ROS function as a distinct mitochondrial retrograde response factor for appropriate cardiogenic specifications and cardiac function in Drosophila embryos

Congenital Heart Diseases (CHDs) are major cause of prenatal and neonatal mortality. These defects occur due to abnormalities during heart formation in utero. The treatments of CHDs pose a major challenge due to lack of in depth understanding of underlying factors. There are certain evidences that suggest abnormal mitochondrial function in these cardiomyopathies. A large percentage of prenatal dilated cardiomyopathies reportedly exhibit defects in mitochondrial oxidative phosphorylation. However, we still have a very little understanding of causal role played by mitochondria in CHDs. Considering the fact that heart has highest density of mitochondria followed by skeletal muscles and central nervous system, investigation of mitochondrial involvement in CHDs is a very intriguing question. The Drosophila embryonic heart is termed as dorsal vessel. It is a simple contractile tube which is similar to the tubular heart of early vertebrate embryo. Despite the evolutionary distance between Drosophila and vertebrates, cardio genesis is governed by similar fundamental mechanisms. The transcription factors as well as signalling pathways involved in cardio genesis are highly conserved among Drosophila and vertebrates. Dorsal vessel is comprised of merely104 cardio blasts; an extremely simple organ; hence enables pinpointing any minor cardiac deformity. The anterior portion is termed aorta whereas posterior portion is termed Heart proper. Hemolymph flows from posterior to anterior of the dorsal vessel. The posterior portion has specific subset of cardio blasts that form the inflow tracts termed as Ostia. Dorsal vessel has segmental repeated pattern of cell types which is a hallmark of insects. The cardio blasts are flanked by pericardial cells which function as nephrocytes. Drosophila can prove to be a good model system to understand cardiac development and function, which can open new doors to understand cardiomyopathies and improve their treatment. In order to perturb mitochondrial complex-I activity during embryonic development, two components of complex-I of ETC; ND75 which is the largest core subunit of complex-I and ND42, an accessory subunit required for the assembly of complex-I of ETC were knocked down at different temporal and spatial windows and embryos were observed for lethality in terms of hatching rate. Severe embryonic lethality was observed in F1 progeny embryos by knocking down ND42 and ND75 with Twist Gal4. Twist Gal4 is the early mesoderm Gal4 driver. Its expression starts at stage 4 when mesoderm begins to form. Live imaging of these embryos indicate cardiac malfunction at stage 17 of embryonic development. Quantitative analysis of cardiac parameters using SOHA (Semi-automated Heartbeat Analysis) confirmed the cardiac defects with respect to different parameters for example Heart rate, Heart period, systole-diastole diameters and systole -diastole intervals. Further investigation of the cardiac defect in knock down embryos at cellular level revealed an intriguing cell specification defect at stage 16 of embryonic development. In wild type stage 16 embryo, cardiac tube is formed of 4 Tinman positive and 2 Seven up positive cardio blasts in each hemi segment. However, in complex-I mutant embryos, we found that this 4+2 pattern of cardio blasts is altered to 2+4 pattern. By tracking this phenotype to the earlier stages, we found that this change in cell identity initiates at stage 13 where some of Tinman positive cells begin to lose Tinman expression and ectopically begins to express Seven up. Therefore, identity of cells is changed due to metabolic disturbances. In the complex-I knockdown embryos, ROS levels were found to be high as indicated by mitoSOX dye and gstD GFP reporter line. Co-immunostaining of Twist protein and gst D GFP showed that ROS levels were only increased in the Twist expressing mesoderm cells at stage 10 of embryonic development, suggesting that knockdown of ND42/ND75 using Twist Gal4 has resulted in elevated ROS levels in mesodermal population. We were able to establish ROS as the key mediator to elicit retrograde response from mitochondria to regulate cell fate specification since over-expression of SOD2 in the background of ND42/ND75 knockdown was able to significantly rescue embryonic lethality, cardiac malfunctioning and cell fate specification defects.