Maintaining PGC-1? expression following pressure overload-induced cardiac hypertrophy preserves angiogenesis but not contractile or mitochondrial function.
ABSTRACT: During pathological hypertrophy, peroxisome proliferator-activated receptor coactivator 1? (PGC-1?) is repressed in concert with reduced mitochondrial oxidative capacity and fatty acid oxidation (FAO). We therefore sought to determine if maintaining or increasing PGC-1? 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-1? genomic locus via a bacterial artificial chromosome (TG) and nontransgenic controls (Cont). PGC-1? 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-1? expression following POH, while preserving myocardial vascularity, does not prevent mitochondrial and contractile dysfunction.
Project description:The aim of this study was to determine whether endogenous GLUT1 induction and the increased glucose utilization that accompanies pressure overload hypertrophy (POH) are required to maintain cardiac function during hemodynamic stress, and to test the hypothesis that lack of GLUT1 will accelerate the transition to heart failure. To determine the contribution of endogenous GLUT1 to the cardiac adaptation to POH, male mice with cardiomyocyte-restricted deletion of the GLUT1 gene (G1KO) and their littermate controls (Cont) were subjected to transverse aortic constriction (TAC). GLUT1 deficiency reduced glycolysis and glucose oxidation by 50%, which was associated with a reciprocal increase in fatty acid oxidation (FAO) relative to controls. Four weeks after TAC, glycolysis increased and FAO decreased by 50% in controls, but were unchanged in G1KO hearts relative to shams. G1KO and controls exhibited equivalent degrees of cardiac hypertrophy, fibrosis, and capillary density loss after TAC. Following TAC, in vivo left ventricular developed pressure was decreased in G1KO hearts relative to controls, but+dP/dt was equivalently reduced in Cont and G1KO mice. Mitochondrial function was equivalently impaired following TAC in both Cont and G1KO hearts. GLUT1 deficiency in cardiomyocytes alters myocardial substrate utilization, but does not substantially exacerbate pressure-overload induced contractile dysfunction or accelerate the progression to heart failure.
Project description: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-1? expression.To determine the role of PGC-1? in maintaining mitochondrial energy metabolism and contractile function in pressure overload hypertrophy.PGC-1? deficient (KO) mice and wildtype (WT) controls 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 nonstressed 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, in relation to WT-TAC, PGC-1? deficient mice exhibited greater degrees of oxidative stress, decreased cardiac efficiency, lower rates of glucose metabolism, and repression of hexokinase II protein.PGC-1? 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.
Project description:Protein posttranslational modifications (PTMs) by O-linked β-N-acetylglucosamine (O-GlcNAc) rise during pressure-overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O-GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O-GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O-GlcNAc levels. We hypothesize that induction of key enzymes augment protein O-GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O-GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1-week TAC (1wTAC) for early hypertrophy, or 6-week TAC (6wTAC) for established hypertrophy. We used western blots to determine O-GlcNAc regulation. To assess the effect of increased protein O-GlcNAcylation with established hypertrophy, mice received thiamet-g (TG) starting 4 weeks after TAC. Protein O-GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O-GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate-limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O-GlcNAc levels but did not affect cardiac mass. In summary, protein O-GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O-GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O-GlcNAc levels during established hypertrophy did not restart hypertrophic growth.
Project description:Hypertrophic stimuli cause transcription of the proto-oncogene c-Myc (Myc). Prior work showed that myocardial knockout of c-Myc (Myc) attenuated hypertrophy and decreased expression of metabolic genes after aortic constriction. Accordingly, we assessed the interplay between Myc, substrate oxidation and cardiac function during early pressure overload hypertrophy. Mice with cardiac specific, inducible Myc knockout (MycKO-TAC) and non-transgenic littermates (Cont-TAC) were subjected to transverse aortic constriction (TAC; n = 7/group). Additional groups underwent sham surgery (Cont-Sham and MycKO-Sham, n = 5 per group). After two weeks, function was measured in isolated working hearts along with substrate fractional contributions to the citric acid cycle by using perfusate with 13C labeled mixed fatty acids, lactate, ketone bodies and unlabeled glucose and insulin. Cardiac function was similar between groups after TAC although +dP/dT and -dP/dT trended towards improvement in MycKO-TAC versus Cont-TAC. In sham hearts, Myc knockout did not affect cardiac function or substrate preferences for the citric acid cycle. However, Myc knockout altered fractional contributions during TAC. The unlabeled fractional contribution increased in MycKO-TAC versus Cont-TAC, whereas ketone and free fatty acid fractional contributions decreased. Additionally, protein posttranslational modifications by O-GlcNAc were significantly greater in Cont-TAC versus both Cont-Sham and MycKO-TAC. In conclusion, Myc alters substrate preferences for the citric acid cycle during early pressure overload hypertrophy without negatively affecting cardiac function. Myc also affects protein posttranslational modifications by O-GlcNAc during hypertrophy, which may regulate Myc-induced metabolic changes.
Project description:RATIONALE:Decreased fatty acid oxidation (FAO) with increased reliance on glucose are hallmarks of metabolic remodeling that occurs in pathological cardiac hypertrophy and is associated with decreased myocardial energetics and impaired cardiac function. To date, it has not been tested whether prevention of the metabolic switch that occurs during the development of cardiac hypertrophy has unequivocal benefits on cardiac function and energetics. OBJECTIVE:Because malonyl CoA production via acetyl CoA carboxylase 2 (ACC2) inhibits the entry of long chain fatty acids into the mitochondria, we hypothesized that mice with a cardiac-specific deletion of ACC2 (ACC2H-/-) would maintain cardiac FAO and improve function and energetics during the development of pressure-overload hypertrophy. METHODS AND RESULTS:ACC2 deletion led to a significant reduction in cardiac malonyl CoA levels. In isolated perfused heart experiments, left ventricular function and oxygen consumption were similar in ACC2H-/- mice despite an ?60% increase in FAO compared with controls (CON). After 8 weeks of pressure overload via transverse aortic constriction (TAC), ACC2H-/- mice exhibited a substrate utilization profile similar to sham animals, whereas CON-TAC hearts had decreased FAO with increased glycolysis and anaplerosis. Myocardial energetics, assessed by 31P nuclear magnetic resonance spectroscopy, and cardiac function were maintained in ACC2H-/- after 8 weeks of TAC. Furthermore, ACC2H-/--TAC demonstrated an attenuation of cardiac hypertrophy with a significant reduction in fibrosis relative to CON-TAC. CONCLUSIONS:These data suggest that reversion to the fetal metabolic profile in chronic pathological hypertrophy is associated with impaired myocardial function and energetics and maintenance of the inherent cardiac metabolic profile and mitochondrial oxidative capacity is a viable therapeutic strategy.
Project description:<h4>Background</h4>The mechanism of cardiac energy production against sustained pressure overload remains to be elucidated.<h4>Methods and results</h4>We generated cardiac-specific kinase-dead (kd) calcium/calmodulin-dependent protein kinase kinase-? (CaMKK?) transgenic (?-MHC CaMKK?kd TG) mice using ?-myosin heavy chain (?-MHC) promoter. Although CaMKK? activity was significantly reduced, these mice had normal cardiac function and morphology at baseline. Here, we show that transverse aortic binding (TAC) in ?-MHC CaMKK?kd TG mice led to accelerated death and left ventricular (LV) dilatation and dysfunction, which was accompanied by significant clinical signs of heart failure. CaMKK? downstream signaling molecules, including adenosine monophosphate-activated protein kinase (AMPK), were also suppressed in ?-MHC CaMKK?kd TG mice compared with wild-type (WT) mice. The expression levels of peroxisome proliferator-activated receptor-? coactivator (PGC)-1?, which is a downstream target of both of CaMKK? and calcium/calmodulin kinases, were also significantly reduced in ?-MHC CaMKK?kd TG mice compared with WT mice after TAC. In accordance with these findings, mitochondrial morphogenesis was damaged and creatine phosphate/?-ATP ratios assessed by magnetic resonance spectroscopy were suppressed in ?-MHC CaMKK?kd TG mice compared with WT mice after TAC.<h4>Conclusions</h4>These data indicate that CaMKK? exerts protective effects on cardiac adaptive energy pooling against pressure-overload possibly through phosphorylation of AMPK and by upregulation of PGC-1?. Thus, CaMKK? may be a therapeutic target for the treatment of heart failure.
Project description:Muscle ring finger-1 (MuRF1) is a muscle-specific protein implicated in the regulation of cardiac myocyte size and contractility. MuRF2, a closely related family member, redundantly interacts with protein substrates, and hetero-dimerizes with MuRF1. Mice lacking either MuRF1 or MuRF2 are phenotypically normal whereas mice lacking both proteins develop a spontaneous cardiac and skeletal muscle hypertrophy indicating cooperative control of muscle mass by MuRF1 and MuRF2. In order to identify the role that MuRF1 plays in regulating cardiac hypertrophy in vivo, we created transgenic mice expressing increased amounts of cardiac MuRF1. Adult MuRF1 transgenic (Tg+) hearts exhibited a non-progressive thinning of the left ventricular wall and a concomitant decrease in cardiac function. Experimental induction of cardiac hypertrophy by trans-aortic constriction (TAC) induced rapid failure of MuRF1 Tg+ hearts. Microarray analysis identified that the levels of genes associated with metabolism (and in particular mitochondrial processes) were significantly altered in MuRF1 Tg+ hearts, both at baseline and during the development of cardiac hypertrophy. Surprisingly, ATP levels in MuRF1 Tg+ mice did not differ from wild type mice despite the depressed contractility following TAC. To explain this discrepancy between the ongoing heart failure and maintained ATP levels in MuRF1 Tg+ hearts, we compared the level and activity of creatine kinase (CK) between wild type and MuRF1 Tg+ hearts. Although mCK and CK-M/B protein levels were unaffected in MuRF1 Tg+ hearts, total CK activity was significantly inhibited. We conclude that MuRF1’s inhibition of CK activity leads to increased susceptibility to heart failure following TAC, demonstrating for the first time that MuRF1 regulates cardiac energetics in vivo. Keywords: Genetic modification, physiological manipulation. Three-condition experiment, MuRF1 Tg+ vs. WT mice. Biological replicates: 4 WT baseline, 3 MuRF1 Tg+ baseline, cardiac overpressure hypertrophy induced by trans-aortic banding at 1 week (3 WT, 3 MurF Tg) and 4 weeks (3 WT, 3 MurF Tg), hearts harvested. One replicate per array.
Project description:Physiological cardiac hypertrophy is associated with mitochondrial adaptations that are characterized by activation of PGC-1alpha and increased fatty acid oxidative (FAO) capacity. It is widely accepted that phosphatidylinositol 3-kinase (PI3K) signaling to Akt1 is required for physiological cardiac growth. However, the signaling pathways that coordinate physiological hypertrophy and metabolic remodeling are incompletely understood. We show here that activation of PI3K is sufficient to increase myocardial FAO capacity and that inhibition of PI3K signaling prevents mitochondrial adaptations in response to physiological hypertrophic stimuli despite increased expression of PGC-1alpha. We also show that activation of the downstream kinase Akt is not required for the mitochondrial adaptations that are secondary to PI3K activation. Thus, in physiological cardiac growth, PI3K is an integrator of cellular growth and metabolic remodeling. Although PI3K signaling to Akt1 is required for cellular growth, Akt-independent pathways mediate the accompanying mitochondrial adaptations.
Project description:Although microRNA-7 (miRNA-7) is known to regulate proliferation of cancer cells by targeting Epidermal growth factor receptor (EGFR/ERBB) family, less is known about its role in cardiac physiology. Transgenic (Tg) mouse with cardiomyocyte-specific overexpression of miRNA-7 was generated to determine its role in cardiac physiology and pathology. Echocardiography on the miRNA-7 Tg mice showed cardiac dilation instead of age-associated physiological cardiac hypertrophy observed in non-Tg control mice. Subjecting miRNA-7 Tg mice to transverse aortic constriction (TAC) resulted in cardiac dilation associated with increased fibrosis bypassing the adaptive cardiac hypertrophic response to TAC. miRNA-7 expression in cardiomyocytes resulted in significant loss of ERBB2 expression with no changes in ERBB1 (EGFR). Cardiac proteomics in the miRNA-7 Tg mice showed significant reduction in mitochondrial membrane structural proteins compared to NTg reflecting role of miRNA-7 beyond the regulation of EGFR/ERRB in mediating cardiac dilation. Consistently, electron microscopy showed that miRNA-7 Tg hearts had disorganized rounded mitochondria that was associated with mitochondrial dysfunction. These findings show that expression of miRNA-7 in the cardiomyocytes results in cardiac dilation instead of adaptive hypertrophic response during aging or to TAC providing insights on yet to be understood role of miRNA-7 in cardiac function.
Project description:PPARdelta (peroxisome proliferator-activated receptor delta) is a regulator of lipid metabolism and has been shown to induce fatty acid oxidation (FAO). PPARdelta transgenic and knock-out mice indicate an involvement of PPARdelta in regulating mitochondrial biogenesis and oxidative capacity; however, the precise mechanisms by which PPARdelta regulates these pathways in skeletal muscle remain unclear. In this study, we determined the effect of selective PPARdelta agonism with the synthetic ligand, GW501516, on FAO and mitochondrial gene expression in vitro and in vivo. Our results show that activation of PPARdelta by GW501516 led to a robust increase in mRNA levels of key lipid metabolism genes. Mitochondrial gene expression and function were not induced under the same conditions. Additionally, the activation of Pdk4 transcription by PPARdelta was coactivated by PGC-1alpha. PGC-1alpha, but not PGC-1beta, was essential for full activation of Cpt-1b and Pdk4 gene expression via PPARdelta agonism. Furthermore, the induction of FAO by PPARdelta agonism was completely abolished in the absence of both PGC-1alpha and PGC-1beta. Conversely, PGC-1alpha-driven FAO was independent of PPARdelta. Neither GW501516 treatment nor knockdown of PPARdelta affects PGC-1alpha-induced mitochondrial gene expression in primary myotubes. These results demonstrate that pharmacological activation of PPARdelta induces FAO via PGC-1alpha. However, PPARdelta agonism does not induce mitochondrial gene expression and function. PGC-1alpha-induced FAO and mitochondrial biogenesis appear to be independent of PPARdelta.