Key points: If skeletal muscle fibres are subjected to excessive activation, or stretched whilst contracting, they subsequently display long‐term reductions in their force response, apparently due in part to structural or molecular changes at the triad junction, where excitation of the surface membrane triggers Ca²⁺ release from the internal Ca²⁺ store. The changes appear to be due to excessive or prolonged increases in intracellular Ca²⁺ levels, which activate Ca²⁺‐dependent proteases known as calpains, but their target proteins are currently unknown. This study shows that excessive muscle stimulation, or directly raising intracellular Ca²⁺ levels, causes calpain activation in tandem with proteolysis of junctophilin, a key protein thought to hold the triad junction together. Proteolysis of junctophilin is also seen in muscle of mice with muscular dystrophy and in cardiac muscle following ischaemic damage. Proteolysis of junctophilin may be a major factor causing muscle weakness and cardiac dysfunction in a range of circumstances. Abstract  Excessive increases in intracellular [Ca²⁺] in skeletal muscle fibres cause failure of excitation–contraction coupling by disrupting communication between the dihydropyridine receptors in the transverse tubular system and the Ca²⁺ release channels (RyRs) in the sarcoplasmic reticulum (SR), but the exact mechanism is unknown. Previous work suggested a possible role of Ca²⁺‐dependent proteolysis in this uncoupling process but found no proteolysis of the dihydropyridine receptors, RyRs or triadin. Junctophilin‐1 (JP1; ∼90 kDa) stabilizes close apposition of the transverse tubular system and SR membranes in adult skeletal muscle; its C‐terminal end is embedded in the SR and its N‐terminal associates with the transverse tubular system membrane. Exposure of skeletal muscle homogenates to precisely set [Ca²⁺] revealed that JP1 undergoes Ca²⁺‐dependent proteolysis over the physiological [Ca²⁺] range in tandem with autolytic activation of endogenous μ‐calpain. Cleavage of JP1 occurs close to the C‐terminal, yielding a ∼75 kDa diffusible fragment and a fixed ∼15 kDa fragment. Depolarization‐induced force responses in rat skinned fibres were abolished following 1 min exposure to 40 μm Ca²⁺, with accompanying loss of full‐length JP1. Supraphysiological stimulation of rat skeletal muscle in vitro by repeated tetanic stimulation in 30 mm caffeine also produced marked proteolysis of JP1 (and not RyR1). In dystrophic mdx mice, JP1 proteolysis is seen in limb muscles at 4 and not at 10 weeks of age. Junctophilin‐2 in cardiac and skeletal muscle also undergoes Ca²⁺‐dependent proteolysis, and junctophilin‐2 levels are reduced following cardiac ischaemia–reperfusion. Junctophilin proteolysis may contribute to skeletal muscle weakness and cardiac dysfunction in a range of circumstances.

Key points: Muscle weakness in old age is due in large part to an overall loss of skeletal muscle tissue, but it remains uncertain how much also stems from alterations in the properties of the individual muscle fibres. This study examined the contractile properties and amount of stored intracellular calcium in single muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) adults. The maximum level of force production (per unit cross‐sectional area) in fast twitch fibres in Old subjects was lower than in Young subjects, and the fibres were also less sensitive to activation by calcium. The amount of calcium stored inside muscle fibres and available to trigger contraction was also lower in both fast‐ and slow‐twitch muscle fibres in the Old subjects. These findings indicate that muscle weakness in old age stems in part from an impaired capacity for force production in the individual muscle fibres. This study examined the contractile properties and sarcoplasmic reticulum (SR) Ca²⁺ content in mechanically skinned vastus lateralis muscle fibres of Old (70 ± 4 years) and Young (22 ± 3 years) humans to investigate whether changes in muscle fibre properties contribute to muscle weakness in old age. In type II fibres of Old subjects, specific force was reduced by ∼17% and Ca²⁺ sensitivity was also reduced (pCa₅₀ decreased ∼0.05 pCa units) relative to that in Young. S‐Glutathionylation of fast troponin I (TnI_f) markedly increased Ca²⁺ sensitivity in type II fibres, but the increase was significantly smaller in Old versus Young (+0.136 and +0.164 pCa unit increases, respectively). Endogenous and maximal SR Ca²⁺ content were significantly smaller in both type I and type II fibres in Old subjects. In fibres of Young, the SR could be nearly fully depleted of Ca²⁺ by a combined caffeine and low Mg²⁺ stimulus, whereas in fibres of Old the amount of non‐releasable Ca²⁺ was significantly increased (by > 12% of endogenous Ca²⁺ content). Western blotting showed an increased proportion of type I fibres in Old subjects, and increased amounts of calsequestrin‐2 and calsequestrin‐like protein. The findings suggest that muscle weakness in old age is probably attributable in part to (i) an increased proportion of type I fibres, (ii) a reduction in both maximum specific force and Ca²⁺ sensitivity in type II fibres, and also a decreased ability of S‐glutathionylation of TnI_f to counter the fatiguing effects of metabolites on Ca²⁺ sensitivity, and (iii) a reduction in the amount of releasable SR Ca²⁺ in both fibre types.