Programme Contents

A slowing of relaxation in EDL muscle from the genetically obese mouse is associated with alterations in SR Ca²⁺ handling

There is growing evidence to suggest that the isometric contractile properties of fast-twitch skeletal muscles from animal models of obesity are different from those of lean controls (Warmington et al., 2000; Bruton et al., 2002; Blazev et al., 2005). In the present study, we specifically investigated the relaxation kinetics of extensor digitorum longus (EDL) muscle isolated from the genetically obese (ob/ob) mouse and its lean counterpart. We also used a single fibre approach to more precisely address any observed differences in relaxation at the whole muscle level.

Male ob/ob and lean mice (18-22 weeks) were killed by halothane overdose in accordance with Victoria University AEEC procedures. EDL muscles were dissected and placed in carbogen bubbled Krebs solution at 25 °C, before being supramaximally stimulated to elicit tetanic (110 Hz) responses, as described previously (Blazev et al., 2005). EDL muscles not incubated in Krebs solution were used to obtain mechanically skinned fibre segments that were electrophoretically typed. Since the EDL muscles of both ob/ob and lean mice contain predominantly type IIB fibres (Blazev et al., 2005) their properties related to Ca²⁺-sensitivity of the contractile apparatus and sarcoplasmic reticulum (SR) Ca²⁺ handling were investigated following the procedures routinely used in our laboratory (Bortolotto et al., 2000, 2001). All results are given as mean ± SEM.

The half relaxation time (1/2 RT) for tetanic responses was significantly slower (p < 0.05) in EDL muscles from ob/ob mouse (n = 8) as compared to lean controls (n = 8) (46.0 ± 2.3 vs 39.9 ± 1.3 ms). There was no difference in the Ca²⁺-sensitivity of the contractile apparatus between IIB fibres isolated from EDL of ob/ob and lean mice, as determined from the pCa (-log₁₀[Ca²⁺]) giving 50% maximum force (i.e., pCa₅₀: 5.77 ± 0.01, n = 14 vs. 5.74 ± 0.02, n = 12). A single fibre investigation of the ability of the SR to maximally sequester Ca²⁺ was carried out by normalising the area under the 30 mM caffeine-induced force response (in the presence of 0.5 mM EGTA and 0.05 mM free Mg²⁺), following maximal SR Ca²⁺ loading at pCa 7.3, to the maximum Ca²⁺-activated force response (F_max). This normalised value for EDL IIB fibres from ob/ob mice (175 ± 16 %F_max.s, n = 8) was significantly smaller than that for fibres from lean mice (268 ± 37 %F_max.s, n = 8), indicating a lower SR Ca²⁺ loading capacity at pCa 7.3 for ob/ob fibres. The lower SR Ca²⁺ content of these fibres was not due to differences in the rate of passive Ca²⁺ leak (% min^-1) from the SR between IIB ob/ob and lean fibres (59.6 ± 11.0, n = 5 vs. 59.3 ± 9.2, n = 6). Furthermore, investigation of slow/fast SR characteristics (Fryer and Stephenson, 1996; Bortolotto et al., 2001) by loading maximally at two different [Ca²⁺], revealed that the ratio (R_6.2/7.3) derived from the area under the force response to 30 mM caffeine following loading at pCa 6.2 versus pCa 7.3 was similar in IIB fibres from ob/ob and lean mice (R_6.2/7.3: 1.50 ± 0.13, n = 7 vs. 1.49 ± 0.11, n = 8). Thus, functional characteristics of the SR Ca²⁺ pumps were the same in these fibres.

Taken together, the above results suggest that the density of the SR Ca²⁺ pumps (expressed per fibre volume) was significantly lower in EDL IIB fibres from ob/ob mice than in fibres from lean mice. This reduces the ability of the SR to sequester Ca²⁺ and return the myoplasmic [Ca²⁺] back to ∼ theoretical resting levels following muscular contraction in ob/ob mice as compared to the lean counterparts, and contributes to the slowing of the 1/2 RT observed in the present study for the tetanic response at the whole muscle level in ob/ob mice.

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