MARF and Opa1 Control Mitochondrial and Cardiac Function in Drosophila

Abstract

Rationale

Mitochondria interact via actions of outer and inner membrane fusion proteins. The role of mitochondrial fusion on functioning of the heart, where mitochondria comprise ~30% of cardiomyocyte volume and their inter-myofilament spatial arrangement with other mitochondria is highly ordered, is unknown.

Objective

Model and analyze mitochondrial fusion defects in Drosophila melanogaster heart tubes with tincΔ4Gal4-directed expression of RNAi for Mitochondrial Assembly Regulatory Factor (MARF) and Optic atrophy 1 (Opa1).

Methods and Results

Live imaging analysis revealed that heart tube-specific knockdown of MARF or Opa1 increases mitochondrial morphometric heterogeneity and induces heart tube dilation with profound contractile impairment. Sarcoplasmic reticular structure was unaffected. Cardiomyocyte expression of human mitofusin (mfn) 1 or 2 rescued MARF RNAi cardiomyopathy, demonstrating functional homology between Drosophila MARF and human mitofusins. Suppressing mitochondrial fusion increased compensatory expression of nuclear-encoded mitochondrial genes, indicating mitochondrial biogenesis. The MARF RNAi cardiomyopathy was prevented by transgenic expression of superoxide dismutase 1.

Conclusions

Mitochondrial fusion is essential to cardiomyocyte mitochondrial function and regeneration. Reactive oxygen species are key mediators of cardiomyopathy in mitochondrial fusion-defective cardiomyocytes. Postulated mitochondrial-ER interactions mediated uniquely by mfn2 appear dispensable to functioning of the fly heart.

INTRODUCTION

Mitochondria generate energy and play central roles governing cell signaling and programmed death ^{1, 2}. Individual mitochondria periodically fuse though the coordinated efforts of three small, membrane-bound GTPases. Mitofusins (Mfn) 1 and 2 on outer mitochondrial membranes are essential for early mitochondrial tethering and outer membrane fusion. Optic Atrophy 1 (Opa1) on inner mitochondrial membranes mediates late fusion. Mitochondria occupy ~30% of the volume of a cardiac myocyte and are highly organized within these cells. Accordingly, cardiac myocytes are highly sensitive to abnormalities of mitochondrial number or function ^{3, 4}. The role of mitochondrial fusion within the highly ordered mitochondrial stacks of cardiomyocytes, and the consequences of altered mitochondrial fusion or mitochondrial-SR tethering in these uniquely structured cells, is unknown.

METHODS

Fly stocks and new transgenic lines

Fly stocks were obtained from the Bloomington Drosophila Stock Center at Indiana University; stock numbers are UAS-GFP (#9899), UAS-mitoGFP (#8442), and UAS-Sod.A (#24750). Rolf Bodmer (Sanford-Burnham Medical Research Institute, La Jolla California) provided tincΔ4Gal4 ⁵. Ming Guo (University of California, Los Angeles) provided MARF RNAi-UAS and Opa1 RNAi-UAS ⁶. hMfn1 and hMfn2 transgenic lines were constructed by subcloning their cDNAs into pUAST. Five independent lines each of hMfn1 and hMfn2 flies were examined; two of each were selected for studies. Adding a cb5 epitope to GFP using PCR mutagenesis and subcloning into pUAST created the ER/SR-GFP fly.

Expanded Methods are available in the Online Data Supplement at http://circres.ahajournals.org.

RESULTS

As a first step to determine the roles of mitochondrial fusion and mitochondrial-SR tethering on cardiac myocyte function we examined hearts of adult Drosophila expressing RNAi for Mitochondrial Assembly Regulatory Factor (MARF), a Drosophila ortholog of mammalian mitofusins ⁶. MARF expression was suppressed 80% by a ubiquitously expressed MARF RNAi (tubulin-Gal4 driver) in 2^nd instar larvae (Fig 1a), but all larvae died before pupation. Therefore, we directed expression of MARF RNAi to cardiomyocytes using tincΔ4-Gal4. Heart tube-specific MARF RNAi flies were viable with normal longevity (Supplemental Figure 1). We examined the consequences of MARF suppression on cardiomyocyte mitochondria using mitochondrial-directed green fluorescent protein (GFP) expressed in cardiomyocytes using tincΔ4-Gal4, and confocal microscopy of live isolated (or for phalloidin staining, formalin-fixed) heart tubes. Wild-type cardiomyocyte mitochondria appeared as relatively homogenous individual organelles arranged longitudinally between myofilaments (Figs 1b–1d). Mitochondria are motile in neurons and fibroblasts ^{7, 8}, but were immobile in cardiomyocytes as determined by time-lapse confocal microscopy of living heart tubes (Supplemental Fig 2). MARF RNAi cardiomyocyte mitochondria tended to cluster in aggregates that distorted the normal myofibrillar architecture (Fig 1c). As is characteristic of mitofusin 1 and 2 double deficient murine embryonic fibroblasts ⁹, higher magnification revealed extensive mitochondrial heterogeneity with both mitochondrial fragments and enlarged organelles (Fig 1d). Mean mitochondrial size was decreased ~30% (Fig 1e).

Figure 1. Suppression of mitochondrial fusion proteins MARF or Opa1 in Drosophila cardiomyocytes alters mitochondrial morphogenesis.

(a) RT-qPCR analysis of Marf and Opa mRNA in Marf RNAi flies. (b–h) Live cell (b, d, f) and fixed heart tube (c, g, h) confocal analysis of mitochondrial (b,c,d,f), t-tubule (g), and SR (h) morphometry and sub-cellular distribution in control (Gal4), MARF RNAi (b–d, g, h) and Opa1 RNAi (f) cardiomyocytes. *=P<0.05 vs Gal4 controls.

We also generated flies in which expression of the mitochondrial inner membrane fusion protein, Opa1, was suppressed in cardiac myocytes. Mitochondrial morphogenesis of Opa1 RNAi cardiomyocytes showed similar abnormalities, with mitochondrial clustering, fragmentation, and a ~30% decrease in mean mitochondrial size (Figs 1e, 1f).

To determine if morphological abnormalities induced by suppressing mitochondrial fusion were specific to mitochondria, we examined two other subcellular structures that are physically and structurally linked to cardiomyocyte mitochondria, t-tubules and sarcoplasmic reticulum (SR) ¹⁰. T-tubules, which are invaginations of the sarcolemma, were visualized with fluorescein-labeled wheat germ agglutinin and were unaffected by MARF suppression (Fig 1g). Cardiomyocyte SR was visualized by tincΔ4-Gal4-driven expression of a GFP-cb5 fusion protein. Wild-type SR has a fine reticular structure (Fig 1h) that was not disrupted MARF RNAi hearts. Thus, suppressing Drosophila cardiomyocyte mitochondrial fusion proteins alters mitochondrial, but not SR or t-tubule, morphology.

The functional consequences of suppressing mitochondrial fusion were assessed using optical coherence tomography of un-anaesthetized adult Drosophila heart tubes ¹¹. Compared to wild type flies, heart tubes of cardiac MARF RNAi flies were dilated and exhibited impaired shortening (Fig 2a–2e) with no change in beating rate (Fig 2b and 2f). Remodeling and contractile impairment induced by MARF RNAi affected the entire heart tube (Fig 2a and Supplemental movies 1 and 2). Opa RNAi heart tubes were also dilated (Fig 2c), but the contractile abnormality and remodeling were more severe (Fig 2a, 2d, and 2e; Supplemental movie 3). Consistent with the more severe cardiomyopathy, life span of Opa1 RNAi flies was reduced by ~25% (Supplemental Fig S1). TUNEL staining did not show evidence for cardiomyocyte death (Supplemental Fig 3).

Figure 2. Cardiomyocyte-specific suppression of mitochondrial fusion proteins induces dilated cardiomyopathy.

(a) Heart tubes expressing soluble GFP are shown at end diastole (left) and end-systole (right). Insets are optical coherence tomographic (OCT) cross sections of heart tubes at the same time in contractile cycle. Arrows indicate level of OCT interrogation. (b) B-mode analyses of heart tubes. EDD indicates end-diastolic dimension; ESD indicates end-systolic dimension. (c–e) Quantitative analysis of OCT imaging studies (mean ± s.e.m.). *=P<0.05 vs Gal4 controls. Experimental n is shown in (c).

These results link mitochondrial structural abnormalities and heart tube dysfunction to deficiency of mitochondrial outer or inner membrane fusion proteins. Whereas Drosophila has only the single outer mitochondrial membrane fusion protein MARF, mammals have two structurally similar mitochondrial outer membrane proteins, Mfn1 and Mfn2. Mfn2 is distinguished from Mfn1 by lower GTPases activity ¹². Both Mfn1 and Mfn2 induce mitochondrial tethering and outer membrane fusion, but in fibroblasts Mfn1 uniquely requires Opa1 ¹³, whereas Mfn2 uniquely mediates mitochondrial-ER tethering ¹⁴. We explored whether these differences in Mfn1 and Mfn2 impacted the cardiomyopathy of MARF deficiency by expressing human (h)Mfn1 and hMfn2 in Drosophila cardiomyocytes. hMfn1 and hMfn2 had little impact on adult Drosophila heart tube dimension or contractile function in the wild-type background (Supplemental Figure 4). However, both hMfn1 (Fig 3a, b) and hMfn2 (Fig 3c, d) improved the cardiomyopathy induced by MARF suppression. The Mfn2 “rescue” was complete, whereas that for Mfn1 was line-dependent. These findings demonstrate that Drosophila MARF is functionally analogous to mammalian mitofusins, and indicate that the unique properties of mammalian Mfn1 and Mfn2 have little impact on fly cardiomyocyte function.

Figure 3. Human mitofusin 1 and mitofusin 2 are functionally analogous to Drosophila MARF.

(a) OCT of heart tubes from control (tincΔ4Gal4), MARF RNAi, and MARF RNAi flies expressing human Mfn1 in cardiomyocytes. Mfn1(1) and Mfn1(2) represent two independent hMfn1 transgenic lines. Group data are in (b). (c, d) Similar studies as in (a, b) with two independent lines of cardiomyocyte hMfn2-expressing flies.

mRNA sequencing of MARF RNAi heart tubes showed abnormal cardiomyocyte expression of skeletal muscle myosin and actin isoforms, recapitulating isoform-switching characteristic of cardiac hypertrophy in mammalian hearts. Gene-Ontology grouping of upregulated Drosophila MARF RNAi heart tube mRNAs (Supplemental Fig 5) revealed disproportionately increased gene expression in three categories, mitochondrial inner membrane, mitochondrial envelope, and carbohydrate metabolism, each of which is rich in nuclear-encoded mitochondrial genes. Thus, cardiomyocyte mitochondrial fusion defects stimulated mitochondrial biogenesis.

Mitochondrial biogenesis can be a compensatory response to eroding mitochondrial function. Mitochondrial production of reactive oxygen species (ROS) is both a cause and consequence of mitochondrial dysfunction ¹⁵. To determine if mitochondrial ROS contributed to MARF RNAi cardiomyopathy, SOD1 was expressed in heart tubes. SOD1 expression did not alter normal heart tube dimension or contraction (Supplemental Fig 4). Strikingly however, SOD normalized heart tube dilatation and contractile dysfunction of MARF RNAi flies (Fig 4a) and markedly improved mitochondrial morphometrics (Fig 4b).

Figure 4. Cardiomyocyte MARF deficiency is rescued by superoxide dismutase.

(a) OCT imaging (top) and quantitative group analysis (bottom) of heart tube dimension and contractile function in control (Gal4), MARF RNAi, and MARF RNAi co-expressing SOD1. (b) Live cell confocal studies of mito-GFP in same groups. Bottom panels are higher magnification from different flies. Bar graph is quantitative data for mitochondrial size. *=P<0.05 vs Gal4 controls.

DISCUSSION

These studies provide the first evidence that mitochondrial fusion occurs in cardiomyocytes and is essential to mitochondrial and heart function. Mitochondrial organization within Drosophila and mammalian cardiomyocytes is highly ordered, with closely packed mitochondria interspersed in “stacks” between the myofilaments. Spatially enforced inter-mitochondrial interactions and limited intra-cellular mitochondrial mobility suggested that mitochondrial tethering proteins, i.e. Drosophila MARF and the mammalian mitofusins, might be superfluous in cardiomyocytes. On the other hand, the postulated critical roles in cardiomyocytes for ER/SR-mitochondrial calcium transport through tight inter-organelle junctions mediated specifically by Mfn2 ^{16, 17} suggest an essential role for Mfn2 independent of, or in addition to, mitochondrial fusion ¹⁸. Based on the current data, we propose that mitochondrial fusion is essential in cardiomyocytes notwithstanding mitochondrial packing, but that specific functions of Mfn2, such as tethering of cardiomyocyte mitochondria to sarcoplasmic reticulum, are dispensable for normal Drosophila heart tube function.

The finding that the MARF RNAi cardiomyopathy is rescued by both hMfn1 and hMfn2 demonstrates that similar mechanisms regulate mitochondrial fusion and functional stability in insects and vertebrates, and has two additional important implications. First, Drosophila MARF is functionally analogous to the mammalian mitofusin proteins. Second, Mfn1 and Mfn2 are largely interchangeable with each other and with MARF in Drosophila heart tubes. Thus, the unique properties of mammalian Mfn1 (higher GTPase activity ¹² and functional interaction with Opa1 ¹³) and Mfn2 (ability to tether mitochondria to ER/SR ¹⁴) are not essential to Drosophila cardiomyocyte health. This does not indicate that Mfn2-mediated mitochondrial-ER/SR interactions don’t occur in cardiomyocytes, or aren’t important for other aspects of cardiomyocyte biology such as programmed cell death ¹⁸, but does suggest that putative mitochondrial-SR interactions mediated by Mfn2 are not essential to normal heart tube function in flies.

Novelty and Significance.

What is known?

• Because of the energy required for life-long cardiac pumping, myocardium is the most mitochondrial-rich tissue.

• Although it has not been studied in cardiac myocytes, in most cell types normal mitochondrial regeneration requires periodic inter-mitochondrial exchange of contents.

• This mitochondrial content exchange process requires mitochondrial tethering and outer membrane fusion mediated by mitofusins 1 and 2, and inner membrane fusion mediated by Optic atrophy 1 (Opa1).

What new information does this article contribute?

• Using the fruit fly heart tube as a model, we show that mitochondrial fusion proteins are necessary to maintain normal cardiac structure and contractile function.

• Genetic complementation to rescue the cardiomyopathy that resulted from suppressing mitochondrial fusion, reveals that the fruit fly and human outer mitochondrial membrane fusion proteins are functionally redundant.

• Through a similar genetic complementation strategy, we show that mitochondrial dysfunction and ROS production are critical mediators of cardiac dysfunction induced by loss of mitochondrial fusion.

Summary .

Mitochondrial fusion is observed in many cells, but its occurrence and functional implications are unknown in cardiomyocytes. Here, genetic inhibition of Drosophila melanogaster cardiomyocyte mitochondrial fusion through RNAi suppression of the mitofusin analog Marf, or inner membrane protein Opa1, produced heterogeneity of mitochondrial size, transcriptional evidence for mitochondrial biogenesis, and dilated cardiomyopathies. Human mitofusins 1 or 2 prevented Marf RNAi cardiomyopathy, demonstrating overlapping functions in Drosophila cardiomyocytes. Marf and Opa1 RNAi cardiomyopathies were rescued by cardiomyocyte expression of superoxide dismutase, implicating reactive oxygen species. Thus, mitochondrial fusion in cardiomyocytes is essential for normal mitochondrial function and to prevent dilated cardiomyopathy.

Non-standard abbreviations and acronyms

MARF

mitochondrial assembly regulatory factor

Opa1

optic atrophy 1

Mfn

mitofusin

ER

endoplasmic reticulum

GFP

green fluorescent protein

OCT

optical coherence tomography