Sarcoplasmic reticulum Ca2+ -induced Ca2+ release regulates class IIa HDAC localization in mouse embryonic cardiomyocytes.

In embryonic cardiomyocytes, sarcoplasmic reticulum (SR)-derived Ca2+ release is required to induce Ca2+ oscillations for contraction and to control cardiac development through Ca2+ -activated pathways. Here, our aim was to study how SR Ca2+ release regulates cytosolic and nuclear Ca2+ distribution and the subsequent effects on the Ca2+ -dependent localization of class IIa histone deacetylases (HDAC) and cardiac-specific gene expression in embryonic cardiomyocytes. Confocal microscopy was used to study changes in Ca2+ -distribution and localization of immunolabeled HDAC4 and HDAC5 upon changes in SR Ca2+ release in mouse embryonic cardiomyocytes. Dynamics of translocation were also observed with a confocal microscope, using HDAC5-green fluorescent protein transfected myocytes. Expression of class IIa HDACs in differentiating myocytes and changes in cardiac-specific gene expression were studied using real-time quantitative PCR. Inhibition of SR Ca2+ release caused a significant decrease in intranuclear Ca2+ concentration, a rapid nuclear import of HDAC5 and subnuclear redistribution of HDAC4. Endogenous localization of HDAC5 and HDAC4 was mostly cytosolic and at the nuclear periphery, respectively. Downregulated expression of cardiac-specific genes was also observed upon SR Ca2+ release inhibition. Electrical stimulation of sarcolemmal Ca2+ influx was not sufficient to rescue either the HDAC localization or the gene expression changes. SR Ca2+ release controls subcellular Ca2+ distribution and regulates localization of HDAC4 and HDAC5 in embryonic cardiomyocytes. Changes in SR Ca2+ release also caused changes in expression of the developmental phase-specific genes, which may be due to the changes in HDAC-localization.

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.

Myogenic skeletal muscle satellite cells communicate by tunnelling nanotubes.

Quiescent satellite cells sit on the surface of the muscle fibres under the basal lamina and are activated by a variety of stimuli to disengage, divide and differentiate into myoblasts that can regenerate or repair muscle fibres. Satellite cells adopt their parent's fibre type and must have some means of communication with the parent fibre. The mechanisms behind this communication are not known. We show here that satellite cells form dynamic connections with muscle fibres and other satellite cells by F-actin based tunnelling nanotubes (TNTs). Our results show that TNTs readily develop between satellite cells and muscle fibres. Once developed, TNTs permit transport of intracellular material, and even cellular organelles such as mitochondria between the muscle fibre and satellite cells. The onset of satellite cell differentiation markers Pax-7 and MyoD expression was slower in satellite cells cultured in the absence than in the presence of muscle cells. Furthermore physical contact between myofibre and satellite cell progeny is required to maintain subtype identity. Our data establish that TNTs constitute an integral part of myogenic cell communication and that physical cellular interaction control myogenic cell fate determination.

Model of excitation-contraction coupling of rat neonatal ventricular myocytes.

The neonatal rat ventricular myocyte culture is one of the most popular experimental cardiac cell models. To our knowledge, the excitation-contraction coupling (ECC) of these cells, i.e., the process linking the electrical activity to the cytosolic Ca2+ transient and contraction, has not been previously analyzed, nor has it been presented as a complete system in detail. Neonatal cardiomyocytes are in the postnatal developmental stage, and therefore, the features of their ECC differ vastly from those of adult ventricular myocytes. We present the first complete analysis of ECC in these cells by characterizing experimentally the action potential and calcium signaling and developing the first mathematical model of ECC in neonatal cardiomyocytes that we know of. We show that in comparison to adult cardiomyocytes, neonatal cardiomyocytes have long action potentials, heterogeneous cytosolic Ca2+ signals, weaker sarcoplasmic reticulum Ca2+ handling, and stronger sarcolemmal Ca2+ handling, with a significant contribution by the Na+/Ca2+ exchanger. The developed model reproduces faithfully the ECC of rat neonatal cardiomyocytes with a novel description of spatial cytosolic [Ca2+] signals. Simulations also demonstrate how an increase in the cell size (hypertrophy) affects the ECC in neonatal cardiomyocytes. This model of ECC in developing cardiomyocytes provides a platform for developing future models of cardiomyocytes at different developmental stages.

Increased mitochondrial Ca2+ and decreased sarcoplasmic reticulum Ca2+ in mitochondrial myopathy.

Genetic mutations that affect mitochondrial function often cause skeletal muscle dysfunction. Here, we used mice with skeletal-muscle-specific disruption of the nuclear gene for mitochondrial transcription factor A (Tfam) to study whether changes in cellular Ca(2+) handling is part of the mechanism of muscle dysfunction in mitochondrial myopathy. Force measurements were combined with measurements of cytosolic Ca(2+), mitochondrial Ca(2+) and membrane potential and reactive oxygen species in intact, adult muscle fibres. The results show reduced sarcoplasmic reticulum (SR) Ca(2+) storage capacity in Tfam KO muscles due to a decreased expression of calsequestrin-1. This resulted in decreased SR Ca(2+) release during contraction and hence lower force production in Tfam KO than in control muscles. Additionally, there were no signs of oxidative stress in Tfam KO cells, whereas they displayed increased mitochondrial [Ca(2+)] during repeated contractions. Mitochondrial [Ca(2+)] remained elevated long after the end of stimulation in muscle cells from terminally ill Tfam KO mice, and the increase was smaller in the presence of the cyclophilin D-binding inhibitor cyclosporin A. The mitochondrial membrane potential in Tfam KO cells did not decrease during repeated contractions. In conclusion, we suggest that the observed changes in Ca(2+) handling are adaptive responses with long-term detrimental effects. Reduced SR Ca(2+) release likely decreases ATP expenditure, but it also induces muscle weakness. Increased [Ca(2+)](mit) will stimulate mitochondrial metabolism acutely but may also trigger cell damage.

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.