Abstract 86: Compromised Sarcolemmal Membrane Repair Contributes to the Progression of Heart Failure

A conserved sarcolemmal membrane repair response exist to counteract membrane damage and restore membrane barrier function in order to maintain normal cellular homeostasis. This response can involve various mechanisms including activation of signaling pathways that trigger vesicular trafficking to the site of injury followed by vesicular fusion with the damaged portion of the membrane to patch the membrane disruption. Previous studies indicate that compromised repair capacity can exacerbate cardiac injury while increasing membrane repair capacity can reduce cardiac pathology. In our studies, membrane repair assays on cardiac and non-cardiac cell lines demonstrated that this process is dependent on activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis through the downstream target Akt1. One mechanism found to increase membrane repair following PI3K/Akt1 activation is elevated exocytotic and endocytotic activity. Further studies indicate that the PI3K/Akt1 pathway is relevant to membrane repair in native hearts. Thick slices of myocardium from explanted human and mouse hearts were probed using multi-photon microscopy to determine the membrane repair capacity. These studies indicate decreased repair capacity in failing human myocardium as well as in mouse hearts following transaortic constriction (TAC). This membrane repair response requires PI3K/Akt1 signaling as genetically modified mice null for Akt1 show compromised sarcolemmal membrane repair. Additionally, PI3K or Akt1 inhibition prevents membrane resealing in non-failing human or mouse myocardium. The compromised membrane repair observed in failing human myocardium can be ameliorated by PI3K or Akt1 agonists. Treatment of TAC mice with multiple therapeutic compounds known to increase membrane repair capacity can minimize the development of structural and functional hallmarks of heart failure. Our results indicate that failing cardiomyocytes exhibit compromised membrane repair and that increased PI3K/Akt1 signaling can increase repair capacity thereby demonstrating potential as a heart failure treatment.

Abstract 235: Sarcolemmal Membrane Associated Protein Isoform 1: a Unique Regulator of Glucose Uptake and Metabolism in the Myocardium

As one of the leading causes of heart disease, diabetes is a problem which needs a solution. Regulation of glucose uptake and metabolism within skeletal and cardiac muscle has proven capable of altering systemic glucose levels and impacting metabolism to potentially improve patient outcomes. Unfortunately, to date, very few muscle specific metabolic regulators are known which can allow us to achieve blood glucose uptake and metabolism. Sarcolemmal Membrane Associated Protein Isoform 1 (SLMAP1) is a novel protein expressed predominantly within muscle tissue. It has been linked to diabetes through animal models, although its role in metabolism remains to be defined. Here we describe a novel role for SLMAP1 in glucose metabolism within the myocardium. We engineered a transgenic (TG) mouse with cardiac specific expression of SLMAP1. Using neonatal cardiomyocytes (NCMs) collected from these mice we performed glucose uptake assays with 2-deoxy-glucose (2DG), measured glycolytic rate using an Extracellular Flux XF24e Bioanalyzer, and analyzed glucose transporter 4 (GLUT4) trafficking using Western Blots, qPCR, and immunofluorescence imaging. NCMs extracted from TG hearts expressing SLMAP1 displayed increased levels of 2DG uptake (93% ± 25%, n=5, P<0.01), basal glycolysis (glycolysis (92 ± 40%, n=5, P<0.05), and maximal glycolysis (75 ± 31%, n=5, P<0.05) compared with wildtype littermates. Confocal microscopy revealed both increased localization of glucose transporter 4 (GLUT4) at the cell surface as well as an expansion of GLUT4 early endosomes in TG NCMs. The data here indicates SLMAP1 as a novel regulator of glucose uptake and metabolism in the myocardium. The targeted expression of SLMAP1 in a muscle specific manner may enhance systemic glycemic control and serve to limit cardiovascular disease in metabolic disorders such as diabetes.

Abstract 400: Regulated Levels of Sarcolemmal Membrane Protein Isoform 3 Impact Cardiac Function in Mice

The sarcolemmal membrane-associated proteins (SLMAPs) are a family of tail-anchored membrane proteins generated by alternative splicing of the SLMAP gene. A ubiquitously expressed SLMAP isoform 3 encompasses an N-terminal FHA domain with extended coiled-coil structure and has been implicated in cell cycle control. Heart function in transgenic mice with cardiac-specific overexpression of SLMAP3 cDNA driven by α myosin heavy chain promoter was evaluated by echocardiography. qPCR and western blot were used to analyze gene and protein expression respectively. Structure and fibrosis was analyzed by H&E and Masson’s Trichrome staining. Function analysis showed a 15% (p<0.05) decrease in ejection fraction and 19% (p<0.05) decrease in fractional shortening in transgenic mice as early as 5 weeks and persisted into old age at 44 weeks. Transgenic mice presented a mild systolic dysfunction and a trend towards dilated cardiomyopathy without any premature death. Natriuretic peptide ANP and BNP levels were not changed and there was no difference in left ventricular mass or activation of the hypertrophic factor Akt1 in SLMAP3 expressing myocardium. However, significant changes in calcium handling proteins with a significant decrease in phosphorylation of phospholamban ser16 (p<0.05) along with a down-regulation of sarco-endoplasmic reticulum Ca2+ ATPase protein and increased ryanodine receptor 2 phosphorylation ser2808 (p<0.05) were noted at 5 weeks of age in transgenic hearts. These data indicate that increased SLMAP3 levels did not influence cardiac remodeling or hypertrophic growth but did impact membrane biology of calcium transport systems in myocardium leading to depressed contractility. Thus regulated levels of SLMAP3 are important to support normal heart function.

Abstract 359: Tripartite Motif Protein 58 (trim58) is a Novel Regulator of Membrane Repair With Increased Expression in Failing Cardiac Muscle

In recent years, members of the tripartite motif-containing (TRIM) family of E3 ubiquitin ligases have been shown to be both positively and negatively regulated in various disease pathologies. TRIM72 (MG53) has been directly linked with regulation of sarcolemmal membrane repair in striated muscle cells, including cardiomyocytes. Recently, we were first to identify that a novel tripartite motif family member, TRIM58, is a negative regulator of the cell membrane repair process in striated muscle cells. Overexpression of TRIM58 decreases the membrane repair capacity of cultured myoblasts as measured by dye occlusion following laser-mediated disruption of the sarcolemmal membrane. We also find that TRIM58 can directly interact with TRIM72/MG53 and alter signaling through phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Protein profiling experiments show that TRIM58 expression increases during the development of heart failure suggesting that TRIM58 may be relevant in the development of cardiac failure. Western blot and histological examination in both human (Fig.1) and mouse transverse aortic constriction (TAC) heart failure samples clearly show an increased expression of TRIM58 in cardiac tissue. Our results suggest that TRIM58 levels might serve as a potential prognostic marker and that TRIM58 may be a therapeutic target for the management of cardiovascular disease.

5′-AMP-activated protein kinase increases glucose uptake independent of GLUT4 translocation in cardiac myocytes

Glucose uptake and glycolysis are increased in the heart during ischemia, and this metabolic alteration constitutes an important contributing factor towards ischemic injury. Therefore, it is important to understand glucose uptake regulation in the ischemic heart. There are primarily 2 glucose transporters controlling glucose uptake into cardiac myocytes: GLUT1 and GLUT4. In the non-ischemic heart, insulin stimulates GLUT4 translocation to the sarcolemmal membrane, while both GLUT1 and GLUT4 translocation can occur following AMPK stimulation. Using a newly developed technique involving [3H]2-deoxyglucose, we measured glucose uptake in H9c2 ventricular myoblasts, and demonstrated that while insulin has no detectable effect on glucose uptake, phenformin-induced AMPK activation increases glucose uptake 2.5-fold. Furthermore, insulin treatment produced no discernible effect on either Akt serine 473 phosphorylation or AMPKα threonine 172 phosphorylation, while treatment with phenformin results in an increase in AMPKα threonine 172 phosphorylation, and a decrease in Akt serine 473 phosphorylation. Visualization of a dsRed-GLUT4 fusion construct in H9c2 cells by laser confocal microscopy showed that unlike insulin, AMPK activation did not redistribute GLUT4 to the sarcolemmal membrane, suggesting that AMPK may regulate glucose uptake via another glucose transporter. These studies suggest that AMPK is a major regulator of glucose uptake in cardiac myocytes.