Abstract
Repetitive low frequency stimulation results in potentiation of twitch force development in fast-twitch skeletal muscle due to myosin regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent skeletal muscle myosin light chain kinase (skMLCK). We generated transgenic mice that express an skMLCK CaM biosensor in skeletal muscle to determine whether skMLCK or CaM is limiting to twitch force potentiation. Three transgenic mouse lines exhibited up to 22-fold increases in skMLCK protein expression in fast-twitch extensor digitorum longus muscle containing type IIa and IIb fibers, with comparable expressions in slow-twitch soleus muscle containing type I and IIa fibers. The high expressing lines showed a more rapid RLC phosphorylation and force potentiation in extensor digitorum longus muscle with low frequency electrical stimulation. Surprisingly, overexpression of skMLCK in soleus muscle did not recapitulate the fast-twitch potentiation response despite marked enhancement of both fast-twitch and slow-twitch RLC phosphorylation. Analysis of calmodulin binding to the biosensor showed a frequency-dependent activation to a maximal extent of 60%. Because skMLCK transgene expression is 22-fold greater than the wild-type kinase, skMLCK rather than calmodulin is normally limiting for RLC phosphorylation and twitch force potentiation. The kinase activation rate (10.6 s(-1)) was only 3.6-fold slower than the contraction rate, whereas the inactivation rate (2.8 s(-1)) was 12-fold slower than relaxation. The slower rate of kinase inactivation in vivo with repetitive contractions provides a biochemical memory via RLC phosphorylation. Importantly, RLC phosphorylation plays a prominent role in skeletal muscle force potentiation of fast-twitch type IIb but not type I or IIa fibers.
Highlights
Measurement of skeletal muscle myosin light chain kinase (skMLCK) Activation by Electrical Stimulation in Intact Skeletal Muscle—Activation of skMLCK was measured in human skeletal ␣-actin (HSA)-skMLCK lumbrical muscles from the hind paw by fluorescence resonance energy transfer (FRET) of the CaM biosensor using procedures similar to those described for kinase activation in bladder smooth muscle [17]
Endogenous expression of skMLCK in soleus muscle was similar between wild-type and transgenic mice from line 1, whereas endogenous skMLCK was not detected in transgenic mice for lines 2 and 3
regulatory light chain (RLC) phosphorylation was significantly greater in soleus that RLCf is phosphorylated in response to electrical stimulamuscle from transgenic versus wild-type mice following 3 and tion [5, 25] and that the magnitude of twitch force potentiation
Summary
We generated transgenic mice that express an skMLCK CaM biosensor in both fast-twitch and slow-twitch skeletal muscle to determine 1) whether skMLCK is a limiting factor for RLC phosphorylation and twitch force potentiation in fast-twitch muscle, 2) whether elevating skMLCK levels in slow-twitch muscle is sufficient to enhance RLC phosphorylation and twitch force potentiation, and 3) whether CaM is rate-limiting to frequency-dependent activation of skMLCK in the intact muscle
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