Aging-affiliated post-translational modifications of skeletal muscle myosin affect biochemical properties, myofibril structure, muscle function, and proteostasis.

The molecular motor myosin is post-translationally modified in its globular head, its S2 hinge, and its thick filament domain during human skeletal muscle aging. To determine the importance of such modifications, we performed an integrative analysis of transgenic Drosophila melanogaster expressing myosin containing post-translational modification mimic mutations. We determined effects on muscle function, myofibril structure, and myosin biochemistry. Modifications in the homozygous state decreased jump muscle function by a third at 3 weeks of age and reduced indirect flight muscle function to negligible levels in young flies, with severe effects on flight muscle myofibril assembly and/or maintenance. Expression of mimic mutations in the heterozygous state or in a wild-type background yielded significant, but less severe, age-dependent effects upon flight muscle structure and function. Modification of the residue in the globular head disabled ATPase activity and in vitro actin filament motility, whereas the S2 hinge mutation reduced actin-activated ATPase activity by 30%. The rod modification diminished filament formation in vitro. The latter mutation also reduced proteostasis, as demonstrated by enhanced accumulation of polyubiquitinated proteins. Overall, we find that mutation of amino acids at sites that are chemically modified during human skeletal muscle aging can disrupt myosin ATPase, myosin filament formation, and/or proteostasis, providing a mechanistic basis for the observed muscle defects. We conclude that age-specific post-translational modifications present in human skeletal muscle are likely to act in a dominant fashion to affect muscle structure and function and may therefore be implicated in degeneration and dysfunction associated with sarcopenia.

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure.

The R249Q mutation in human ß-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a ß-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent ß-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the ß-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

The R369 Myosin Residue within Loop 4 Is Critical for Actin Binding and Muscle Function in Drosophila.

The myosin molecular motor interacts with actin filaments in an ATP-dependent manner to yield muscle contraction. Myosin heavy chain residue R369 is located within loop 4 at the actin-tropomyosin interface of myosin's upper 50 kDa subdomain. To probe the importance of R369, we introduced a histidine mutation of that residue into Drosophila myosin and implemented an integrative approach to determine effects at the biochemical, cellular, and whole organism levels. Substituting the similarly charged but bulkier histidine residue reduces maximal actin binding in vitro without affecting myosin ATPase activity. R369H mutants exhibit impaired flight ability that is dominant in heterozygotes and progressive with age in homozygotes. Indirect flight muscle ultrastructure is normal in mutant homozygotes, suggesting that assembly defects or structural deterioration of myofibrils are not causative of reduced flight. Jump ability is also reduced in homozygotes. In contrast to these skeletal muscle defects, R369H mutants show normal heart ultrastructure and function, suggesting that this residue is differentially sensitive to perturbation in different myosin isoforms or muscle types. Overall, our findings indicate that R369 is an actin binding residue that is critical for myosin function in skeletal muscles, and suggest that more severe perturbations at this residue may cause human myopathies through a similar mechanism.

Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila.

Dilated cardiomyopathy (DCM), a life-threatening disease characterized by pathological heart enlargement, can be caused by myosin mutations that reduce contractile function. To better define the mechanistic basis of this disease, we employed the powerful genetic and integrative approaches available in Drosophila melanogaster. To this end, we generated and analyzed the first fly model of human myosin-induced DCM. The model reproduces the S532P human ß-cardiac myosin heavy chain DCM mutation, which is located within an actin-binding region of the motor domain. In concordance with the mutation's location at the actomyosin interface, steady-state ATPase and muscle mechanics experiments revealed that the S532P mutation reduces the rates of actin-dependent ATPase activity and actin binding and increases the rate of actin detachment. The depressed function of this myosin form reduces the number of cross-bridges during active wing beating, the power output of indirect flight muscles, and flight ability. Further, S532P mutant hearts exhibit cardiac dilation that is mutant gene dose-dependent. Our study shows that Drosophila can faithfully model various aspects of human DCM phenotypes and suggests that impaired actomyosin interactions in S532P myosin induce contractile deficits that trigger the disease.

Prolonged myosin binding increases muscle stiffness in Drosophila models of Freeman-Sheldon syndrome.

Freeman-Sheldon syndrome (FSS) is characterized by congenital contractures resulting from dominant point mutations in the embryonic isoform of muscle myosin. To investigate its disease mechanism, we used Drosophila models expressing FSS myosin mutations Y583S or T178I in their flight and jump muscles. We isolated these muscles from heterozygous mutant Drosophila and performed skinned fiber mechanics. The most striking mechanical alteration was an increase in active muscle stiffness. Y583S/+ and T178I/+ fibers' elastic moduli increased 70 and 77%, respectively. Increased stiffness contributed to decreased power generation, 49 and 66%, as a result of increased work absorbed during the lengthening portion of the contractile cycle. Slower muscle kinetics also contributed to the mutant phenotype, as shown by 17 and 32% decreases in optimal frequency for power generation, and 27 and 41% slower muscle apparent rate constant 2πb. Combined with previous measurements of slower in vitro actin motility, our results suggest a rate reduction of at least one strongly bound cross-bridge cycle transition that increases the time myosin spends strongly bound to actin, ton. Increased ton was further supported by decreased ATP affinity and a 16% slowing of jump muscle relaxation rate in T178I heterozygotes. Impaired muscle function caused diminished flight and jump ability of Y583S/+ and T178I/+ Drosophila. Based on our results, assuming that our model system mimics human skeletal muscle, we propose that one mechanism driving FSS is elevated muscle stiffness arising from prolonged ton in developing muscle fibers.

Educating the Next Generation of Undergraduate URM Cancer Scientists: Results and Lessons Learned from a Cancer Research Partnership Scholar Program.

To improve cancer disparities among under-represented minority (URM) populations, better representation of URM individuals in cancer research is needed. The San Diego State University and University of California San Diego Moores Cancer Center Partnership is addressing cancer disparities through an educational program targeting undergraduate URM students. The Partnership provides a paid intensive summer research internship enriched with year-round activities that include educational sessions, a journal club, mentorship, social activities, and poster sessions and presentations. Program evaluation through follow-up surveys, focus groups, and other formal and informal feedback, including advisory and program steering committees, are used to improve the program. Long-term follow-up among scholars (minimum of 10 years) provides data to evaluate the program's long-term impact on scholars' education and career path. Since 2016, 63 URM undergraduate students participated in the scholar program. At the year-2 follow-up (2016 cohort; n = 12), 50% had completed their Graduate Record Examination (GRE) and/or applied to graduate or medical school. Lessons learned during the course of the program led to implementation of changes to provide a better learning experience and increase overall program satisfaction. Updates were made to recruitment timeline, improvements of the recruitment processes, refinement of the program contracts and onboarding meetings, identification of essential program coordinator skills and responsibilities, adjustments to program components, and establishment of a well-mapped and scheduled evaluation plan. The Partnership identified best practices and lessons learned for implementing lab-based internship scholar programs in biomedical and public health fields that could be considered in other programs.

Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain.

We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal ß-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3-encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D ) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD ) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2 ) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3-encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.

Drosophila myosin mutants model the disparate severity of type 1 and type 2B distal arthrogryposis and indicate an enhanced actin affinity mechanism.

BACKGROUND: Distal arthrogryposis (DA) is a group of autosomal dominant skeletal muscle diseases characterized by congenital contractures of distal limb joints. The most common cause of DA is a mutation of the embryonic myosin heavy chain gene, MYH3. Human phenotypes of DA are divided into the weakest form-DA1, a moderately severe form-DA2B (Sheldon-Hall Syndrome), and a severe DA disorder-DA2A (Freeman-Sheldon Syndrome). As models of DA1 and DA2B do not exist, their disease mechanisms are poorly understood. METHODS: We produced the first models of myosin-based DA1 (F437I) and DA2B (A234T) using transgenic Drosophila melanogaster and performed an integrative analysis of the effects of the mutations. Assessments included lifespan, locomotion, ultrastructural analysis, muscle mechanics, ATPase activity, in vitro motility, and protein modeling. RESULTS: We observed significant defects in DA1 and DA2B Drosophila flight and jump ability, as well as myofibril assembly and stability, with homozygotes displaying more severe phenotypes than heterozygotes. Notably, DA2B flies showed dramatically stronger phenotypic defects compared to DA1 flies, mirroring the human condition. Mechanical studies of indirect flight muscle fibers from DA1 heterozygotes revealed reduced power output along with increased stiffness and force production, compared to wild-type controls. Further, isolated DA1 myosin showed significantly reduced myosin ATPase activity and in vitro actin filament motility. These data in conjunction with our sinusoidal analysis of fibers suggest prolonged myosin binding to actin and a slowed step associated with Pi release and/or the power stroke. Our results are supported by molecular modeling studies, which indicate that the F437I and A234T mutations affect specific amino acid residue interactions within the myosin motor domain that may alter interaction with actin and nucleotide. CONCLUSIONS: The allele-specific ultrastructural and locomotory defects in our Drosophila DA1 and DA2B models are concordant with the differential severity of the human diseases. Further, the mechanical and biochemical defects engendered by the DA1 mutation reveal that power production, fiber stiffness, and nucleotide handling are aberrant in F437I muscle and myosin. The defects observed in our DA1 and DA2B Drosophila models provide insight into DA phenotypes in humans, suggesting that contractures arise from prolonged actomyosin interactions.

X-ray Crystallographic and Molecular Dynamic Analyses of Drosophila melanogaster Embryonic Muscle Myosin Define Domains Responsible for Isoform-Specific Properties.

Drosophila melanogaster is a powerful system for characterizing alternative myosin isoforms and modeling muscle diseases, but high-resolution structures of fruit fly contractile proteins have not been determined. Here we report the first x-ray crystal structure of an insect myosin: the D melanogaster skeletal muscle myosin II embryonic isoform (EMB). Using our system for recombinant expression of myosin heavy chain (MHC) proteins in whole transgenic flies, we prepared and crystallized stable proteolytic S1-like fragments containing the entire EMB motor domain bound to an essential light chain. We solved the x-ray crystal structure by molecular replacement and refined the resulting model against diffraction data to 2.2 Å resolution. The protein is captured in two slightly different renditions of the rigor-like conformation with a citrate of crystallization at the nucleotide binding site and exhibits structural features common to myosins of diverse classes from all kingdoms of life. All atom molecular dynamics simulations on EMB in its nucleotide-free state and a derivative homology model containing 61 amino acid substitutions unique to the indirect flight muscle isoform (IFI) suggest that differences in the identity of residues within the relay and the converter that are encoded for by MHC alternative exons 9 and 11, respectively, directly contribute to increased mobility of these regions in IFI relative to EMB. This suggests the possibility that alternative folding or conformational stability within these regions contribute to the observed functional differences in Drosophila EMB and IFI myosins.

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is a genetic disease that causes thickening of the heart's ventricular walls and is a leading cause of sudden cardiac death. HCM is caused by missense mutations in muscle proteins including myosin, but how these mutations alter muscle mechanical performance in largely unknown. We investigated the disease mechanism for HCM myosin mutation R249Q by expressing it in the indirect flight muscle of Drosophila melanogaster and measuring alterations to muscle and flight performance. Muscle mechanical analysis revealed R249Q decreased muscle power production due to slower muscle kinetics and decreased force production; force production was reduced because fewer mutant myosin cross-bridges were bound simultaneously to actin. This work does not support the commonly proposed hypothesis that myosin HCM mutations increase muscle contractility, or causes a gain in function; instead, it suggests that for some myosin HCM mutations, hypertrophy is a compensation for decreased contractility. ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, we expressed an HCM myosin mutation, R249Q, in Drosophila indirect flight muscle (IFM) and assessed myofibril structure, skinned fibre mechanical properties, and flight ability. Mechanics experiments were performed on fibres dissected from 2-h-old adult flies, prior to degradation of IFM myofilament structure, which started at 2 days old and increased with age. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. The muscle apparent rate constant 2πb decreased 33% for homozygous but increased for heterozygous fibres. The apparent rate constant 2πc was greater for homozygous fibres. This indicates that R249Q myosin is slowing attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, our results do not support the increased contractility hypothesis. Instead, our results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.

Suppression of myopathic lamin mutations by muscle-specific activation of AMPK and modulation of downstream signaling.

Laminopathies are diseases caused by dominant mutations in the human LMNA gene encoding A-type lamins. Lamins are intermediate filaments that line the inner nuclear membrane, provide structural support for the nucleus and regulate gene expression. Drosophila melanogaster models of skeletal muscle laminopathies were developed to investigate the pathological defects caused by mutant lamins and identify potential therapeutic targets. Human disease-causing LMNA mutations were modeled in Drosophila Lamin C (LamC) and expressed in indirect flight muscle (IFM). IFM-specific expression of mutant, but not wild-type LamC, caused held-up wings indicative of myofibrillar defects. Analyses of the muscles revealed cytoplasmic aggregates of nuclear envelope (NE) proteins, nuclear and mitochondrial dysmorphology, myofibrillar disorganization and up-regulation of the autophagy cargo receptor p62. We hypothesized that the cytoplasmic aggregates of NE proteins trigger signaling pathways that alter cellular homeostasis, causing muscle dysfunction. In support of this hypothesis, transcriptomics data from human muscle biopsy tissue revealed misregulation of the AMP-activated protein kinase (AMPK)/4E-binding protein 1 (4E-BP1)/autophagy/proteostatic pathways. Ribosomal protein S6K (S6K) messenger RNA (mRNA) levels were increased and AMPKα and mRNAs encoding downstream targets were decreased in muscles expressing mutant LMNA relative controls. The Drosophila laminopathy models were used to determine if altering the levels of these factors modulated muscle pathology. Muscle-specific over-expression of AMPKα and down-stream targets 4E-BP, Forkhead box transcription factors O (Foxo) and Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), as well as inhibition of S6K, suppressed the held-up wing phenotype, myofibrillar defects and LamC aggregation. These findings provide novel insights on mutant LMNA-based disease mechanisms and identify potential targets for drug therapy.

Reductions in ATPase activity, actin sliding velocity, and myofibril stability yield muscle dysfunction in Drosophila models of myosin-based Freeman-Sheldon syndrome.

Using Drosophila melanogaster, we created the first animal models for myosin-based Freeman-Sheldon syndrome (FSS), a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus our Drosophila models mimic the human condition in that FSS mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide-binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in FSS patients.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy.

K146N is a dominant mutation in human ß-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

Interacting-heads motif has been conserved as a mechanism of myosin II inhibition since before the origin of animals.

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.

TRiC/CCT chaperonins are essential for maintaining myofibril organization, cardiac physiological rhythm, and lifespan.

We recently reported that CCT chaperonin subunits are upregulated in a cardiac-specific manner under time-restricted feeding (TRF) [Gill S et al. (2015) Science 347, 1265-1269], suggesting that TRiC/CCT has a heart-specific function. To understand the CCT chaperonin function in cardiomyocytes, we performed its cardiac-specific knock-down in the Drosophila melanogaster model. This resulted in disorganization of cardiac actin- and myosin-containing myofibrils and severe physiological dysfunction, including restricted heart diameters, elevated cardiac dysrhythmia and compromised cardiac performance. We also noted that cardiac-specific knock-down of CCT chaperonin significantly shortens lifespans. Additionally, disruption of circadian rhythm yields further deterioration of cardiac function of hypomorphic CCT mutants. Our analysis reveals that both the orchestration of protein folding and circadian rhythms mediated by CCT chaperonin are critical for maintaining heart contractility.

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the ß-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects.

A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in individuals with IBM3.

Expression patterns of cardiac aging in Drosophila.

Aging causes cardiac dysfunction, often leading to heart failure and death. The molecular basis of age-associated changes in cardiac structure and function is largely unknown. The fruit fly, Drosophila melanogaster, is well-suited to investigate the genetics of cardiac aging. Flies age rapidly over the course of weeks, benefit from many tools to easily manipulate their genome, and their heart has significant genetic and phenotypic similarities to the human heart. Here, we performed a cardiac-specific gene expression study on aging Drosophila and carried out a comparative meta-analysis with published rodent data. Pathway level transcriptome comparisons suggest that age-related, extra-cellular matrix remodeling and alterations in mitochondrial metabolism, protein handling, and contractile functions are conserved between Drosophila and rodent hearts. However, expression of only a few individual genes similarly changed over time between and even within species. We also examined gene expression in single fly hearts and found significant variability as has been reported in rodents. We propose that individuals may arrive at similar cardiac aging phenotypes via dissimilar transcriptional changes, including those in transcription factors and micro-RNAs. Finally, our data suggest the transcription factor Odd-skipped, which is essential for normal heart development, is also a crucial regulator of cardiac aging.

A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila.

An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.

Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy.

AIMS: Heart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance, and actin cytoskeletal reorganization. Profilin is a well-conserved, ubiquitously expressed, multifunctional actin-binding protein, and its role in cardiomyocytes is largely unknown. Given its involvement in vascular hypertrophy, we aimed to test the hypothesis that profilin-1 is a key mediator of cardiomyocyte-specific hypertrophic remodelling. METHODS AND RESULTS: Profilin-1 was elevated in multiple mouse models of hypertrophy, and a cardiomyocyte-specific increase of profilin in Drosophila resulted in significantly larger heart tube dimensions. Moreover, adenovirus-mediated overexpression of profilin-1 in neonatal rat ventricular myocytes (NRVMs) induced a hypertrophic response, measured by increased myocyte size and gene expression. Profilin-1 silencing suppressed the response in NRVMs stimulated with phenylephrine or endothelin-1. Mechanistically, we found that profilin-1 regulates hypertrophy, in part, through activation of the ERK1/2 signalling cascade. Confocal microscopy showed that profilin localized to the Z-line of Drosophila myofibrils under normal conditions and accumulated near the M-line when overexpressed. Elevated profilin levels resulted in elongated sarcomeres, myofibrillar disorganization, and sarcomeric disarray, which correlated with impaired muscle function. CONCLUSION: Our results identify novel roles for profilin as an important mediator of cardiomyocyte hypertrophy. We show that overexpression of profilin is sufficient to induce cardiomyocyte hypertrophy and sarcomeric remodelling, and silencing of profilin attenuates the hypertrophic response.