Rationale Pressure overload cardiac hypertrophy, a risk factor for heart failure, is associated with reduced mitochondrial fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS) proteins that correlate in rodents with reduced PGC-1α expression. Objective To determine the role of PGC-1β in maintaining mitochondrial energy metabolism and contractile function in pressure overload hypertrophy. Methods and Results PGC-1β deficient (KO) mice and wildtype controls (WT) were subjected to transverse aortic constriction (TAC). Although LV function was modestly reduced in young KO hearts, there was no further decline with age so that LV function was similar between KO and WT when TAC was performed. WT – TAC mice developed relatively compensated LVH, despite reduced mitochondrial function and repression of OXPHOS and FAO genes. In non-stressed KO hearts, OXPHOS gene expression and palmitoyl-carnitine supported mitochondrial function were reduced to the same extent as banded WT, but FAO gene expression was normal. Following TAC, KO mice progressed more rapidly to heart failure and developed more severe mitochondrial dysfunction, despite a similar overall pattern of repression of OXPHOS and FAO genes as WT – TAC. However, relative to WT TAC, PGC-1β deficient mice exhibited greater degrees of oxidative stress, decreased cardiac efficiency, lower rates of glucose metabolism and repression of hexokinase II protein. Conclusions PGC-1β plays an important role in maintaining baseline mitochondrial function and cardiac contractile function following pressure overload hypertrophy by preserving glucose metabolism and preventing oxidative stress.

BackgroundIncreased glucose transporter 1 (GLUT1) expression and glucose utilization that accompany pressure overload‐induced hypertrophy (POH) are believed to be cardioprotective. Moreover, it has been shown that lifelong transgenic overexpression of GLUT1 in the heart prevents cardiac dysfunction after aortic constriction. The relevance of this model to clinical practice is unclear because of the life‐long duration of increased glucose metabolism. Therefore, we sought to determine if a short‐term increase in GLUT1‐mediated myocardial glucose uptake would still confer cardioprotection if overexpression occurred at the onset of POH.Methods and ResultsMice with cardiomyocyte‐specific inducible overexpression of a hemagglutinin (HA)‐tagged GLUT1 transgene (G1HA) and their controls (Cont) were subjected to transverse aortic constriction (TAC) 2 days after transgene induction with doxycycline (DOX). Analysis was performed 4 weeks after TAC. Mitochondrial function, adenosine triphosphate (ATP) synthesis, and mRNA expression of oxidative phosphorylation (OXPHOS) genes were reduced in Cont mice, but were maintained in concert with increased glucose utilization in G1HA following TAC. Despite attenuated adverse remodeling in G1HA relative to control TAC mice, cardiac hypertrophy was exacerbated in these mice, and positive dP/dt (in vivo) and cardiac power (ex vivo) were equivalently decreased in Cont and G1HA TAC mice compared to shams, consistent with left ventricular dysfunction. O‐GlcNAcylation of Ca2+ cycling proteins was increased in G1HA TAC hearts.ConclusionsShort‐term cardiac specific induction of GLUT1 at the onset of POH preserves mitochondrial function and attenuates pathological remodeling, but exacerbates the hypertrophic phenotype and is insufficient to prevent POH‐induced cardiac contractile dysfunction, possibly due to impaired calcium cycling.

Mechanistic target of rapamycin (Mtor) is required for embryonic inner cell mass proliferation during early development. However, Mtor expression levels are very low in the mouse heart during embryogenesis. To determine if Mtor plays a role during mouse cardiac development, cardiomyocyte specific Mtor deletion was achieved using α myosin heavy chain (α-MHC) driven Cre recombinase. Initial mosaic expression of Cre between embryonic day (E) 10.5 and E11.5 eliminated a subset of cardiomyocytes with high Cre activity by apoptosis and reduced overall cardiac proliferative capacity. The remaining cardiomyocytes proliferated and expanded normally. However loss of 50% of cardiomyocytes defined a threshold that impairs the ability of the embryonic heart to sustain the embryo’s circulatory requirements. As a result 92% of embryos with cardiomyocyte Mtor deficiency died by the end of gestation. Thus Mtor is required for survival and proliferation of cardiomyocytes in the developing heart.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear whether these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy, we generated transgenic mice with inducible cardiomyocyte-specific expression of the GLUT4. We examined mice rendered hyperglycemic following lowdose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in nondiabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucosedependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest that reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

During pathological hypertrophy, peroxisome proliferator-activated receptor coactivator 1α (PGC-1α) is repressed in concert with reduced mitochondrial oxidative capacity and fatty acid oxidation (FAO). We therefore sought to determine if maintaining or increasing PGC-1α levels in the context of pressure overload hypertrophy (POH) would preserve mitochondrial function and prevent contractile dysfunction. Pathological cardiac hypertrophy was induced using 4 wk of transverse aortic constriction (TAC) in mice overexpressing the human PGC-1α genomic locus via a bacterial artificial chromosome (TG) and nontransgenic controls (Cont). PGC-1α levels were increased by 40% in TG mice and were sustained following TAC. Although TAC-induced repression of FAO genes and oxidative phosphorylation (oxphos) genes was prevented in TG mice, mitochondrial function and ATP synthesis were equivalently impaired in Cont and TG mice after TAC. Contractile function was also equally impaired in Cont and TG mice following TAC, as demonstrated by decreased +dP/dt and ejection fraction and increased left ventricular developed pressure and end diastolic pressure. Conversely, capillary density was preserved, in concert with increased VEGF expression, while apoptosis and fibrosis were reduced in TG relative to Cont mice after TAC. Hence, sustaining physiological levels of PGC-1α expression following POH, while preserving myocardial vascularity, does not prevent mitochondrial and contractile dysfunction.

Diabetes increases mortality and accelerates left ventricular (LV) dysfunction following myocardial infarction (MI). This study sought to determine the impact of impaired myocardial insulin signaling, in the absence of diabetes, on the development of LV dysfunction following MI. Mice with cardiomyocyte-restricted knock out of the insulin receptor (CIRKO) and wild type (WT) mice were subjected to proximal left coronary artery ligation (MI) and followed for 14 days. Despite equivalent infarct size, mortality was increased in CIRKO-MI vs. WT-MI mice (68 % vs. 40 %, respectively). In surviving mice, LV ejection fraction and dP/dt were reduced by > 40% in CIRKO-MI vs. WT-MI. Relative to shams, isometric developed tension in LV papillary muscles increased in WT-MI but not in CIRKO-MI. Time to peak tension and relaxation times were prolonged in CIRKO-MI vs. WT-MI suggesting impaired, load-independent myocardial contractile function. To elucidate mechanisms for impaired LV contractility, mitochondrial function was examined in permeabilized cardiac fibers. Whereas maximal ADP-stimulated mitochondrial O 2 consumption rates (V ADP ) with palmitoyl carnitine were unchanged in WT-MI mice relative to sham-operated animals, V ADP was significantly reduced in CIRKO-MI (13.17 ± 0.94 vs. 9.14 ± 0.88 nmol O 2 /min/mgdw, p<0.05). Relative to WT-MI, expression levels of GLUT4, PPAR-α, SERCA2, and the FA-Oxidation genes MCAD, LCAD, CPT2 and the electron transfer flavoprotein ETFDH were repressed in CIRKO-MI. Thus reduced insulin action in cardiac myocytes accelerates post-MI LV dysfunction, due in part to a rapid decline in mitochondrial FA oxidative capacity, which combined with limited glucose transport capacity may reduce substrate utilization and availability.

These studies investigate the role of uncoupling protein 3 (UCP3) in cardiac energy metabolism, cardiac O2 consumption (MVO2), cardiac efficiency (CE), and mitochondrial uncoupling in high fat (HF)–fed or leptin-deficient mice. UCP3KO and wild-type (WT) mice were fed normal chow or HF diets for 10 weeks. Substrate utilization rates, MVO2, CE, and mitochondrial uncoupling were measured in perfused working hearts and saponin-permeabilized cardiac fibers, respectively. Similar analyses were performed in hearts of ob/ob mice lacking UCP3 (U3OB mice). HF increased cardiac UCP3 protein. However, fatty acid (FA) oxidation rates were similarly increased by HF diet in WT and UCP3KO mice. By contrast, MVO2 increased in WT, but not in UCP3KO with HF, leading to increased CE in UCP3KO mice. Consistent with increased CE, mitochondrial coupling was increased in the hearts of HF-fed UCP3KO mice. Unexpectedly, UCP3 deletion in ob/ob mice reduced FA oxidation but had no effect on MVO2 or CE. In addition, FA-induced mitochondrial uncoupling was similarly enhanced in U3OB compared with ob/ob hearts and was associated with elevated mitochondrial thioesterase-1 protein content. These studies show that although UCP3 may mediate mitochondrial uncoupling and reduced CE after HF feeding, it does not mediate uncoupling in leptin-deficient states.

Nitroalkene derivatives of nitro-oleic acid (OA-NO2) are endogenous lipid products with potent anti-inflammatory properties in vitro. The present study was undertaken to evaluate the in vivo anti-inflammatory effect of OA-NO2 in mice given LPS. Two days before LPS administration, C57BL/6J mice were chronically infused with vehicle (LPS vehicle) or OA-NO2 (LPS OA-NO2) at 200 microg x kg(-1) x day(-1) via osmotic minipumps; LPS was administered via a single intraperitoneal (ip) injection (10 mg/kg in saline). A third group received an ip injection of saline without LPS or OA-NO2 and served as controls. At 18 h of LPS administration, LPS vehicle mice displayed multiorgan dysfunction as evidenced by elevated plasma urea and creatinine (kidney), aspartate aminotransferase (AST) and alanine aminotransferase (ALT; liver), and lactate dehydrogenase (LDH) and reduced ejection fraction (heart). In contrast, the severity of multiorgan dysfunction was less in LPS OA-NO2 animals. The levels of circulating TNF-alpha and renal TNF-alpha mRNA expression, together with renal mRNA expression of monocyte chemoattractant protein-1, ICAM-1, and VCAM-1, and with renal mRNA and protein expression of inducible nitric oxide synthase and cyclooxygenase 2, and renal cGMP and PGE2 contents, were greater in LPS vehicle vs. control mice, but were attenuated in LPS OA-NO2 animals. Similar patterns of changes in the expression of inflammatory mediators were observed in the liver. Together, pretreatment with OA-NO2 ameliorated the inflammatory response and multiorgan injury in endotoxin-induced endotoxemia in mice.