Changes in contractile activation characteristics of rat fast and slow skeletal muscle fibres during regeneration

Abstract

Damaged skeletal muscle fibres are replaced with new contractile units via muscle regeneration. Regenerating muscle fibres synthesize functionally distinct isoforms of contractile and regulatory proteins but little is known of their functional properties during the regeneration process. An advantage of utilizing single muscle fibre preparations is that assessment of their function is based on the overall characteristics of the contractile apparatus and regulatory system and as such, these preparations are sensitive in revealing not only coarse, but also subtle functional differences between muscle fibres. We examined the Ca(2+)- and Sr(2+)-activated contractile characteristics of permeabilized fibres from rat fast-twitch (extensor digitorum longus) and slow-twitch (soleus) muscles at 7, 14 and 21 days following myotoxic injury, to test the hypothesis that fibres from regenerating fast and slow muscles have different functional characteristics to fibres from uninjured muscles. Regenerating muscle fibres had approximately 10% of the maximal force producing capacity (P(o)) of control (uninjured) fibres, and an altered sensitivity to Ca(2+) and Sr(2+) at 7 days post-injury. Increased force production and a shift in Ca(2+) sensitivity consistent with fibre maturation were observed during regeneration such that P(o) was restored to 36-45% of that in control fibres by 21 days, and sensitivity to Ca(2+) and Sr(2+) was similar to that of control (uninjured) fibres. The findings support the hypothesis that regenerating muscle fibres have different contractile activation characteristics compared with mature fibres, and that they adopt properties of mature fast- or slow-twitch muscle fibres in a progressive manner as the regeneration process is completed.

Figure 1. Raw force traces showing Ca²⁺ and Sr²⁺ activation of isolated permeabilized fibres

Raw force traces showing Ca²⁺ and Sr²⁺ activation of isolated permeabilized fibres from EDL (A) and soleus (B) muscles of the rat at 7 days post-injury (upper panel in each case) and from uninjured muscles (lower panel in each case). Note the obvious differences in absolute force producing capacity between injured/regenerating and uninjured fibres. Filled arrowheads indicate the sequential transfer of an isolated fibre segment into solutions of differing [Ca²⁺] (or [Sr²⁺]). The numbers accompanying each arrow refer to the estimated pCa or pSr in each case. The open arrow indicates fibre is bathed in pre-activating (low-relaxing) solution. For each activation sequence, a fibre was always transferred from relaxing solution to pre-activation solution and incubated for 1 min before being transferred into activating solutions of differing [Ca²⁺] and [Sr²⁺].

Figure 2. Force–pCa and force–pSr relations for permeabilized muscle fibres from uninjured and regenerating muscles

Force–pCa and force–pSr relations for permeabilized muscle fibres from uninjured (dashed lines) and regenerating EDL (A) and soleus (B) limb muscles (continuous lines) examined 7, 14 and 21 days post-injury. The symbols represent actual data points (means ± s.e.m.) and the curves represent the lines of best fit (see Methods). Arrows indicate the general shift in force–pCa (filled symbols) and force–pSr relations (open symbols) from the uninjured state during the time course of regeneration. The numbers of fibres in each case are those described in Tables 1 and 2 for each time point.

Figure 3. MHC isoform composition of uninjured and regenerating muscles

A, electrophoretic separation of myosin heavy chain (MyHC) isoforms in homogenates prepared from control (uninjured) and regenerating (7 days post-injury) EDL and soleus muscles. Note decreased type IIb MyHC in regenerating EDL muscle compared with control (uninjured) EDL muscle, and increased type II MyHC isoform expression in regenerating soleus muscles compared with control (uninjured) muscles. B and C, MyHC isoform composition of uninjured and regenerating EDL (B) and soleus (C) muscles examined at 7, 14 and 21 days post-injury. Analysis is based on densitometric evaluation of polyacrylamide gel electrophoretic separation of MyHC isoforms from muscle.