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Background In heart failure the myocardium becomes insulin resistant which negatively influences cardiac energy metabolism and function, while increasing cardiac insulin signalling improves cardiac function and prevents adverse remodelling in the failing heart. Glucagon’s action on cardiac glucose and lipid homeostasis counteract that of insulin’s action. We hypothesised that pharmacological antagonism of myocardial glucagon action, using a human monoclonal antibody (mAb A) against glucagon receptor (GCGR), a G-protein coupled receptor, will enhance insulin sensitivity and improve cardiac energy metabolism and function post myocardial infarction (MI). Methods Male C57BL/6 mice were subjected to a permanent left anterior descending coronary artery ligation to induce MI, following which they received either saline or mAb A (4 mg kg−1 week−1 starting at 1 week post-MI) for 3 weeks. Results Echocardiographic assessment at 4 weeks post-MI showed that mAb A treatment improved % ejection fraction (40.0 ± 2.3% vs 30.7 ± 1.7% in vehicle-treated MI heart, p

Background: During the development of heart failure cardiac fuel metabolism switches from predominantly fatty acid oxidation (FAO) to increased reliance on glucose, especially glycolysis. Mechanisms responsible for the switch are poorly understood but appear to be coupled with impaired mitochondrial function. We recently demonstrated that increased glucose metabolism is required for cardiomyocytes growth during pathological remodeling. Hypothesis: Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) in hypertrophied hearts suppresses ATP synthesis and shifts cardiac metabolism from fatty acid oxidation towards glucose metabolism. Methods and Results: We report that ATPIF1 expression is upregulated in cardiomyocytes and mouse hearts undergoing pathological hypertrophy. Using genetic models of ATPIF1 gain- and loss-of-function in cardiomyocytes and in mouse hearts,we find that upregulation of ATPIF1 in cardiac hypertrophy inhibits ATP synthesis. Furthermore, quantitative analysis of chemical crosslinking by mass spectrometry revealed that increased expression of ATPIF1 promoted the formation of F o F 1 -ATP synthase nonproductive tetramer. Impairment of F o F 1 -ATP synthase function in respiring mitochondria increasedROS generation resulting in transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the switch to glycolysis in pressure overload induced cardiac hypertrophy. Conclusions: We show that upregulation of ATPIF1 drives glucose metabolism at the expense of energy supply during the pathological growth of cardiomyocytes. Our study proposes a central role of ATP synthase in toggling anabolic and catabolic metabolism during pathological remodeling, illustrating a new concept for metabolic reprogramming of the heart.

Aging-associated diseases, including cardiac dysfunction, are increasingly common in the population. However, the mechanisms of physiologic aging in general, and cardiac aging in particular, remain poorly understood. While effective medical interventions are available for some kinds of heart failure, one age-related impairment, diastolic dysfunction in Heart Failure with Preserved Ejection Fraction (HFpEF) is lacking a clinically effective treatment. Using the model of naturally aging mice and rats, we show direct evidence of increased proton leak in the aged heart mitochondria. Moreover, we identified ANT1 as mediating the increased proton permeability of old cardiomyocytes. Most importantly, the tetra-peptide drug SS-31 (elamipretide) prevents age-related excess proton entry, decreases the mitochondrial flash activity and mitochondrial permeability transition pore (mPTP) opening and rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome. Our results uncover a novel mechanism of age-related cardiac dysfunction and elucidate how SS-31 is able to reverse this clinically important complication of cardiac aging. Significance Aging is the greatest risk factor for cardiac dysfunction, including Heart Failure with Preserved Ejection Fraction (HFpEF). Unfortunately, the mechanisms of cardiac aging remain elusive, and there are no effective pharmacologic therapies for HFpEF. Here, we show direct evidence of increased proton leak in aged cardiac mitochondria and have identified ANT1 as mediating the increased proton permeability of old cardiomyocytes. Moreover, the mitochondrial-targeted tetra-peptide SS-31 (elamipretide) prevents the age-related excess proton entry and rejuvenates mitochondrial function by direct association with ANT1 and the mitochondrial ATP synthasome, resulting in alleviation of diastolic dysfunction in old mice. Our results unmask a novel mechanism of cardiac aging and elucidate how SS-31 reverses this clinically important complication of aging.

In hypertrophic hearts, autophagic flux insufficiency is recognized as a key pathology leading to maladaptive cardiac remodeling and heart failure. This study aimed to illuminate the cardioprotective role and mechanisms of a new myokine and adipokine, irisin, in cardiac hypertrophy and remodeling. Adult male wild-type, mouse-FNDC5 (irisin-precursor)-knockout and FNDC5 transgenic mice received 4 weeks of transverse aortic constriction (TAC) alone or combined with intraperitoneal injection of chloroquine diphosphate (CQ). Endogenous FNDC5 ablation aggravated and exogenous FNDC5 overexpression attenuated the TAC-induced hypertrophic damage in the heart, which was comparable to the protection of irisin against cardiomyocyte hypertrophy induced by angiotensin II (Ang II) or phenylephrine (PE). Accumulated autophagosome and impaired autophagy flux occurred in the TAC-treated myocardium and Ang II- or PE-insulted cardiomyocytes. Irisin deficiency caused reduced autophagy and aggravated autophagy flux failure, whereas irisin overexpression or supplementation induced protective autophagy and improved autophagy flux, which were reversed by autophagy inhibitors Atg5 siRNA, 3-MA and CQ. Irisin boosted the activity of only AMPK but not Akt and MAPK family members in hypertrophic hearts and cultured cardiomyocytes and further activated ULK1 at Ser555 but not Ser757 and did not affect the mTOR-S6K axis. Blockage of AMPK and ULK1 with compund C and SBI-0206965, respectively, both abrogated irisin's protection against cardiomyocyte hypertrophic injury and reversed its induction of both autophagy and autophagy flux. Our results suggest that irisin protects against pressure overload-induced cardiac hypertrophy by inducing protective autophagy and autophagy flux via activating AMPK-ULK1 signaling.

Phosphatidylinositide 3-kinase (PI3K) signaling plays a critical role in maintaining normal cardiac structure and function. PI3Kα and PI3Kγ are the dominant cardiac isoforms and have both adaptive and maladaptive roles in heart disease. Broad spectrum PI3K inhibitors are emerging as potential new chemotherapeutic agents which may have deleterious long-term effects on the heart. We created a double mutant (PI3KDM) model by crossing p110γ −/− (PI3KγKO) with cardiac-specific PI3KαDN mice and studied cardiac structure and function at 1-year of age. Pressure–volume loop analysis and echocardiographic assessment showed PI3KDM mice developed marked impairment in systolic function while the wildtype, PI3KαDN, and PI3KγKO mice maintained normal systolic and diastolic function at 1-year of age. The PI3KDM hearts displayed increased expression of disease markers, increased myocardial fibrosis and matrix metalloproteinase (MMP) activity, depolymerization of intracellular F-actin, loss of phospho(threonine-308)-Akt, and normalization of phospho-Erk1/2 signaling. Dual loss of PI3Kα and PI3Kγ isoforms results in an age-dependent cardiomyopathy implying that long-term exposure to pan-PI3K inhibitors may lead to severe cardiotoxicity.

The heart is an excitable organ that undergoes spontaneous force generation and relaxation cycles driven by excitation–contraction (EC) coupling. A fraction of the oscillating cytosolic Ca 2 + during each heartbeat is taken up by mitochondria to stimulate mitochondrial metabolism, the major source of energy in the heart. Whether the mitochondrial metabolism is regulated individually during EC coupling and whether this heterogeneous regulation bears any physiological or pathological relevance have not been studied. Here, we developed a novel approach to determine the regulation of individual mitochondrial metabolism during cardiac EC coupling. Through monitoring superoxide flashes, which are stochastic and bursting superoxide production events arising from increased metabolism in individual mitochondria, we found that EC coupling stimulated the metabolism in individual mitochondria as indicated by significantly increased superoxide flash activity during electrical stimulation of the cultured intact myocytes or perfused heart. Mechanistically, cytosolic calcium transients promoted individual mitochondria to take up calcium via mitochondrial calcium uniporter, which subsequently triggered transient opening of the permeability transition pore and stimulated metabolism and bursting superoxide flash in that mitochondrion. The bursting superoxide, in turn, promoted local calcium release. In the early stage of heart failure, EC coupling regulation of superoxide flashes was compromised. This study highlights the heterogeneity in the regulation of cardiac mitochondrial metabolism, which may contribute to local redox signaling.

Angiotensin-converting enzyme 2 (ACE2) is a monocarboxypeptidase that metabolizes several vasoactive peptides, including angiotensin II (Ang-II; a vasoconstrictive/proliferative peptide), which it converts to Ang-(1–7). Ang-(1–7) acts through the Mas receptor to mediate vasodilatory/antiproliferative actions. The renin–angiotensin system involving the ACE–Ang-II–Ang-II type-1 receptor (AT1R) axis is antagonized by the ACE2–Ang-(1–7)–Mas receptor axis. Loss of ACE2 enhances adverse remodeling and susceptibility to pressure and volume overload. Human recombinant ACE2 may act to suppress myocardial hypertrophy, fibrosis, inflammation, and diastolic dysfunction in heart failure patients. The ACE2–Ang-(1–7)–Mas axis may present a new therapeutic target for the treatment of heart failure patients. This review is mainly focused on the analysis of ACE2, including its influence and potentially positive effects, as well as the potential use of human recombinant ACE2 as a novel therapy for the treatment cardiovascular diseases, such as hypertension and heart failure.

Defective intracellular calcium (Ca2 +) handling is implicated in the pathogenesis of heart failure. Novel approaches targeting both cardiac Ca2 + release and reuptake processes, such as S100A1, have the potential to rescue the function of failing cardiac myocytes. Here, we show that two members of the S100 Ca2 + binding protein family, S100A2 and S100A6 that share high sequence homology, differentially influence cardiac Ca2 + handling and contractility. Cardiac gene expression of S100A2 significantly enhanced both contractile and relaxation performance of rodent and canine cardiac myocytes, mimicking the functional effects of its cardiac homologue, S100A1. To interrogate mechanism, Ca2 + spark frequency, a measure of the gating of the ryanodine receptor Ca2 + release channel, was found to be significantly increased by S100A2. Therapeutic testing showed that S100A2 rescued the contractile defects of failing cardiac myocytes. In contrast, cardiac expression of S100A6 had no significant effects on contractility or Ca2 + handling. These data reveal novel differential effects of S100 proteins on cardiac myocyte performance that may be useful in application to diseased cardiac muscle.

Rationale Diastolic dysfunction is a common feature in many heart failure patients with preserved ejection fraction and has been associated with altered myocardial metabolism in hypertensive and diabetic patients. Therefore, metabolic interventions to improve diastolic function are warranted. In mice with a germline cardiac-specific deletion of acetyl CoA carboxylase 2 (ACC2), systolic dysfunction induced by pressure-overload was prevented by maintaining cardiac fatty acid oxidation (FAO). However, it has not been evaluated whether this strategy would prevent the development of diastolic dysfunction in the adult heart. Objective To test the hypothesis that augmenting cardiac FAO is protective against angiotensin II (AngII)-induced diastolic dysfunction in an adult mouse heart. Methods and results We generated a mouse model to induce cardiac-specific deletion of ACC2 in adult mice. Tamoxifen treatment (20 mg/kg/day for 5 days) was sufficient to delete ACC2 protein and increase cardiac FAO by 50% in ACC2 flox/flox-MerCreMer+ mice (iKO). After 4 weeks of AngII (1.1 mg/kg/day), delivered by osmotic mini-pumps, iKO mice showed normalized E/E′ and E′/A′ ratios compared to AngII treated controls (CON). The prevention of diastolic dysfunction in iKO-AngII was accompanied by maintained FAO and reduced glycolysis and anaplerosis. Furthermore, iKO-AngII hearts had a ~ 50% attenuation of cardiac hypertrophy and fibrosis compared to CON. In addition, maintenance of FAO in iKO hearts suppressed AngII-associated increases in oxidative stress and sustained mitochondrial respiratory complex activities. Conclusion These data demonstrate that impaired FAO is a contributor to the development of diastolic dysfunction induced by AngII. Maintenance of FAO in this model leads to an attenuation of hypertrophy, reduces fibrosis, suppresses increases in oxidative stress, and maintains mitochondrial function. Therefore, targeting mitochondrial FAO is a promising therapeutic strategy for the treatment of diastolic dysfunction.

Rationale: The classic phagocyte nicotinamide adenine dinucleotide phosphate oxidase (gp91 phox or Nox2) is expressed in the heart. Nox2 activation requires membrane translocation of the p47 phox subunit and is linked to heart failure. We hypothesized that loss of p47 phox subunit will result in decreased reactive oxygen species production and resistance to heart failure. Objective: To define the role of p47 phox in pressure overload–induced biomechanical stress. Methods and Results: Eight-week-old male p47 phox null (p47 phox knockout [KO]), Nox2 null (Nox2KO), and wild-type mice were subjected to transverse aortic constriction–induced pressure overload. Contrary to our hypothesis, p47 phox KO mice showed markedly worsened systolic dysfunction in response to pressure overload at 5 and 9 weeks after transverse aortic constriction compared with wild-type–transverse aortic constriction mice. We found that biomechanical stress upregulated N-cadherin and β-catenin in p47 phox KO hearts but disrupted the actin filament cytoskeleton and reduced phosphorylation of focal adhesion kinase. p47 phox interacts with cytosolic cortactin by coimmunoprecipitation and double immunofluorescence staining in murine and human hearts and translocated to the membrane on biomechanical stress where cortactin interacted with N-cadherin, resulting in adaptive cytoskeletal remodeling. However, p47 phox KO hearts showed impaired interaction of cortactin with N-cadherin, resulting in loss of biomechanical stress–induced actin polymerization and cytoskeletal remodeling. In contrast, Nox2 does not interact with cortactin, and Nox2-deficient hearts were protected from pressure overload–induced adverse myocardial and intracellular cytoskeletal remodeling. Conclusions: We showed a novel role of p47 phox subunit beyond and independent of nicotinamide adenine dinucleotide phosphate oxidase activity as a regulator of cortactin and adaptive cytoskeletal remodeling, leading to a paradoxically enhanced susceptibility to biomechanical stress and heart failure.

Iron-overload cardiomyopathy is a prevalent cause of heart failure on a world-wide basis and is a major cause of mortality and morbidity in patients with secondary iron-overload and genetic hemochromatosis. We investigated the therapeutic effects of resveratrol in acquired and genetic models of iron-overload cardiomyopathy. Murine iron-overload models showed cardiac iron-overload, increased oxidative stress, altered Ca2+ homeostasis and myocardial fibrosis resulting in heart disease. Iron-overload increased nuclear and acetylated levels of FOXO1 with corresponding inverse changes in SIRT1 levels in the heart corrected by resveratrol therapy. Resveratrol, reduced the pathological remodeling and improved cardiac function in murine models of acquired and genetic iron-overload at varying stages of iron-overload. Echocardiography and hemodynamic analysis revealed a complete normalization of iron-overload mediated diastolic and systolic dysfunction in response to resveratrol therapy. Myocardial SERCA2a levels were reduced in iron-overloaded hearts and resveratrol therapy restored SERCA2a levels and corrected altered Ca2+ homeostasis. Iron-mediated pro-oxidant and pro-fibrotic effects in human and murine cardiomyocytes and cardiofibroblasts were suppressed by resveratrol which correlated with reduction in iron-induced myocardial oxidative stress and myocardial fibrosis. Resveratrol represents a clinically and economically feasible therapeutic intervention to reduce the global burden from iron-overload cardiomyopathy at early and chronic stages of iron-overload.

A novel angiotensin-converting enzyme (ACE) homolog, named ACE2, is a monocarboxypeptidase which metabolizes several peptides. ACE2 degrades Angiotensin (Ang) II, a peptide with vasoconstrictive/proliferative effects, to generate Ang-(1-7), which acting through its receptor Mas exerts vasodilatory/anti-proliferative actions. In addition, as ACE2 is a multifunctional enzyme and its actions on other vasoactive peptides can also contribute to its vasoactive effects including the apelin-13 and apelin-17 peptides. The discovery of ACE2 corroborates the establishment of two counter-regulatory arms within the renin-angiotensin system. The first one is formed by the classical pathway involving the ACE-Ang II-AT(1) receptor axis and the second arm is constituted by the ACE2-Ang 1-7/Mas receptor axis. Loss of ACE2 enhances the adverse pathological remodeling susceptibility to pressure-overload and myocardial infarction. ACE2 is also a negative regulator of Ang II-induced myocardial hypertrophy, fibrosis, and diastolic dysfunction. The ACE2-Ang 1-7/Mas axis may represent new possibilities for developing novel therapeutic strategies for the treatment of hypertension and cardiovascular diseases. In this review, we will summarize the biochemical and pathophysiological aspects of ACE2 with a focus on its role in diastolic and systolic heart failure.

Cardiac remodeling involves molecular, cellular, and interstitial changes that manifest clinically as changes in size, shape, and function of the heart after injury or stress stimulation and was initially coined to describe the prominent changes that occur after myocardial infarction (MI) [1]. Adverse ventricular remodeling during the healing phase after acute MI continues to be an important problem that impacts adult cardiology practice. Indeed, coronary artery disease is now the most common cause of heart failure in westernized countries and is rising in prevalence in India and China. Most clinicians and cardiovascular researchers recognize that significant left ventricular (LV) remodeling occurs during infarct healing, and optimal healing is critical for survival with a favorable outcome. Over the last three decades, several laboratories have been actively searching for specific molecular targets that may lead to the development of therapies and strategies to optimize postinfarct healing, prevent adverse remodeling, and improve clinical outcome. However, specific therapy to optimize healing and prevent adverse post-MI remodeling is currently lacking and only suboptimal medical therapy currently exists. Hearts continue to enlarge after MI, and heart failure is a growing burden. At present, ginseng is one of the most extensively used alternative medications throughout the world and has appeared in the pharmacopoeias of several western countries, including the United States and European countries with indications for cardiovascular diseases and other conditions [2]. In this issue, Yin H et al. showed that the active ingredient of the ginseng extract, ginsenoside-Rg1, enhances angiogenesis and reduces adverse ventricular remodeling in a rat model of MI [3]. Ginsenoside-Rg1 is the most prevalent and active constituent of ginseng. Three major targets of adverse myocardial remodeling were the key mediators of the cardioprotective effects seen with ginsenoside-Rg1administration: stimulation of the PI3K/ Akt [and inhibition of p38 mitogen-activated protein kinase (MAPK)] pathway with improvement in post-MI angiogenesis and reduction in myocardial fibrosis (Fig. 1). These beneficial effects resulted in reduced LV dilation and a modest improvement in systolic function based on the assessment of fractional shortening and ejection fraction. PI3K inhibition using LY294002 also reduced the phosphorylation of the p38 MAPK pathway which is known to play a key role in inducing inflammatory cytokines and apoptotic cell death. The PI3K/Akt pathways mediate cardioprotective effects in a wide variety of pathological conditions including important anti-apoptotic effects and modulation of cellular growth and metabolism [4]. However, pharmacological inhibition of PI3K signaling using LY294002 cannot distinguish isoform-specific effects of PI3K signaling which is well established to play a differential role in myocardial ischemic injury [4]. In addition, LY294002 is a potent blocker of voltage-gated K channels in cardiomyocytes [5]. Thus, off-target effects Clinical implications: Yin H et al. Ginsenoside-Rg1 enhances angiogenesis and ameliorates ventricular remodeling in a rat model of myocardial infarction. S. Bodiga :G. Y. Oudit (*) Division of Cardiology, Department of Medicine, Mazankowski Alberta Heart Institute, University of Alberta, Edmonton, Alberta T6G 2S2, Canada e-mail: gavin.oudit@ualberta.ca

Aims Angiotensin-converting enzyme 2 (ACE2) is an important negative regulator of the renin-angiotensin system. Loss of ACE2 enhances the susceptibility to heart disease but the mechanism remains elusive. We hypothesized that ACE2 deficiency activates the NADPH oxidase system in pressure overload-induced heart failure. Methods and results Using the aortic constriction model, we subjected wild-type ( Ace2+/y ), ACE2 knockout (ACE2KO, Ace2−/y ), p47phox knockout (p47phoxKO, p47phox− /−), and ACE2/p47phox double KO mice to pressure overload. We examined changes in peptide levels, NADPH oxidase activity, gene expression, matrix metalloproteinases (MMP) activity, pathological signalling, and heart function. Loss of ACE2 resulted in enhanced susceptibility to biomechanical stress leading to eccentric remodelling, increased pathological hypertrophy, and worsening of systolic performance. Myocardial angiotensin II (Ang II) levels were increased, whereas Ang 1–7 levels were lowered. Activation of Ang II-stimulated signalling pathways in the ACE2-deficient myocardium was associated with increased expression and phosphorylation of p47phox, NADPH oxidase activity, and superoxide generation, leading to enhanced MMP-mediated degradation of the extracellular matrix. Additional loss of p47phox in the ACE2KO mice normalized the increased NADPH oxidase activity, superoxide production, and systolic dysfunction following pressure overload. Ang 1–7 supplementation suppressed the increased NADPH oxidase and rescued the early dilated cardiomyopathy in pressure-overloaded ACE2KO mice. Conclusion In the absence of ACE2, biomechanical stress triggers activation of the myocardial NAPDH oxidase system with a critical role of the p47phox subunit. Increased production of superoxide, activation of MMP, and pathological signalling leads to severe adverse myocardial remodelling and dysfunction in ACE2KO mice.

Acquired and inherited diseases of the heart represent a major health care issue in this country and throughout the World. Clinical medicine has made important advancements in the past quarter century to enable several effective treatment regimes for cardiac patients. Nevertheless, it is apparent that even with the best care, current treatment strategies and therapeutics are inadequate for treating heart disease, leaving it arguably the most pressing health issue today. In this context it is important to seek new approaches to redress the functional deficits in failing myocardium. This review focuses on several recent gene, cell and chemical-based experimental therapeutics currently being developed in the laboratory for potential translation to patient care. For example, new advances in bio-sensing inducible gene expression systems offer the potential for designer cardio-protective proteins to be expressed only during hypoxia/ischemia in the heart. Stem cells continue to offer the promise of cardiac repair, and some recent advances are discussed here. In addition, discovery and applications of synthetic polymers are presented as a chemical-based strategy for acute and chronic treatment of diseased and failing cardiac tissue. Collectively, these approaches serve as the front lines in basic biomedical research, with an eye toward translation of these findings to clinically meaningful applications in cardiac disease.

Mineralocorticoid receptor (MR) blockade has been shown to suppress cardiac hypertrophy and remodeling in animal models of pressure overload (POL). This study aims to determine whether MR deficiency in myeloid cells modulates aortic constriction-induced cardiovascular injuries. Myeloid MR knockout (MMRKO) mice and littermate control mice were subjected to abdominal aortic constriction (AAC) or sham operation. We found that AAC-induced cardiac hypertrophy and fibrosis were significantly attenuated in MMRKO mice. Expression of genes important in generating reactive oxygen species was decreased in MMRKO mice, while that of manganese superoxide dismutase increased. Furthermore, expression of genes important in cardiac metabolism was increased in MMRKO hearts. Macrophage infiltration in the heart was inhibited and expression of inflammatory genes was decreased in MMRKO mice. In addition, aortic fibrosis and inflammation were attenuated in MMRKO mice. Taken together, our data indicated that MR deficiency in myeloid cells effectively attenuated aortic constriction-induced cardiac hypertrophy and fibrosis, as well as aortic fibrosis and inflammation.

Oxidative stress plays a key pathogenic role in experimental and human heart failure. However, the source of ROS (reactive oxygen species) is a key determinant of the cardiac adaptation to pathological stressors. In the present study, we have shown that human dilated cardiomyopathy is associated with increased NOX2 (NADPH oxidase 2) levels, increased oxidative stress with adverse myocardial remodelling and activation of MAPKs (mitogen-activated protein kinases). Advanced heart failure in mice was also associated with increased NOX2 levels. Furthermore, we have utilized the pressure-overload model to examine the role of NOX2 in advanced heart failure. Increased cardiomyocyte hypertrophy and myocardial fibrosis in response to pressure overload correlated with increased oxidative stress, and loss of NOX2 prevented the increase in oxidative stress, development of cardiomyocyte hypertrophy, myocardial fibrosis and increased myocardial MMP (matrix metalloproteinase) activity in response to pressure overload. Consistent with these findings, expression of disease markers revealed a marked suppression of atrial natriuretic factor, β-myosin heavy chain, B-type natriuretic peptide and α-skeletal actin expression in pressure-overloaded hearts from NOX2-deficient mice. Activation of MAPK signalling, a well-known mediator of pathological remodelling, was lowered in hearts from NOX2-deficient mice in response to pressure overload. Functional assessment using transthoracic echocardiography and invasive pressure–volume loop analysis showed a marked protection in diastolic and systolic dysfunction in pressure-overloaded hearts from NOX2-deficient mice. Loss of NOX2 prevented oxidative stress in heart disease and resulted in sustained protection from the progression to advanced heart failure. Our results support a key pathogenic role of NOX2 in murine and human heart failure, and specific therapy antagonizing NOX2 activity may have therapeutic effects in advanced heart failure.

Angiotensin-converting enzyme 2 (ACE2) metabolizes Ang II into Ang 1-7 thereby negatively regulating the renin-angiotensin system. However, heart disease in humans and in animal models is associated with only a partial loss of ACE2. ACE2 is an X-linked gene; and as such, we tested the clinical relevance of a partial loss of ACE2 by using female ACE2(+/+) (wildtype) and ACE2(+/-) (heterozygote) mice. Pressure overload in ACE2(+/-) mice resulted in greater LV dilation and worsening systolic and diastolic dysfunction. These changes were associated with increased myocardial fibrosis, hypertrophy, and upregulation of pathological gene expression. In response to Ang II infusion, there was increased NADPH oxidase activity and myocardial fibrosis resulting in the worsening of Ang II-induced diastolic dysfunction with a preserved systolic function. Ang II-mediated cellular effects in cultured adult ACE2(+/-) cardiomyocytes and cardiofibroblasts were exacerbated. Ang II-mediated pathological signaling worsened in ACE2(+/-) hearts characterized by an increase in the phosphorylation of ERK1/2 and JNK1/2 and STAT-3 pathways. The ACE2(+/-) mice showed an exacerbated pressor response with increased vascular fibrosis and stiffness. Vascular superoxide and nitrotyrosine levels were increased in ACE2(+/-) vessels consistent with increased vascular oxidative stress. These changes occurred with increased renal fibrosis and superoxide production. Partial heterozygote loss of ACE2 is sufficient to increase the susceptibility to heart disease secondary to pressure overload and Ang II infusion.Heart disease in humans with idiopathic dilated cardiomyopathy is associated with a partial loss of ACE2. Heterozygote female ACE2 mutant mice showed enhanced susceptibility to pressure overload-induced heart disease. Heterozygote female ACE2 mutant mice showed enhanced susceptibility to Ang II-induced heart and vascular diseases. Partial loss of ACE2 is sufficient to enhance the susceptibility to heart disease.

Background Coronary artery disease leading to myocardial ischemia is the most common cause of heart failure. Apelin ( APLN ), the endogenous peptide ligand of the APJ receptor, has emerged as a novel regulator of the cardiovascular system. Methods and Results Here we show a critical role of APLN in myocardial infarction ( MI ) and ischemia‐reperfusion ( IR ) injury in patients and animal models. Myocardial APLN levels were reduced in patients with ischemic heart failure. Loss of APLN increased MI ‐related mortality, infarct size, and inflammation with drastic reductions in prosurvival pathways resulting in greater systolic dysfunction and heart failure. APLN deficiency decreased vascular sprouting, impaired sprouting of human endothelial progenitor cells, and compromised in vivo myocardial angiogenesis. Lack of APLN enhanced susceptibility to ischemic injury and compromised functional recovery following ex vivo and in vivo IR injury. We designed and synthesized two novel APLN analogues resistant to angiotensin converting enzyme 2 cleavage and identified one analogue, which mimicked the function of APLN , to be markedly protective against ex vivo and in vivo myocardial IR injury linked to greater activation of survival pathways and promotion of angiogenesis. Conclusions APLN is a critical regulator of the myocardial response to infarction and ischemia and pharmacologically targeting this pathway is feasible and represents a new class of potential therapeutic agents.

SummaryMitochondrial respiratory dysfunction is linked to the pathogenesis of multiple diseases, including heart failure, but the specific mechanisms for this link remain largely elusive. We modeled the impairment of mitochondrial respiration by the inactivation of the Ndufs4 gene, a protein critical for complex I assembly, in the mouse heart (cKO). Although complex I-supported respiration decreased by >40%, the cKO mice maintained normal cardiac function in vivo and high-energy phosphate content in isolated perfused hearts. However, the cKO mice developed accelerated heart failure after pressure overload or repeated pregnancy. Decreased NAD+/NADH ratio by complex I deficiency inhibited Sirt3 activity, leading to an increase in protein acetylation and sensitization of the permeability transition in mitochondria (mPTP). NAD+ precursor supplementation to cKO mice partially normalized the NAD+/NADH ratio, protein acetylation, and mPTP sensitivity. These findings describe a mechanism connecting mitochondrial dysfunction to the susceptibility to diseases and propose a potential therapeutic target.

Diastolic dysfunction is characterized by slow or incomplete relaxation of the ventricles during diastole, and is an important contributor to heart failure pathophysiology. Clinical symptoms include fatigue, shortness of breath, and pulmonary and peripheral edema, all contributing to decreased quality of life and poor prognosis. There are currently no therapies available that directly target the heart pump defects in diastolic function. Calcium mishandling is a hallmark of heart disease and has been the subject of a large body of research. Efforts are ongoing in a number of gene therapy approaches to normalize the function of calcium handling proteins such as sarcoplasmic reticulum calcium ATPase. An alternative approach to address calcium mishandling in diastolic dysfunction is to introduce calcium buffers to facilitate relaxation of the heart. Parvalbumin is a calcium binding protein found in fast-twitch skeletal muscle and not normally expressed in the heart. Gene transfer of parvalbumin into normal and diseased cardiac myocytes increases relaxation rate but also markedly decreases contraction amplitude. Although parvalbumin binds calcium in a delayed manner, it is not delayed enough to preserve full contractility. Factors contributing to the temporal nature of calcium buffering by parvalbumin are discussed in relation to remediation of diastolic dysfunction. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Angiotensin-converting enzyme 2 (ACE2)/Ang II/Ang 1–7 and the apelin/APJ are two important peptide systems which exert diverse effects on the cardiovascular system. ACE2 is a key negative regulator of the renin-angiotensin system (RAS) where it metabolizes angiotensin (Ang) II into Ang 1–7, an endogenous antagonist of Ang II. Both the prolonged activation of RAS and the loss of ACE2 can be detrimental as they lead to functional deterioration of the heart and progression of cardiac, renal, and vascular diseases. Recombinant human ACE2 in an animal model of ACE2 knockout mice lowers Ang II. These interactions neutralize the pressor and subpressor pathologic effects of Ang II by producing Ang 1–7 levelsin vivo, that might be cardiovascular protective. ACE2 hydrolyzes apelin to Ang II and, therefore, is responsible for the degradation of both peptides. Apelin has emerged as a promising peptide biomarker of heart failure. The serum level of apelin in cardiovascular diseases tends to be decreased. Apelin is recognized as an imperative controller of systemic blood pressure and myocardium contractility. Dysregulation of the apelin/APJ system may be involved in the predisposition to cardiovascular diseases, and enhancing apelin action may have important therapeutic effects.

Loss of angiotensin (Ang)-converting enzyme 2 (ACE2) and inability to metabolize Ang II to Ang 1-7 perpetuate the actions of Ang II after biomechanical stress and exacerbate early adverse myocardial remodeling. Ang receptor blockers are known to antagonize the effect of Ang II by blocking Ang II type 1 receptor (AT 1 R) and also by upregulating the ACE2 expression. We directly compare the benefits of AT 1 R blockade versus enhancing Ang 1-7 action in pressure-overload–induced heart failure in ACE2 knockout mice. AT 1 R blockade and Ang 1-7 both resulted in marked recovery of systolic dysfunction in pressure-overloaded ACE2-null mice. Similarly, both therapies attenuated the increase in NADPH oxidase activation by downregulating the expression of Nox2 and p47 phox subunits and also by limiting the p47 phox phosphorylation. Biomechanical stress-induced increase in protein kinase C-α expression and phosphorylation of extracellular signal–regulated kinase 1/2, signal transducer and activator of transcription 3, Akt, and glycogen synthase kinase 3β were normalized by irbesartan and Ang 1-7. Ang receptor blocker and Ang 1-7 effectively reduced matrix metalloproteinase 2 activation and matrix metalloproteinase 9 levels. Ang II–mediated cellular effects in cultured adult cardiomyocytes and cardiofibrolasts isolated from pressure-overloaded ACE2-null hearts were inhibited to similar degree by AT 1 R blockade and stimulation with Ang 1-7. Thus, treatment with the AT 1 R blocker irbesartan and Ang 1-7 prevented the cardiac hypertrophy and improved cardiac remodeling in pressure-overloaded ACE2-null mice by suppressing NADPH oxidase and normalizing pathological signaling pathways.

Oxygen metabolism is a strong predictor of the general health and fitness of an organism. In this study, we hypothesized that a divergence in intrinsic aerobic fitness would co-segregate with susceptibility for cardiovascular dysfunction. To test this hypothesis, cardiac function was assessed in rats specifically selected over nineteen generations for their low (LCR) and high (HCR) intrinsic aerobic running capacity. As an integrative marker of native aerobic capacity, run time to exhaustion between LCR and HCR rats had markedly diverged by 436% at generation nineteen of artificial selection. In vivo assessment of baseline cardiac function by echocardiography and catheter-based conductance micromanometry showed no marked difference in cardiac performance. However, when challenged by exposure to acute hypoxia, cardiac pump failure occurred significantly earlier in LCR rats compared to HCR animals. Acute cardiac decompensation in LCR rats was exclusively due to the development of intractable irregular ventricular contractions. Analysis of isolated cardiac myocytes showed significantly slower sarcomeric relaxation and delayed kinetics of calcium cycling in LCR myocytes compared to HCR myocytes. This study also revealed that artificial selection for low native aerobic capacity is a novel pathologic stimulus that results in myosin heavy chain isoform switching in the heart as shown by increased levels of beta-MHC in LCR rats. Together, these results provide evidence that alterations in sub-cellular calcium handling and MHC isoform composition are associated with susceptibility to compensatory cardiac remodeling and hypoxia induced pump failure in animals with low intrinsic aerobic capacity.

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+binding affinities that account for delayed Ca2+buffering in response to the intracellular Ca2+transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+content without otherwise altering intracellular Ca2+homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+buffers with modified Mg2+and Ca2+affinities to limit systolic Ca2+buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.

The cytosolic Ca2+/Mg2+-binding protein α-parvalbumin (α-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range, α-Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv's Mg2+ affinity may lower the effective concentration of Parv ([Parv]) to speed relaxation and, thus, limit Parv-mediated depressed contraction. Naturally occurring α/β-Parv isoforms show divergence in amino acid primary structure (57% homology) and cation-binding affinities, with β-Parv having an estimated 16% greater Mg2+ affinity and ∼200% greater Ca2+ affinity than α-Parv. We tested the hypothesis that, at the same or lower estimated [Parv], mechanical relaxation rate would be more significantly accelerated by β-Parv than by α-Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Relaxation properties were significantly slowed in adult cardiac myocytes isolated from DS rats compared with controls: time from peak contraction to 50% relaxation was 57 ± 2 vs. 49 ± 2 (SE) ms ( P < 0.05), validating this model system. DS cardiac myocytes were subsequently transduced with α- or β-Parv adenoviral vectors. Upon Parv gene transfer, β-Parv caused significantly faster relaxation than α-Parv ( P < 0.05), even though estimated [β-Parv] was ∼10% of [α-Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.