Binding Of Myosin To Actin Research Articles

Within Thermal Scales: The Kinetic and Energetic Pull of Chemical Entropy.

Biological systems are fundamentally containers of thermally fluctuating atoms that through unknown mechanisms are structurally layered across many thermal scales from atoms to amino acids to primary, secondary, and tertiary structures to functional proteins to functional macromolecular assemblies and up. Understanding how the irreversible kinetics (i.e., the arrow of time) of biological systems emerge from the equilibrium kinetics of constituent structures defined on smaller thermal scales is central to describing biological function. Muscle's irreversible power stroke - with its mechanochemistry defined on both the thermal scale of muscle and the thermal scale of myosin motors - provides a clear solution to this problem. Individual myosin motors function as reversible force-generating switches induced by actin binding and gated by the release of inorganic phosphate, P i . As shown in a companion article, when N individual switches thermally scale up to an ensemble of N switches in muscle, the entropy of a binary system of switches is created. We have shown in muscle that a change in state of this binary system of switches entropically drives actin-myosin binding (the switch) and muscle's irreversible power stroke, and that this simple two-state model accurately accounts for most key aspects of muscle contraction. Extending this observation beyond muscle, here I show that the chemical kinetics of an ensemble of N molecules differs fundamentally from a conventional chemical analysis of N individual molecules, describing irreversible chemical reactions as being pulled into the future by the a priori defined entropy of a binary system rather than being pushed forward by the physical occupancy of chemical states (e.g., mass action).

Act88F pseudo-acetylation of lysine 50 (K50) negatively regulates muscle function in Drosophila

Non-histone post-translational modifications (PTMs) are an increasingly important area of study that contribute to our understanding for protein function in the control of health and disease. Protein PTMs, such as acetylation, are emerging as key regulators of striated muscle function, yet our understanding of how non-histone protein acetylation affects muscle physiology remains fragmentary. Previous reports from our lab identified lysine (K) 52 of skeletal muscle alpha actin (ACTA1) to be acetylated, yet no reports have shown a role for ACTA1 acetylation in muscle function. Act88F is 93% homologous to ACTA1 and K50 is identical to K52 of ACTA1. Thus, for these studies, we mutated K50 with a glutamine (Q) on the Act88F gene in the indirect flight muscle (IFM) of Drosophila to mimic acetylation. We examined physiological function (i.e. flight and climbing) as well as biophysical changes between acetylated Act88F and the associated myofibrillar proteins involved in contraction and relaxation (myosin, tropomyosin, and troponin). We report that Act88F K50Q pseudo-acetylation resulted in a flightless phenotype and decreased climbing ability. In addition, we report that Act88F K50Q flies had increased IFM damage as examined via fluorescent imaging. Interestingly, Act88F K50Q did not change actin-myosin binding, actin sliding velocity, or Ca2+ sensitivity in actin-motility assays. However, Act88F pseudo-acetylation did decrease actin filament length, but only after interacting with myosin, implying that Act88F acetylation increases actin filament breaking. These data suggest that Act88F acetylation at K50 worsens muscle function by destabilizing filamentous actin, resulting in muscle tearing and damage to the IFM, which results in loss of flight. This work suggests that actin acetylation, at least on lysine 50, in response to environmental stressors or diet could greatly change muscle function and sarcomere integrity. This research was funded by the Dennis Meiss & Janet Ralston Fund for Nutri-epigenetic Research, the National Institute for General Medical Sciences (NIGMS) of the NIH (P20 GM130459), the National Heart, Lung, and Blood Institute of the NIH (R15 HL143496), National Institute of Aging of the NIH (R21 AG077248) and NSF EPSCOR Track II (OIA-1826801) to B.S.F. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Myosin inhibitor reverses hypertrophic cardiomyopathy in genotypically diverse pediatric iPSC-cardiomyocytes to mirror variant correction

Pathogenic variants in MYH7 and MYBPC3 account for the majority of hypertrophic cardiomyopathy (HCM). Targeted drugs like myosin ATPase inhibitors have not been evaluated in children. We generate patient and variant-corrected iPSC-cardiomyocytes (CMs) from pediatric HCM patients harboring single variants in MYH7 (V606M; R453C), MYBPC3 (G148R) or digenic variants (MYBPC3 P955fs, TNNI3 A157V). We also generate CMs harboring MYBPC3 mono- and biallelic variants using CRISPR editing of a healthy control. Compared with isogenic and healthy controls, variant-positive CMs show sarcomere disorganization, higher contractility, calcium transients, and ATPase activity. However, only MYH7 and biallelic MYBPC3 variant-positive CMs show stronger myosin-actin binding. Targeted myosin ATPase inhibitors show complete rescue of the phenotype in variant-positive CMs and in cardiac Biowires to mirror isogenic controls. The response is superior to verapamil or metoprolol. Myosin inhibitors can be effective in genotypically diverse HCM highlighting the need for myosin inhibitor drug trials in pediatric HCM.

Abstract 17019: The Missense W792R Mutation in C6 Domain of Cardiac Myosin Binding Protein-C Alters Morphology and Contractility in Mouse Myocardium

Background: Mutations in cardiac myosin binding protein C (cMyBP-C) are a common cause of hypertrophic cardiomyopathy (HCM) in humans. While many known cMyBP-C mutations lead to haploinsufficiency, others lead to amino acid substitutions which may impact protein function in unique ways The W792R mutation introduces a charge change and is known to be pathogenic in humans. This mutation, located remotely from both the regulatory amino terminus, and the C-terminus, has an unknown impact on the final protein structure and function. Goal: Generate a murine knock-in model to fully characterize the impact of the W792R mutation on cardiac morphology and function in vivo. Methods: We generated a W792R knock-in (KI) mouse and produced heterozygous (HT) and homozygous (KI) offspring, and wild-type (WT) littermate controls. We performed histology, RNA and protein expression analyses, and mechanics on isolated trabeculae at postnatal day 12 (KI n=5; HT n=3; and LC n=4). One-way ANOVA used to compare KI and HT to WT samples, with p-values < ; 0.05 as significant. Results: The KI mice did not live past ~20 days old with massive hypertrophy, dilated atrium, myofibrillar disarray and fibrosis. Full-length mutant protein was expressed and incorporated into the sarcomere. Survival and morphology in the HT was similar to WT. Post-natal day 10 KI mice demonstrated severely enlarged atria and ventricles, interstitial fibrosis, a trend towards increased Ca 2+ sensitivity (pCA 50 KI 5.8 vs HT 5.65 vs LC 5.67 mM; p > 0.05), a trend towards increased cross-bridge cycling kinetics (k tr KI 24.00 + 2.42 vs HT 24.33 + 1.08 vs LC 21.25 + 0.87; p > 0.05) and increased levels of β-myosin heavy chain expression compared to both LC and HT mice. Conclusions: In our mouse HCM model, homozygous expression of the W792R-cMyBP-C mutation recapitulates severe human HCM and provides mechanistic insights into the pathology of this mutation. The trend towards accelerated crossbridge kinetics suggest that the W792R mutation disrupts the inhibitory effects of cMyBP-C on myosin-actin binding implicating the potential application of current myosin ATPase inhibitors in the treatment of patients carrying this mutation. Ongoing work will expand sample sizes to evaluate significance of findings.

Characterizing the functional impact of embryonic myosin mutations in stem cell-derived skeletal muscle.

Divergent contractile properties of different myosins suggest that each isoform performs distinct functions in embryonic and adult skeletal muscles. To better understand the role of embryonic myosin heavy chain 3 (MYH3) in regulating skeletal muscle pattern formation, growth and regeneration, our group investigated the functional defects caused by multiple mutations. Our team generated human induced pluripotent stem cell (iPSC) lines bearing T178I or R672C mutations in MYH3. Human iPSCs bearing these mutations were differentiated into skeletal muscle and evaluated for differences in the structure, maturation and functional performance of the sarcomere. Isolated myofibril mechanics showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to controls. Myofibril preparations were also used to compare ATP binding rates and showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. When incorporated into 3D engineered tissues, these changes in MYH3 mechanics resulted in slower relaxation kinetics and an incomplete relaxation of the tissue in response to repetitive tetanic stimulation, which may explain the emergence of developmental contractures in patients possessing MYH3 mutations. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the β-sheet and SH-Helix than in controls. Mutations also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

Embryonic myosin mutations alter the mechanics and maturation of HiPSC derived skeletal muscle.

Distal arthrogryposis (DA) is a skeletal muscle disorder characterized by joint contractures predominantly localized in the distal extremities. DA associated syndromes like Freeman-Sheldon syndrome (FSS) are linked to mutations in the MYH3 gene that encodes the embryonic skeletal muscle myosin. To study the mutation and its effect on developing muscle, we generated human induced pluripotent stem cell (hiPSC) lines bearing T178I or R672C MYH3 mutations. hiPSC were differentiated into skeletal muscle and evaluated for differences in the maturation of the contractile unit and its functional performance. Preliminary myofibril mechanics measurements showed T178I myotubes generated significantly greater specific force compared to control myofibrils. Diseased myofibrils also expressed significantly slower rates of relaxation and prolonged thin filament deactivation compared to control myofibrils. Myofibril preparations were also used to compare ATP binding rates across conditions. Preliminary results showed homozygous R672C myofibrils have faster ATP binding rates compared to heterozygous and control preparations. Mutations in MYH3 were also associated with alterations in myosin isoform content, with homozygous R672C having reduced MYH3 levels in day 7 cells. Molecular dynamics simulations of R672C and T178I mutations in the post rigor state showed greater separation between the beta-sheet and SH-helix than in controls. Mutation also disrupted local salt bridges and hydrogen bond formation in some residues that help stabilize the nucleotide binding pocket and converter-connected helix. Interrupted structural communication between the nucleotide binding pocket and surrounding functional regions may underly the impaired relaxation phenotype. Ongoing studies will further characterize the effect of mutation on actin-myosin binding, crossbridge cycling kinetics, the maturation of skeletal myotubes over time, and myosin isoform switching dynamics.

A rational approach to selective targeting of skeletal myosin.

Altered actin-myosin binding in the sarcomere has been linked to congenital skeletal and cardiac myopathies characterised by muscular weakness.1Since the discovery of omecamtiv mecarbil (OM)2, the direct targeting of the myosin II motor protein has been considered as a promising strategy to restore normal muscle function. While there has been success in the development of cardiac myosin drugs, with one of them recently achieving FDA approval,3 selective targeting of skeletal myosin has been less explored. Here, we used a range of molecular modelling techniques to compare the structure and dynamics of the OM binding site in cardiac and in fast type IIa skeletal myosin.4 We found important differences in the shape and size of the binding pocket in the two isoforms, which we relate to small but significant differences in its amino acidic composition. Moreover, inclusion of protein dynamics turned out to be essential to explain the observed selectivity of OM for the cardiac isoform. Information from our modelling is currently being used to identify novel compounds that can selectively bind skeletal myosin, and potentially treat congenital myopathies where muscle weakness is related to myosin loss-of-function.

Abstract 15182: Recruiting Super-Relaxed Myosin to Restore Contractility in Human Right Heart Failure

Intro: Right ventricular (RV) dysfunction worsens outcomes in pulmonary hypertension (PH) due to heart failure with reduced ejection fraction (HF), yet underlying mechanisms are unknown. We previously reported RV myofilament max calcium-activated tension (T max ) is reduced in human PH-HF. Here, we reveal that this decline is due to more myosin heads in the super-relaxed (SRX) state which lack force-production due to low ATP turnover. Methods: Frozen RV myocardium from 25 PH-HF and 9 control patients were permeabilized and myocyte mechanics and small angle X-ray diffraction measured. Results: RV T max (16±4 vs. 23±3 mN/mm 2 , p<1x10 -15 ) and peak power (0.06±0.01 vs. 0.11±0.03, p=7.9x10 -5 ) were 50% lower in HF. Unsupervised machine learning (infinite mixture Dirichlet process) using mechanics identified two HF subgroups distinguished by RV T max : Group 1 with slightly reduced T max (19±1 mN/mm 2 , p=3.8x10 -6 ) and Group 2 with 40% reduced T max (14±3 mN/mm 2 , p<1x10 -15 ), with Group 2 exhibiting clinically overt RV failure vs. Group 1 (right atrial pressure, RAP 14±6 vs. 9±4, p=0.01; RAP/pulmonary capillary wedge ratio 0.7±0.3 vs. 0.4±0.2, p=0.047). To test if low RV T max was associated with more SRX myosin, X-ray diffraction patterns from RV myofibers were used to determine the equatorial intensity ratio (I 1,1 /I 1,0 ), an indicator of myosin proximity to the thick filament backbone. The ratio was lower (more SRX), which was associated with reduced myosin ATP turnover in Group 2 vs. both 1 (p=9.0x10 -7 ) and controls (p=1.8x10 -9 ). We then tested the effect of SRX on T max by perturbing the proportion of SRX myosin using mavacamten (M), a myosin inhibitor that stabilizes the SRX state, and deoxy-ATP (dATP), a nucleotide that destabilizes SRX. In group 2, dATP augmented T max by 30% (p=0.0002) while M had no effect (p=0.1); meanwhile in group 1, M suppressed T max by 30% (p=0.0002) while dATP had no effect (p=0.9). Finally, a myosin activator EMD-57033, which increases probability of actin-myosin binding, augmented T max (correlation between ΔSRX-ΔT max r=-0.55, p=0.01). Conclusion: We find increased SRX myosin contributes to RV myofilament and chamber-level failure in PH-HF. Small molecules that recruit SRX myosin may have therapeutic benefit in patients with RV dysfunction.

Skeletal muscle alpha actin (ACTA1) acetylation negatively regulates muscle function in response to obesity

Post‐translational modifications (PTMs) of proteins are an increasingly important area of study that contributes to our understanding for protein function in the control of health and disease. Acetylation, for example, has long been associated with histone modifications to control gene expression, yet emerging omics data demonstrates that acetylation occurs across the proteome. Our most recent work further shows global shifts in non‐histone protein acetylation within the left ventricle (LV) in response to diet‐induced obesity. Despite these advances in omics technologies, our understanding for how non‐histone protein acetylation regulates cardiac biology remains a ‘black box’. In this study, we examined the functional importance for skeletal muscle alpha actin (ACTA1) acetylation in the control of muscle function. For this, we used actin‐motility assays to examine the biophysical changes between acetylated ACTA1 and the other myofibrillar proteins involved in cardiac contraction and relaxation (e.g. myosin, tropomyosin). Here, we show that ACTA1 acetylation increased actin‐myosin binding, decreased actin sliding velocity and increased Ca2+ sensitivity. These data suggest that ACTA1 acetylation significantly alters muscle function. Of note, we previously published that lysine (K) residue 50 (K50) of ACTA1 was acetylated, similar to other proteomics reports. Interestingly, the positively charged K50 on ACTA1 sits juxtaposed to the negatively charged aspartic acid (D) residue 391 of the cardiac myosin binding protein (cMy‐BP). cMy‐BP is essential for actin‐myosin interactions, and this suggests an important role for ACTA1 K50 acetylation in the control of muscle contraction. Thus, we knocked out endogenous actin (Act88F) from the indirect flight muscles (IFMs) of D. melanogaster and inserted an Act88F K50Q mutant, which mimics acetylation specifically at this site. Previous reports have shown that IFMs behave similarly to cardiac muscle. Phenotypically, the K50Q flies behave similar to their wild‐type counterparts. However, current studies are underway to examine changes in flight as well as purify the K50Q actin for actin‐motility assays to examine biophysical changes in myofibrillar proteins. Combined, our data suggests that ACTA1 acetylation negatively impacts muscle function in response to obesity, current work will determine the implications for K50 actin acetylation in this phenotype.

A Novel Missense Variant in Actin Binding Domain of MYH7 Is Associated With Left Ventricular Noncompaction.

Cardiomyopathies are a group of common heart disorders that affect numerous people worldwide. Left ventricular non-compaction (LVNC) is a structural disorder of the ventricular wall, categorized as a type of cardiomyopathy that mostly caused by genetic disorders. Genetic variations are underlying causes of developmental deformation of the heart wall and the resultant contractile insufficiency. Here, we investigated a family with several affected members exhibiting LVNC phenotype. By whole-exome sequencing (WES) of three affected members, we identified a novel heterozygous missense variant (c.1963C>A:p.Leu655Met) in the gene encoding myosin heavy chain 7 (MYH7). This gene is evolutionary conserved among different organisms. We identified MYH7 as a highly enriched myosin, compared to other types of myosin heavy chains, in skeletal and cardiac muscles. Furthermore, MYH7 was among a few classes of MYH in mouse heart that highly expresses from early embryonic to adult stages. In silico predictions showed an altered actin-myosin binding, resulting in weaker binding energy that can cause LVNC. Moreover, CRISPR/Cas9 mediated MYH7 knockout in zebrafish caused impaired cardiovascular development. Altogether, these findings provide the first evidence for involvement of p.Leu655Met missense variant in the incidence of LVNC, most probably through actin-myosin binding defects during ventricular wall morphogenesis.

Evaluation of ultrasound-assisted L-histidine marination on beef M. semitendinosus: Insight into meat quality and actomyosin properties

This paper aimed to evaluate the effects of ultrasound-assisted L-histidine marination (UMH) on meat quality and actomyosin properties of beef M. semitendinosus. Our results found that UMH treatment effectively avoided excessive liquid withdrawal, and disrupted myofibril integrity by modifying the water distribution and weakening connection of actin-myosin with increased muscle pH. The ultrasound-treated sample provided more opportunity for the filtration of L-histidine to intervene the isoelectric point and conformation of muscle protein. The activated caspase-3 and changes of ATPase activity in UMH-treated meat accelerated the postmortem ageing, and L-histidine might competitively inhibit the actin-myosin binding by the imidazole group. UMH decreased the surface hydrophobicity by shielding hydrophobic area and unfolding the actomyosin structure. In addition, the increased actomyosin solubility with smaller particle size enhanced the SH content for better cross-linking of myosin tail, and formation of heat-set gelling protein structure. Therefore, UMH treatment manifested the potential to improve beef quality.

The Central Role of the F-Actin Surface in Myosin Force Generation.

Simple SummaryAlthough actin is a highly conserved protein, it is involved in many diverse cellular processes. Actin owes its diversity of function to its ability to bind to a host of actin-binding proteins (ABPs) that localize across its surface. Among the most studied ABPs is the molecular motor, myosin. Myosin generates force on actin filaments by pairing ATP hydrolysis, product release, and actin-binding to the conformational changes that lead to movement. Central to this process is the progression of myosin binding to the actin surface as it moves through its ATPase cycle. During binding, actin acts as a myosin ATPase activator, catalyzing essential hydrolysis release steps. Here, we use the current model of actin-myosin binding as a roadmap to describe the portions of the actin-myosin interface that are sequentially formed throughout the motor cycle. At each step, we compare the interactions of a diverse set of high-resolution actin-myosin cryo-electron microscopy structures to define what portions of the interface are conserved and which are isoform-specific.Actin is one of the most abundant and versatile proteins in eukaryotic cells. As discussed in many contributions to this Special Issue, its transition from a monomeric G-actin to a filamentous F-actin form plays a critical role in a variety of cellular processes, including control of cell shape and cell motility. Once polymerized from G-actin, F-actin forms the central core of muscle-thin filaments and acts as molecular tracks for myosin-based motor activity. The ATP-dependent cross-bridge cycle of myosin attachment and detachment drives the sliding of myosin thick filaments past thin filaments in muscle and the translocation of cargo in somatic cells. The variation in actin function is dependent on the variation in muscle and non-muscle myosin isoform behavior as well as interactions with a plethora of additional actin-binding proteins. Extensive work has been devoted to defining the kinetics of actin-based force generation powered by the ATPase activity of myosin. In addition, over the past decade, cryo-electron microscopy has revealed the atomic-evel details of the binding of myosin isoforms on the F-actin surface. Most accounts of the structural interactions between myosin and actin are described from the perspective of the myosin molecule. Here, we discuss myosin-binding to actin as viewed from the actin surface. We then describe conserved structural features of actin required for the binding of all or most myosin isoforms while also noting specific interactions unique to myosin isoforms.

Can a chloride channel blocker mitigate muscle fatigue?

Can a chloride channel blocker mitigate muscle fatigue?

Saturation of Actin-Myosin Kinetics and Mechanics

Actin-myosin kinetics and mechanics are well-characterized at the level of individual myosin molecules; however, the collective behavior of an ensemble of myosin molecules differs significantly from that of the sum of single myosin molecules. For instance, actin movement generated by one myosin molecule is associated with actin-myosin binding, or attachment, whereas actin movement generated by many myosin molecules, N, is influenced by actin-myosin detachment. Major gaps exist in our understanding of the basic chemical kinetics underlying ensemble myosin mechanics.

Collective Force Generator Model of Muscle Contraction

Muscle contraction results from cyclic interactions of actin-myosin binding coupled to the catalyzed hydrolysis of ATP. Although the mechanics and kinetics of muscle myosins are well established, it is less evident how these single molecule behaviors scale up to the collective behaviors of many myosin molecules working together. We have developed a computer simulation of collective force generation by myosin molecules based on well-established kinetic and mechanical properties of single myosin molecules and use an in-vitro motility assay to test model predictions. A collective force model predicts that even in in vitro motility assays myosin heads collectively generate force in common compliant elements and at sufficiently high numbers, N, reach a stall force Consistent with our collective force model, we show that ATPase activity in a motility assay increases linearly at low myosin density, saturating with actin sliding velocities, V, at high myosin densities. We show that activation of V upon addition of inorganic phosphate, Pi, results from Pi decreasing the auto-inhibiting collective force, which in turn activates the ATPase activity. Collective force generation by myosin results in unexpected emergent mechanical behaviors that offer new interpretations for how muscle works.

Abstract MP102: Skeletal Muscle Alpha Actin Acetylation Enhances Myosin Binding and Increases Calcium Sensitivity in vitro

Actin and myosin are key proteins for muscle contraction/relaxation, while tropomyosin regulates actin-myosin interactions in the presence or absence of calcium. Mutations or dysregulation of any of these proteins results in cardiomyopathy. We previously showed that skeletal muscle alpha actin (ACTA1) was significantly deacetylated on lysine residues, K52, K317, and K328 in remodeled left ventricular tissue of obese mice. Computational modeling suggests that the positively charged lysine residues K328 and K317 of ACTA1 can interact electrostatically with the negatively charged glutamic acid residue E181 of tropomyosin and E286 of myosin. As acetylation is predicted to neutralize the positively charged lysine, ACTA1 acetylation would be postulated to decrease actin-myosin or actin-tropomyosin electrostatic interactions. To test this hypothesis, we used an in vitro actin motility assay to determine myosin sliding velocity, calcium sensitivity, and attachment/detachment kinetics of acetylated/deacetylated ACTA1. In addition, an actin binding protein spin-down assay was used to determine actin-myosin binding affinity using skeletal and cardiac myosin. In these assays, ACTA1 was chemically acetylated with acetic anhydride. In vitro actin motility analysis showed a significant decrease in sliding velocity with acetylated ACTA1 and skeletal myosin (1.709±0.210 μm/s) compared with deacetylated ACTA1 (4.427±0.275 μm/s). A similar significant decrease was also noted with cardiac myosin. Further analysis showed a significant increase in calcium sensitivity with ACTA1 acetylation (3.197x10-7 Kd compared to deacetylated ACTA1 1.191x10-6 Kd) and a loss of tropomyosin regulation with increasing ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced actin binding affinity to cardiac and skeletal myosin. Investigation of attachment/detachment kinetics are currently underway. These data suggest that ACTA1 acetylation disrupts tropomyosin’s ability to inhibit myosin binding in the absence of calcium and further regulates actin-myosin interactions. Lastly, these data highlight acetylation as an additional post-translational modification outside of phosphorylation in the regulation of muscle contraction.

Diabetes with Heart Failure Increases Methylglyoxal Modifications in the Sarcomere Which Inhibit Function

Methylglyoxal (MG), a glycolysis byproduct, is significantly elevated in diabetes and modifies proteins by reacting with arginine and lysine residues to form irreversible carbonyl adducts. The effect of MG on the cardiac myofilament is explored here. Myofilament was enriched from LV tissue from patients who had diabetes and heart failure (dbHF), dilated cardiomyopathy (DCM), and non-failing (NF) control hearts. dbHF patients exhibited a ∼40% increase in MG-modifications on myofilament proteins compared to NF and DCM hearts. Skinned myocytes from NF hearts exhibited decreased Ca2+ sensitivity when exposed to MG, while myocytes from dbHF patients were resistant, suggesting dbHF myocytes were already affected by MG-induced decreased myofilament function. Mass spectrometry on LV tissue identified novel MG-modifications on actin and myosin that suggested MG might depress actin-myosin binding. Cosedimentation of myosin with bare actin showed MG had no effect on their direct binding. However, myosin cosedimentation with native thin filaments (NTF) was decreased with MG, suggesting that actin-myosin binding is depressed only when tropomyosin and the troponin complex are present. Tropomyosin/troponin were directly assed for MG-modifications by MS and none were found. The in vitro motility assay showed MG depressed Ca2+-sensitivity when either NTF or myosin were independently exposed. These data suggest MG-modifications on either actin or myosin depress function by interfering with how these proteins interact with tropomyosin or the troponin complex. Given the increased MG-modifications in dbHF patients, we hypothesized they may be pathogenic. Indeed, in a pre-diabetic mouse model (high fat diet for 12 weeks), while there were no overt health issues, there was a 17% increase in myofilament MG-modifications. Overall, these results show a potential underlying mechanism by which diabetes causes heart failure through MG-modifications of the myofilament, having important implications for treatment and/or prevention.

Development of an Integrated Human Model of Electrophysiology and Actin-Myosin Binding to Describe Sarcomere Force Generation in Cardiomyocytes

We have developed an integrated human model which links detailed sub-models of sarcomere electrophysiology and actin-myosin kinetics to describe sarcomere force generation. While many published quantitative models connect cardiac electrophysiology to force generation or full heart mechanics, these models typically include a lumped representation of actin-myosin mechanics without sufficient granularity. In order to mechanistically represent both heart failure conditions and treatment strategies which operate at the actin-myosin level, we have developed an integrated multiscale model of sarcomere force generation. Our platform connects published electrophysiology and lumped force generation models to an actin-myosin (AM) model developed based on published data on cardiac myosin modulators. Specifically, the AM model was calibrated to published in vitro data of phosphate release and ATP turnover, and can successfully reproduce the molecular effect of cardiac myosin modulation. We connected this in vitro model with healthy and heart failure phenotypes to describe cardiac force generation before and after therapy. With this multiscale approach we can also simulate sarcomere length-dependent activation, calcium-force relationships, and determine how these simulations are affected in failing myocardium after treatment with cardiac modulators. We demonstrate that models calibrated to data at one scale often need to be adjusted in the context of a multiscale model. For example, the calcium transient produced from a published heart failure electrophysiology model generates little force in the integrated multiscale model, underscoring the continuing challenge of linking in vitro experimental efforts to clinical outputs. We discuss our methods to connect information across scales into one coherent mathematical framework. The final model describes how specific therapeutic interventions at different stages of the actin-myosin cycle will alter sarcomere force generation, which will propagate to changes in clinical outcomes like ejection fraction.

Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition

Introduction: For the past decades long-term survival of oral squamous cell carcinoma (OSCC) patients remain unchanged which provides a wide platform for research. Caldesmon (CaD), an actin-myosin binding protein, has a contributing role in cytoskeleton modulation and cell motility. The aim of this study is to evaluate the expression of CaD in metastatic and non-metastatic OSCC and to discuss the possible role of CaD in epithelial mesenchymal transition. Materials and Methods: Archival blocks of 25 metastatic and 25 non-metastatic OSCC patients are included where CaD expression is evaluated immunohistochemically. Results: Overall expression and staining intensity of CaD were statistically significant in different grades of metastatic and non-metastatic OSCC. In summary, high expression of CaD is observed in increasing grades of OSCC but the metastatic potential of the tumour doesn’t seem to have any relation with CaD.

Mecanismos regulatorios del tono vascular pulmonar neonatal. Una perspectiva molecular

The extrauterine-milieu adaptation includes a considerable increase in PaO2, that specifically induces structural and vasoactive changes at pulmonary circulation. Such changes transform a poor irrigated circulation into a circulation that receive ∼100% of neonatal cardiac output, supporting the normal alveolar-capillary gas exchange. Local regulation of basal neonatal pulmonary circulation is maintaining by a delicate equilibrium between vasoconstrictor and vasodilator agents. This equilibrium, allows to maintain the pulmonary circulation as an hemodynamic system with a high blood flow and a low vascular resistance. Vasocontrictors action allows actin and light-chain myosin interaction. Two main pathways induced this effect in smooth muscle cell: a) a calcium dependent pathway, that increases intracellular calcium, facilitating actin – myosin binding, and b) the independent calcium pathway, which achieves through consecutive phosphorylation reactions sensitize the proteins involved, promoting the binding of actin and light-chain myosin. These actions are mediated by agonists produced mainly in the pulmonary endothelium, such as endothelin-1 and thromboxane, or by agonists from other cell types such as serotonin. Vasodilator agents regulate the vasoconstrictor response, mainly by inhibiting signals that induce calcium-independent vasoconstriction, through activation of protein kinases, which in turn will inhibit the function of ROCK kinase, one of the last effectors of vasoconstriction before formation of the actin and light-chain myosin binding. This review will focus on describing these mechanisms of primal importance in the first hours of our lives as independent individuals.