Electron microscope studies have shown that the switched-off state of myosin II in muscle involves intramolecular interaction between the two heads of myosin and between one head and the tail. The interaction, seen in both myosin filaments and isolated molecules, inhibits activity by blocking actin-binding and ATPase sites on myosin. This interacting-heads motif is highly conserved, occurring in invertebrates and vertebrates, in striated, smooth, and nonmuscle myosin IIs, and in myosins regulated by both Ca²⁺ binding and regulatory light-chain phosphorylation. Our goal was to determine how early this motif arose by studying the structure of inhibited myosin II molecules from primitive animals and from earlier, unicellular species that predate animals. Myosin II from Cnidaria (sea anemones, jellyfish), the most primitive animals with muscles, and Porifera (sponges), the most primitive of all animals (lacking muscle tissue) showed the same interacting-heads structure as myosins from higher animals, confirming the early origin of the motif. The social amoeba Dictyostelium discoideum showed a similar, but modified, version of the motif, while the amoeba Acanthamoeba castellanii and fission yeast (Schizosaccharomyces pombe) showed no head–head interaction, consistent with the different sequences and regulatory mechanisms of these myosins compared with animal myosin IIs. Our results suggest that head–head/head–tail interactions have been conserved, with slight modifications, as a mechanism for regulating myosin II activity from the emergence of the first animals and before. The early origins of these interactions highlight their importance in generating the inhibited (relaxed) state of myosin in muscle and nonmuscle cells. Significance All animals have the ability to move. Myosin II is the motor protein that generates this movement by powering muscular contraction; it also drives motility in nonmuscle cells. In relaxed muscle and in quiescent nonmuscle cells, myosin II is switched off by intramolecular interactions between its heads that inhibit its activity. This interacting-heads motif (IHM) is a fundamental contributor to contractile regulation. Given its importance in cell contractility, we wanted to determine when the IHM first evolved. Using electron microscopy, image averaging, and sequence analysis of myosin II from primitive organisms, we show that the IHM has existed since the earliest animals and before. This ancient origin highlights the central role of the IHM in regulating myosin II function.

Amyloid-like inclusions have been associated with Huntington's disease (HD), which is caused by expanded polyglutamine repeats in the Huntingtin protein. HD patients exhibit a high incidence of cardiovascular events, presumably as a result of accumulation of toxic amyloid-like inclusions. We have generated a Drosophila model of cardiac amyloidosis that exhibits accumulation of PolyQ aggregates and oxidative stress in myocardial cells, upon heart-specific expression of Huntingtin protein fragments (Htt-PolyQ) with disease-causing poly-glutamine repeats (PolyQ-46, PolyQ-72, and PolyQ-102). Cardiac expression of GFP-tagged Htt-PolyQs resulted in PolyQ length-dependent functional defects that included increased incidence of arrhythmias and extreme cardiac dilation, accompanied by a significant decrease in contractility. Structural and ultrastructural analysis of the myocardial cells revealed reduced myofibrillar content, myofibrillar disorganization, mitochondrial defects and the presence of PolyQ-GFP positive aggregates. Cardiac-specific expression of disease causing Poly-Q also shortens lifespan of flies dramatically. To further confirm the involvement of oxidative stress or protein unfolding and to understand the mechanism of PolyQ induced cardiomyopathy, we co-expressed expanded PolyQ-72 with the antioxidant superoxide dismutase (SOD) or the myosin chaperone UNC-45. Co-expression of SOD suppressed PolyQ-72 induced mitochondrial defects and partially suppressed aggregation as well as myofibrillar disorganization. However, co-expression of UNC-45 dramatically suppressed PolyQ-72 induced aggregation and partially suppressed myofibrillar disorganization. Moreover, co-expression of both UNC-45 and SOD more efficiently suppressed GFP-positive aggregates, myofibrillar disorganization and physiological cardiac defects induced by PolyQ-72 than did either treatment alone. Our results demonstrate that mutant-PolyQ induces aggregates, disrupts the sarcomeric organization of contractile proteins, leads to mitochondrial dysfunction and increases oxidative stress in cardiomyocytes leading to abnormal cardiac function. We conclude that modulation of both protein unfolding and oxidative stress pathways in the Drosophila heart model can ameliorate the detrimental PolyQ effects, thus providing unique insights into the genetic mechanisms underlying amyloid-induced cardiac failure in HD patients. Huntington's disease (HD) is associated with amyloid-like inclusions in the brain and heart, and accumulation of amyloid protein is associated with neurodegeneration and cardiomyopathy. Recent studies suggest that HD patients show increased susceptibility to cardiac failure. However, the mechanisms by which disease-causing poly-glutamine repeats (PolyQ) cause heart dysfunction in these patients are unclear. We have developed a novel Drosophila heart model that exhibits significant GFP-positive aggregates upon HD-causing PolyQ expression in myocardial cells resulting in PolyQ length-dependent physiological defects. Modulation of protein folding and oxidative stress pathways in this system reduced the number of aggregates and reversed the cardiac dysfunction in response to expression of disease-causing PolyQ. The ability to explore PolyQ-associated mechanisms of cardiomyopathy in a genetically tractable whole organism, Drosophila melanogaster, promises to provide novel insights into the relationship between amyloid accumulation and heart dysfunction. Our findings not only impact the understanding of PolyQ-induced cardiomyopathy but also other human cardiac diseases associated with oxidative stress, mitochondrial defects and protein homeostasis.

By comparing the structure of wild-type and mutant muscle myosin heavy chain (MHC) genes of Drosophila melanogaster, we have identified the defect in the homozygous-viable, flightless mutant Mhc10. The mutation is within the 3' splice acceptor of an alternative exon (exon 15a) that encodes the central region of the MHC hinge. The splice acceptor defect prevents the accumulation of mRNAs containing exon 15a, whereas transcripts with a divergent copy of this exon (exon 15b) are unaffected by the mutation. In situ hybridization and Northern blot analysis of wild-type organisms reveals that exon 15b is used in larval MHCs, whereas exons 15a and/or 15b are used in adult tissues. Because Mhc10 mutants fail to accumulate transcripts encoding MHC protein with hinge region a, analysis of their muscle-specific reduction in thick filament number serves as a sensitive assay system for determining the pattern of accumulation of MHCs with alternative hinge regions. Electron microscopic comparisons of various muscles from wild-type and Mhc10 adults reveals that those that contract rapidly or develop high levels of tension utilize only hinge region a, those that contract at moderate rates accumulate MHCs of both types, and those that are slowly contracting have MHCs with hinge region b. The presence of alternative hinge-coding exons and their highly tissue-specific usage suggests that this portion of the MHC molecule is important to the isoform-specific properties of MHC that lead to the different physiological and ultrastructural characteristics of various Drosophila muscle types. The absence of other alternative exons in the rod-coding region, aside from those shown previously to encode alternative carboxyl termini, demonstrates that the bulk of the myosin rod is not involved in the generation of isoform-specific properties of the MHC molecule.

We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC-null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.