Dissociation of mitochondrial from sarcoplasmic reticular stress in Drosophila cardiomyopathy induced by molecularly distinct mitochondrial fusion defects

Abstract

Mitochondrial dynamism (fusion and fission) is responsible for remodeling interconnected mitochondrial networks in some cell types. Adult cardiac myocytes lack mitochondrial networks, and their mitochondria are inherently "fragmented". Mitochondrial fusion/fission is so infrequent in cardiomyocytes as to not be observable under normal conditions, suggesting that mitochondrial dynamism may be dispensable in this cell type. However, we previously observed that cardiomyocyte-specific genetic suppression of mitochondrial fusion factors optic atrophy 1 (Opa1) and mitofusin/MARF evokes cardiomyopathy in Drosophila hearts. We posited that fusion-mediated remodeling of mitochondria may be critical for cardiac homeostasis, although never directly observed. Alternately, we considered that inner membrane Opa1 and outer membrane mitofusin/MARF might have other as-yet poorly described roles that affect mitochondrial and cardiac function. Here we compared heart tube function in three models of mitochondrial fragmentation in Drosophila cardiomyocytes: Drp1 expression, Opa1 RNAi, and mitofusin MARF RNA1. Mitochondrial fragmentation evoked by enhanced Drp1-mediated fission did not adversely impact heart tube function. In contrast, RNAi-mediated suppression of either Opa1 or mitofusin/MARF induced cardiac dysfunction associated with mitochondrial depolarization and ROS production. Inhibiting ROS by overexpressing superoxide dismutase (SOD) or suppressing ROMO1 prevented mitochondrial and heart tube dysfunction provoked by Opa1 RNAi, but not by mitofusin/MARF RNAi. In contrast, enhancing the ability of endoplasmic/sarcoplasmic reticulum to handle stress by expressing Xbp1 rescued the cardiomyopathy of mitofusin/MARF insufficiency without improving that caused by Opa1 deficiency. We conclude that decreased mitochondrial size is not inherently detrimental to cardiomyocytes. Rather, preservation of mitochondrial function by Opa1 located on the inner mitochondrial membrane, and prevention of ER stress by mitofusin/MARF located on the outer mitochondrial membrane, are central functions of these "mitochondrial fusion proteins".

Keywords: Drosophila; Endoplasmic reticular stress; Mitochondrial fusion; Mitofusin; Optic Atrophy 1; Reactive oxygen species.

Figure 1. Cardiomyocyte-specific expression of Drp1 induces mitochondrial fragmentation without heart tube dysfunction

A. Mitochondrial size in representative control (tincΔ4-Gal4/+; Ctrl) and cardiac Drp1 transgenic (TG) cardiomyocytes by confocal analysis of cardiac-expressed mito-GFP fluorescence. White scale bar is 20 μm. B. Group histogram data for mitochondrial area (left graph), cumulative distribution curve (inset) and comparison of the median mitochondrial area (bar graph) of control (Ctrl) and Drp1 overexpressing (Drp1 TG) flies. C. Drp1 expression in isolated heart tubes. Left, mRNA by RT-qPCR; right, immunoblot analysis. D. Optical coherence tomography (OCT) of heart tubes. Group quantitative data for end-systolic dimension (ESD) and fractional shortening are to the right. E. Representative images and quantitative data (right) for tincΔ4-Gal4-driven mito-GFP (green) and TMRE (red) individual and merged confocal micrographs.

Figure 2. Expression of transgenic SOD1 and SOD2 rescue the cardiac dysfunction evoked by Opa1 knockdown

A. OCT of heart tube contractions in Ctrl (tincΔ4-Gal4/+), and strains with cardiac specific expression of Opa1 RNAi, transgenic SOD1, transgenic SOD2, Opa1 RNAi along with SOD1(TG) and Opa1 RNAi along with SOD2 (TG), driven by tincΔ4-Gal4. B. Bar graphs showing group mean OCT data for ESD and percent fractional shortening for the groups in A. C. Mitochondrial size as per Figure 1.

Figure 3. Cardiac dysfunction evoked by MARF knockdown is unaffected by over-expression of transgenic SOD1 or SOD2

A. and B. OCT and C. mitochondrial size studies as in Figure 2, except for cardiomyocyte-specific mitofusin/MARF deficiency.

Figure 4. Expression of transgenic SOD alleviates the cardiomyocyte mitochondrial membrane potential loss and suppresses ROS production in Opa1 but not in MARF knockdowns

A and B. Representative images tincΔ4-Gal4–driven mito-GFP (green) and TMRE (red) double stained images of Opa1 RNAi +/− SOD1 (A) and MARF RNA1 +/− SOD1 (B). To the right are group quantitative data from TMRE images (left panels) and for the corresponding ROS studies (right panels). C and D. Mitochondrial polarization status as a function of organelle size in Opa1 (C) and mitofusin/MARF (D) knockdown heart tubes. Insets show individual data points (n=3 hearts per group; *=P<0.05). White scale bar is 12 μm.

Figure 5. Decreasing mitochondrial ROS improves cardiac function in Opa1 RNAi, but not mitofusin/MARF RNAi, flies

A. Representative images from OCT studies of contracting heart tubes. B. Group mean OCT data. C. and D. Group mean histogram data for mitochondrial area (left), cumulative distribution curve (middle) and median mitochondrial area (right) for MARF RNAi (C) and Opa1 RNAi (D) with and without ROMO1 RNAi.

Figure 6. ER stress response gene Xbp1 suppresses cardiac dysfunction induced by mitofusin/MARF knockdown, but not by Opa1 knockdown

A. Representative OCT images. B. Group mean OCT data. C. Mitochondrial morphology as in Figure 4.

Figure 7. Fusion factor-specific restoration of mitochondrial membrane potential by cardiomyocyte-specific expression of ROMO1 RNAi or Xbp1 transgene

A. Representative double stained images of tincΔ4-Gal4–driven mito-GFP (green) and TMRE (red) showing depolarized mitochondria (with less red staining). MARF and Opa1 knockdowns strains show increased number of mitochondria which are not stained by TMRE. Expression of the transgenic Romo1 RNAi decreases the number of depolarized mitochondria in the Opa1 but not in the MARF knockdown. B. Group quantitative data of stained mitochondria in heart specific expression of Romo1 RNAi in the Opa1 knockdown. C. Group quantitative data of heart specific expression of Romo1 RNAi or Xbp1 (TG) in the MARF knockdown flies. *=P<0.05; ns=non-significant.