We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor substances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to approximately 50%. This suggests that the Ca2+-calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the nucleus upon vasopressin stimulation. Inhibition of protein kinase C (PKC) isoforms by bisindolylmaleimide had no significant influence on human ALC-1 promoter activity. Thus, our results demonstrate a dominant role of Ca2+-calmodulin-dependent signaling pathways in the regulation of human ALC-1 expression.
Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for beta-myosin heavy chain (MHC) and alpha-MHC in the neonatal rat heart: antisense MHC mRNA expression of alpha-MHC and beta-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the beta-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and beta-MHC consisted of approximately 2,700 bp, suggesting a higher probability of alpha-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of alpha-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of alpha-MHC was 52.0 +/- 2.6% on the sense mRNA but only 36.3 +/- 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart.
Analysis of mRNA by Northern blot and reverse transcription-polymerase chain reaction demonstrated the expression of sense and considerable amounts of naturally occurring antisense mRNA for beta-myosin heavy chain (MHC) and alpha-MHC in the neonatal rat heart: antisense MHC mRNA expression of alpha-MHC and beta-MHC was approximately half of the corresponding sense MHC mRNA expression. Using a computational approach, we could identify a reverse Pol II promoter in the beta-MHC gene. Both sense and antisense MHC mRNA demonstrated similar sizes of approximately 6,000 bp in the Northern blot. Alpha-MHC antisense mRNA consisted of approximately 3,700 bp of complementary exon sequences and beta-MHC consisted of approximately 2,700 bp, suggesting a higher probability of alpha-MHC mRNA dimerization. Hence, sense mRNA transcripts and protein of alpha-MHC should exist at different relative levels in the neonatal state. In fact, the relative proportion of alpha-MHC was 52.0 +/- 2.6% on the sense mRNA but only 36.3 +/- 1.8% on the protein level. Because of its high abundance in the heart, we suggest that in the neonatal heart naturally occurring antisense mRNA may play a role in the regulation of MHC expression and, therefore, in the control of the energetical and contractile behaviour of the heart.
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