Summary Elevated levels of branched-chain amino acids (BCAAs) have recently been implicated in the development of cardiovascular and metabolic diseases but the molecular mechanisms are unknown. In a mouse model of impaired BCAA catabolism (KO), we found that chronic accumulation of BCAAs suppressed glucose metabolism and sensitized the heart to ischemic injury. High levels of BCAAs selectively disrupted mitochondrial pyruvate utilization through inhibition of pyruvate dehydrogenase complex (PDH) activity. Furthermore, downregulation of hexosamine biosynthetic pathway in KO hearts decreased protein O-linked-N-acetylglucosamine (O-GlcNAc) modification and inactivated PDH resulting in significant decreases in glucose oxidation. Although the metabolic remodeling in KO did not affect baseline cardiac energetics or function, it rendered the heart vulnerable to ischemia-reperfusion injury. Promoting BCAA catabolism or normalizing glucose utilization by overexpressing GLUT1 in the KO heart rescued the metabolic and functional outcome. These observations revealed a novel role of BCAA catabolism in regulating cardiac metabolism and stress response.
Cardiovascular sequelae including diabetic cardiomyopathy constitute the major cause of death in diabetic patients. Although several factors may contribute to the development of this cardiomyopathy, the underlying molecular/cellular mechanisms leading to cardiac dysfunction are still partially understood. Recently, a novel paradigm for the role of the adipocytokine resistin in diabetes has emerged. Resistin has been proposed to be a link between obesity, insulin resistance and diabetes. Using microarray analysis, we have recently found that cardiomyocytes isolated from type 2 diabetic hearts express high levels of resistin. However, the function of resistin with respect to cardiac function is unknown. In this study we show that resistin is not only expressed in the heart, but also promotes cardiac hypertrophy. Adenovirus-mediated overexpression of resistin in cultured neonatal rat ventricular myocytes (NRVM) significantly increased sarcomere organization and cell size, increased protein synthesis and increased the expression of atrial natriuretic factor and β-myosin heavy chain. Overexpression of resistin in NRVM was also associated with activation of the mitogenactivated protein (MAP) kinases, ERK1/2 and p38, as well as increased Ser-636 phosphorylation of insulin receptor substrate-1 (IRS-1), indicating that IRS-1/MAPK pathway may be involved in the observed hypertrophic response. Overexpression of resistin in adult cultured cardiomyocytes significantly altered myocyte mechanics by depressing cell contractility as well as contraction and relaxation velocities. Intracellular Ca 2+ measurements showed slower Ca 2+ transients decay in resistin-transduced myocytes compared to controls, suggesting impaired cytoplasmic Ca 2+ clearing or alterations in myofilament activation. We conclude that resistin overexpression alters cardiac contractility, confers to primary cardiomyocytes all the features of the hypertrophic phenotype and promotes cardiac hypertrophy possibly via the IRS-1/MAPK pathway.
We have previously reported that resistin induces hypertrophy and impairs contractility in isolated rat cardiomyocytes. To examine the long-term cardiovascular effects of resistin, we induced in vivo overexpression of resistin using adeno-associated virus serotype 9 injected by tail vein in rats and compared to control animals. Ten weeks after viral injection, overexpression of resistin was associated with increased ratio of left ventricular (LV) weight/body weight increased end-systolic LV volume and significant decrease in LV contractility, measured by the end-systolic pressure volume relationship slope in LV pressure volume loops, compared to controls. At the molecular level, mRNA expression of ANF and β-MHC, and protein levels of phospholamban were increased in the resistin group without a change in the level of SERCA2a protein expression. Increased fibrosis by histology, associated with increased mRNA levels of collagen, fibronectin and connective tissue growth factor were observed in the resistin-overexpressing hearts. Resistin overexpression was also associated with increased apoptosis in vivo, along with an apoptotic molecular phenotype in vivo and in vitro. Resistin-overexpressing LV tissue had higher levels of TNF-α receptor 1 and iNOS, and reduced levels of eNOS. Cardiomyocytes overexpressing resistin in vitro produced larger amounts of TNFα in the medium, had increased phosphorylation of IκBα and displayed increased intracellular reactive oxygen species (ROS) content with increased expression and activity of ROS-producing NADPH oxidases compared to controls. Long-term resistin overexpression is associated with a complex phenotype of oxidative stress, inflammation, fibrosis, apoptosis and myocardial remodeling and dysfunction in rats. This phenotype recapitulates key features of diabetic cardiomyopathy.
Rationale AMP-activated protein kinase (AMPK) is a master regulator of cell metabolism and an attractive drug target for cancer, metabolic and cardiovascular diseases. Point mutations in the regulatory γ2-subunit of AMPK (encoded by Prkag2 gene) caused a unique form of human cardiomyopathy characterized by cardiac hypertrophy, ventricular pre-excitation and glycogen storage. Understanding the disease mechanisms of Prkag2 cardiomyopathy is not only beneficial for the patients but also critical to the utility of AMPK as a drug target. Objective We sought to identify the pro-growth signaling pathway(s) triggered by Prkag2 mutation and to distinguish it from the secondary response to glycogen storage. Methods and Results In a mouse model of N488I mutation of the Prkag2 (R2M), we rescued the glycogen storage phenotype by genetic inhibition of glucose-6-phosphate stimulated glycogen synthase activity. Ablation of glycogen storage eliminated the ventricular pre-excitation but did not affect the excessive cardiac growth in R2M mice. The pro-growth effect in R2M hearts was mediated via increased insulin sensitivity and hyperactivity of Akt, resulting in activation of mTOR and inactivation of FoxO signaling pathways. Consequently, cardiac myocyte proliferation during the postnatal period was enhanced in R2M hearts followed by hypertrophic growth in adult hearts. Inhibition of mTOR activity by rapamycin or restoration of FoxO activity by overexpressing FoxO1 rescued the abnormal cardiac growth. Conclusions Our study reveals a novel mechanism for Prkag2 cardiomyopathy independent of glycogen storage. The role of γ2-AMPK in cell growth also has broad implications in cardiac development, growth and regeneration.
AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis and multiple biological processes in cell growth and survival, hence an attractive drug target. AMPK is a heterotrimeric protein consisting of α catalytic, β and γ regulatory subunits; two isoforms of each subunit are present in the heart. Studies using both genetic and pharmacological approaches have demonstrated important roles of AMPK in protecting the heart during ischemia/reperfusion injury as well as in pathological hypertrophy and failure. There is also emerging evidence suggesting isoform-specific function of AMPK, e.g. mutations of the γ2 subunit cause human cardiomyopathy. Thus, strategies avoiding the undesirable effects of altering γ2-AMPK activity, such as isoform selective activation of AMPK may lead to cardioprotective therapies with greater efficacy and safety. Keywords AMPK; energy homeostasis; drug target; isoform; cardiomyopathy AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase and is phylogenetically conserved from yeast to mammals. AMPK is sensitive to a broad spectrum of stresses, especially those that cause changes in cellular energy status. Activation of AMPK stimulates ATP-generating pathways, such as glucose uptake, glycolysis and fatty acid oxidation, and inhibits ATP-consuming pathways, such as fatty acid and cholesterol synthesis [1,2]. Therefore, the kinase is originally considered a master metabolic switcher that controls the cellular and whole body energy homeostasis. More recent work has also revealed an important role of AMPK in cell growth and survival through its interactions with mitochondrial biogenesis, protein synthesis and degradation pathways [3][4][5]. Thus, there has been intense interest in targeting AMPK for the treatment of multiple prevalent diseases, such as diabetes and obesity, cancer and cardiovascular diseases. AMPK structure and activityAMPK is a heterotrimeric complex composed of a catalytic α-subunit and two regulatory β-and γ-subunits. Each subunit exists in multiple isoforms encoded by separate genes (α1, α2, © 2010 Elsevier Ltd. All rights reserved.Correspondence: Rong Tian, MD, PhD, Mitochondria and Metabolism Center, University of Washington School of Medicine, 815 Mercer St., Seattle, WA 98109, rongtian@u.washington.edu . Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript β1, β2, γ1, γ2, and γ3), and their combination give rise to a variety of AMPK holoenzymes ( Figure 1). Phosphorylation of Threonine 172 in the cataly...
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