Mitochondria Association to Calcium Release Units is Controlled by Age and Muscle Activity

BACKGROUND: At the most basic level, skeletal muscle contraction requires Ca(2+) and ATP and, thus, is under direct control of two important intracellular organelles: Ca(2+) release units (CRUs) - specialized intracellular junctions, also named triads, which are involved in excitation-contraction (EC) coupling - and mitochondria, the organelles deputed to produce the energy required for most cellular functions (i.e. aerobic ATP production). It is now becoming clear that: a) CRUs and mitochondria interact functionally and structurally, as entry of Ca(2+) into the mitochondrial matrix is required to stimulate the respiratory chain, and increase production of ATP (Fig. 1) (Sembrowich et al. 1985 (1); Brookes et al. 2004 (2); Rossi et al. 2009) (3); b) we recently discovered that, in adult skeletal muscle fibers, mitochondria and CRUs are placed in close proximity to each other (Fig. 2) and structurally linked by small strands called tethers (Fig. 3) (Boncompagni et al. 2009)(4). SCIENTIFIC HYPOTHESIS OF THE STUDY: Miss-function of mitochondria and functional/structural changes affecting the EC coupling apparatus have been both proposed to contribute to the age-related decline of skeletal muscle performance (Delbono et al. 1995 (5); Boncompagni et al. 2006 (6)). In this study, we tested the following hypothesis: muscle activity improves/maintains the correct association between CRUs and mitochondria, which is challenged by ageing and inactivity. EXPERIMENTAL PLAN: We have studied the morphology, frequency, and sarcomericlocalization of both CRUs and mitochondria using light, confocal, and electron microscopy (EM) in: a) Extensor Digitorum Longus (EDL) muscles from adult (3-12 months of age) and ageing (≥24 months of age) wild type (WT) mice; and b) in human biopsies from sedentary elderly subjects (70 ± 5 years) and age matched sportmen (69 ± 4 years of age) to determine how EC coupling and mitochondrial apparatuses are affected by age and exercise. RESULTS A: Studies in mice revealed that: a) the number of CRUs/100µm(2) (measured in longitudinal EM sections) in aging mice decreases significantly compared to adult mice: 87.4 ± 30.3 vs. 74.1 ± 25.1, respectively (p<0.01); b) the number of mitochondria-profiles/100µm(2) also decreases with age: 52.9 ± 23.5 vs. 42.8 ± 21.0, respectively (p<0.01); c) in ageing fibers mitochondria are more frequently found at the A band of the sarcomere (5.9 ± 3.3 vs. 1.5 ± 5.3), i.e. away from CRUs. The miss-placement of mitochondria is likely the results of the decreased frequency of tethers: in ageing fibers their number decreased with age from 14/100µm(2) in adult vs. 6/100µm(2) in ageing mice. The above changes taken together caused a significant decrease in the number CRUs-mitochondria couples/100µm(2): 37.4 ± 17.4 vs. 27.0 ± 15.8 (a decrease of ~27%). This reduction of CRU/mitochondria couples may significantly contribute to the decrease of specific force and endurance of skeletal muscle associated to ageing. A manuscript containing these data was recently submitted for publication (Pietrangelo & D’Incecco et al. submitted). In order to determine if the structural changes described above are caused by ageing itself or if inactivity plays also a central role in the progressive decay of EC coupling and mitochondrial apparatuses, we also studied mice that had access to running wheels for the second part of their lives (from 1 to 2 years of age). Results collected from these (unpublished) findings indicated that exercise improves number of mitochondria (a), their position with respect to sarcomeric striation (b), and their association to CRUs (c): a) number of mitochondria: 49.3 ± 19.8* / 100µm(2); b) number of mitochondria at the A band: 2.0 ± 4.2* / 100µm(2); number of CRUs-mitochondria couples: 35.0 ± 16.8* / 100µm(2). Please compare these data with results from sedentary mice reported above (differences were all highly significant: *p<0.01). RESULTS B: Studies in human Vastus Lateralis biopsies from sedentary elderly subjects confirmed general findings collected in mice (i.e. decrease in frequency of both CRUs and mitochondria and partial miss-placement of mitochondria). In human studies (see Figure 4), we compared samples from two groups of elderly individuals (all males): sedentary subjects (70 ± 5 years) or sportmen (69 ± 4 years of age), i.e. individuals who regularly exercised in the last several years of their lives. These studies revealed that both CRUs and mitochondria increase with exercise, mitochondria more than CRUs. Number of CRUs / 100µm(2): 20.3 ± 10.0 in sedentary vs. 21.6 ± 10.8 in sportsmen; number of mitochondria / 100µm(2): 37.1 ± 18.3 in sedentary vs. 52.0 ± 21.3 in sportsmen. The combined increase of both CRUs and mitochondria resulted in largely increased frequency of CRU/mitochondria pairs / 100µm(2): 5.9 ± 5.5 in sedentary vs. 11.1 ± 8.3 in sportsmen. These studies have been recently published in a larger study including, beside the just described EM analysis, also functional and histological data (Zampieri et al. 2015)7 DISCUSSION AND PERSPECTIVES: The results collected in our studies suggest that structure/organization of both EC coupling and mitochondrial apparatus: a) is affected by age; b) is better preserved by exercise. In conclusion, the dramatic age-related decay affecting mitochondrial and EC coupling apparatuses in skeletal muscle of mice and humans are, at least in part, caused by inactivity due to changes in life style.