Kayla M Pound

Recruitment of Compensatory Pathways to Sustain Oxidative Flux With Reduced Carnitine Palmitoyltransferase I Activity Characterizes Inefficiency in Energy Metabolism in Hypertrophied Hearts

Background— Transport rates of long-chain free fatty acids into mitochondria via carnitine palmitoyltransferase I relative to overall oxidative rates in hypertrophied hearts remain poorly understood. Furthermore, the extent of glucose oxidation, despite increased glycolysis in hypertrophy, remains controversial. The present study explores potential compensatory mechanisms to sustain tricarboxylic acid cycle flux that resolve the apparent discrepancy of reduced fatty acid oxidation without increased glucose oxidation through pyruvate dehydrogenase complex in the energy-poor, hypertrophied heart. Methods and Results— We studied flux through the oxidative metabolism of intact adult rat hearts subjected to 10 weeks of pressure overload (hypertrophied; n=9) or sham operation (sham; n=8) using dynamic 13C–nuclear magnetic resonance. Isolated hearts were perfused with [2,4,6,8,10,12,14,16-13C8] palmitate (0.4 mmol/L) plus glucose (5 mmol/L) in a 14.1-T nuclear magnetic resonance magnet. At similar tricarboxylic acid cycle rates, flux through carnitine palmitoyltransferase I was 23% lower in hypertrophied (P<0.04) compared with sham hearts and corresponded to a shift toward increased expression of the L–carnitine palmitoyltransferase I isoform. Glucose oxidation via pyruvate dehydrogenase complex did not compensate for reduced palmitate oxidation rates. However, hypertrophied rats displayed an 83% increase in anaplerotic flux into the tricarboxylic acid cycle (P<0.03) that was supported by glycolytic pyruvate, coincident with increased mRNA transcript levels for malic enzyme. Conclusions— In cardiac hypertrophy, fatty acid oxidation rates are reduced, whereas compensatory increases in anaplerosis maintain tricarboxylic acid cycle flux and account for a greater portion of glucose oxidation than previously recognized. The shift away from acetyl coenzyme A production toward carbon influx via anaplerosis bypasses energy, yielding reactions contributing to a less energy-efficient heart.

Substrate–Enzyme Competition Attenuates Upregulated Anaplerotic Flux Through Malic Enzyme in Hypertrophied Rat Heart and Restores Triacylglyceride Content. Attenuating Upregulated Anaplerosis in Hypertrophy

Recent work identifies the recruitment of alternate routes for carbohydrate oxidation, other than pyruvate dehydrogenase (PDH), in hypertrophied heart. Increased carboxylation of pyruvate via cytosolic malic enzyme (ME), producing malate, enables “anaplerotic” influx of carbon into the citric acid cycle. In addition to inefficient NADH production from pyruvate fueling this anaplerosis, ME also consumes NADPH necessary for lipogenesis. Thus, we tested the balance between PDH and ME fluxes in hypertrophied hearts and examined whether low triacylglyceride (TAG) was linked to ME-catalyzed anaplerosis. Sham-operated (SHAM) and aortic banded rat hearts (HYP) were perfused with buffer containing either 13C-palmitate plus glucose or 13C glucose plus palmitate for 30 minutes. Hearts remained untreated or received dichloroacetate (DCA) to activate PDH and increase substrate competition with ME. HYP showed a 13% to 26% reduction in rate pressure product (RPP) and impaired dP/dt versus SHAM (P<0.05). DCA did not affect RPP but normalized dP/dt in HYP. HYP had elevated ME expression with a 90% elevation in anaplerosis over SHAM. Increasing competition from PDH reduced anaplerosis in HYP+DCA by 18%. Correspondingly, malate was 2.2-fold greater in HYP than SHAM but was lowered with PDH activation: HYP=1419±220 nmol/g dry weight; HYP+DCA=343±56 nmol/g dry weight. TAG content in HYP (9.7±0.7 &mgr;mol/g dry weight) was lower than SHAM (13.5±1.0 &mgr;mol/g dry weight). Interestingly, reduced anaplerosis in HYP+DCA corresponded with normalized TAG (14.9±0.6 &mgr;mol/g dry weight) and improved contractility. Thus, we have determined partial reversibility of increased anaplerosis in HYP. The findings suggest anaplerosis through NADPH-dependent, cytosolic ME limits TAG formation in hypertrophied hearts.

Sexual dimorphism in cardiac triacylglyceride dynamics in mice on long term caloric restriction

Human studies indicate augmented myocardial lipid metabolism in females, and that sex and obesity interact to predict myocardial fatty acid oxidation and storage. Altered lipid dynamics precede cardiomyopathies, and many studies now address high fat diets. Conversely, caloric restriction (CR), is the most studied model for longevity and stress resistance, including protection against myocardial ischemia. However, no information exists on the effects of long-term caloric restriction (CR) on triacylglyceride (TAG) content and dynamics in the heart. This study explored the effects of CR, sex and age on TAG dynamics in mouse hearts. Male and female SVJ129 mice were fed either normal (ND) or CR diet for 3 or 10 months. In 5-month-old mice, CR similarly decreased cardiac TAG in males (ND: 25.5±4.5 nmol/mg protein; CR: 12.6±2.7, P<0.05) and females (ND: 30.1±4.4; CR: 13.7±1.2) (no significant differences in TAG content were seen between sexes). CR reduced the contribution of exogenous palmitate to oxidative metabolism in males and females, by 15% and 11% respectively, versus ND, without affecting cardiac workload. CR also induced a larger reduction in TAG turnover in male (68%) than female hearts (38%). Interestingly, in 5 month old male mice, CR reproduced the lower TAG turnover rates of middle-aged males (ND 13-month-old male=423±76 nmol/mg protein/min). Thus, long term CR reduces TAG pool dynamics. Despite reduced content, hearts of female mice subjected to CR retained a more dynamic TAG pool than males, while males respond with greater metabolic remodeling of cardiac lipid dynamics.

SERCA1 expression enhances the metabolic efficiency of improved contractility in post-ischemic heart.

Myocardial stunning is characterized by a metabolic uncoupling from function as mitochondrial tricarboxylic acid (TCA) cycle and oxygen consumption remain normal despite reduced contractility. Overexpression of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1) in hearts has recently been reported to reduce dysfunction at reperfusion. In this study we determine whether the metabolic coupling to function improves with SERCA treatment. PBS (control) or adenovirus carrying the cDNA for SERCA1 was delivered via coronary perfusion in vivo to Sprague-Dawley rat hearts. Three days following gene transfer, isolated hearts were perfused with 0.4 mM [2,4,6,8,10,12,14,16-13C8] palmitate and 5 mM glucose, and subjected to 15-min ischemia followed by 40-min reperfusion. Consistent with myocardial stunning, rate pressure product (RPP) and left ventricular developed pressure (LVDP) were depressed 30-40% (p<0.05) in the PBS group. With SERCA1 overexpression, dP/dt was 20% greater than controls (p<0.05), and LVDP and RPP recovered to pre-ischemic values. From dynamic 13C NMR, TCA cycle flux at reperfusion was similar to pre-ischemic values for both groups. Therefore, the efficiency of coupling between cardiac work and TCA cycle flux was restored with SERCA1 treatment. Oxidative efficiency was also enhanced with SERCA1 as cytosolic NADH transport into the mitochondria was significantly greater compared to the PBS group. In addition, the phosphocreatine to ATP ratio (PCr/ATP) was not compromised with SERCA1 expression, despite enhanced function, and depressed fatty acid oxidation at 40-min reperfusion in the PBS group was not reversed with SERCA1. These data demonstrate that metabolic coupling and NADH transport are significantly improved with SERCA1 treatment.