Publications by authors named "Guy Salama"

76 Publications

Cardiac natriuretic peptide deficiency sensitizes the heart to stress induced ventricular arrhythmias via impaired CREB signaling.

Cardiovasc Res 2021 Jul 30. Epub 2021 Jul 30.

Pittsburgh Heart, Lung, Blood, and Vascular Medicine Institute and Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine and University of Pittsburgh Medical Center, Pittsburgh, PA, USA.

Aims: The cardiac natriuretic peptides (atrial natriuretic peptide [ANP] and B-type natriuretic peptide [BNP]) are important regulators of cardiovascular physiology, with reduced natriuretic peptide (NP) activity linked to multiple human cardiovascular diseases. We hypothesized that deficiency of either ANP or BNP would lead to similar changes in left ventricular structure and function given their shared receptor affinities.

Methods And Results: We directly compared murine models deficient of ANP or BNP in the same genetic backgrounds (C57BL6/J) and environments. We evaluated control, ANP deficient (Nppa-/-) or BNP deficient (Nppb-/-) mice under unstressed conditions and multiple forms of pathological myocardial stress. Survival, myocardial structure, function and electrophysiology, tissue histology, and biochemical analyses were evaluated in the groups. In vitro validation of our findings was performed using human derived induced pluripotent stem cell cardiomyocytes (iPS-CM). In the unstressed state, both ANP and BNP deficient mice displayed mild ventricular hypertrophy which did not increase up to 1 year of life. NP-deficient mice exposed to acute myocardial stress secondary to thoracic aortic constriction (TAC) had similar pathological myocardial remodeling but a significant increase in sudden death. We discovered that the NP-deficient mice are more susceptible to stress induced ventricular arrhythmias using both in vivo and ex vivo models. Mechanistically, deficiency of either ANP or BNP led to reduced myocardial cGMP levels and reduced phosphorylation of the cAMP response element-binding protein (CREBS133) transcriptional regulator. Selective CREB inhibition sensitized wild type hearts to stress induced ventricular arrhythmias. ANP and BNP regulate cardiomyocyte CREBS133 phosphorylation through a cGMP-dependent protein kinase 1 (PKG1) and p38 mitogen activated protein kinase (p38 MAPK) signaling cascade.

Conclusions: Our data show that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels to regulate cardiomyocyte p38 MAPK and CREB activity. Cardiac natriuretic peptide deficiency leads to a reduction in CREB signaling which sensitizes the heart to stress induced ventricular arrhythmias.

Translational Perspective: Our study found that ANP or BNP deficiency leads to increased sudden death and ventricular arrhythmias after acute myocardial stress in murine models. We discovered that ANP and BNP act in a non-redundant fashion to maintain myocardial cGMP levels and uncovered a unique role for these peptides in regulating cardiomyocyte p38 MAPK and CREB signaling through a cGMP-PKG1 pathway. Importantly, this signaling pathway was conserved in human cardiomyocytes. This study provides mechanistic insight into how modulating natriuretic peptide levels in human heart failure patients reduces sudden death and mortality.
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http://dx.doi.org/10.1093/cvr/cvab257DOI Listing
July 2021

Relaxin Inhibits Ventricular Arrhythmia and Asystole in Rats With Pulmonary Arterial Hypertension.

Front Cardiovasc Med 2021 6;8:668222. Epub 2021 Jul 6.

Department of Medicine, University of Pittsburgh, Pittsburgh, PA, United States.

Pulmonary arterial hypertension (PAH) leads to right ventricular cardiomyopathy and cardiac dysfunctions where in the clinical setting, cardiac arrest is the likely cause of death, in ~70% of PAH patients. We investigated the cardiac phenotype of PAH hearts and tested the hypothesis that the insulin-like hormone, Relaxin could prevent maladaptive cardiac remodeling and protect against cardiac dysfunctions in a PAH animal model. PAH was induced in rats with sugen (20 mg/kg), hypoxia then normoxia (3-weeks/each); relaxin (RLX = 0, 30 or 400 μg/kg/day, ≥ 6/group) was delivered subcutaneously (6-weeks) with implanted osmotic mini-pumps. Right ventricle (RV) hemodynamics and Doppler-flow measurements were followed by cardiac isolation, optical mapping, and arrhythmia phenotype. Sugen-hypoxia (SuHx) treated rats developed PAH characterized by higher RV systolic pressures (50 ± 19 vs. 22 ± 5 mmHg), hypertrophy, reduced stroke volume, ventricular fibrillation (VF) ( = 6/11) and bradycardia/arrest ( = 5/11); both cardiac phenotypes were suppressed with dithiothreitol (DTT = 1 mM) ( = 0/2/group) or RLX (low or high dose, = 0/6/group). PAH hearts developed increased fibrosis that was reversed by RLX-HD, but not RLX-LD. Relaxin decreased Nrf2 and glutathione transferases but not glutathione-reductase. High-dose RLX improved pulmonary arterial compliance (measured by Doppler flow), suppressed VF even after burst-pacing, = 2/6). Relaxin suppressed VF and asystole through electrical remodeling and by reversing thiol oxidative stress. For the first time, we showed two cardiac phenotypes in PAH animals and their prevention by RLX. Relaxin may modulate maladaptive cardiac remodeling in PAH and protect against arrhythmia and cardiac arrest.
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http://dx.doi.org/10.3389/fcvm.2021.668222DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8290063PMC
July 2021

Relaxin abrogates genomic remodeling of the aged heart.

Vitam Horm 2021 18;115:419-448. Epub 2021 Feb 18.

Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, PA, United States.

"Healthy" aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in clinical trials, Relaxin-therapy for 2-days reduced mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short lifespan (2-3h) but long-lasting protective actions suggested that relaxin acts at a genomic level to reverse maladaptive remodeling in AF, HF and aging. Our recent studies showed that a 2-week treatment with Relaxin (0.4mg/kg/day) of aged (24months old F-344 rats) increases the expression of voltage-gated Na channels (mRNA, Nav1.5 and I), connexin-43, abrogates inflammatory and immune responses and reverses myocardial fibrosis and cellular hypertrophy of the aged hearts. Relaxin acts directly at a wide range of cell types in the cardiovascular system that express its cognate GPCR receptor, RXFP1. RNA-seq analysis of young and aged hearts with and without Relaxin treatment revealed that "normal" aging altered the expression of ~10% of genes expressed in the ventricles, including: ion channels, components of fibrosis, hemodynamic biomarkers, immune and inflammatory responses which were reversed by Relaxin. The extensive cardiovascular remodeling caused by Relaxin was mediated through the activation of the Wnt/β-catenin signaling pathway which was otherwise suppressed by in adult cardiomyocytes intracellular by cytosolic Dickkopf1 (Dkk1). Wnt/β-catenin signaling is a mechanism that can explain the pleiotropic actions of Relaxin and the marked reversal of genomic changes that occur in aged hearts.
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http://dx.doi.org/10.1016/bs.vh.2020.12.017DOI Listing
February 2021

Do sex hormones impact stress responses by modulating the cellular composition of the myocardium?

Cardiovasc Res 2021 Aug;117(10):2140-2142

Department of Medicine, University of Pittsburgh, 3550 Terrace Street, S628 Scaife Hall, Pittsburgh, PA 15261, USA.

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http://dx.doi.org/10.1093/cvr/cvab072DOI Listing
August 2021

Relaxin reverses maladaptive remodeling of the aged heart through Wnt-signaling.

Sci Rep 2019 12 6;9(1):18545. Epub 2019 Dec 6.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, 15261, USA.

Healthy aging results in cardiac structural and electrical remodeling that increases susceptibility to cardiovascular diseases. Relaxin, an insulin-like hormone, suppresses atrial fibrillation, inflammation and fibrosis in aged rats but the mechanisms-of-action are unknown. Here we show that relaxin treatment of aged rats reverses pathological electrical remodeling (increasing Nav1.5 expression and localization of Connexin43 to intercalated disks) by activating canonical Wnt signaling. In isolated adult ventricular myocytes, relaxin upregulated Nav1.5 (EC = 1.3 nM) by a mechanism inhibited by the addition of Dickkopf-1. Furthermore, relaxin increased the levels of connexin43, Wnt1, and cytosolic and nuclear β-catenin. Treatment with Wnt1 or CHIR-99021 (a GSK3β inhibitor) mimicked the relaxin effects. In isolated fibroblasts, relaxin blocked TGFβ-induced collagen elevation in a Wnt dependent manner. These findings demonstrate a close interplay between relaxin and Wnt-signaling resulting in myocardial remodeling and reveals a fundamental mechanism of great therapeutic potential.
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http://dx.doi.org/10.1038/s41598-019-53867-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897890PMC
December 2019

Identification of peripheral neural circuits that regulate heart rate using optogenetic and viral vector strategies.

Nat Commun 2019 04 26;10(1):1944. Epub 2019 Apr 26.

Cardiac Arrhythmia Center and Neurocardiology Research Program of Excellence, David Geffen School of Medicine, University of California - Los Angeles (UCLA), Los Angeles, CA, 90095, USA.

Heart rate is under the precise control of the autonomic nervous system. However, the wiring of peripheral neural circuits that regulate heart rate is poorly understood. Here, we develop a clearing-imaging-analysis pipeline to visualize innervation of intact hearts in 3D and employed a multi-technique approach to map parasympathetic and sympathetic neural circuits that control heart rate in mice. We identify cholinergic neurons and noradrenergic neurons in an intrinsic cardiac ganglion and the stellate ganglia, respectively, that project to the sinoatrial node. We also report that the heart rate response to optogenetic versus electrical stimulation of the vagus nerve displays different temporal characteristics and that vagal afferents enhance parasympathetic and reduce sympathetic tone to the heart via central mechanisms. Our findings provide new insights into neural regulation of heart rate, and our methodology to study cardiac circuits can be readily used to interrogate neural control of other visceral organs.
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http://dx.doi.org/10.1038/s41467-019-09770-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486614PMC
April 2019

Cardioprotective actions of relaxin.

Mol Cell Endocrinol 2019 05 6;487:45-53. Epub 2019 Jan 6.

Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, PA, 15261, USA; McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, 15261, USA. Electronic address:

Relaxin is a hormone of pregnancy first discovered for its ability to induce ligament relaxation in nonpregnant guinea pig and is important for softening of the birth canal during parturition, decidualization, implantation, nipple development and increased maternal renal perfusion, glomerular filtration, and cardiac output. Subsequently, relaxin has been shown to exert multiple beneficial cardiovascular effects during pathological events such as hypertension, atrial fibrillation, heart failure and myocardial infarction, including suppression of arrhythmia and inflammation, and reversal of fibrosis. Despite extensive studies, the mechanisms underlying relaxin's effects are not well understood. Relaxin signals primarily through its G protein coupled receptor, the relaxin family peptide receptor-1, to activate multiple signaling pathways and this review summarizes our understanding of these pathways as they relate to the cardioprotective actions of relaxin, focusing on relaxin's anti-fibrotic, anti-arrhythmic and anti-inflammatory properties. Further, this review includes a brief overview of relaxin in clinical trials for heart failure and progress in the development of relaxin mimetics.
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http://dx.doi.org/10.1016/j.mce.2018.12.016DOI Listing
May 2019

Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling.

Sci Rep 2018 05 8;8(1):7145. Epub 2018 May 8.

Institute of Physiology I, Life&Brain Center, Medical Faculty, University of Bonn, Sigmund Freud Str. 25, 53127, Bonn, Germany.

Ventricular tachycardia (VT) is the most common and potentially lethal complication following myocardial infarction (MI). Biological correction of the conduction inhomogeneity that underlies re-entry could be a major advance in infarction therapy. As minimal increases in conduction of infarcted tissue markedly influence VT susceptibility, we reasoned that enhanced propagation of the electrical signal between non-excitable cells within a resolving infarct might comprise a simple means to decrease post-infarction arrhythmia risk. We therefore tested lentivirus-mediated delivery of the gap-junction protein Connexin 43 (Cx43) into acute myocardial lesions. Cx43 was expressed in (myo)fibroblasts and CD45 cells within the scar and provided prominent and long lasting arrhythmia protection in vivo. Optical mapping of Cx43 injected hearts revealed enhanced conduction velocity within the scar, indicating Cx43-mediated electrical coupling between myocytes and (myo)fibroblasts. Thus, Cx43 gene therapy, by direct in vivo transduction of non-cardiomyocytes, comprises a simple and clinically applicable biological therapy that markedly reduces post-infarction VT.
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http://dx.doi.org/10.1038/s41598-018-25147-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5940892PMC
May 2018

Relaxin reverses inflammatory and immune signals in aged hearts.

PLoS One 2018 18;13(1):e0190935. Epub 2018 Jan 18.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, United States of America.

Background: 'Healthy' aging drives structural and functional changes in the heart including maladaptive electrical remodeling, fibrosis and inflammation, which lower the threshold for cardiovascular diseases such as heart failure (HF) and atrial fibrillation (AF). Despite mixed results in recent clinical trials, Relaxin-therapy for 2-days could reduce mortality by 37% at 180-days post-treatment, in patients with acute decompensated HF. Relaxin's short life-span (hours) but long-lasting protective actions led us to test the hypothesis that relaxin acts at a genomic level to reverse maladaptive remodeling in aging and HF.

Methods And Results: Young (9-month) and aged (24-month), male and female F-344/Brown Norway rats were treated with relaxin (0.4 mg/kg/day) for 2-weeks delivered by subcutaneous osmotic mini-pumps or with sodium acetate (controls). The genomic effects of aging and relaxin were evaluated by extracting RNA from the left ventricles and analyzing genomic changes by RNA-sequencing, Ingenuity Pathway Analysis, MetaCore and tissue immunohistochemistry. We found that aging promotes a native inflammatory response with distinct sex-differences and relaxin suppresses transcription of multiple genes and signaling pathways associated with inflammation and HF in both genders. In addition, aging significantly increased: macrophage infiltration and atrial natriuretic peptide levels in female ventricles, and activation of the complement cascade, whereas relaxin reversed these age-related effects.

Conclusion: These data support the hypothesis that relaxin alters gene transcription and suppresses inflammatory pathways and genes associated with HF and aging. Relaxin's suppression of inflammation and fibrosis supports its potential as a therapy for cardiovascular and inflammation-related diseases, such as HF, AF and diabetes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190935PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5773192PMC
February 2018

Estradiol up-regulates L-type Ca channels via membrane-bound estrogen receptor/phosphoinositide-3-kinase/Akt/cAMP response element-binding protein signaling pathway.

Heart Rhythm 2018 05 9;15(5):741-749. Epub 2018 Jan 9.

Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, Pennsylvania. Electronic address:

Background: In long QT syndrome type 2, women are more prone than men to the lethal arrhythmia torsades de pointes. We previously reported that 17β-estradiol (E2) up-regulates L-type Ca channels and current (I) (∼30%) in rabbit ventricular myocytes by a classic genomic mechanism mediated by estrogen receptor-α (ERα). In long QT syndrome type 2 (I blockade or bradycardia), the higher Ca influx via I causes Ca overload, spontaneous sarcoplasmic reticulum Ca release, and reactivation of I that triggers early afterdepolarizations and torsades de pointes.

Objective: The purpose of this study was to investigate the molecular mechanisms whereby E2 up-regulates I, which are poorly understood.

Methods: H9C2 and rat myocytes were incubated with E2 ± ER antagonist, or inhibitors of downstream transcription factors, for 24 hours, followed by western blots of Cav1.2α1C and voltage-clamp measurements of I.

Results: Incubation of H9C2 cells with E2 (10-100 nM) increased I density and Cav1.2α1C expression, which were suppressed by the ER antagonist ICI182,780 (1 μM). Enhanced I and Cav1.2α1C expression by E2 was suppressed by inhibitors of phosphoinositide-3-kinase (Pi3K) (30 μM LY294002; P <.05) and Akt (5 μM MK2206) but not of mitogen-activated protein kinase (5 μM U0126) or protein kinase A (1 μM KT5720). E2 incubation increased p-CREB via the Pi3K/Akt pathway, reached a peak in 20 minutes (3-fold), and leveled off to 1.5-fold 24 hours later. Furthermore, a CREB decoy oligonucleotide inhibited E2-induced Cav1.2α1C expression, whereas membrane-impermeable E2 (E2-bovine serum albumin) was equally effective at Cav1.2α1C up-regulation as E2.

Conclusion: Estradiol up-regulates Cav1.2α1C and I via plasma membrane ER and by activating Pi3K, Akt, and CREB signaling. The promoter regions of the CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding sites for p-CREB and ERα, which suggests a synergistic regulation by these pathways.
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http://dx.doi.org/10.1016/j.hrthm.2018.01.019DOI Listing
May 2018

A model of cardiac ryanodine receptor gating predicts experimental Ca-dynamics and Ca-triggered arrhythmia in the long QT syndrome.

Chaos 2017 Sep;27(9):093940

Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

Abnormal Ca handling is well-established as the trigger of cardiac arrhythmia in catecholaminergic polymorphic ventricular tachycardia and digoxin toxicity, but its role remains controversial in Torsade de Pointes (TdP), the arrhythmia associated with the long QT syndrome (LQTS). Recent experimental results show that early afterdepolarizations (EADs) that initiate TdP are caused by spontaneous (non-voltage-triggered) Ca release from Ca-overloaded sarcoplasmic reticulum (SR) rather than the activation of the L-type Ca-channel window current. In bradycardia and long QT type 2 (LQT2), a second, non-voltage triggered cytosolic Ca elevation increases gradually in amplitude, occurs before overt voltage instability, and then precedes the rise of EADs. Here, we used a modified Shannon-Puglisi-Bers model of rabbit ventricular myocytes to reproduce experimental Ca dynamics in bradycardia and LQT2. Abnormal systolic Ca-oscillations and EADs caused by SR Ca-release are reproduced in a modified 0-dimensional model, where 3 gates in series control the ryanodine receptor (RyR2) conductance. Two gates control RyR2 activation and inactivation and sense cytosolic Ca while a third gate senses luminal junctional SR Ca. The model predicts EADs in bradycardia and low extracellular [K] and cessation of SR Ca-release terminate salvos of EADs. Ca-waves, systolic cell-synchronous Ca-release, and multifocal diastolic Ca release seen in subcellular Ca-mapping experiments are observed in the 2-dimensional version of the model. These results support the role of SR Ca-overload, abnormal SR Ca-release, and the subsequent activation of the electrogenic Na/Ca-exchanger as the mechanism of TdP. The model offers new insights into the genesis of cardiac arrhythmia and new therapeutic strategies.
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http://dx.doi.org/10.1063/1.5000711DOI Listing
September 2017

Genomic upregulation of cardiac Cav1.2α and NCX1 by estrogen in women.

Biol Sex Differ 2017 08 14;8(1):26. Epub 2017 Aug 14.

Department of Bioengineering and the Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, PA, 15261, USA.

Background: Women have a higher risk of lethal arrhythmias than men in long QT syndrome type 2 (LQTS2), but the mechanisms remain uncertain due to the limited availability of healthy control human tissue. We have previously reported that in female rabbits, estrogen increases arrhythmia risk in drug-induced LQTS2 by upregulating L-type Ca (I) and sodium-calcium exchange (I) currents at the base of the epicardium by a genomic mechanism. This study investigates if the effects of estrogen on rabbit I and I apply to human hearts.

Methods: Postmortem human left ventricular tissue samples were probed with selective antibodies for regional heterogeneities of ion channel protein expression and compared to rabbit myocardium. Functionally, I and I were measured from female and male cardiomyocytes derived from human induced pluripotent stem cells (iPS-CMs) with the voltage-clamp technique from control and estrogen-treated iPS-CMs.

Results: In women (n = 12), Cav1.2α (primary subunit of the L-type calcium channel protein 1) and NCX1 (sodium-calcium exchange protein) levels were higher at the base than apex of the epicardium (40 ± 14 and 81 ± 30%, respectively, P < 0.05), but not in men (n = 6) or postmenopausal women (n = 6). Similarly, in cardiomyocytes derived from female human iPS-CMs, estrogen (1 nM, 1-2 days) increased I (31%, P < 0.05) and I (7.5-fold, - 90 mV, P < 0.01) and their mRNA levels (P < 0.05). Moreover, in male human iPS-CMs, estrogen failed to alter I and I.

Conclusions: The results show that estrogen upregulates cardiac I and I in women through genomic mechanisms that account for sex differences in Ca handling and spatial heterogeneities of repolarization due to base-apex heterogeneities of Cav1.2α and NCX1. By analogy with rabbit studies, these effects account for human sex-difference in arrhythmia risk.
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http://dx.doi.org/10.1186/s13293-017-0148-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557418PMC
August 2017

Gene-Targeted Mice with the Human Troponin T R141W Mutation Develop Dilated Cardiomyopathy with Calcium Desensitization.

PLoS One 2016 9;11(12):e0167681. Epub 2016 Dec 9.

UPMC Heart and Vascular Institute and Division of Cardiology, Department of Medicine, University of Pittsburgh, Pittsburgh, PA, United States of America.

Most studies of the mechanisms leading to hereditary dilated cardiomyopathy (DCM) have been performed in reconstituted in vitro systems. Genetically engineered murine models offer the opportunity to dissect these mechanisms in vivo. We generated a gene-targeted knock-in murine model of the autosomal dominant Arg141Trp (R141W) mutation in Tnnt2, which was first described in a human family with DCM. Mice heterozygous for the mutation (Tnnt2R141W/+) recapitulated the human phenotype, developing left ventricular dilation and reduced contractility. There was a gene dosage effect, so that the phenotype in Tnnt2R141W/+mice was attenuated by transgenic overexpression of wildtype Tnnt2 mRNA transcript. Male mice exhibited poorer survival than females. Biomechanical studies on skinned fibers from Tnnt2R141W/+ hearts showed a significant decrease in pCa50 (-log[Ca2+] required for generation of 50% of maximal force) relative to wildtype hearts, indicating Ca2+ desensitization. Optical mapping studies of Langendorff-perfused Tnnt2R141W/+ hearts showed marked increases in diastolic and peak systolic intracellular Ca2+ ([Ca2+]i), and prolonged systolic rise and diastolic fall of [Ca2+]i. Perfused Tnnt2R141W/+ hearts had slower intrinsic rates in sinus rhythm and reduced peak heart rates in response to isoproterenol. Tnnt2R141W/+ hearts exhibited a reduction in phosphorylated phospholamban relative to wildtype mice. However, crossing Tnnt2R141W/+ mice with phospholamban knockout (Pln-/-) mice, which exhibit increased Ca2+ transients and contractility, had no effect on the DCM phenotype. We conclude that the Tnnt2 R141W mutation causes a Ca2+ desensitization and mice adapt by increasing Ca2+-transient amplitudes, which impairs Ca2+ handling dynamics, metabolism and responses to β-adrenergic activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0167681PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5147943PMC
July 2017

Treatment of catecholaminergic polymorphic ventricular tachycardia in mice using novel RyR2-modifying drugs.

Int J Cardiol 2017 Jan 29;227:668-673. Epub 2016 Oct 29.

Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA; Department of Medicine (Cardiology), Baylor College of Medicine, Houston, TX 77030, USA; Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA. Electronic address:

Rationale: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal arrhythmic disorder caused by mutations in the type-2 ryanodine receptor (RyR2). Mutant RyR2 cause abnormal Ca leak from the sarcoplasmic reticulum (SR), which is associated with the development of arrhythmias.

Objective: To determine whether derivatives of tetracaine, a local anesthetic drug with known RyR2 inhibiting action, could prevent CPVT induction by suppression of RyR2-mediated SR Ca leak.

Methods And Results: Confocal microscopy was used to assess the effects of tetracaine and 9 derivatives (EL1-EL9) on spontaneous Ca sparks in ventricular myocytes isolated from RyR2-R176Q/+ mice with CPVT. Whereas each derivative suppressed the Ca spark frequency, derivative EL9 was most effective at the screening dose of 500nmol/L. At this high dose, the Ca transient amplitude was not affected in myocytes from WT or R176Q/+ mice. The IC of EL9 was determined to be 13nmol/L, which is about 400× time lower than known RyR2 stabilizer K201. EL9 prevented the induction of ventricular tachycardia observed in placebo-treated R176Q/+ mice, without affecting heart rate or cardiac contractility.

Conclusions: Tetracaine derivatives represent a novel class of RyR2 stabilizing drugs that could be used for the treatment of the potentially fatal disorder catecholaminergic polymorphic ventricular tachycardia.
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http://dx.doi.org/10.1016/j.ijcard.2016.10.078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5164850PMC
January 2017

Relaxin suppresses atrial fibrillation in aged rats by reversing fibrosis and upregulating Na+ channels.

Heart Rhythm 2016 Apr 19;13(4):983-91. Epub 2015 Dec 19.

University of Pittsburgh School of Medicine, Heart and Vascular Institute, Pittsburgh, Pennsylvania,; University of Pittsburgh Department of Bioengineering, and the McGowan Institute for Regenerative Medicine, Pittsburgh, Pennsylvania. Electronic address:

Background: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly patients and has been correlated with enhanced age-dependent atrial fibrosis. Reversal of atrial fibrosis has been proposed as therapeutic strategy to suppress AF.

Objective: To test the ability of relaxin to reverse age-dependent atrial fibrosis and suppress AF.

Methods: Aged F-344 rats (24 months old) were treated with subcutaneous infusion of vehicle or relaxin (0.4 mg/kg/day) for 2 weeks. Rat hearts were excised, perfused on a Langendorff apparatus, and stained with voltage and Ca(2+) indicator dyes. Optical mapping and programmed electrical stimulation was used to test arrhythmia vulnerability and changes in electrophysiological characteristics. Changes in protein expression and Na(+) current density (INa) were measured by tissue immunofluorescence and whole-cell patch clamp technique.

Results: In aged rats, sustained AF was readily induced with a premature pulse (n = 7/8) and relaxin treatment suppressed sustained AF by a premature impulse or burst pacing (n = 1/6) (P < .01). Relaxin significantly increased atrial action potential conduction velocity and decreased atrial fibrosis. Relaxin treatment increased Nav1.5 expression (n = 6; 36% ± 10%) and decreased total collagen and collagen I (n = 5-6; 55%-66% ± 15%) in aged atria (P < .05) and decreased collagen I and III and TGF-β1 mRNA (P < .05). Voltage-clamp experiments demonstrated that relaxin treatment (100 nM for 2 days) increased atrial INa by 46% ± 4% (n = 12-13/group, P < .02).

Conclusion: Relaxin suppresses AF through an increase in atrial conduction velocity by decreasing atrial fibrosis and increasing INa. These data provide compelling evidence that relaxin may serve as an effective therapy to manage AF in geriatric patients by reversing fibrosis and modulating cardiac ionic currents.
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http://dx.doi.org/10.1016/j.hrthm.2015.12.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801709PMC
April 2016

The link between abnormal calcium handling and electrical instability in acquired long QT syndrome--Does calcium precipitate arrhythmic storms?

Prog Biophys Mol Biol 2016 Jan 26;120(1-3):210-21. Epub 2015 Nov 26.

Department of Medicine, Heart and Vascular Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address:

Release of Ca(2+) ions from sarcoplasmic reticulum (SR) into myocyte cytoplasm and their binding to troponin C is the final signal form myocardial contraction. Synchronous contraction of ventricular myocytes is necessary for efficient cardiac pumping function. This requires both shuttling of Ca(2+) between SR and cytoplasm in individual myocytes, and organ-level synchronization of this process by means of electrical coupling among ventricular myocytes. Abnormal Ca(2+) release from SR causes arrhythmias in the setting of CPVT (catecholaminergic polymorphic ventricular tachycardia) and digoxin toxicity. Recent optical mapping data indicate that abnormal Ca(2+) handling causes arrhythmias in models of both repolarization impairment and profound bradycardia. The mechanisms involve dynamic spatial heterogeneity of myocardial Ca(2+) handling preceding arrhythmia onset, cell-synchronous systolic secondary Ca(2+) elevation (SSCE), as well as more complex abnormalities of intracellular Ca(2+) handling detected by subcellular optical mapping in Langendorff-perfused hearts. The regional heterogeneities in Ca(2+) handling cause action potential (AP) heterogeneities through sodium-calcium exchange (NCX) activation and eventually overwhelm electrical coupling of the tissue. Divergent Ca(2+) dynamics among different myocardial regions leads to temporal instability of AP duration and - on the patient level - in T wave lability. Although T-wave alternans has been linked to cardiac arrhythmias, non-alternans lability is observed in pre-clinical models of the long QT syndrome (LQTS) and CPVT, and in LQTS patients. Analysis of T wave lability may provide a real-time window on the abnormal Ca(2+) dynamics causing specific arrhythmias such as Torsade de Pointes (TdP).
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http://dx.doi.org/10.1016/j.pbiomolbio.2015.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808372PMC
January 2016

Synchronous systolic subcellular Ca2+-elevations underlie ventricular arrhythmia in drug-induced long QT type 2.

Circ Arrhythm Electrophysiol 2015 Jun 26;8(3):703-12. Epub 2015 Feb 26.

From the Department of Bioengineering (J.J.K., G.S.), and Department of Medicine, Heart and Vascular Institute (J.J.K., J.N., Q.L., G.S.), University of Pittsburgh, PA; and Tsinghua University School of Medicine, China (Q.L).

Background: Repolarization delay is a common clinical problem, which can promote ventricular arrhythmias. In myocytes, abnormal sarcoplasmic reticulum Ca(2+)-release is proposed as the mechanism that causes early afterdepolarizations, the cellular equivalent of ectopic-activity in drug-induced long-QT syndrome. A crucial missing link is how such a stochastic process can overcome the source-sink mismatch to depolarize sufficient ventricular tissue to initiate arrhythmias.

Methods And Results: Optical maps of action potentials and Ca(2+)-transients from Langendorff rabbit hearts were measured at low (150×150 μm(2)/pixel) and high (1.5×1.5 μm(2)/pixel) resolution before and during arrhythmias. Drug-induced long QT type 2, elicited with dofetilide inhibition of IKr (the rapid component of rectifying K+ current), produced spontaneous Ca(2+)-elevations during diastole and systole, before the onset of arrhythmias. Diastolic Ca(2+-)waves appeared randomly, propagated within individual myocytes, were out-of-phase with adjacent myocytes, and often died-out. Systolic secondary Ca(2+-)elevations were synchronous within individual myocytes, appeared 188±30 ms after the action potential-upstroke, occurred during high cytosolic Ca(2+) (40%-60% of peak-Ca(2+)-transients), appeared first in small islands (0.5×0.5 mm(2)) that enlarged and spread throughout the epicardium. Synchronous systolic Ca(2+-)elevations preceded voltage-depolarizations (9.2±5 ms; n=5) and produced pronounced Spatial Heterogeneities of Ca(2+)-transient-durations and action potential-durations. Early afterdepolarizations originating from sites with the steepest gradients of membrane-potential propagated and initiated arrhythmias. Interestingly, more complex subcellular Ca(2+)-dynamics (multiple chaotic Ca(2+)-waves) occurred during arrhythmias. K201, a ryanodine receptor stabilizer, eliminated Ca(2+)-elevations and arrhythmias.

Conclusions: The results indicate that systolic and diastolic Ca(2+)-elevations emanate from sarcoplasmic reticulum Ca(2+)-release and systolic Ca(2+)-elevations are synchronous because of high cytosolic and luminal-sarcoplasmic reticulum Ca(2+), which overcomes source-sink mismatch to trigger arrhythmias in intact hearts.
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http://dx.doi.org/10.1161/CIRCEP.114.002214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4472565PMC
June 2015

Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells.

J Mol Cell Cardiol 2015 Apr 30;81:81-93. Epub 2015 Jan 30.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address:

Background And Objectives: The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain.

Methods: Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions.

Results: The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1μM) or caffeine (100μM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2μM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2μM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules.

Conclusions: Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.
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http://dx.doi.org/10.1016/j.yjmcc.2015.01.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4409767PMC
April 2015

Computational and experimental characterization of a fluorescent dye for detection of potassium ion concentration.

J Phys Chem A 2014 Oct 2;118(42):9837-43. Epub 2014 Oct 2.

Chemistry Departmentand ‡Molecular Biosensor and Imaging Center, Carnegie Mellon University , Pittsburgh, Pennsylvania 15213, United States.

The fluorescence of the SKC-513 ((E)-N-(9-(4-(1,4,7,10,13-pentaoxa-16-azacyclooctadecan-16-yl)phenyl)-6-(butyl(3-sulfopropyl)amino)-3H-xanthen-3-ylidene)-N-(3-sulfopropyl)butan-1-aminium) dye is shown experimentally to have high sensitivity to binding of the K(+) ion. Computations are used to explore the potential origins of this sensitivity and to make some suggestions regarding structural improvements. In the absence of K(+), excitation is to two nearly degenerate states, a neutral (N) excited state with a high oscillator strength, and a charge-transfer (CT) state with a lower oscillator strength. Binding of K(+) destabilizes the CT state, raising its energy far above the N state. The increase in fluorescence quantum yield upon binding of K(+) is attributed to the increased energy of the CT state suppressing a nonradiative pathway mediated by the CT state. The near degeneracy of the N and CT excited states can be understood by considering SKC-513 as a reduced symmetry version of a parent molecule with 3-fold symmetry. Computations show that acceptor-donor substituents can be used to alter the relative energies of the N and CT state, whereas a methylene spacer between the heterocycle and phenylene groups can be used to increase the coupling between these states. These modifications provide synthetic handles with which to optimize the dye for K(+) detection.
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http://dx.doi.org/10.1021/jp507552qDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207556PMC
October 2014

Sex differences in the mechanisms underlying long QT syndrome.

Am J Physiol Heart Circ Physiol 2014 Sep 27;307(5):H640-8. Epub 2014 Jun 27.

Center for Cellular and Systems Electrophysiology, University at Buffalo, State University of New York, Buffalo, New York; Department of Obstetrics and Gynecology, University at Buffalo, State University of New York, Buffalo, New York; and Department of Physiology and Biophysics, University at Buffalo, State University of New York, Buffalo, New York

Sexual dimorphism is a well-established phenomenon, but its degree varies tremendously among species. Since the early days of Einthoven's development of the three-lead galvanometer ECG, we have known there are marked differences in QT intervals of men and women. It required over a century to appreciate the profound implications of sex-based electrophysiological differences in QT interval on the panoply of sex differences with respect to arrhythmia risk, drug sensitivity, and treatment modalities. Little is known about the fundamental mechanism responsible for sex differences in electrical substrate of the human heart, in large part due to the lack of tissue availability. Animal models are an important research tool, but species differences in the sexual dimorphism of the QT interval, the ionic currents underlying the cardiac repolarization, and effects of sex steroids make it difficult to interpolate animal to human sex differences. In addition, in some species, different strains of the same animal model yield conflicting data. Each model has its strengths, such as ease of genetic manipulation in mice or size in dogs. However, many animals do not reproduce the sexual dimorphism of QT seen in humans. To match sex linked prolongation of QT interval and arrhythmogenic phenotype, the current data suggest that the rabbit may be best suited to provide insight into sex differences in humans. In the future, emerging technologies such as induced pluripotent stem cell derived cardiac myocyte systems may offer the opportunity to study sex differences in a controlled hormonal situation in the context of a sex specific human model system.
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http://dx.doi.org/10.1152/ajpheart.00864.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4187395PMC
September 2014

Engineered Human Muscle Tissue from Skeletal Muscle Derived Stem Cells and Induced Pluripotent Stem Cell Derived Cardiac Cells.

Int J Tissue Eng 2013 Sep;2013:198762

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15219, USA ; Department of Developmental Biology, University of Pittsburgh, Pittsburgh, PA 15224, USA ; McGowan Institute of Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA 15219, USA ; Rangos Research Center, Room 8121, 4401 Pennsylvania Avenue, Pittsburgh, PA 15224, USA.

During development, cardiac and skeletal muscle share major transcription factors and sarcomere proteins which were generally regarded as specific to either cardiac or skeletal muscle but not both in terminally differentiated adult cardiac or skeletal muscle. Here, we investigated whether artificial muscle constructed from human skeletal muscle derived stem cells (MDSCs) recapitulates developmental similarities between cardiac and skeletal muscle. We constructed 3-dimensional collagen-based engineered muscle tissue (EMT) using MDSCs (MDSC-EMT) and compared the biochemical and contractile properties with EMT using induced pluripotent stem (iPS) cell-derived cardiac cells (iPS-EMT). Both MDSC-EMT and iPS-EMT expressed cardiac specific troponins, fast skeletal muscle myosin heavy chain, and connexin-43 mimicking developing cardiac or skeletal muscle. At the transcriptional level, MDSC-EMT and iPS-EMT upregulated both cardiac and skeletal muscle-specific genes and expressed Nkx2.5 and Myo-D proteins. MDSC-EMT displayed intracellular calcium ion transients and responses to isoproterenol. Contractile force measurements of MDSC-EMT demonstrated functional properties of immature cardiac and skeletal muscle in both tissues. Results suggest that the EMT from MDSCs mimics developing cardiac and skeletal muscle and can serve as a useful functioning striated muscle model for investigation of stem cell differentiation and therapeutic options of MDSCs for cardiac repair.
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http://dx.doi.org/10.1155/2013/198762DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3984572PMC
September 2013

Repopulation of decellularized mouse heart with human induced pluripotent stem cell-derived cardiovascular progenitor cells.

Nat Commun 2013 ;4:2307

Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15201, USA.

Heart disease is the leading cause of death in the world. Heart tissue engineering holds a great promise for future heart disease therapy by building personalized heart tissues. Here we create heart constructs by repopulating decellularized mouse hearts with human induced pluripotent stem cell-derived multipotential cardiovascular progenitor cells. We show that the seeded multipotential cardiovascular progenitor cells migrate, proliferate and differentiate in situ into cardiomyocytes, smooth muscle cells and endothelial cells to reconstruct the decellularized hearts. After 20 days of perfusion, the engineered heart tissues exhibit spontaneous contractions, generate mechanical force and are responsive to drugs. In addition, we observe that heart extracellular matrix promoted cardiomyocyte proliferation, differentiation and myofilament formation from the repopulated human multipotential cardiovascular progenitor cells. Our novel strategy to engineer personalized heart constructs could benefit the study of early heart formation or may find application in preclinical testing.
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http://dx.doi.org/10.1038/ncomms3307DOI Listing
April 2014

Relaxin suppresses atrial fibrillation by reversing fibrosis and myocyte hypertrophy and increasing conduction velocity and sodium current in spontaneously hypertensive rat hearts.

Circ Res 2013 Jul 7;113(3):313-21. Epub 2013 Jun 7.

Department of Bioengineering, Medicine, University of Pittsburgh Medical Center Heart and Vascular Institute, and Developmental Biology, University of Pittsburgh, Pittsburgh, PA, USA.

Rationale: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy.

Objective: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR).

Methods And Results: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca²⁺ transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P<0.01 versus WKY), steeper CV restitution kinetics, greater collagen deposition, higher levels of transcripts for transforming growth factor-β, metalloproteinase-2, metalloproteinase-9, collagen I/III, and reduced connexin 43 phosphorylation (P<0.05 versus WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5) and SHR+V (n=4/4), but not in WKY (n=0/5) and SHR+RLX (n=1/8; P<0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV restitution kinetics, reduced action potential duration at 90% recovery to baseline, increased CV (P<0.01), and reversed atrial hypertrophy (P<0.05). Independent of antifibrotic actions, RLX (0.1 µmol/L) increased Na⁺ current density, INa (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P<0.01).

Conclusions: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing INa. The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.
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http://dx.doi.org/10.1161/CIRCRESAHA.113.301646DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774019PMC
July 2013

Bradycardia alters Ca(2+) dynamics enhancing dispersion of repolarization and arrhythmia risk.

Am J Physiol Heart Circ Physiol 2013 Mar 11;304(6):H848-60. Epub 2013 Jan 11.

Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Bradycardia prolongs action potential (AP) durations (APD adaptation), enhances dispersion of repolarization (DOR), and promotes tachyarrhythmias. Yet, the mechanisms responsible for enhanced DOR and tachyarrhythmias remain largely unexplored. Ca(2+) transients and APs were measured optically from Langendorff rabbit hearts at high (150 × 150 μm(2)) or low (1.5 × 1.5 cm(2)) magnification while pacing at a physiological (120 beats/min) or a slow heart rate (SHR = 50 beats/min). Western blots and pharmacological interventions were used to elucidate the regional effects of bradycardia. As a result, bradycardia (SHR 50 beats/min) increased APDs gradually (time constant τf→s = 48 ± 9.2 s) and caused a secondary Ca(2+) release (SCR) from the sarcoplasmic reticulum during AP plateaus, occurring at the base on average of 184.4 ± 9.7 ms after the Ca(2+) transient upstroke. In subcellular imaging, SCRs were temporally synchronous and spatially homogeneous within myocytes. In diastole, SHR elicited variable asynchronous sarcoplasmic reticulum Ca(2+) release events leading to subcellular Ca(2+) waves, detectable only at high magnification. SCR was regionally heterogeneous, correlated with APD prolongation (P < 0.01, n = 5), enhanced DOR (r = 0.9277 ± 0.03, n = 7), and was gradually reversed by pacing at 120 beats/min along with APD shortening (P < 0.05, n = 5). A stabilizer of leaky ryanodine receptors (RyR2), 3-(4-benzylcyclohexyl)-1-(7-methoxy-2,3-dihydrobenzo[f][1,4]thiazepin-4(5H)-yl)propan-1-one (K201; 1 μM), suppressed SCR and reduced APD at the base, thereby reducing DOR (P < 0.02, n = 5). Ventricular ectopy induced by bradycardia (n = 5/15) was suppressed by K201. Western blot analysis revealed spatial differences of voltage-gated L-type Ca(2+) channel protein (Cav1.2α), Na(+)-Ca(2+) exchange (NCX1), voltage-gated Na(+) channel (Nav1.5), and rabbit ether-a-go-go-related (rERG) protein [but not RyR2 or sarcoplasmic reticulum Ca(2+) ATPase 2a] that correlate with the SCR distribution and explain the molecular basis for SCR heterogeneities. In conclusion, acute bradycardia elicits synchronized subcellular SCRs of sufficient magnitude to overcome the source-sink mismatch and to promote afterdepolarizations.
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http://dx.doi.org/10.1152/ajpheart.00787.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602772PMC
March 2013

High-purity enrichment of functional cardiovascular cells from human iPS cells.

Cardiovasc Res 2012 Aug 6;95(3):327-35. Epub 2012 Jun 6.

Department of Developmental Biology, University of Pittsburgh School of Medicine, 530 45th Street, Rangos Research Center, Pittsburgh, PA 15201, USA.

Aims: A variety of human inherited heart diseases affect the normal functions of cardiomyocytes (CMs), endothelial cells (ECs), or smooth muscle cells (SMCs). To study human heart disease and generate cardiac cells for basic and translational research, an efficient strategy is needed for production of cardiac lineages from human stem cells. In the present study, a highly reproducible method was developed that can simultaneously enrich a large number of CMs and cardiac SMCs and ECs from human induced pluripotent stem (iPS) cells with high purity.

Methods And Results: Human multipotent cardiovascular progenitor cells were generated from human iPS cells, followed by selective differentiation of the multipotent cardiovascular progenitor cells into CMs, ECs, and SMCs. With further fluorescence-activated cell sorting, each of the three cardiovascular cell types could be enriched with high purity (>90%). These enriched cardiovascular cells exhibited specific gene expression signatures and normal functions when assayed both in vitro and in vivo. Moreover, CMs purified from iPS cells derived from a patient with LEOPARD syndrome, a disease characterized by cardiac hypertrophy, showed the expected up-regulated expression of genes associated with cardiac hypertrophy.

Conclusions: Overall, our technical advance provides the means for generating a renewable resource of pure human cardiovascular cells that can be used to dissect the mechanisms of human inherited heart disease and for the future development of drug and cell therapeutics for heart disease.
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http://dx.doi.org/10.1093/cvr/cvs185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4415083PMC
August 2012

Ranolazine stabilizes cardiac ryanodine receptors: a novel mechanism for the suppression of early afterdepolarization and torsades de pointes in long QT type 2.

Heart Rhythm 2012 Jun 11;9(6):953-60. Epub 2012 Jan 11.

Department of Bioengineering, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA.

Background: Ranolazine (Ran) is known to inhibit multiple targets, including the late Na(+)current, the rapid delayed rectifying K(+)current, the L-type Ca(2+)current, and fatty acid metabolism. Functionally, Ran suppresses early afterdepolarization (EADs) and torsades de pointes (TdP) in drug-induced long QT type 2 (LQT2) presumably by decreasing intracellular [Na(+)](i) and Ca(2+)overload. However, simulations of EADs in LQT2 failed to predict their suppression by Ran.

Objective: To elucidate the mechanism(s) whereby Ran alters cardiac action potentials (APs) and cytosolic Ca(2+)transients and suppresses EADs and TdP in LQT2.

Methods: The known effects of Ran were included in simulations (Shannon and Mahajan models) of rabbit ventricular APs and Ca(2+)transients in control and LQT2 models and compared with experimental optical mapping data from Langendorff rabbit hearts treated with E4031 (0.5 μM) to block the rapid delayed rectifying K(+)current. Direct effects of Ran on cardiac ryanodine receptors (RyR2) were investigated in single channels and changes in Ca(2+)-dependent high-affinity ryanodine binding.

Results: Ran (10 μM) alone prolonged action potential durations (206 ± 4.6 to 240 ± 7.8 ms; P <0.05); E4031 prolonged action potential durations (204 ± 6 to 546 ± 35 ms; P <0.05) and elicited EADs and TdP that were suppressed by Ran (10 μM; n = 7 of 7 hearts). Simulations (Shannon but not Mahajan model) closely reproduced experimental data except for EAD suppression by Ran. Ran reduced open probability (P(o)) of RyR2 (half maximal inhibitory concentration = 10 ± 3 μM; n = 7) in bilayers and shifted half maximal effective concentration for Ca(2+)-dependent ryanodine binding from 0.42 ± 0.02 to 0.64 ± 0.02 μM with 30 μM Ran.

Conclusions: Ran reduces P(o) of RyR2, desensitizes Ca(2+)-dependent RyR2 activation, and inhibits Ca(i) oscillations, which represents a novel mechanism for its suppression of EADs and TdP.
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http://dx.doi.org/10.1016/j.hrthm.2012.01.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335957PMC
June 2012

Oestrogen upregulates L-type Ca²⁺ channels via oestrogen-receptor- by a regional genomic mechanism in female rabbit hearts.

J Physiol 2012 Feb 28;590(3):493-508. Epub 2011 Nov 28.

University of Pittsburgh, Department of Medicine, Cardiovascular Institute, 3550 Terrace Street, Suite S 628 Scaife Hall, Pittsburgh, PA 15261, USA.

In type-2 long QT (LQT2), adult women and adolescent boys have a higher risk of lethal arrhythmias, called Torsades de pointes (TdP), compared to the opposite sex. In rabbit hearts, similar sex- and age-dependent TdP risks were attributed to higher expression levels of L-type Ca(2+) channels and Na(+)-Ca(2+) exchanger, at the base of the female epicardium. Here, the effects of oestrogen and progesterone are investigated to elucidate the mechanisms whereby I(Ca,L) density is upregulated in adult female rabbit hearts. I(Ca,L) density was measured by the whole-cell patch-clamp technique on days 0-3 in cardiomyocytes isolated from the base and apex of adult female epicardium. Peak I(Ca,L) was 28% higher at the base than apex (P < 0.01) and decreased gradually (days 0-3), becoming similar to apex myocytes, which had stable currents for 3 days. Incubation with oestrogen (E2, 0.1-1.0 nm) increased I(Ca,L) (∼2-fold) in female base but not endo-, apex or male myocytes. Progesterone (0.1-10 μm) had no effect at base myocytes. An agonist of the α- (PPT, 5 nm) but not the β- (DPN, 5 nm) subtype oestrogen receptor (ERα/ERβ) upregulated I(Ca,L) like E2. Western blots detected similar levels of ERα and ERβ in male and female hearts at the base and apex. E2 increased Cav1.2α (immunocytochemistry) and mRNA (RT-PCR) levels but did not change I(Ca,L) kinetics. I(Ca,L) upregulation by E2 was suppressed by the ER antagonist ICI 182,780 (10 μm) or by inhibition of transcription (actinomycin D, 4 μm) or protein biosynthesis (cycloheximide, 70 μm). Therefore, E2 upregulates I(Ca,L) by a regional genomic mechanism involving ERα which is a known determinant of sex differences in TdP risk in LQT2.
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http://dx.doi.org/10.1113/jphysiol.2011.219501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379696PMC
February 2012

Mathematical modeling mechanisms of arrhythmias in transgenic mouse heart overexpressing TNF-α.

Am J Physiol Heart Circ Physiol 2012 Feb 11;302(4):H934-52. Epub 2011 Nov 11.

Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.

Transgenic mice overexpressing tumor necrosis factor-α (TNF-α mice) possess many of the features of human heart failure, such as dilated cardiomyopathy, impaired Ca(2+) handling, arrhythmias, and decreased survival. Although TNF-α mice have been studied extensively with a number of experimental methods, the mechanisms of heart failure are not completely understood. We created a mathematical model that reproduced experimentally observed changes in the action potential (AP) and Ca(2+) handling of isolated TNF-α mice ventricular myocytes. To study the contribution of the differences in ion currents, AP, Ca(2+) handling, and intercellular coupling to the development of arrhythmias in TNF-α mice, we further created several multicellular model tissues with combinations of wild-type (WT)/reduced gap junction conductance, WT/prolonged AP, and WT/decreased Na(+) current (I(Na)) amplitude. All model tissues were examined for susceptibility to Ca(2+) alternans, AP propagation block, and reentry. Our modeling results demonstrated that, similar to experimental data in TNF-α mice, Ca(2+) alternans in TNF-α tissues developed at longer basic cycle lengths. The greater susceptibility to Ca(2+) alternans was attributed to the prolonged AP, resulting in larger inactivation of I(Na), and to the decreased SR Ca(2+) uptake and corresponding smaller SR Ca(2+) load. Simulations demonstrated that AP prolongation induces an increased susceptibility to AP propagation block. Programmed stimulation of the model tissues with a premature impulse showed that reduced gap junction conduction increased the vulnerable window for initiation reentry, supporting the idea that reduced intercellular coupling is the major factor for reentrant arrhythmias in TNF-α mice.
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http://dx.doi.org/10.1152/ajpheart.00493.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3360583PMC
February 2012
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