a restrictive cardiomyopathy for which diuretics are frequently used, but vasodilators have classically been relatively contraindicated due to side effects of hypotension. In the setting of decompensated heart failure, this may not be the case. We report a man with advanced cardiac amyloidosis who presented to the hospital with decompensated heart failure, in part, due to elevated systemic vascular resistance. Through the use of invasive hemodynamic testing, we were able to demonstrate an increase in cardiac output in response to a nitroprusside challenge. In turn, the patient had an improvement in his symptoms and was sent home on afterload reducing medications. This discerns a subpopulation of cardiac amyloidosis patients in decompensated heart failure who benefit from medications that reduce systemic vascular resistance, and can benefit from hemodynamic testing, especially when diuretics fail to control symptoms. Learning objective: Medications that cause peripheral vasodilation are standard therapy for patients with reduced ejection fraction, however, they are seldom used for patients with cardiac amyloidosis due to adverse effects. In some cases, there may be value in using hemodynamic measurements in patients with advanced cardiac amyloidosis to guide management as some patients may have hemodynamics that resemble those of systolic heart failure. This may offer a novel approach to symptomatic treatment of advanced cardiac amyloidosis.

SGLT2 inhibitor ertugliflozin decreases elevated intracellular sodium, and improves energetics and contractile function in diabetic cardiomyopathy.

BACKGROUND: Elevated myocardial intracellular sodium ([Na+]i) was shown to decrease mitochondrial calcium ([Ca2+]MITO) via mitochondrial sodium/calcium exchanger (NCXMITO), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na+]i in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na+]i. METHODS: Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by 31P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCXMITO inhibitor. Myocardial [Na+]i was measured by 23Na NMR spectroscopy. RESULTS: HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na+]i was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na+]i to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts. CONCLUSIONS: Elevated myocardial [Na+]i contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na+]i and/or NCXMITO may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na+]i.

Genetic loss of insulin receptors worsens cardiac efficiency in diabetes.

AIMS: To determine the contribution of insulin signaling versus systemic metabolism to metabolic and mitochondrial alterations in type 1 diabetic hearts and test the hypothesis that antecedent mitochondrial dysfunction contributes to impaired cardiac efficiency (CE) in diabetes. METHODS AND RESULTS: Control mice (WT) and mice with cardiomyocyte-restricted deletion of insulin receptors (CIRKO) were rendered diabetic with streptozotocin (WT-STZ and CIRKO-STZ, respectively), non-diabetic controls received vehicle (citrate buffer). Cardiac function was determined by echocardiography; myocardial metabolism, oxygen consumption (MVO(2)) and CE were determined in isolated perfused hearts; mitochondrial function was determined in permeabilized cardiac fibers and mitochondrial proteomics by liquid chromatography mass spectrometry. Pyruvate supported respiration and ATP synthesis were equivalently reduced by diabetes and genotype, with synergistic impairment in ATP synthesis in CIRKO-STZ. In contrast, fatty acid delivery and utilization was increased by diabetes irrespective of genotype, but not in non-diabetic CIRKO. Diabetes and genotype synergistically increased MVO(2) in CIRKO-STZ, leading to reduced CE. Irrespective of diabetes, genotype impaired ATP/O ratios in mitochondria exposed to palmitoyl carnitine, consistent with mitochondrial uncoupling. Proteomics revealed reduced content of fatty acid oxidation proteins in CIRKO mitochondria, which were induced by diabetes, whereas tricarboxylic acid cycle and oxidative phosphorylation proteins were reduced both in CIRKO mitochondria and by diabetes. CONCLUSIONS: Deficient insulin signaling and diabetes mediate distinct effects on cardiac mitochondria. Antecedent loss of insulin signaling markedly impairs CE when diabetes is induced, via mechanisms that may be secondary to mitochondrial uncoupling and increased FA utilization.

Mitochondrial transporter ATP binding cassette mitochondrial erythroid is a novel gene required for cardiac recovery after ischemia/reperfusion.

BACKGROUND: Oxidative stress and mitochondrial dysfunction are central mediators of cardiac dysfunction after ischemia/reperfusion. ATP binding cassette mitochondrial erythroid (ABC-me; ABCB10; mABC2) is a mitochondrial transporter highly induced during erythroid differentiation and predominantly expressed in bone marrow, liver, and heart. Until now, ABC-me function in heart was unknown. Several lines of evidence demonstrate that the yeast ortholog of ABC-me protects against increased oxidative stress. Therefore, ABC-me is a potential modulator of the outcome of ischemia/reperfusion in the heart. METHODS AND RESULTS: Mice harboring 1 functional allele of ABC-me (ABC-me(+/-)) were generated by replacing ABC-me exons 2 and 3 with a neomycin resistance cassette. Cardiac function was assessed with Langendorff perfusion and echocardiography. Under basal conditions, ABC-me(+/-) mice had normal heart structure, hemodynamic function, mitochondrial respiration, and oxidative status. However, after ischemia/reperfusion, the recovery of hemodynamic function was reduced by 50% in ABC-me(+/-) hearts as a result of impairments in both systolic and diastolic function. This reduction was associated with impaired mitochondrial bioenergetic function and with oxidative damage to both mitochondrial lipids and sarcoplasmic reticulum calcium ATPase after reperfusion. Treatment of ABC-me(+/-) hearts with the superoxide dismutase/catalase mimetic EUK-207 prevented oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and restored mitochondrial and cardiac function to wild-type levels after reperfusion. CONCLUSIONS: Inactivation of 1 allele of ABC-me increases the susceptibility to oxidative stress induced by ischemia/reperfusion, leading to increased oxidative damage to mitochondria and sarcoplasmic reticulum calcium ATPase and to impaired functional recovery. Thus, ABC-me is a novel gene that determines the ability to tolerate cardiac ischemia/reperfusion.

Ivabradine in the Management of COVID-19-related Cardiovascular Complications: A Perspective.

Besides acute respiratory distress syndrome, acute cardiac injury is a major complication in severe coronavirus disease 2019 (COVID-19) and is associated with a poor clinical outcome. Acute cardiac injury with COVID-19 can be of various etiologies, including myocardial ischemia or infarction and myocarditis, and may compromise cardiac function, resulting in acute heart failure or cardiogenic shock. Systemic inflammatory response increases heart rate (HR), which disrupts the myocardial oxygen supply/demand balance and worsens cardiac energy efficiency, thus further deteriorating the cardiac performance of the injured myocardium. In fact, the combination of elevated resting HR and markers of inflammation synergistically predicts adverse cardiovascular prognosis. Thus, targeted HR reduction may potentially be of benefit in cardiovascular pathologies associated with COVID-19. Ivabradine is a drug that selectively reduces HR via If current inhibition in the sinoatrial node without a negative effect on inotropy. Besides selective HR reduction, ivabradine was found to exert various beneficial pleiotropic effects, either HR-dependent or HR-independent, including anti-inflammatory, anti-atherosclerotic, anti-oxidant and antiproliferative actions and the attenuation of endothelial dysfunction and neurohumoral activation. Cardioprotection by ivabradine has already been indicated in cardiovascular pathologies that are prevalent with COVID-19, including myocarditis, acute coronary syndrome, cardiogenic shock or cardiac dysautonomia. Here, we suggest that ivabradine may be beneficial in the management of COVID-19- related cardiovascular complications.

Novel Small-Molecule Troponin Activator Increases Cardiac Contractile Function Without Negative Impact on Energetics.

BACKGROUND: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents-myotropes-activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. METHODS: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by 31P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. RESULTS: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG~ATP) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG~ATP. CONCLUSIONS: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Effects of Sodium-Glucose Linked Transporter 2 Inhibition With Ertugliflozin on Mitochondrial Function, Energetics, and Metabolic Gene Expression in the Presence and Absence of Diabetes Mellitus in Mice.

Background Inhibitors of the sodium-glucose linked transporter 2 improve cardiovascular outcomes in patients with or without type 2 diabetes mellitus, but the effects on cardiac energetics and mitochondrial function are unknown. We assessed the effects of sodium-glucose linked transporter 2 inhibition on mitochondrial function, high-energy phosphates, and genes encoding mitochondrial proteins in hearts of mice with and without diet-induced diabetic cardiomyopathy. Methods and Results Mice fed a control diet or a high-fat, high-sucrose diet received ertugliflozin mixed with the diet (0.5 mg/g of diet) for 4 months. Isolated mitochondria were assessed for functional capacity. High-energy phosphates were assessed by 31P nuclear magnetic resonance spectroscopy concurrently with contractile performance in isolated beating hearts. The high-fat, high-sucrose diet caused myocardial hypertrophy, diastolic dysfunction, mitochondrial dysfunction, and impaired energetic response, all of which were prevented by ertugliflozin. With both diets, ertugliflozin caused supernormalization of contractile reserve, as measured by rate×pressure product at high work demand. Likewise, the myocardial gene sets most enriched by ertugliflozin were for oxidative phosphorylation and fatty acid metabolism, both of which were enriched independent of diet. Conclusions Ertugliflozin not only prevented high-fat, high-sucrose-induced pathological cardiac remodeling, but improved contractile reserve and induced the expression of oxidative phosphorylation and fatty acid metabolism gene sets independent of diabetic status. These effects of sodium-glucose linked transporter 2 inhibition on cardiac energetics and metabolism may contribute to improved structure and function in cardiac diseases associated with mitochondrial dysfunction, such as heart failure.

Aberrant activation of AMP-activated protein kinase remodels metabolic network in favor of cardiac glycogen storage.

AMP-activated protein kinase (AMPK) responds to impaired cellular energy status by stimulating substrate metabolism for ATP generation. Mutation of the gamma2 regulatory subunit of AMPK in humans renders the kinase insensitive to energy status and causes glycogen storage cardiomyopathy via unknown mechanisms. Using transgenic mice expressing one of the mutant gamma2 subunits (N488I) in the heart, we found that aberrant high activity of AMPK in the absence of energy deficit caused extensive remodeling of the substrate metabolism pathways to accommodate increases in both glucose uptake and fatty acid oxidation in the hearts of gamma2 mutant mice via distinct, yet synergistic mechanisms resulting in selective fuel storage as glycogen. Increased glucose entry in the gamma2 mutant mouse hearts was directed through the remodeled metabolic network toward glycogen synthesis and, at a substantially higher glycogen level, recycled through the glycogen pool to enter glycolysis. Thus, the metabolic consequences of chronic activation of AMPK in the absence of energy deficiency is distinct from those previously reported during stress conditions. These findings are of particular importance in considering AMPK as a target for the treatment of metabolic diseases.

Differential Effects of Sacubitril/Valsartan on Diastolic Function in Mice With Obesity-Related Metabolic Heart Disease.

Mice with obesity and metabolic heart disease (MHD) due to a high-fat, high-sucrose diet were treated with placebo, a clinically relevant dose of sacubitril (SAC)/valsartan (VAL), or an equivalent dose of VAL for 4 months. There were striking differences between SAC/VAL and VAL with regard to: 1) diastolic dysfunction; 2) interstitial fibrosis; and to a lesser degree; 3) oxidative stress-all of which were more favorably affected by SAC/VAL. SAC/VAL and VAL similarly attenuated myocardial hypertrophy and improved myocardial energetics. In mice with obesity-related MHD, neprilysin inhibition exerts favorable effects on diastolic function.

A novel tracer for in vivo optical imaging of fatty acid metabolism in the heart and brown adipose tissue.

Multiplexed imaging is essential for the evaluation of substrate utilization in metabolically active organs, such as the heart and brown adipose tissue (BAT), where substrate preference changes in pathophysiologic states. Optical imaging provides a useful platform because of its low cost, high throughput and intrinsic ability to perform composite readouts. However, the paucity of probes available for in vivo use has limited optical methods to image substrate metabolism. Here, we present a novel near-infrared (NIR) free fatty acid (FFA) tracer suitable for in vivo imaging of deep tissues such as the heart. Using click chemistry, Alexa Fluor 647 DIBO Alkyne was conjugated to palmitic acid. Mice injected with 0.05 nmol/g bodyweight of the conjugate (AlexaFFA) were subjected to conditions known to increase FFA uptake in the heart (fasting) and BAT [cold exposure and injection with the ß3 adrenergic agonist CL 316, 243(CL)]. Organs were subsequently imaged both ex vivo and in vivo to quantify AlexaFFA uptake. The blood kinetics of AlexaFFA followed a two-compartment model with an initial fast compartment half-life of 0.14 h and a subsequent slow compartment half-life of 5.2 h, consistent with reversible protein binding. Ex vivo fluorescence imaging after overnight cold exposure and fasting produced a significant increase in AlexaFFA uptake in the heart (58 ± 12%) and BAT (278 ± 19%) compared to warm/fed animals. In vivo imaging of the heart and BAT after exposure to CL and fasting showed a significant increase in AlexaFFA uptake in the heart (48 ± 20%) and BAT (40 ± 10%) compared to saline-injected/fed mice. We present a novel near-infrared FFA tracer, AlexaFFA, that is suitable for in vivo quantification of FFA metabolism and can be applied in the context of a low cost, high throughput, and multiplexed optical imaging platform.

Use of Ventilatory Efficiency Slope as a Marker for Increased Mortality in Wild-Type Transthyretin Cardiac Amyloidosis.

Wild-type transthyretin amyloidosis (ATTRwt) results in an infiltrative cardiomyopathy often culminating in symptomatic heart failure. The use of cardiopulmonary exercise testing (CPET) in determining outcomes in ATTRwt cardiac amyloidosis is unknown. Given the emergence of novel therapies to treat transthyretin amyloidosis, we sought to investigate the utility of CPET on outcomes in patients with ATTRwt cardiomyopathy. Fifty-six patients, with biopsy and immunohistochemically proved ATTRwt, were enrolled between 2005 and 2015, as part of an NIH ATTRwt substudy at the Boston University Amyloidosis Center. Patients were prospectively studied, which included laboratory tests, electrocardiogram, echocardiography, in addition to CPET. In this cohort of ATTRwt patients who performed CPET were elderly, all were male, and predominantly white (69.9%). The overall median survival was 59.01 months (95% confidence interval [CI] 49.29 to 88.69). By multivariate analysis, C-reactive protein (CRP; hazard ratio [HR] 1.10 [1.03 to 1.18]), decreased sodium (HR 0.75 [0.58 to 0.97]), creatinine (HR 7.48 [2.44 to 22.98]) and VE/VCO2 (HR 1.10 [1.05 to 1.16]) were significant risk factors for mortality (p <0.05). Peak VO2 was insignificant by both univariate and multivariate analyses. ATTRwt patients with VE/VCO2 >40 had a worse median survival of 38.54 months (95% CI 32.63 to 51.47) versus 88.69 months (95% CI 56.26 to 89.49) than patients with VE/VCO2 slope ≤40. Receiver-operating characteristic curve showed that the combination of VE/VCO2, CRP, sodium, and creatinine (Area under the ROC Curve [AUC], 0.89) predicted 1-year mortality in ATTRwt cardiac amyloidosis. In conclusion, increased VE/VCO2, in combination with CRP, sodium, and creatinine, may identify patients at increased risk of death in ATTRwt cardiomyopathy. VE/VCO2 might have a role in objectively assessing therapeutic response in ATTRwt cardiac amyloidosis.