Muscle stem cells contribute to myofibres in sedentary adult mice

Abstract

Skeletal muscle is essential for mobility, stability and whole body metabolism, and muscle loss, for instance, during sarcopenia, has profound consequences. Satellite cells (muscle stem cells) have been hypothesized, but not yet demonstrated, to contribute to muscle homeostasis and a decline in their contribution to myofibre homeostasis to play a part in sarcopenia. To test their role in muscle maintenance, we genetically labelled and ablated satellite cells in adult sedentary mice. We demonstrate via genetic lineage experiments that, even in the absence of injury, satellite cells contribute to myofibres in all adult muscles, although the extent and timing differs. However, genetic ablation experiments showed that satellite cells are not globally required to maintain myofibre cross-sectional area of uninjured adult muscle.

Figure 1. Satellite cell number differs between muscles and with age

(a–g) Representative cross-sections showing Pax7+ satellite cells in EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM at 12 and 20 months in control Pax7^+/+;Rosa^DTA/+ mice. Scale bar = 100 µm. Mean number of Pax7+ satellite cells per volume (mm³) of muscle at 12 months (dark blue, n = 6 EDL, TA, n = 7 other muscles) and 20 months (light blue, n = 3 EDL, n = 5 other muscles). Data are expressed as mean ± 1 s.e.m for all graphs. Two-tailed student t-test, *p≤0.05.

Figure 2. Satellite cells contribute to myofibers in all muscles

(a) Schematic showing genetic labeling of satellite cells with GFP at 6 months and harvested at either 12 or 20 months. (d–i, k, m) Representative cross-sections of EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM of 12 or 20 month Pax7^CreERT2/+;Rosa^mTmG/+ mice showing GFP+ myofibers to which satellite cells have contributed. It should be noted that the percentage of satellite cell contribution maybe inflated in EOM due to the small size of its myofibers, which may reduce dilution of the GFP signal. Scale bar = 100 µm. (j, l) Adjacent sections in soleus and diaphragm identifying slow MyHCI+ myofibers. White arrows indicate fast GFP+MyHCI−fibers, orange arrows indicate slow GFP+MyHCI+ fibers. Green graphs show %GFP+ myofibers at 12 months (dark green, n = 4 for each muscle) or 20 months (light green, n = 6 for each muscle). Orange graphs show %GFP+ MyHCI+ myofibers in soleus (dark orange, n = 4 at 12 months; light orange, n = 6 at 20 months) and diaphragm (dark orange, n = 3 at 12 months; light orange, n = 5 at 20 months). Data are expressed as mean ± 1 s.e.m for all graphs. Two-tailed student t-test, *p≤0.05, **p<0.01 ***p<0.001.

Figure 3. Myofibers decline in size in most limb muscles with age

(a–g) Representative cross-sections showing outlined laminin+ myofibers in EDL, TA, gastrocnemius, plantaris, soleus, diaphragm, and EOM at 12 and 20 months in control Pax7^+/+;Rosa^DTA/+ mice. Slow MyHCI+ fibers (red) are identified in (e) soleus and (f) diaphragm. Scale bar = 100 µm for all panels. Histograms generated by MuscleQNT show relative frequency of myofibers sizes in muscles at 12 months (dark blue, n = 7 for each muscle) and 20 months (light blue, n = 4 for EDL, n = 5 for other muscles). Additional histograms for (e) soleus and (f) diaphragm show relative frequency of fast MyHCI− and slow MyHCI+ myofibers in muscles at 12 and 20 months. Permutation tests were conducted to determine whether counts of myofibers were significantly different in a particular bin between 12 and 20 month muscles (see Methods). Black asterisks indicate an empirical p value of <0.05 and gray asterisks a value of <0.10 (see Methods).

Figure 4. Testing satellite cell role in maintaining size of limb myofibers

(a) Schematic showing ablation of satellite cells at 6 months and harvested at either 12 or 20 months. (b–f) Representative cross-sections showing outlined laminin+ myofibers in limb muscles EDL, TA, gastrocnemius, plantaris, and soleus at 12 and 20 months in Pax7^CreERT2/+;Rosa^DTA/+ mice (see Figure 3 for representative cross-sections of Pax7^+/+;Rosa^DTA/+ mice). Histograms generated by MuscleQNT show relative frequency of myofibers sizes in muscles at 12 months (dark blue, control Pax7^+/+, n = 7, for each muscle; red satellite cell ablated Pax7^CreERT2/+, n = 8 for each muscle) and 20 months (light blue, control Pax7^+/+, n = 4 for EDL, n = 5 for other muscles; pink, satellite cell ablated Pax7^creERT2/+, n = 6 for each muscle). Scale bar = 100 µm for all panels. (g) Additional histograms for the soleus show frequency of fast MyHCI− and slow MyHCI+ myofibers. Permutation tests (see Methods) were conducted to determine whether counts of myofibers were significantly different in a particular bin between control and satellite cell ablated muscles. Black asterisks indicate an empirical p value of <0.05 and gray asterisks a value of <0.10 (see Methods). Note that the MuscleQNT parameters slightly differ between 12 and 20 month TA, gastrocnemius, plantaris, and diaphragm muscles; to explicitly compare the CSA of 12 and 20 month control of muscles see Figure 3. (h) Myonuclei are significantly longer (two-tailed student t-test, **p<0.01) and number of myonuclei are significantly reduced in 20 month versus 12 month EDL, but not in plantaris (two-tailed student t-test, *p<0.05).

Figure 5. Testing satellite cell role in maintaining EOM and diaphragm myofibers

(a, c) Representative cross-sections showing outlined laminin+ myofibers in diaphragm and EOM at 12 and 20 months in Pax7^CreERT2/+;Rosa^DTA/+ mice (see Figure 3 for representative cross-sections of Pax7^+/+;Rosa^DTA/+ mice). Histograms generated by MuscleQNT show relative frequency of myofibers sizes in muscles at 12 months (dark blue, control Pax7^+/+, n = 7; red, satellite cell ablated Pax7^creERT2/+, n = 8) and 20 months (light blue, control Pax7^+/+, n = 5, pink, satellite cell ablated Pax7^creERT2/+, n = 6) Scale bar = 100 µm in both panels. (b) Additional histograms for the diaphragm show frequency of fast MyHCI− and slow MyHCI+ myofibers. Permutation tests (see Methods) were conducted to determine whether counts of myofibers were significantly different in a particular bins between control and satellite cell ablated muscles. Black asterisks indicate an empirical p value of <0.05 and gray asterisks a value of <0.10 (see Methods). (d) Myonuclei are significantly longer at 12 months (two-tailed student t-test, *p<0.05), but number of myonuclei do not change between 12 and 20 months in the diaphragm (two-tailed student t-test).

Figure 6. Summary of satellite cell contribution to adult uninjured muscle

Pax7+ satellite cells, labeled at 6 months, contribute to myofibers in all muscles between 6 and 12 months. After 12 months there is no appreciable satellite cell contribution in most limb muscles (TA, plantaris, soleus, and EDL), but there is continued contribution in the diaphragm and EOM (also gastrocnemius, not shown). Pax7+ satellite cells are not required to maintain myofiber cross-sectional area of most muscles, but may help maintain myofiber CSA in EOM at 12 months and EDL at 20 months (red boxes). In EOM potentially Pax7-Pitx2+ (blue circles) satellite cells, as suggested by, compensate when Pax7+ satellites are ablated. Pax7+ satellite cells are shown as green circles in left panels and as brown circles in right panels.