Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca2+ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca2+ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament's relaxed state resulting in an increased number of crossbridges during Ca2+ activation. Additionally, in the low Ca2+-relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca2+, and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

The structure of the native cardiac thin filament at systolic Ca2+ levels.

Every heartbeat relies on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca2+ levels. Thin filaments are comprised of two actin strands, each harboring equally separated troponin complexes, which bind Ca2+ to move tropomyosin cables away from the myosin binding sites and, thus, activate systolic contraction. Recently, structures of thin filaments obtained at low (pCa ∼9) or high (pCa ∼3) Ca2+ levels revealed the transition between the Ca2+-free and Ca2+-bound states. However, in working cardiac muscle, Ca2+ levels fluctuate at intermediate values between pCa ∼6 and pCa ∼7. The structure of the thin filament at physiological Ca2+ levels is unknown. We used cryoelectron microscopy and statistical analysis to reveal the structure of the cardiac thin filament at systolic pCa = 5.8. We show that the two strands of the thin filament consist of a mixture of regulatory units, which are composed of Ca2+-free, Ca2+-bound, or mixed (e.g., Ca2+ free on one side and Ca2+ bound on the other side) troponin complexes. We traced troponin complex conformations along and across individual thin filaments to directly determine the structural composition of the cardiac native thin filament at systolic Ca2+ levels. We demonstrate that the two thin filament strands are activated stochastically with short-range cooperativity evident only on one of the two strands. Our findings suggest a mechanism by which cardiac muscle is regulated by narrow range Ca2+ fluctuations.

Fast skeletal myosin-binding protein-C regulates fast skeletal muscle contraction.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2-/-) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2-/- mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2-/- mice. Small-angle X-ray diffraction of C2-/- EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2-/- EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2-/- mice. Finally, C2-/- muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle.

Negative Charges Introduced Near the IT Helix of Cardiac Troponin T Stabilize the Active State of Actin Filaments.

The disordered and basic C-terminal 14 residues of human troponin T (TnT) are essential for full inhibition of actomyosin ATPase activity at low Ca2+ levels and for limiting activation at saturating Ca2+. In previous studies, stepwise truncation of the C-terminal region of TnT increased activity in proportion to the number of positive charges eliminated. To define key basic residues more closely, we generated phosphomimetic-like mutants of TnT. Phosphomimetic mutants were chosen because of reports that phosphorylation of TnT, including sites within the C terminal region, depressed activity, contrary to our expectations. Four constructs were made where one or more Ser and Thr residues were replaced with Asp residues. The S275D and T277D mutants, near the IT helix and adjacent to basic residues, produced the greatest activation of ATPase rates in solution; the effects of the S275D mutant were recapitulated in muscle fiber preparations with enhanced myofilament Ca2+ sensitivity. Actin filaments containing S275D TnT were also shown to be incapable of populating the inactive state at low Ca2+ levels. Actin filaments containing both S275D/T284D were not statistically different from those containing only S275D in both solution and cardiac muscle preparation studies. Finally, actin filaments containing T284D TnT, closer to the C-terminus and not adjacent to a basic residue, had the smallest effect on activity. Thus, the effects of negative charge placement in the C-terminal region of TnT were greatest near the IT helix and adjacent to a basic residue.

Nucleus Mechanosensing in Cardiomyocytes.

Cardiac muscle contraction is distinct from the contraction of other muscle types. The heart continuously undergoes contraction-relaxation cycles throughout an animal's lifespan. It must respond to constantly varying physical and energetic burdens over the short term on a beat-to-beat basis and relies on different mechanisms over the long term. Muscle contractility is based on actin and myosin interactions that are regulated by cytoplasmic calcium ions. Genetic variants of sarcomeric proteins can lead to the pathophysiological development of cardiac dysfunction. The sarcomere is physically connected to other cytoskeletal components. Actin filaments, microtubules and desmin proteins are responsible for these interactions. Therefore, mechanical as well as biochemical signals from sarcomeric contractions are transmitted to and sensed by other parts of the cardiomyocyte, particularly the nucleus which can respond to these stimuli. Proteins anchored to the nuclear envelope display a broad response which remodels the structure of the nucleus. In this review, we examine the central aspects of mechanotransduction in the cardiomyocyte where the transmission of mechanical signals to the nucleus can result in changes in gene expression and nucleus morphology. The correlation of nucleus sensing and dysfunction of sarcomeric proteins may assist the understanding of a wide range of functional responses in the progress of cardiomyopathic diseases.

Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2.

The ACTN2 gene encodes α-actinin 2, located in the Z-disc of the sarcomeres in striated muscle. In this study, we sought to investigate the effects of an ACTN2 missense variant of unknown significance (p.A868T) on cardiac muscle structure and function. Left ventricular free wall samples were obtained at the time of cardiac transplantation from a heart failure patient with the ACTN2 A868T heterozygous variant. This variant is in the EF 3-4 domain known to interact with titin and α-actinin. At the ultrastructural level, ACTN2 A868T cardiac samples presented small structural changes in cardiomyocytes when compared to healthy donor samples. However, contractile mechanics of permeabilized ACTN2 A868T variant cardiac tissue displayed higher myofilament Ca2+ sensitivity of isometric force, reduced sinusoidal stiffness, and faster rates of tension redevelopment at all Ca2+ levels. Small-angle X-ray diffraction indicated increased separation between thick and thin filaments, possibly contributing to changes in muscle kinetics. Molecular dynamics simulations indicated that while the mutation does not significantly impact the structure of α-actinin on its own, it likely alters the conformation associated with titin binding. Our results can be explained by two Z-disc mediated communication pathways: one pathway that involves α-actinin's interaction with actin, affecting thin filament regulation, and the other pathway that involves α-actinin's interaction with titin, affecting thick filament activation. This work establishes the role of α-actinin 2 in modulating cross-bridge kinetics and force development in the human myocardium as well as how it can be involved in the development of cardiac disease.

Myofibril orientation as a metric for characterizing heart disease.

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 because of 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 with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length of 1.9 µm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 µm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 µm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent x-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases.

Arrhythmogenic Cardiomyopathy: Exercise Pitfalls, Role of Connexin-43, and Moving beyond Antiarrhythmics.

Arrhythmogenic Cardiomyopathy (ACM), a Mendelian disorder that can affect both left and right ventricles, is most often associated with pathogenic desmosomal variants that can lead to fibrofatty replacement of the myocardium, a pathological hallmark of this disease. Current therapies are aimed to prevent the worsening of disease phenotypes and sudden cardiac death (SCD). Despite the use of implantable cardioverter defibrillators (ICDs) there is no present therapy that would mitigate the loss in electrical signal and propagation by these fibrofatty barriers. Recent studies have shown the influence of forced vs. voluntary exercise in a variety of healthy and diseased mice; more specifically, that exercised mice show increased Connexin-43 (Cx43) expression levels. Fascinatingly, increased Cx43 expression ameliorated the abnormal electrical signal conduction in the myocardium of diseased mice. These findings point to a major translational pitfall in current therapeutics for ACM patients, who are advised to completely cease exercising and already demonstrate reduced Cx43 levels at the myocyte intercalated disc. Considering cardiac dysfunction in ACM arises from the loss of cardiomyocytes and electrical signal conduction abnormalities, an increase in Cx43 expression-promoted by low to moderate intensity exercise and/or gene therapy-could very well improve cardiac function in ACM patients.

Efficacy and Safety of Angiotensin Receptor Blockers in a Pre-Clinical Model of Arrhythmogenic Cardiomyopathy.

Arrhythmogenic Cardiomyopathy (ACM) is a familial heart disease, characterized by contractile dysfunction, ventricular arrhythmias (VAs), and the risk of sudden cardiac death. Currently, implantable cardioverter defibrillators and antiarrhythmics are the mainstays in ACM therapeutics. Angiotensin receptor blockers (ARBs) have been highlighted in the treatment of heart diseases, including ACM. Yet, recent research has additionally implicated ARBs in the genesis of VAs and myocardial lipolysis via the peroxisome proliferator-activated receptor gamma (PPARγ) pathway. The latter is of particular interest, as fibrofatty infiltration is a pathological hallmark in ACM. Here, we tested two ARBs, Valsartan and Telmisartan, and the PPAR agonist, Rosiglitazone, in an animal model of ACM, homozygous Desmoglein-2 mutant mice (Dsg2mut/mut). Cardiac function, premature ventricular contractions (PVCs), fibrofatty scars, PPARα/γ protein levels, and PPAR-mediated mRNA transcripts were assessed. Of note, not a single mouse treated with Rosiglitazone made it to the study endpoint (i.e., 100% mortality: n = 5/5). Telmisartan-treated Dsg2mut/mut mice displayed the preservation of contractile function (percent ejection fraction [%EF]; 74.8 ± 6.8%EF) compared to Vehicle- (42.5 ± 5.6%EF) and Valsartan-treated (63.1 ± 4.4%EF) mice. However, Telmisartan-treated Dsg2mut/mut mice showed increased cardiac wall motion abnormalities, augmented %PVCs, electrocardiographic repolarization/depolarization abnormalities, larger fibrotic lesions, and increased expression of PPARy-regulated gene transcripts compared to their Dsg2mut/mut counterparts. Alternatively, Valsartan-treated Dsg2mut/mut mice harbored fewer myocardial scars, reduced %PVC, and increased Wnt-mediated transcripts. Considering our findings, caution should be taken by physicians when prescribing medications that may increase PPARy signaling in patients with ACM.

A comprehensive guide to genetic variants and post-translational modifications of cardiac troponin C.

Familial cardiomyopathy is an inherited disease that affects the structure and function of heart muscle and has an extreme range of phenotypes. Among the millions of affected individuals, patients with hypertrophic (HCM), dilated (DCM), or left ventricular non-compaction (LVNC) cardiomyopathy can experience morphologic changes of the heart which lead to sudden death in the most detrimental cases. TNNC1, the gene that codes for cardiac troponin C (cTnC), is a sarcomere gene associated with cardiomyopathies in which probands exhibit young age of presentation and high death, transplant or ventricular fibrillation events relative to TNNT2 and TNNI3 probands. Using GnomAD, ClinVar, UniProt and PhosphoSitePlus databases and published literature, an extensive list to date of identified genetic variants in TNNC1 and post-translational modifications (PTMs) in cTnC was compiled. Additionally, a recent cryo-EM structure of the cardiac thin filament regulatory unit was used to localize each functionally studied amino acid variant and each PTM (acetylation, glycation, s-nitrosylation, phosphorylation) in the structure of cTnC. TNNC1 has a large number of variants (> 100) relative to other genes of the same transcript size. Surprisingly, the mapped variant amino acids and PTMs are distributed throughout the cTnC structure. While many cardiomyopathy-associated variants are localized in α-helical regions of cTnC, this was not statistically significant χ2 (p = 0.72). Exploring the variants in TNNC1 and PTMs of cTnC in the contexts of cardiomyopathy association, physiological modulation and potential non-canonical roles provides insights into the normal function of cTnC along with the many facets of TNNC1 as a cardiomyopathic gene.

Mandibular muscle troponin of the Florida carpenter ant Camponotus floridanus: extending our insights into invertebrate Ca2+ regulation.

Ants use their mandibles for a variety of functions and behaviors. We investigated mandibular muscle structure and function from major workers of the Florida carpenter ant Camponotus floridanus: force-pCa relation and velocity of unloaded shortening of single, permeabilized fibres, primary sequences of troponin subunits (TnC, TnI and TnT) from a mandibular muscle cDNA library, and muscle fibre ultrastructure. From the mechanical measurements, we found Ca2+-sensitivity of isometric force was markedly shifted rightward compared with vertebrate striated muscle. From the troponin sequence results, we identified features that could explain the rightward shift of Ca2+-activation: the N-helix of TnC is effectively absent and three of the four EF-hands of TnC (sites I, II and III) do not adhere to canonical sequence rules for divalent cation binding; two alternatively spliced isoforms of TnI were identified with the alternatively spliced exon occurring in the region of the IT-arm α-helical coiled-coil, and the N-terminal extension of TnI may be involved in modulation of regulation, as in mammalian cardiac muscle; and TnT has a Glu-rich C-terminus. In addition, a structural homology model was built of C. floridanus troponin on the thin filament. From analysis of electron micrographs, we found thick filaments are almost as long as the 6.8 µm sarcomeres, have diameter of ~ 16 nm, and typical center-to-center spacing of ~ 46 nm. These results have implications for the mechanisms by which mandibular muscle fibres perform such a variety of functions, and how the structure of the troponin complex aids in these tasks.

Eliminating the First Inactive State and Stabilizing the Active State of the Cardiac Regulatory System Alters Behavior in Solution and in Ordered Systems.

Calcium binding to troponin C (TnC) is insufficient for full activation of myosin ATPase activity by actin-tropomyosin-troponin. Previous attempts to investigate full activation utilized ATP-free myosin or chemically modified myosin to stabilize the active state of regulated actin. We utilized the Δ14-TnT and the A8V-TnC mutants to stabilize the activated state at saturating Ca2+ and to eliminate one of the inactive states at low Ca2+. The observed effects differed in solution studies and in the more ordered in vitro motility assay and in skinned cardiac muscle preparations. At saturating Ca2+, full activation with Δ14-TnT·A8V-TnC decreased the apparent KM for actin-activated ATPase activity compared to bare actin filaments. Rates of in vitro motility increased at both high and low Ca2+ with Δ14-TnT; the maximum shortening speed at high Ca2+ increased 1.8-fold. Cardiac muscle preparations exhibited increased Ca2+ sensitivity and large increases in resting force with either Δ14-TnT or Δ14-TnT·A8V-TnC. We also observed a significant increase in the maximal rate of tension redevelopment. The results of full activation with Ca2+ and Δ14-TnT·A8V-TnC confirmed and extended several earlier observations using other means of reaching full activation. Furthermore, at low Ca2+, elimination of the first inactive state led to partial activation. This work also confirms, in three distinct experimental systems, that troponin is able to stabilize the active state of actin-tropomyosin-troponin without the need for high-affinity myosin binding. The results are relevant to the reason for two inactive states and for the role of force producing myosin in regulation.

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium.

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+ Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

The intrinsically disordered C terminus of troponin T binds to troponin C to modulate myocardial force generation.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca2+-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca2+ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC-cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

Cardiomyocyte nuclearity and ploidy: when is double trouble?

Considerable effort has gone into investigating mechanisms that underlie the developmental transition in which mammalian cardiomyocytes (CMs) switch from being able to proliferate during development, to essentially having lost that ability at maturity. This problem is interesting not only for scientific curiosity, but also for its clinical relevance because controlling the ability of mature CMs to replicate would provide a much-needed approach for restoring cardiac function in damaged hearts. In this review, we focus on the propensity of mature mammalian CMs to be multinucleated and polyploid, and the extent to which this may be necessary for normal physiology yet possibly disadvantageous in some circumstances. In this context, we explore whether the concept of the myonuclear domain (MND) in multinucleated skeletal muscle fibers might apply to cardiomyocytes, and whether cardio-MND size might be related to the transition of CMs to become multinuclear. Nuclei in CMs are almost certainly integrators of not only biochemical, but also-because of their central location within the myofibrils-mechanical information, and this multimodal, integrative function in adult CMs-involving molecules that have been extensively studied along with newly identified possibilities-could influence both gene expression as well as replication of the genome and the nuclei themselves.

Structural and functional impact of troponin C-mediated Ca2+ sensitization on myofilament lattice spacing and cross-bridge mechanics in mouse cardiac muscle.

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca2+ sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca2+-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca2+ sensitizer. Our findings suggest that both methods of increasing myofilament Ca2+ sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (kTR); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I1,1/I1,0 ratio from X-ray diffraction. Computational modeling suggests that Ca2+ sensitization through this cTnC mutation or bepridil accelerates kTR primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in kTR, we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca2+ activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca2+ sensitization may act through stabilization of cTnC's D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca2+ activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.

Enhanced troponin I binding explains the functional changes produced by the hypertrophic cardiomyopathy mutation A8V of cardiac troponin C.

Higher affinity for TnI explains how troponin C (TnC) carrying a causative hypertrophic cardiomyopathy mutation, TnC(A8V), sensitizes muscle cells to Ca(2+). Muscle fibers reconstituted with TnC(A8V) require ∼2.3-fold less [Ca(2+)] to achieve 50% maximum-tension compared to fibers reconstituted with wild-type TnC (TnC(WT)). Binding measurements rule out a significant change in N-terminus Ca(2+)-affinity of isolated TnC(A8V), and TnC(A8V) binds the switch-peptide of troponin-I (TnI(sp)) ∼1.6-fold more strongly than TnC(WT); thus we model the TnC-TnI(sp) interaction as competing with the TnI-actin interaction. Tension data are well-fit by a model constrained to conditions in which the affinity of TnC(A8V) for TnI(sp) is 1.5-1.7-fold higher than that of TnC(WT) at all [Ca(2+)]. Mean ATPase rates of reconstituted cardiac myofibrils is greater for TnC(A8V) than TnC(WT) at all [Ca(2+)], with statistically significant differences in the means at higher [Ca(2+)]. To probe TnC-TnI interaction in low Ca(2+), displacement of bis-ANS from TnI was monitored as a function of TnC. Whereas Ca(2+)-TnC(WT) displaces significantly more bis-ANS than Mg(2+)-TnC(WT), Ca(2+)-TnC(A8V) displaces probe equivalently to Mg(2+)-TnC(A8V) and Ca(2+)-TnC(WT), consistent with stronger Ca(2+)-independent TnC(A8V)-TnI(sp). A Matlab program for computing theoretical activation is reported. Our work suggests that contractility is constantly above normal in hearts made hypertrophic by TnC(A8V).

Altered signaling pathways linked to angiotensin II underpin the upregulation of renal Na(+)-ATPase in chronically undernourished rats.

This study has investigated the participation of altered signaling linked to angiotensin II (Ang II) that could be associated with increased Na(+) reabsorption in renal proximal tubules during chronic undernutrition. A multideficient chow for rats (basic regional diet, BRD) was used, which mimics several human diets widely taken in developing countries. The Vmax of the ouabain-resistant Na(+)-ATPase resident in the basolateral membranes increased >3-fold (P<0.001) accompanied by an increase in Na(+) affinity from 4.0 to 0.2mM (P<0.001). BRD rats had a >3-fold acceleration of the formation of phosphorylated intermediates in the early stage of the catalytic cycle (in the E1 conformation) (P<0.001). Immunostaining showed a huge increase in Ang II-positive cells in the cortical tubulointerstitium neighboring the basolateral membranes (>6-fold, P<0.001). PKC isoforms (α, ε, λ, ζ), Ang II type 1 receptors and PP2A were upregulated in BRD rats (in %): 55 (P<0.001); 35 (P<0.01); 125, 55, 11 and 30 (P<0.001). PKA was downregulated by 55% (P<0.001). With NetPhosK 1.0 and NetPhos 2.0, we detected 4 high-score (>0.70) regulatory phosphorylation sites for PKC and 1 for PKA in the primary sequence of the Na(+)-ATPase α-subunit, which are located in domains that are key for Na(+) binding and catalysis. Therefore, chronic undernutrition stimulates tubulointerstitial activity of Ang II and impairs PKC- and PKA-mediated regulatory phosphorylation, which culminates in an exaggerated Na(+) reabsorption across the proximal tubular epithelium.

NFĸB signaling drives myocardial injury via CCR2+ macrophages in a preclinical model of arrhythmogenic cardiomyopathy.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2+ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2+ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2mut/mut mice were dependent on CCR2+ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.