Bay K 8644 induced necrosis in murine skeletal muscle in vitro: myofibre breakdown precedes significant alterations of intracellular [Ca] or pH

Summary

The effect of the Ca²⁺-channel agonist Bay K 8644 (1 μmol/l) on the ultrastructure, Ca²⁺-homeostasis, pH and membrane potential of murine diaphragm muscle, in vitro, has been investigated. Treatment with Bay K 8644 in a standard physiological saline, for 1–2 h, induced swelling of the muscle mitochondria and minor damage to the myofibrils. Ultrastructural Ca-localisation by antimonate precipitation revealed no differences between treated and control preparations. Accompanying the structural changes there was a small, non-significant increase in muscle Ca content. In EGTA-buffered (Ca-free) standard saline the induction of damage was not inhibited. When [K⁺]_o was raised to 20 mmol/l, a procedure that approximately halved the resting potential, Bay K 8644 induced severe ultrastructural damage within 1 h, and complete cellular necrosis within 2 h. Induction of myopathy was unaffected by synaptic blockade (150 μmol/ld-tubocurarine). Necrosis was accompanied by depolarisation of membrane potential (Em) and increased antimonate precipitation in the sarcoplasm, and was abolished by buffering of [Ca²⁺]_o with EGTA. However, muscles did not develop tension and measurements of both total Ca and [Ca²⁺]_i suggest that cellular Ca²⁺ buffering was not seriously impaired until 2 h after Bay K 8644 application. Measurement of sarcoplasmic pH revealed no significant change during fibre necrosis. It is proposed that in partially depolarised preparations Bay K 8644 acts on a Ca²⁺-channels in the cell membrane, probably the T-tubules, to induce muscle necrosis through enhanced influx of Ca²⁺. However, muscle necrosis occurs before significant elevation of [Ca²⁺]_i and does not require sarcoplasmic acidification.