Lourdes Figueroa

Measurement of RyR permeability reveals a role of calsequestrin in termination of SR Ca 2+ release in skeletal muscle

The mechanisms that terminate Ca2+ release from the sarcoplasmic reticulum are not fully understood. D4cpv-Casq1 (Sztretye et al. 2011. J. Gen. Physiol. doi:10.1085/jgp.201010591) was used in mouse skeletal muscle cells under voltage clamp to measure free Ca2+ concentration inside the sarcoplasmic reticulum (SR), [Ca2+]SR, simultaneously with that in the cytosol, [Ca2+]c, during the response to long-lasting depolarization of the plasma membrane. The ratio of Ca2+ release flux (derived from [Ca2+]c(t)) over the gradient that drives it (essentially equal to [Ca2+]SR) provided directly, for the first time, a dynamic measure of the permeability to Ca2+ of the releasing SR membrane. During maximal depolarization, flux rapidly rises to a peak and then decays. Before 0.5 s, [Ca2+]SR stabilized at ∼35% of its resting level; depletion was therefore incomplete. By 0.4 s of depolarization, the measured permeability decayed to ∼10% of maximum, indicating ryanodine receptor channel closure. Inactivation of the t tubule voltage sensor was immeasurably small by this time and thus not a significant factor in channel closure. In cells of mice null for Casq1, permeability did not decrease in the same way, indicating that calsequestrin (Casq) is essential in the mechanism of channel closure and termination of Ca2+ release. The absence of this mechanism explains why the total amount of calcium releasable by depolarization is not greatly reduced in Casq-null muscle (Royer et al. 2010. J. Gen. Physiol. doi:10.1085/jgp.201010454). When the fast buffer BAPTA was introduced in the cytosol, release flux became more intense, and the SR emptied earlier. The consequent reduction in permeability accelerated as well, reaching comparable decay at earlier times but comparable levels of depletion. This observation indicates that [Ca2+]SR, sensed by Casq and transmitted to the channels presumably via connecting proteins, is determinant to cause the closure that terminates Ca2+ release.

D4cpv-calsequestrin: a sensitive ratiometric biosensor accurately targeted to the calcium store of skeletal muscle.

Current fluorescent monitors of free [Ca2+] in the sarcoplasmic reticulum (SR) of skeletal muscle cells are of limited quantitative value. They provide either a nonratio signal that is difficult to calibrate and is not specific or, in the case of Forster resonant energy transfer (FRET) biosensors, a signal of small dynamic range, which may be degraded further by imperfect targeting and interference from endogenous ligands of calsequestrin. We describe a novel tool that uses the cameleon D4cpv, which has a greater dynamic range and lower susceptibility to endogenous ligands than earlier cameleons. D4cpv was targeted to the SR by fusion with the cDNA of calsequestrin 1 or a variant that binds less Ca2+. “D4cpv-Casq1,” expressed in adult mouse at concentrations up to 22 µmole/liter of muscle cell, displayed the accurate targeting of calsequestrin and stayed inside cells after permeabilization of surface and t system membranes, which confirmed its strict targeting. FRET ratio changes of D4cpv-Casq1 were calibrated inside cells, with an effective KD of 222 µM and a dynamic range [(Rmax − Rmin)/Rmin] of 2.5, which are improvements over comparable sensors. Both the maximal ratio, Rmax, and its resting value were slightly lower in areas of high expression, a variation that was inversely correlated to distance from the sites of protein synthesis. The average [Ca2+]SR in 74 viable cells at rest was 416 µM. The distribution of individual ratio values was Gaussian, but that of the calculated [Ca2+]SR was skewed, with a tail of very large values, up to 6 mM. Model calculations reproduce this skewness as the consequence of quantifiably small variations in biosensor performance. Local variability, a perceived weakness of biosensors, thus becomes quantifiable. It is demonstrably small in D4cpv. D4cpv-Casq1 therefore provides substantial improvements in sensitivity, specificity, and reproducibility over existing monitors of SR free Ca2+ concentration.

Synthetic localized calcium transients directly probe signalling mechanisms in skeletal muscle

•  The signal for skeletal muscle contraction is a rapid increase in cytosolic Ca2+ concentration, which requires the coordinated opening of ryanodine receptor (RyR) channels in the sarcoplasmic reticulum. •  Channel opening is controlled by voltage‐sensing dihydropyridine receptors (DHPRs) of plasma membrane and T tubules. Whether or not their signal is amplified by Ca2+‐induced Ca2+ release (CICR) is controversial. •  We used two‐photon lysis of an advanced Ca2+ cage to produce local Ca2+ concentration transients that were large, fast, reproducible and quantifiable, while monitoring the cellular response with a dual confocal laser scanner. •  Single frog muscle cells in physiological solutions responded to transients greater than 0.28 μm with propagated CICR waves. •  Mouse cells did not respond to stimuli up to 8 μm, unless channel opening drugs were present. •  We conclude that CICR contributes to physiological Ca2+ release in frog but not mouse muscle. •  Mice and presumably other mammals do have a capability for CICR that is normally inhibited. It could be manifested under special circumstances, including diseases.

The couplonopathies: A comparative approach to a class of diseases of skeletal and cardiac muscle.

A novel category of diseases of striated muscle is proposed, the couplonopathies, as those that affect components of the couplon and thereby alter its operation. Couplons are the functional units of intracellular calcium release in excitation–contraction coupling. They comprise dihydropyridine receptors, ryanodine receptors (Ca2+ release channels), and a growing list of ancillary proteins whose alteration may lead to disease. Within a generally similar plan, the couplons of skeletal and cardiac muscle show, in a few places, marked structural divergence associated with critical differences in the mechanisms whereby they fulfill their signaling role. Most important among these are the presence of a mechanical or allosteric communication between voltage sensors and Ca2+ release channels, exclusive to the skeletal couplon, and the smaller capacity of the Ca stores in cardiac muscle, which results in greater swings of store concentration during physiological function. Consideration of these structural and functional differences affords insights into the pathogenesis of several couplonopathies. The exclusive mechanical connection of the skeletal couplon explains differences in pathogenesis between malignant hyperthermia (MH) and catecholaminergic polymorphic ventricular tachycardia (CPVT), conditions most commonly caused by mutations in homologous regions of the skeletal and cardiac Ca2+ release channels. Based on mechanistic considerations applicable to both couplons, we identify the plasmalemma as a site of secondary modifications, typically an increase in store-operated calcium entry, that are relevant in MH pathogenesis. Similar considerations help explain the different consequences that mutations in triadin and calsequestrin have in these two tissues. As more information is gathered on the composition of cardiac and skeletal couplons, this comparative and mechanistic approach to couplonopathies should be useful to understand pathogenesis, clarify diagnosis, and propose tissue-specific drug development.

Dynamic measurement of the calcium buffering properties of the sarcoplasmic reticulum in mouse skeletal muscle

•  The rise in cytosolic calcium ion concentration that triggers muscle contraction requires release of a large amount of calcium from the cellular store, sarcoplasmic reticulum (SR), where it is stored bound, largely to the protein calsequestrin. •  Binding of calcium by calsequestrin is a complex process, believed to involve changes in protein conformation and aggregation. We want to know to what extent these properties, observed in vitro, apply inside cells. •  We measured the calcium buffering power of the SR, defined as the ratio of change in total SR calcium by change in free [Ca2+]SR, in muscle cells of wild type or calsequestrin‐lacking mice, using two different methods to monitor [Ca2+]SR and deriving changes in total SR calcium content from simultaneous measurements of cytosolic [Ca2+]. •  The average buffering power during a large, depleting calcium release event was 157 in the wild type and 40 in the calsequestrin‐null mice, suggesting that three‐quarters of the calcium released normally comes from the calsequestrin‐bound pool. •  The Ca2+ buffering ability of the SR is different from that of the equivalent concentration of calsequestrin in aqueous solution, the SR exhibiting greater affinity and cooperativity. We conclude that calsequestrin adopts different properties inside cells. •  SR buffering power depends on the SR Ca2+ load and on the rate of its changes, a dependence that could be, at least in part, explained by the unique Ca2+ binding properties of calsequestrin. •  This study reveals Ca2+ buffering as a highly dynamic process, marking it as both a vulnerable link in diseases that involve loss of control of Ca2+ release, and a candidate for further study and intervention.

Altered Ca2+ concentration, permeability and buffering in the myofibre Ca2+ store of a mouse model of malignant hyperthermia

•  Malignant Hyperthermia (MH) affects the Ca2+ movements that control muscle contraction. We measured Ca2+ movements in skeletal muscle of “Y522S” mice, with a tyrosine‐to‐serine mutation in the RyR channel that causes MH in mice and humans. •  In YS cells, [Ca2+] inside the Ca2+ store (sarcoplasmic reticulum, SR) was 45% of that in the wild type (WT), but the SR membrane permeability increased 2‐fold, resulting in Ca2+ release of initially normal value. •  During Ca2+release, cytosolic [Ca2+] and SR Ca2+ buffering power evolved differently in YS and WT. These variables became similar in WT exposed to BAPTA, an inhibitor of Ca2+‐dependent inactivation (CDI) of the RyR, suggesting that tyrosine 522 is involved in CDI. •  Similar paradoxical observations in YS and WT cells with reduced content of the SR protein calsequestrin, revealed the importance of balance between SR Ca permeability (increased in YS) and storage capability (decreased when calsequestrin is low).

Calsequestrin depolymerizes when calcium is depleted in the sarcoplasmic reticulum of working muscle.

Significance We show that calsequestrin, the main Ca2+ storing protein of muscle, is polymerized inside the sarcoplasmic reticulum (SR) and its mobility increases greatly upon SR depletion, indicating depolymerization. Deep depletion causes massive calsequestrin migration and radical SR remodeling, often accompanied by a surge in intra-SR free Ca2+. The changes in calsequestrin polymerization observed in aqueous solutions therefore also occur in vivo. These changes help explain some uniquely advantageous properties of the SR as a source of calcium for contractile activation. The results support untested hypotheses about additional calsequestrin roles in the control of channel gating and facilitation of calcium flux. They also provide insights on the consequences of calsequestrin mutations for functional competence and structural stability of skeletal and cardiac muscle. Calsequestrin, the only known protein with cyclical storage and supply of calcium as main role, is proposed to have other functions, which remain unproven. Voluntary movement and the heart beat require this calcium flow to be massive and fast. How does calsequestrin do it? To bind large amounts of calcium in vitro, calsequestrin must polymerize and then depolymerize to release it. Does this rule apply inside the sarcoplasmic reticulum (SR) of a working cell? We answered using fluorescently tagged calsequestrin expressed in muscles of mice. By FRAP and imaging we monitored mobility of calsequestrin as [Ca2+] in the SR--measured with a calsequestrin-fused biosensor--was lowered. We found that calsequestrin is polymerized within the SR at rest and that it depolymerized as [Ca2+] went down: fully when calcium depletion was maximal (a condition achieved with an SR calcium channel opening drug) and partially when depletion was limited (a condition imposed by fatiguing stimulation, long-lasting depolarization, or low drug concentrations). With fluorescence and electron microscopic imaging we demonstrated massive movements of calsequestrin accompanied by drastic morphological SR changes in fully depleted cells. When cells were partially depleted no remodeling was found. The present results support the proposed role of calsequestrin in termination of calcium release by conformationally inducing closure of SR channels. A channel closing switch operated by calsequestrin depolymerization will limit depletion, thereby preventing full disassembly of the polymeric calsequestrin network and catastrophic structural changes in the SR.

Confocal Imaging of Transmembrane Voltage by SEER of di-8-ANEPPS

Imaging, optical mapping, and optical multisite recording of transmembrane potential (Vm) are essential for studying excitable cells and systems. The naphthylstyryl voltage-sensitive dyes, including di-8-ANEPPS, shift both their fluorescence excitation and emission spectra upon changes in Vm. Accordingly, they have been used for monitoring Vm in nonratioing and both emission and excitation ratioing modes. Their changes in fluorescence are usually much less than 10% per 100 mV. Conventional ratioing increases sensitivity to between 3 and 15% per 100 mV. Low sensitivity limits the value of these dyes, especially when imaged with low light systems like confocal scanners. Here we demonstrate the improvement afforded by shifted excitation and emission ratioing (SEER) as applied to imaging membrane potential in flexor digitorum brevis muscle fibers of adult mice. SEER—the ratioing of two images of fluorescence, obtained with different excitation wavelengths in different emission bands—was implemented in two commercial confocal systems. A conventional pinhole scanner, affording optimal setting of emission bands but less than ideal excitation wavelengths, achieved a sensitivity of up to 27% per 100 mV, nearly doubling the value found by conventional ratioing of the same data. A better pair of excitation lights should increase the sensitivity further, to 35% per 100 mV. The maximum acquisition rate with this system was 1 kHz. A fast “slit scanner” increased the effective rate to 8 kHz, but sensitivity was lower. In its high-sensitivity implementation, the technique demonstrated progressive deterioration of action potentials upon fatiguing tetani induced by stimulation patterns at >40 Hz, thereby identifying action potential decay as a contributor to fatigue onset. Using the fast implementation, we could image for the first time an action potential simultaneously at multiple locations along the t-tubule system. These images resolved the radially varying lag associated with propagation at a finite velocity.

Abnormal calcium signalling and the caffeine–halothane contracture test

Background: The variable clinical presentation of malignant hyperthermia (MH), a disorder of calcium signalling, hinders its diagnosis and management. Diagnosis relies on the caffeine–halothane contracture test, measuring contraction forces upon exposure of muscle to caffeine or halothane (FC and FH, respectively). Patients with above‐threshold FC or FH are diagnosed as MH susceptible. Many patients test positive to halothane only (termed ‘HH’). Our objective was to determine the characteristics of these HH patients, including their clinical symptoms and features of cytosolic Ca2+ signalling related to excitation–contraction coupling in myotubes. Methods: After institutional ethics committee approval, recruited patients undergoing contracture testing at Torontos MH centre were assigned to three groups: HH, doubly positive (HS), and negative patients (HN). A clinical index was assembled from musculoskeletal symptoms and signs. An analogous calcium index summarised four measures in cultured myotubes: resting [Ca2+]cytosol, frequency of spontaneous cytosolic Ca2+ events, Ca2+ waves, and cell‐wide Ca2+ spikes after electrical stimulation. Results: The highest values of both indexes were found in the HH group; the differences in calcium index between HH and the other groups were statistically significant. The principal component analysis confirmed the unique cell‐level features of the HH group, and identified elevated resting [Ca2+]cytosol and spontaneous event frequency as the defining HH characteristics. Conclusions: These findings suggest that HH pathogenesis stems from excess Ca2+ leak through sarcoplasmic reticulum channels. This identifies HH as a separate diagnostic group and opens their condition to treatment based on understanding of pathophysiological mechanisms.

Using two dyes with the same fluorophore to monitor cellular calcium concentration in an extended range.

We extend the sensitivity of quantitative concentration imaging to an approximately 1000-fold range of concentrations by a method that uses two fluorescent dyes with the same fluorophore, having different affinity for the monitored species. While the formulation and illustration refer to a monitor of calcium concentration, the method is applicable to any species that binds to multiple indicators with the same spectral properties. The use of a common fluorophore has the virtue of leaving vast regions of the electromagnetic spectrum available for other applications. We provide the exact analytic expression relating measured fluorescence to [Ca2+] at equilibrium and an approximate analytic expression that does not require the equilibrium assumption. The sensitivity of the method is calculated numerically for two useful dye pairs. As illustrative application of the enhanced measurement, we use fluo-4 and fluo-4FF to image the calcium wave produced by a cardiac myocyte in response to a small artificial calcium spark.