Structural and contractile alterations in skeletal muscles from the genetically obese (ob/ob) mouse: impairment or adaptation?

Obesity studies using humans are confounded by variables that are difficult to control and are restricted by ethical considerations. Given that they display many of the common attributes of obese humans, animal models of obesity have become invaluable tools for studying the etiology and related pathologies of the obese condition. Obese animals have been often reported to exhibit marked reductions in skeletal muscle size, but currently available data on the contractile function of skeletal muscles in rodent models of obesity are scarce and often conflicting. Therefore, the overall aim of this study was to further knowledge and understanding of the functional status of skeletal muscle in ob/ob mouse, a commonly used model of obesity, by employing a combination of biochemical and physiological methods and whole muscle and single fibre approaches. The animals were used at an age where there is no evidence of hyperglycemia or hypertension, thereby allowing the study of obesity without the often concurrent syndromes of diabetes and hypertension. The muscles examined included two hind limb muscles [extensor digitorum longus (EDL) – predominately locomotor; soleus (SOL) – predominately postural) and one trunk muscle [sternomastoid (SM) – involved in head/neck motion and respiratory assistance]. The data generated in the series of studies comprising this work is summarised as follows: 1. The EDL, SM and SOL muscles of ob/ob mice and controls were examined with respect to size [mass, muscle mass-to-body mass ratio, cross-sectional area (CSA)], fibre CSA, protein content, myosin heavy chain (MHC) content, MHC isoform composition and fibre type composition. Compared with the controls, all three muscles from ob/ob mice were smaller in size, with EDL and SM being the most affected. The CSA values for fibres from the predominant fibre types in EDL and SM muscles (IIB and IIB+IID) were smaller than those for fibres from control muscles, and the fibre size differences were consistent with the differences in muscle size. No differences between EDL, SM and SOL muscles from ob/ob and lean mice were found with respect to total protein content and MHC content (both normalised to muscle mass). Electrophoretic analyses of MHC isoform composition in whole muscle homogenates and single muscle fibres showed a shift towards slower MHC isoforms and a greater proportion of hybrid fibres in all three muscles of ob/ob mice, which suggest that skeletal muscles of ob/ob mice may follow a different pattern of development or undergo an obesity-related structural remodeling which does not involve changes in protein content (Chapter 3). 2. Isometric contractile characteristics [twitch and tetanic force-generating capacity and kinetics; force-frequency relationship] of EDL, SM and SOL muscles from ob/ob and lean mice were explored using an isolated whole muscle preparation. The following statistically significant differences were observed for muscles from ob/ob mice when compared to muscles from lean mice: (i) lower force-generating capacity (Po/body mass) for EDL, SM and SOL muscles; (ii) slower kinetics of the twitch response (W50 value) for EDL and SOL muscles; (iii) lower rate of force development of the tetanic response for EDL muscle; (iv) slower relaxation kinetics of the tetanic response for EDL and SOL muscles; (v) a leftward shift in the force-frequency relationship for EDL and SOL muscles. (Chapter 4). 3. The possibility that skeletal muscles of ob/ob mice displayed slower relaxation kinetics because of functional alterations in the contractile apparatus and/or SR function was explored using a single fibre approach. In these experiments, the following parameters were probed in mechanically skinned type IIB fibres (the predominant fibre type in both muscle groups) from EDL muscles of ob/ob and lean mice: (i) Ca2+ sensitivity (pCa50; pCa10; Hill coefficient: where pCa = -log10[Ca2+]); (ii) endogenous SR Ca2+ content; (iii) SR Ca2+ leak rate; (iv) maximal SR Ca2+ loading capacity at two different [Ca2+] (an indicator of slow/fast SR properties); and, (v) maximal SR Ca2+ loading at pCa 7.3, normalised to maximum Ca2+-activated force (an indicator of the density of SR Ca2+ pumps in the fibre). The data showed similarities in all the parameters except (v), suggesting that EDL type IIB fibres from ob/ob mice possess a lower density of the SR Ca2+ pumps per fibre volume. A lower density of SR Ca2+ pumps would reduce the ability of the SR to sequester Ca2+ and return [Ca2+]i back to resting levels following a contractile response, thereby slowing relaxation. (Chapter 5). 4. The fatigability characteristics [fatigue resistance; recovery of peak tetanic force at 5 min and 60 min after cessation of fatiguing exercise; loss in the ability to generate force at stimulation frequencies of 5 to 90 (or 110) Hz, at 60 min after cessation of fatiguing exercise] of EDL, SM and SOL muscles from ob/ob and lean mice were explored using isolated whole muscle preparations and a fatiguing protocol that elicited a state of low frequency fatigue in all three muscles. As fatigability of the muscle is determined, in part, by the rate of energy supply relative to the rate of energy consumption, ob/ob and lean mice were also compared with respect to myofibrillar ATPase activity (determined in EDL type IIB fibres – the predominant fibre type) and lactate dehydrogenase (LDH) isoenzyme profile (determined in EDL, SM and SOL muscle homogenates). Musclespecific decreases in fatigability were observed in muscles from ob/ob mice, with: (i) EDL displaying greater fatigue resistance; (ii) EDL exhibiting greater recovery of peak tetanic force at 5 min post-fatiguing exercise; (iii) EDL and SOL displaying greater recovery of force at 60 min post-fatiguing exercise; and (iv) EDL and SOL exhibiting a lower loss in the ability to generate force at low stimulation frequencies (greater than or equal to 30 Hz). The decreased fatigability of EDL muscles from ob/ob mice could not be related to lower myofibrillar ATPase activity in type IIB fibres. No simple relationship could be established between the fatigability characteristics of EDL, SM and SOL muscles of ob/ob mice and their LDH isoenzyme profile. This is because the LDH isoenzyme data showed a shift towards a more aerobic-oxidative phenotype for all three muscles of ob/ob mice, including SM which showed no changes in fatigability characteristics. (Chapter 6). The data collected on EDL, SM and SOL muscles from lean mice revealed a number of notable inter-muscle differences in these animals: (i) SOL contains a larger proportion of hybrid fibres than EDL and SM (Chapter 3); (ii) SM has a lower force-generating capacity, as indicated by (peak tetanic force)/(estimated physiological cross-sectional area), than EDL and SOL (Chapter 4); (iii) SM displays faster contractile kinetics than EDL and produces a different force-frequency curve (Chapter 4); and (iv) compared with SOL muscle, EDL and SM display a markedly greater loss of ability to develop force when stimulated at higher stimulation frequencies (greather than or equal to 50 Hz) at 60 min recovery from fatiguing exercise (Chapter 6). As EDL and SM muscles contain similar fibre type composition, their differences in contractile characteristics must be related to other factors, such as (but not limited to) differences in anatomical location and physiological function