Actin forms many cytostructural elements, including the backbone of striated muscle thin filaments. Our mechanistic understanding of most actin-dependent processes remains limited, partly due to difficulty with recombinant actin expression. While fully-functional actin cannot be produced in bacteria, it can be generated using baculovirus expression systems. Here, we created an additional tool for recombinant actin production, for hypothesis testing and for resolving the molecular properties and cellular effects of unique variants. Specifically, we engineered Drosophila that can express untagged actins exclusively in indirect flight muscles (IFMs). Act88F encodes sarcomeric IFM actin. Attempts to rescue IFM function (i.e. flight) in Act88F-nulls, by inserting transgenic Act88F (Act88FTG-WT) ectopically throughout the genome have failed, potentially due to ineffective cis/trans regulation of Act88FTG-WT expression. To circumvent these problems, we used CRISPR-editing and introduced an attP landing site directly into Act88F, to disrupt the endogenous gene and serve as a target for site-specific transgene insertion. attP-mediated disruption of Act88F yielded flightlessness and actin-free IFMs. Transformants expressing GFP or Act88FTG-WT, from the endogenous Act88F integration locus, demonstrated robust IFM-specific protein production and restored flight, respectively. Having established the expression system, we designed Drosophila mutants to test the roles of specific actin residues in tropomyosin/myosin-binding and nemaline myopathy pathogenesis. Both Act88FTG-R147Q and Act88F TG-F352Striggered IFM hypercontraction and impaired flight. Finally, we determined whether mammalian actin could be generated and observed high-level expression of human α-cardiac actin (ACTC1), which behaved indistinguishably from bovine ACTC1 in unregulated and regulated motility (IVM) assays. We are currently comparing IVM properties of IFM-purified vs. baculovirus/Sf12-purified ACTC1. Overall, our novel fly line permits transgenic actin expression in mature muscle fibers that are themselves amenable to structural and functional analyses, and serves as a cost-effective, perpetual source of ample protein for biophysical experimentation.

OBJECTIVE:Cardiac troponin I (cTnI) is an essential physiological and pathological regulator of cardiac relaxation. Significant to this regulation, the post-translational modification of cTnI through phosphorylation functions as a key mechanism to accelerate myofibril relaxation. Similar to phosphorylation, post-translational modification by acetylation alters amino acid charge and protein function. Recent studies have demonstrated that the acetylation of cardiac myofibril proteins accelerates relaxation and that cTnI is acetylated in the heart. These findings highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to changes in myofilament, myofibril, and/or cellular mechanics. The objective of this study was to determine the effects of mimicking acetylation at a single site of cTnI (lysine-132; K132) on myofilament, myofibril, and cellular mechanics and elucidate its influence on molecular function. METHODS:To determine if pseudo-acetylation of cTnI at 132 modulates thin filament regulation of the acto-myosin interaction, we reconstituted thin filaments containing WT or K132Q (to mimic acetylation) cTnI and assessed in vitro motility. To test if mimicking acetylation at K132 alters cellular relaxation, adult rat ventricular cardiomyocytes were infected with adenoviral constructs expressing either cTnI K132Q or K132 replaced with arginine (K132R; to prevent acetylation) and cell shortening and isolated myofibril mechanics were measured. Finally, to confirm that changes in cell shortening and myofibril mechanics were directly due to pseudo-acetylation of cTnI at K132, we exchanged troponin containing WT or K132Q cTnI into isolated myofibrils and measured myofibril mechanical properties. RESULTS:Reconstituted thin filaments containing K132Q cTnI exhibited decreased calcium sensitivity compared to thin filaments reconstituted with WT cTnI. Cardiomyocytes expressing K132Q cTnI had faster relengthening and myofibrils isolated from these cells had faster relaxation along with decreased calcium sensitivity compared to cardiomyocytes expressing WT or K132R cTnI. Myofibrils exchanged with K132Q cTnI ex vivo demonstrated faster relaxation and decreased calcium sensitivity. CONCLUSIONS:Our results indicate for the first time that mimicking acetylation of a specific cTnI lysine accelerates myofilament, myofibril, and myocyte relaxation. This work underscores the importance of understanding how acetylation of specific sarcomeric proteins affects cardiac homeostasis and disease and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.

Striated muscle contraction is regulated by Ca2+-dependent modulation of myosin cross-bridge binding to F-actin by the thin filament troponin (Tn)-tropomyosin (Tm) complex. In the absence of Ca2+, Tn binds to actin and constrains Tm to an azimuthal location where it sterically occludes myosin binding sites along the thin filament surface. This limits force production and promotes muscle relaxation. In addition to Tn-actin interactions, inhibitory Tm positioning requires associations between other thin filament constituents. For example, the actin 'A-triad', composed of residues K326, K328 and R147, forms numerous, highly favourable electrostatic contacts with Tm that are critical for establishing its inhibitory azimuthal binding position. Here, we review recent findings, including the identification and interrogation of modifications within and proximal to the A-triad that are associated with disease and/or altered muscle behaviour, which highlight the surface feature's role in F-actin-Tm interactions and contractile regulation.

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.

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.

TnT1, the N-terminal fragment of troponin-T (TnT), contributes directly to contractile inhibition, independent of other troponin subunits. Impaired relaxation and diastolic dysfunction are hallmarks of hypertrophic (HCM) and restrictive (RCM) cardiomyopathies, while dilated cardiomyopathy (DCM) is characterized by reduced systolic force. We tested the hypothesis that disease-causing mutations within TnT1's highly-conserved tropomyosin-binding element affect its role in confining tropomyosin to an inhibitory position along F-actin and, thereby, alter the regulation of actomyosin force generation. In vitro, in situ, and in vivo experiments were conducted to measure the effects of human HCM/RCM TnT1 peptides on myosin-driven F-actin-tropomyosin sliding, and TnT variants on Drosophila cardiac performance. Motility results confirmed that TnT1 enhances tropomyosin's native inhibitory properties. However, compared to control, the HCM/RCM peptides were significantly less effective at preventing force-generating myosin interactions with actin-tropomyosin-TnT1 filaments. Likewise, fly hearts over-expressing mutant TnTs displayed impaired relaxation and restrictive physiology that were predominantly due to excessive, unimpeded myosin cycling even under ultra-low intracellular Ca2+ conditions. Mutant myofibrils displayed elevated resting tension, also because of tropomyosin mispositioning and strong actomyosin binding. Conversely, DCM-causing TnT variants prompted cardiac dilation in flies, consistent with depressed force production resulting from reinforced inhibitory tropomyosin positioning. We are currently analyzing the effects of particular mutations in rat ventricular cardiomyocytes and myofibrils. Electrostatic contacts mediate F-actin-tropomyosin binding and confine tropomyosin to a default location where it impedes actomyosin-dependent force generation. We propose that TnT1's association with tropomyosin optimizes the formation of F-actin-tropomyosin contacts, and thereby promotes thin filament inhibition. Thus, the mutations may disrupt TnT's normal contribution to myocardial relaxation by weakening (HCM/RCM) or strengthening (DCM) TnT1-Tpm binding and, consequently, destabilizing or promoting tropomyosin's inhibitory positioning respectively, potentially serving as the most proximal cause of pathology.

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification that neutralizes lysine's positive charge. Positively charged residues on actin, particularly Lys326 and Lys328, are predicted to form critical electrostatic interactions with tropomyosin (Tpm) that promote its binding to filamentous (F)-actin and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm's position along F-actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys326 and Lys328 are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, nonspecific actin acetylation did not disrupt characteristic F-actin-Tpm binding. However, it significantly reduced Tpm-mediated inhibition of myosin attachment, as reflected by increased F-actin-Tpm motility that persisted in the presence of Tn and submaximal Ca2+ Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys326 and Lys328, also resulted in less inhibited F-actin-Tpm, implying that modifying only these residues influences Tpm's location and, potentially, thin filament regulation. To unequivocally determine the residue-specific consequences of acetylation on Tn-Tpm-based regulation of actomyosin activity, we assessed the effects of K326Q and K328Q acetyl (Ac)-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

Lysine deacetylases (KDAC's) catalyze the removal of acetyl groups from lysine side chains, while lysine acetylases (KAT's) perform the reverse reaction. KDAC inhibition was shown to increase lysine acetylation of muscle proteins, and was associated with enhanced Ca2+-sensitivity in skinned fibers and increased relaxation rate in isolated myofibrils. Interestingly, the KAT enzyme, PCAF, directly acetylates myosin in vitro, and actin has been implicated as a potential target of PCAF. Therefore, we attempted to acetylate actin with PCAF, in vitro, to determine its potential effect(s) on muscle function. PCAF underwent autoacetylation and bound to actin, yet failed to acetylate it. Chemical treatment of actin with acetic anhydride, however, increased lysine acetylation roughly 200-fold. Surprisingly, in vitro motility (IVM) of (100 μg/ml myosin concentration) and tropomyosin affinity for acetylated F-actin were indistinguishable from unacetylated control. However, IVM of acetylated F-actin, performed at low myosin concentration (12.5 μg/ml) and in the presence of tropomyosin, revealed a significant 63% increase in motile filaments relative to unacetylated control (p < 0.0001), suggesting that actin acetylation reduces tropomyosin-mediated inhibition of myosin binding. Since K326 and K328 have been reported as the most reactive lysines on actin, are acetylated in vivo, and pseudo-acetylation of both modulates muscle contraction, we assessed the effect of K326Q and K328Q pseudo-acetylated actin on Ca2+-mediated thin filament activation via regulated IVM. Although reconstituted thin filaments containing K326Q (pCa50 = 6.36 ± 0.03) did not alter Ca2+-sensitivity relative to WT (pCa50 = 6.37 ± 0.03), K328Q induced a significant leftward shift in Ca2+-sensitivity (ΔpCa50 = 0.19; p < 0.0001), consistent with a loss in tropomyosin-mediated inhibition. The data indicate that actin K328 acetylation modulates contractile function, which makes it a potential mechanism for regulating muscle performance in vivo.

Myosin storage myopathy (MSM) is a predominantly autosomal dominant, congenital skeletal muscle disorder caused by missense mutations in the slow muscle/β-cardiac myosin heavy chain (MHC) rod. MSM is characterized by subsarcolemmal accumulations of β-MHC that have a hyaline appearance. Cases of MSM with cardiomyopathy have been reported. The mutations disrupt hydropathy or charge of residues in the heptad repeat, altering interactions that stabilize the rod's coiled-coil dimers and thick filaments.We have previously demonstrated in vitro that purified mutant MHC showed a reduced ability to form myosin filaments and that L1793P and E1883K filaments were less stable in limited proteolysis experiments. We have now generated heterozygous Drosophila MSM models to perform physiological, structural, and biophysical analysis of the effects of L1793P, R1845W, and E1883K mutations on diverse muscles. Flight and jump ability were highly compromised in mutant heterozygotes. Confocal and electron microscopy revealedsevere disruption of indirect flight musclemorphology and myofibrillar disarray/degeneration with hyaline-like inclusions in pupal muscles. We are currently performing super-resolution confocal microscopy to identify possible in vivo thick filament length discrepancies. Further, cryo-electron microscopy three-dimensional image reconstruction of native thick filaments will explore potential ultrastructural aberrations in filament backbones and molecular packing. Semi-automated heartbeat analysis of mutant heterozygotes revealed restrictive cardiac physiology and diastolic dysfunction, which worsened with age. Lifespans of MSM mutants were reduced relative to control. Thus, our heterozygous Drosophila models phenocopy afflicted patients’ skeletal and cardiac muscle structure and function. Going forward, our novel fly models will be used to screen for in vivo modifiers of mutant muscle performance.

Gain-of-function mutations in sarcomeric genes often manifest as hypercontractility-associated myopathies. Two such diseases are Hypertrophic (HCM) and Restrictive cardiomyopathy (RCM). These are clinically heterogeneous, autosomal dominant disorders characterized by hyperdynamic myocardial contraction and impaired relaxation, with (HCM) or without (RCM) hypertrophy. The current study focuses on charge-altering HCM/RCM-causing troponin-T (TNNT2) missense mutations, K124N, R130C, E136K, in the highly conserved N-terminal (TnT1) tropomyosin (Tm)-binding element. We have conducted in vitro, in situ and in vivo experiments using human TnT1 peptides in a minimalistic four-element motility assay, physiological assessment of surgically exposed transgenic Drosophila hearts by SOHA (Semi-automated Optical Heartbeat Analysis) and live fluorescence imaging of intact fly cardiac tubes, respectively. This is the first report of an in vivo/in situ study on these mutations. We tested the hypothesis that these mutations limit TnT1's ability to help confine Tm to the “B-state”, where it sterically blocks acto-myosin binding during diastole. We observed increased F-actin-Tm-TnT1 sliding velocities with all three mutant peptides in the in vitro motility assay, implying disinhibited acto-myosin interactions. Transgenic Drosophila hearts over-expressing mutant TnT's showed cardiac restriction, impaired relaxation and reduced cardiac output. To elucidate underlying mechanisms, we treated the heart tubes sequentially with EGTA-AM (to chelate intracellular Ca2+) and blebbistatin (to inhibit myosin), and measured live-cell responses consistent with impaired Ca2+ homeostasis, as well as Ca2+-independent cross-bridge cycling at rest. Additionally, we isolated single myofibrils from a novel CRISPR-Cas9 fly line, engineered to express the E136K RCM mutation, and observed elevated resting tension relative to controls. Our results confirm our hypothesis that these cardiomyopathy mutations disrupt TnT's normal contribution to myocardial relaxation, which likely serves as the mechanistic basis of pathology in our unique fly models and, potentially, in human patients.

Mutations in sarcomeric proteins are heritable causes of dilated cardiomyopathy (DCM). α-Tropomyosin (TPM1) mutations have previously been reported in both hypertrophic cardiomyopathy and DCM. We report phenotypic features and in vitro functional assays of a TPM1 sequence variant in four-generation family of Mediterranean ancestry associated with a novel TPM1 variant, D55N. We assessed the functional role of TPM1 D55N variant in association with DCM. Methods: Clinical evaluation and family history was obtained on the proband and family members. After genetic counseling, testing was performed in clinical laboratories and pedigree analysis was conducted (Progeny software). In vitro functional assessment the mutation measured the actin binding capacity of bacterially expressed Tm WT vs Tm D55N using actin co-sedimentation assays. Results:The age of diagnosis of clinical DCM in the family ranged from the first to the seventh decade of life, including two individuals who underwent heart transplant in the first and second decades. The D55N sequence variant was identified in 7 living individuals. The pattern of inheritance of DCM in this family was autosomal dominant with a 71% penetrance of disease. Actin binding assay shows that the binding ability of α-Tropomyosin to filamentous actin (F-actin) filaments increased significantly, an average of 64.5%±5.4 s.e.m at any given concentration (0.25 μM to 2 μM) (n=3 ANOVA p<0.05) in the presence of the mutation. Further functional assessment of the mutants will be undertaken. Conclusions: Genetic sequencing and initial functional analyses strongly suggest a pathogenic role for TPM1 D55N mutation in DCM which increases the af?nity of a-Tm binding to F-actin.

Supranormal contractile properties are frequently associated with cardiac diseases. Anesthetic agents, including propofol, can depress myocardial contraction. We tested the hypothesis that fropofol, a propofol derivative, reduces force development in cardiac muscles via inhibition of cross-bridge cycling and may therefore have therapeutic potential. Force and intracellular Ca2+ concentration ([Ca2+]i) transients of rat trabecular muscles were determined. Myofilament ATPase, actin-activated myosin ATPase, and velocity of actin filaments propelled by myosin were also measured. Fropofol dose dependently decreased force without altering [Ca2+]i in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca2+-activated force ( Fmax) and increased the [Ca2+]i required for 50% of Fmax (Ca50) at steady state without affecting the Hill coefficient in both intact and skinned cardiac fibers. The drug also depressed cardiac myofibrillar and actin-activated myosin ATPase activity. In vitro actin sliding velocity was significantly reduced when fropofol was introduced during rigor binding of cross-bridges. The data suggest that the depressing effects of fropofol on cardiac contractility are likely to be related to direct targeting of actomyosin interactions. From a clinical standpoint, these findings are particularly significant, given that fropofol is a nonanesthetic small molecule that decreases myocardial contractility specifically and thus may be useful in the treatment of hypercontractile cardiac disorders.-Ren, X., Schmidt, W., Huang, Y., Lu, H., Liu, W., Bu, W., Eckenhoff, R., Cammarato, A., Gao, W. D. Fropofol decreases force development in cardiac muscle.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle characterized by ventricular wall thickening, hyperdynamic contractile properties, and impaired relaxation. It is commonly caused by mutations in sarcomeric proteins. The alpha-cardiac actin ACTC M305L HCM mutation is located near the nucleotide-binding site and residues predicted to help confine tropomyosin to an inhibitory position along thin filaments. We generated several Drosophila models of the disorder to evaluate the effects of the lesion from the organ through molecular level. In all muscles tested, phenotypic disturbances correlated with mutant protein load. For example, relative to wildtype actin, when highly overexpressed in the fly heart, the variant significantly reduced cardiac output, prolonged systole, and restricted diastolic volumes. Preliminary force measurements from jump muscle fibers corroborated a dose-dependent, M305L actin-induced pathological effect. Moreover, we observed greater incremental decreases in flight ability with increasing amounts of mutant versus wildtype actin expression in indirect flight muscles (IFM). Flight was abolished when the mutant was highly overexpressed and destructive myosin-induced hypercontraction was evident, suggesting disrupted contractile regulation and poorly inhibited actomyosin associations. However, regulated in vitro motility parameters, including Vmax, nH, and pCa50, of thin filaments reconstituted from M305L or wildtype IFM actin and bovine cardiac troponin-tropomyosin, were indistinguishable. Likewise, no overt differences in computationally-derived electrostatic interaction energy landscapes between M305L and wildtype actin-tropomyosin filaments were observed, suggesting unperturbed tropomyosin-positioning in the absence of troponin. Nonetheless, over sparsely populated beds of myosin, a significantly higher percentage of troponin-free M305L versus wildtype actin-tropomyosin filaments were motile, implying tropomyosin is less effective at blocking actomyosin associations on mutant filaments. Mispositioning of tropomyosin may contribute to the supranormal contraction and compromised relaxation observed in our in vivo models and, potentially, in human patients.

Restrictive (RCM) and hypertrophic (HCM) cardiomyopathies are heterogeneous diseases of the heart, characterized by impaired relaxation and diastolic dysfunction, without/with ventricular wall thickening. The pathogenesis can be attributed to hyperdynamic contractile function of mutant sarcomeres, which is considered the most proximal effect of disease-causing mutations. ∼100 causative thin filament mutations have been identified, of which 15% occur in cardiac troponin-T (cTnT/TNNT2), the tropomyosin-binding subunit of the regulatory troponin complex. Of these, 70% are clustered in the N-terminal cTnT1 tail, which contains a highly conserved tropomyosin binding element spanning residues 112-136. At low Ca2+ concentrations, troponin constrains tropomyosin to the blocked/B-state, occluding acto-myosin interactions. cTnT1 contributes to B-state formation and, thus, contractile inhibition. Here, we investigated three charge-altering mutations in invariant cTnT1 residues: E136K (RCM-causing), K124N, R130C (HCM-causing). We hypothesize that these mutations weaken cTnT1's inherent inhibitory properties via altered cTnT1-tropomyosin binding, leading to destabilization of the blocked/B-state and excessive acto-myosin cycling. Human cTnT1 was cloned, mutagenized, expressed, purified, and reconstituted with vertebrate F-actin-tropomyosin to determine the effects of the mutations on in vitro motility. We verified the inhibitory effect of WT-TnT1 on F-actin-tropomyosin sliding speed. All 3 mutant-cTnT1-actin-tropomyosin filaments showed significantly higher sliding speeds versus control: E136K (1.15±0.22 vs. 1.00±0.24μm/s), K124N (1.21±0.21 vs. 1.00±0.18μm/s) and R130C (1.63±0.51 vs. 1.00±0.18μm/s). We also examined sliding speeds as a function of myosin concentration, and observed differences between WT and mutant filaments to be amplified at 50-75μg/ml. Additionally, there was a 6-12% increase in the number of moving versus non-moving mutant filaments. These data suggest a destabilized cTnT1-mediated B-state, which could contribute to the cardiac hypercontractility and, importantly, impaired relaxation observed in patients. We are currently investigating the effects of these mutations on cTnT1-tropomyosin binding affinities.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease of the heart. HCM mutations in sarcomeric genes often engender hypercontractile myocardium that displays increased Ca2+-sensitivity of force, enhanced cross-bridge cycling, and incomplete relaxation, which precede abnormal wall thickening. The alpha-cardiac actin (ACTC) Ala295Ser substitution causes a highly penetrant disease with diverse phenotypes. To help resolve the mechanistic basis of the disorder, we generated several Drosophila models that limit genetic diversity and pathological complexity. We created lines with inducible wildtype UAS-Act57BWTand mutant UAS-Act57BA295Scardiac actin transgenes to examine the effects of A295S actin on the fly heart. High-speed video microscopy and motion analysis of beating hearts revealed A295S actin expression significantly reduced diastolic volumes and prolonged systolic intervals, consistent with elevated contractile properties at rest and during contraction. In situ treatment of Act57BA295S hearts with blebbistatin, under low intracellular Ca2+ conditions, caused significantly larger increases in myocyte length vs. Act57BWTcontrols. These data suggest A295S actin induces excessive thin filament disinhibition, myosin binding, and resting tension. We also expressed indirect flight muscle (IFM)-targeted Act88FWT or Act88FA295S transgenes in an IFM actin-null background. Confocal microscopy resolved hypercontracted Act88FA295S fibers. Reconstituted Act88FA295S thin filaments, using bovine cardiac troponin-tropomyosin, displayed significantly enhanced Ca2+-sensitivity of activation vs. control filaments in regulated in vitro motility experiments. Finally, in silico modelling revealed the A295S mutation influences the orientation of nearby actin residues that mediate critical electrostatic interactions with tropomyosin. Such mutation-induced propagated effects likely lower the energy required to relieve tropomyosin-mediated inhibition and promote force production. Overall, enhanced contractile activity from the molecular through the cellular level is consistent with the early hyperdynamic contractile properties frequently associated with HCM and could trigger ACTC A295S-associated pathology. This work was supported by R37HL36153 and 1R01HL124091.

K326 and K328 of actin establish critical electrostatic interactions with negatively charged amino acids of tropomyosin. Recently, these residues were shown to be acetylated in guinea pig hearts, a post-translational modification (PTM) that could weaken actin-tropomyosin contacts, and possibly modulate contraction. Expression of K326Q, K328Q, and K326Q/K328Q acetyl-mimetic cardiac actin, in indirect flight muscles of Drosophila, resulted in severe hypercontraction when modest amounts (∼20%) of K328Q actin were present. Furthermore, beating fly hearts expressing similar levels of K328Q or K326Q/K328Q actin displayed significantly prolonged systolic intervals (204±0.01 and 211±0.01 msec respectively) relative to controls (161±0.01 msec, p<0.001). Despite nearly equivalent baseline shortening velocities for WT- and K328Q-expressing hearts (450±24.2 vs. 449±23.9 µm/s respectively), exposure to a viscous afterload resulted in an approximate 45% reduction in velocity for control myocardium compared to only 30% for K328Q-containing hearts (p<0.05). Thin filaments were reconstituted using vertebrate cardiac troponin-tropomyosin and K328Q, K326Q or WT actin. The pCa50, calculated from in vitro sliding velocities, was significantly leftward-shifted for K328Q relative to WT thin filaments (6.25±0.03 vs. 6.06±0.02 µm/sec, p<0.001), while that for K326Q filaments was indistinguishable. These results are consistent with acetylation-induced increases in crossbridge cycling, power production and sensitivity of force development. Lastly, to determine if the PTM may be a regulatory mechanism exploited in vivo, we measured the relative amounts of acetylated actin in guinea pig and human ventricles during cardiac failure. We discovered ∼75% more acetylated actin in guinea pig hearts subjected to aortic constriction (p<0.001), and ∼21% more in human dilated cardiomyopathy samples (p<0.05), relative to controls. In sum, acetylation of actin K326 and/or K328 could be a novel means to enhance performance in failing hearts. This work was supported by 5T32HL007227-38 and 1R01HL124091.

Nemaline myopathy is a congenital disorder characterized by muscle weakness. Dominant-negative disease-causing mutations have been identified in various genes that encode sarcomeric thin filament proteins. of these, mutations in skeletal muscle α-actin (ACTA1) account for ∼25% of all reported cases. To investigate the myopathic phenotypes from the level of whole muscle down through the level of single molecules, we generated multiple transgenic Drosophila fly lines expressing three different mutations - V37L, D288G and F352S - in Act88F, the indirect flight muscle (IFM) actin gene. A gradation in flight behavior was observed (V37L>D288G>F352S), which correlated with the observed differences in muscle fiber morphology. While most of these mutations depress muscle function in patients, the F352S lesion elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function. Confocal and electron microscopy revealed breaks in mutant F352S IFM fibers and extensive myofibrillar and myofilamentous disarray. Z-lines showed streaming and formed "zebra bodies". The microscopic alterations are consistent with excessive acto-myosin interactions, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of vertebrate tropomyosin and troponin. However, relative to control, F352S heterozygous filaments showed a significant (p<0.01) increase in calcium sensitivity (pCa50 = 5.99±0.03 vs. 6.11±0.03), consistent with a mutation-induced gain in molecular function. Going forward, our novel Drosophila models will be used to screen for in vivo modifiers of mutant muscle performance. Supported by NIH R01HL124091 to AC.

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.

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.

Nemaline myopathy is a congenital human muscle disorder characterized by masticatory, limb and respiratory weakness. Dominant negative disease-causing mutations have been identified in ten genes that notably encode sarcomeric thin filament proteins including α actin (ACTA1). While most of these mutations cause a loss of muscle function, the F352S actin mutation elevates myosin cross-bridge strain and steady-state isometric force production and, thereby, apparently increases contractile function in humans. To investigate the F352S-induced myopathic cascade from the level of single molecules through the level of whole muscle we generated transgenic Drosophila that express the mutation in Act88F, the indirect flight muscle (IFM) actin gene. Mutant heterozygotes were flightless. Fluorescent and electron microscopy revealed breaks in mutant IFM fibers and extensive myofibrillar disarray. For example, the highly ordered double hexagonal thin/thick filament lattice, at the periphery of myofibrils, was distorted with myofilaments occasionally losing longitudinal orientation. Z-lines showed streaming and formed “zebra bodies”. The microscopic alterations are consistent with inappropriate acto-myosin interaction, unevenly distributed force, and destructive hypercontraction. Interestingly, in vitro sliding velocities of purified IFM F-actin from wild-type (3.17±0.12 μm/s) vs. F352S mutant heterozygous (2.88±0.09 μm/s, mean±SEM) flies did not significantly differ. Similarly, no differences in maximum calcium-activated velocity (3.88±0.11 vs. 3.77±0.10 μm/s) or in cooperativity (nH = 2.13±0.27 vs. 2.62±0.46) were observed for F-actin in the presence of tropomyosin and troponin. However, F352S heterozygous filaments showed a significant (p < 0.01) increase in calcium sensitivity relative to control (pCa50 = 6.11 +/- 0.03 vs. 5.99 +/- 0.03), consistent with mutation-induced gain in molecular function. Going forward, our novel Drosophila model will be used to screen for in vivo modifiers of mutant muscle performance. Supported by NIH 2PO1 AR052354 (P.D. Allen PI; CFA Core D).