Detection of Calcium Sparks in Intact and Permeabilized Skeletal Muscle Fibers

Ca2+ sparks are the elementary units of Ca2+ signaling in striated muscle fibers that appear as highly localized Ca2+ release events through ryanodine receptor (RyR) Ca2+ release channels in the sarcoplasmic reticulum (SR). While these events are commonly observed in resting cardiac myocytes, they are rarely seen in resting skeletal muscle fibers. Since Ca2+ spark analysis can provide extensive data on the Ca2+ handling characteritsics of normal and diseased striated muscle, there has been interest in developing methods for observing Ca2+ sparks in skeletal muscle. Previously, we discovered that stress generated by osmotic pressure changes induces a robust Ca2+ spark response confined in close spatial proximity to the sarcolemmal membrane in wild-type intact mammalian muscles. Our studies showed these peripheral Ca2+ sparks (PCS) were altered in dystrophic or aged skeletal muscles. Other methods to induce Ca2+ sparks include permeabilization of the sarcolemmal membrane with detergents, such as saponin. In this chapter, we will discuss the methods for isolation of muscle fibers, the techniques for inducing Ca2+ sparks in these isolated fibers, and provide guidance on the analysis of data from these experiments.