Deactivation of the PPARaPGC1a Complex during Pathologic Cardiac Hypertrophic Growth

The next step was to determine whether, as predicted by the alteration in FAO enzyme expression, the activity of PPARa and its coactivator PGC-1a is altered in the hypertrophied heart in parallel with the metabolic changes. The results of several studies of rodent models of pressure overload hypertrophy have now demonstrated that the cardiac expression of the genes encoding PPARa and PGC-1 a are down-regulated within 7 days of the onset of ventricular pressure overload (Fig. 4B) (Sack et al. 1997; Barger et al. 2000). Studies have shown that steady-state nuclear levels of PPARa protein are decreased in the pathologically hypertrophied heart, whereas the levels of its transcriptional antagonist,

COUP-TF, are increased (Sack et al. 1997). In addition, studies performed in cardiac myocytes in culture revealed that the deactivation of PPARa-mediated control of M-CPT I gene transcription following exposure to a hyper-trophic agonist occurs within 24 hours, a surprisingly rapid response (Barger et al. 2000). This latter observation suggested that posttranslational mechanisms also served to rapidly deactivate PPARa during hypertrophic growth. Indeed, further studies demonstrated that activation of extracellular regulated kinase-mitogen-activated protein kinase (ERK-MAPK) leads to a rapid reduction in PPARa trans-activation following exposure to hypertrophic agonists such as the aj-adrenergic agonist phenylephrine. Last, recent studies have shown that in contrast to pressure overload-induced cardiac hypertrophy, the expression of the PPARa and PGC-1a genes is maintained at high levels in physiologic cardiac hypertrophy due to exercise training (A.R. Wende and D.P. Kelly, unpubl.). In summary, the PPARa/PGC-1a complex is deactivated at the level of gene expression and posttranslationally following the onset of pressure overload but not in physiologic forms of hypertrophic growth (Fig. 4C).

Do PPARa-mediated Alterations in Cardiac Energy Metabolism Influence the Cardiac Hypertrophic Phenotype?

Several lines of evidence suggest that alterations in cardiac energy substrate utilization are linked directly to hy-pertrophic growth programs. First, as noted above, children with inborn errors in the FAO pathway develop cardiac hypertrophy. Second, pharmacologic inhibition of CPT I and other FAO enzymes causes cardiac hypertrophy in cell culture and in vivo (Litwin et al. 1990; Vetter et al. 1995). Several recent studies have added further support to the notion that derangements in myocyte lipid metabolism serve as primary triggers of cardiac hypertrophy. A recent study by Schaffer and coworkers (Chiu et al. 2001) has provided evidence that abnormalities in my-ocardial lipid homeostasis, such as might occur when the capacity for FAO is diminished, lead to hypertrophic growth. Mice with cardiac-specific overexpression of acyl-CoA synthetase (ACS) develop cardiac hypertrophy associated with neutral lipid accumulation within my-ocytes. These results suggest that lipid-mediated signaling pathways may trigger a growth response. A second study in humans has suggested that the activity of PPARa may modify the cardiac hypertrophic response. Jamshidi and colleagues (Jamshidi et al. 2002; Kelly 2002) found that a single nucleotide polymorphism within the PPARa gene is associated with the degree of left ventricular hypertrophy due to exercise training in British Army volunteers. In addition, this same study found that PPARa genotype was a determinant of the degree of left ventricular hypertrophy caused by hypertension in a large cohort. Interestingly, the latter association was only observed in males. Taken together, these results suggest that PPARa-mediated alterations in FAO influence the hy-pertrophic response.

Deactivation of the PPARa/RXRa Complex in the Hypoxic Cardiac Myocyte

A critical cellular adaptive response to conditions of reduced oxygen availability involves the suppression of cellular energy consumption and production (Fahey and Lister 1989; Hochachka et al. 1996). Under hypoxic conditions, decreased oxygen consumption is achieved in part by increasing glycolysis while down-regulating mitochondrial FAO flux (Abdel-aleem et al. 1998; Rumsey et al. 1999). This metabolic switch, which is similar to that of the pathologically hypertrophied heart, would also reduce the generation of potentially toxic reactive species within the mitochondrion in hypoxic conditions. Recently, studies performed with neonatal cardiac myocytes in culture have shown that the PPARa-mediated activation of M-CPT I is diminished following exposure to hypoxic conditions (Huss et al. 2001). Gel mobility shift studies demonstrated that exposure to hypoxia leads to a reduction in PPARa/RXR DNA-binding activity. However, the acute reduction in PPARa/RXR activity is not caused by altered levels of PPARa as occurs in the hy-pertrophied heart. Rather, the levels of RXRa are reduced by hypoxic exposure. These results indicate that short-term (24 hours) exposure to hypoxic conditions leads to a rapid fall in the availability of RXRa, the obligate PPARa partner, effectively deactivating the PPARa-me-diated control of its target genes involved in FAO. In a separate study performed in vivo, longer periods of hy-poxia (days) were shown to cause a reduction in PPARa gene expression (Razeghi et al. 2001), identifying a second mechanism whereby hypoxia alters PPARa signaling.

DERANGEMENTS IN MYOCARDIAL

ENERGY METABOLISM IN THE DIABETIC HEART: THE ROLE OF PATHOLOGIC ACTIVATION OF THE PPARa/PGC-1a REGULATORY PATHWAY

The PPARa Gene Regulatory Pathway Is Activated in the Diabetic Heart

Diabetes mellitus is associated with increased cardiac morbidity and mortality (Kannel et al. 1974). Cardiomyopathy commonly occurs in diabetics independent of known risk factors such as coronary disease or hypertension (Rubler et al. 1972). Although little is known about the pathogenesis of diabetic cardiomyopathy, evidence is emerging that functional abnormalities are directly related to derangements in myocardial energy metabolism (Rodrigues et al. 1995; Stanley et al. 1997). In diabetes, the capacity of the heart to switch between utilization of fatty acids and glucose is severely constrained because the uptake and utilization of glucose is dependent on an intact insulin signaling pathway (Fig. 1). Accordingly, in the uncontrolled diabetic state, the heart relies almost exclusively on FAO to fulfill its ATP requirements (Rodrigues et al. 1995; Stanley et al. 1997; Belke et al. 2000). The chronic dependence of the diabetic heart on mito-chondrial FAO could have detrimental consequences, in-

Figure 4. Down-regulation of PPARa and FAO enzyme gene expression in the hypertrophied and failing heart. (A) FAO enzyme (MCAD and LCHAD) levels in LV from failing human hearts. Representative autoradiographs of northern (top) and western (bottom two rows) blot analyses performed with total RNA or protein prepared from LV of (normal LV function) controls and age-matched subjects with heart failure. Northern blot analyses were performed using radiolabeled cDNA probes listed at left. Western blot analyses were performed with a polyclonal anti-MCAD antibody and actin control antibody. (B) The PPARa pathway is deactivated in pressure overload-induced hypertrophied mouse heart. Representative autoradiographs of northern blot analyses performed with RNA from the LV of mice 7 days after placement of a constricting band around the transverse aortic arch (Band) or sham operation (Sham). A schematic of the aortic arch banding procedure is shown at top. Abbreviations: (MCAD) medium-chain acyl-CoA dehydrogenase; (LCHAD) 3-OH long-chain acyl-CoA dehydrogenase; (ANF) atrial natriuretic factor; (cTNI) cardiac troponin I; (ATPasee) ATPase subunit e; (M-CPT I) muscle-type carnitine palmitoyltransferase I; (ACO) acyl-CoA oxidase. (C) Schematic of the energy substrate and transcriptional switches known to occur during normal postnatal physiologic cardiac hypertrophic growth compared to the pathologic form caused by pressure overload. The boxes at the bottom denote chief source of energy. The deactivation of PPARa and PGC-1 occurs at both transcriptional and posttranscriptional levels as described in the text.

Control Heart Failure

MCAD LCHAD ANF GAPDH

cTNI I ATPasee band-

band-

sham Band Sham Band sham Band Sham Band

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Postnatal

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Hypertrophy t PPARa t PGC-1a

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Fatty Acid Oxidation

Glycolysis

MCAD Prot

cluding increased myocardial oxygen demand and accumulation of toxic intermediates derived from increased cellular fatty acid uptake and catabolism.

We have recently shown that the myocardial expression of PPARa, PGC-1a, and multiple downstream PPARa target genes involved in mitochondrial FAO is abnormally elevated in the hearts of mice with insulin-deficient or insulin-resistant forms of diabetes (Finck et al. 2002). Interestingly, the expression of the downstream PPARa target genes is not increased in the hearts of diabetic PPARa-null mice (B.N. Finck and D.P. Kelly, un-publ.). These results suggest that the increased capacity of the diabetic heart for fatty acid utilization is mediated, at least in part, by chronic activation of the PPARa gene regulatory pathway. To evaluate the potential role of chronic activation of the PPARa regulatory pathway in the development of the metabolic and functional disturbances of the diabetic heart, a transgenic mouse model with cardiac-restricted overexpression of PPARa was established. This transgenic model allowed the evaluation of the effects of PPARa on cardiac metabolism and function in the absence of systemic abnormalities related to the diabetic state. Mice transgenic for a PPARa cDNA downstream from the cardiac a-myosin heavy-chain promoter (MHC-PPAR mice) were generated (Fig. 5). As predicted, MHC-PPAR mice exhibited increased expres sion of PPARa target genes involved in the cellular uptake and oxidation of fatty acids (Finck et al. 2002). Interestingly, the expression of genes involved in glucose uptake (GLUT4), glycolysis, and glucose oxidation were coordinately down-regulated. These results provide evidence that primary activation of the fatty acid utilization pathway via PPARa leads to a counter-regulatory reduction in the expression of genes involved in glucose utilization. The mechanism for the transcriptional repression of glucose utilization enzymes by PPARa overexpression is unknown but is likely mediated through indirect pathways, given that the majority of these genes are not known to be direct PPARa targets.

Several lines of evidence demonstrated that, as predicted by the derangements in gene expression, myocar-dial metabolism is altered in the MHC-PPAR heart. First, micro-positron emission tomographic (micro-PET) studies of cardiac substrate uptake were performed with intact MHC-PPAR mice. The micro-PET studies revealed that the myocardial uptake of the tracer [11C]palmitate was increased and [18F]fluorodeoxyglucose (FDG) import was reduced in MHC-PPAR mouse heart (Fig. 6A). Second, analyses of substrate utilization rates using isolated working hearts from MHC-PPAR and nontransgenic littermate mice demonstrated that palmitate oxidation rates were increased ~65%, whereas glucose oxidation was reduced by over 60% in MHC-PPAR hearts compared to controls (Fig. 6B). Taken together with the gene expression data, these results indicate that the metabolic phenotype of the MHC-PPAR heart is remarkably similar to that of the diabetic heart.

/

aMHC Promoter

I PPARa

NTG 404-4

404-11 402-2

404-3

PPARa mRNA

PPARa protein

PPARa protein

FLAG-PPARa mRNA

FLAG-PPARa mRNA

Figure 5. Generation of transgenic mouse lines that overexpress PPARa in a cardiac-restricted manner (MHC-PPAR mice). The schematic at the top depicts the MHC-PPAR construct used to generate transgenic mice with cardiac-specific overexpression of PPARa. A FLAG-tagged PPARa cDNA was inserted downstream from the a-cardiac myosin heavy-chain promoter. Four independent lines of mice transgenic for the MHC-PPAR construct were established (404-4, 404-11, 402-2, 404-3). Representative autoradiographs of northern (top) and western (middle) blot studies performed with heart samples from 6-week-old mice from each transgenic line are displayed. At the exposure shown, endogenous PPARa could not be detected in nontrans-genic (NTG) samples. FLAG-PPARa protein was detected in the transgenic mice using an antibody directed against PPARa. Cardiac-specific expression of FLAG-PPARa mRNA was confirmed by northern blot analysis performed with multiple tissues. Abbreviations: (NTG) nontransgenic; (H) heart; (BAT) brown adipose tissue; (SM) skeletal muscle; (K) kidney; (L) liver.

MHC-PPAR Mice Provide Evidence for a Link between Metabolic and Functional Derangements in the Diabetic Heart

The MHC-PPAR mice afforded the opportunity to determine whether altered myocardial metabolism due to chronic activation of the PPARa pathway leads to functional abnormalities known to occur in the diabetic heart (diabetic cardiomyopathy). The initial stages of diabetic cardiomyopathy are characterized by ventricular hypertrophy and diastolic dysfunction. In severe cases, cardiac abnormalities can progress to systolic ventricular dysfunction and overt congestive heart failure. Indeed, MHC-PPAR mice exhibit an increased biventricular weight/body weight ratio in a pattern dependent on the level of transgene expression (Finck et al. 2002). In addition, MHC-PPAR mice exhibit reduced ventricular systolic function and chamber dilatation as determined by echocardiography (Fig. 7). The degree of ventricular dysfunction in MHC-PPAR mice was worsened when the mice were rendered insulin-deficient by administration of the pancreatic islet cell toxin streptozotocin (B.N. Finck and D.P. Kelly, unpubl.). In summary, the MHC-PPAR mice develop both metabolic and functional characteristics of the diabetic heart, indicating that primary derangements in mitochondrial substrate utilization can lead to functional consequences. Although the heightened rate of mitochondrial FAO in the diabetic heart likely represents an adaptive response in the short term, the dysfunctional phenotype of the MHC-PPAR heart suggests that this metabolic alteration may become maladaptive, contributing to the development of diabetic cardiomyopathy.

The Potential Role of "Lipotoxicity" in the Development of Cardiomyopathy in MHC-PPAR Mice: Relevance to the Diabetic State

How do the metabolic derangements of the MHC-PPAR heart or the diabetic heart lead to cardiac hypertrophy and dysfunction? A clue was provided by the observation that in hearts of diabetic animals, histologic analyses often reveal evidence of lipid droplet accumulation within myocytes (Murthy and Shipp 1977; Paulson and Crass 1982; Zhou et al. 2000). Indeed, recent evidence indicates that lipid accretion in the myocyte may trigger hypertrophic growth and apoptosis (Chiu et al. 2001). It is possible that the increased rate of myocardial fatty acid uptake, especially in the context of elevated circulating plasma lipids as seen in the uncontrolled diabetic, outpaces the increased oxidative capacity of the diabetic cardiomyocyte. This possibility was evaluated in MHC-PPAR mice by subjecting them to a short-term fast to acutely elevate circulating free fatty acid levels. Histo-logic studies using oil red O staining and quantitative

Palmitate Oxidation

Palmitate Oxidation

Figure 6. Myocardial palmitate utilization is increased and glucose utilization reduced in MHC-PPAR mice. (A) Panels at left contain representative images of nC-palmitate and 18F-fluorodeoxyglucose (FDG) uptake into myocardium as determined by micropositron emission tomography (microPET) in nontransgenic and MHC-PPAR littermate mice. The relative amount of tracer uptake into the mouse heart 15 seconds after bolus injection of C-palmitate or 18F-FDG into the jugular vein is indicated by the color scale (0-100). The arrow indicates the cardiac field. As shown in the upper panel, the color field is increased into the red scale in the hearts of MHC-PPAR mice infused with 11C-palmitate, which is indicative of enhanced myocardial uptake of FA. Conversely, uptake of 18F-FDG is substantially lower in hearts of MHC-PPAR mice compared to NTG littermates. (B) Myocardial palmitate oxidation is increased and glucose oxidation reduced in MHC-PPAR mice. The oxidation of [9,10-3H]palmitate and [U-C]glucose was assessed in isolated working hearts of MHC-PPAR or nontransgenic littermate mice. Bars represent mean (± s.e.m.) oxidation rates expressed as nmole of substrate oxidized/min/g dry mass. *p<0.05 versus nontransgenic littermate mice. (A, Reprinted, with permission, from Finck et al. 2002.)

Figure 6. Myocardial palmitate utilization is increased and glucose utilization reduced in MHC-PPAR mice. (A) Panels at left contain representative images of nC-palmitate and 18F-fluorodeoxyglucose (FDG) uptake into myocardium as determined by micropositron emission tomography (microPET) in nontransgenic and MHC-PPAR littermate mice. The relative amount of tracer uptake into the mouse heart 15 seconds after bolus injection of C-palmitate or 18F-FDG into the jugular vein is indicated by the color scale (0-100). The arrow indicates the cardiac field. As shown in the upper panel, the color field is increased into the red scale in the hearts of MHC-PPAR mice infused with 11C-palmitate, which is indicative of enhanced myocardial uptake of FA. Conversely, uptake of 18F-FDG is substantially lower in hearts of MHC-PPAR mice compared to NTG littermates. (B) Myocardial palmitate oxidation is increased and glucose oxidation reduced in MHC-PPAR mice. The oxidation of [9,10-3H]palmitate and [U-C]glucose was assessed in isolated working hearts of MHC-PPAR or nontransgenic littermate mice. Bars represent mean (± s.e.m.) oxidation rates expressed as nmole of substrate oxidized/min/g dry mass. *p<0.05 versus nontransgenic littermate mice. (A, Reprinted, with permission, from Finck et al. 2002.)

analyses of myocardial triacylglyceride (TAG) using electrospray ionization mass spectrometry (ESIMS) revealed markedly increased TAG levels in fasted MHC-PPAR mice (Fig. 8) (Finck et al. 2002). A similar pattern of myocardial lipid accumulation was observed in diabetic wild-type mice, albeit of lesser magnitude. Strikingly, the combination of insulin deficiency and the MHC-PPAR genotype leads to a massive increase in my-ocardial TAG content (B.N. Finck and D.P. Kelly, un-publ.). These results suggest that abnormalities in my-ocardial lipid balance lead to ventricular hypertrophy and dysfunction in the diabetic heart.

Blood Pressure Health

Blood Pressure Health

Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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