Cardiac resynchronization therapy using bi-ventricular pacing is proven effective in the management of heart failure (HF) with a wide QRS-complex. In the absence of QRS prolongation, however, device-based resynchronization is reported unsuitable. As an alternative, the present study tests a regenerative cell-based approach in the setting of narrow QRS-complex HF.

Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an "anomalous," nonmonotonic mole-fraction dependence in the presence of certain sodium-potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac's preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation-likely steric-associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K(+) for one Na(+) in the wild-type (WT) channel, increasing the relative likelihood of Na(+) occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na-K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels.

Contractile discordance exacerbates cardiac dysfunction, aggravating heart failure outcome. Dissecting the genesis of mechanical dyssynchrony would enable an early diagnosis before advanced disease.

Endothelial to mesenchymal transition (EndMT) plays a major role during development, and also contributes to several adult cardiovascular diseases. Importantly, mesenchymal cells including fibroblasts are prominent in atherosclerosis, with key functions including regulation of: inflammation, matrix and collagen production, and plaque structural integrity. However, little is known about the origins of atherosclerosis-associated fibroblasts. Here we show using endothelial-specific lineage-tracking that EndMT-derived fibroblast-like cells are common in atherosclerotic lesions, with EndMT-derived cells expressing a range of fibroblast-specific markers. In vitro modelling confirms that EndMT is driven by TGF-β signalling, oxidative stress and hypoxia; all hallmarks of atherosclerosis. 'Transitioning' cells are readily detected in human plaques co-expressing endothelial and fibroblast/mesenchymal proteins, indicative of EndMT. The extent of EndMT correlates with an unstable plaque phenotype, which appears driven by altered collagen-MMP production in EndMT-derived cells. We conclude that EndMT contributes to atherosclerotic patho-biology and is associated with complex plaques that may be related to clinical events.

Na(V)1.5 is a mechanosensitive voltage-gated Na(+) channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na(+) current and delayed rectifier (I(Kr)) currents. Recently, ranolazine was also shown to be an inhibitor of Na(V)1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na(+) current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na(+) current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.

Cardiomyocyte calcium overloading has been implicated in the pathogenesis of Duchenne muscular dystrophy (DMD) heart disease. The cardiac isoform of sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a major role in removing cytosolic calcium during heart muscle relaxation. Here, we tested the hypothesis that SERCA2a over-expression may mitigate electrocardiography (ECG) abnormalities in old female mdx mice, a murine model of DMD cardiomyopathy.

Prolongation of the action potential duration (APD) has consistently been observed in experimental models of cardiac hypertrophy and failure as well as in humans and is partially attributed to a reduction of a hyperpolarizing current provided by the calcium-independent transient outward K(+) channel (I(to)). In the present study, we examined the effects of manipulating ion channel currents (I(to) and sodium/calcium exchanger (NCX)) and the associated alterations in action potential duration on cardiomyocyte hypertrophy and signaling induced by angiotensin II (AngII). Our aim was to examined whether distinct patterns of intracellular calcium manipulation could generate distinct patterns of MAPkinase activation and cellular hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) were infected with Ad. beta-gal/GFP, Ad.Kv4.3, Ad.Kv4.3 antisense or Ad.NCX adenoviruses and hypertrophy induced by incubation with AngII. Overexpression of Kv4.3 increased I(to) density, shortened APD, decreased Ca(2+) influx and inhibited AngII-induced (3)H-leucine incorporation and ANF and beta-MHC expression. These hypertrophic changes were also paralleled by blockade of ERK MAP kinases activation as well as calcineurin expression. These electrical and hypertrophic changes produced by overexpression of Kv4.3 were completely and significantly reversed by Kv4.3 antisense and NCX gene transfer. Our findings indicate that AngII-mediated hypertrophy response in NRVMs can be abrogated by an enhancement of I(to) function through overexpression of Kv4.3 and that modulation of action potential duration can be important in the development of cardiac hypertrophy.

Embryonic stem cells possess a pluripotent transcriptional background with the developmental capacity for distinct cell fates. Simultaneous expression of genetic elements for multiple outcomes obscures cascades relevant to specific cell phenotypes. To map molecular patterns critical to cardiogenesis, we interrogated gene expression in stem cells undergoing guided differentiation, and defined a genomic paradigm responsible for confinement of pluripotency.

Response to stem cell therapy in heart failure is heterogeneous, warranting a better understanding of outcome predictors. This study assessed left ventricular volume, a surrogate of disease severity, on cell therapy benefit. Small to large infarctions were induced in murine hearts to model moderate, advanced, and end-stage ischemic cardiomyopathy. At 1 month postinfarction, cardiomyopathic cohorts with comparable left ventricular enlargement and dysfunction were randomized 1:1 to those that either received sham treatment or epicardial delivery of cardiopoietic stem cells (CP). Progressive dilation and pump failure consistently developed in sham. In comparison, CP treatment produced significant benefit at 1 month post-therapy, albeit with an efficacy impacted by cardiomyopathic stage. Advanced ischemic cardiomyopathy was the most responsive to CP-mediated salvage, exhibiting both structural and functional restitution, with proteome deconvolution substantiating that cell therapy reversed infarction-induced remodeling of functional pathways. Moderate cardiomyopathy was less responsive to CP therapy, improving contractility but without reversing preexistent heart enlargement. In end-stage disease, CP therapy showed the least benefit. This proof-of-concept study thus demonstrates an optimal window, or "Goldilocks principle," of left ventricular enlargement for maximized stem cell-based cardiac repair. Disease severity grading, prior to cell therapy, should be considered to inform regenerative medicine interventions.

Cardiopoietic stem cells have reached advanced clinical testing for ischemic heart failure. To profile their molecular influence on recipient hearts, systems proteomics was here applied in a chronic model of infarction randomized with and without human cardiopoietic stem cell treatment. Multidimensional label-free tandem mass spectrometry resolved and quantified 3987 proteins constituting the cardiac proteome. Infarction altered 450 proteins, reduced to 283 by stem cell treatment. Notably, cell therapy non-stochastically reversed a majority of infarction-provoked changes, remediating 85% of disease-affected protein clusters. Pathway and network analysis decoded functional reorganization, distinguished by prioritization of vasculogenesis, cardiac development, organ regeneration, and differentiation. Subproteome restoration nullified adverse ischemic effects, validated by echo-/electro-cardiographic documentation of improved cardiac chamber size, reduced QT prolongation and augmented ejection fraction post-cell therapy. Collectively, cardiopoietic stem cell intervention transitioned infarcted hearts from a cardiomyopathic trajectory towards pre-disease. Systems proteomics thus offers utility to delineate and interpret complex molecular regenerative outcomes.

Tendon injuries are among the most common and severe hand injuries with a high demand for functional recovery. Stem cells have been identified and isolated from different species and a variety of tissues for the sake of regenerative medicine. Recently, turkey has been suggested as a potential new large animal model for flexor tendon-related research. However, turkey tissue-specific stem cells have not been investigated. Here, we presented the isolation and verification of tendon-derived stem cells (TDSCs) from 6- to 8-month-old heritage-breed turkey. TDSCs were isolated from turkey flexor tendon by plating nucleated cells at the determined optimal density. Approximately 4% of the nucleated cells demonstrated clonogenicity, high proliferation rate, and trilineage differentiation potential after induction culturing. These cells expressed surface antigens CD90, CD105, and CD44, but did not express CD45. There was a high level of gene expression of tenogenic markers in TDSCs, including mohawk, collagen type I, tenascin C, and elastin. Turkey TDSCs also expressed transcription factors PouV, Nanog, and Sox2, which are critically involved in the regulation of stemness. The successful isolation of tendon-derived stem cells from turkey was beneficial for future studies in tendon tissue engineering and would help in the development of new treatment for tendon diseases using this novel animal model.

Stem cell paracrine activity is implicated in cardiac repair. Linkage between secretome functionality and therapeutic outcome was here interrogated by systems analytics of biobanked human cardiopoietic cells, a regenerative biologic in advanced clinical trials. Protein chip array identified 155 proteins differentially secreted by cardiopoietic cells with clinical benefit, expanded into a 520 node network, collectively revealing inherent vasculogenic properties along with cardiac and smooth muscle differentiation and development. Next generation RNA sequencing, refined by pathway analysis, pinpointed miR-146 dependent regulation upstream of the decoded secretome. Intracellular and extracellular integration unmasked commonality across cardio-vasculogenic processes. Mirroring the secretome pattern, infarcted hearts benefiting from cardiopoietic cell therapy restored the disease proteome engaging cardiovascular system functions. The cardiopoietic cell secretome thus confers a therapeutic molecular imprint on recipient hearts, with response informed by predictive systems profiling.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

The intrinsic healing following tendon injury is ideal, in which tendon progenitor cells proliferate and migrate to the injury site to directly bridge or regenerate tendon tissue. However, the mechanism determining why and how those cells are attracted to the injury site for tendon healing is not understood. Since the tenocytes near the injury site go through apoptosis or necrosis following injury, we hypothesized that secretions from injured tenocytes might have biological effects on cell proliferation and migration to enhance tendon healing. Tenocyte apoptosis was induced by 24 h cell starvation. Apoptotic body-rich media (T-ABRM) and apoptotic body-depleted media (T-ABDM) were collected from culture media after centrifuging. Tenocytes and bone marrow-derived stem cells (BMDSCs) were isolated and cultured with the following four media: (1) T-ABRM, (2) T-ABDM, (3) GDF-5, or (4) basal medium with 2% fetal calf serum (FCS). The cell activities and functions were evaluated. Both T-ABRM and T-ABDM treatments significantly stimulated the cell proliferation, migration, and extracellular matrix synthesis for both tenocytes and BMDSCs compared to the control groups (GDF-5 and basal medium). However, cell proliferation, migration, and extracellular matrix production of T-ABRM-treated cells were significantly higher than the T-ABDM, which indicates the apoptotic bodies are critical for cell activities. Our study revealed the possible mechanism of the intrinsic healing of the tendon in which apoptotic bodies, in the process of apoptosis, following tendon injury promote tenocyte and stromal cell proliferation, migration, and production. Future studies should analyze the components of the apoptotic bodies that play this role, and, thus, the targeting of therapeutics can be developed.

LeuT-like fold Na-dependent secondary active transporters form a large family of integral membrane proteins that transport various substrates against their concentration gradient across lipid membranes, using the free energy stored in the downhill concentration gradient of sodium ions. These transporters play an active role in synaptic transmission, the delivery of key nutrients, and the maintenance of osmotic pressure inside the cell. It is generally believed that binding of an ion and/or a substrate drives the conformational dynamics of the transporter. However, the exact mechanism for converting ion binding into useful work has yet to be established. Using a multi-dimensional path sampling (string-method) followed by all-atom free energy simulations, we established the principal thermodynamic and kinetic components governing the ion-dependent conformational dynamics of a LeuT-like fold transporter, the sodium/benzyl-hydantoin symporter Mhp1, for an entire conformational cycle. We found that inward-facing and outward-facing states of Mhp1 display nearly the same free energies with an ion absent from the Na2 site conserved across the LeuT-like fold transporters. The barrier separating an apo-state from inward-facing or outward-facing states of the transporter is very low, suggesting stochastic gating in the absence of ion/substrate bound. In contrast, the binding of a Na2 ion shifts the free energy stabilizing the outward-facing state and promoting substrate binding. Our results indicate that ion binding to the Na2 site may also play a key role in the intracellular thin gate dynamics modulation by altering its interactions with the transmembrane helix 5 (TM5). The Potential of Mean Force (PMF) computations for a substrate entrance displays two energy minima that correspond to the locations of the main binding site S1 and proposed allosteric S2 binding site. However, it was found that substrate's binds to the site S1 ∼5 kcal/mol more favorable than that to the site S2 for all studied bound combinations of ions and a substrate.

Background The Purkinje network appears to play a pivotal role in the triggering as well as maintenance of ventricular fibrillation. Irreversible electroporation ( IRE ) using direct current has shown promise as a nonthermal ablation modality in the heart, but its ability to target and ablate the Purkinje tissue is undefined. Our aim was to investigate the potential for selective ablation of Purkinje/fascicular fibers using IRE . Methods and Results In an ex vivo Langendorff model of canine heart (n=8), direct current was delivered in a unipolar manner at various dosages from 750 to 2500 V, in 10 pulses with a 90-μs duration at a frequency of 1 Hz. The window of ventricular fibrillation vulnerability was assessed before and after delivery of electroporation energy using a shock on T-wave method. IRE consistently eradicated all Purkinje potentials at voltages between 750 and 2500 V (minimum field strength of 250-833 V/cm). The ventricular electrogram amplitude was only minimally reduced by ablation: 0.6±2.3 mV ( P=0.03). In 4 hearts after IRE delivery, ventricular fibrillation could not be reinduced. At baseline, the lower limit of vulnerability to ventricular fibrillation was 1.8±0.4 J, and the upper limit of vulnerability was 19.5±3.0 J. The window of vulnerability was 17.8±2.9 J. Delivery of electroporation energy significantly reduced the window of vulnerability to 5.7±2.9 J ( P=0.0003), with a postablation lower limit of vulnerability=7.3±2.63 J, and the upper limit of vulnerability=18.8±5.2 J. Conclusions Our study highlights that Purkinje tissue can be ablated with IRE without any evidence of underlying myocardial damage.

Neurotransmitter:sodium symporters (NSSs) terminate neurotransmission by Na(+)-dependent reuptake of released neurotransmitters. Previous studies suggested that Na(+)-binding reconfigures dynamically coupled structural elements in an allosteric interaction network (AIN) responsible for function-related conformational changes, but the intramolecular pathway of this mechanism has remained uncharted. We describe a new approach for the modeling and analysis of intramolecular dynamics in the bacterial NSS homolog LeuT. From microsecond-scale molecular dynamics simulations and cognate experimental verifications in both LeuT and human dopamine transporter (hDAT), we apply the novel method to identify the composition and the dynamic properties of their conserved AIN. In LeuT, two different perturbations disrupting Na(+) binding and transport (i.e. replacing Na(+) with Li(+) or the Y268A mutation at the intracellular gate) affect the AIN in strikingly similar ways. In contrast, other mutations that affect the intracellular gate (i.e. R5A and D369A) do not significantly impair Na(+) cooperativity and transport. Our analysis shows these perturbations to have much lesser effects on the AIN, underscoring the sensitivity of this novel method to the mechanistic nature of the perturbation. Notably, this set of observations holds as well for hDAT, where the aligned Y335A, R60A, and D436A mutations also produce different impacts on Na(+) dependence. Thus, the detailed AIN generated from our method is shown to connect Na(+) binding with global conformational changes that are critical for the transport mechanism. That the AIN between the Na(+) binding sites and the intracellular gate in bacterial LeuT resembles that in eukaryotic hDAT highlights the conservation of allosteric pathways underlying NSS function.

Fibroblast behavior and cell-matrix interactions of cells from normal and idiopathic carpal tunnel syndrome (CTS) subsynovial connective tissue (SSCT) with and without Triamcinolone Acetonide (TA) were compared in this study. A cell-seeded gel contraction model was applied to investigate the effect of steroid treatment on SSCT fibroblast gene expression and function.

Stem cell-based therapy has been applied to accelerate the revitalization of allograft tendon into a viable and functional tendon. Although many authors have proposed different methods to help the seeded stem cell distribution in the decellularized allograft, limited success has been achieved as tendon is a high dense connective tissue. We hypothesized that bone marrow stromal cells (BMSCs), seeded through the lateral slit, can regenerate the decellularized tendon (DCT) graft. The cell proliferation, cell viability, and tendon-specific gene expression are increased with the seeded cell density.

The flexor digitorum superficialis (FDS) and flexor digitorum profundus (FDP) are critical for finger flexion. Although research has recently focused on these tendons' coactivity, their contributions in different tasks remain unclear. This study created a novel simultaneous approach to investigate the coactivity between the tendons and to clarify their contributions in different tasks.