Increased Calcium Sensitivity Research Articles

Ultrasound-guided gluteal nerves electrical stimulation to enhance strength and power in individuals with chronic knee pain: a randomized controlled pilot trial

IntroductionVarious pathophysiological contexts can be accompanied by weakness, arthrogenic muscle inhibition, and even disability. In this scenario, peripheral nerve stimulation has been studied not only for pain management but also for the improvement of neuromuscular parameters. For this purpose, the use of Transcutaneous Electrical Nerve Stimulation (TENS) has typically been investigated, but recently, the use of ultrasound-guided percutaneous peripheral nerve stimulation (pPNS) has gained popularity. In this regard, electrical stimulation has a predisposition to activate Type II muscle fibers and has been shown to be capable of generating short-term potentiation by increasing calcium sensitivity. However, the evidence of pPNS applied in humans investigating such variables is rather limited.ObjectivesThis pilot study aimed to assess the feasibility of the methodology and explore the potential of pPNS in enhancing hip extension performance in individuals suffering from knee pain, comparing it with TENS.MethodsTwelve participants were divided into pPNS and TENS groups, undergoing pre- and post-intervention assessments of peak concentric power (W), strength (N), execution speed (m/s), and one-repetition maximum (1RM) (kg) estimation. For pPNS, two needles were positioned adjacent to the superior and inferior gluteal nerves under ultrasound guidance. For TENS, electrodes were positioned between the posterosuperior iliac spine and the ischial tuberosity, and halfway between the posterosuperior iliac spine and the greater trochanter. The interventions consisted of 10 stimulations of 10 s at a frequency of 10 Hz with a pulse width of 240 μs, with rest intervals of 10 s between stimulations.ResultsPeripheral nerve stimulation significantly improved concentric power at 30% (p = 0.03) and 50% (p = 0.03) of 1RM, surpassing TENS, which showed minimal changes. No significant strength differences were observed post-intervention in either group.ConclusionThis work presents evidence where pPNS applied to the gluteal nerves results in an enhanced performance of hip extension at submaximal loads. However, this improvement does not seem to be reflected in short-term changes in the estimation of the 1RM by the force-velocity profile.

Proteolytic degradation of atrial sarcomere proteins underlies contractile defects in atrial fibrillation.

Atrial fibrillation (AFib) is the most common cardiac rhythm disturbance, often treated via electrical cardioversion. Following rhythm restoration, a period of depressed mechanical function known as atrial stunning occurs, suggesting that defects in contractility occur in AFib and are revealed upon restoration of rhythm. This project aims to define the contractile remodeling that occurs in AFib. To assess contractile function, we used a canine atrial tachypacing model of induced AFib. Mass spectrometry analysis showed dysregulation of contractile proteins in samples from AFib compared to sinus rhythm atria. Atrial cardiomyocytes showed reduced force of contraction, decreased resting tension, and increased calcium sensitivity in skinned single cardiomyocyte studies. These alterations correlated with degradation of myofilament proteins including myosin heavy chain altering force of contraction, titin altering resting tension, and TnI altering calcium sensitivity. We measured degradation of other myofilament proteins including cMyBP-C and actininshowing significant degradation in the AFib samples compared to sinus rhythm atria. Many of the protein degradation products appeared as discrete cleavage products that are generated by calpain proteolysis. We assessed calpain activity and found it to be significantly increased. These results provide an understanding of the contractile remodeling that occurs in AFib and provide insight into the molecular explanation for atrial stunning and the increased risk of atrial thrombus and stroke in AFib.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

Enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction in patient-specific induced pluripotent stem cell-derived cardiomyocytes with MYH7 mutation

Hypertrophic cardiomyopathy (HCM), the most common inherited heart disease, is frequently caused by mutations in the β-cardiac myosin heavy chain gene (MYH7). Abnormal calcium handling and diastolic dysfunction are archetypical features of HCM caused by MYH7 gene mutations. However, the mechanism of how MYH7 mutations leads to these features remains unclear, which inhibits the development of effective therapies. Initially, cardiomyocytes were generated from induced pluripotent stem cells from an eight-year-old girl diagnosed with HCM carrying a MYH7(C.1063 G>A) heterozygous mutation(mutant-iPSC-CMs) and mutation-corrected isogenic iPSCs(control-iPSC-CMs) in the present study. Next, we compared phenotype of mutant-iPSC-CMs to that of control-iPSC-CMs, by assessing their morphology, hypertrophy-related genes expression, calcium handling, diastolic function and myofilament calcium sensitivity at days 15 and 40 respectively. Finally, to better understand increased myofilament Ca2+ sensitivity as a central mechanism of central pathogenicity in HCM, inhibition of calcium sensitivity with mavacamten can improveed cardiomyocyte hypertrophy. Mutant-iPSC-CMs exhibited enlarged areas, increased sarcomere disarray, enhanced expression of hypertrophy-related genes proteins, abnormal calcium handling, diastolic dysfunction and increased myofilament calcium sensitivity at day 40, but only significant increase in calcium sensitivity and mild diastolic dysfunction at day 15. Increased calcium sensitivity by levosimendan aggravates cardiomyocyte hypertrophy phenotypes such as expression of hypertrophy-related genes, abnormal calcium handling and diastolic dysfunction, while inhibition of calcium sensitivity significantly improves cardiomyocyte hypertrophy phenotypes in mutant-iPSC-CMs, suggesting increased myofilament calcium sensitivity is the primary mechanisms for MYH7 mutations pathogenesis. Our studies have uncovered a pathogenic mechanism of HCM caused by MYH7 gene mutations through which enhanced myofilament calcium sensitivity aggravates abnormal calcium handling and diastolic dysfunction. Correction of the myofilament calcium sensitivity was found to be an effective method for treating the development of HCM phenotype in vitro.

Abstract 18778: Patient-Derived Stem Cell Models of Pediatric Restrictive Cardiomyopathy Exhibit Increased Resting Tension and Cardiomyocyte Calcium Sensitivity

Introduction: Pediatric Restrictive Cardiomyopathy (RCM) is a genetic cardiomyopathy resulting from profoundly increased stiffness of the wall of the cardiac ventricle with normal or low normal contractility. Treatment options are limited, with most patients necessitating heart transplant. Therapies that can improve diastolic function are needed for this severe disease. However, development of novel therapies has been limited by a paucity of models of this rare disease. Here, we develop two novel models of pediatric RCM and use novel high-throughput functional analysis to measure diastolic dysfunction and defects in calcium handling. Hypothesis: Patient-derived human induced pluripotent stem cell (hiPSC) models of pediatric RCM will exhibit increased resting tension indicative of diastolic dysfunction, as well as increased calcium sensitivity. Methods: Cardiomyocytes were differentiated from hiPSCs derived from two pediatric patients with RCM, as well as a paired isogenic CRISPR-corrected lines. Cells were replated onto polyacrylamide gels and stained with calcium sensitive dyes, and contractility, resting tension, and calcium sensitivity were measured using a novel high-throughput functional imaging platform. Results: RCM cells from both patients showed elevated contractility, resting tension, and calcium sensitivity compared to their isogenic controls. Conclusions: Patient-derived RCM cells show alterations in calcium sensitivity and resting tension that recapitulate and explain the diastolic dysfunction seen in patients. These lines, combined with the high-throughput measurement platform described here, will provide a powerful platform for exploring novel therapeutic strategies for this severe disease.

Abstract EC.04: The Effects Of Cardiac Troponin Activator CK-358 On Cardiac Contractility In A Mouse Model Of Genetic Dilated Cardiomyopathy

Genetic dilated cardiomyopathy (DCM) is one of the most common causes of heart failure. However, most treatments do not address the underlying contractile deficit caused by the disease. In an effort to create more targeted DCM therapeutics, novel small molecule drugs known as sarcomeric modulators have been of recent interest. In a recent study, a cardiac troponin activator was shown to improve cardiac muscle contractility without adversely affecting energetics or diastolic function. Here, we aim to elucidate the mechanism by which the cardiac troponin activator CK-3827358 (CK-358) is able to recover contractile function in a mouse model of DCM with a mutation at residue 61 of cardiac troponin C (cTnC I61Q). We measured steady state force and k tr , a measure of crossbridge cycling kinetics, in demembranated ventricular tissue from control and cTnC I61Q mice in solutions with increasing concentrations of calcium, ranging from pCa 9 to pCa 4. The cTnC I61Q mutation significantly decreased calcium sensitivity versus control (pCa 50 : 5.48 ± 0.06 vs 5.75 ± 0.01). Compared to controls, CK-358 (1 μM) significantly increased calcium sensitivity (pCa 50 ) in tissue from both cTnC I61Q and control mice (6.03 ± 0.06 and 6.50 ± 0.18, P=0.005) without any change in maximum force. CK-358 also significantly increased k tr , possibly suggesting activation of thin filaments. Next, we used a fluorescently labeled human cardiac troponin C (HcTnC) conjugated to N,N′-dimethyl-N-(iodoacetyl)-N′-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) combined with subunits T and I to make a full HcTn complex. We performed calcium titrations with the HcTn-IANBD and saw no change in fluorescence in the control protein with and without CK-358. Finally, we performed HcTnC titrations with HcTnI and saw no change in fluorescence at maximum, sub-maximum, and minimum calcium concentrations. Overall, we demonstrate that cardiac troponin activator CK-358 did not alter calcium binding to HcTnC or its interaction with HcTnI. However, it restores the calcium sensitivity of cTnC I61Q DCM ventricular sarcomeres. Future studies will examine the effects of CK-358 on calcium binding in decorated thin filaments and measure activation using stopped flow spectroscopy in this model of DCM.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 μM.

Clinical and functional characterization of a long survivor congenital titinopathy patient with a novel metatranscript-only titin variant

Congenital titinopathies are an emerging group of a potentially severe form of congenital myopathies caused by biallelic mutations in titin, encoding the largest existing human protein involved in the formation and stability of sarcomeres. In this study we describe a patient with a congenital myopathy characterized by multiple contractures, a rigid spine, non progressive muscular weakness, and a novel homozygous TTN pathogenic variant in a metatranscript-only exon: the c.36400A > T, p.Lys12134*. Muscle biopsies showed increased internalized nuclei, variability in fiber size, mild fibrosis, type 1 fiber predominance, and a slight increase in the number of satellite cells. RNA studies revealed the retention of intron 170 and 171 in the open reading frame, and immunoflourescence and western blot studies, a normal titin content. Single fiber functional studies showed a slight decrease in absolute maximal force and a cross-sectional area with no decreases in tension, suggesting that weakness is not sarcomere-based but due to hypotrophy. Passive properties of single fibers were not affected, but the observed increased calcium sensitivity of force generation might contribute to the contractural phenotype and rigid spine of the patient. Our findings provide evidence for a pathogenic, causative role of a metatranscript-only titin variant in a long survivor congenital titinopathy patient with distal arthrogryposis and rigid spine.

Conversion of a Cardiac Muscle Modulator from an Inhibitor to an Activator.

The binding of calcium to cardiac troponin C (cTnC) enhances the binding of troponin I (cTnI) switch region to the regulatory domain of cTnC (cNTnC) and triggers muscle contraction. Several molecules alter the response of the sarcomere by targeting this interface; virtually all have an aromatic core that binds to the hydrophobic pocket of cNTnC and an aliphatic tail that interacts with the switch region of cTnI. W7 has been extensively studied, and the positively charged tail has been shown to be important for its inhibitory action. Herein we investigate the importance of the aromatic core of W7 by synthesizing compounds that have the core region of calcium activator dfbp-o with various lengths of the same tail (D-series). These compounds all bind more tightly to cNTnC-cTnI chimera (cChimera) than the analogous W-series compounds and show increased calcium sensitivity of force generation and ATPase activity, demonstrating that the cardiovascular system is tightly balanced.

Proteomic signatures predict depressed cooperativity and increased calcium sensitivity in heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) affects over half of all heart failure patients. We recently reported several deficits in the tension-calcium relationship from isolated skinned cardiomyocytes from HFpEF patients, including depressed cooperativity, increased calcium sensitivity, and depressed maximum tension. Here, we sought to identify whether proteomic signatures associate with these deficits. We obtained total proteomic profiles, consisting of 2250 proteins, from right ventricular endomyocardial biopsies in 43 patients. Machine learning based cluster analysis revealed two clusters of proteomic profiles, Group 1 (n=31) and Group 2 (n=12). Compared to Group 1, patients in Group 2 had depressed E/A ratio (p=0.048) and smaller left ventricles (p=0.02) on echocardiography, right ventricular dysfunction (p=0.08) on right heart catheterization, and worsened HF symptoms based on New York Heart Association functional class (p=0.054). Analysis of tension-calcium relationships revealed that myocytes from Group 2 had a higher Hill coefficient (p=0.0001), indicative of better cooperativity, and increased calcium sensitivity (p=0.001), but no differences in maximum tension. We next identified which proteins correlated with Hill coefficient and calcium sensitivity using univariate regression. We found that 330 proteins negatively correlated with Hill coefficient, while 298 proteins positivity correlated with calcium sensitivity, corresponding more to Group 2. Gene ontology pathway analysis revealed these proteins (either set) were enriched for metabolic processes, including fatty acid metabolism, mitochondrial respiration, and cellular biosynthetic processes. This poses an intriguing linkage between downregulation of mitochondrial metabolic processes and enhanced cooperativity and calcium sensitivity. The latter is reported in HF with reduced EF, but the former seems unique to HFpEF. Metabolite perturbations may affect sarcomere post-translational modification, driving shifts in cooperativity and calcium sensitivity. Further studies are ongoing to identify the cause of these myofilament calcium-tension changes and implications.

Elucidating the mechanism of Danicamtiv on force, kinetics, and myosin structure and function.

Myosin modulators are a novel class of small molecules that alter cardiac contractility. Omecamtiv mecarbil, the first identified myosin activator, showed only modest clinical benefits in systolic heart failure patients. Thus, there is an urgency to develop alternative myosin activators. Danicamtiv (Dani) has emerged as a potential candidate; however, a detailed mechanism is not known. Here, we aim to elucidate the mechanism of Dani on contractile function in pig cardiac muscle. Demembranated ventricular tissues show a significant 0.1 pCa unit increase in calcium sensitivity and 10% increase in maximal force after incubation in 1 µM Dani. The most potent effects occur in submaximal calcium concentrations, leading to a flattening of the force-calcium relationship, suggesting decreased cooperativity. Maximal rates of tension redevelopment are decreased by approximately 60% with Dani. Isolated cardiac myofibrils provide details about contractile kinetics. Experiments with 1 µM Dani show a 49% decrease in fast-phase relaxation kinetics. Slow-phase isometric relaxation exhibits 47% slower crossbridge detachment rate and 34% longer thin filament deactivation. Next, we assess ATP utilization in the crossbridge cycle. Filament sliding velocity slows 55% on addition of 0.5 µM Dani, similar to the effect of ADP on velocity. The effects of Dani and ADP are not additive suggesting a similar mode of action. ATP binding is unaltered up to 10 µM Dani using stopped flow spectroscopy. Results of X-ray diffraction studies of porcine myocardium at rest show an increase in equatorial intensity ratio (I1,1/I1,0) in response to 50 µM Dani, reflecting an increased proximity of myosin heads to the thin filament. In conclusion, we hypothesize that Dani primes the thick filament for activation and alters relaxation through inhibited ATP hydrolysis product release. Future studies will test these hypotheses.

Atrial cardiomyocytes from a canine model of atrial fibrillation demonstrate reduced force development and altered expression of contractile proteins.

Atrial fibrillation (AF) is the most common cardiac rhythm disturbance and associates with worse outcomes in cardiac contractile-related diseases including cardiomyopathy. As most studies surrounding AF focus on the electrophysiology of the disease, there is little known about cardiomyocyte contractile function in AF. We used atrial tissue from an established atrial tachypacing canine model of AF. Whole protein lysates were collected from the atria to conduct mass spectrometry analysis showing dysregulation of numerous molecular pathways, including contractility. Skinned atrial cardiomyocytes were isolated from the tissue for force calcium and passive tension experiments. Force calcium experiments showed a significant reduction in the maximum force of contraction of AF cardiomyocytes. To explain this, myosin heavy chain (MHC) isoform expression was evaluated because isoform switching is known to alter force of contraction. Decreased percent beta expression was found, explaining the reduction of force due to MHC alpha's capacity for reduced force of contraction. AF cardiomyocytes showed increased calcium sensitivity of force development, which may translate to prolonged contraction with slower relaxation in vivo. A significant reduction in S100A1 was seen in the AF samples via mass spectrometry, which is associated with increased calcium sensitivity. Prolonged contraction along with reduced force of each contraction could support the development of atrial clots. Atrial cardiomyocytes from the AF hearts demonstrated a significant reduction in the passive tension. Evaluation of titin isoform expression, which is known to alter passive tension, showed increased titin degradation, supporting the capacity for atrial dilation in vivo. Discerning the role of contractility in the pathology of atrial fibrillation could provide novel avenues of AF treatment, especially in instances where it is associated with contractile diseases.

Predictive genotype-phenotype correlation of four tropomyosin-1 variants of unknown significance.

Cardiomyopathies affect nearly 20% of humanity worldwide and are often caused by mutations in heart muscle genes that encode cardiac thin filament (TF) proteins. The on-off switching of cardiac muscle contraction is controlled by the TF proteins troponin and tropomyosin responding to alterations in sarcoplasmic calcium levels. In the absence of calcium, troponin-tropomyosin inhibits actomyosin interactions and induces muscle relaxation, whereas the addition of calcium relieves this constraint leading to contraction. Many mutations in TF proteins that are linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we applied an interdisciplinary approach to correlate atomic-level alterations in tropomyosin structure to myofilament and tissue-level function to better understand mechanisms of disease. Twenty variants of unknown significance (VUS) in TPM1 from ClinVar were screened computationally using molecular dynamics to predict effects on tropomyosin structure and dynamics. Four candidate variants (A102D, D258E, K233N, and A239T) were identified and selected for further functional studies using in vitro motility assays and human engineered heart tissue (EHT) mechanics. The variants associated with hypertrophic cardiomyopathy, D258E and A102D, increased calcium sensitivity, while K233N, a VUS associated with dilated cardiomyopathy, lead to a decrease in calcium sensitivity. EHTs expressing A102D displayed a nearly 50% increase in peak isometric twitch force and delayed relaxation and D258E-EHTs also slowed relaxation. These phenotypes are consistent with the increased calcium sensitivity observed in the in vitro motility assay and with clinical manifestations of hypertrophic cardiomyopathy. Meanwhile, DCM-associated variants K233N and A239T caused dramatic and significant reductions in isometric twitch force when expressed in EHTs. Our approach reveals an important step toward predictive genotype-phenotype correlation and has the potential to benefit patients by accelerating the characterization and classification of tropomyosin variants.

239 VASOSPASTIC ANGINA: A CASE OF COMPLETE FUNCTIONAL ASSESSMENT

Abstract Introduction Vasospastic angina is defined as a focal or diffuse spasm of the arterial wall of an epicardial coronary artery due to muscle cells hyperreactivity caused by an increased calcium sensitivity. Clinical presentation typically consists in chronic story of rest and/or effort typical chest pain, episodes of ST elevated or not ST elevated myocardial infarction, arrhythmias or cardiac arrest; an increased vagal tone, drugs, allergic reactions or the exposure to extreme cold also can trigger vasospasm. Clinical case 73 years old dyslipidemic woman; in past medical history thyroidectomy and radiotherapy for carcinoma and right quadrantectomy with radiotherapy and hormonal therapy for breast cancer. In 2017 admission to cath-lab for non-ST elevation myocardial infarction (NSTEMI) with the evidence of mild left coronary artery disease without critical obstructive stenosis (MINOCA). For the persisting of effort angina and dyspnea (NYHA and CCS II-III), started Diltiazem therapy (60 mg x3) with clinical benefit. In 2020, due to the onset of new episodes of angina, started Ranolazine therapy. In June 2022, for the worsening of clinical symptoms, was evaluated with a Coronary CT, that showed a 65% stenosis at right coronary-acute marginal bifurcation and mild disease on left coronary artery, and then was admitted for an elective coronary angiography, with no evidence of epicardial artery critical stenosis. At the functional evaluation FFR, CFR and IMR (>0.80, 2.5 and 18) demonstrated normal coronary flow reserve and good microcirculation function. For the suspicion of vasospastic angina, acetylcholine test was performed with the evidence of mid-distal left anterior descending artery total occlusion, angina onset and ECG changes, promptly regressed with nitrates administration (Figure 1). Patient was discharged with the optimization of medical therapy with Diltiazem 120 mg x3, with clinical benefit. Conclusions To date vasospastic angina remains underdiagnosed. Functional evaluation of coronary arteries plays a fundamental role in the diagnostic algorithm of the disease and should always be investigated in case of patients with typical symptoms and the absence of critical epicardial stenosis. Further investigations are required to better explain pathogenesis and to identify affected patients in shorter times to ensure early optimized medical therapy.

Computational Exploration and Characterization of Potential Calcium Sensitizing Mutations in Cardiac Troponin C.

Calcium-dependent heart muscle contraction is regulated by the cardiac troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). cNTnC contains one calcium binding site (site II), and altered calcium binding in this site has been studied for decades. It has been previously shown that cNTnC mutants, which increase calcium sensitization may have therapeutic benefits, such as restoring cardiac muscle contractility and functionality post-myocardial infarction events. Here, we computationally characterized eight mutations for their potential effects on calcium binding affinity in site II of cNTnC. We utilized two distinct methods to estimate calcium binding: adaptive steered molecular dynamics (ASMD) and thermodynamic integration (TI). We observed a sensitizing trend for all mutations based on the employed ASMD methodology. The TI results showed excellent agreement with experimentally known calcium binding affinities in wild-type cNTnC. Based on the TI results, five mutants were predicted to increase calcium sensitivity in site II. This study presents an interesting comparison of the two computational methods, which have both been shown to be valuable tools in characterizing the impacts of calcium sensitivity in mutant cNTnC systems.

Abstract 12713: Increased Calcium Sensitivity in Hypoplastic Left Heart Syndrome Hearts With Systolic Failure

Introduction: Systolic failure of the systemic right ventricle (RV) in children with hypoplastic left heart syndrome (HLHS) is a significant cause of death and a growing indication for heart transplantation. However, the underlying etiology of RV systolic failure in this population remains ill-defined. Hypothesis: We hypothesize that cardiomyocyte autonomous defects in contractility, characterized by increased calcium sensitivity, may contribute to systolic failure in HLHS. Methods: Explanted RV tissue was collected and flash frozen at the time of heart transplant in pediatric subjects with HLHS. HLHS subjects were classified by their primary indication for transplant: HLHS subjects with normal systolic function (HLHS-NF, N=2) were transplanted secondary to refractory protein losing enteropathy, and HLHS-F subjects (N=9) had end-stage heart failure due to decreased systolic function. Cardiomyocytes were isolated from frozen RV tissue, permeabilized, and glued to a force transducer and length controller. Force generated by the cardiomyocyte at different calcium concentrations was measured and force-calcium dose response curves were generated (modified Hill equation). pCa50 denotes the negative log of calcium concentration required to generate 50% maximal tension. Results: Less calcium is required to generate 50% maximal tension in HLHS-F cardiomyocytes (n=33 cardiomyocytes) when compared to HLHS-NF (n=5 cardiomyocytes), demonstrated by a higher pCa50 (5.62 vs. 5.46, p=0.027). Maximal force generation is similar between HLHS-NF and HLHS-F cardiomyocytes (55.5 mN/mm2 vs. 54.8 mN/mm2, p=0.96). Conclusions: Cardiomyocytes isolated from HLHS-F demonstrate increased calcium sensitivity when compared to those from HLHS-NF. Although future studies are needed, the presence of intrinsic alterations in sarcomeric properties in HLHS-F supports the hypothesis that cardiomyocyte autonomous defects may contribute to systolic failure in HLHS.

Abstract 15247: A Novel Patient-Derived Stem Cell Model of Restrictive Cardiomyopathy Exhibits Impaired Cardiomyocyte Relaxation Secondary to Increased Myocardial Calcium Sensitivity

Introduction: Restrictive Cardiomyopathy (RCM) is a rare disease characterized by profound, isolated defects in myocardial relaxation, normal or near-normal myocardial wall thickness, and preserved contractile function. Outcomes for patients with RCM are poor, with increased morbidity and mortality compared to other forms of cardiomyopathy. In some patients, RCM can be caused by genetic mutations, notably in genes encoding for components of the sarcomere. While many mutations have been described in RCM patients, the molecular and cellular mechanisms leading from these mutations to the patient’s physiological phenotype are poorly described. Hypothesis: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients will recapitulate the myocardial relaxation defects seen in RCM and provide a novel in vitro model for investigating the molecular mechanisms underpinning this disease. Methods: Induced pluripotent stem cells (iPSCs) were derived from a pediatric patient with RCM caused by a TNNT2 R94C mutation. CRISPR was used to correct the mutation, and cellular mechanics and calcium handling properties were compared to the patient line using a novel high-throughput traction-force imaging platform. Transcriptional profiles were compared using RNA-seq. Results: RCM cells carrying the TNNT2 R94C mutation show increased calcium sensitivity leading to increased resting tension at baseline, as well as prolonged calcium kinetics leading to prolonged relaxation times. Gene expression profiling reveals changes in expression of ECM genes, which may further contribute to the impaired myocardial relaxation seen in RCM. Conclusions: Patient-derived hiPSC-CMs from an RCM patient recapitulate the relaxation defects seen in RCM and highlight the central role of increased calcium sensitivity in impaired myocardial relaxation.

Abstract P2113: A Novel Patient-derived Stem Cell Model Of Restrictive Cardiomyopathy Exhibits Impaired Cardiomyocyte Relaxation Secondary To Increased Myocardial Calcium Sensitivity

Introduction: Restrictive Cardiomyopathy (RCM) is a rare disease characterized by profound, isolated defects in myocardial relaxation, normal or near-normal myocardial wall thickness, and preserved contractile function. Outcomes for patients with RCM are poor, with increased morbidity and mortality compared to other forms of cardiomyopathy. In some patients, RCM can be caused by genetic mutations, notably in genes encoding for components of the sarcomere. While many mutations have been described in RCM patients, the molecular and cellular mechanisms leading from these mutations to the patient’s physiological phenotype are poorly described. Hypothesis: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) derived from RCM patients will recapitulate the myocardial relaxation defects seen in RCM and provide a novel in vitro model for investigating the molecular mechanisms underpinning this disease. Methods: Induced pluripotent stem cells (iPSCs) were derived from a pediatric patient with RCM caused by a TNNT2 R94C mutation. CRISPR was used to correct the mutation, and cellular mechanics and calcium handling properties were compared to the patient line using a novel high-throughput traction-force imaging platform. Transcriptional profiles were compared using RNA-seq. Results: RCM cells carrying the TNNT2 R94C mutation show increased calcium sensitivity leading to increased resting tension at baseline, as well as prolonged calcium kinetics leading to prolonged relaxation times. Gene expression profiling reveals changes in expression of ECM genes, which may further contribute to the impaired myocardial relaxation seen in RCM. Conclusions: Patient-derived hiPSC-CMs from an RCM patient recapitulate the relaxation defects seen in RCM and highlight the central role of increased calcium sensitivity in impaired myocardial relaxation.

Abstract P1061: Crosstalk Between Acetylation And Methylglyoxal Modifications On Myofilament Proteins Affects Sarcomere Function

Diabetes doubles the risk of developing heart failure, independent of coronary artery disease and hypertension. The mechanisms connecting these two diseases are poorly understood, although we have previously shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis byproduct that can irreversibly modify lysine (K) and arginine (R) residues in a process called glycation. We found MGO glycation of the myofilament decreased calcium sensitivity and maximum calcium activated force in diabetes. Here we hypothesized MGO may further impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to impact cardiac function. To assess this hypothesis, non-failing human left ventricular (LV) myocardial tissue was exposed to MGO (100 μM) or vehicle and then the treated with acetic anhydride in acetonitrile (300 μM, Ac 2 O-ACN) overnight. Ac 2 O-ACN increased myofilament protein acetylation in both groups, but this effect was greatly reduced in samples pre-treated with MGO, suggesting these two PTMs do compete for a subset of lysine residues. To discover the specific sites of this acetylation-glycation crosstalk, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes, which revealed sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Importantly, these glycation-acetylation crosstalk sites were found in human samples and the data suggest the balance of these may change in disease states. To explore the functional consequence(s) of this crosstalk, we incubated human LV isolated skinned myocytes with Ac 2 O-ACN (300 μM, overnight), which enhanced function by significantly increasing calcium sensitivity. However, pre-treatment with MGO (100 μM) completely blocked this functional effect of acetylation. Together, these data demonstrate that glycation and acetylation compete for certain myofilament lysine residues, and that glycation can block the beneficial functional effects of acetylation. These results provide insight into the molecular mechanisms behind diabetic cardiomyopathy and the utility of targeting acetylation clinically.

HDAC Inhibition Regulates Cardiac Function by Increasing Myofilament Calcium Sensitivity and Decreasing Diastolic Tension.

We recently established a large animal model that recapitulates key clinical features of heart failure with preserved ejection fraction (HFpEF) and tested the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). SAHA reversed and prevented the development of cardiopulmonary impairment. This study evaluated the effects of SAHA at the level of cardiomyocyte and contractile protein function to understand how it modulates cardiac function. Both isolated adult feline ventricular cardiomyocytes (AFVM) and left ventricle (LV) trabeculae isolated from non-failing donors were treated with SAHA or vehicle before recording functional data. Skinned myocytes were isolated from AFVM and human trabeculae to assess myofilament function. SAHA-treated AFVM had increased contractility and improved relaxation kinetics but no difference in peak calcium transients, with increased calcium sensitivity and decreased passive stiffness of myofilaments. Mass spectrometry analysis revealed increased acetylation of the myosin regulatory light chain with SAHA treatment. SAHA-treated human trabeculae had decreased diastolic tension and increased developed force. Myofilaments isolated from human trabeculae had increased calcium sensitivity and decreased passive stiffness. These findings suggest that SAHA has an important role in the direct control of cardiac function at the level of the cardiomyocyte and myofilament by increasing myofilament calcium sensitivity and reducing diastolic tension.