Perspective Perspectives on: Local calcium signaling Subcellular Ca2+ signaling in the heart: the role of ryanodine receptor sensitivity Benjamin L. Prosser,1 Christopher W. Ward,1,2 and W.J. Lederer1 1Center for Biomedical Engineering and Technology, BioMET, and 2School of Nursing, University of Maryland, Baltimore, MD 21201 2+ 2+ Local Ca signaling in the heart enables the triggering elevated [Ca ]i? Does the cardiac cytoskeleton play both and regulation of the time-dependent changes of intra- organizational and dynamic roles in the regulation of 2+ 2+ 2+ 2+ cellular Ca concentration ([Ca ]i) to be flexibly mod- Ca sparks? How does SR Ca instability arise, and how ulated with a high degree of stability and safety. The does it lead to the generation of cardiac arrhythmias? elementary physiological and pathophysiological com- How can mathematical modeling contribute to such in- ponents of subcellular Ca2+ signaling are regulated vestigations? How does Ca2+ signaling dysfunction influ- through a process of Ca2+-induced Ca2+ release (CICR), ence or underlie disease progression? We present here not by store overload–induced Ca2+ release (SOICR). some organizing principles that should help to resolve The central element of CICR regulation is the sensitivity several issues and lay the foundation for future studies. of the SR Ca2+ release channel, the RYR type 2 (RYR2), to 2+ [Ca ]i. Here, in terms of past discoveries and future Spatial organization of the heart cell and the control 2+ 2+ work, we discuss how RYR2 sensitivity to [Ca ]i is in­ of Ca sparks fluenced by key factors, including SR luminal Ca2+ Subcellular anatomy: key to Ca2+ signaling stability. The SR 2+ ([Ca ]SR), RYR2 phosphorylation, mutations in RYR2 includes two primary components, the jSR and the lon- and its interacting proteins, as well as cellular stretch. gitudinal SR (also called the free SR or the “network” Finally, we explore the pathophysiological consequences SR; see Fig. 1) (Brochet et al., 2005). In addition, the SR of dysfunctional tuning of these regulatory factors in is connected to the ER and the nuclear Ca2+ store (Wu the context of dystrophic cardiomyopathy. and Bers, 2006), thus making the entire Ca2+ storage The spatial organization of the cardiac ventricular network fully interconnected. The primary Ca2+ release 2+ myocyte enables precise regulation of the cardiac [Ca ]i sites are located at the jSR, an extremely small pancake- transient (Bers, 2001; Cheng and Lederer, 2008). The shaped sub-volume in the system (each jSR contains 1 details of this regulation at the subcellular level, however, attoliter, 1 × 1018 l, a size 4,000 times smaller than a remain both controversial and exciting. There is consen- Ca2+ spark). On one face, the jSR contains a paracrystal- 2+ 2+ sus that the “global” or cell-wide [Ca ]i signal in ventric- line cluster of 10–300 SR Ca release channels, the The Journal of General Physiology ular myocytes is central to cardiac function; it underlies RYR2s that span 15 nm (the “subspace”) to the surface contraction and contributes to the regulation of electri- membrane (i.e., the transverse tubule [TT] or surface sar- cal activity. Despite the certainty and clarity of these colemmal [SL] membranes) (Franzini-Armstrong et al., contributions, the subcellular details of Ca2+ signaling 1999; Baddeley et al., 2009). The other face of the jSR remain unsettled. How do subcellular organelles con- points toward the M line and is tens of nanometers away tribute to Ca2+ sparks? Although it is clear that sparks from the Z-disk end of the mitochondria and is con- originate almost exclusively at the junctional SR (jSR), nected through the thin nanoscopic tubules that consti- many aspects of Ca2+ spark signaling are still being inves- tute the longitudinal SR. The L-type Ca2+ channels (or tigated. For example, do mitochondria serve as dynamic dihydropyridine receptors) are transmembrane pro- Ca2+ stores? What “triggers” spontaneous Ca2+ sparks teins that span the SL and TT membranes facing the versus synchronized sparks? How does a propagating subspace and thus appose the jSR. The lumen of the jSR Ca2+ wave arise? What sustains the propagating wave of also contains the Ca2+-binding protein calsequestrin type 2 (CASQ2) and other regulatory proteins, such as junctin, triadin, junctophilin, and a small amount of Correspondence to W.J. Lederer: j­l­e­d­@­u­m­a­r­y­l­a 2+ Abbreviations used in this paper: AnkB, ankyrin B; [Ca ]i, intracellular 2+ 2+ 2+ 2+ © 2010 Prosser et al. This article is distributed under the terms of an Attribution– Ca ; [Ca ]SR, SR luminal Ca ; CASQ2, calsequestrin type 2; CICR, Ca - induced Ca2+ release; DMD, Duchenne muscular dystrophy; jSR, junctional Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publi- cation date (see http://www.rupress.org/terms). After six months it is available under a SR; ROS, reactive oxygen species; RYR2, RYR type 2; SL, surface sarcolemmal; Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, SOICR, store overload–induced Ca2+ release; TT, transverse tubule. as described at http://creativecommons.org/licenses/by-nc-sa/3.0/). The Rockefeller University Press $30.00 J. Gen. Physiol. Vol. 136 No. 2 135–142 www.jgp.org/cgi/doi/10.1085/jgp.201010406 135 other proteins including the ER Ca2+-binding and chaper- was more challenging. Mathematical modeling of spark one protein calreticulin (Bers, 1991; Györke et al., 2007). behavior suggests that if a significant fraction of the local The brief opening of either an L-type Ca2+ channel or jSR Ca2+ is depleted during the Ca2+ spark (between 50 an RYR2 under diastolic conditions leads to a local ele- and 90%), a robust termination of the spark will be seen 2+ 2+ vation of Ca in the subspace ([Ca ]subspace) from the (Sobie et al., 2002). Importantly, this occurs without the 2+ normal [Ca ]i of 100 nM to 10 µM (Cannell and Soeller, need for “inactivation” of the RYR2s, unlike in other 1997; Soeller and Cannell, 1997; Sobie et al., 2002). models (Stern et al., 1999). In support of this model, 2+ This elevation of [Ca ]subspace, albeit brief (<1 ms), is there is no experimental evidence to date that suggests sufficient to activate the RYR2 cluster to produce a Ca2+ rapid “fateful” inactivation of RYR2s (Liu et al., 2010). 2+ 2+ 2+ 2+ spark, a process termed Ca -induced Ca release, or The [Ca ]SR depletion hypothesis regarding Ca spark CICR (Fabiato, 1983; Cheng et al., 1993; Cannell et al., termination has been supported by the observation of 1994a,b; Sobie et al., 2002; Cheng and Lederer, 2008; Ca2+ “blinks” (Brochet et al., 2005). A Ca2+ blink is the 2+ 2+ Györke and Terentyev, 2008; Liu et al., 2010). The spa- [Ca ]SR depletion signal that occurs when a Ca tial organization of the jSR enables reliable activation of spark takes place. It is the reciprocal signal of a Ca2+ spark; 2+ 2+ Ca sparks by action potentials, but the relative insensi- specifically, [Ca ]SR depletion is seen whenever sparks tivity of RYR2s to calcium protects the cell from instabil- occur because the Ca2+ flux that underlies the Ca2+ ity and enables Ca2+ sparks during diastole to remain spark comes from the jSR lumen. SR Ca2+ leak is the isolated from neighboring jSR spark sites. Thus, Ca2+ term of art now used to describe the non-synchronized sparks do not normally activate other Ca2+ sparks (Cheng loss of Ca2+ from the SR, which may occur as Ca2+ sparks et al., 1993). High gain in the signaling pathway is thus or as very small release events that may be invisible when created by the spatial organization of RYR2s in a cluster, viewed with a confocal microscope (Sobie et al., 2006). and stability is maintained by the relative insensitivity of How Ca2+ leak may occur, and how this leak may influence 2+ 2+ the RYR2s to [Ca ]i (Cheng and Lederer, 2008). Ca instability and cardiac arrhythmogenesis, is actively under examination (Wehrens et al., 2003; Lehnart et al., Ca2+ sparks, Ca2+ blinks, and SR Ca2+ leak. The “life cycle” 2006, 2008; Sobie et al., 2006). of a Ca2+ spark provides clues to its regulation. Should 2+ 2+ 2+ 2+ [Ca ]subspace increase to 10 µM, there is good probabil- Triggering Ca sparks and Ca waves. A Ca spark is the ity that a Ca2+ spark will arise through CICR as noted local Ca2+ signal produced by an ensemble of RYR2s at a above. Although Ca2+ spark termination is seen robustly single jSR (Cheng and Lederer, 2008). Diastolic sparks in experiments, our understanding of how this occurs can occur when a single RYR2 opens probabilistically Figure 1. RYR2s in cardiac ventricular myocytes. (A) Diagram of heart cell illustrating the locations of the TTs and SL with respect to the L-type Ca2+ channels (LCC), the junctional SR (jSR), the longitudinal SR, SERCA, RYR2 cluster, subspace, the Na+/Ca2+ exchanger (NCX), the plasmalemmal Ca2+ ATPase (PMCA), and a mitochondrion. (B) The dashed box (in A) is enlarged to reveal some of the 2+ + changes that may occur in DMD. Micro-ruptures may lead to an increase in [Ca ]i and [Na ]i, as may hyperactivity of stretch-activated channels (SAC); overexpression of caveoli, a putative site of NADPH oxidase, may also be involved. (C) Diagrammatic illustration how RYR2 2+ Po sensitivity to [Ca ]i may be shifted to A (increase in sensitivity) or shifted to B (decrease in sensitivity). See Table I. 136 Regulation of local Ca2+ signaling in the heart 2+ and increases [Ca ]subspace to a high level (i.e., 10 µM), a jSR cluster.