Impaired voluntary running capacity of creatine kinase-deficient mice

Abstract

The creatine kinase system (CK) is important for energy delivery in skeletal and cardiac muscles. The two main isoforms of this enzyme, cytosolic MM-CK and mitochondrial mi-CK, are expressed in a developmental and muscle-type specific manner. Mice deficient in one or both of these isoforms are viable and fertile but exhibit profound functional, metabolic and structural muscle remodelling that primarily affects fast skeletal muscles, which show an increased contribution of oxidative metabolism to contractile function. However, the consequences of these alterations in terms of physical capabilities have not yet been characterized. Consequently, we compared the voluntary exercise capacity of 9-month-old male wild-type (WT), M-CK knockout (M-CK(-/-)), and M-CK and mi-CK double knockout (CK(-/-)) mice, using cages equipped with running wheels. Exercise performance, calculated by total distance covered and by work done during the training period, was more than 10-fold lower in CK(-/-) mice than controls, with M-CK(-/-) mice exhibiting intermediate performance. Similarly, the mean distance run per activation was lower in M-CK(-/-) and even lower in CK(-/-) mice. However, the maximal running speed (V(max)) was lower only for CK(-/-) mice. This was accompanied by severe skeletal muscle mass decrease in CK(-/-) mice, with signs of histological damage that included enlarged interstitial areas, aggregations of mononuclear cells in the interstitium, heterogeneity of myofibre size and the presence of very small fibres. No overt sign of cardiac dysfunction was observed by magnetic resonance imaging during dobutamine stimulation. These results show that metabolic failure induced by CK deficiency profoundly affects the ability of mice to engage in chronic bouts of endurance running exercise and that this decrease in performance is also associated with muscle wasting.

Figure 1. Weekly wheel-running parameters for wild-type (WT), cytosolic creatine kinase (M-CK^−/−) and cytosolic and mitochondrial creatine kinase (CK^−/−) deficient mice

A, work performed was much higher in WT than in CK^−/− with intermediate values in M-CK^−/− mice. B, the results were identical for the mean daily distance. C, the maximal running speed was significantly higher in WT and MM-CK^−/− than in CK^−/− mice. D, distance per run increased considerably over time in control mice but stayed low in CK^−/− mice and improved slightly with time in M-CK^−/−. *,†P < 0.05; **,††P < 0.01; ***,†††P < 0.001. *Significantly different from control mice; †significantly different from CK^−/− mice.

Figure 2. Mitochondrial enzyme content of soleus and gastrocnemius muscles of sedentary or active, WT or CK^−/− mice

Citrate synthase (CS) is an enzyme of the Krebs cycle and cytochrome c oxidase (COX) is the complex IV of the respiratory chain. Small changes in mitochondrial enzyme activities are observed in soleus, both in response to CK deficiency and activity. In gastrocnemius muscle, a dramatic increase in mitochondrial enzymes is present in CK deficiency in both sedentary and active animals. *P < 0.05; ***P < 0.001. *Significantly different from control mice.

Figure 3. Myosin heavy chain (MHC) content (%) of soleus (A), gastrocnemius (B) and plantaris (C) muscles of sedentary or active, WT or CK^−/− mice

*,†P < 0.05; **,††P < 0.01; ***,†††P < 0.001. *Significantly different WT versus CK^−/− mice; †significantly different sedentary versus active mice.

Figure 4. Light microscopy cross-sections of soleus (A–D) and plantaris muscles (E–H) taken from WT (A and E) and CK^−/− mice (B–D and F–H)

Transverse sections (H&E staining) show enlarged interstitial areas, endomysial infiltrates of mononucleated cells (arrow heads), heterogeneity in myofibre size with small fibres close to large rounded myofibres (D), features of degenerating fibres (filled stars), myofibres with internal nuclei (black arrows), focal and large areas of connective tissue (F and G) and histological structures with degenerative features that are difficult to identify (white arrows). Connective tissue was shown to extend around myofibres and to induce fibrous coating (arrow heads). Scale bar, 50 μm.

Figure 5. MRI determination of kinetics of contraction of CK^−/− mice

Mid-ventricular image of a CK^−/− mouse heart under dobutamine-induced stress in end systole (A) and in end diastole (B) used to measure contraction kinetics (slice thickness, 0.5 mm). C, LV surface–time curve of a CK^−/− mouse at rest and under dobutamine-induced stress: the slice area of the LV cavity is plotted as a function of time after the R wave, allowing calculation of the maximal rates of ventricular filling and ejection.

Figure 6. Correlation between muscle mass and exercise capacity

A strong correlation is observed between the between the relative weights of soleus (A) or plantaris (B) at the end of the exercise period and the total running distance, taken as an index of voluntary exercise capacity of all groups of mice.