Detection of calcium sparks in intact and permeabilized skeletal muscle fibers

Ca ²⁺ sparks are the elementary units of Ca ²⁺ signaling in striated muscle fibers that appear as highly localized Ca ²⁺ release events through ryanodine receptor (RyR) Ca ²⁺ 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 Ca ²⁺ spark analysis can provide extensive data on the Ca ²⁺ handling characteritsics of normal and diseased striated muscle, there has been interest in developing methods for observing Ca ²⁺ sparks in skeletal muscle. Previously, we discovered that stress generated by osmotic pressure changes induces a robust Ca ²⁺ spark response confined in close spatial proximity to the sarcolemmal membrane in wild-type intact mammalian muscles. Our studies showed these peripheral Ca ²⁺ sparks (PCS) were altered in dystrophic or aged skeletal muscles. Other methods to induce Ca ²⁺ 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 Ca ²⁺ sparks in these isolated fibers, and provide guidance on the analysis of data from these experiments.