A method to measure myocardial calcium handling in adult Drosophila

Abstract

Rationale: Normal cardiac physiology requires highly regulated cytosolic Ca 2+concentrations and abnormalities in Ca handling are associated with heart failure. The majority of approaches to identifying the components that regulate intracellular Ca dynamics rely on cells in culture, mouse models, and human samples. However, a genetically robust system for unbiased screens of mutations that affect Ca handling remains a challenge. Objective: We sought to develop a new method to measure myocardial Ca cycling in adult Drosophila and determine whether cardiomyopathic fly hearts recapitulate aspects of diseased mammalian myocardium. Methods and results: Using engineered transgenic Drosophila that have cardiac-specific expression of Ca-sensing fluorescent protein, GCaMP2, we developed methods to measure parameters associated with myocardial Ca handling. The following key observations were identified: (1) Control w Drosophila hearts have readily measureable Ca-dependent fluorescent signals that are dependent on L-type Ca channels and SR Ca stores and originate from rostral and caudal pacemakers. (2) A fly mutant, held-up (hdp), that has a point mutation in troponin I and has a dilated cardiomyopathic phenotype demonstrates abnormalities in myocardial Ca handling that include increases in the duration of the 50% rise in intensity to peak intensity, the half-time of fluorescence decline from peak, the full duration at half-maximal intensity, and decreases in the linear slope of decay from 80% to 20% intensity decay. (3) Hearts from hdp mutants had reductions in caffeine-induced Ca increases and reductions in ryanodine receptor (RyR) without changes in L-type Ca channel transcripts in comparison with w. Conclusions: Our results show that the cardiac-specific expression of GCaMP2 provides a means of characterizing propagating Ca transients in adult fly hearts. Moreover, the adult fruit fly heart recapitulates several aspects of Ca regulation observed in mammalian myocardium. A mutation in Drosophila that causes an enlarged cardiac chamber and impaired contractile function is associated with abnormalities in the cytosolic Ca transient as well as changes in transcript levels of proteins associated with Ca handling. This new methodology has the potential to permit an examination of evolutionarily conserved myocardial Ca-handing mechanisms by applying the vast resources available in the fly genomics community to conduct genetic screens to identify new genes involved in generated Ca transients and arrhythmias. © 2011 American Heart Association, Inc.