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Introduction: The development of patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offers an opportunity to study genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM), one of the most common inherited cardiac diseases. However, immaturity of the iPSC-CMs and the lack of a multicellular composition pose concerns over its faithfulness in disease modeling and its utility in developing mechanism-specific treatment. Methods: The Biowire platform was used to generate 3D engineered cardiac tissues (ECTs) using HCM patient-derived iPSC-CMs carrying a ß-myosin mutation (MYH7-R403Q) and its isogenic control (WT), withal ECTs contained healthy human cardiac fibroblasts. ECTs were subjected to electro-mechanical maturation for 6 weeks before being used in HCM phenotype studies. Results: Both WT and R403Q ECTs exhibited mature cardiac phenotypes, including a lack of automaticity and a ventricular-like action potential (AP) with a resting membrane potential < -75 mV. Compared to WT, R403Q ECTs demonstrated many HCM-associated pathological changes including increased tissue size and cell volume, shortened sarcomere length and disorganized sarcomere structure. In functional assays, R403Q ECTs showed increased twitch amplitude, slower contractile kinetics, a less pronounced force-frequency relationship, a smaller post-rest potentiation, prolonged AP durations, and slower Ca2+ transient decay time. Finally, we observed downregulation of calcium handling genes and upregulation of NPPB in R403Q vs. WT ECTs. In an HCM phenotype prevention experiment, ECTs were treated for 5-weeks with 250 nM mavacamten or a vehicle control. We found that chronic mavacamten treatment of R403Q ECTs: (i) shortened relaxation time, (ii) reduced APD90 prolongation, (iii) upregulated ADRB2, ATP2A2, RYR2, and CACNA1C, (iv) decreased B-type natriuretic peptide (BNP) mRNA and protein expression levels, and (v) increased sarcomere length and reduced sarcomere disarray. Discussion: Taken together, we demonstrated R403Q ECTs generated in the Biowire platform recapitulated many cardiac hypertrophy phenotypes and that chronic mavacamten treatment prevented much of the pathology. This demonstrates that the Biowire ECTs are well-suited to phenotypic-based drug discovery in a human-relevant disease model.

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The role of C-type natriuretic peptide (CNP) in the regulation of cardiac function in humans remains to be established as previous investigations have been confined to animal model systems. Here, we used well-characterized engineered cardiac tissues (ECTs) generated from human stem cell-derived cardiomyocytes and fibroblasts to study the acute effects of CNP on contractility. Application of CNP elicited a positive inotropic response as evidenced by increases in maximum twitch amplitude, maximum contraction slope and maximum calcium amplitude. This inotropic response was accompanied by a positive lusitropic response as demonstrated by reductions in time from peak contraction to 90% of relaxation and time from peak calcium transient to 90% of decay that paralleled increases in maximum contraction decay slope and maximum calcium decay slope. To establish translatability, CNP-induced changes in contractility were also assessed in rat ex vivo (isolated heart) and in vivo models. Here, the effects on force kinetics observed in ECTs mirrored those observed in both the ex vivo and in vivo model systems, whereas the increase in maximal force generation with CNP application was only detected in ECTs. In conclusion, CNP induces a positive inotropic and lusitropic response in ECTs, thus supporting an important role for CNP in the regulation of human cardiac function. The high degree of translatability between ECTs, ex vivo and in vivo models further supports a regulatory role for CNP and expands the current understanding of the translational value of human ECTs. NEW FINDINGS: What is the central question of this study? What are the acute responses to C-type natriuretic peptide (CNP) in human-engineered cardiac tissues (ECTs) on cardiac function and how well do they translate to matched concentrations in animal ex vivo and in vivo models? What is the main finding and its importance? Acute stimulation of ECTs with CNP induced positive lusitropic and inotropic effects on cardiac contractility, which closely reflected the changes observed in rat ex vivo and in vivo cardiac models. These findings support an important role for CNP in the regulation of human cardiac function and highlight the translational value of ECTs.

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Cardiac contractility modulation (CCM) is a medical device therapy whereby non-excitatory electrical stimulations are delivered to the myocardium during the absolute refractory period to enhance cardiac function. We previously evaluated the effects of the standard CCM pulse parameters in isolated rabbit ventricular cardiomyocytes and 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, on flexible substrate. In the present study, we sought to extend these results to human 3D microphysiological systems to develop a robust model to evaluate various clinical CCM pulse parameters in vitro. HiPSC-CMs were studied in conventional 2D monolayer format, on stiff substrate (i.e., glass), and as 3D human engineered cardiac tissues (ECTs). Cardiac contractile properties were evaluated by video (i.e., pixel) and force-based analysis. CCM pulses were assessed at varying electrical 'doses' using a commercial pulse generator. A robust CCM contractile response was observed for 3D ECTs. Under comparable conditions, conventional 2D monolayer hiPSC-CMs, on stiff substrate, displayed no contractile response. 3D ECTs displayed enhanced contractile properties including increased contraction amplitude (i.e., force), and accelerated contraction and relaxation slopes under standard acute CCM stimulation. Moreover, 3D ECTs displayed enhanced contractility in a CCM pulse parameter-dependent manner by adjustment of CCM pulse delay, duration, amplitude, and number relative to baseline. The observed acute effects subsided when the CCM stimulation was stopped and gradually returned to baseline. These data represent the first study of CCM in 3D hiPSC-CM models and provide a nonclinical tool to assess various CCM device signals in 3D human cardiac tissues prior to in vivo animal studies. Moreover, this work provides a foundation to evaluate the effects of additional cardiac medical devices in 3D ECTs.

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Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the ß-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

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Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

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Heart failure (HF) and subarachnoid hemorrhage (SAH) chronically reduce cerebral perfusion, which negatively affects clinical outcome. This work demonstrates a strong relationship between cerebral artery cystic fibrosis transmembrane conductance regulator (CFTR) expression and altered cerebrovascular reactivity in HF and SAH. In HF and SAH, CFTR corrector compounds (C18 or lumacaftor) normalize pathological alterations in cerebral artery CFTR expression, vascular reactivity, and cerebral perfusion, without affecting systemic hemodynamic parameters. This normalization correlates with reduced neuronal injury. Therefore, CFTR therapeutics have emerged as valuable clinical tools to manage cerebrovascular dysfunction, impaired cerebral perfusion, and neuronal injury.

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BACKGROUND: The SCN5A mutation, P1332L, is linked to a malignant form of congenital long QT syndrome, type 3 (LQT3), and affected patients are highly responsive to the Na+ channel blocking drug, mexiletine. In contrast, A647D is an atypical SCN5A mutation causing Brugada syndrome. An asymptomatic male with both P1332L and A647D presented with varying P wave/QRS aberrancy and mild QTc prolongation which did not shorten measurably with mexiletine. OBJECTIVE: We characterized the biophysical properties of P1332L, A647D and wild-type (WT) Na+ channels as well as their combinations in order to understand our proband's phenotype and to guide mexilitine therapy. METHODS: Na+ channel biophysics and mexilitine-binding kinetics were assessed using heterologous expression studies in CHO-K1 cells and human ventricular myocyte modeling. RESULTS: Compared to WT, P1332L channels displayed a hyperpolarizing shift in inactivation, slower inactivation and prominent late Na+ currents (INa). While A647D had no effect on the biophysical properties of INa, it reduced peak and late INa density when co-expressed with either WT or P1332L. Additionally, while P1332L channels had greater sensitivity to block by mexiletine compared to WT, this was reduced in the presence of A647D. Modelling studies revealed that mixing P1332L with A647D channels, action potential durations were shortened compared to P1332L, while peak INa was reduced compared to either A647D coexpressing with WT or WT alone. CONCLUSIONS: While A647D mitigates the lethal LQT3 phenotype seen with P1332L, it also reduces mexilitine sensitivity and decreases INa density. These results explain our proband's mild repolarization abnormality and prominent conduction defect in the atria and ventricles, but also suggest that expression of P1332L with A647D yields a novel disease phenotype for which mexiletine pharmacotherapy is no longer suitable.

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Defining the roadblocks responsible for cell cycle arrest in adult cardiomyocytes lies at the core of developing cardiac regenerative therapies. p53 and Mdm2 are crucial mediators of cell cycle arrest in proliferative cell types, however, little is known about their function in regulating homeostasis and proliferation in terminally differentiated cell types, like cardiomyocytes. To explore this, we generated a cardiac-specific conditional deletion of p53 and Mdm2 (DKO) in adult mice. Herein we describe the development of a dilated cardiomyopathy, in the absence of cardiac hypertrophy. In addition, DKO hearts exhibited a significant increase in cardiomyocyte proliferation. Further evaluation showed that proliferation was mediated by a significant increase in Cdk2 and cyclin E with downregulation of p21Cip1 and p27Kip1. Comparison of miRNA expression profiles from DKO mouse hearts and controls revealed 11 miRNAs that were downregulated in the DKO hearts and enriched for mRNA targets involved in cell cycle regulation. Knockdown of these miRNAs in neonatal rat cardiomyocytes significantly increased cytokinesis with an upregulation in the expression of crucial cell cycle regulators. These results illustrate the importance of the cooperative activities of p53 and Mdm2 in a network of miRNAs that function to impose a barrier against aberrant cardiomyocyte cell cycle re-entry to maintain cardiac homeostasis.

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Tumour necrosis factor (TNF) is a ubiquitously expressed cytokine with functions beyond the immune system. In several diseases, the induction of TNF expression in resistance artery smooth muscle cells enhances microvascular myogenic vasoconstriction and perturbs blood flow. This pathological role prompted our hypothesis that constitutively expressed TNF regulates myogenic signalling and systemic haemodynamics under non-pathological settings. Here we show that acutely deleting the TNF gene in smooth muscle cells or pharmacologically scavenging TNF with etanercept (ETN) reduces blood pressure and resistance artery myogenic responsiveness; the latter effect is conserved across five species, including humans. Changes in transmural pressure are transduced into intracellular signals by membrane-bound TNF (mTNF) that connect to a canonical myogenic signalling pathway. Our data positions mTNF 'reverse signalling' as an integral element of a microvascular mechanosensor; pathologic or therapeutic perturbations of TNF signalling, therefore, necessarily affect microvascular tone and systemic haemodynamics.

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The fast transient outward potassium current (Ito,f) plays a critical role in the electrical and contractile properties of the myocardium. Ito,f channels are formed by the co-assembly of the pore-forming α-subunits, Kv4.2 and Kv4.3, together with the accessory ß-subunit KChIP2. Reductions of Ito,f are common in the diseased heart, which is also associated with enhanced stimulation of ß-adrenergic receptors (ß-ARs). We used cultured neonatal rat ventricular myocytes to examine how chronic ß-AR stimulation decreases Ito,f. To determine which downstream pathways mediate these Ito,f changes, adenoviral infections were used to inhibit CaMKIIδc, CaMKIIδb, calcineurin, or nuclear factor κB (NF-κB). We observed that chronic ß-AR stimulation with isoproterenol (ISO) for 48 h reduced Ito,f along with mRNA expression of all three of its subunits (Kv4.2, Kv4.3, and KChIP2). Inhibiting either CaMKIIδc nor CaMKIIδb did not prevent the ISO-mediated Ito,f reductions, even though CaMKIIδc and CaMKIIδb clearly regulated Ito,f and the mRNA expression of its subunits. Likewise, calcineurin inhibition did not prevent the Ito,f reductions induced by ß-AR stimulation despite strongly modulating Ito,f and subunit mRNA expression. In contrast, NF-κB inhibition partly rescued the ISO-mediated Ito,f reductions in association with restoration of KChIP2 mRNA expression. Consistent with these observations, KChIP2 promoter activity was reduced by p65 as well as ß-AR stimulation. In conclusion, NF-κB, and not CaMKIIδ or calcineurin, partly mediates the Ito,f reductions induced by chronic ß-AR stimulation. Both mRNA and KChIP2 promoter data suggest that the ISO-induced Ito,f reductions are, in part, mediated through reduced KChIP2 transcription caused by NF-κB activation.

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The Ca(2+)-independent transient outward K(+) current (I(to)) plays a critical role in underlying phase 1 of repolarization of the cardiac action potential and, as a result, is central to modulating excitation-contraction coupling and propensity for arrhythmia. Additionally, I(to) and its molecular constituents are consistently reduced in cardiac hypertrophy and heart failure. In this review, we discuss the physiological role of I(to) as well as the molecular basis of this current in human and canine hearts, in which I(to) has been thoroughly studied. In particular, we discuss the role of Ito; in the action potential and the mechanisms by which I(to) modulates excitation-contraction coupling. We also describe the effects of mutations in the subunits constituting the Ito channel as well as the role of I(to) in the failing myocardium. Finally, we review pharmacological modulation of I(to) and discuss the evidence supporting the hypothesis that restoration of I(to) in the setting of heart failure may be therapeutically beneficial by enhancing excitation-contraction coupling and cardiac function.

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Atrial fibrillation (AF) is the most common supraventricular arrhythmia that, for unknown reasons, is linked to intense endurance exercise. Our studies reveal that 6 weeks of swimming or treadmill exercise improves heart pump function and reduces heart-rates. Exercise also increases vulnerability to AF in association with inflammation, fibrosis, increased vagal tone, slowed conduction velocity, prolonged cardiomyocyte action potentials and RyR2 phosphorylation (CamKII-dependent S2814) in the atria, without corresponding alterations in the ventricles. Microarray results suggest the involvement of the inflammatory cytokine, TNFα, in exercised-induced atrial remodelling. Accordingly, exercise induces TNFα-dependent activation of both NFκB and p38MAPK, while TNFα inhibition (with etanercept), TNFα gene ablation, or p38 inhibition, prevents atrial structural remodelling and AF vulnerability in response to exercise, without affecting the beneficial physiological changes. Our results identify TNFα as a key factor in the pathology of intense exercise-induced AF.

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Directed differentiation protocols enable derivation of cardiomyocytes from human pluripotent stem cells (hPSCs) and permit engineering of human myocardium in vitro. However, hPSC-derived cardiomyocytes are reflective of very early human development, limiting their utility in the generation of in vitro models of mature myocardium. Here we describe a platform that combines three-dimensional cell cultivation with electrical stimulation to mature hPSC-derived cardiac tissues. We used quantitative structural, molecular and electrophysiological analyses to explain the responses of immature human myocardium to electrical stimulation and pacing. We demonstrated that the engineered platform allows for the generation of three-dimensional, aligned cardiac tissues (biowires) with frequent striations. Biowires submitted to electrical stimulation had markedly increased myofibril ultrastructural organization, elevated conduction velocity and improved both electrophysiological and Ca(2+) handling properties compared to nonstimulated controls. These changes were in agreement with cardiomyocyte maturation and were dependent on the stimulation rate.

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Phospholamban (PLN) is a 52 amino acid integral membrane protein of the sarcoplasmic reticulum (SR) that exists in both monomeric and pentameric forms. In its unphosphorylated state, PLN inhibits the SR Ca(2+) ATPase (SERCA). This inhibition is relieved when PLN is phosphorylated as a result of ß-adrenergic stimulation of the heart. Consistent with some predictions from molecular models and from functional studies of PLN incorporated into planar lipid bilayers, it has also been postulated that pentameric PLN can also form ion-selective channels. Other molecular models contradict this hypothesis, however. In the work reported here, we used the Ca(2+)-sensitive fluorescent dye Fura-2, to examine the passive Ca(2+) permeability of the SR membrane in vesicles derived from cardiac ventricle. We have found that phosphorylation of PLN by protein kinase A (PKA) leads to an increase in the rate of Ca(2+) leak from Ca(2+)-loaded SR vesicles. This enhanced rate of Ca(2+) leak from the SR is also observed when SR vesicles are incubated with a PLN specific antibody (A1) that mimics phosphorylation of PLN. The ryanodine receptor blocker ruthenium red does not affect the increased rate of Ca(2+) leak from the SR after PLN phosphorylation with PKA or after exposure to A1 antibody, arguing against a possible role of ryanodine receptors in mediating the enhanced leak. Our results are consistent with the hypothesis that phosphorylated PLN forms or regulates a Ca(2+) leak pathway in cardiac SR membranes in situ.

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Inorganic polyphosphate (poly P) is a polymer made from as few as 10 to several hundred phosphate molecules linked by phosphoanhydride bonds similar to ATP. Poly P is ubiquitous in all mammalian organisms, where it plays multiple physiological roles. The metabolism of poly P in mammalian organisms is not well understood. We have examined the mechanism of poly P production and the role of this polymer in cell energy metabolism. Poly P levels in mitochondria and intact cells were estimated using a fluorescent molecular probe, 4',6-diamidino-2-phenylindole. Poly P levels were dependent on the metabolic state of the mitochondria. Poly P levels were increased by substrates of respiration and in turn reduced by mitochondrial inhibitor (rotenone) or an uncoupler (carbonyl cyanide p-trifluoromethoxyphenylhydrazone). Oligomycin, an inhibitor of mitochondrial ATP-synthase, blocked the production of poly P. Enzymatic depletion of poly P from cells significantly altered the rate of ATP metabolism. We propose the existence of a feedback mechanism where poly P production and cell energy metabolism regulate each other.

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Ca(2+) transport by the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) is sensitive to monovalent cations. Possible K(+) binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca(2+) transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca(2+) transport correlated in most cases with their direct effects on SERCA. Choline(+), however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca(2+) transport. Of the monovalent cations tested, only Cs(+) significantly affected the Hill coefficient of Ca(2+) transport (n(H)). An increase in n(H) from approximately 2 in K(+) to approximately 3 in Cs(+) was seen in all of the forms of SERCA examined. The effects of Cs(+) on the maximum velocity of Ca(2+) uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K(+) binding sites of the different forms of the protein.

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Anion and cation channels present in the sarcoplasmic reticulum (SR) are believed to be necessary to maintain the electroneutrality of SR membrane during Ca(2+) uptake by the SR Ca(2+) pump (SERCA). Here we incorporated canine cardiac SR ion channels into lipid bilayers and studied the effects of tamoxifen and other antiestrogens on these channels. A Cl(-) channel was identified exhibiting multiple subconductance levels which could be divided into two primary conductance bands. Tamoxifen decreases the time the channel spends in its higher, voltage-sensitive band and the mean channel current. The lower, voltage-insensitive, conductance band is not affected by tamoxifen, nor is a K(+) channel present in the cardiac SR preparation. By examining SR Ca(2+) uptake, SERCA ATPase activity, and SR ion channels in the same preparation, we also estimated SERCA transport current, SR Cl(-) and K(+) currents, and the density of SERCA, Cl(-), and K(+) channels in cardiac SR membranes.

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Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4',6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the microg/ml range. Here, we report that long-wavelength excitation (> or = 400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.

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Replacement of K(+) with Cs(+) on the cytoplasmic side of the sarcoplasmic reticulum (SR) membrane reduces the maximum velocity (V(max)) of Ca(2+) uptake into the SR of saponin-permeabilized rat ventricular myocytes. To compare the sensitivity of the cardiac and smooth muscle/non-muscle forms of the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a and -2b respectively) to replacement of K(+) with Cs(+), SERCA2a and SERCA2b were expressed in HEK-293 cells. Ca(2+) uptake into HEK cell microsomes was inhibited by replacement of extravesicular K(+) with Cs(+) (V(max) of SERCA2a-mediated Ca(2+) uptake in CsCl was 80% of that in KCl; V(max) of SERCA2b-mediated uptake was 70% of that in KCl). The Ca(2+) sensitivity of uptake was decreased for both SERCA2a- and SERCA2b-mediated uptake and the Hill coefficients were increased in the presence of CsCl. The effects of Cs(+) on uptake were associated with direct inhibition of the ATPase activity of SERCA2a and SERCA2b. Our results indicate that cation binding sites are present in both SERCA2 isoforms, although the extent to which SERCA2b is inhibited by K(+) replacement is greater than that of SERCA2a or SERCA1. Consideration of these results and the recent molecular modeling work of others suggests that monovalent cations could interact with the Ca(2+) binding region of SERCA.

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