Natasha H. Banke

The transcriptional coactivator PGC-1α is essential for maximal and efficient cardiac mitochondrial fatty acid oxidation and lipid homeostasis

High-capacity mitochondrial ATP production is essential for normal function of the adult heart, and evidence is emerging that mitochondrial derangements occur in common myocardial diseases. Previous overexpression studies have shown that the inducible transcriptional coactivator peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is capable of activating postnatal cardiac myocyte mitochondrial biogenesis. Recently, we generated mice deficient in PGC-1alpha (PGC-1alpha(-/-) mice), which survive with modestly blunted postnatal cardiac growth. To determine if PGC-1alpha is essential for normal cardiac energy metabolic capacity, mitochondrial function experiments were performed on saponin-permeabilized myocardial fibers from PGC-1alpha(-/-) mice. These experiments demonstrated reduced maximal (state 3) palmitoyl-l-carnitine respiration and increased maximal (state 3) pyruvate respiration in PGC-1alpha(-/-) mice compared with PGC-1alpha(+/+) controls. ATP synthesis rates obtained during maximal (state 3) respiration in permeabilized myocardial fibers were reduced for PGC-1alpha(-/-) mice, whereas ATP produced per oxygen consumed (ATP/O), a measure of metabolic efficiency, was decreased by 58% for PGC-1alpha(-/-) fibers. Ex vivo isolated working heart experiments demonstrated that PGC-1alpha(-/-) mice exhibited lower cardiac power, reduced palmitate oxidation, and increased reliance on glucose oxidation, with the latter likely a compensatory response. (13)C NMR revealed that hearts from PGC-1alpha(-/-) mice exhibited a limited capacity to recruit triglyceride as a source for lipid oxidation during beta-adrenergic challenge. Consistent with reduced mitochondrial fatty acid oxidative enzyme gene expression, the total triglyceride content was greater in hearts of PGC-1alpha(-/-) mice relative to PGC-1alpha(+/+) following a fast. Overall, these results demonstrate that PGC-1alpha is essential for the maintenance of maximal, efficient cardiac mitochondrial fatty acid oxidation, ATP synthesis, and myocardial lipid homeostasis.

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.

Early impairment of transmural principal strains in the left ventricular wall after short-term, high-fat feeding of mice predisposed to cardiac steatosis.

Background—Myocardial lipid accumulation precedes some cardiomyopathies, but little is known of concurrent effects on ventricular mechanics. We tested the hypothesis that intramyocardial lipid accumulation during a short-term, high-fat diet (HFD) affects 2-dimensional strains in the heart. We examined the hearts of nontransgenic (NTG) mice and of transgenic mice predisposed to elevated triacylglyceride (TAG) storage linked to low-level overexpression of peroxisome proliferator activated receptor (PPAR-&agr;). Methods and Results—Myocardial lipid and transmural principal strains E1 and E2 were determined in vivo with 1H magnetic resonance spectroscopy/imaging before and after 2 weeks of an HFD in both PPAR-&agr; and NTG littermate mice. Baseline lipid was elevated in PPAR-&agr; compared with NTG mice. An HFD increased mobile lipid by 174% in NTG mice (P<0.05) and by 79% in PPAR-&agr; mice (P<0.05). After an HFD, lipid and TAG were higher in PPAR-&agr; versus NTG mice by 63% and 81%, respectively. However, TAG in PPAR-&agr; mice after an HFD was similar to TAG in PPAR-&agr; mice fed a regular diet, suggesting that the magnetic resonance spectroscopy signal from lipid is not exclusive to TAG. Only at the highest lipid contents, achieved in PPAR-&agr; mice, were strains affected. Endocardial strain was most compromised, with a negative correlation to lipid (P<0.05). Conclusions—A short-term HFD elevated myocardial lipid measures as determined by magnetic resonance spectroscopy, which became dissociated from TAG content in hearts predisposed to cardiac steatosis. The increased lipid was associated with concurrent, transmural reductions in E1 and E2 strains across the left ventricular wall. Strains were attenuated at the highest levels of lipid accumulation, suggesting a threshold response. Thus, 2-dimensional strains are impaired early and without left ventricular diastolic dysfunction, owing to cardiac steatosis.

Multiphasic triacylglycerol dynamics in the intact heart during acute in vivo overexpression of CD36

Cardiac triacylglycerol (TAG) stores buffer the intracellular availability of long chain fatty acid (LCFA) that act as nuclear receptor ligands, substrate for lipotoxic derivatives, and high energy-yield fuel. The kinetic characteristics of TAG turnover and homeostatic mechanisms linking uptake and storage dynamics in hearts have until now remained elusive. This work examines TAG pool dynamics in the intact beating heart, under normal conditions and in response to acute gene expression-induced changes in CD36. Dynamic mode 13C NMR elucidated multiple kinetic processes in 13C-palmitate incorporation into TAG: an initial, saturable exponential component and a slower linear rate. Although previous work indicates the linear component to reflect TAG turnover, we hypothesized the saturable exponential to reflect transport of LCFA across the sarcolemma. Thus, we overexpressed the LCFA transporter CD36 through cardiac-specific adenoviral infection in vivo. Within 72 h, CD36 expression was increased 40% in intact hearts, accelerating the exponential phase relative to PBS-infused hearts. TAG turnover also increased with elevations in adipose triglyceride lipase (ATGL) and a modest increase in diacylglycerol acyltransferase 1 (DGAT1), without a significant expansion of the intracellular lipid pools. The results demonstrate a dynamic system of reciprocal gene regulation that couples saturable LCFA uptake across the sarcolemma to TAG synthesis/lipolysis rates.

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.

Impaired cytosolic NADH shuttling and elevated UCP3 contribute to inefficient citric acid cycle flux support of postischemic cardiac work in diabetic hearts.

Diabetic hearts are subject to more extensive ischemia/reperfusion (ISC/REP) damage. This study examined the efficiency of citric acid cycle (CAC) flux and the transfer of cytosolic reducing equivalents into the mitochondria for oxidative support of cardiac work following ISC/REP in hearts of c57bl/6 (NORM) and type 2 diabetic, db/db mouse hearts. Flux through the CAC and malate-aspartate shuttle (MA) were monitored via dynamic (13)C NMR of isolated hearts perfused with (13)C palmitate+glucose. MA flux was lower in db/db than NORM. Oxoglutarate malate carrier (OMC) was elevated in the db/db heart, suggesting a compensatory response to low NADHc. Baseline CAC flux per unit work (rate-pressure-product, RPP) was similar between NORM and db/db, but ISC/REP reduced the efficiency of CAC flux/RPP by 20% in db/db. ISC/REP also increased UCP3 transcription, indicating potential for greater uncoupling. Therefore, ISC/REP induces inefficient carbon utilization through the CAC in hearts of diabetic mice due to the combined inefficiencies in NADHc transfer per OMC content and increased uncoupling via UCP3. Ischemia and reperfusion exacerbated pre-existing mitochondrial defects and metabolic limitations in the cytosol of diabetic hearts. These limitations and defects render diabetic hearts more susceptible to inefficient carbon fuel utilization for oxidative energy metabolism.

Acute liver carnitine palmitoyltransferase I overexpression recapitulates reduced palmitate oxidation of cardiac hypertrophy.

Rationale: Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts. Objective: This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts. Methods and Results: L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 &mgr;mol/min per gram of dry weight, mean±SE) vs PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling. Conclusions: The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.

Early Impairment of Transmural Principal Strains in the Left Ventricle Wall Following Short-Term, High Fat Feeding of Mice Predisposed to Cardiac Steatosis

Background—Myocardial lipid accumulation precedes some cardiomyopathies, but little is known of concurrent effects on ventricular mechanics. We tested the hypothesis that intramyocardial lipid accumulation during a short-term, high-fat diet (HFD) affects 2-dimensional strains in the heart. We examined the hearts of nontransgenic (NTG) mice and of transgenic mice predisposed to elevated triacylglyceride (TAG) storage linked to low-level overexpression of peroxisome proliferator activated receptor (PPAR-&agr;). Methods and Results—Myocardial lipid and transmural principal strains E1 and E2 were determined in vivo with 1H magnetic resonance spectroscopy/imaging before and after 2 weeks of an HFD in both PPAR-&agr; and NTG littermate mice. Baseline lipid was elevated in PPAR-&agr; compared with NTG mice. An HFD increased mobile lipid by 174% in NTG mice (P<0.05) and by 79% in PPAR-&agr; mice (P<0.05). After an HFD, lipid and TAG were higher in PPAR-&agr; versus NTG mice by 63% and 81%, respectively. However, TAG in PPAR-&agr; mice after an HFD was similar to TAG in PPAR-&agr; mice fed a regular diet, suggesting that the magnetic resonance spectroscopy signal from lipid is not exclusive to TAG. Only at the highest lipid contents, achieved in PPAR-&agr; mice, were strains affected. Endocardial strain was most compromised, with a negative correlation to lipid (P<0.05). Conclusions—A short-term HFD elevated myocardial lipid measures as determined by magnetic resonance spectroscopy, which became dissociated from TAG content in hearts predisposed to cardiac steatosis. The increased lipid was associated with concurrent, transmural reductions in E1 and E2 strains across the left ventricular wall. Strains were attenuated at the highest levels of lipid accumulation, suggesting a threshold response. Thus, 2-dimensional strains are impaired early and without left ventricular diastolic dysfunction, owing to cardiac steatosis.

Peroxisome proliferator-activated receptor-α expression induces alterations in cardiac myofilaments in a pressure-overload model of hypertrophy

Although alterations in fatty acid (FA) metabolism have been shown to have a negative impact on contractility of the hypertrophied heart, the targets of action remain elusive. In this study we compared the function of skinned fiber bundles from transgenic (Tg) mice that overexpress a relatively low level of the peroxisome proliferator-activated receptor α (PPARα), and nontransgenic (NTg) littermates. The mice (NTg-T and Tg-T) were stressed by transverse aortic constriction (TAC) and compared with shams (NTg-S and Tg-S). There was an approximate 4-fold increase in PPARα expression in Tg-S compared with NTg-S, but Tg-T hearts showed the same PPARα expression as NTg-T. Expression of PPARα did not alter the hypertrophic response to TAC but did reduce ejection fraction (EF) in Tg-T hearts compared with other groups. The rate of actomyosin ATP hydrolysis was significantly higher in Tg-S skinned fiber bundles compared with all other groups. Tg-T hearts showed an increase in phosphorylation of specific sites on cardiac myosin binding protein-C (cMyBP-C) and β-myosin heavy chain isoform. These results advance our understanding of potential signaling to the myofilaments induced by altered FA metabolism under normal and pathological states. We demonstrate that chronic and transient PPARα activation during pathological stress alters myofilament response to Ca2+ through a mechanism that is possibly mediated by MyBP-C phosphorylation and myosin heavy chain isoforms.NEW & NOTEWORTHY Data presented here demonstrate novel signaling to sarcomeric proteins by chronic alterations in fatty acid metabolism induced by PPARα. The mechanism involves modifications of key myofilament regulatory proteins modifying cross-bridge dynamics with differential effects in controls and hearts stressed by pressure overload.

Acute L-CPT1 Overexpression Recapitulates Reduced Palmitate Oxidation of Cardiac Hypertrophy

Rationale: Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts. Objective: This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts. Methods and Results: L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 &mgr;mol/min per gram of dry weight, mean±SE) vs PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling. Conclusions: The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.