J. Michael O'Donnell

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.

Preferential Oxidation of Triacylglyceride-Derived Fatty Acids in Heart Is Augmented by the Nuclear Receptor PPARα

Rationale: Long chain fatty acids (LCFAs) are the preferred substrate for energy provision in hearts. However, the contribution of endogenous triacylglyceride (TAG) turnover to LCFA oxidation and the overall dependence of mitochondrial oxidation on endogenous lipid is largely unstudied. Objective: We sought to determine the role of TAG turnover in supporting LCFA oxidation and the influence of the lipid-activated nuclear receptor, proliferator-activated receptor (PPAR)&agr;, on this balance. Methods and Results: Palmitoyl turnover within TAG and palmitate oxidation rates were quantified in isolated hearts, from normal mice (nontransgenic) and mice with cardiac-specific overexpression of PPAR&agr; (MHC-PPAR&agr;). Turnover of palmitoyl units within TAG, and thus palmitoyl-coenzyme A recycling, in nontransgenic (4.5±2.3 &mgr;mol/min per gram dry weight) was 3.75-fold faster than palmitate oxidation (1.2±0.4). This high rate of palmitoyl unit turnover indicates preferential oxidation of palmitoyl units derived from TAG in normal hearts. PPAR&agr; overexpression augmented TAG turnover 3-fold over nontransgenic hearts, despite similar fractions of acetyl-coenzyme A synthesis from palmitate and oxygen use at the same workload. Palmitoyl turnover within TAG of MHC-PPAR&agr; hearts (16.2±2.9, P<0.05) was 12.5-fold faster than oxidation (1.3±0.2). Elevated TAG turnover in MHC-PPAR&agr; correlated with increased mRNA for enzymes involved in both TAG synthesis, Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), Dgat1 (diacylglycerol acetyltransferase 1), and Agpat3 (1-acylglycerol-3-phospate O-acyltransferase 3), and lipolysis, Pnliprp1 (pancreatic lipase related protein 1). Conclusions: The role of endogenous TAG in supporting &bgr;-oxidation in the normal heart is much more dynamic than previously thought, and lipolysis provides the bulk of LCFA for oxidation. Accelerated palmitoyl turnover in TAG, attributable to chronic PPAR&agr; activation, results in near requisite oxidation of LCFAs from TAG.

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.

Efficient, cardiac-specific adenoviral gene transfer in rat heart by isolated retrograde perfusion in vivo

While a number of virus-based delivery schemes have been developed for myocardial gene transfer, no technique has proven capable of modifying a majority of cardiac myocytes rapidly and homogeneously in the in vivo rat model, and most schemes result in significant infection of the liver and other organs. However, adenoviral delivery to the excised heart during retrograde perfusion can produce 67–92% efficient gene transfer. In this study, we adapt this isolation/perfusion scheme to the heart in vivo. We isolated the heart in vivo by simultaneously clamping all vessels to/from the heart. The heart was then continuously retrograde perfused through a catheter positioned in the aortic root. A second catheter in the right ventricle provided a path for efflux. After perfusing the heart for 7.5 min with calcium-free Tyrode solution followed by 90 s no-flow viral exposure (AdV.cmv.LacZ; 1012 viral particles/ml), gene transfer efficiency was 60% compared to 5% by a conventional cross-clamp technique. Infection of peripheral organs was dramatically reduced. Given the prevalence of the rat in so many models of heart disease, this enhancement of infection represents an advancement in viral-based delivery of exogenous genes to heart for the study of gene therapy in vivo.

Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo

Adenoviral gene transfer of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a to the hypertrophic heart in vivo has been consistently reported to lead to enhanced myocardial contractility. It is unknown if the faster skeletal muscle isoform, SERCA1, expressed in the whole heart in early failure, leads to similar improvements and whether metabolic requirements are maintained during an adrenergic challenge. In this study, Ad.cmv.SERCA1 was delivered in vivo to aortic banded and sham-operated Sprague-Dawley rat hearts. The total SERCA content increased 34%. At 48-72 h posttransfer, echocardiograms were acquired, hearts were excised and retrograded perfused, and hemodynamics were measured parallel to NMR measures of the phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) and energy substrate selection at basal and high workloads (isoproterenol). In the Langendorff mode, the rate-pressure product was enhanced 27% with SERCA1 in hypertrophic hearts and 10% in shams. The adrenergic response to isoproterenol was significantly potentiated in both groups with SERCA1. 31P NMR analysis of PCr/ATP revealed that the ratio remained low in the hypertrophic group with SERCA1 overexpression and was not further compromised with adrenergic challenge. 13C NMR analysis revealed fat and carbohydrate oxidation were unaffected at basal with SERCA1 expression; however, there was a shift from fats to carbohydrates at higher workloads with SERCA1 in both groups. Transport of NADH-reducing equivalents into the mitochondria via the alpha-ketoglutamate-malate transporter was not affected by either SERCA1 overexpression or adrenergic challenge in both groups. Echocardiograms revealed an important distinction between in vivo versus ex vivo data. In contrast to previous SERCA2a studies, the echocardiogram data revealed that SERCA1 expression compromised function (fractional shortening) in the hypertrophic group. Shams were unaffected. While our ex vivo findings support much of the earlier cardiomyocyte and transgenic data, the in vivo data challenge previous reports of improved cardiac function in heart failure models after SERCA intervention.

A novel pharmacological strategy by PTEN inhibition for improving metabolic resuscitation and survival after mouse cardiac arrest.

Sudden cardiac arrest (SCA) is a leading cause of death in the United States. Despite return of spontaneous circulation, patients die due to post-SCA syndrome that includes myocardial dysfunction, brain injury, impaired metabolism, and inflammation. No medications improve SCA survival. Our prior work suggests that optimal Akt activation is critical for cooling protection and SCA recovery. Here, we investigate a small inhibitor of PTEN, an Akt-related phosphatase present in heart and brain, as a potential therapy in improving cardiac and neurological recovery after SCA. Anesthetized adult female wild-type C57BL/6 mice were randomized to pretreatment of VO-OHpic (VO) 30 min before SCA or vehicle control. Mice underwent 8 min of KCl-induced asystolic arrest followed by CPR. Resuscitated animals were hemodynamically monitored for 2 h and observed for 72 h. Outcomes included heart pressure-volume loops, energetics (phosphocreatine and ATP from (31)P NMR), protein phosphorylation of Akt, GSK3β, pyruvate dehydrogenase (PDH) and phospholamban, circulating inflammatory cytokines, plasma lactate, and glucose as measures of systemic metabolic recovery. VO reduced deterioration of left ventricular maximum pressure, maximum rate of change in the left ventricular pressure, and Petco2 and improved 72 h neurological intact survival (50% vs. 10%; P < 0.05). It reduced plasma lactate, glucose, IL-1β, and Pre-B cell colony enhancing factor, while increasing IL-10. VO increased phosphorylation of Akt and GSK3β in both heart and brain, and cardiac phospholamban phosphorylation while reducing p-PDH. Moreover, VO improved cardiac bioenergetic recovery. We concluded that pharmacologic PTEN inhibition enhances Akt activation, improving metabolic, cardiovascular, and neurologic recovery with increased survival after SCA. PTEN inhibitors may be a novel pharmacologic strategy for treating SCA.

Multiphasic Regulation of Systemic and Peripheral Organ Metabolic Responses to Cardiac Hypertrophy.

Background— Reduced fat oxidation in hypertrophied hearts coincides with a shift of carnitine palmitoyl transferase I from muscle to increased liver isoforms. Acutely increased carnitine palmitoyl transferase I in normal rodent hearts has been shown to recapitulate the reduced fat oxidation and elevated atrial natriuretic peptide message of cardiac hypertrophy. Methods and Results— Because of the potential for reduced fat oxidation to affect cardiac atrial natriuretic peptide, and thus, induce adipose lipolysis, we studied peripheral and systemic metabolism in male C57BL/6 mice model of transverse aortic constriction in which left ventricular hypertrophy occurred by 2 weeks without functional decline until 16 weeks (ejection fraction, −45.6%; fractional shortening, −22.6%). We report the first evidence for initially improved glucose tolerance and insulin sensitivity in response to 2 weeks transverse aortic constriction versus sham, linked to enhanced insulin signaling in liver and visceral adipose tissue (epididymal white adipose tissue [WAT]), reduced WAT inflammation, elevated adiponectin, mulitilocular subcutaneous adipose tissue (inguinal WAT) with upregulated oxidative/thermogenic gene expression, and downregulated lipolysis and lipogenesis genes in epididymal WAT. By 6 weeks transverse aortic constriction, the metabolic profile reversed with impaired insulin sensitivity and glucose tolerance, reduced insulin signaling in liver, epididymal WAT and heart, and downregulation of oxidative enzymes in brown adipose tissue and oxidative and lipogenic genes in inguinal WAT. Conclusions— Changes in insulin signaling, circulating natriuretic peptides and adipokines, and varied expression of adipose genes associated with altered insulin response/glucose handling and thermogenesis occurred prior to any functional decline in transverse aortic constriction hearts. The findings demonstrate multiphasic responses in extracardiac metabolism to pathogenic cardiac stress, with early iWAT browning providing potential metabolic benefits.