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After cardiac ischemia, long-chain fatty acids, such as palmitate, increase in plasma and heart. Palmitate has previously been shown to cause apoptosis in cardiac myocytes. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. Phosphatidylserine translocation and caspase 3-like activity confirmed the apoptotic action of palmitate. Cytosolic cytochrome cwas detected at 8 h and plateaued at 12 h. The mitochondrial membrane potential (ΔΨ) in tetramethylrhodamine ethyl ester-loaded cardiac myocytes decreased significantly in individual mitochondria by 8 h. This loss was heterogeneous, with a few energized mitochondria per myocyte remaining at 24 h. Total ATP levels remained high at 16 h. The ΔΨ loss was delayed by cyclosporin A, a mitochondrial permeability transition inhibitor. Mitochondrial swelling accompanied changes in ΔΨ. Carnitine palmitoyltransferase I activity fell at 16 h; this decline was accompanied by ceramide increases that paralleled decreased complex III activity. We conclude that carnitine palmitoyltransferase I inhibition, ceramide accumulation, and complex III inhibition are downstream events in cardiac apoptosis mediated by palmitate and occur independent of events leading to caspase 3-like activation.

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The saturated fatty acid palmitate induces apoptosis in neonatal rat cardiomyocytes. This apoptosis is associated with early mitochondrial release of cytochrome c and a subsequent loss of mitochondrial membrane potential. Recent reports implicate a role for reactive oxygen species (ROS) in palmitate-induced apoptosis. We studied the role of ROS in palmitate-induced apoptosis in the neonatal rat cardiomyocyte and report no evidence of ROS involvement. ROS production, nitric oxide production, and nuclear factor-B activation were not increased above those observed using the nonapoptotic fatty acid oleate. Indeed, the production of ROS was significantly higher in cells treated with oleate. Furthermore, the presence of antioxidants and ROS scavengers did not attenuate the induction of apoptosis by palmitate. Variations in the fatty acid-to-albumin ratio from 2:1 to 7:1 had no effect on the absence of ROS production or on the extent of apoptosis. No evidence was found for an increase in oxidative protein modification in palmitate-treated cells. Our results lead us to conclude that oxidative stress does not play a role in palmitate-induced apoptosis.antioxidants; nitric oxide; mitochondria; nuclear factor-B

Transcriptional regulation of nuclear encoded mitochondrial proteins is dependent on nuclear transcription factors that act on genes encoding key components of mitochondrial transcription, replication, and heme biosynthetic machinery. Cellular factors that target expression of proteins to the heart have been well characterized with respect to excitation-contraction coupling. No information currently exists that examines whether parallel transcriptional mechanisms regulate nuclear encoded expression of heart-specific mitochondrial isoforms. The muscle CPT-I␤ isoform in heart is a TATAless gene that uses Sp-1 proteins to support basal expression. The rat cardiac fatty acid response element (؊301/؊289), previously characterized in the human gene, is responsive to oleic acid following serum deprivation. Deletion and mutational analysis of the 5-flanking sequence of the carnitine palmitoyltransferase I␤ (CPT-I␤) gene defines regulatory regions in the ؊391/ ؉80 promoter luciferase construct. When deleted or mutated constructs were individually transfected into cardiac myocytes, CPT-I/luciferase reporter gene expression was significantly depressed at sites involving a putative MEF2 sequence downstream from the fatty acid response element and a cluster of heart-specific regulatory regions flanked by two Sp1 elements. Each site demonstrated binding to cardiac nuclear proteins and competition specificity (or supershifts) with oligonucleotides and antibodies. Individual expression vectors for Nkx2.5, serum response factor (SRF), and GATA4 enhanced CPT-I reporter gene expression 4 -36-fold in CV-1 cells. Although cotransfection of Nkx and SRF produced additive luciferase expression, the combination of SRF and GATA-4 cotransfection resulted in synergistic activation of CPT-I␤. The results demonstrate that SRF and the tissue-restricted isoform, GATA-4, drive robust gene transcription of a mitochondrial protein highly expressed in heart. Expression of nuclear and mitochondrial encoded expression of respiratory chain subunits occurs despite physical separation of transcriptional events within separate genomes. Stimulation and coordination of mitochondrial gene expression from these two sites is accomplished by the nuclear respiratory factors, NRF-1 and NRF-2 (1, 2). Using electrical stimulation to produce hypertrophic growth of neonatal cardiac myocytes, the transcriptional activation of cytochrome c is preceded by induction of NRF-1 mRNA (3). This observation is consistent with NRF-1 induction as a prerequisite for synthesis of respiratory chain components. These basic insights into cellular factors that link nuclear events to mitochondrial gene activation are critical for adaptation to environmental stresses and the necessity for enhanced energy production.In contrast to subcellular coordination of mitochondrial biogenesis and respiratory chain synthesis, less is known concerning tissue-specific transcriptional regulation of nuclear encoded genes involved in energy metabolism. These genes are particularly important in cardiac...

Electrical stimulation of neonatal rat cardiac myocytes in culture produces increases in myocyte size (hypertrophy) and organization of actin into myofibrillar arrays. The maturation of the cells is associated with enhanced contractile parameters and cellular calcium content. The numbers and intensity of cellular mitochondrial profiles increase, as measured by scanning laser confocal microscopy. Consistent with the hypertrophic response is increased cellular content of ␤-myosin heavy chain and cytochrome oxidase subunit Va messages, as well as increases in cytochrome oxidase activity in the stimulated cardiac myocytes. Myocyte contractile capacity is associated with increased expression of the muscle carnitine palmitoyltransferase (CPT-I) isoform as measured by Northern analysis, immunoblotting, and altered sensitivity of CPT-I activity to malonyl-CoA in the stimulated cells. The data suggest that a switch from the liver isoform of CPT-I, prominent in the neonatal rat heart, to the muscle CPT-I which predominates in adult rat heart, takes place in the neonatal cardiac myocytes over the same time period as the hypertrophic-mediated changes in myofibrillar assembly and increased contractile activity.