Cardiac Myocytes from Newborn Mice (original) (raw)

Nuclear and cytosolic calcium are regulated independently

Proceedings of the National Academy of Sciences, 2003

Nuclear calcium (Ca 2؉ ) regulates a number of important cellular processes, including gene transcription, growth, and apoptosis. However, it is unclear whether Ca 2؉ signaling is regulated differently in the nucleus and cytosol. To investigate this possibility, we examined subcellular mechanisms of Ca 2؉ release in the HepG2 liver cell line. The type II isoform of the inositol 1,4,5-trisphosphate (InsP 3) receptor (InsP 3R) was expressed to a similar extent in the endoplasmic reticulum and nucleus, whereas the type III InsP 3R was concentrated in the endoplasmic reticulum, and the type I isoform was not expressed. Ca 2؉ signals induced by low InsP3 concentrations started earlier or were larger in the nucleus than in the cytosol, indicating higher sensitivity of nuclear Ca 2؉ stores for InsP3. Nuclear InsP3R channels were active at lower InsP 3 concentrations than InsP3R from cytosol. Enriched expression of type II InsP 3R in the nucleus results in greater sensitivity of the nucleus to InsP 3, thus providing a mechanism for independent regulation of Ca 2؉ -dependent processes in this cellular compartment.

Local InsP3-dependent perinuclear Ca2+ signaling in cardiac myocyte excitation-transcription coupling

Journal of Clinical Investigation, 2006

Previous work showed that calmodulin (CaM) and Ca 2+ -CaM-dependent protein kinase II (CaMKII) are somehow involved in cardiac hypertrophic signaling, that inositol 1,4,5-trisphosphate receptors (InsP 3 Rs) in ventricular myocytes are mainly in the nuclear envelope, where they associate with CaMKII, and that class II histone deacetylases (e.g., HDAC5) suppress hypertrophic gene transcription. Furthermore, HDAC phosphorylation in response to neurohumoral stimuli that induce hypertrophy, such as endothelin-1 (ET-1), activates HDAC nuclear export, thereby regulating cardiac myocyte transcription. Here we demonstrate a detailed mechanistic convergence of these 3 issues in adult ventricular myocytes. We show that ET-1, which activates plasmalemmal G protein-coupled receptors and InsP 3 production, elicits local nuclear envelope Ca 2+ release via InsP 3 R. This local Ca 2+ release activates nuclear CaMKII, which triggers HDAC5 phosphorylation and nuclear export (derepressing transcription). Remarkably, this Ca 2+ -dependent pathway cannot be activated by the global Ca 2+ transients that cause contraction at each heartbeat. This novel local Ca 2+ signaling in excitation-transcription coupling is analogous to but separate (and insulated) from that involved in excitation-contraction coupling. Thus, myocytes can distinguish simultaneous local and global Ca 2+ signals involved in contractile activation from those targeting gene expression.

Nuclear Ca2+ sparks and waves mediated by inositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyocytes

Cell Calcium, 2008

Dynamic nuclear Ca 2+ signals play pivotal roles in diverse cellular functions including gene transcription, cell growth, differentiation, and apoptosis. Here we report a novel nuclear Ca 2+ regulatory mechanism mediated by inositol 1,4,5-trisphosphate receptors (IP 3 Rs) around the nucleus in developing cardiac myocytes. Activation of IP 3 Rs by ␣ 1 -adrenergic receptor (␣ 1 AR) stimulation or by IP 3 application (in saponinpermeabilized cells) increases Ca 2+ spark frequency preferentially in the region around the nucleus in neonatal rat ventricular myocytes. A nuclear enrichment of IP 3 R distribution supports the higher responsiveness of Ca 2+ release in this particular region. Strikingly, we observed "nuclear Ca 2+ waves" that engulf the entire nucleus without spreading into the bulk cytosol. ␣ 1 AR stimulation enhances the occurrence of nuclear Ca 2+ waves and confers them the ability to trigger cytosolic Ca 2+ waves via IP 3 R-dependent pathways. This finding accounts, at least partly, for a profound frequency-dependent modulation of global Ca 2+ oscillations during ␣ 1 AR stimulation. Thus, IP 3 R-mediated Ca 2+ waves traveling in the nuclear region provide active, autonomous regulation of nuclear Ca 2+ signaling, which provides for not only the local signal transduction, but also a pacemaker to drive global Ca 2+ transient in the context of ␣ 1 AR stimulation in developing cardiac myocytes.

Elevated InsP3R expression underlies enhanced calcium fluxes and spontaneous extra-systolic calcium release events in hypertrophic cardiac myocytes

Channels, 2010

C ardiac hypertrophy is associated with profound remodeling of Ca 2+ signaling pathways. During the early, compensated stages of hypertrophy, Ca 2+ fluxes may be enhanced to facilitate greater contraction, whereas as the hypertrophic heart decompensates, Ca 2+ homeostatic mechanisms are dysregulated leading to decreased contractility, arrhythmia and death. Although ryanodine receptor Ca 2+ release channels (RyR) on the sarcoplasmic reticulum (SR) intracellular Ca 2+ store are primarily responsible for the Ca 2+ flux that induces myocyte contraction, a role for Ca 2+ release via the inositol 1,4,5-trisphosphate receptor (InsP 3 R) in cardiac physiology has also emerged. Specifically, InsP 3 -induced Ca 2+ signals generated following myocyte stimulation with an InsP 3 -generating agonist (e.g., endothelin, ET-1), lead to modulation of Ca 2+ signals associated with excitation-contraction coupling (ECC) and the induction of spontaneous Ca 2+ release events that cause cellular arrhythmia. Using myocytes from spontaneously hypertensive rats (SHR), we recently reported that expression of the type 2 InsP 3 R (InsP 3 R2) is significantly increased during hypertrophy. Notably, this increased expression was restricted to the junctional SR in close proximity to RyRs. There, enhanced Ca 2+ release via InsP 3 Rs serves to sensitize neighboring RyRs causing an augmentation of Ca 2+ fluxes during ECC as well as an increase in non-triggered Ca 2+ release events. This manuscript has been published online, prior to printing. Once the issue is complete and page numbers have been assigned, the citation will change accordingly.

Calcium sensing receptor regulates cardiomyocyte function through nuclear calcium

Cell Biology International, 2012

Nuclear Ca 2+ plays a pivotal role in the regulation of gene expression. IP 3 (inositol-1,4,5-trisphosphate) is an important regulator of nuclear Ca 2+. We hypothesized that the CaR (calcium sensing receptor) stimulates nuclear Ca 2+ release through IICR (IP 3-induced calcium release) from perinuclear stores. Spontaneous Ca 2+ oscillations and the spark frequency of nuclear Ca 2+ were measured simultaneously in NRVMs (neonatal rat ventricular myocytes) using confocal imaging. CaR-induced nuclear Ca 2+ release through IICR was abolished by inhibition of CaR and IP 3 Rs (IP 3 receptors). However, no effect on the inhibition of RyRs (ryanodine receptors) was detected. The results suggest that CaR specifically modulates nuclear Ca 2+ signalling through the IP 3 R pathway. Interestingly, nuclear Ca 2+ was released from perinuclear stores by CaR activator-induced cardiomyocyte hypertrophy through the Ca 2+-dependent phosphatase CaN (calcineurin)/ NFAT (nuclear factor of activated T-cells) pathway. We have also demonstrated that the activation of the CaR increased the NRVM protein content, enlarged cell size and stimulated CaN expression and NFAT nuclear translocation in NRVMs. Thus, CaR enhances the nuclear Ca 2+ transient in NRVMs by increasing fractional Ca 2+ release from perinuclear stores, which is involved in cardiac hypertrophy through the CaN/NFAT pathway.

Role of inositol 1,4,5-trisphosphate in the regulation of ventricular Ca2+ signaling in intact mouse heart

Journal of Molecular and Cellular Cardiology, 2012

Inositol 1,4,5-trisphosphate (InsP 3 R)-mediated Ca 2+ signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP 3-mediated Ca 2+ signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP 3-sensitive intracellular Ca 2+ signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP 3 ] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP 3. Our results indicate that the basal [Ca 2+ ] increased after the flash photolysis of caged InsP 3 without affecting the action potential (AP)-induced Ca 2+ transients. The amplitude of the basal [Ca 2+ ] elevation depended on the intracellular [InsP 3 ] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP 3-induced Ca 2+ release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca 2+ release from both RyR-and InsP 3 R-containing Ca 2+ stores, suggesting that these pools have similar Ca 2+ reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP 3 was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitationcontraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.

Calcium signaling in the nucleusThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell

Canadian Journal of Physiology and Pharmacology, 2006

Cytosolic Ca 2+ is a versatile secondary messenger that regulates a wide range of cellular activities. In the past decade, evidence has accumulated that free Ca 2+ within the nucleus also plays an important messenger function. Here we review the mechanisms and effects of Ca 2+ signals within the nucleus. In particular, evidence is reviewed that the nucleus contains the machinery necessary for production of inositol 1,4,5-trisphosphate and for inositol 1,4,5-trisphosphate receptor-mediated Ca 2+ release. The role of Ca 2+ signals within the nucleus is discussed including regulation of such critical cell functions as gene expression, activation of kinases, and permeability of nuclear pores.

1,4,5-Inositol Trisphosphate-Operated Intracellular Ca 2+ Stores and Angiotensin-II/Endothelin-1 Signaling Pathway Are Functional in Human Embryonic Stem Cell-Derived Cardiomyocytes

STEM CELLS, 2008

On the basis of previous findings suggesting that in human embryonic stem cell-derived cardiomyocytes (hESC-CM) the sarcoplasmic reticulum Ca 2؉ -induced release of calcium machinery is either absent or immature, in the present study we tested the hypothesis that hESC-CM contain fully functional 1,4,5-inositol trisphosphate (1,4,5-IP 3 )-operated intracellular Ca 2؉ ([Ca 2؉ ] i ) stores that can be mobilized upon appropriate physiological stimuli. To test this hypothesis we investigated the effects of angiotensin-II (AT-II) and endothelin-1 (ET-1), which activate the 1,4,5-IP 3 pathway, on [Ca 2؉ ] i transients and contractions in beating clusters of hESC-CM. Our major findings were that in paced hESC-CM both AT-II and ET-1 (10 ؊9 to 10 ؊7 M) increased the contraction amplitude and the maximal rates of contraction and relaxation. In addition, AT-II (10 ؊9 to 10 ؊7 M) increased the [Ca 2؉ ] i transient amplitude. The involvement of 1,4,5-IP 3dependent intracellular Ca 2؉ release in the inotropic effect of AT-II was supported by the findings that (a) hESC-CM express AT-II, ET-1, and 1,4,5-IP 3 receptors determined by immunofluorescence staining, and (b) the effects of AT-II were blocked by 2 M 2-aminoethoxyphenyl borate (a 1,4,5-IP 3 receptor blocker) and U73122 (a phospholipase C blocker). In conclusion, these findings demonstrate for the first time that hESC-CM exhibit functional AT-II and ET-1 signaling pathways, as well as 1,4,5-IP 3 -operated releasable Ca 2؉ stores.