Store-operated Ca2+ entry (SOCE) is an essential Ca2+ signaling mechanism present in most animal cells. SOCE refers to Ca2+ influx that is activated by depletion of sarco/endoplasmic reticulum (S/ER) Ca2+ stores. The main components of SOCE are STIM and Orai. STIM proteins function as S/ER Ca2+ sensors, and upon S/ER Ca2+ depletion STIM rearranges to S/ER-plasma membrane junctions and activates Orai Ca2+ influx channels. Studies have implicated SOCE in cardiac hypertrophy pathogenesis, but SOCE's role in normal heart physiology remains poorly understood. We therefore analyzed heart-specific SOCE function in Drosophila, a powerful animal model of cardiac physiology. We show that heart-specific suppression of Stim and Orai in larvae and adults resulted in reduced contractility consistent with dilated cardiomyopathy. Myofibers were also highly disorganized in Stim and Orai RNAi hearts, reflecting possible decompensation or upregulated stress signaling. Furthermore, we show that reduced heart function due to SOCE suppression adversely affected animal viability, as heart specific Stim and Orai RNAi animals exhibited significant delays in post-embryonic development and adults died earlier than controls. Collectively, our results demonstrate that SOCE is essential for physiological heart function, and establish Drosophila as an important model for understanding the role of SOCE in cardiac pathophysiology.

Heart failure is often preceded by pathological cardiac hypertrophy, a thickening of the heart musculature driven by complex gene regulatory and signaling processes. The Drosophila heart has great potential as a genetic model for deciphering the underlying mechanisms of cardiac hypertrophy. However, current methods for evaluating hypertrophy of the Drosophila heart are laborious and difficult to carry out reproducibly. Here we demonstrate that micro-computerized tomography (microCT) is an accessible, highly reproducible method for non-destructive, quantitative analysis of Drosophila heart morphology and size. To validate our microCT approach for analyzing Drosophila cardiac hypertrophy, we show that expression of constitutively active Ras (Ras85DV12), previously shown to cause hypertrophy of the fly heart, results in significant thickening of both adult and larval heart walls when measured from microCT images. We then show using microCT analysis that genetic upregulation of store-operated Ca2+ entry (SOCE) driven by expression of constitutively active Stim (StimCA) or Orai (OraiCA) proteins also results in significant hypertrophy of the Drosophila heart, through a process that specifically depends on Orai Ca2+ influx channels. Intravital imaging of heart contractility revealed significantly reduced end diastolic and systolic dimensions in StimCA and OraiCA expressing hearts, consistent with the hypertrophic phenotype. These results demonstrate that increased SOCE activity is an important driver of hypertrophic cardiomyocyte growth, and demonstrate how microCT analysis combined with tractable genetic tools in Drosophila can be used to delineate molecular signaling processes that underlie cardiac hypertrophy and heart failure.

In this study, we examine the cause and progression of mitochondrial diseases linked to the loss of mtRNase P, a three-protein complex responsible for processing and cleaving mitochondrial transfer RNAs (tRNA) from their nascent transcripts. When mtRNase P function is missing, mature mitochondrial tRNA levels are decreased, resulting in mitochondrial dysfunction. mtRNase P is composed of Mitochondrial RNase P Protein (MRPP) 1, 2, and 3. MRPP1 and 2 have their own enzymatic activity separate from MRPP3, which is the endonuclease responsible for cleaving tRNA. Human mutations in all subunits cause mitochondrial disease. The loss of mitochondrial function can cause devastating, often multisystemic failures. When mitochondria do not provide enough energy and metabolites, the result can be skeletal muscle weakness, cardiomyopathy, and heart arrhythmias. These symptoms are complex and often difficult to interpret, making disease models useful for diagnosing disease onset and progression. Previously, we identified Drosophila orthologs of each mtRNase P subunit (Roswell/MRPP1, Scully/MRPP2, Mulder/MRPP3) and found that the loss of each subunit causes lethality and decreased mitochondrial tRNA processing in vivo. Here, we use Drosophila to model mtRNase P mitochondrial diseases by reducing the level of each subunit in skeletal and heart muscle using tissue-specific RNAi knockdown. We find that mtRNase P reduction in skeletal muscle decreases adult eclosion and causes reduced muscle mass and function. Adult flies exhibit significant age-progressive locomotor defects. Cardiac-specific mtRNase P knockdowns reduce fly lifespan for Roswell and Scully, but not Mulder. Using intravital imaging, we find that adult hearts have impaired contractility and exhibit substantial arrhythmia. This occurs for roswell and mulder knockdowns, but with little effect for scully. The phenotypes shown here are similar to those exhibited by patients with mitochondrial disease, including disease caused by mutations in MRPP1 and 2. These findings also suggest that skeletal and cardiac deficiencies induced by mtRNase P loss are differentially affected by the three subunits. These differences could have implications for disease progression in skeletal and heart muscle and shed light on how the enzyme complex functions in different tissues.