Fura-2 fluorescent technique for the assessment of Ca²⁺ homeostasis in cardiomyocytes

Abstract

Ca²⁺ homeostasis plays a pivotal role in maintaining cell growth and function. Many heart diseases are related to the abnormalities in Ca²⁺ mobilization and extrusion. Ca²⁺-sensitive fluorescent dyes have been used successfully to estimate intracellular free Ca²⁺ ([Ca²⁺]ⁱ) level and the mechanisms of Ca²⁺ movements in living cells. This article is focused on the methodology involving the use of Fura-2/AM or free Fura-2 to measure agonist-induced Ca²⁺ mobilization as well as the mechanisms of changes in [Ca²⁺]. in cardiomyocytes. Methods involving Fura-2 technique for the measurement of Ca²⁺ extrusion from the cells and Ca²⁺ reuptake by sarcoplasmic reticulum (SR) are also described. The prevention of KCl-induced increase in the intracellular Ca²⁺ is shown by chelating the extracellular Ca²⁺ with EGTA or by the presence of Ca²⁺-channel inhibitors such as verapamil and diltiazem. The involvement of SR in the ATP-induced increase in intracellular Ca²⁺ is illustrated by the use of Ca²⁺-pump inhibitors, thapsigargin and cyclopiazonic acid as well as ryanodine which deplete the SR Ca²⁺ storage. The use of 2-nitro-4-carboxyphenyl N,N-diphenyl carbamate (NCDC), an inhibitor of inositol 1,4,5-trisphosphate (IP3) production, is described for the attenuation of phosphatidic acid (PA) induced increase in Ca²⁺-mobilization. The increase in intracellular Ca²⁺ in cardiomyocytes by PA, unlike that by KC1 or ATP, was observed in diabetic myocardium. Thus, it appears that the Fura-2 method for the measurement of Ca²⁺ homeostasis in cardiomyocytes is useful in studying the pathophysiology and pharmacology of Ca²⁺ movements. (Mol Cell Biochem 172: 149–157, 1997)