TNNT2 mutations in the tropomyosin binding region of TNT1 disrupt its role in contractile inhibition and stimulate cardiac dysfunction.

Muscle contraction is regulated by the movement of end-to-end-linked troponin-tropomyosin complexes over the thin filament surface, which uncovers or blocks myosin binding sites along F-actin. The N-terminal half of troponin T (TnT), TNT1, independently promotes tropomyosin-based, steric inhibition of acto-myosin associations, in vitro. Recent structural models additionally suggest TNT1 may restrain the uniform, regulatory translocation of tropomyosin. Therefore, TnT potentially contributes to striated muscle relaxation; however, the in vivo functional relevance and molecular basis of this noncanonical role remain unclear. Impaired relaxation is a hallmark of hypertrophic and restrictive cardiomyopathies (HCM and RCM). Investigating the effects of cardiomyopathy-causing mutations could help clarify TNT1's enigmatic inhibitory property. We tested the hypothesis that coupling of TNT1 with tropomyosin's end-to-end overlap region helps anchor tropomyosin to an inhibitory position on F-actin, where it deters myosin binding at rest, and that, correspondingly, cross-bridge cycling is defectively suppressed under diastolic/low Ca2+ conditions in the presence of HCM/RCM lesions. The impact of TNT1 mutations on Drosophila cardiac performance, rat myofibrillar and cardiomyocyte properties, and human TNT1's propensity to inhibit myosin-driven, F-actin-tropomyosin motility were evaluated. Our data collectively demonstrate that removing conserved, charged residues in TNT1's tropomyosin-binding domain impairs TnT's contribution to inhibitory tropomyosin positioning and relaxation. Thus, TNT1 may modulate acto-myosin activity by optimizing F-actin-tropomyosin interfacial contacts and by binding to actin, which restrict tropomyosin's movement to activating configurations. HCM/RCM mutations, therefore, highlight TNT1's essential role in contractile regulation by diminishing its tropomyosin-anchoring effects, potentially serving as the initial trigger of pathology in our animal models and humans.

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 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.

Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model.

Dysregulation of L-type Ca2+ channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-α1D and Ca-α1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca2+ channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca2+ channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca2+ currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca2+ currents recorded in individual cells when Ca2+ channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca2+-dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca2+ channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies.

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.

CaMKII oxidation is a critical performance/disease trade-off acquired at the dawn of vertebrate evolution.

Antagonistic pleiotropy is a foundational theory that predicts aging-related diseases are the result of evolved genetic traits conferring advantages early in life. Here we examine CaMKII, a pluripotent signaling molecule that contributes to common aging-related diseases, and find that its activation by reactive oxygen species (ROS) was acquired more than half-a-billion years ago along the vertebrate stem lineage. Functional experiments using genetically engineered mice and flies reveal ancestral vertebrates were poised to benefit from the union of ROS and CaMKII, which conferred physiological advantage by allowing ROS to increase intracellular Ca2+ and activate transcriptional programs important for exercise and immunity. Enhanced sensitivity to the adverse effects of ROS in diseases and aging is thus a trade-off for positive traits that facilitated the early and continued evolutionary success of vertebrates.

A role for actin flexibility in thin filament-mediated contractile regulation and myopathy.

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.

MICAL1 constrains cardiac stress responses and protects against disease by oxidizing CaMKII.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity. MICAL1, a methionine monooxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin-binding (CaM-binding) domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1 in mice caused cardiac arrhythmias and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight-or-flight responses in mice, strikingly impaired heart function in Drosophila melanogaster, and caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Quantifying Tissue-Specific Overexpression of FOXO in Drosophila via mRNA Fluorescence In Situ Hybridization Using Branched DNA Probe Technology.

While the highly conserved FOXO transcription factors have been studied in Drosophila melanogaster for decades, the ability to accurately control and measure their tissue-specific expression is often cumbersome due to a lack of reagents and to limited, nonhomogeneous samples. The need for quantitation within a distinct cell type is particularly important because transcription factors must be expressed in specific amounts to perform their functions properly. However, the inherent heterogeneity of many samples can make evaluating cell-specific FOXO and/or FOXO load difficult. Here, we describe an extremely sensitive fluorescence in situ hybridization (FISH) approach for visualizing and quantifying multiple mRNAs with single-cell resolution in adult Drosophila cardiomyocytes. The procedure relies upon branched DNA technology, which allows several fluorescent molecules to label an individual transcript, drastically increasing the signal-to-noise ratio compared to other FISH assays. This protocol can be modified for use in various small animal models, tissue types, and for assorted nucleic acids.

Imaging neural activity in the ventral nerve cord of behaving adult Drosophila.

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because limb premotor and motor circuits have been largely inaccessible for large-scale recordings in intact, moving animals-a constraint that is true for both vertebrate and invertebrate models. Here, we introduce a method for 2-photon functional imaging from the ventral nerve cord (VNC) of behaving adult Drosophila melanogaster. We use this method to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a previously uncharacterized class of locomotion-associated A1 descending neurons. Finally, we develop a genetic reagent to destroy the indirect flight muscles and to facilitate experimental access to the VNC. Taken together, these approaches enable the direct investigation of circuits associated with complex limb movements.

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.

Distortion of the Actin A-Triad Results in Contractile Disinhibition and Cardiomyopathy.

Striated muscle contraction is regulated by the movement of tropomyosin over the thin filament surface, which blocks or exposes myosin binding sites on actin. Findings suggest that electrostatic contacts, particularly those between K326, K328, and R147 on actin and tropomyosin, establish an energetically favorable F-actin-tropomyosin configuration, with tropomyosin positioned in a location that impedes actomyosin associations and promotes relaxation. Here, we provide data that directly support a vital role for these actin residues, termed the A-triad, in tropomyosin positioning in intact functioning muscle. By examining the effects of an A295S α-cardiac actin hypertrophic cardiomyopathy-causing mutation, over a range of increasingly complex in silico, in vitro, and in vivo Drosophila muscle models, we propose that subtle A-triad-tropomyosin perturbation can destabilize thin filament regulation, which leads to hypercontractility and triggers disease. Our efforts increase understanding of basic thin filament biology and help unravel the mechanistic basis of a complex cardiac disorder.

Modest overexpression of FOXO maintains cardiac proteostasis and ameliorates age-associated functional decline.

Heart performance declines with age. Impaired protein quality control (PQC), due to reduced ubiquitin-proteasome system (UPS) activity, autophagic function, and/or chaperone-mediated protein refolding, contributes to cardiac deterioration. The transcription factor FOXO participates in regulating genes involved in PQC, senescence, and numerous other processes. Here, a comprehensive approach, involving molecular genetics, novel assays to probe insect cardiac physiology, and bioinformatics, was utilized to investigate the influence of heart-restricted manipulation of dFOXO expression in the rapidly aging Drosophila melanogaster model. Modest dFOXO overexpression was cardioprotective, ameliorating nonpathological functional decline with age. This was accompanied by increased expression of genes associated predominantly with the UPS, relative to other PQC components, which was validated by a significant decrease in ubiquitinated proteins. RNAi knockdown of UPS candidates accordingly compromised myocardial physiology in young flies. Conversely, excessive dFOXO overexpression or suppression proved detrimental to heart function and/or organismal development. This study highlights D. melanogaster as a model of cardiac aging and FOXO as a tightly regulated mediator of proteostasis and heart performance over time.

Cardiac-Restricted Expression of VCP/TER94 RNAi or Disease Alleles Perturbs Drosophila Heart Structure and Impairs Function.

Valosin-containing protein (VCP) is a highly conserved mechanoenzyme that helps maintain protein homeostasis in all cells and serves specialized functions in distinct cell types. In skeletal muscle, it is critical for myofibrillogenesis and atrophy. However, little is known about VCP's role(s) in the heart. Its functional diversity is determined by differential binding of distinct cofactors/adapters, which is likely disrupted during disease. VCP mutations cause multisystem proteinopathy (MSP), a pleiotropic degenerative disorder that involves inclusion body myopathy. MSP patients display progressive muscle weakness. They also exhibit cardiomyopathy and die from cardiac and respiratory failure, which are consistent with critical myocardial roles for the enzyme. Nonetheless, efficient models to interrogate VCP in cardiac muscle remain underdeveloped and poorly studied. Here, we investigated the significance of VCP and mutant VCP in the Drosophila heart. Cardiac-restricted RNAi-mediated knockdown of TER94, the Drosophila VCP homolog, severely perturbed myofibrillar organization and heart function in adult flies. Furthermore, expression of MSP disease-causing alleles engendered cardiomyopathy in adults and structural defects in embryonic hearts. Drosophila may therefore serve as a valuable model for examining role(s) of VCP in cardiogenesis and for identifying novel heart-specific VCP interactions, which when disrupted via mutation, contribute to or elicit cardiac pathology.

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.

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.

Pseudo-acetylation of K326 and K328 of actin disrupts Drosophila melanogaster indirect flight muscle structure and performance.

In striated muscle tropomyosin (Tm) extends along the length of F-actin-containing thin filaments. Its location governs access of myosin binding sites on actin and, hence, force production. Intermolecular electrostatic associations are believed to mediate critical interactions between the proteins. For example, actin residues K326, K328, and R147 were predicted to establish contacts with E181 of Tm. Moreover, K328 also potentially forms direct interactions with E286 of myosin when the motor is strongly bound. Recently, LC-MS/MS analysis of the cardiac acetyl-lysine proteome revealed K326 and K328 of actin were acetylated, a post-translational modification (PTM) that masks the residues' inherent positive charges. Here, we tested the hypothesis that by removing the vital actin charges at residues 326 and 328, the PTM would perturb Tm positioning and/or strong myosin binding as manifested by altered skeletal muscle function and structure in the Drosophila melanogaster model system. Transgenic flies were created that permit tissue-specific expression of K326Q, K328Q, or K326Q/K328Q acetyl-mimetic actin and of wild-type actin via the UAS-GAL4 bipartite expression system. Compared to wild-type actin, muscle-restricted expression of mutant actin had a dose-dependent effect on flight ability. Moreover, excessive K328Q and K326Q/K328Q actin overexpression induced indirect flight muscle degeneration, a phenotype consistent with hypercontraction observed in other Drosophila myofibrillar mutants. Based on F-actin-Tm and F-actin-Tm-myosin models and on our physiological data, we conclude that acetylating K326 and K328 of actin alters electrostatic associations with Tm and/or myosin and thereby augments contractile properties. Our findings highlight the utility of Drosophila as a model that permits efficient targeted design and assessment of molecular and tissue-specific responses to muscle protein modifications, in vivo.