Long-chain fatty acid-induced changes in gene expression in neonatal cardiac myocytes

Abstract

Long-chain fatty acids are the most important substrates for the heart. In addition, they have been shown to affect signalling pathways and gene expression. To explore the effects of long-chain fatty acids on cardiac gene expression, neonatal rat ventricular myocytes were cultured for 48 h with either glucose (10 m M ), fatty acids (palmitic and oleic acid, 0.25 m M each), or a combination of both as exogenous substrates. Exposure to fatty acids (both in the absence or presence of glucose) neither affected cellular morphology and protein content nor induced alterations in the expression of phenotypic marker genes like atrial natriuretic factor and the Ca-ATPase SERCA2. However, incubation with fatty acids (with or without glucose) resulted in up to 4-fold increases of the mRNA levels of fatty acid translocase (FAT/CD36), heart-type fatty acid-binding protein, acyl-CoA synthetase, and long-chain acyl-CoA dehydrogenase. In contrast, the expression of genes coding for proteins involved in glucose uptake and metabolism, i.e., glucose transporter GLUT4, hexokinase II, and glyceraldehyde 3-phosphate dehydrogenase, remained constant or even declined under these conditions. These changes corresponded with a 60% increase in cardiomyocyte fatty acid oxidation capacity. Interestingly, the peroxisome proliferator-activated receptor-␣ (PPAR ␣ )-ligand Wy 14,643, but not the PPAR ␥ -ligand ciglitazone, also resulted in increased mRNA levels of genes involved in fatty acid metabolism.In conclusion, fatty acids specifically and co-ordinately up-regulate transcription of genes coding for proteins involved in cardiac fatty acid transport and metabolism, most likely through activation of PPAR ␣ . -van der Lee, K. A

Fasting-induced changes in the expression of genes controlling substrate metabolism in the rat heart

“…This speculation is supported by previous in vitro observations. We found that the mere addition of fatty acids to the culture medium of neonatal cardiomyocytes leads to a selective and coordinate induction of the expression of proteins involved in fatty acid uptake and metabolism, thereby showing that fatty acids themselves are able to modulate gene expression in cardiac myocytes (7).…”

“…Ten g of total RNA was size-fractionated on a denaturing gel, transferred to a nylon membrane (Hybond-NX, Amersham, Slough, UK), and fixed with standard techniques. Following prehybridization, the filters were probed with fragments of cDNA of GLUT4, hexokinase II (HKII), FAT/CD36, H-FABP, acyl-CoA synthetase (ACS), longchain acyl-CoA dehydrogenase (LCAD), or uncoupling protein-2 (UCP-2), as described previously (7,12). A 2.1 kb EcoRI fragment of rat glucose transporter GLUT1 cDNA (a gift of Dr. A. Zorzano, University of Barcelona, Spain), a 0.6 kb BglII fragment of rat hexokinase I (HKI; a gift of Dr. J. E. Wilson, Michigan State University, USA), a 0.5 kb KpnI-BamHI fragment of muscletype carnitine palmitoyl transferase 1 (mCPT1; kindly provided by Dr. F. van de Leij, University of Groningen, The Netherlands), a PstI fragment of mouse peroxisome proliferator-activated receptor ␣ (PPAR ␣ ; kindly provided by Dr. J. Auwerx, Institute Pasteur, Lille, France), and a 1.5 kb BamHI fragment of rat PPAR ␤ / ␦ (a gift of Dr. P. A. Grimaldi, University of Nice, France) were also used for hybridization, as well as a 1.0 kb fragment of rat CS generated via reverse transcriptase PCR using for ward (5 Ј -ATCCGTTTCCGAGGCTWY AGT-3 Ј ) and reversed (5 Ј -CGTGAY ACCCCRAACAGGACY-3 Ј ) primers, and a 0.7 kb fragment of rat UCP-3 also generated via RT-PCR using (5 Ј -GGCCATCCTCCG GAA CCATGG-3 Ј ) and (5 Ј -GCGGCCTGCTTGCCTTGTTCA-3 Ј ) as forward and reversed primers, respectively.…”

“…Recently we have demonstrated that long-term exposure of primary cultures of rat neonatal cardiac myocytes to fatty acids resulted in an increased fatty acid oxidation capacity (7). This was accompanied by a specific and coordinated up-regulation of the expression of genes involved in cellular fatty acid transport and metabolism, whereas the expression of the insulin-sensitive glucose transporter GLUT4 declined (7,8).…”

“…Recently we have demonstrated that long-term exposure of primary cultures of rat neonatal cardiac myocytes to fatty acids resulted in an increased fatty acid oxidation capacity (7). This was accompanied by a specific and coordinated up-regulation of the expression of genes involved in cellular fatty acid transport and metabolism, whereas the expression of the insulin-sensitive glucose transporter GLUT4 declined (7,8). These data point to metabolic plasticity of cardiac muscle cells, that is, the ability to respond to changes in substrate supply by adjusting the expression of genes encoding proteins involved in substrate transport and metabolism.…”

Long-chain fatty acids modify hypertrophic responses of cultured primary neonatal cardiomyocytes

“…In these two cases, the final concentration of FA in the medium was 160 and 40 times lower, respectively, than the concentrations used in the present study. In some other cases, neonatal cardiomyocytes have been cultured in the presence of FA-BSA complexes at concentrations similar to that used in the present study, but hypertrophy responses were not the primary focus of these particular studies (23)(24)(25). Nonetheless, chronic palmitate:oleate-BSA has been shown to induce many phenotypic changes in cardiomyocytes, including an increase in the rate of FA oxidation, an increase in the mRNA concentration of several genes involved in FA metabolism, and a decrease in the mRNA concentration of several genes involved in glucose metabolism (23).…”