Effects of ryanodine receptor agonist 4-chloro-m-cresol on myoplasmic free Ca²⁺ concentration and force of contraction in mouse skeletal muscle

In single mouse skeletal muscle fibers injected with fluorescent Ca²⁺ indicator Indo-1, 4-chloro-m-cresol (chlorocresol, 4-CmC) and its lipophilic analogue 4-chloro-3-ethylphenol (4-CEP) increased resting myoplasmic free [Ca²⁺] ([Ca²⁺](i)) in a dose-dependent manner. In this regard, 4-CEP was more potent than 4-CmC and both were more potent than caffeine. High concentrations of 4-CmC (1 mM) or 4-CEP (500 μM) caused large and irreversible increase in resting [Ca²⁺](i) leading to contracture. 4-CmC potentiated the [Ca²⁺](i) increase and force of contraction induced by tetanic stimulation. Unlike caffeine, 4-CmC did not affect the activity of sarcoplasmic reticulum Ca²⁺ pump or the myofibrillar Ca²⁺ sensitivity. A low concentration of 4-CEP (20 μM) had no effect on resting [Ca²⁺](i) on its own, but it enhanced the resting [Ca²⁺](i) increase induced by caffeine and also potentiated the [Ca²⁺](i) increase and contraction induced by tetanic stimulation. However, a relatively high concentration of 4-CEP (200 μM) inhibited tetanic stimulation-induced [Ca²⁺](i) increase and contraction. Dantrolene, a muscle relaxant, inhibited 4-CmC-induced [Ca²⁺](i) increase under resting conditions. However, when 4-CEP was applied in the presence of dantrolene, there was an exaggerated increase in [Ca²⁺](i). We conclude that 4-CmC and 4-CEP are potent agonists that can increase [Ca²⁺](i) rapidly and reversibly by activating ryanodine receptors in situ in intact skeletal muscle fibers. These compounds, specially 4-CmC, may be useful for mechanistic and functional studies of ryanodine receptors and excitation-contraction coupling in skeletal muscles.