A Method to Measure Myocardial Calcium Handling in Adult Drosophila

Abstract

Rationale Normal cardiac physiology requires highly regulated cytosolic Ca2+ concentrations and abnormalities in Ca2+ handling are associated with heart failure. The majority of approaches to identify the components that regulate intracellular Ca2+ dynamics rely on cells in culture, mouse models, and human samples. However, a genetically robust system for unbiased screens of mutations that affect Ca2+ handling remains a challenge. Objective We sought to develop a new method to measure myocardial Ca2+ 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 Ca2+ sensing fluorescent protein, GCaMP2, we developed methods to measure parameters associated with myocardial Ca2+ handling. The following key observations were identified: (1) Control w1118 Drosophila hearts have readily measureable Ca2+-dependent fluorescent signals that are dependent on L-type Ca2+ channels and SR Ca2+ stores and originate from rostral and caudal pacemakers; (2) A fly mutant, held-up2 (hdp2) that has a point mutation in Troponin I and has a dilated cardiomyopathic phenotype demonstrates abnormalities in myocardial Ca2+ 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 (FDHM) and decreases in the linear slope of decay from 80% to 20% intensity decay; and (3) Hearts from hdp2 mutants had reductions in caffeine-induced Ca2+ increases and reductions in ryanodine receptor (RyR) without changes in L-type Ca2+ channel transcripts compared to w1118. Conclusions Our results show that the cardiac-specific expression of GCaMP2 provides a means to characterize propagating Ca2+ transients in adult fly hearts. Moreover, the adult fruit fly heart recapitulates several aspects of Ca2+ 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 Ca2+ transient as well as changes in transcript levels of proteins associated with Ca2+ handling. This new methodology has the potential to permit an examination of evolutionarily conserved myocardial Ca2+ handing mechanisms by applying the vast resources available in the fly genomics community to conduct genetic screens to identify new genes involved in generated Ca2+ transients and arrhythmias.

Enhanced Assessment of Contractile Dynamics in Drosophila Hearts

“…Phase analysis showed thapsigargin treatment selectively prolonged the relaxation phase and slightly reduced the contraction phase at 30 min compared with controls (Figure 2B). These data are consistent with the increased Ca 2+ transient observed in response to similar thapsigargin treatments by Lin et al (10) and suggest that SOHA v.3 upgrades, in combination with the semi-intact hear t preparation, may be useful to screen for drugs that modulate repolarization reserve in cardiac tissue.…”

A Drosophila melanogaster Model of Diastolic Dysfunction and Cardiomyopathy Based on Impaired Troponin-T Function

“…Quantitative polymerase chain reaction analysis was performed to assay possible changes in the Ca 2+ -handling biosignature of up 101 relative to Canton-S hearts (Online Figure II). 71 No significant differences were identified between the lines in transcript levels of L-type Ca 2+ channels, ryanodine receptors, SERCA, Na/Ca exchangers, or in inositol-3-phosphate receptors. Although this assay does not preclude possible post-transcriptional or post-translational modifications that could influence the encoded proteins, nor does it completely rule out all potential adaptive Ca 2+ responses, the results suggest the cardiac phenotype we observe is primarily due to the direct, Ca 2+ -independent, C-state promoting effect the up 101 TnT mutation exerts on thin filaments.…”

“…69,70 Moreover, a Drosophila TnI mutant, which is characterized by an enlarged cardiac chamber and cardiac contractile dysfunction, displays abnormalities in the cytosolic Ca 2+ transient as well as changes in transcription of proteins associated with Ca 2+ handling. 71 Therefore, the up 101 TnT mutant may similarly exhibit downstream alterations in Ca 2+ kinetics and homeostasis that potentially contribute to the observed cardiac phenotype. Quantitative polymerase chain reaction analysis was performed to assay possible changes in the Ca 2+ -handling biosignature of up 101 relative to Canton-S hearts (Online Figure II).…”

Animal Models of Heart Failure

“…The readers should be aware that fly 137 and fish 138 models are available. These animal models are particularly well suited for studies exploring the role(s) of specific genes in the development, progression, or prevention of HF.…”