Major findings

In the study, we have identified the location of p16Ink4a+ senescent cells in human skeletal muscle, and measured the impact of resistance exercise on the quantity of p16Ink4a+ senescent cells under two different protein supplemented conditions. The main findings of the study are as follows: 1) No senescent myofibers are detected in the skeletal muscle of young men aged between 20-25 y; 2) Most of the senescent cells found around muscle fibers are endothelial progenitor cells; 3) A single bout of resistance exercise reduces the senescent endothelial progenitor cells significantly in challenged muscle and maintains at low levels for 48 h; 4) Resistance exercise with low protein availability is associated with greater increases in macrophage infiltration and further depletion of senescent endothelial progenitor cells in muscle tissue during recovery, but prevents muscle hypertrophy for a long term. Taken together, these data suggest that senescence cell clearance and muscle mass increment are associated with the magnitude of muscle inflammation after resistance exercise, which can be influenced by protein supplementation around exercise.

It is not surprising that more than 40% of CD34+ endothelial progenitor cells in skeletal muscle of healthy young men showed various degrees of P16Ink4a+ signals. The average lifespan of endothelial cells in cardiac muscle of animals lasts only ~2 weeks [2], suggesting that this cell type ages rapidly compared with myofibers in muscle tissue [1]. Endothelial cells in skeletal muscle may be viewed as a collection of fit and unfit cell populations, based on their capability to survive against physical challenges. Resistance exercise may weakens those unfit cells, leading to presentations of danger-associated molecular pattern to attract immune attack from phagocytic macrophage [14]. The selective scavenging mechanism by innate immunity may help to reinforce a multicellular system by recovering exercise muscle tissue with younger and healthier cell population for better survival against next similar challenges. Eliminating p16Ink4a+ senescent cells is known to significantly increases lifespan in mice [3]. Given the fact that resistance exercise increases muscle damage, the present results on senescent cell elimination provide an explanation on how a destructive challenge may bring benefit for human survival [15].

Resistance exercise immediately increased presences of leukocyte and phagocytic macrophage in challenged skeletal muscle [8]. The rapid decline of senescent endothelial progenitor cells after exercise may have been associated with the combined results of increased phagocytosis [7] and homing of new circulating endothelial progenitor cells (or termed blood outgrowth endothelial cells from bone marrow) in capillaries of challenged muscle [16,17]. Macrophage activation is a fast acting mechanism to clear the senescent cells in muscle tissue [10,18]. Selective elimination of human senescent cells by phagocytic macrophage has been reported [7], which can occur at an extremely fast pace. Our observation on different magnitudes of p16Ink4a+ senescent cell clearance in muscle tissue after resistance training under low and high protein trials fits well with the difference in the amount of CD68+ macrophage infiltration within tissue. An acute episode of tissue ischemia during eccentric muscle contractions may increase homing of circulating endothelial progenitor cells to challenged sites [16].

The current result suggests that low protein availability delays resolution of muscle inflammation and decreases muscle mass increment after resistance training. Inflammation is essential for muscle regeneration induced by weight loading [9,10]. During the 48-h recovery after resistance exercise, high protein supplementation attenuates increases in CD68+ macrophage infiltration and promotes earlier presence of CD163+ macrophage in the exercised skeletal muscle 48 h after workout, compared with low protein supplemented condition. The surge of regenerative CD163+ macrophage in challenged muscle occurs nearing completion of the phagocytic phase of inflammation [9]. Therefore, the result of the study suggests that increasing protein availability accelerates completion of the inflammation program in exercised skeletal muscle for recovery. Protein and amino acids are primary nitrogen source of nucleotide synthesis for DNA replication during cell proliferation [19], which is essential for regenerating endothelial cell during post-exercise recovery. Amino acids have been shown to promote endothelial cell proliferation in vitro [20]. The rate of endothelial cell proliferation in capillaries can occur at a very fast rate with a doubling rate around a day [21]. Prolonged CD68+ macrophage infiltration with low protein availability may cause more phagocytosis in challenged tissue to extract endogenous nitrogen source from existing muscle tissue in response to the increasing demand on DNA synthesis during regeneration. This may explain the results on insignificant muscle mass increment under low protein supplementation following a 12-week of resistance training. It is not new that delayed protein supplementation after resistance exercise can have a negative effect on muscle hypertrophy in a long term [12,13]. Therefore, the present data on more CD68+ macrophage and less CD163+ macrophage in human skeletal muscle 48 h after resistance exercise may be an indicative of delayed resolution of muscle inflammation due to low protein availability.

The major limitation of the study is that only a single type of training has been used to investigate the senescent cell clearance effect and the data should not be generalized to other exercises. Additional studies on exercise regimen that employs more concentric muscle contractions, such as swimming, cycling, and aerobic exercise, are required.