Grant: Discovery Grants Program - Individual

This application is for funding to do work examining the energetic, mechanistic and functional aspects of activity dependent potentiation (or simply ""potentiation"") of mouse skeletal muscle. Potentiation is defined as the increase in muscle force observed during or following contractile activity of fast twitch muscles. Potentiation is a characteristic property of the fast twitch phenotype and these muscle types may often operate in this state in vivo. Potentiation is readily induced in fast twitch muscle and different forms of potentiation exist. For example, staircase potentiation is the progressive increase in force observed during prolonged, low frequency stimulation while posttetanic potentiation (PTP) is the increase in force observed following brief but high frequency stimulation. In both cases, the primary intracellular mechanism for potentiation is phosphorylation of the regulatory light chain (RLC) subunits contained by fast myosin types. The phosphate content of the RLC is highly regulated with the same calcium signals that regulate muscle force also activate a skeletal myosin light chain kinase enzyme that catalyzes the transfer of a phosphate to the RLC. The removal of the phosphate is unregulated and occurs at a steady rate; thus the phosphate content of the RLC varies directly with contractile activity. Phosphorylation of the RLC increases the calcium sensitivity of the contractile apparatus, thus accounting for how and why this molecular mechanism potentiates force. Indeed, studies performed on skeletal myosin light chain kinase knockout mice show that, in the absence of RLC phosphorylation, PTP is completely absent and staircase potentiation is greatly reduced (~ 50%). This interesting outcome indicates that even though RLC phosphorylation may be the primary mechanism for PTP, secondary mechanisms may be involved in staircase potentiation. This secondary mechanism may be stimulation - induced increase in resting calcium levels although the mechanism by which this happens is unknown. Interestingly, in addition to increasing force, potentiation may also increase the energetic cost of contraction although the relative contribution of RLC phosphorylation versus altered resting calcium homeostasis to this important effect is unknown. One of the main objectives of this grant application are to examine the influence of potentiation on the energetics of muscle contraction. The novel aspect of these studies is that we will examine this issue using muscles devoid of skeletal myosin light chain kinase (""knockouts"") and thus which do not display stimulation induced increases in RLC phosphorylation. Thus, use of these unique muscles will provide definitive answers as to how or if potentiation alters the energetic demands of mouse muscle and whether this change can be attributed to RLC phosphorylation or to altered calcium homeostasis. In other experiments, we will compare the force responses of normal and skeletal myosin light chain kinase knockout muscles to determine the relative contribution of these respective mechanisms to potentiation under a variety of conditions. A related objective of this research program is to separate out the mechanical / functional consequences of RLC phosphorylation versus altered resting calcium. In other experiments we will attempt to determine whether the potentiation observed in the lumbrical, a muscle with which we can quantify intracellular calcium during contraction, can be uncoupled from changes in resting calcium and also to determine how this change potentiates force in the absence of RLC phosphorylation. In summary, this work utilizing normal and knockout muscles (with and without RLC phosphorylation, respectively) will answer many questions related to potentiation.