Title: Calsequestrin, triadin and more: the molecules that modulate calcium release in cardiac and skeletal muscle
Abstract: Calcium signals play a role in every cell where functional switching takes place. These signals were demonstrated first in striated muscles, where they control the force-generating interaction among contractile filaments, the function named excitation–contraction (EC) coupling. The three main molecular players that cooperate to produce these signals are the Ca2+ release channel of the sarcoplasmic reticulum (or RyR), the voltage sensor (or DHPR) – a molecule resident in the plasma membrane and its invaginations, which transduces the membrane potential changes to a control signal for the RyR – and the ATP-powered pump that brings Ca2+ back into the storage organelle. Melzer et al. (1995), Bers (2002) and Guatimosim et al. (2002) provide useful background information on skeletal and cardiac muscle EC coupling. The chemical identities and basic functions and interactions of these molecules were established long ago. More recently it has become obvious that correct signalling for EC coupling requires the presence and adequate functioning of a number of other proteins, which are either integral to the SR membrane, lumenal – resident inside the SR – or, in the special but perhaps not unique case of Orai1, present in the plasma and t-tubule membranes. Features and roles of these players were discussed in the Journal of Physiology Symposium Calsequestrin, triadin and more: the proteins that modulate calcium release in cardiac and skeletal muscle. The symposium was planned with the intent to compare skeletal and cardiac muscle side by side, a strategy that continues to provide advantages in understanding of function and its correlation with structure. It was interesting to consider among other questions how homologous proteins, such as triadin, junctin and calsequestrin, are adapted to the specific functional requirements of these two muscle types and whether the molecular differences between protein isoforms can account for the observed differences in their function. An additional goal was to discuss the human diseases (and corresponding animal models) associated with abnormalities in some of these molecules. The studies addressed in the symposium have the potential to help manage diseases or functional deficits. At the same time, the study of altered function or structure in patients with inheritable mutations (or models with similar changes) constitutes a productive approach to clarifying structure–function relationships. The present issue of The Journal includes one review article by each of the seven symposium speakers and one brief Perspective by each of the six discussants that completed the programme. In the first article, Susan Treves and collaborators provide a broad overview of SR proteins that have been found and identified in SR vesicular fractions. In particular, they distinguish between proteins considered major (on the basis of their quantity) and minor, and then proceed to discuss five of these minor components. The attention then moves to calsequestrin (Casq), the main lumenal Ca2+ binding protein. Bjorn Knollmann focuses on the insights provided by gene-targeted Casq2 (cardiac isoform) knock-out models that replicate cases of complete absence of Casq2 observed in humans. He concludes that the contribution of this molecule to Ca2+ storage and release is dispensable, but recognizes the stabilizing role of the protein, made patent by the catecholaminergic polymorphic ventricular tachycardia (CPVT) syndrome associated with its absence. In the first Perspective, Gerhard Meissner approaches a broad swathe of questions, regarding the roles of Casq1, Casq2 and Triadin, through the lens of his studies using silencing of the respective genes in myogenic cells in culture. The silencing approach is valued as an alternative to gene ablation, which provides less opportunity for compensatory or other confounding changes, and in the hands of these investigators resulted in nearly complete deletion of the targeted protein. Sandor Györke and coworkers reexamine the role of Casq2 in cardiac muscle and the pathogenesis of CPVT. They discuss mechanisms whereby a reduction in its concentration will allow for premature Ca2+ release and associated arrhythmias. They stress the importance of 'lumenal Ca2+-dependent deactivation', whereby release channels are closed and resistant to activation when [Ca2+]SR falls below a threshold. They conclude that reduction or alteration in Casq2 may disrupt this stabilizing mechanism, via the reduced buffering and/or associated changes in the ability of Casq2 to act as lumenal Ca2+ sensor. Two articles and one Perspective examine the roles of calsequestrin in skeletal muscle. Feliciano Protasi and collaborators review the properties of a model mouse lacking Casq1. Most interestingly, they find in male mice a susceptibility to trigger lethal episodes resembling malignant hyperthermia (MH) in response to volatile anaesthetics and heat stress, thus accentuating the parallels between the roles of calsequestrin in skeletal and cardiac muscle. Leandro Royer and Eduardo Ríos recall a number of functional observations indicating that the Ca2+ buffering properties of the SR are strongly non-linear, and are apparently optimized to support Ca2+ release when the SR lumenal [Ca2+] is close to the values found at rest. They discuss evidence that these properties result from the presence of calsequestrin and require its correct aggregation and polymerization inside the SR. In discussing the contributions on calsequestrin, David MacLennan and Wayne Chen were struck by the similarities between the consequences of its ablation in cardiac and skeletal muscle, and mutations of the RyR channels in both tissues. From this parallel they derive a model of pathogenesis that result in premature activation of Ca2+ release by the lumenal Ca2+, activation that is promoted either by reduction in Ca2+ buffering (when Casq is altered) or destabilizing mutations of the RyRs. Triadin and junctin (JNC) are integral SR membrane proteins that interact with both the release channel and calsequestrin. The possible roles of triadin are reviewed by Isabelle Marty and colleagues. They propose that the protein modulates the RyR's gating function, so that any modification of its expression levels will reduce Ca2+ release. They also suggest a novel concept: that triadin connects the RyR and associated proteins to the microtubule network, a connection that could be necessary to preserve both junctional structure and normal function. Both the functional role of triadin as adjuvant of Ca2+ release and its structural role as a putative anchoring protein are examined by Paul Allen. Based on multiple lines of evidence, including his own studies of a triadin KO mouse, he candidly and forcefully concludes that triadin is 'useful but not essential'. The examination of the roles of these proteins is continued by Tracy Pritchard and Litsa Kranias, who focus on junctin and the histidine-rich Ca2+ binding protein (HRC). Both proteins are involved in ternary or quaternary interactions (with the release channel and the SR Ca2+ pump in the case of HRC). The endowment of both is reduced in the failing heart. The review is notable for discussing altered Ca2+ cycling in heart failure and proposing an intriguing pathogenetic model that involves these two proteins. Angela Dulhunty and colleagues continue the exploration of the roles of junctin, initiated by Pritchard and Kranias. They emphasize the differences between JNC and triadin, proposing that JNC has a unique role in the stabilization of a closed release channel at low luminal [Ca2+]SR, related perhaps to this protein's ability (which triadin lacks) to bind simultaneously to calsequestrin and the release channel. One topic that appears repeatedly in different articles is the existence of alternative sources of Ca2+, which become manifest upon the many manipulations that result in the ablation, partial or total, of calsequestrin. These sources may be enhanced by the cell upon the disappearance of Casq, or may preexist in the wild-type. Influx of extracellular Ca2+ might help support Ca2+ release. In skeletal muscle this contribution is known to be minimal during a single action potential, but could become important during sustained activation, thus postponing or counteracting the SR depletion that is believed to contribute to muscle fatigue. Bob Dirksen reviewed recent advances in our understanding of function and molecular makeup of the so-called store operated Ca2+ entry (SOCE) pathway, including the demonstration in skeletal muscle of the two main components that constitute this pathway in non-excitable cells: STIM1 as the SR Ca2+ sensor and Orai1 as the periplasmic Ca2+ entry channel. Finally, Paul Rosenberg, whose laboratory provided the first evidence of the involvement of the STIM1–Orai1 complex in the SOCE pathway of skeletal muscle (Stiber et al. 2008), discusses in his Perspective possible approaches to unravel the mechanisms leading from the genotype of mice models without STIM1 or Orai1 to the corresponding phenotypes, which include multiple deficits in tissues and systems other than muscle. The collective contemplation of these articles yields strong pointers to future directions in research. Both calsequestrin and triadin are now known to be non-essential – life without them is possible, which calls for defining their roles quantitatively. For calsequestrin this means quantifying its capacity as a Ca2+ reservoir, as well as the relative relevance of the mechanisms that substitute for it upon its ablation. Additionally, conflicting views will have to be resolved regarding the relevance of its role as Ca2+ sensor and modulator of channel opening. Triadin's newly described structural roles open a new avenue to be explored. The studies of the minor proteins are likely to reveal entirely new roles, either in function or maintenance of junctional structures. The studies of basic function and mechanism will rapidly improve our understanding of inheritable diseases, as well as conditions of functional deterioration like heart failure, muscle fatigue, and the decay associated with ageing. Finally, the studies of SOCE, enhanced by new knowledge at the molecular level, are likely to radically change our views of long-term Ca2+ homeostasis in muscle function.