In Situ Calibration of Nucleoplasmic versus Cytoplasmic Ca2+ Concentration in Adult Cardiomyocytes

Quantification of subcellularly resolved Ca2+ signals in cardiomyocytes is essential for understanding Ca2+ fluxes in excitation-contraction and excitation-transcription coupling. The properties of fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca2+-dependent fluorescence changes into [Ca2+] changes. Therefore, we determined the in situ characteristics of a frequently used Ca2+ indicator, Fluo-4, and a ratiometric Ca2+ indicator, Asante Calcium Red, and evaluated their use for reporting and quantifying cytoplasmic and nucleoplasmic Ca2+ signals in isolated cardiomyocytes. Ca2+ calibration curves revealed significant differences in the apparent Ca2+ dissociation constants of Fluo-4 and Asante Calcium Red between cytoplasm and nucleoplasm. These parameters were used for transformation of fluorescence into nucleoplasmic and cytoplasmic [Ca2+]. Resting and diastolic [Ca2+] were always higher in the nucleoplasm. Systolic [Ca2+] was usually higher in the cytoplasm, but some cells (15%) exhibited higher systolic [Ca2+] in the nucleoplasm. Ca2+ store depletion or blockade of Ca2+ leak pathways eliminated the resting [Ca2+] gradient between nucleoplasm and cytoplasm, whereas inhibition of inositol 1,4,5-trisphosphate receptors by 2-APB reversed it. The results suggest the presence of significant nucleoplasmic-to-cytoplasmic [Ca2+] gradients in resting myocytes and during the cardiac cycle. Nucleoplasmic [Ca2+] in cardiomyocytes may be regulated via two mechanisms: diffusion from the cytoplasm and active Ca2+ release via inositol 1,4,5-trisphosphate receptors from perinuclear Ca2+ stores

Na+-dependent SR Ca2+ overload induces arrhythmogenic events in mouse cardiomyocytes with a human CPVT mutation

Aims Mutations in the cardiac ryanodine receptor Ca2+ release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca2+ release in response to SR Ca2+ overload during β-adrenergic stimulation. However, whether elevated SR Ca2+ content—in the absence of protein kinase A activation—affects RyR2 function and arrhythmogenesis in CPVT remains elusive. Methods and results Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2R4496C+/−) associated with CPVT were investigated in the absence and presence of 1 µmol/L JTV-519 (RyR2 stabilizer) followed by 100 µmol/L ouabain intervention to increase cytosolic [Na+] and SR Ca2+ load. Changes in membrane potential and intracellular [Ca2+] were monitored with whole-cell patch-clamping and confocal Ca2+ imaging, respectively. At baseline, action potentials (APs), Ca2+ transients, fractional SR Ca2+ release, and SR Ca2+ load were comparable in wild-type (WT) and RyR2R4496C+/− myocytes. Ouabain evoked significant increases in diastolic [Ca2+], peak systolic [Ca2+], fractional SR Ca2+ release, and SR Ca2+ content that were quantitatively similar in WT and RyR2R4496C+/− myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca2+ waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2R4496C+/− when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events. Conclusion The elevation of SR Ca2+ load—in the absence of β-adrenergic stimulation—is sufficient to increase the propensity for triggered arrhythmias in RyR2R4496C+/− cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias

Early Remodeling of Perinuclear Ca2+ Stores and Nucleoplasmic Ca2+ Signaling During the Development of Hypertrophy and Heart Failure

Background—A hallmark of heart failure is impaired cytoplasmic Ca2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results—Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca2+ transporters. These changes were associated with altered nucleoplasmic Ca2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca2+ levels with diminished and prolonged nuclear Ca2+ transients and slowed intranuclear Ca2+ diffusion. Altered nucleoplasmic Ca2+ levels were translated to higher activation of nuclear Ca2+/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca2+ changes that are typical of heart failure. Conclusions-During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling

Emerging roles of inositol 1,4,5-trisphosphate signaling in cardiac myocytes

Inositol 1,4,5-trisphosphate (IP3) is a ubiquitous intracellular messenger regulating diverse functions in almost all mammalian cell types. It is generated by membrane receptors that couple to phospholipase C (PLC), an enzyme which liberates IP3 from phosphatidylinositol 4,5-bisphosphate (PIP2). The major action of IP3, which is hydrophilic and thus translocates from the membrane into the cytoplasm, is to induce Ca2+ release from endogenous stores through IP3 receptors (IP3Rs). Cardiac excitation-contraction coupling relies largely on ryanodine receptor (RyR)-induced Ca2+ release from the sarcoplasmic reticulum. Myocytes express a significantly larger number of RyRs compared to IP3Rs (~100:1), and furthermore they experience substantial fluxes of Ca2+ with each heartbeat. Therefore, the role of IP3 and IP3-mediated Ca2+ signaling in cardiac myocytes has long been enigmatic. Recent evidence, however, indicates that despite their paucity cardiac IP3Rs may play crucial roles in regulating diverse cardiac functions. Strategic localization of IP3Rs in cytoplasmic compartments and the nucleus enables them to participate in subsarcolemmal, bulk cytoplasmic and nuclear Ca2+ signaling in embryonic stem cell-derived and neonatal cardiomyocytes, and in adult cardiac myocytes from the atria and ventricles. Intriguingly, expression of both IP3Rs and membrane receptors that couple to PLC/IP3 signaling is altered in cardiac disease such as atrial fibrillation or heart failure, suggesting the involvement of IP3 signaling in the pathology of these diseases. Thus, IP3 exerts important physiological and pathological functions in the heart, ranging from the regulation of pacemaking, excitation-contraction and excitation-transcription coupling to the initiation and/or progression of arrhythmias, hypertrophy and heart failure