NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma

Regulation of cardiac ryanodine receptor function by the cyclic-GMP dependent protein kinase G

Background: The cGMP-dependent protein kinase G (PKG) phosphorylates the cardiac ryanodine receptor (RyR2) in vitro. We aimed to determine whether modulation of endogenous PKG alters RyR2-mediated spontaneous Ca2+ release and whether this effect is linked to a change in RyR2 phosphorylation. Methods: & Results: Human embryonic kidney (HEK293) cells with inducible RyR2 expression were treated with the cGMP analogue 8-Br-cGMP (100 μM) to activate endogenous PKG. In cells transfected with luminal Ca2+ sensor, D1ER, PKG activation significantly reduced the threshold for RyR2-mediated spontaneous Ca2+ release (93.9 ± 0.4% of store size with vehicle vs. 91.7 ± 0.8% with 8-Br-cGMP, P = 0.04). Mutation of the proposed PKG phosphorylation sites, S2808 and S2030, either individually or as a combination, prevented the decrease in Ca2+ release threshold induced by endogenous PKG activation. Interestingly, despite a functional dependence on expression of RyR2 phosphorylation sites, 8-Br-cGMP activation of PKG did not promote a detectable change in S2808 phosphorylation (P = 0.9). Paradoxically, pharmacological inhibition of PKG with KT 5823 (1 μM) also reduced the threshold for spontaneous Ca2+ release through RyR2 without affecting S2808 phosphorylation. Silencing RNA knockdown of endogenous PKG expression also had no quantifiable effect on RyR2 S2808 phosphorylation (P = 0.9). However, unlike PKG inhibition with KT 5823, PKG knockdown did not alter spontaneous Ca2+ release propensity or luminal Ca2+ handling. Conclusion: In an intact cell model, activation of endogenous PKG reduces the threshold for RyR2-mediated spontaneous Ca2+ release in a manner dependent on the RyR2 phosphorylation sites S2808 and S2030. This study clarifies the regulation of RyR2 Ca2+ release by endogenous PKG and functionally implicates the role of RyR2 phosphorylation.Fil: Gonano, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; Argentina. University of Otago; Nueva ZelandaFil: Aitken Buck, Hamish M.. University of Otago; Nueva ZelandaFil: Chakraborty, Akash D.. University of Otago; Nueva ZelandaFil: Worthington, Luke P. I.. University of Otago; Nueva ZelandaFil: Cully, Tanya R.. University of Otago; Nueva ZelandaFil: Lamberts, Regis R.. University of Otago; Nueva ZelandaFil: Vila Petroff, Martin Gerarde. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani". Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Centro de Investigaciones Cardiovasculares "Dr. Horacio Eugenio Cingolani"; ArgentinaFil: Jones, Peter P.. University of Otago; Nueva Zeland

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 homeostasis that may lead to the pathology of the muscle. Here we examine the activation of store overload-induced Ca release (SOICR) in mdx muscle. The activation of SOICR is associated with the depolymerization of the sarcoplasmic reticulum (SR) Ca buffer calsequestrin and the reduction of SR Ca buffering power (BSR). The role of SOICR in healthy and dystrophic muscle is unclear. Using skinned fibers we show that lowering the Mg concentration can activate discrete Ca 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-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 than in WT. The consequence of delayed SOICR and leaky RyRs is prolonged high BSR and a reduction in free Ca 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 entry

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

Skeletal muscle fibres are large and highly elongated cells specialized for producing the force required for posture and movement. The process of controlling the production of force within the muscle, known as excitation-contraction coupling, requires virtually simultaneous release of large amounts of Ca from the sarcoplasmic reticulum (SR) at the level of every sarcomere within the muscle fibre. Here we imaged Ca movements within the SR, tubular (t-) system and in the cytoplasm to observe that the SR of skeletal muscle is a connected network capable of allowing diffusion of Ca within its lumen to promote the propagation of Ca release throughout the fibre under conditions where inhibition of SR ryanodine receptors (RyRs) was reduced. Reduction of cytoplasmic [Mg] ([Mg]) induced a leak of Ca through RyRs, causing a reduction in SR Ca buffering power argued to be due to a breakdown of SR calsequestrin polymers, leading to a local elevation of [Ca]. The local rise in [Ca], an intra-SR Ca transient, induced a local diffusely rising [Ca]. A prolonged Ca wave lasting tens of seconds or more was generated from these events. Ca waves were dependent on the diffusion of Ca within the lumen of the SR and ended as [Ca] dropped to low levels to inactivate RyRs. Inactivation of RyRs allowed re-accumulation of [Ca] and the activation of secondary Ca waves in the persistent presence of low [Mg] if the threshold [Ca] for RyR opening could be reached. Secondary Ca waves occurred without an abrupt reduction in SR Ca buffering power. Ca release and wave propagation occurred in the absence of Ca-induced Ca release. These observations are consistent with the activation of Ca release through RyRs of lowered cytoplasmic inhibition by [Ca] or store overload-induced Ca release. Restitution of SR Ca buffering power to its initially high value required imposing normal resting ionic conditions in the cytoplasm, which re-imposed the normal resting inhibition on the RyRs, allowing [Ca] to return to endogenous levels without activation of store overload-induced Ca release. These results are discussed in the context of how pathophysiological Ca release such as that occurring in malignant hyperthermia can be generated