Barium chloride injures myofibers through calcium-induced proteolysis with fragmentation of motor nerves and microvessels.

BACKGROUND

Local injection of BaCl is an established model of acute injury to study the regeneration of skeletal muscle. However, the mechanism by which BaCl causes muscle injury is unresolved. Because Ba inhibits K channels, we hypothesized that BaCl induces myofiber depolarization leading to Ca overload, proteolysis, and membrane disruption. While BaCl spares resident satellite cells, its effect on other tissue components integral to contractile function has not been defined. We therefore asked whether motor nerves and microvessels, which control and supply myofibers, are injured by BaCl treatment.

METHODS

The intact extensor digitorum longus (EDL) muscle was isolated from male mice (aged 3-4 months) and irrigated with physiological salt solution (PSS) at 37 °C. Myofiber membrane potential (V) was recorded using sharp microelectrodes while intracellular calcium concentration ([Ca]) was evaluated with Fura 2 dye. Isometric force production of EDL was measured in situ, proteolytic activity was quantified by calpain degradation of αII-spectrin, and membrane disruption was marked by nuclear staining with propidium iodide (PI). To test for effects on motor nerves and microvessels, tibialis anterior or gluteus maximus muscles were injected with 1.2% BaCl (50-75 μL) in vivo followed by immunostaining to evaluate the integrity of respective tissue elements post injury. Data were analyzed using Students t test and analysis of variance with P ≤ 0.05 considered statistically significant.

RESULTS

Addition of 1.2% BaCl to PSS depolarized myofibers from - 79 ± 3 mV to - 17 ± 7 mV with a corresponding rise in [Ca]; isometric force transiently increased from 7.4 ± 0.1 g to 11.1 ± 0.4 g. Following 1 h of BaCl exposure, 92 ± 3% of myonuclei stained with PI (vs. 8 ± 3% in controls) with enhanced cleavage of αII-spectrin. Eliminating Ca from PSS prevented the rise in [Ca] and ameliorated myonuclear staining with PI during BaCl exposure. Motor axons and capillary networks appeared fragmented within 24 h following injection of 1.2% BaCl and morphological integrity deteriorated through 72 h.

CONCLUSIONS

BaCl injures myofibers through depolarization of the sarcolemma, causing Ca overload with transient contraction, leading to proteolysis and membrane rupture. Motor innervation and capillarity appear disrupted concomitant with myofiber damage, further compromising muscle integrity.