Ca²⁺ itself or Ca²⁺-dependent signaling pathways play fundamental roles in various cellular processes from cell growth to death. The most representative example can be found in skeletal muscle cells where a well-timed and adequate supply of Ca²⁺ is required for coordinated Ca²⁺-dependent skeletal muscle functions, such as the interactions of contractile proteins during contraction. Intracellular Ca²⁺ movements between the cytosol and sarcoplasmic reticulum (SR) are strictly regulated to maintain the appropriate Ca²⁺ supply in skeletal muscle cells. Added to intracellular Ca²⁺ movements, the contribution of extracellular Ca²⁺ entry to skeletal muscle functions and its significance have been continuously studied since the early 1990s. Here, studies on the roles of channel proteins that mediate extracellular Ca²⁺ entry into skeletal muscle cells using skeletal myoblasts, myotubes, fibers, tissue, or skeletal muscle-originated cell lines are reviewed with special attention to the proposed functions of transient receptor potential canonical proteins (TRPCs) as store-operated Ca²⁺ entry (SOCE) channels under normal conditions and the potential abnormal properties of TRPCs in muscle diseases such as Duchenne muscular dystrophy (DMD).

Aging causes skeletal muscles to become atrophied, weak, and easily fatigued. Here, we have tested the hypothesis that normal aging in skeletal muscle cells is associated with Ca²⁺ intracellular dyshomeostasis and oxidative stress. Intracellular Ca²⁺ concentration ([Ca²⁺]_i), resting intracellular Na⁺ concentration ([Na⁺]_i) and reactive oxygen species (ROS) production were measured in vivo (superficial gastrocnemius fibers) using double-barreled ion-selective microelectrodes, and in vitro [isolated single flexor digitorum brevis fibers] using fluorescent ROS sensor CM-H2DCFDA in young (3 months of age), middle-aged (12 months of age), and aged (24 months of age) mice. We found an age-related increase in [Ca²⁺]_i from 121 ± 4 nM in young muscle cells which rose to 255 ± 36 nM in middle-aged and to 409 ± 25 nM in aged cells. [Na⁺]_i also showed an age-dependent elevation, increasing from 8 ± 0.5 mM in young muscle fibers, to 12 ± 1 mM in middle-aged and to 17 ± 1 mM in old muscle fibers. Using the fluorescent ROS sensor CM-H2DCFDA we found that these increases in intracellular cation concentrations were associated with significantly increased basal ROS production as demonstrated by age related increases in the rate of dichlorodihydrofluorescein fluorescence. To determine is this could be modified by reducing ROS and/or blocking sarcolemmal Ca²⁺ influx we administered flufenamic acid (FFA), a non-steroidal anti-inflammatory drug which is also a non-selective blocker of the transient receptor potential canonical channels (TRPCs), for 4 weeks to determine if this would have a beneficial effect. FFA treatment reduced both basal ROS production and muscle [Ca²⁺]_i and [Na⁺]_i in middle-aged and aged muscle fibers compared to fibers and muscles of untreated 12 and 24-months old mice. [Ca²⁺]_i was reduced to 134 ± 8 nM in middle-aged muscle and to 246 ± 40 nM in muscle from aged mice. Likewise [Na⁺]_i was reduced to 9 ± 0.7 mM in middle-aged muscles and to 13 ± 1 mM in muscle from aged mice. FFA treatment also reduced age associated increases in plasma interleukin 6 and tumor necrosis factor-alpha (TNF-α) concentrations which were elevated in 12 and 24-months old mice compared to young mice and decreased age-related muscle damage as indicated by a reduction in serum creatine kinase (CK) activity. Our data provides a direct demonstration that normal aging is associated with a significant elevation [Ca²⁺]_i, [Na⁺]_i, and intracellular ROS production in skeletal muscle fibers. Furthermore, the fact that FFA reduced the intracellular [Ca²⁺], [Na⁺], and ROS production as well as the elevated IL6, TNF-α, and CK levels, led us to suggest that its pharmacological effect may be related to its action both as a TRPC channel blocker and as an anti-inflammatory.

Background: Until recently, the mechanism for the malignant hyperthermia crisis has been attributed solely to sustained massive Ca²⁺ release from the sarcoplasmic reticulum on exposure to triggering agents. This study tested the hypothesis that transient receptor potential cation (TRPC) channels are important contributors to the Ca²⁺ dyshomeostasis in a mouse model relevant to malignant hyperthermia. Methods: This study examined the mechanisms responsible for Ca²⁺ dyshomeostasis in RYR1-p.G2435R mouse muscles and muscle cells using calcium and sodium ion selective microelectrodes, manganese quench of Fura2 fluorescence, and Western blots. Results: RYR1-p.G2435R mouse muscle cells have chronically elevated intracellular resting calcium and sodium and rate of manganese quench (homozygous greater than heterozygous) compared with wild-type muscles. After exposure to 1-oleoyl-2-acetyl-sn-glycerol, a TRPC3/6 activator, increases in intracellular resting calcium/sodium were significantly greater in RYR1-p.G2435R muscles (from 153 ± 11 nM/10 ± 0.5 mM to 304 ± 45 nM/14.2 ± 0.7 mM in heterozygotes P < 0.001] and from 251 ± 25 nM/13.9 ± 0.5 mM to 534 ± 64 nM/20.9 ± 1.5 mM in homozygotes [P < 0.001] compared with 123 ± 3 nM/8 ± 0.1 mM to 196 ± 27 nM/9.4 ± 0.7 mM in wild type). These increases were inhibited both by simply removing extracellular Ca²⁺ and by exposure to either a nonspecific (gadolinium) or a newly available, more specific pharmacologic agent (SAR7334) to block TRPC6- and TRPC3-mediated cation influx into cells. Furthermore, local pretreatment with SAR7334 partially decreased the elevation of intracellular resting calcium that is seen in RYR1-p.G2435R muscles during exposure to halothane. Western blot analysis showed that expression of TRPC3 and TRPC6 were significantly increased in RYR1-p.G2435R muscles in a gene–dose–dependent manner, supporting their being a primary molecular basis for increased sarcolemmal cation influx. Conclusions: Muscle cells in knock-in mice expressing the RYR1-p.G2435R mutation are hypersensitive to TRPC3/6 activators. This hypersensitivity can be negated with pharmacologic agents that block TRPC3/6 activity. This reinforces the working hypothesis that transient receptor potential cation channels play a critical role in causing intracellular calcium and sodium overload in malignant hyperthermia–susceptible muscle, both at rest and during the malignant hyperthermia crisis. Editor’s Perspective: What We Already Know about This Topic: What This Article Tells Us That Is New:

Background: Postoperative cognitive dysfunction (POCD) is a common disorder after general anesthesia in elderly patients, the precise mechanisms of which remain unclear. Methods: We investigated the effect of isoflurane with or without dantrolene pretreatment on intracellular calcium concentration ([Ca²⁺]_i), reactive oxygen species (ROS) production, cellular lactate dehydrogenase (LDH) leak, calpain activity, and cognitive function using the Morris water maze test of young (3 months), middle-aged (12–13 months), and aged (24–25 months) C57BL6/J mice. Results: Aged cortical and hippocampal neurons showed chronically elevated [Ca²⁺]_i compared to young neurons. Furthermore, aged hippocampal neurons exhibited higher ROS production, increased LDH leak, and elevated calpain activity. Exposure to isoflurane exacerbated these markers in aged neurons, contributing to increased cognitive deficits in aged mice. Dantrolene pretreatment reduced [Ca²⁺]_i for all age groups and prevented or significantly mitigated the effects of isoflurane on [Ca²⁺]_i, ROS production, LDH leak, and calpain activity in aged neurons. Dantrolene also normalized or improved age-associated cognitive deficits and mitigated the cognitive deficits caused by isoflurane. Conclusions: These findings suggest that isoflurane-induced cytotoxicity and cognitive decline in aging are linked to disruptions in neuronal intracellular processes, highlighting the reduction of [Ca²⁺]_i as a potential therapeutic intervention.