the myriad of intracellular signaling pathways involving intracellular Ca2+ means Ca2+ plays a role in everything from cellular metabolism to cell death. In heart muscle Ca2+ is primarily known for its role in cellular contraction. Regulation of cardiac muscle contractility is achieved through modulation of the spatiotemporal nature of intracellular Ca2+ signals, operating in areas of restricted diffusion or microdomains, close to Ca2+ release sites. This Viewpoint article briefly integrates experimental evidence in support of this concept.
Excitation-contraction (E-C) coupling in cardiac muscle is the transduction mechanism linking the action potential to cell shortening that occurs in response to a transient rise in intracellular calcium (Ca2+i). Activation of the L-type Ca2+ current (ICa,L) triggers a larger release of Ca2+ from the sarcoplasmic reticulum (SR) store via Ca2+-induced Ca2+ release (CICR) due to activation of the SR Ca2+ release channel, the ryanodine receptor (RyR2), on the SR membrane (2, 9, 10). This Ca2+ reaches the myofilaments, which results in contraction of the cell and the generation of force.
Since the first observations of elementary Ca2+ release events or Ca2+ sparks in cardiac muscle (5) it has become evident that unitary events are important in shaping global Ca2+ signaling in the heart such that coordinated release of Ca2+ from the SR reflects the temporal and spatial summation of Ca2+ spark activity. Coordinated Ca2+ release activity from the SR is initially injected into the small volume of the junctional cleft (described below), in advance of reaching the myofilaments. This creates an area of high Ca2+ (a microdomain) that is spatially and temporally distinct from that found globally (Fig. 1). This creates unique opportunities for the regulation of Ca2+ signaling due to modulation of either the rapid local release flux or the spatial compartmentalization of areas of high Ca2+, which prevents regenerative (and arrhythmic) Ca2+ release (Ca2+ waves). In the junctional cleft region of the ventricular cell these two situations occur in concert, enabling the cell to use the Ca2+ release activity in a regulatory capacity by virtue of proximity and/or sensitivity of the signaling pathway to the high Ca2+ levels in the microdomain environment. Thus temporal or spatial compartmentalization of Ca2+ signals is a key feature for the regulation of ventricular contractility. This Viewpoint article will explore the evidence and advances in experimental thinking in support of cellular mechanisms contributing to and using this spatiotemporal Ca2+ activity, and therefore, local Ca2+ signaling in cardiac ventricular myocytes during the cardiac cycle, which are confined specifically to the area of the junctional cleft.
CA2+ MICRODOMAINS IN THE JUNCTIONAL CLEFT OF THE VENTRICULAR CELL
The primary functional prerequisite for localized Ca2+ signaling is the generation of a Ca2+ microdomain, an area of Ca2+ within the cell exhibiting steep concentration gradients with the surrounding intracellular areas. The spatial and temporal generation of a Ca2+ microdomain is a product of the activity and location of Ca2+ flux pathways on both extracellular and intracellular membranes and the interaction of these pathways with endogenous buffer systems that shape the spatial and temporal spread of Ca2+ in the cell. In the ventricular myocyte, this requirement is met by the activity and location of ICa,L and RyR2 to the extracellular and intracellular membranes, respectively. A unique feature of the mammalian ventricular cardiac cell is the close proximity between sarcolemmal (ICa,L) and SR (RyR2) Ca2+ release pathways. This structural arrangement is brought about by the close juxtaposition between the t-tubule membrane (which contains a large proportion of ICa,L; Ref. 21) and the SR membrane, forming the dyadic or junctional cleft, a narrow region 10–20 nm wide (15, 16). The clustering of ICa,L and RyR2 is termed a couplon, which are located throughout the dyadic or junctional cleft. Thus the fluxes of Ca2+ and the diffusion within this space is restricted not only by endogenous buffer systems but also by the physical constraints of the t-tubule and SR membranes. This unique arrangement serves to enhance the concentration of Ca2+ in this microdomain during systole; predictions from modeling studies suggest rises in the region up to 100 μM within the limited volume of the junctional cleft within a few milliseconds (22, 26, 38).
EXOGENOUS CA2+ BUFFERS
Endogenous buffer systems contribute to limiting the spatial and temporal spread of a Ca2+ signal. The activity of intracellular buffers may be quantitatively described by the length constant (λ) of the buffer species. This is the effective distance a Ca2+ ion travels before it is bound by the buffer and is related to the rate of binding of Ca2+ to the buffer and its concentration: for any given buffer concentration, a slower rate of binding means Ca2+ will be able to travel further before becoming chelated. Differences in endogenous buffer systems can be overcome in an experimental setting by introducing exogenous buffers into cardiac cells (often in combination with whole cell voltage clamp where the patch pipette has direct access with the cell interior). These compounds have more closely approximated buffering parameters: buffering by BAPTA (which has a short length constant, ∼10 nm at 10 mM) spatially restricts Ca2+ to a greater extent than EGTA, which has a longer length constant (∼100 nm at 10 mM). The relevance of these concepts and their use in identifying processes governed by Ca2+ microdomains in ventricular cells cannot be underestimated (42) since the sensitivity of Ca2+-dependent processes to an exogenous buffer is a key defining characteristic of processes regulated by localized Ca2+ signaling in cardiac cells. Fowler et al. (13) recently demonstrated the retention of rapid Ca2+-sensitive inactivation kinetics of ICa,L despite increases of endogenous EGTA to 50 mM (length constant of ∼50 nm), which abolishes the global intracellular Ca2+ transient during stimulation. This effect is also reported by Sham (36) and Brette et al. (3). ICa,L therefore represents an ideal bioassay of localized Ca2+ in the ventricular cell, since its inactivation kinetics are exquisitely sensitive to Ca2+ in the vicinity of the channel (17, 20, 44) and since its location, opposed to the SR Ca2+ release pathway, affords it privileged access to localized Ca2+ released from the SR (35). Since inactivation of the channel is not affected by high levels of buffering, this suggests that ICa,L and RyR2 are intimately opposed.
Song and coworkers (39) used the slow Ca2+ buffer EGTA to shed light on the SR Ca2+ release process. They combined a low-affinity Ca2+-sensitive indicator with mM concentrations of EGTA to resolve triggered localized SR Ca2+ release events in cardiac myocytes, referred to as Ca2+ spikes. These rapid SR Ca2+ release events (∼10–15 ms time to peak) provide a readout of local SR Ca2+ flux due to EGTA filtering out changes in global Ca2+ but leaving SR release intact. This method has shown that the synchrony of Ca2+ spikes plays a significant role in efficient E-C coupling in cardiac muscle.
PHYSIOLOGICAL PROCESSES DRIVEN BY THE CLEFT MICRODOMAIN
Many Ca2+-sensitive processes and effectors are intrinsically associated and regulated by Ca2+ signaling in the ventricular microdomain.
Ca2+ release and contraction.
The close apposition of ICa,L and RyR2 in the couplon has significant functional impact. This arrangement offers an efficient method of delivering enough Ca2+ to RyR2 on stimulation to activate Ca2+ release from the SR and hence contraction. In an elegant study, data from the laboratory of Zahradnikova and coworkers (31; see also 4, 14) recently demonstrated the functionality of the couplon and local Ca2+ release in directly regulating global Ca2+ release and contractility. Their results indicate that eight L-type Ca2+ channels, from a cluster of 20–40 LTCCs, are required to be open to trigger CICR, measured using Ca2+ spikes, from a cluster of RyRs in the couplon. Thus alterations to the number of active L-type channels (and adjacent RyRs) in the couplon has a direct influence on local Ca2+ release activity and thus the regulation of global Ca2+ signaling and contractility.
The mechanisms underpinning inotropic interventions also appear to operate within the ventricular microdomain, since these mechanisms are robustly intact even in the presence of exogenous Ca2+ buffers (see rationale above). For example, β-adrenergic stimulation increases the open probability of unitary ICa,L (iCa,L), increasing local Ca2+ flux and increasing the synchronicity of local Ca2+ release (31, 40), enhancing both the amplitude and time course of the resulting cellular Ca2+ transient. Similarly, ICa,L has been shown to be modulated locally by activation of β2-adrenergic stimulation in contrast to global responses mediated by β1-receptors (6). Additionally, the plasticity of ICa,L, in particular its response to increases in pacing frequency, results in an increase in current amplitude and a slowing of inactivation (current facilitation) (19, 28, 47). This behavior, which involves CaMKII (13), is not dependent on a global Ca2+ signal, strongly suggesting that this regulatory pathway is confined to a spatially and temporally discrete, local Ca2+-signaling mechanism. This conclusion is supported by modeling studies that demonstrate the suitability of the ventricular microdomain, but not the cytosol, for activating CaMKII (34).
An example of this is the local association of the Ca2+-sensitive protein calmodulin (CaM), which is constitutively tethered to the cytosolic C-terminus of the L-type Ca2+ channel (7, 8, 27, 46). Privileged access to Ca2+ in the vicinity of the channel during activation (both from flux through the channel and Ca2+ released from the SR; see above) results in channel inactivation driven by conformational changes to CaM (29, 30, 33, 44). On a beat to beat basis this limits Ca2+ influx and prevents sustained increases in SR Ca2+ load. A further example is demonstrated by the Ca2+-sensitive peptide sorcin. Sorcin is a 21.6-KDa penta EF hand Ca2+-binding protein expressed in cardiac tissue and demonstrates association with the dyad region (24) and binding to the L-type Ca2+ channel (25). Recent evidence has demonstrated that modulation of ICa,L by sorcin is insensitive to high concentrations of Ca2+ buffer (13), suggesting that endogenously it operates via the Ca2+ microdomain although it is unclear whether this mechanism requires cyclical or sustained alterations to intracellular Ca2+.
Interaction of ion channel pathways in the junctional microdomain.
There may be Ca2+-sensitive feedback between ion flux pathways via the junctional microdomain. For example, ICa,L Ca2+-sensitive inactivation kinetics are modulated in the absence of NCX activity (32), suggesting that the exchanger plays a role in controlling Ca2+ in the localized region of the channel. It is tempting to conclude that this is a local component contributing to global Ca2+ flux balance (45). Abolition of this effect occurred in the presence of BAPTA, a buffer with a short length constant, suggesting that these two pathways operate via a localized Ca2+ microdomain. A functional estimate of the separation of these two pathways has yet to be elucidated.
THE ROLE OF THE T-TUBULES
The juxtaposition of ICa,L and RyR2 brought about by the t-tubule system in mammalian ventricular cells implicates the t-tubules as a significant contributory factor in the generation of a Ca2+ microdomain in ventricular tissue and therefore in the regulation of contractility. Indeed, Ca2+ release is attenuated and ICa,L facilitation is abolished in the absence of a t-system (11). Accumulating evidence has demonstrated changes to the t-system in heart failure (1), which may alter the synchrony of Ca2+ release (12, 18, 23), particularly as a result of t-tubule reorganization, which results in the breakdown of couplons as RyRs become “orphaned” as clusters of L-type Ca2+ channels migrate away from the SR as the t-tubules remodel (41). Shorofsky et al. (37) report alterations to Ca2+ sparks during cardiac hypertrophy and many studies demonstrate alterations in Ca2+ release as a result of heart failure. These changes suggest that cardiac pathology affects localized Ca2+ signaling, but a detailed investigation of these alterations has yet to be performed. Thus changes to t-tubule density, their structure and their function may have important consequences for localized Ca2+-dependent regulation of cardiac contractility as a result of cardiac pathology.
The demonstration of localized Ca2+ signaling in cardiac ventricular cells underpins the importance of these signals for efficient E-C coupling and cardiac function. The details of localized Ca2+ signaling remain to be fully elucidated and the extent and interdependence of signaling pathways will reveal the complexity of local regulation of E-C coupling in cardiac tissue and how these processes are modified during cardiac pathology.
The authors gratefully acknowledge the support of the British Heart Foundation. M. R. Fowler is in receipt of an Intermediate Research Fellowship (FS/06/027).
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