Ca2+-calmodulin-dependent protein kinase II represses cardiac transcription of the L-type calcium channel alpha(1C)-subunit gene (Cacna1c) by DREAM translocation.

Recent studies have demonstrated that changes in the activity of calcium-calmodulin-dependent protein kinase II (CaMKII) induce a unique cardiomyocyte phenotype through the regulation of specific genes involved in excitation-contraction (E-C)-coupling. To explain the transcriptional effects of CaMKII we identified a novel CaMKII-dependent pathway for controlling the expression of the pore-forming α-subunit (Cav1.2) of the L-type calcium channel (LTCC) in cardiac myocytes. We show that overexpression of either cytosolic (δC) or nuclear (δB) CaMKII isoforms selectively downregulate the expression of the Cav1.2. Pharmacological inhibition of CaMKII activity induced measurable changes in LTCC current density and subsequent changes in cardiomyocyte calcium signalling in less than 24 h. The effect of CaMKII on the α1C-subunit gene (Cacna1c) promoter was abolished by deletion of the downstream regulatory element (DRE), which binds transcriptional repressor DREAM/calsenilin/KChIP3. Imaging DREAM-GFP (green fluorescent protein)-expressing cardiomyocytes showed that CaMKII potentiates the calcium-induced nuclear translocation of DREAM. Thereby CaMKII increases DREAM binding to the DRE consensus sequence of the endogenous Cacna1c gene. By mathematical modelling we demonstrate that the LTCC downregulation through the Ca2+-CaMKII-DREAM cascade constitutes a physiological feedback mechanism enabling cardiomyocytes to adjust the calcium intrusion through LTCCs to the amount of intracellular calcium detected by CaMKII.

Mitochondrial uncoupling downregulates calsequestrin expression and reduces SR Ca2+ stores in cardiomyocytes.

AIMS: Mitochondrial cardiomyopathy is associated with deleterious remodelling of cardiomyocyte Ca(2+) signalling that is partly due to the suppressed expression of the sarcoplasmic reticulum (SR) Ca(2+) buffer calsequestrin (CASQ2). This study was aimed at determining whether CASQ2 downregulation is directly caused by impaired mitochondrial function. METHODS AND RESULTS: Mitochondrial stress was induced in cultured neonatal rat cardiomyocytes by means of the mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Ca(2+) transients and reactive oxygen species (ROS) were measured by confocal microscopy using the indicators fluo-4 and MitoSOX red, respectively. Mitochondrial stress led to concentration-dependent downregulation of calsequestrin (CASQ2) and changes in the Ca(2+) signals of the cardiomyocytes that were accompanied by reduction in SR Ca(2+) content and amplitude and duration of Ca(2+) sparks. Caspase 3, p38, and p53 inhibitors had no effect on FCCP-induced CASQ2 downregulation; however, it was attenuated by the ROS scavenger N-acetylcysteine (NAC). Importantly, NAC not only decreased FCCP-induced ROS production, but it also restored the Ca(2+) signals, SR Ca(2+) content, and Ca(2+) spark properties to control levels. CONCLUSION: Mitochondrial uncoupling results in fast transcriptional changes in CASQ2 expression that manifest as compromised Ca(2+) signalling, and these changes can be prevented by ROS scavengers. As impaired mitochondrial function has been implicated in several cardiac pathologies as well as in normal ageing, the mechanisms described here might be involved in a wide spectrum of cardiac conditions.

Calcium-calmodulin kinase II is the common factor in calcium-dependent cardiac expression and secretion of A- and B-type natriuretic peptides.

Peptides derived from the precursor of A- and B-type natriuretic peptides (ANP and BNP) are powerful clinical markers of cardiac hypertrophy and dysfunction. It is known that many stimuli affecting the intracellular calcium concentration also induce ANP and BNP secretion. It was our intention to study the mechanisms by which calcium regulates the secretion of ANP and BNP. The effects of pacing and calcium-calmodulin kinase II activity on natriuretic peptide secretion were studied in isolated perfused rat atria and cultured rat neonatal cardiomyocytes. In isolated rat atrium pacing induced an increase in diastolic, systolic, and averaged intracellular free calcium concentration and a frequency-dependent increase in the secretion of both ANP and BNP. The molar ratio of the secreted natriuretic peptides (ANP to BNP) remained nearly constant ( approximately 1000) at all the pacing frequencies tested (1, 3, 6, and 8 Hz). Calmodulin kinase II inhibitor KN-93 (3 mum) did not affect intracellular free calcium concentration but showed a frequency-dependent inhibitory effect on ANP and BNP secretion without a change in ANP to BNP ratio. In the neonatal cardiomyocytes, KN-93 (3 mum) suppressed the secretion and gene expression of both ANP and BNP. Overexpression of constitutively active (T286D) or nuclear (delta(B)) calcium-calmodulin kinase II induced an increase in ANP and BNP gene expression. The results indicate that the calcium-dependent secretion and gene expression of A- and B-type natriuretic peptides are similarly regulated by calmodulin kinase II-dependent mechanisms. This is a plausible mechanism contributing to exercise-induced natriuretic peptide secretion and the augmented secretion in heart dysfunction due to impaired calcium handling.

Hypoxia inducible factor regulates the cardiac expression and secretion of apelin.

Apelin and its G-protein-coupled receptor APJ have various beneficial effects on cardiac function and blood pressure. The mechanisms that regulate apelin gene expression are not known. Because apelin gene expression has been shown to increase in cardiac ischemia, we investigated if apelin (Apln) gene expression was sensitive to hypoxia. Here we show that hypoxia increases the apelin expression in rat myocardium and in cultured cardiomyocytes. Pharmacological activation of hypoxia inducible factor by desferrioxamine (DFO) or expression of a constitutively active form of HIF-1alpha increased apelin expression in cardiomyocyte cultures. The induction of apelin by hypoxia was abolished on transient expression of the HIF inhibitory PAS protein in cardiomyocytes. Increased apelin expression induced by hypoxia or DFO was accompanied by the processing of the cellular storage form proapelin into smaller apelin peptides and increased secretion of these biologically active forms of apelin. In a rat in vivo model, acute myocardial infarction (24 h) led to a transient increase in ventricular apelin mRNA levels. Our results indicate that apelin gene is regulated by hypoxia in cardiac myocytes via the HIF pathway, suggesting a role for apelin as a potential marker for acute cardiac hypoxia with a possible compensatory role in myocardial tissue suffering from oxygen deprivation.