Role of fatty acids and peroxisome proliferator-activated receptor alpha in cardiac muscle

The aim of this project is to investigate the effects of increased fatty acid (FA) levels in cardiac muscle. Several lines of evidence point to a role of FAs in the pathogenesis of cardiomyopathy. Impaired degradation of FAs caused by inborn errors of metabolism results in cardiomyopathy pointing to a direct toxic effect. FA could also play a role in the development of diabetic cardiomyopathy since in an insulin-deficient state, energy production is shifted almost exclusively towards beta-oxidation of free FA (FFA). This concept has been supported by results from experimental animal models of cardiomyopathy and diabetes. The peroxisome proliferator activated receptor, (PPAR-alpha) is one of the key regulatory elements of the cellular adaptive mechanism in response to increased FA levels. In addition to hypolipidemic drugs and other synthetic compounds, FA are potent PPAR-alpha activators and increase transcription of peroxisomal, mitochondrial and cytochrome P450 target genes. Although humans are refractory to most classes of peroxisome proliferators with respect to peroxisome proliferation, PPAR-alpha is expressed and proliferator-responsive elements have been identified in potential target genes. Mice with targeted disruption of PPAR-alpha do not display the peroxisome proliferator pleiotropic response but are viable, fertile and exhibit no gross phenotypic defects although some changes in lipid metabolism cap be detected. In, previous experiments using transgenic mice overexpressing human lipoprotein lipase (hLPL) under the control of a muscle specific promotor, we have been able to show that increased intracellular FFAs in skeletal muscle cause proliferation of peroxisomes and mitochondria as well as an increase in marker enzyme activity. In contrast, no changes in PPAR-alpha mRNA was found. In addition, myopathy characterised by glycogen storage, muscle fibre degeneration, and fibre atrophy with centralisation of nuclei, myopathies was noted. The effects of FFAs on cardiac muscle will. be investigated using several transgenic mouse models. In preliminary experiments using PPAR-alpha deficient mice overexpressing hLPL under control of a muscle specific promoter, we observed premature death of male animals. PlasmaFFA, were elevated and glucose levels decreased but not significantly different from PPAR-alpha-/- animals. In perfused isolated hearts, left ventricular developed pressure was lower indicating reduced myocardial function. Using mice with tissue specific expression of hLPL, FFA are increased in cardiac or skeletal and cardiac muscle to directly test their effect on the heart functionally and pathohistologically. By crossbreeding these mice with PPAR- alpha-/- mice. PPAR-alpha mediated effects can be discerned. Cardiac muscle will be investigated on histological, ultrastructural and molecular level. Functional analysis will include inotropic state, cardiac rhythm, and coronary flow. As metabolic parameters, fatty acids, acylcarnitines, glucose, insulin, glycogen and plasmalogens will be examined, Expression of FA metabolising enzymes will be investigated on protein and mRNA level. In addition, changes in expression patterns of proteins and mRNA species will be studied in cardiomyocytes. Furthermore it will be attempted to reverse the observed gender specific effect of reduced cardiac function and premature death. We believe that these mice constitute a powerful tool to further probe the role of FFA in cardiac disease and that the results of our investigation could also be of importance for the explanation of the role of FAs in human cardiomyopathies such as diabetic cardiomyopathy.

The aim of this project was to investigate the effects of increased fatty acid (FA) levels in cardiac muscle. Several lines of evidence point to a role of FAs in the pathogenesis of cardiomyopathy. Impaired degradation of FAs caused by inborn errors of metabolism results in cardiomyopathy pointing to a direct toxic effect. FA could also play a role in the development of diabetic cardiomyopathy since in an insulin-deficient state, energy production is shifted almost exclusively towards ß-oxidation of free FA (FFA). This concept has been supported by results from experimental animal models of cardiomyopathy and diabetes. To elucidate the role of PPARa in FA metabolism in muscle, transgenic mouse lines were generated that express human LPL in skeletal and cardiac muscle and lack PPARa , a transcription factor which is believed to be a key regulator of FA metabolism. LPL overexpression in muscle tissue of male mice results in growth retardation and reduces their life span. Additional disruption of PPARa shortens it even further. However, only the combination of both manipulations results in myocardial and coronary dysfunction. These effects are accompanied by metabolic changes that are likely to affect heart function, namely a LPL- and PPAR-/-dependent deprivation of FA. However, this deprivation is not compensated for by heightened FA transport rate into myocardial cells. The functional impairments appear not to be related to excessive lipid accumulation following disruption of PPARa . This suggests that in situations where FFA are the primary source of energy production (such as fasting and diabetes), cardiomyopathy can also be caused by pathogenic mechanisms other than lipid accumulation. Therefore, combining genetic modifications such as overexpression of LPL and knockout of PPARa may be a valuable novel approach for the elucidation of physiological modulations, pathological changes, and interactions in cellular lipid metabolism. Preliminary results indicate that several additional genes might play a role in the establishment of metabolic cardiomyopathies. Increased levels of triglyceride rich lipoproteins provoke lipid accumulation in the artery wall, triggering early inflammatory responses central to atherosclerosis such as endothelial adhesion molecule expression. The endogenous mechanisms limiting such reactions remain are largely unknown. Since LPL plays a central role in lipid metabolism by hydrolyzing triglyceride rich lipoproteins and releasing FA, we investigated its role in vitro as well as in vivo. We found that LPL action could reverse early events associated with atherosclerosis. In fact, these LPL effects were absent in endothelial cells isolated from PPARa-deficient mice. This suggests a novel anti- inflammatory role for LPL. In vivo, transgenic mice over-expressing LPL had increased peroxisome proliferation but not in the genetic absence of PPARa . Although human plasma possesses minimal PPARa activators despite its abundant free FA, marked PPARa activation is seen with human plasma after LPL is added in vitro or systemically released in vivo. Interestingly, fasting, a state dependent on intracellular triglyceride utilization, induces PPARa expression. Thus, PPARa would appear an integral adaptive response to energy supply and demand, either induced by fasting or activated through LPL after feeding. These data suggest a novel pathway in which the key lipolytic enzyme LPL can act on circulating lipoproteins to generate PPARa ligands, providing a potentially important link between lipoprotein metabolism and PPARa effects which might reduce the risk for atherosclerosis.