The effects of mechanical stretch on the immunological and anti-inflammatory properties of skeletal muscle cells: An in vitro study of potential mechanisms that affect response to exercise therapy

The contraction of skeletal muscle during exercise that occurs in the absence of muscle damage has been demonstrated to yield a significant increase in the release of potentially anti-inflammatory myokine interleukin-6 (IL-6). Increased generation of IL-6 may be valuable in managing inflammatory states, such as chronic kidney disease (CKD). However, the IL-6 output of CKD patients during muscular exercise may be unusually weak, due to factors such as metabolic acidosis.

In CKD, physical exertion has been reported to lower levels of amino acids in muscles in vivo. This effect may be attributable to uraemic metabolic acidosis and exercise-induced acidosis impairing mitogen-activated protein kinases (MAPKs) such as p38, which normally activate the amino acid transporter SNAT2. The SNAT2 transporter protein is also known to be directly and rapidly inhibited by low pH. The resulting intracellular amino acid depletion could, in turn, result in inhibited protein synthesis, and hence reduced production of IL-6.

This study sought to test the following hypotheses: (1) higher levels of IL-6 expression occur in mechanically stressed skeletal muscle cells following in vitro mechanical stretch, arising from the activation of MAPKs, followed by activation of SNAT2; and (2) low pH blocks this process.

Experiments on L6 rat skeletal muscle cells showed that drug inhibition of MAPKs inhibited SNAT2 transport activity, confirming that MAPKs play a role in the regulation of SNAT2 and, by extension, in the availability of amino acids for protein synthesis.

Interestingly, while P-p38 and P-JNK activation significantly increased after cyclic stretching of 1-10 minutes duration, SNAT2 activity significantly increased only transiently i.e. after periods of stretching lasting 1 minute, suggesting that this transporter may paradoxically be inhibited by prolonged stretching. In addition, mRNA of IL-6 was increased after 30 minutes of intermittent stretching and, (as previously reported elsewhere) by 6 hours of Ca2+ loading of the cells with ionomycin which activates p38.

The siRNA silencing of SNAT2 in L6 myoblasts decreased gene expression of the mRNA of IL-6, suggesting that SNAT2 can influence IL-6 expression. As SNAT2 and MAPKs, particularly p38 and JNK, are inhibited at low pH, it would be predicted that low pH (pH 7.1) would decrease IL-6 expression. However, both with and without ionomycin, low pH failed to decrease IL-6 mRNA; indeed with ionomycin low pH strongly activated IL-6 expression.It is concluded that even though the acidosis-sensing amino acid transporter SNAT2 may play some role in regulating IL-6 expression (as previously hypothesised), a distinct and novel stimulatory effect of low pH over-rides this and maybe a more important regulator of IL-6 expression in L6 skeletal muscle cells.