Mosquitoes are vectors for some of the most devastating diseases on the planet. Given the centrality of acoustic sensing in the precopulatory behavior of these vectors, the use of an exogenous acoustic stimulus offers the potential of interfering with the courtship behavior of these insects. Previous research on the acoustotactic response of mosquitoes has been conducted on tethered preparations using low-intensity sound stimuli. To quantify differences in acoustotactic responses between mosquitos of distinct sex and species, we examined the effects of incidental sound stimuli on the flight behavior of free-flying male vs. female Aedes aegypti and Anopheles gambiae mosquitoes. The key variables were sound frequency (100–1000 Hz) and intensity (67–103 dB, measured at 12.5 cm from the source), and the acoustotactic response was measured in terms of the relative increase in flight speed in response to the stimulus. The data show, for the first time, significant sex- and species-specific differences in acoustotactic responses. A. aegypti exhibited a greater response to sound stimulus compared to An. gambiae, and the response also extended over a larger range of frequencies. Furthermore, the males of both species displayed a greater acoustotactic response than females, with An. gambiae females exhibiting minimal response to sound.

Myopathies are among the major causes of mortality in the world. There is no complete cure for this heterogeneous group of diseases, but a sensitive, specific, and fast diagnostic tool may improve therapy effectiveness. In this study, Raman spectroscopy is applied to discriminate between muscle mutants in Drosophila on the basis of associated changes at the molecular level. Raman spectra were collected from indirect flight muscles of mutants, upheld(1) (up(1)), heldup(2) (hdp(2)), myosin heavy chain(7) (Mhc(7)), actin88F(KM88) (Act88F(KM88)), upheld(101) (up(101)), and Canton-S (CS) control group, for both 2 and 12 days old flies. Difference spectra (mutant minus control) of all the mutants showed an increase in nucleic acid and β-sheet and/or random coil protein content along with a decrease in α-helix protein. Interestingly, the 12th day samples of up(1) and Act88F(KM88) showed significantly higher levels of glycogen and carotenoids than CS. A principal components based linear discriminant analysis classification model was developed based on multidimensional Raman spectra, which classified the mutants according to their pathophysiology and yielded an overall accuracy of 97% and 93% for 2 and 12 days old flies, respectively. The up(1) and Act88F(KM88) (nemaline-myopathy) mutants form a group that is clearly separated in a linear discriminant plane from up(101) and hdp(2) (cardiomyopathy) mutants. Notably, Raman spectra from a human sample with nemaline-myopathy formed a cluster with the corresponding Drosophila mutant (up(1)). In conclusion, this is the first demonstration in which myopathies, despite their heterogeneity, were screened on the basis of biochemical differences using Raman spectroscopy.

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformationdependent 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.

MicroRNAs (miRNAs) are small noncoding endogenous RNAs, typically 21–23 nucleotides long, that regulate gene expression, usually post-transcriptionally, by binding to the 3′-UTR of target mRNA, thus blocking translation. The expression of several miRNAs is significantly altered during cardiac hypertrophy, myocardial ischemia, fibrosis, heart failure, and other cardiac myopathies. Recent studies have implicated miRNA-9 (miR-9) in myocardial hypertrophy. However, a detailed mechanism remains obscure. In this study, we have addressed the roles of miR-9 in muscle development and function using a genetically tractable model system, the indirect flight muscles (IFMs) of Drosophila melanogaster. Bioinformatics analysis identified 135 potential miR-9a targets, of which 27 genes were associated with Drosophila muscle development. Troponin-T (TnT) was identified as major structural gene target of miR-9a. We show that flies overexpressing miR-9a in the IFMs have abnormal wing position and are flightless. These flies also exhibit a loss of muscle integrity and sarcomeric organization causing an abnormal muscle condition known as “hypercontraction.” Additionally, miR-9a overexpression resulted in the reduction of TnT protein levels while transcript levels were unaffected. Furthermore, muscle abnormalities associated with miR-9a overexpression were completely rescued by overexpression of TnT transgenes which lacked the miR-9a binding site. These findings indicate that miR-9a interacts with the 3′-UTR of the TnT mRNA and downregulates the TnT protein levels by translational repression. The reduction in TnT levels leads to a cooperative downregulation of other thin filament structural proteins. Our findings have implications for understanding the cellular pathophysiology of cardiomyopathies associated with miR-9 overexpression.

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.

Recent proteomics studies of vertebrate striated muscle have identified lysine acetylation at several sites on actin. Acetylation is a reversible post-translational modification (PTM) that neutralizes lysine’s positive charge. Actin’s positively charged residues, particularly K326 and K328, are predicted to form several, critical electrostatic interactions with tropomyosin (Tpm) that promote its binding and bias Tpm to an azimuthal location where it impedes myosin attachment. The troponin (Tn) complex also influences Tpm’s position along filamentous (F-) actin as a function of Ca2+ to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, K326 and K328 on sarcomeric actin are among the documented acetylated residues. Using an acetic anhydride-based labeling approach, we showed that excessive, non-specific 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 K326 and K328, also resulted in less inhibited F-actin-Tpm, implying that modifying these residues only, 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 Ac-mimetic actin on Ca2+-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca2+ sensitivity of activation relative to control. Together our findings suggest that actin acetylation, particularly K328, modulates muscle contraction via disrupting inhibitory Tpm positioning.

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