Immunolocalization of type 2 inositol 1,4,5-trisphosphate receptors in cardiac myocytes from newborn mice

Abstract

The precise localization and role of inositol 1,4,5-trisphosphate (InsP(3)) receptors (InsP(3)Rs) in cardiac muscle cells are largely unknown. It is believed that waves and oscillations in cytosolic free calcium triggered by activation of InsP(3)Rs underlie modifications of cellular responses that lead to changes in gene expression in other cells. However, how changes in cytosolic calcium alter gene expression in cardiac cells is unknown. Moreover, it is unclear how changes in cytosolic calcium that alter gene expression do so independently of effects of calcium on other cellular functions, such as contraction. Here we show that InsP(3)R type 2 is the only isoform present in cardiac myocytes isolated from neonatal mouse ventricles. We also show that type 2 InsP(3)Rs are associated with the nucleus and that activation of type 2 InsP(3)Rs with endothelin-1 or phenylephrine selectively increases transcription of atrial natriuretic factor and skeletal alpha-actin. Type 2 InsP(3)Rs are also in striations. Activation of InsP(3)Rs with adenophostin A in permeabilized cells induced calcium release in the nuclear domain and other regions of the cell away from the nucleus. Agonist-induced increase in gene expression and calcium release were blocked by the InsP(3)R inhibitors 2-aminoethoxydiphenyl borate and xestospongin C. The spatial separation of type 2 InsP(3)Rs provides support for the concept that microdomains of calcium discretely alter various cell processes. Our experiments suggest that calcium released by InsP(3)Rs in the nuclear domain provides a direct mechanism for the control of gene expression, whereas release of calcium in the cytoplasm may modulate other processes, such as contraction.

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

“…Consistent with this model, it has been demonstrated that activation of IP 3 Rs in rat neonatal ventricular myocytes using a membrane-permeant IP 3 analogue or an α 1 -adrenergic agonist causes perinuclear Ca 2+ release events, and that blocking IP 3 R opening decreases hypertrophic growth in response to phenylephrine stimulation [109]. Similar findings were made using mouse neonatal cells stimulated with ET-1 and phenylephrine; nuclear-associated IP 3 Rs triggered nucleoplasmic Ca 2+ signals and hypertrophic gene transcription [157]. Further work is required to establish whether this scheme is relevant in vivo.…”

“…However, the concept that IP 3 Rs impact specifically on nuclear Ca 2+ signaling is gaining momentum. Numerous recent reports examining Ca 2+ signaling in neonatal [103,108,109,157], atrial [71,72,137] and ventricular myocytes [140] have demonstrated specific effects of IP 3 -generating agonists or IP 3 itself on nucleoplasmic Ca 2+ signals. Coupling these data with the observations that IP 3 is necessary for some forms of hypertrophy [109,157,158] raises the possibility that IP 3 Rs are key regulators of cardiac myocyte gene transcription and fate.…”

An update on nuclear calcium signalling

“…Together with the finding that inhibiting InsP 3 R opening prevented hypertrophic growth in response to phenylephrine stimulation (Luo et al, 2006), it is logical to conclude that phenylephrine promotes hypertrophy by activating perinuclear InsP 3 Rs and, consequently, causing nucleoplasmic Ca 2+ signals. Comparable observations have been made in cardiac cells stimulated with another InsP 3 -generating agonist, endothelin-1; InsP 3 Rs in close proximity to the NE triggered nucleoplasmic Ca 2+ signals and hypertrophic gene transcription, whereas EC-coupling did not (Garcia et al, 2004;Higazi et al, 2009). Although InsP 3 Rs conduct significantly less Ca 2+ and are expressed at levels that are ~100-fold lower than RyRs, the activation of InsP 3 Rs can clearly have a dramatic effect on gene transcription in cardiac myocytes owing to their specific impact on nuclear Ca 2+ .…”

“…The presence of small InsP 3 -sensitive vesicular stores could help to explain the curious spotted distribution of nuclear InsP 3 R staining that is sometimes observed when using InsP 3 R-specific antibodies (particularly type-2-InsP 3 R-specific antibodies) (Garcia et al, 2004;Laflamme et al, 2002;Leite et al, 2003). If this is the case, these vesicular structures could represent nuclear InsP 3 -sensitive Ca 2+ stores that might operate independently of cytosolic Ca 2+ channels.…”

“…RyRs are therefore considered to be the main Ca 2+ -release channel in the heart. However, cardiac myocytes also express InsP 3 Rs , and recent studies of neonatal (Garcia et al, 2004;Guatimosim et al, 2008;Luo et al, 2008b;Luo et al, 2006), atrial Kockskamper et al, 2008a;Zima et al, 2007) and ventricular myocytes (Higazi et al, 2009; demonstrated that InsP 3 -generating agonists, or InsP 3 itself, have specific effects on nucleoplasmic Ca 2+ signals. Indeed, it appears that a substantial proportion of InsP 3 Rs in neonatal and adult cardiomyocytes is expressed on membranes close to the nucleus or on the NE (Bare et al, 2005;Higazi et al, 2009;Liu et al, 2001) (Fig.…”