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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 Ca(2+)/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.

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 Ca(2+) 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., The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation is located near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here the authors assessed M305L actin in vivo, in vitro, and in silico to characterize emergent pathological properties and define the mechanistic basis of disease.

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.

Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been due to a paucity of methods for objective and quantitative analysis. Here we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority as compared to conventional histological assessments. Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length (SL) of 1.9 μm, the angle σ was 0.23±0.01 rad, decreased to 0.19±0.01 rad at a SL of 2.1 μm, and further decreased to 0.15±0.01 rad at a SL of 2.3 μm (pStatement of SignificanceWe introduce a precise and quantitative approach to directly measure myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions as a novel tool for phenotypically characterizing striated muscle systems. We use this technique to demonstrate that myocardium from disease model organisms and failing human myocardium suffers from greater myofibrillar disorientation compared to healthy controls. We also demonstrate that excellent diffraction patterns can be obtained from frozen and thawed human myocardium. Given the ready availability of frozen human heart tissue in tissue banks, this capability opens up a large space of potential experiments relating sarcomere structure to dysfunction in cardiac disorders.

Rationale: Myocyte disarray is a hallmark of cardiomyopathy. However, the orientation of individual myofibrils and myofilaments and how their alignment may be altered in disease progression have been largely underexplored. This oversight has been predominantly due to a paucity of methods for objective and quantitative analysis. Objective: To introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near physiological conditions and demonstrate its superiority versus traditional histological assessments. Methods and Results: Using small-angle X-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in skinned, relaxed, wildtype mouse myocardium by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length (SL) of 1.9 μm, the angle σ was 0.23±0.01 rad, decreased to 0.19±0.01 rad at a SL of 2.1 μm, and further decreased to 0.15±0.01 rad at a SL of 2.3 μm (p Conclusions: Our method for assessing myofibrillar orientation limits the artifacts introduced by traditional histological processing and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent X-ray diffraction patterns from frozen, biopsied human myocardium opens up new avenues of inquiry regarding the relation of myofibrillar structure to function in health and disease.

Patients with left heart failure and reduced ejection fraction (HFrEF) have variable RV failure that, if present, drastically worsens outcomes. In a cohort of 21 HFrEF patients from two hospital sites, we have previously shown (Aslam et al, Eur J HF; 2020: volume 23, pages 339-341) that like global function, RV myocyte maximum calcium-activated myocyte tension (T max ) is quite variable (COV 27%). To determine if a relationship between RV myocyte function and indices of RV chamber function exists, we trained a random forest classifier based on 41 clinical variables, including hemodynamic, laboratory, and echocardiographic data, and queried the importance of each. This revealed that the most predictive model for reduced T max was based on the pulmonary artery pulsatility index (PAPi), an established clinical index of RV failure. To gain insight into potential mechanisms for depressed T max in HFrEF patients with a low PAPi, we obtained small angle x-ray diffraction patterns in 5 HFrEF patients with depressed PAPi and T max and compared this to 5 non-failing (NF) controls. The equatorial intensity ratio I(1,1)/I(1,0) was reduced in low T max RV muscle fibers vs. controls (0.250.06 vs. 0.180.02, Pmax to test if this association applies. These findings focus attention on thick filament structural and configuration abnormalities as potential culprits underlying RV disease in HFrEF. Further studies using novel sarcomere enhancers will test if these changes can be remedied, and if so, in which patients.

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 β-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.

Aging is associated with a decline in heart function across the tissue, cellular, and molecular levels. The risk of cardiovascular disease grows significantly over time, and as developed countries continue to see an increase in lifespan, the cost of cardiovascular healthcare for the elderly will undoubtedly rise. The molecular basis for cardiac function deterioration with age is multifaceted and not entirely clear, and there is a limit to what investigations can be performed on human subjects or mammalian models. Drosophila melanogaster has emerged as a useful model organism for studying aging in a short timeframe, benefitting from a suite of molecular and genetic tools and displaying highly conserved traits of cardiac senescence. Here, we discuss recent advances in our understanding of cardiac aging and how the fruit fly has aided in these developments.

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., Natural selection may favor traits underlying aging-related diseases if they benefit the young. Wang et al. find that oxidative activation of CaMKII provides physiological benefits critical to the initial and continued success of vertebrates but at the cost of disease, frailty, and shortened lifespan.

Tobacman and Cammarato identified locations of pathogenic and nonpathogenic troponin mutations in a thin-filament atomic model. Both mutation types had nonrandom distributions. 95% of pathogenic sites were in troponin regions that inhibit contraction via direct contacts with actin or tropomyosin., Reversible Ca2+ binding to troponin is the primary on-off switch of the contractile apparatus of striated muscles, including the heart. Dominant missense mutations in human cardiac troponin genes are among the causes of hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy. Structural understanding of troponin action has recently advanced considerably via electron microscopy and molecular dynamics studies of the thin filament. As a result, it is now possible to examine cardiomyopathy-inducing troponin mutations in thin-filament structural context, and from that to seek new insight into pathogenesis and into the troponin regulatory mechanism. We compiled from consortium reports a representative set of troponin mutation sites whose pathogenicity was determined using standardized clinical genetics criteria. Another set of sites, apparently tolerant of amino acid substitutions, was compiled from the gnomAD v2 database. Pathogenic substitutions occurred predominantly in the areas of troponin that contact actin or tropomyosin, including, but not limited to, two regions of newly proposed structure and long-known implication in cardiomyopathy: the C-terminal third of troponin I and a part of the troponin T N terminus. The pathogenic mutations were located in troponin regions that prevent contraction under low Ca2+ concentration conditions. These regions contribute to Ca2+-regulated steric hindrance of myosin by the combined effects of troponin and tropomyosin. Loss-of-function mutations within these parts of troponin result in loss of inhibition, consistent with the hypercontractile phenotype characteristic of HCM. Notably, pathogenic mutations are absent in our dataset from the Ca2+-binding, activation-producing troponin C (TnC) N-lobe, which controls contraction by a multi-faceted mechanism. Apparently benign mutations are also diminished in the TnC N-lobe, suggesting mutations are poorly tolerated in that critical domain.

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.

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 Lys(326) and Lys(328), 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 Ca(2+) to regulate exposure of myosin-binding sites and, thus, myosin cross-bridge recruitment and force production. Interestingly, Lys(326) and Lys(328) 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 Ca(2+). Furthermore, decreasing the extent of chemical acetylation, to presumptively target highly reactive Lys(326) and Lys(328), 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 Ca(2+)-dependent, in vitro motility parameters of reconstituted thin filaments (RTFs). Incorporation of K328Q actin significantly enhanced Ca(2+) sensitivity of RTF activation relative to control. Together, our findings suggest that actin acetylation, especially Lys(328), modulates muscle contraction via disrupting inhibitory Tpm positioning.

The identification of genetic variants that predispose individuals to cardiovascular disease and a better understanding of their targets would be highly advantageous. Genome-wide association studies have identified variants that associate with QT-interval length (a measure of myocardial repolarization). Three of the strongest associating variants (single-nucleotide polymorphisms) are located in the putative promotor region of CNOT1, a gene encoding the central CNOT1 subunit of CCR4-NOT: a multifunctional, conserved complex regulating gene expression and mRNA stability and turnover. We isolated the minimum fragment of the CNOT1 promoter containing all three variants from individuals homozygous for the QT risk alleles and demonstrated that the haplotype associating with longer QT interval caused reduced reporter expression in a cardiac cell line, suggesting that reduced CNOT1 expression might contribute to abnormal QT intervals. Systematic siRNA-mediated knockdown of CCR4-NOT components in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) revealed that silencing CNOT1 and other CCR4-NOT genes reduced their proliferative capacity. Silencing CNOT7 also shortened action potential duration. Furthermore, the cardiac-specific knockdown of Drosophila orthologs of CCR4-NOT genes in vivo (CNOT1/Not1 and CNOT7/8/Pop2) was either lethal or resulted in dilated cardiomyopathy, reduced contractility or a propensity for arrhythmia. Silencing CNOT2/Not2, CNOT4/Not4 and CNOT6/6L/twin also affected cardiac chamber size and contractility. Developmental studies suggested that CNOT1/Not1 and CNOT7/8/Pop2 are required during cardiac remodeling from larval to adult stages. To summarize, we have demonstrated how disease-associated genes identified by GWAS can be investigated by combining human cardiomyocyte cell-based and whole-organism in vivo heart models. Our results also suggest a potential link of CNOT1 and CNOT7/8 to QT alterations and further establish a crucial role of the CCR4-NOT complex in heart development and function. This article has an associated First Person interview with the first author of the paper., Summary: Genome-wide association studies combined with in vitro human cardiac cell assays and a model organism suitable for heart studies in vivo connect CNOT1, CNOT7 and overall the CCR4-NOT complex to human heart disease and morbidity.

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 storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the β-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.

Background Heart failure with preserved ejection fraction (HFpEF) constitutes half of all HF and lacks effective therapy, yet myocardial biological data from HFpEF patients is nearly non-existent. Methods We prospectively obtained right ventricular septal endomyocardial biopsies in HFpEF. Myocardial tissue from HFpEF (n=41), heart failure with reduced ejection fraction (HFrEF, n=30) and control (CON, n=24) were analyzed by RNA sequencing. Results HFpEF patients were older, more often African American (68%), with more diabetes (63%) and higher body mass index (median 41 kg/m2 [36-46]), compared to HFrEF. Agnostic principal component and hierarchical gene clustering analyses separated HFpEF, HFrEF and CON with minimal overlap (Figure 1A, 1B). HFpEF uniquely upregulated genes clustered in mitochondrial ATP synthesis/electron transport pathways that correlated with obesity; these pathways were downregulated in HFrEF. HFpEF uniquely down-regulated genes in endoplasmic reticulum stress, autophagy, and angiogenesis, uncorrelated with obesity. A subset of downregulated genes in HFpEF (upregulated in HFrEF) were functionally tested by knock-down in Drosophila; 65% altering fractional shortening and/or delaying relaxation. Three transcriptome-derived HFpEF subgroups were identified with distinct clinical correlates and clinical outcomes (Figure 1C, 1D): 1) mostly male with pulmonary hypertension, elevated cardiac biomarkers, altered stress/sarcomere genes, and worse event-free survival; 2) female with low cardiac biomarkers and inflammatory signaling; and 3) mixed sex with altered extracellular matrix genes. Obesity, hypertension, and hypertrophy were similar. Conclusions HFpEF has a distinct transcriptional profile from HFrEF, involving pathways both dependent and independent of obesity. These transcriptome signatures defined molecular-subgroups within HFpEF and highlight new signaling targets that may prove valuable for precision therapeutics.

Objective: Cardiac troponin I (cTnI) is an essential regulator of cardiac contractility and relaxation. Mutations within key regions of this regulator lead to cardiomyopathies. Further, post-translational modification of cTnI through phosphorylation impacts myofibril relaxation and calcium sensitivity. Recent studies have also demonstrated that myofibril proteins are acetylated leading to faster relaxation. These studies highlight the potential significance of myofilament acetylation; however, it is not known if site-specific acetylation of cTnI can lead to mechanical changes in the myofibril. The objective of this study was to determine if acetylation at a single site of cTnI (lysine-132; K132) is sufficient to alter myofibril protein mechanics. Methods: Adult rat ventricular cardiomyocytes infected with adenoviral constructs expressing either cTnI K132 replaced with glutamine (K132Q; to mimic acetylation) or K132 replaced with arginine (K132R; to prevent acetylation) were subjected to cell contractility and isolated myofibril mechanic measurements. Additionally, skinned myofibrils were exchanged with troponin containing wildtype (WT) or K132Q cTnI and mechanics assessed. Finally, dynamics of reconstituted thin filaments containing WT or K132Q cTnI were assessed by in vitro motility assay. Results: Cardiomyocytes expressing cTnI K132Q relaxed faster and had decreased calcium sensitivity compared to WT cTnI at the whole cell and myofibril level. Relaxation or calcium sensitivity did not differ between cardiomyocytes infected with WT cTnI and cTnI K132R. Myofibrils with cTnI K132Q exchanged ex vivo demonstrate faster relaxation and decreased calcium sensitivity as well as decreased motility. Conclusions: Our results indicate for the first time that acetylation at a specific cTnI lysine can alter myofibril relaxation and calcium sensitivity. This work underscores the importance of understanding how acetylation affects specific sarcomeric proteins in the context of cardiac disease, and suggests that modulation of myofilament lysine acetylation may represent a novel therapeutic target to alter cardiac relaxation.

Establishing connections between changes in linear DNA sequences and complex downstream mesoscopic pathology remains a major challenge in biology. Herein, we report a novel, multi-modal and multiscale imaging approach for comprehensive assessment of cardiovascular physiology in Drosophila melanogaster. We employed high-speed angiography, optical coherence tomography (OCT) and confocal microscopy to reveal functional and structural abnormalities in the hdp2 mutant, pre-pupal heart tube and aorta relative to controls. hdp2 harbor a mutation in wupA, which encodes an ortholog of human troponin I (TNNI3). TNNI3 variants frequently engender cardiomyopathy. We demonstrate that the hdp2 aortic and cardiac muscle walls are disrupted and that shorter sarcomeres are associated with smaller, stiffer aortas, which consequently result in increased flow and pulse wave velocities. The mutant hearts also displayed diastolic and latent systolic dysfunction. We conclude that hdp2 pre-pupal hearts are exposed to increased afterload due to aortic hypoplasia. This may in turn contribute to diastolic and subtle systolic dysfunction via vascular-heart tube interaction, which describes the effect of the arterial loading system on cardiac function. Ultimately, the cardiovascular pathophysiology caused by a point mutation in a sarcomeric protein demonstrates that complex and dynamic micro- and mesoscopic phenotypes can be mechanistically explained in a gene sequence- and molecular-specific manner.

Reactive oxygen species (ROS) contribute to health and disease. CaMKII is a widely expressed enzyme whose activation by oxidation of regulatory domain methionines (ox-CaMKII) contributes to cardiovascular disease, asthma, and cancer. Here we integrate comparative genomic and experimental data to show that CaMKII activation by ROS arose more than half-a-billion years ago on the vertebrate stem lineage where it constituted a bridge between ROS and increased intracellular Ca2+ release, exercise responsive gene transcription, and improved performance in skeletal muscle. These enhancements to fight-or-flight physiology were likely key in facilitating a well-evidenced shift in the behavioural ecology of our immediate chordate ancestors, and, in turn, the evolutionary success of vertebrates. Still, the ox-CaMKII innovation for augmenting performance must be considered a critical evolutionary trade-off, as it rendered us more susceptible to common and often fatal diseases linked to excessive ROS.

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.

Striated muscle contraction is regulated by Ca(2+)-dependent modulation of myosin cross-bridge binding to F-actin by the thin filament troponin (Tn)-tropomyosin (Tm) complex. In the absence of Ca(2+), 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 favorable 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 behavior, which highlight the surface feature’s role in F-actin-Tm interactions and contractile regulation.

Valosin Containing Protein (VCP)/p97 is a AAA-ATPase with functions in vast cellular protein quality control processes, including targeting of misfolded or aggregated proteins for degradation by the ubiquitin proteasome system and autophagy. Mutations in VCP cause a multisystem degenerative proteinopathy disorder that includes pathologies of the nervous system, skeletal muscle, bone, and heart. However, the molecular function of VCP in myocytes is unknown. We generated cardiomyocyte-specific transgenic mice overexpressing wildtype VCP or a VCP K524A mutant with deficient ATPase activity. Mice overexpressing wildtype VCP exhibit normal cardiac structure and function while mutant VCP overexpressing mice develop cardiomyopathy and have elevated levels of ubiquitinated proteins in the heart. Additionally, we generated transgenic flies overexpressing wildtype VCP or VCP K524A in muscle. Flies overexpressing the VCP ATPase-deficient mutant have reduced flight ability at two days of age and are unable to fly at seven days of age, suggesting conserved indispensable homeostatic functions for VCP in heart and skeletal muscle. Moreover, mouse hearts and Drosophila indirect flight muscle overexpressing the ATPase-deficient VCP mutant exhibit profound ultrastructural abnormalities consistent with dysregulation of proteostasis. Extensive proteomics in Drosophila and in mouse heart identified conserved interactions of VCP with protein complexes that suggest unique functions for VCP in regulating novel quality control pathways in muscle. These data and novel regulatory relationships will be presented, which implicate important and evolutionarily conserved functions for VCP and suggest molecular mechanisms that underlie the molecular etiology of multisystem proteinopathy disorders.

K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined howDrosophilamuscle 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 mutatedDrosophilahearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

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 Ca(2+) concentration ([Ca(2+)](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 [Ca(2+)](i) in normal and pressure-induced hypertrophied-hypercontractile muscles. Similarly, fropofol depressed maximum Ca(2+)-activated force (F(max)) and increased the [Ca(2+)](i) required for 50% of F(max) (Ca(50)) 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.

Summary: Lipotoxic cardiomyopathy (LCM) is characterized by abnormal myocardial accumulation of lipids, including ceramide; however, the contribution of ceramide to the etiology of LCM is unclear. Here, we investigated the association of ceramide metabolism and ceramide-interacting proteins (CIPs) in LCM in the Drosophila heart model. We find that ceramide feeding or ceramide-elevating genetic manipulations are strongly associated with cardiac dilation and defects in contractility. High ceramide-associated LCM is prevented by inhibiting ceramide synthesis, establishing a robust model of direct ceramide-associated LCM, corroborating previous indirect evidence in mammals. We identified several CIPs from mouse heart and Drosophila extracts, including caspase activator Annexin-X, myosin chaperone Unc-45, and lipogenic enzyme FASN1, and remarkably, their cardiac-specific manipulation can prevent LCM. Collectively, these data suggest that high ceramide-associated lipotoxicity is mediated, in part, through altering caspase activation, sarcomeric maintenance, and lipogenesis, thus providing evidence for conserved mechanisms in LCM pathogenesis in mammals. : Lipotoxic cardiomyopathy (LCM) is characterized by abnormal myocardial accumulation of lipids, including ceramide. Here, Walls et al. find that ceramide feeding or ceramide-elevating genetic manipulations induce LCM. They identified several ceramide-interacting proteins, whose subsequent cardiac-specific manipulation can prevent LCM by altering caspase activation, sarcomeric maintenance, and lipogenesis. Keywords: heart, sphingolipids, Drosophila, diabetic cardiac disease, myriocin, apoptosis, lipogenesis, Unc-45, Annexin, FASN

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because the portion of the nervous system that contains limb premotor and motor circuits is largely inaccessible to large-scale recording techniques 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 of behaving adultDrosophila 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 novel class of locomotion-associated descending neurons. This new approach enables the direct investigation of circuits associated with complex limb movements.