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.

Pathologic hypertrophy of the heart is regulated through membranebound receptors and intracellular signaling pathways that function, in part, by altering Ca 2+ handling and Ca 2+ -dependent signaling effectors. Transient receptor potential canonical (TRPC) channels are important mediators of Ca 2+ -dependent signal transduction that can sense stretch or activation of membrane-bound receptors. Here we generated cardiac-specific transgenic mice that express dominant-negative (dn) TRPC3, dnTRPC6, or dnTRPC4 toward blocking the activity of the TRPC3/6/7 or TRPC1/4/5 subfamily of channels in the heart. Remarkably, all three dn transgenic strategies attenuated the cardiac hypertrophic response following either neuroendocrine agonist infusion or pressure-overload stimulation. dnTRPC transgenic mice also were partially protected from loss of cardiac functional performance following long-term pressure-overload stimulation. Importantly, adult myocytes isolated from hypertrophic WT hearts showed a unique Ca 2+ influx activity under store-depleted conditions that was not observed in myocytes from hypertrophied dnTRPC3, dnTRPC6, or dnTRPC4 hearts. Moreover, dnTRPC4 inhibited the activity of the TRPC3/6/7 subfamily in the heart, suggesting that these two subfamilies function in coordinated complexes. Mechanistically, inhibition of TRPC channels in transgenic mice or in cultured neonatal myocytes significantly reduced activity in the calcineurin-nuclear factor of activated T cells (NFAT), a known Ca 2+ -dependent hypertrophy-inducing pathway. Thus, TRPC channels are necessary mediators of pathologic cardiac hypertrophy, in part through a calcineurin-NFAT signaling pathway.calcium | heart | signaling | calcineurin P athologic cardiac hypertrophy, an enlargement of the adult heart caused by disease-inducing stimuli, can cause sudden death and is a leading predictor for the development of heart failure (1). The growth of individual myocytes is programmed by neuroendocrine factors that signal through membrane-bound receptors leading to activation of signal-transduction pathways and alterations in gene expression (2). Augmentation in intracellular Ca 2+ is thought to be critically involved in signaling cardiac hypertrophy, in part through the Ca 2+ -activated protein phosphatase calcineurin, which leads to activation of the transcription factor, nuclear factor of activated T cells (NFAT), which induces hypertrophic response genes (3). Transient receptor potential canonical (TRPC) channels are cation-selective influx channels that can initiate cardiac hypertrophy when overexpressed, in part because of increased Ca 2+ influx and calcineurin activation (4-7). Functional TRPC channels are comprised of homo-or heterotetramers between either TRPC1/4/5 or TRPC3/ 6/7 subfamily members, although overexpression of any one subunit alone can produce enhanced currents (8). In general, TRPC3/ 6/7 are activated by diacylglycerol (DAG) generated by G proteincoupled receptors (GPCR)/Gαq/phospholipase C signaling, and TRPC1/4/5 can be activated by d...

Abstract-Previous ventricular myocyte studies indicated that ryanodine receptors (RyRs) are in the sarcoplasmic reticulum (SR) and are critical in excitation-contraction coupling, whereas the inositol trisphosphate (InsP 3 ) receptors are separately localized on the nuclear envelope (NucEn) and involved in nuclear Ca 2ϩ signaling. Here, we find that both caffeine and InsP 3 receptor agonists deplete free [Ca 2ϩ ] inside both SR and NucEn. Fluorescence recovery after photobleach (FRAP) was measured using the low-affinity Ca 2ϩ indicator Fluo-5N trapped inside the SR and NucEn (where its fluorescence is high because [Ca 2ϩ ] is Ϸ1 mmol/L). After Fluo-5N photobleach in one end of the cell, FRAP occurred, accompanied by fluorescence decline in the unbleached end with similar time constants (Ϸ2 minutes) until fluorescence regained spatial uniformity. Notably, SR and NucEn fluorescence recovered simultaneously in the bleached end. Ca 2ϩ diffusion inside the SR-NucEn was also measured. SR Ca 2ϩ -ATPase was completely blocked but without acute SR Ca 2ϩ depletion. Then caffeine was applied locally to one end of the myocyte. In the caffeine-exposed end, free SR [Ca 2ϩ Intracellular Ca 2ϩ stores include SR (endoplasmic reticulum [ER] in nonmuscle cells) and nuclear envelope (NucEn). Free [Ca 2ϩ ] inside these stores can be Ϸ1 mmol/L, which is similar to that in the extracellular space. 3 The SR is composed of junctional SR (jSR), which is covered by RyRs and faces toward the T tubules, and free SR (fSR), which contains SR-ER Ca 2ϩ -ATPase (SERCA). 4 The NucEn surrounds the nucleus and has both an inner and an outer membrane and a space in between where [Ca 2ϩ ] can be millimolar. Recently, we found that cardiac myocytes use local Ca 2ϩ release from inositol trisphosphate (InsP 3 ) receptors (InsP 3 R) in the NucEn to respond to the neurohumoral stimuli in excitation-transcription coupling (ETC). 5 Endothelin-1 (ET-1) application caused InsP 3 to activate local Ca 2ϩ release from the NucEn via InsP 3 R, which activated calmodulin and CaMKII to phosphorylate histone deacetylase-5 (causing its nuclear export) and activation of transcription. 5 Notably, this pathway could only be activated by Ca 2ϩ from local InsP 3 R (not global Ca 2ϩ transients), presumably because calmodulin and CaMKII physically associate with the InsP 3 R at the NucEn. 5,6 Indeed, in ventricular myocytes, RyRs are mainly in SR, whereas InsP 3 R are mainly in NucEn. 6 Thus, SR and NucEn differ structurally and functionally and may reflect discrete physically different Ca 2ϩ stores. In addition, the relatively rigid sarcomeric organization in striated muscle with a dense protein mesh at the Z-lines raises the possibility that local SR within a given sarcomere could serve its particular sarcomere but not necessarily communicate with neighboring sarcomeres either longitudinally or transversely. The main aim of the present study was to determine whether different SR regions and NucEn constitute discrete separate Ca 2ϩ stores. In some cell types (...

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