Dichotomy of Ca²⁺ in the heart: contraction versus intracellular signaling

Abstract

Ca²⁺ plays a pivotal role in both excitation-contraction coupling (ECC) and activation of Ca²⁺-dependent signaling pathways. One of the remaining questions in cardiac biology is how Ca²⁺-dependent signaling pathways are regulated under conditions of continual Ca²⁺ transients that mediate cardiac contraction during each heartbeat. Ca²⁺-calmodulin–dependent protein kinase II (CaMKII) activation and its ability to regulate histone deacetylase 5 (HDAC5) nuclear shuttling represent a critical Ca²⁺-dependent signaling circuit for controlling cardiac hypertrophy and heart failure, yet the mechanism of activation by Ca²⁺ is not known. In this issue of the JCI, Wu et al. convincingly demonstrate that the inositol 1,4,5-trisphosphate receptor (InsP₃R) is involved in local control of Ca²⁺ for activating CaMKII in the nuclear envelope of adult ventricular cardiac myocytes. The overall paradigm that is demonstrated is the best example of a molecular mechanism whereby signaling is directly regulated by a local Ca²⁺ pool that is disparate or geometrically insensitive to cytosolic Ca²⁺ underlying each contractile cycle.

The physiologic relevance of “reactive” Ca²⁺ signaling in the heart is uncertain, given the highly specialized manner in which Ca²⁺ cycling is tightly controlled through the membrane compartments/domains, channels, and pumps that underlie excitation-contraction coupling (ECC). For example, in response to depolarization of the sarcolemma, Ca²⁺ enters through the voltage-dependent L-type Ca²⁺ channel, which directly stimulates adjacent ryanodine receptors embedded within the sarcoplasmic reticulum (SR). This priming Ca²⁺ from the L-type channel induces a much larger release of Ca²⁺ from ryanodine receptors that increase intracellular Ca²⁺ concentration by more than 10-fold to induce contraction. During diastole, Ca²⁺ is removed from the cytoplasm by resequestration back into the SR through the action of SR/ER Ca²⁺-ATPase (SERCA) as well as extrusion from the cell through the action of the Na⁺/Ca²⁺ exchanger within the sarcolemma (Figure 1). Thus, ECC-mediated Ca²⁺ cycling accounts for nearly all routinely detectable Ca²⁺ alterations that occur within a cardiomyocyte, which makes it difficult to explain how Ca²⁺-activated signaling proteins function in this background. Also to be considered, it would make little sense to regulate Ca²⁺-dependent signaling proteins through changes in Ca²⁺ cycling associated with ECC since such a mechanism would not afford a disconnection between inotropy and signaling. Indeed, near maximal Ca²⁺ cycling due to phospholamban deletion in mice does not induce cardiac hypertrophy or otherwise predispose the heart to dysfunction that would be associated with activation of reactive signaling pathways, indicating that inotropic drive is not a direct source of Ca²⁺ for intracellular signaling pathways (1). Thus, specialized pools of Ca²⁺ that are location specific or somehow buffered from cytoplasmic Ca²⁺ need to be evoked to account for the regulation of Ca²⁺-sensitive signaling proteins, such as Ca²⁺-calmodulin–dependent protein kinase (CaMK), calcineurin, and PKC. However, the existence of microdomains or region-specific buffering of Ca²⁺ to control signaling proteins has not been convincingly demonstrated to date in cardiac myocytes. In this issue of the JCI, Wu et al. report on their employment of a series of pharmacologic, molecular, and genetic manipulations to convincingly demonstrate that inositol 1,4,5-trisphosphate receptor–based (InsP₃R-based) alterations in Ca²⁺ levels regulate CaMKII activation and its ability to control histone deacetylase 5 (HDAC5) translocation, thus suggesting a Ca²⁺-dependent reactive signaling circuit that is controlled outside of ECC-based Ca²⁺ cycling (2).

Figure 1.

Schematic of potential Ca²⁺ sources that might be specialized to regulate reactive signaling pathways in cardiac myocytes. (i) Some L-type Ca²⁺ channels (I_Ca,L) are not associated with the junctional complex and hence could be involved in providing a local Ca²⁺ signal in specific membrane-associated compartments. (ii) T-type Ca²⁺ channels (I_Ca,T) are reexpressed in hypertrophic states where they could provide Ca²⁺ in specific microenvironments associated with the sarcolemma to affect reactive signaling pathways. (iii) Capacitative or store-operated Ca²⁺ entry through transient receptor potential (TRP) channels, alone or in conjunction with (iv) InsP₃R-mediated release of Ca²⁺ from the ER/nuclear envelope, could also provide a highly localized Ca²⁺ pool for controlling reactive signaling pathways in cardiac myocytes. Signaling from G protein–coupled receptors (GPCRs) activates PLC and generates InsP₃, causing a perinuclear Ca²⁺ signal through the InsP₃R, resulting in CamKII activation and HDAC5 nuclear export, as proposed by Wu et al. (2). IP₃, InsP₃; IP₃R, InsP₃R; PLN, phospholamban; NCX, Na⁺/Ca²⁺ exchanger; RyR, ryanodine receptor; CaM, calmodulin; SERCA, SR/ER Ca²⁺-ATPase; P, phosphate.

Ca²⁺-sensitive reactive signaling pathways in cardiac myocytes

In response to disease-causing stimuli, the myocardium is directly influenced by neuroendocrine secreted growth factors and/or cytokines that induce ventricular remodeling, hypertrophic enlargement of myocytes, and alterations in the viability of myocytes. Many of these neuroendocrine factors (e.g., angiotensin II and endothelin-1) signal through G protein–coupled receptors on cardiac myocytes to induce phospholipase C (PLC) activation, which in turn generates InsP₃ and diacylglycerol (DAG) (3). In most cell types, InsP₃ generation in turn leads to release of Ca²⁺ from the endoplasmic reticulum through the InsP₃R channel, although it is uncertain whether InsP₃R activity significantly regulates Ca²⁺ release in adult ventricular cardiac myocytes. It is known that the heart generates InsP₃ and that the type 2 InsP₃R (InsP₃R2) is present and localized around the nucleus of ventricular myocytes while atrial myocytes also likely contain InsP₃Rs in the junctional SR where they can affect ECC (4, 5).

Neuroendocrine factors and cytokines promote activation of a number of intracellular signaling pathways in cardiac myocytes, including MAPK, calcineurin, PKC, CaMK, and IGF-1 pathway constituents (3). Three of these signaling factors, calcineurin, CaMK, and PKCα, -β, and -γ, require increases in Ca²⁺ to become activated although, as discussed above, the source of such Ca²⁺ in cardiac myocytes has yet to be determined. In most other cell types, InsP₃R-regulated Ca²⁺ mobilization in association with store-operated Ca²⁺ entry is required for activation of Ca²⁺-dependent signaling effectors (6). The importance of understanding such regulation in cardiac myocytes is underscored by the observation that both calcineurin and CaMK are potent inducers of the hypertrophic response (7–9). Hence, careful elucidation of the mechanism whereby calcineurin and CaMK become activated in cardiac myocytes should suggest the pool(s) of Ca²⁺ that function in reactive signaling.

Potential Ca²⁺ pools for mediating reactive signaling

The cardiac myocyte contains an organized array of sarcolemmal invaginations referred to as the T tubule network that closely apposes the SR as a means of distributing membrane depolarization and coordinated Ca²⁺ release throughout the cytoplasm. The complexity of this T tubule network as well as regions of the SR/ER that includes the nuclear envelope could easily accommodate more specialized Ca²⁺ regulatory domains that function outside of ECC and serve a signaling function. At least 4 different regulatory mechanisms for compartmentalizing Ca²⁺ outside of ECC have been hypothesized. One possible source of reactive Ca²⁺ is the voltage-regulated L-type Ca²⁺ channel itself, which normally triggers Ca²⁺-induced Ca²⁺ release and contraction. However, a percentage of L-type Ca²⁺ channels are not associated with the junctional complex and Ca²⁺-induced Ca²⁺ release, but instead could be dedicated to special membrane domains, such as lipid rafts to mediate a signaling function (Figure 1, i). Indeed, overexpression of the pore-forming subunit of the L-type Ca²⁺ channel in the hearts of transgenic mice led to late-onset cardiac hypertrophy (10). Another potential source of Ca²⁺ for reactive signaling is the voltage-dependent current mediated by T-type Ca²⁺ channels (Figure 1, ii). T-type channels are not normally expressed in mature ventricular myocytes, but expression is present in immature myocytes as well as in adult ventricular myocytes from hypertrophied and failing hearts (11–13). In other cell types, T-type currents play important roles in regulating reactive signaling and cellular proliferation, and more interestingly, these channels are associated with specialized membrane structures such as lipid rafts (14). A third possible source of reactive Ca²⁺ in myocytes is associated with members of the transient receptor potential channel that are thought to mediate store-operated Ca²⁺ entry and are expressed in the heart (15) (Figure 1, iii). Indeed, previous work has suggested that neonatal and/or adult rat cardiac myocytes are capable of store-operated Ca²⁺ entry once depleted with PLC-dependent agonists (16–18). Moreover, Hunton and colleagues showed that general inhibitors of store-operated Ca²⁺ entry could reduce calcineurin activation in cardiac myocytes (17). Indeed, in nonmyocytes, calcineurin signaling is prominently regulated by store-operated Ca²⁺ entry (19). In general, store-operated Ca²⁺ entry is associated or coupled with PLC-dependent InsP₃R Ca²⁺ release. Thus, the fourth potential source of Ca²⁺ for reactive signaling is InsP₃R dependent (Figure 1, iv). In adult ventricular cardiac myocytes, InsP₃R2 is expressed and primarily localized to the nuclear envelope (5). Interestingly, InsP₃R2 is physically associated with CaMKII and thus could serve a more specialized role in providing a local pool of Ca²⁺ to activate this kinase in or around the nucleus (Figure 1, iv).

InsP₃R signaling may be the missing link

In this issue of the JCI, Wu et al. provide convincing evidence that Ca²⁺ from an InsP₃R-dependent store regulates activation of HDAC5 nuclear export through CaMKII in adult ventricular cardiomyocytes (2). HDAC4/5 activity and nuclear occupancy are directly regulated by CaMK-, PKC-, and protein kinase D–mediated (PKD-mediated) phosphorylation (20, 21). That this regulatory relationship is central to the cardiac hypertrophic response is consistent with the observation that Hdac9- and Hdac5-null mice each develop exaggerated hypertrophy following pressure overload or when crossed with the calcineurin transgene (22, 23). Here, the authors demonstrate that the G protein–coupled agonist (PLC-activating) endothelin-1 induced export of HDAC5 from adult cardiac myocytes without affecting total cytosolic Ca²⁺ concentration and that this export was blocked with an InsP₃R inhibitor or stimulated directly with an InsP₃R agonist (2). More convincingly, HDAC5 nuclear export after endothelin-1 stimulation is completely absent in adult cardiac myocytes from Ip3r2 gene–targeted mice. These results unequivocally demonstrate that HDAC5 regulation depends in part on InsP₃R signaling. Assessment of Ca²⁺ levels within the cell using Fluo-5N suggested that an InsP₃ signal induces Ca²⁺ mobilization in the perinuclear area where the InsP₃R2 is localized in the nuclear envelope. Thus, HDAC5 nuclear export, which permits gene activation and induction of the hypertrophic response, can be directly regulated by an InsP₃-dependent pathway through a region-specific pool of Ca²⁺ within or near the nuclear envelope. This movement of HDAC5 in association with InsP₃R-dependent Ca²⁺ mobilization was partially mediated by CaMK activation since pharmacologic inhibition blocked approximately half of the HDAC5 nuclear export. Thus, the current work by Wu et al. establishes at least 1 paradigm whereby a specialized pool of Ca²⁺ can regulate a hypertrophic signaling circuit within a ventricular cardiac myocyte independent of contractile Ca²⁺.

As with most landmark studies, Wu et al. raises a number of important issues. First, it is interesting that pacing of adult cardiac myocytes up to 2 Hz did not cause HDAC5 nuclear export, suggesting that ECC-mediated Ca²⁺ fluxing has little effect on this regulatory pathway (2). In contrast, pacing of skeletal muscle myotubes enhanced Ca²⁺ transients enough to induce nuclear export of HDAC4 as well as nuclear factor of activated T cells (NFAT), a sensor of calcineurin signaling (24, 25). These later observations suggest that contractile Ca²⁺ can set in motion 2 separate Ca²⁺-dependent signaling pathways in skeletal muscle myotubes although this paradigm does not seem to apply to adult cardiac myocytes. Indeed, calcineurin does not even regulate hypertrophy in skeletal muscle as it does in heart muscle, demonstrating profound differences in signaling between these tissues (26). Hence, cardiac myocytes appear to be unique in their ability to compartmentalize or control Ca²⁺ such that the ECC-based pool is distinct from the pool that regulates reactive signaling.

One such Ca²⁺ microenvironment in the adult ventricular cardiac myocytes appears to be dependent on the InsP₃R localized to the perinuclear region. Indeed, CaMKII is directly complexed with the InsP₃R in cardiac myocytes, suggesting a mechanism whereby CaMKII might only respond to exceedingly high Ca²⁺ levels in the microenvironment of the InsP₃R and hence be insensitive to total intracellular Ca²⁺ levels that cannot reach such a putative threshold. For example, such a microenvironment exists at the junction between the sarcolemma and SR, where local Ca²⁺ regulates the independent activation of each ryanodine receptor (27). While Wu et al. only evaluated HDAC5 nuclear export as regulated by CaMKII (2), this paradigm should be evaluated for other signaling circuits that are controlled by Ca²⁺, such as other class II HDACs, calcineurin, and/or PKC isoforms. Similarly, it would also be interesting to determine if CaMKII and HDAC5 can be regulated by other potential microdomains of Ca²⁺ within the cardiac myocyte so that a hierarchy or specialization of Ca²⁺ microdomains might be established. Thus, the results of Wu et al. have not only established 1 Ca²⁺-dependent regulatory paradigm, but they have also provided the conceptual framework for further parsing contractile versus reactive signaling Ca²⁺ in the heart.