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Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that translocates to the nucleus to regulate transcription factors in different tissues or pathologic states. Although studied extensively in cancer, its biological role in the heart remains unresolved. PKM1 is more abundant than the PKM2 isoform in cardiomyocytes, and thus, we speculated that PKM2 is not genetically redundant to PKM1 and may be critical in regulating cardiomyocyte-specific transcription factors important for cardiac survival. Here, we showed that nuclear PKM2 ( S37 P-PKM2) in cardiomyocytes interacts with prosurvival and proapoptotic transcription factors, including GATA4, GATA6, and P53. Cardiomyocyte-specific PKM2-deficient mice ( Pkm2 Mut Cre + ) developed age-dependent dilated cardiac dysfunction and had decreased amounts of GATA4 and GATA6 (GATA4/6) but increased amounts of P53 compared to Control Cre +  hearts. Nuclear PKM2 prevented caspase-1-dependent cleavage and degradation of GATA4/6 while also providing a molecular platform for MDM2-mediated reduction of P53. In a preclinical heart failure mouse model, nuclear PKM2 and GATA4/6 were decreased, whereas P53 was increased in cardiomyocytes. Loss of nuclear PKM2 was ubiquitination dependent and associated with the induction of the E3 ubiquitin ligase TRIM35. In mice, cardiomyocyte-specific TRIM35 overexpression resulted in decreased S37 P-PKM2 and GATA4/6 along with increased P53 in cardiomyocytes compared to littermate controls and similar cardiac dysfunction to Pkm2 Mut Cre +  mice. In patients with dilated left ventricles, increase in TRIM35 was associated with decreased S37 P-PKM2 and GATA4/6 and increased P53. This study supports a previously unrecognized role for PKM2 as a molecular platform that mediates cell signaling events essential for cardiac survival.

Obesity is increasing in prevalence and is strongly associated with metabolic and cardiovascular disorders. The renin-angiotensin system (RAS) has emerged as a key pathogenic mechanism for these disorders; angiotensin (Ang)-converting enzyme 2 (ACE2) negatively regulates RAS by metabolizing Ang II into Ang 1-7. We studied the role of ACE2 in obesity-mediated cardiac dysfunction. ACE2 null (ACE2KO) and wild-type (WT) mice were fed a high-fat diet (HFD) or a control diet and studied at 6 months of age. Loss of ACE2 resulted in decreased weight gain but increased glucose intolerance, epicardial adipose tissue (EAT) inflammation, and polarization of macrophages into a proinflammatory phenotype in response to HFD. Similarly, human EAT in patients with obesity and heart failure displayed a proinflammatory macrophage phenotype. Exacerbated EAT inflammation in ACE2KO-HFD mice was associated with decreased myocardial adiponectin, decreased phosphorylation of AMPK, increased cardiac steatosis and lipotoxicity, and myocardial insulin resistance, which worsened heart function. Ang 1-7 (24 µg/kg/h) administered to ACE2KO-HFD mice resulted in ameliorated EAT inflammation and reduced cardiac steatosis and lipotoxicity, resulting in normalization of heart failure. In conclusion, ACE2 plays a novel role in heart disease associated with obesity wherein ACE2 negatively regulates obesity-induced EAT inflammation and cardiac insulin resistance., (© 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

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.

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.

Background: Activation of the renin-angiotensin and sympathetic nervous systems may alter the cardiac energy substrate preference, thereby contributing to the progression of heart failure with normal ejection fraction. We assessed the qualitative and quantitative effects of angiotensin II (Ang II) and the α-adrenergic agonist, phenylephrine (PE), on cardiac energy metabolism in experimental models of hypertrophy and diastolic dysfunction and the role of the Ang II type 1 receptor., Methods and Results: Ang II (1.5 mg·kg(-1)·day(-1)) or PE (40 mg·kg(-1)·day(-1)) was administered to 9-week-old male C57/BL6 wild-type mice for 14 days via implanted microosmotic pumps. Echocardiography showed concentric hypertrophy and diastolic dysfunction, with preserved systolic function in Ang II- and PE-treated mice. Ang II induced marked reduction in cardiac glucose oxidation and lactate oxidation, with no change in glycolysis and fatty acid β-oxidation. Tricarboxylic acid acetyl coenzyme A production and ATP production were reduced in response to Ang II. Cardiac pyruvate dehydrogenase kinase 4 expression was upregulated by Ang II and PE, resulting in a reduction in the pyruvate dehydrogenase activity, the rate-limiting step for carbohydrate oxidation. Pyruvate dehydrogenase kinase 4 upregulation correlated with the activation of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F pathway in response to Ang II. Ang II type 1 receptor blockade normalized the activation of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F pathway and prevented the reduction in glucose oxidation but increased fatty acid oxidation., Conclusions: Ang II- and PE-induced hypertrophy and diastolic dysfunction is associated with reduced glucose oxidation because of the cyclin/cyclin-dependent kinase-retinoblastoma protein-E2F-induced upregulation of pyruvate dehydrogenase kinase 4, and targeting these pathways may provide novel therapy for heart failure with normal ejection fraction.

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.

Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics.Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells and developed robust engineered heart tissues by seeding induced pluripotent stem cell-derived cardiomyocytes into a laser-cut scaffold possessing native cardiac fiber alignment to study human cardiac mechanobiology at both the cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype by gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in 5 families bearing distinct sarcomeric mutations.Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain (MYH7) led to increased force generation, which, when coupled with slower twitch relaxation, destabilized the MLP (muscle LIM protein) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric muscle LIM protein level caused disinhibition of calcineurin-nuclear factor of activated T-cells signaling, which promoted cardiac hypertrophy. We demonstrate that the common muscle LIM protein-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations.Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP-mechanosensory complex. [ABSTRACT FROM AUTHOR]