Tanya R. Cully

Upregulation of store-operated Ca2+ entry in dystrophic mdx mouse muscle

Store-operated Ca(2+) entry (SOCE) is an important mechanism in virtually all cells. In adult skeletal muscle, this mechanism is highly specialized for the rapid delivery of Ca(2+) from the transverse tubule into the junctional cleft during periods of depleting Ca(2+) release. In dystrophic muscle fibers, SOCE may be a source of Ca(2+) overload, leading to cell necrosis. However, this possibility is yet to be examined in an adult fiber during Ca(2+) release. To examine this, Ca(2+) in the tubular system and cytoplasm were simultaneously imaged during direct release of Ca(2+) from sarcoplasmic reticulum (SR) in skeletal muscle fibers from healthy (wild-type, WT) and dystrophic mdx mouse. The mdx fibers were found to have normal activation and deactivation properties of SOCE. However, a depression of the cytoplasmic Ca(2+) transient in mdx compared with WT fibers was observed, as was a shift in the SOCE activation and deactivation thresholds to higher SR Ca(2+) concentrations ([Ca(2+)](SR)). The shift in SOCE activation and deactivation thresholds was accompanied by an approximately threefold increase in STIM1 and Orai1 proteins in dystrophic muscle. While the mdx fibers can introduce more Ca(2+) into the fiber for an equivalent depletion of [Ca(2+)](SR) via SOCE, it remains unclear whether this is deleterious.

Ultra-rapid activation and deactivation of store-operated Ca2+ entry in skeletal muscle

Skeletal muscle is highly specialized for the rapid delivery of Ca(2+) to the contractile apparatus during excitation-contraction coupling (EC coupling). Previous studies have shown the presence of a relatively fast-activated store-operated Ca(2+) entry (SOCE) mechanism (<1s) to be present in skeletal muscle, unlike the situation occurring in non-excitable cells. We simultaneously imaged [Ca(2+)] in the t-system and cytoplasm in mechanically skinned fibers during SR Ca(2+) release and observed both cell-wide Ca(2+) release and Ca(2+) waves. SOCE activation followed cell-wide Ca(2+) release from high sarcoplasmic reticulum (SR) [Ca(2+)] ([Ca(2+)](SR)) by seconds, consistent with depletion of [Ca(2+)](SR) to an absolute threshold for SOCE and an unformed SOCE complex at high [Ca(2+)](SR). Ca(2+) waves occurred at low [Ca(2+)](SR), close to the threshold for SOCE, minimizing the time between Ca(2+) release and Ca(2+) influx. Local activation of SOCE during Ca(2+) waves occurred in approximately 27ms following local initiation of SR depletion indicating a steep relationship between [Ca(2+)](SR) and SOCE activation. Most of this delay was due to slow release of Ca(2+) from SR, leaving only milliseconds at most for the activation of Ca(2+) entry following store depletion. SOCE was also observed to deactivate effectively instantly during store refilling at low [Ca(2+)](SR). These rapid kinetics of SOCE persisted as subsequent Ca(2+) waves propagated along the fiber. Thus we show for the first time millisecond activation and deactivation of SOCE during low amplitude [Ca(2+)](SR) oscillations at low [Ca(2+)](SR). To account for the observed Ca(2+) movements we propose the SOCE complex forms during the progressive depletion of [Ca(2+)](SR) prior to reaching the activation threshold of SOCE and this complex remains stable at low [Ca(2+)](SR).

Evidence That the EphA2 Receptor Exacerbates Ischemic Brain Injury

Ephrin (Eph) signaling within the central nervous system is known to modulate axon guidance, synaptic plasticity, and to promote long-term potentiation. We investigated the potential involvement of EphA2 receptors in ischemic stroke-induced brain inflammation in a mouse model of focal stroke. Cerebral ischemia was induced in male C57Bl6/J wild-type (WT) and EphA2-deficient (EphA2−/−) mice by middle cerebral artery occlusion (MCAO; 60 min), followed by reperfusion (24 or 72 h). Brain infarction was measured using triphenyltetrazolium chloride staining. Neurological deficit scores and brain infarct volumes were significantly less in EphA2−/− mice compared with WT controls. This protection by EphA2 deletion was associated with a comparative decrease in brain edema, blood-brain barrier damage, MMP-9 expression and leukocyte infiltration, and higher expression levels of the tight junction protein, zona occludens-1. Moreover, EphA2−/− brains had significantly lower levels of the pro-apoptotic proteins, cleaved caspase-3 and BAX, and higher levels of the anti-apoptotic protein, Bcl-2 as compared to WT group. We confirmed that isolated WT cortical neurons express the EphA2 receptor and its ligands (ephrin-A1–A3). Furthermore, expression of all four proteins was increased in WT primary cortical neurons following 24 h of glucose deprivation, and in the brains of WT mice following stroke. Glucose deprivation induced less cell death in primary neurons from EphA2−/− compared with WT mice. In conclusion, our data provide the first evidence that the EphA2 receptor directly contributes to blood-brain barrier damage and neuronal death following ischemic stroke.

Longitudinal and transversal propagation of excitation along the tubular system of rat fast‐twitch muscle fibres studied by high speed confocal microscopy

Non‐technical summary  Contraction of the vertebrate skeletal muscle is dependent on the excitation of the highly specialized plasma membrane of individual muscle fibres. Most of the plasma membrane covers an extensive, structured network of narrow invaginating tubules, but little is known about how excitation propagates along the tubular network of mammalian skeletal muscle fibres in the radial and longitudinal directions with respect to the fibre axis. Here we show that excitation can take multiple pathways through the internal network of tubules in fast‐twitch muscle fibres of the rat and determine the rates that excitation can spread within the internalized membrane tubules and show how this can change during muscle fatigue. Our results increase our understanding of mammalian skeletal muscle function in health, fatigue and some disease states.

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.

Store‐operated Ca2+ entry is not required for store refilling in skeletal muscle

The present review describes store‐operated Ca2+ entry (SOCE) in skeletal muscle. Fundamental discoveries in the field of skeletal muscle SOCE are described and the techniques that were used to make these. The advantages and limitations in these techniques are discussed to provide a means of questioning and determining the physiological role(s) of SOCE in skeletal muscle. It is concluded that SOCE has little or no role in the filling of the sarcoplasmic reticulum with Ca2+ at rest or during a single contracture. It is likely that SOCE is activated during fatigue, although direct measurements of SOCE are lacking and the physiological significance remains uncertain.

Activation and propagation of Ca2+ release from inside the sarcoplasmic reticulum network of mammalian skeletal muscle

Prolonged Ca2+ transients and Ca2+ waves have been observed in mammalian skeletal muscle upon reducing the normal resting inhibition of the ryanodine receptor (RyR)/Ca2+ release channels of the sarcoplasmic reticulum (SR) by lowering cytoplasmic [Mg2+] ([Mg2+]cyto). The mechanism driving Ca2+ release under these conditions may have pathophysiological implications but is poorly understood. The lowering of [Mg2+]cyto induced a leak of Ca2+ from the SR that triggered a local reduction of the SR Ca2+ buffering power to induce the rise in [Ca2+]SR, an intra‐SR Ca2+ transient. Prolonged Ca2+ transients always developed following this event and evolved as propagating Ca2+ waves. No classical hallmarks of cytoplasmic Ca2+ propagation mechanisms were observed in conjunction with these waves. In conjunction with the spread of Ca2+ release, Ca2+ was observed to diffuse through the SR away from the site of the intra‐SR Ca2+ transient. Ca2+ release was sustained by the action of SR Ca2+ pumps, presumably to maintain [Ca2+]SR above an inactivation threshold for closure of the RyRs. Inactivation of prolonged Ca2+ release allowed [Ca2+]SR to recover to threshold for activation of further Ca2+ waves, which were always briefer in duration than the initial Ca2+ release transients, consistent with a reduced SR Ca2+ buffering capacity. These observations reveal activation of Ca2+ release in mammalian skeletal muscle by [Ca2+]SR in the absence of the normal resting inhibition of the RyR and changes in voltage. Diffusion of Ca2+ within SR promotes propagation of Ca2+ release.

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20–30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ⩽1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.

A quantitative description of tubular system Ca2+ handling in fast‐ and slow‐twitch muscle fibres

Current methods do not allow a quantitative description of Ca2+ movements across the tubular (t‐) system membrane without isolating the membranes from their native skeletal muscle fibre. Here we present a fluorescence‐based method that allows determination of the t‐system [Ca2+] transients and derivation of t‐system Ca2+ fluxes in mechanically skinned skeletal muscle fibres. Differences in t‐system Ca2+‐handling properties between fast‐ and slow‐twitch fibres from rat muscle are resolved for the first time using this new technique. The method can be used to study Ca2+ handling of the t‐system and allows direct comparisons of t‐system Ca2+ transients and Ca2+ fluxes between groups of fibres and fibres from different strains of animals.

Leaky ryanodine receptors delay the activation of store overload-induced Ca2+ release, a mechanism underlying malignant hyperthermia-like events in dystrophic muscle

The mouse model of Duchenne muscular dystrophy, the mdx mouse, displays changes in Ca(2+)homeostasis that may lead to the pathology of the muscle. Here we examine the activation of store overload-induced Ca(2+)release (SOICR) in mdx muscle. The activation of SOICR is associated with the depolymerization of the sarcoplasmic reticulum (SR) Ca(2+)buffer calsequestrin and the reduction of SR Ca(2+)buffering power (BSR). The role of SOICR in healthy and dystrophic muscle is unclear. Using skinned fibers we show that lowering the Mg(2+)concentration can activate discrete Ca(2+)release events that did not necessarily lead to activation of SOICR. However, SOICR waves could propagate into these fiber segments. The average delay to activation of SOICR in mdx fibers was longer than in wild-type (WT) fibers. In the lowered Ca(2+)-buffered environment following large SOICR events, brief waves in mdx fibers displayed a low amplitude and propagation rate, in contrast to WT fibers that showed a range of amplitudes correlated with wave propagation rate. The distinct properties of SOICR in mdx fibers were consistent with a ryanodine receptor (RyR) that was leakier to Ca(2+)than in WT. The consequence of delayed SOICR and leaky RyRs is prolonged high BSRand a reduction in free Ca(2+)concentration inside the SR as total SR calcium drops. We present a hypothesis that SOICR activation is required in healthy muscle and that this mechanism works suboptimally in mdx fibers to fail to limit the activation of store-operated Ca(2+)entry.