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2023 Acuerdos transformativos CRUE, Abnormal endothelin-1 (ET-1) activity is involved in the pathogenesis of vascular diseases such as essential and pulmonary arterial hypertension, coronary artery disease, and cerebrovascular disease, blockade of ET receptors having shown efficacy in clinical assays and experimental models of hypertension. Augmented Ca2+ influx and changes in Ca2+ sensitization associated with arterial vasoconstriction underlie increased systemic vascular resistance in hypertension. Since peripheral resistance arteries play a key role in blood pressure regulation, we aimed to determine here the specific Ca2+ signaling mechanisms linked to the ET receptor-mediated vasoconstriction in resistance arteries and their selective regulation by protein kinase C (PKC), Rho kinase (RhoK), the phosphatidylinositol 3-kinase (PI3K) and the mitogen-activated protein kinase (MAPK). ET-1-induced contraction was mediated by the endothelin ETA receptor with a minor contribution of vascular smooth muscle (VSM) endothelin ETB receptors. ET receptor activation elicited Ca2+ mobilization from intracellular stores, extracellular Ca2+ influx and Ca2+ sensitization associated with contraction in resistance arteries. Vasoconstriction induced by ET-1 was largely dependent on activation of canonical transient receptor potential channel 3 (TRPC3) and extracellular Ca2+ influx through nifedipine-sensitive voltage-dependent Ca2+ channels. PI3K inhibition reduced intracellular Ca2+ mobilization and Ca2+ entry without altering vasoconstriction elicited by ET-1, while PKC has dual opposite actions by enhancing Ca2+ influx associated with contraction, and by inhibiting Ca2+ release from intracellular stores. RhoK was a major determinant of the enhanced sensitivity of the contractile filaments underlying ET-1 vasoconstriction, with also a modulatory positive action on Ca2+ influx and intracellular Ca2+ release. Augmented RhoK and PKC activities are involved in vascular dysfunction in hypertension and vascular complicati, Ministerio de Ciencia e Innovación (España), European Commission, Sección Deptal. de Fisiología (Farmacia), Fac. de Farmacia, TRUE, pub, APC financiada por la UCM

Despite numerous efforts to generate mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), cells often remain immature, electrically isolated, and may not reflect adult biology. Conductive polymers are attractive candidates to facilitate electrical communication between hPSC-CMs, especially at sub-confluent cell densities or diseased cells lacking cell-cell junctions. Here we electrospun conductive polymers to create a conductive fiber mesh and assess if electrical signal propagation is improved in hPSC-CMs seeded on the mesh network. Matrix characterization indicated fiber structure remained stable over weeks in buffer, scaffold stiffness remained near in vivo cardiac stiffness, and electrical conductivity scaled with conductive polymer concentration. Cells remained adherent and viable on the scaffolds for at least 5 days. Transcriptomic profiling of hPSC-CMs cultured on conductive substrates for 3 days showed upregulation of cardiac and muscle-related genes versus non-conductive fibers. Structural proteins were more organized and calcium handling was improved on conductive substrates, even at sub-confluent cell densities; prolonged culture on conductive scaffolds improved membrane depolarization compared to non-conductive substrates. Taken together, these data suggest that blended, conductive scaffolds are stable, supportive of electrical coupling in hPSC-CMs, and promote maturation, which may improve our ability to model cardiac diseases and develop targeted therapies.

Cholecalciferol improves insulin signaling and glucose metabolism in the heart and reduces circulating non-esterified fatty acids. Cholecalciferol effects on the cardiac fatty acid (FA) metabolism and the consequences on calcium handling were examined. Blood lipid profile was determined. Western blot and qRT-PCR were used to examine protein and mRNA expression. Cholecalciferoltreated rats had increased acetyl CoA carboxylase 2 protein expression and decreased expression of malonyl CoA decarboxylase. In addition, the expression of uncoupling protein 3 was elevated. Also, the level of peroxisome proliferator-activated receptor-gamma coactivator in the nucleus of heart cells was increased along with the level of sarcoplasmic/endoplasmic reticulum Ca2+ATPase in the microsomal fraction. In parallel, the L-type calcium channel and ryanodine receptor expression was reduced. In the heart of healthy rats, cholecalciferol affects proteins regulating malonyl CoA availability and intracellular Ca2+ handling proteins.

Lo scompenso cardiaco è una delle principali cause di morte improvvisa nei paesi sviluppati ed è noto che il cuore scompensato sia caratterizzato da una ridotta contrattilità causata da un’alterazione della dinamica del calcio tra il sarcoplasma e il reticolo sarcoplasmatico. In questo ambito, istaroxime è un farmaco in fase 2 preclinica che combina l’inibizione della pompa Na+/K+ e la stimolazione di SERCA2a, mostrando effetti promettenti per il trattamento dello scompenso cardiaco acuto. Tuttavia, l’uso di istaroxime è limitato al trattamento i.v. a causa della sua breve emivita (⁓ 1 h) e dalla sua estensiva metabolizzazione a livello epatico portando alla formazione di una molecola chiamata PST3093. Il primo obiettivo del mio progetto di tesi mirava allo studio del PST3093, il principale metabolita di istaroxime, per capire se fosse dotato di attività farmacologica e per comprendere meglio gli effetti in vivo dell’istaroxime. Sulla base dei risultati ottenuti, il secondo obiettivo era quello di sviluppare degli analoghi del PST3093, stimolatori di SERCA2a e dotati di un gruppo metabolicamente stabile per la somministrazione orale. Effetti in vivo e in vitro dei derivati del PST3093 venivano valutati in un modello di ratto con cardiomiopatia diabetica indotta da streptozotocina (STZ), in quanto caratterizzato da disfunzione diastolica associata a down-regolazione di SERCA2a. Innanzitutto, venivano studiati gli effetti del PST3093 sull’attività di SERCA2a e della pompa Na+/K+, sulla dinamica del calcio intracellulare in miociti ventricolari isolati e sull’emodinamica nei ratti STZ. A differenza di istaroxime, il PST3093 risultava uno stimolatore “selettivo” di SERCA2a, privo di effetti sulla pompa Na+/K+ ̧ con un profilo meno aritmogenico del composto di origine. Il PST3093 risultava attivo a concentrazioni nanomolari in preparazioni cardiache di cavia e di ratto STZ e, similmente a istaroxime, stimolava SERCA2a solo in presenza del fosfolambano, diminuendon, Heart failure (HF) is one of the leading causes of sudden death in developed countries and it is known that failing hearts are characterized by reduced contractile properties caused by impaired Ca2+ cycling between the sarcoplasm and sarcoplasmic reticulum (SR). In this field, istaroxime is a small-molecule drug under phase 2 clinical trial that, combining inhibition of Na+/K+ ATPase and SERCA2a stimulation, shows an interesting profile for acute HF treatment. However, istaroxime use is restricted to acute i.v. infusion because of its plasma half-life of about 1 hour in humans and its extensive hepatic metabolism to a molecule, named PST3093. The first aim of my thesis project dealt with the investigation whether PST3093, the main metabolite of istaroxime, may, on its own, be endowed with pharmacological activity and at least partially explain in vivo istaroxime effects. In light of the results, the second aim was to develop PST3093 analogues with metabolically stable groups, with the purpose to generate orally administrable SERCA2a stimulators. In vivo and in vitro effects of PST3093 follow-on compounds were evaluated by using streptozotocin (STZ)-treated rats developing diabetic cardiomyopathy with diastolic dysfunction associated to SERCA2a downregulation. Firstly, we characterized PST3093 effects on SERCA2a and Na+/K+ ATPase activities, intracellular Ca2+ dynamics in isolated ventricular myocytes and in vivo hemodynamic effects in STZ rats. At variance with its parent compound, PST3093 is a “selective” (i.e. devoid of Na+/K+ ATPase inhibition) SERCA2a stimulator, showing a safer profile than istaroxime. It is active at nanomolar concentrations in cardiac preparations from normal guinea pig and STZ rats and, similarly to istaroxime, it stimulates SERCA2a only in the presence of phospholamban (PLN), thus relieving its inhibitory activity on SERCA2a. In-vivo PST3093 i.v. infusion (acute effects) in STZ rats improved overall cardiac performance and reversed most STZ

Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum–adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

[Abstract] Specialized proresolving mediators and, in particular, 5(S), (6)R, 7-trihydroxyheptanoic acid methyl ester (BML-111) emerge as new therapeutic tools to prevent cardiac dysfunction and deleterious cardiac damage associated with myocarditis progression. The cardioprotective role of BML-111 is mainly caused by the prevention of increased oxidative stress and nuclear factor erythroid-derived 2-like 2 (NRF2) down-regulation induced by myocarditis. At the molecular level, BML-111 activates NRF2 signaling, which prevents sarcoplasmic reticulum–adenosine triphosphatase 2A down-regulation and Ca2+ mishandling, and attenuates the cardiac dysfunction and tissue damage induced by myocarditis.

Aims: Diabetic cardiomyopathy is a multifactorial disease characterized by an early onset of diastolic dysfunction (DD) that precedes the development of systolic impairment. Mechanisms that can restore cardiac relaxation improving intracellular Ca2+ dynamics represent a promising therapeutic approach for cardiovascular diseases associated to DD. Istaroxime has the dual properties to accelerate Ca2+ uptake into sarcoplasmic reticulum (SR) through the SR Ca2+ pump (SERCA2a) stimulation and to inhibit Na+/K+ ATPase (NKA). This project aims to characterize istaroxime effects at a concentration (100 nmol/L) marginally affecting NKA, in order to highlight its effects dependent on the stimulation of SERCA2a in an animal model of mild diabetes. Methods and results: Streptozotocin (STZ) treated diabetic rats were studied at 9 weeks after STZ injection in comparison to controls (CTR). Istaroxime effects were evaluated in vivo and in left ventricular (LV) preparations. STZ animals showed 1) marked DD not associated to cardiac fibrosis, 2) LV mass reduction associated to reduced LV cell dimension and T-tubules loss, 3) reduced LV SERCA2 protein level and activity and 4) slower SR Ca2+ uptake rate, 5) LV action potential (AP) prolongation and increased short-term variability (STV) of AP duration, 6) increased diastolic Ca2+, and 7) unaltered SR Ca2+ content and stability in intact cells. Acute istaroxime infusion (0.11 mg/kg/min for 15 min) reduced DD in STZ rats. Accordingly, in STZ myocytes istaroxime (100 nmol/L) stimulated SERCA2a activity and blunted STZ-induced abnormalities in LV Ca2+ dynamics. In CTR myocytes, istaroxime increased diastolic Ca2+ level due to NKA blockade albeit minimal, while its effects on SERCA2a were almost absent. Conclusions: SERCA2a stimulation by istaroxime improved STZ-induced DD and intracellular Ca2+ handling anomalies. Thus, SERCA2a stimulation can be considered a promising therapeutic approach for DD treatment. Translational perspective: Defi

[EN] During atrial fibrillation, cardiac tissue undergoes different remodeling processes at different scales from the molecular level to the tissue level. One central player that contributes to both electrical and structural remodeling is the myofibroblast. Based on recent experimental evidence on myofibroblasts' ability to contract, we extended a biophysical myofibroblast model with Ca2+ handling components and studied the effect on cellular and tissue electrophysiology. Using genetic algorithms, we fitted the myofibroblast model parameters to the existing in vitro data. In silico experiments showed that Ca2+ currents can explain the experimentally observed variability regarding the myofibroblast resting membrane potential. The presence of an L-type Ca2+ current can trigger automaticity in the myofibroblast with a cycle length of 799.9 ms. Myocyte action potentials were prolonged when coupled to myofibroblasts with Ca2+ handling machinery. Different spatial myofibroblast distribution patterns increased the vulnerable window to induce arrhythmia from 12 ms in non-fibrotic tissue to 22 & PLUSMN; 2.5 ms and altered the reentry dynamics. Our findings suggest that Ca2+ handling can considerably affect myofibroblast electrophysiology and alter the electrical propagation in atrial tissue composed of myocytes coupled with myofibroblasts. These findings can inform experimental validation experiments to further elucidate the role of myofibroblast Ca2+ handling in atrial arrhythmogenesis.

[EN] During atrial fibrillation, cardiac tissue undergoes different remodeling processes at different scales from the molecular level to the tissue level. One central player that contributes to both electrical and structural remodeling is the myofibroblast. Based on recent experimental evidence on myofibroblasts' ability to contract, we extended a biophysical myofibroblast model with Ca2+ handling components and studied the effect on cellular and tissue electrophysiology. Using genetic algorithms, we fitted the myofibroblast model parameters to the existing in vitro data. In silico experiments showed that Ca2+ currents can explain the experimentally observed variability regarding the myofibroblast resting membrane potential. The presence of an L-type Ca2+ current can trigger automaticity in the myofibroblast with a cycle length of 799.9 ms. Myocyte action potentials were prolonged when coupled to myofibroblasts with Ca2+ handling machinery. Different spatial myofibroblast distribution patterns increased the vulnerable window to induce arrhythmia from 12 ms in non-fibrotic tissue to 22 & PLUSMN; 2.5 ms and altered the reentry dynamics. Our findings suggest that Ca2+ handling can considerably affect myofibroblast electrophysiology and alter the electrical propagation in atrial tissue composed of myocytes coupled with myofibroblasts. These findings can inform experimental validation experiments to further elucidate the role of myofibroblast Ca2+ handling in atrial arrhythmogenesis.

[EN] During atrial fibrillation, cardiac tissue undergoes different remodeling processes at different scales from the molecular level to the tissue level. One central player that contributes to both electrical and structural remodeling is the myofibroblast. Based on recent experimental evidence on myofibroblasts' ability to contract, we extended a biophysical myofibroblast model with Ca2+ handling components and studied the effect on cellular and tissue electrophysiology. Using genetic algorithms, we fitted the myofibroblast model parameters to the existing in vitro data. In silico experiments showed that Ca2+ currents can explain the experimentally observed variability regarding the myofibroblast resting membrane potential. The presence of an L-type Ca2+ current can trigger automaticity in the myofibroblast with a cycle length of 799.9 ms. Myocyte action potentials were prolonged when coupled to myofibroblasts with Ca2+ handling machinery. Different spatial myofibroblast distribution patterns increased the vulnerable window to induce arrhythmia from 12 ms in non-fibrotic tissue to 22 & PLUSMN; 2.5 ms and altered the reentry dynamics. Our findings suggest that Ca2+ handling can considerably affect myofibroblast electrophysiology and alter the electrical propagation in atrial tissue composed of myocytes coupled with myofibroblasts. These findings can inform experimental validation experiments to further elucidate the role of myofibroblast Ca2+ handling in atrial arrhythmogenesis.

Background: Combined treatment with anthracyclines (e.g., doxorubicin; Dox) and trastuzumab (Trz), a humanized anti-human epidermal growth factor receptor 2 (HER2; ErbB2) antibody, in patients with HER2-positive cancer is limited by cardiotoxicity, as manifested by contractile dysfunction and arrhythmia. The respective roles of the two agents in the cardiotoxicity of the combined therapy are incompletely understood. Objective: To assess cardiac performance, T-tubule organization, electrophysiological changes and intracellular Ca2+ handling in cardiac myocytes (CMs) using an in vivo rat model of Dox/Trz-related cardiotoxicity. Methods and Results: Adult rats received 6 doses of either Dox or Trz, or the two agents sequentially. Dox-mediated left ventricular (LV) dysfunction was aggravated by Trz administration. Dox treatment, but not Trz, induced T-tubule disarray. Moreover, Dox, but not Trz monotherapy, induced prolonged action potential duration (APD), increased incidence of delayed afterdepolarizations (DADs) and beat-to-beat variability of repolarization (BVR), and slower Ca2+ transient decay. Although APD, DADs, BVR and Ca2+ transient decay recovered over time after the cessation of Dox treatment, subsequent Trz administration exacerbated these abnormalities. Trz, but not Dox, reduced Ca2+ transient amplitude and SR Ca2+ content, although only Dox treatment was associated with SERCA downregulation. Finally, Dox treatment increased Ca2+ spark frequency, resting Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and long-lasting Ca2+ release events (so-called Ca2+ “embers”), partially reproduced by Trz treatment. Conclusion: These results suggest that in vivo Dox but not Trz administration causes T-tubule disarray and pronounced changes in electrical activity of CMs. While adaptive changes may account for normal AP shape and reduced DADs late after Dox administration, subsequent Trz administration interferes with such adaptive changes. Intracellular Ca2+ handling

Ca2+ mishandling is a common feature in several cardiovascular diseases such as heart failure (HF). In many cases, impairment of key players in intracellular Ca2+ homeostasis has been identified as the underlying mechanism of cardiac dysfunction and cardiac arrhythmias associated with HF. In this review, we summarize primary novel findings related to Ca2+ mishandling in HF progression. HF research has increasingly focused on the identification of new targets and the contribution of their role in Ca2+ handling to the progression of the disease. Recent research studies have identified potential targets in three major emerging areas implicated in regulation of Ca2+ handling: the innate immune system, bone metabolism factors and post-translational modification of key proteins involved in regulation of Ca2+ handling. Here, we describe their possible contributions to the progression of HF.

BACKGROUND: Cardiac Bridging Integrator 1 (cBIN1) is a membrane deformation protein that generates calcium microdomains at cardiomyocyte t-tubules, whose transcription is reduced in heart failure, and is released into blood. cBIN1 score (CS), an inverse index of plasma cBIN1, measures cellular myocardial remodeling. In patients with heart failure with preserved ejection fraction (HFpEF), CS diagnoses ambulatory heart failure and prognosticates hospitalization. The performance of CS has not been tested in patients with heart failure with reduced ejection fraction (HFrEF). METHODS AND RESULTS: CS was determined from plasma of patients recruited in a prospective study. Two comparative cohorts consisted of 158 ambulatory HFrEF patients (left ventricular ejection fraction (LVEF) ≤ 40%, 57 ± 10 years, 80% men) and 115 age and sex matched volunteers with no known history of HF. N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations were also analyzed for comparison. CS follows a normal distribution with a median of 0 in the controls, which increases to a median of 1.9 (p < 0.0001) in HFrEF patients. CS correlates with clinically assessed New York Heart Association Class (p = 0.007). During 1-year follow-up, a high CS (≥ 1.9) in patients predicts increased cardiovascular events (43% vs. 26%, p = 0.01, hazard ratio 1.9). Compared to a model with demographics, clinical risk factors, and NT-proBNP, adding CS to the model improved the overall continuous net reclassification improvement (NRI 0.64; 95% CI 0.18-1.10; p = 0.006). Although performance for diagnosis and prognosis was similar to CS, NT-proBNP did not prognosticate between patients whose NT-proBNP values were > 400 pg/ml. CONCLUSION: CS, which is mechanistically distinct from NT-proBNP, successfully differentiates myocardial health between patients with HFrEF and matched controls. A high CS reflects advanced NYHA stage, pathologic cardiac muscle remodeling, and predicts 1-year risk of cardiovascular

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Although biomimetic stimuli, such as microgroove-induced alignment (mu), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined mu-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-beta signaling was downregulated; the TGF-beta receptor agonist and antagonist TGF-beta 1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also i

Although biomimetic stimuli, such as microgroove-induced alignment (mu), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined mu-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-beta signaling was downregulated; the TGF-beta receptor agonist and antagonist TGF-beta 1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also i

Excessive iron accumulation in the heart causes iron overload cardiomyopathy (IOC), which initially presents as diastolic dysfunction and arrhythmia but progresses to systolic dysfunction and end-stage heart failure when left untreated. However, the mechanisms of iron-related cardiac injury and how iron accumulates in human cardiomyocytes are not well understood. Herein, using human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we model IOC and screen for drugs to rescue the iron overload phenotypes. Human iPSC-CMs under excess iron exposure recapitulate early-stage IOC, including oxidative stress, arrhythmia, and contractile dysfunction. We find that iron-induced changes in calcium kinetics play a critical role in dysregulation of CM functions. We identify that ebselen, a selective divalent metal transporter 1 (DMT1) inhibitor and antioxidant, could prevent the observed iron overload phenotypes, supporting the role of DMT1 in iron uptake into the human myocardium. These results suggest that ebselen may be a potential preventive and therapeutic agent for treating patients with secondary iron overload.

Although biomimetic stimuli, such as microgroove-induced alignment (mu), triiodothyronine (T3) induction, and electrical conditioning (EC), have been reported to promote maturation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), a systematic examination of their combinatorial effects on engineered cardiac tissue constructs and the underlying molecular pathways has not been reported. Herein, human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) were used to generate a micro-patterned human ventricular cardiac anisotropic sheets (hvCAS) for studying the physiological effects of combinatorial treatments by a range of functional, calcium (Ca2+)-handling, and molecular analyses. High-resolution optical mapping showed that combined mu-T3-EC treatment of hvCAS increased the conduction velocity, anisotropic ratio, and proportion of mature quiescent-yet-excitable preparations by 2. 3-, 1. 8-, and 5-fold (>70%), respectively. Such electrophysiological changes could be attributed to an increase in inward sodium current density and a decrease in funny current densities, which is consistent with the observed up- and downregulated SCN1B and HCN2/4 transcripts, respectively. Furthermore, Ca2+-handling transcripts encoding for phospholamban (PLN) and sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) were upregulated, and this led to faster upstroke and decay kinetics of Ca2+-transients. RNA-sequencing and pathway mapping of T3-EC-treated hvCAS revealed that the TGF-beta signaling was downregulated; the TGF-beta receptor agonist and antagonist TGF-beta 1 and SB431542 partially reversed T3-EC induced quiescence and reduced spontaneous contractions, respectively. Taken together, we concluded that topographical cues alone primed cardiac tissue constructs for augmented electrophysiological and calcium handling by T3-EC. Not only do these studies improve our understanding of hPSC-CM biology, but the orchestration of these pro-maturational factors also i

Aims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of PluricyteVR CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. Methods and Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp results experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2þ measurements included Ca2þtransients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in PluricyteVR CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2þ-induced Ca2þ release, response to caffeine and excitation-contraction coupling gain characterized the cellular Ca2þ-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single PluricyteVR CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. Conclusion Taking their ion-current characteristics and Ca2þ handling into account, PluricyteVR CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhyt

Cardiovascular diseases (CVDs) underlie a high rate of mortality worldwide. Both acquired and inherited CVDs are closely related to ionic channelopathies, compromising the expression and function of a wide number of ion channels. Ca2+ mishandling has been associated with several CVDs, such as those involved in metabolic disorders, hypertrophy, heart failure, and several inherited heart diseases that implicate cardiac dysfunction and arrhythmias (Kushnir et al., 2018). Moreover, the archives of biomedical and life sciences are showing a multitude of contributions related to this topic, demonstrating the high interest of the scientific community in this specific research field. Indeed, Ca2+ is the key regulator of the Ca2+ induced Ca2+ release (CICR) process, the main actor in cardiac excitation-contraction coupling (ECC) (Fabiato, 1983). Briefly, ECC begins with membrane depolarization of cardiomyocytes, activating sarcolemmal L-type voltagedependent Ca2+ channels, thus promoting an inward Ca2+ current (ICaL) that activates the intracellular Ca2+ channel/ryanodine receptor type 2 (RyR2), located at the sarcoplasmic reticulum (SR), triggering a transient increase of cytosolic Ca2+ levels, and eliciting cell contraction. To get new insights into these processes, Aguilar-Sanchez et al. evaluated in an elegant study whether the autonomous system regulates ECC in endocardial and epicardial layers from intact beating mouse hearts. The authors recorded action potentials (APs), Ca2+ transients, and Ca2+ currents in whole hearts, demonstrating that the Ca2+ influx occurring in AP phase 1, and not in phase 2, triggers ECC (Aguilar-Sanchez et al.).

Cardiac and skeletal muscle cell contraction is a result of excitation-contraction coupling (ECC), where an electrical signal leads to a rise in intracellular calcium levels and contraction. This process is carefully regulated to meet physiological demand and heavily dependent on an adequate energy supply. Disturbed ECC can have severe consequences on muscle cell function and underlies many cardiac and skeletal muscle pathologies. Cell stress, changing intracellular Ca²⁺ concentrations, and calcium signal dynamics can all play a role in the transcriptional regulation of genes involved in myocyte Ca²⁺-handling. In this thesis project, the transcriptional control of ECC was studied in skeletal and cardiac myocytes. Skeletal myocyte calsequestrin (CASQ1) was downregulated in a mouse model of mitochondrial myopathy and it contributed to the decreased SR Ca²⁺ load and impaired Ca²⁺ handling in Tfam-/- skeletal myocytes. In cultured neonatal cardiomyocytes, mitochondrial uncoupler FCCP-induced mitochondrial dysfunction led to downregulation of cardiac calsequestrin (CASQ2) and similarly impaired Ca²⁺ handling. Whereas there was no increase in reactive oxygen species (ROS) levels in Tfam-/- myocytes, cultured cells exposed to FCCP did display increased ROS, an effect that was counteracted by coexposure with the ROS scavenger (NAC). NAC attenuated FCCP-induced CASQ2 downregulation and restored Ca²⁺ handling. Therefore, mitochondrial dysfunction led to CASQ1/2 downregulation and impaired Ca²⁺ handling in these two cell types, but by different mechanisms. This project also looked at the role of Ca²⁺ dynamics on the transcriptional regulation of Ca²⁺ handling genes. Increased intracellular Ca²⁺ levels and β-adrenergic stimulation of cardiomyocytes activate Ca²⁺-calmodulin kinase II (CaMKII) and can trigger hypertrophic remodeling. It was found that CaMKII downregulated expression of the L-type Ca²⁺ channel α1c-subunit (Cacna1c) in cultured cardiomyocytes. An, Tiivistelmä Sydän- ja luustolihassolujen supistuminen on seurausta ärsytys-supistuskytkennästä (ECC), jossa sähköinen ärsytys kohottaa solunsisäistä kalsiumpitoisuutta ja aiheuttaa supistuksen. Tätä säädellään tarkasti fysiologisen tarpeen mukaan, ja se riippuu riittävästä energian saannista. Häiriintynyt ECC voi aiheuttaa vakavia seurauksia lihassolujen toiminnalle, ja se on mukana monien sydän- ja luustolihasten sairauksien synnyssä. Tässä tutkimuksessa ECC:n transkriptionaalista säätelyä tutkittiin luustolihasten ja sydämen lihassoluissa. Luustolihassolujen kalsekvestriinin (CASQ1) väheneminen pienensi SR:n Ca²⁺-määrää mitokondrioiden myopatian hiirimallissa ja heikensi Ca²⁺-tasapainon ylläpitoa Tfam-/--luustolihassoluissa. Viljellyissä vastasyntyneiden kammio-sydänlihassoluissa mitokondrio-irtikytkijän FCCP:n aiheuttama mitokondrioiden toimintahäiriö johti sydämen kalsekvestriinin (CASQ2) vähenemiseen ja heikensi samalla tavalla Ca²⁺-tasapainon ylläpitoa. Vaikka Tfam-/--myosyyteissä reaktiivisten happilajien (ROS) tasot eivät olleet koholla, FCCP:lle altistetuissa viljellyissä soluissa ROS kuitenkin lisääntyi. Vaikutusta esti ROS-puhdistaja NAC, joka heikensi FCCP:n aiheuttamaa CASQ2:n laskua ja palautti Ca²⁺-säätelyn normaaliksi. Mitokondrioiden toimintahäiriö siis johti CASQ1/2:n vähenemiseen ja Ca²⁺-säätelyn heikentymiseen molemmissa solutyypeissä, mutta eri mekanismeilla. Tässä tutkimuksessa tarkasteltiin myös Ca²⁺-dynamiikan osuutta Ca²⁺-tasapainoon osallistuvien geenien transkription säätelyssä. Lisääntynyt solunsisäinen Ca²⁺-taso ja sydänlihassolujen β-adrenerginen stimulointi aktivoivat Ca²⁺-kalmoduliinikinaasi II:n (CaMKII), ja ne voivat laukaista sydämen hypertrofisen uudelleenmuovautumisen. Havaittiin, että CaMKII vähensi L-tyypin Ca²⁺-kanavan a1c-alayksikön (Cacna1c) ilmentymistä viljellyissä sydänlihassoluissa. Promoottorianalyysi osoitti tämän johtuvan transkription repressorin DREAM:n sitoutumisesta oletettuun DRE:hen (alavirrassa sijaitseva

Background: Diastolic dysfunction is an important contributor to many cardiac pathologies including diabetic cardiomyopathy and heart failure with preserved ejection fraction. It is characterised by ventricular stiffness, inadequate filling of the ventricles and elevated ventricular pressure. In addition to extracellular influences, evidence suggests that a cardiomyocyte specific intrinsic stiffness may also be an important contributor to diastolic dysfunction, but the mechanisms are not well understood. This might be partly due to the lack of specific animal models available to study underlying mechanisms, in particular in HFpEF. The aim of this thesis was to evaluate cellular and molecular mechanisms of diastolic dysfunction in a model of type 1 diabetes and in a newly characterised model of HFpEF, the Hypertrophic Heart Rat (HHR). Research questions: Q1. Can measurement of in vitro intact cardiomyocyte stiffness be correlated with in vivo diastolic function to determine whether cellular stiffness contributes to cardiac diastolic dysfunction in pathological settings? (Chapter 2) Q2. What are the subcellular mechanisms that contribute to increased stiffness in a pathological model of diastolic dysfunction? (Chapter 3) Q3. Can the Hypertrophic Heart Rat be used as a novel rodent model of HFpEF and what is the underlying cardiomyocyte pathophysiology driving diastolic dysfunction in HFpEF? (Chapter 4) Methods: Type 1 diabetes was induced in Sprague Dawley rats using a single dosage of Streptozotocin. The Hypertrophic Heart Rat (HHR) was characterised and utilised as a model of HFpEF. Echocardiography was used to assess in vivo heart function in diabetic and HFpEF rats. Surface electrocardiogram recordings were performed to assess in vivo electrical activity in HFpEF rats. Cardiomyocytes were isolated by collagenase dissociation. Under loaded conditions glass fibers were attached (MyoTak) at the cell longitudinal surface, and paced cardiomyocytes (1Hz, 2.0mM Ca2+, 37°

[EN] Heart failure (HF) is characterized by altered Ca2+ cycling, resulting in cardiac contractile dysfunction. Failing myocytes undergo electrophysiological remodeling, which is known to be the main cause of abnormal Ca2+ homeostasis. However, structural remodeling, specifically proliferating fibroblasts coupled to myocytes in the failing heart, could also contribute to Ca2+ cycling impairment. The goal of the present study was to systematically analyze the mechanisms by which myocyte-fibroblast coupling could affect Ca2+ dynamics in normal conditions and in HF. Simulations of healthy and failing human myocytes were performed using established mathematical models, and cells were either isolated or coupled to fibroblasts. Univariate and multivariate sensitivity analyses were performed to quantify effects of ion transport pathways on biomarkers computed from intracellular [Ca2+] waveforms. Variability in ion channels and pumps was imposed and populations of models were analyzed to determine effects on Ca2+ dynamics. Our results suggest that both univariate and multivariate sensitivity analyses are valuable methodologies to shed light into the ionic mechanisms underlying Ca2+ impairment in HF, although differences between the two methodologies are observed at high parameter variability. These can result from either the fact that multivariate analyses take into account ion channels or non-linear effects of ion transport pathways on Ca2+ dynamics. Coupling either healthy or failing myocytes to fibroblasts decreased Ca2+ transients due to an indirect sink effect on action potential and thus on Ca2+ related currents. Simulations that investigated restoration of normal physiology in failing myocytes showed that Ca2+ cycling can be normalized by increasing SERCA and L-type Ca2+ current activity while decreasing Na+-Ca2+ exchange and SR Ca2+ leak. Changes required to normalize action potentials in failing myocytes depended on whether myocytes were coupled to fibroblasts. In

[EN] Heart failure (HF) is characterized by altered Ca2+ cycling, resulting in cardiac contractile dysfunction. Failing myocytes undergo electrophysiological remodeling, which is known to be the main cause of abnormal Ca2+ homeostasis. However, structural remodeling, specifically proliferating fibroblasts coupled to myocytes in the failing heart, could also contribute to Ca2+ cycling impairment. The goal of the present study was to systematically analyze the mechanisms by which myocyte-fibroblast coupling could affect Ca2+ dynamics in normal conditions and in HF. Simulations of healthy and failing human myocytes were performed using established mathematical models, and cells were either isolated or coupled to fibroblasts. Univariate and multivariate sensitivity analyses were performed to quantify effects of ion transport pathways on biomarkers computed from intracellular [Ca2+] waveforms. Variability in ion channels and pumps was imposed and populations of models were analyzed to determine effects on Ca2+ dynamics. Our results suggest that both univariate and multivariate sensitivity analyses are valuable methodologies to shed light into the ionic mechanisms underlying Ca2+ impairment in HF, although differences between the two methodologies are observed at high parameter variability. These can result from either the fact that multivariate analyses take into account ion channels or non-linear effects of ion transport pathways on Ca2+ dynamics. Coupling either healthy or failing myocytes to fibroblasts decreased Ca2+ transients due to an indirect sink effect on action potential and thus on Ca2+ related currents. Simulations that investigated restoration of normal physiology in failing myocytes showed that Ca2+ cycling can be normalized by increasing SERCA and L-type Ca2+ current activity while decreasing Na+-Ca2+ exchange and SR Ca2+ leak. Changes required to normalize action potentials in failing myocytes depended on whether myocytes were coupled to fibroblasts. In

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, were these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.

The occurrence of arrhythmia is often related to basic heart rate. Prognostic significance is associated with such a relationship; furthermore, heart rate modulation may result as an ancillary effect of therapy, or be considered as a therapeutic tool. This review discusses the cellular mechanisms underlying arrhythmia occurrence during tachycardia or bradycardia, considering rate changes per se or as a mirror of autonomic modulation. Besides the influence of steady-state heart rate, dynamic aspects of changes in rate and autonomic balance are considered. The discussion leads to the conclusion that the prognostic significance of arrhythmia relationship with heart rate, and the consequence of heart rate on arrhythmogenesis, may vary according to the substrate present in the specific case and should be considered accordingly.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare genetic disease caused by defective prelamin A processing, leading to nuclear lamina alterations, severe cardiovascular pathology, and premature death. Prelamin A alterations also occur in physiological aging. It remains unknown how defective prelamin A processing affects the cardiac rhythm. We show age-dependent cardiac repolarization abnormalities in HGPS patients that are also present in the Zmpste24-/- mouse model of HGPS. Challenge of Zmpste24-/- mice with the ß-adrenergic agonist isoproterenol did not trigger ventricular arrhythmia but caused bradycardia-related premature ventricular complexes and slow-rate polymorphic ventricular rhythms during recovery. Patch-clamping in Zmpste24-/- cardiomyocytes revealed prolonged calcium-transient duration and reduced sarcoplasmic reticulum calcium loading and release, consistent with the absence of isoproterenol-induced ventricular arrhythmia. Zmpste24-/- progeroid mice also developed severe fibrosis-unrelated bradycardia and PQ interval and QRS complex prolongation. These conduction defects were accompanied by overt mislocalization of the gap junction protein connexin43 (Cx43). Remarkably, Cx43 mislocalization was also evident in autopsied left ventricle tissue from HGPS patients, suggesting intercellular connectivity alterations at late stages of the disease. The similarities between HGPS patients and progeroid mice reported here strongly suggest that defective cardiac repolarization and cardiomyocyte connectivity are important abnormalities in the HGPS pathogenesis that increase the risk of arrhythmia and premature death., Peer Reviewed, Postprint (published version)

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.

Tightly regulated Ca(2+) homeostasis is a prerequisite for proper cardiac function. To dissect the regulatory network of cardiac Ca(2+) handling, we performed a chemical suppressor screen on zebrafish tremblor embryos, which suffer from Ca(2+) extrusion defects. Efsevin was identified based on its potent activity to restore coordinated contractions in tremblor. We show that efsevin binds to VDAC2, potentiates mitochondrial Ca(2+) uptake and accelerates the transfer of Ca(2+) from intracellular stores into mitochondria. In cardiomyocytes, efsevin restricts the temporal and spatial boundaries of Ca(2+) sparks and thereby inhibits Ca(2+) overload-induced erratic Ca(2+) waves and irregular contractions. We further show that overexpression of VDAC2 recapitulates the suppressive effect of efsevin on tremblor embryos whereas VDAC2 deficiency attenuates efsevin's rescue effect and that VDAC2 functions synergistically with MCU to suppress cardiac fibrillation in tremblor. Together, these findings demonstrate a critical modulatory role for VDAC2-dependent mitochondrial Ca(2+) uptake in the regulation of cardiac rhythmicity.

AimsTo evaluate the ability of N-acetylglucosamine (GlcNAc) decorated nanoparticles and their cargo to modulate calcium handling in failing cardiac myocytes (CMs).Materials & methodsPrimary CMs isolated from normal and failing hearts were treated with GlcNAc nanoparticles in order to assess the ability of the nanoparticles and their cargo to correct dysfunctional calcium handling in failing myocytes.Results & conclusionGlcNAc particles reduced aberrant calcium release in failing CMs and restored sarcomere function. Additionally, encapsulation of a small calcium-modulating protein, S100A1, in GlcNAc nanoparticles also showed improved calcium regulation. Thus, the development of our bioactive nanoparticle allows for a 'two-hit' treatment, by which the cargo and also the nanoparticle itself can modulate intracellular protein activity.

Rationale: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca2+ ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca2+ load and Ca2+ leak. Conversely, PLN-KO accelerates Ca2+ sequestration and aborts arrhythmogenic spontaneous Ca2+ waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca2+-triggered arrhythmias. Objective: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. Methods and Results: We generated a PLN-deficient, RyR2-mutant mouse model (PLN−/−/RyR2-R4496C+/−) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT–associated RyR2-R4496C mutant mice. Ca2+ imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/− ventricular myocytes during sarcoplasmic reticulum Ca2+ overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca2+ sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca2+ overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca2+ ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li+ failed to convert mini-waves to cell-wide SCWs in PLN−/−/RyR2-R4496C+/− ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. Conclusions: Our results demonstrate that despite severe sarcoplasmic reticulum Ca2+ leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca2+ sequestration represents an effective approach for suppressing, Peer Reviewed, Postprint (published version)

Objective: Istaroxime (PST2744) is a novel inotropic agent which enhances SERCA2 activity. We investigated istaroxime effect on Ca(2+) handling abnormalities in myocardial hypertrophy/failure (HF). Methods: guinea-pig myocytes were studied 12 weeks after aortic banding (AoB) and compared to those of sham-operated animals (sham). The gain of calcium-induced Ca(2+) release (CICR), sarcoplasmic reticulum (SR) Ca(2+) content, Na(+)/Ca(2+) exchanger (NCX) function and the rate of SR reloading after caffeine-induced depletion (SR Ca(2+) uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca(2+) and membrane currents. Results: HF characterization: AoB caused hypertrophy and failure in 100% and 25% of animals respectively. While CICR-gain during constant pacing was preserved, SR Ca(2+) content and SR Ca(2+) uptake were strongly depressed. Resting Ca(2+) and the slope of the INCX/Ca(2+) relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca(2+) content and SR Ca(2+) uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca(2+) uptake in AoB myocytes. Istaroxime increased resting Ca(2+) and the slope of the INCX/Ca(2+) relationship similarly in sham and AoB myocytes. Istraoxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Conclusions: Clear-cut abnormalities in Ca(2+) handling occurred in this model of hypertrophy with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.

In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously suppor

In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously suppor

Relatively limited information is available regarding the mechanisms controlling vasomotricity in human vessels. Isolated vessels obtained from patients undergoing surgery were used to characterize the role of endothelial factors and to study coupling mechanisms between receptors, intracellular calcium, and contraction. However, these investigations are limited by the availability of tissues and many uncontrolled factors. Cultured human vascular cells were also used, were these cells rapidly lose at least some of their differentiated characters. Recently, a human blood vessel equivalent was constructed in vitro from cultured cells, using tissue engineering. This technique allowed us to obtain vessel equivalents containing intima, media, and adventitia layers or tubular media layer only. Contraction and rises in intracellular calcium produced by agonists were studied, indicating that such human vessel equivalents may provide valuable models for pharmacological studies.