A role of palmitoylcarnitine in the onset of arrhythmias and deterioration of mechanical function in the postischemic heart is suggested. Patients with genetic carnitine palmitoyltransferase (CPT) deficiencies die in cardiac arrhythmia. Morbidity due to these deficiencies is likely underestimated. The hear contains two isoforms of CPT-I, both of which produce palmitoylcarnitine for beta-oxidation. In adults, the liver isoform constitutes 2-3% of heart activity and is 100-fold less sensitive than the muscle isoform to its inhibitor, malonyl-CoA. Liver CPT-I fluctuates in activity with inhibitor sensitivity depending on diet or hormones. Liver CPT-I contributes 25% to neonatal CPT-I. Electrical stimulation of neonatal cardiac myocytes produces cellular maturation that is accompanied by proliferation and differentiation of mitochondria, the latter exemplified by isoform switching from the liver CPT-I to adult muscle CPT-I. The ability to follow sequential gene activation is a unique feature of this cardiac myocyte system and will allow us to elucidate the pathway(s) involved in the differentiation response. To understand the mechanism of CPT-I switching, [we have determined the rat muscle CPT-I gene structure and identified the minimal promoter by sequential deletion analysis.] The role of transcriptional activity in the increased muscle CPT-mRNA will be tested using 5 'flanking sequences and mutational analysis of transfected pCPT-I.Luc constructs, DNase footprinting and gel band shift assay. Novel proteins involved in isoform switching will be characterized by cloning and/or purification. [To follow the cellular integration involved in the switch to the highly malonyl-CoA sensitive isoform, the model of electrical stimulation will be used to follow maturation of the malonyl-CoA sensitive isoform, the model of electrical stimulation will be used to follow maturation of the malonyl-CoA synthetic pathway. The role of contractile activity) and fatty acids in the control of malonyl-CoA production and beta-oxidation [during mitochondrial differentiation] will be examined by measuring the effects of glucose and fatty acids on the AMP/kinase/acetyl-CoA carboxylase phosphorylation cascade. We will test the hypothesis that [as the mitochondria increase and mature, greater proportions of] myocyte malonyl-CoA is present in the mitochondrial compartment as a result of intramitochondrial synthesis of malonyl-CoA by propionyl-CoA carboxylase using acetyl-CoA as a substrate. Location of a malonyl-CoA compartment that is inaccessible to CPT-I will shed light on the inconsistency between the high malonyl CoA content in heart and reliance on beta-oxidation.
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