Calcium Transient Duration Research Articles

B-PO04-010 THE EFFECT OF CHRONIC NICOTINE EXPOSURE ON CARDIAC ELECTROPHYSIOLOGY IN THE RABBIT HEART

Tobacco smoke exposure is associated with increased risk for atrial and ventricular arrhythmias. However, the individual components of tobacco smoke that may promote arrhythmias and the underlying electrophysiological mechanisms responsible are unknown. Nicotine (NIC) is a major component of tobacco smoke and emerging tobacco products, including e-cigarettes. To determine the effects of chronic NIC exposure on autonomic and electrophysiological remodeling in the rabbit heart. Fully innervated hearts from rabbits exposed to NIC (21mg/day, transdermal patch, 28 days, n=7) or control (CT, n=4) rabbit hearts were optically mapped using voltage- (RH237) and calcium-sensitive (Rhod2-AM) indicators. Sympathetic nerve stimulation (SNS) was performed with electrical stimulation at the first thoracic vertebrae in which voltage was kept constant (7 V) and frequency was increased in 0.5 Hz increments until a 15% increase in heart rate (HR) was achieved. B-adrenergic (B-AR) responsiveness was evaluated with norepinephrine (500nM, NE). Baseline HR was higher in NIC vs. CT (215.8 BPM vs. 151 BPM, p<0.05). SNS stimulation thresholds necessary to produce an increase in HR were higher in NIC vs. CT (4.17 Hz vs. 1.38 Hz, p<0.05). Maximal HR increase after 40s of SNS trended lower in NIC vs. CT (19.38 vs. 54.30% increase, p=0.1). SNS also tended to produce a smaller change in action potential duration (APD80: -8.14 vs. -16.68% decrease, p=NS) and in calcium transient duration (CaTD80: -7.80 vs. - 20.74% decrease, p=NS) in NIC vs. CT. B-AR responsiveness was decreased in NIC vs. CT (33.38 vs. 75.77% increase in HR with NE, p=0.12). These results suggest that chronic NIC exposure may cause baseline sympathetic hyperactivity reduced B-AR sensitivity. Findings from this study will have significant implications for youth and young adults who represent the fastest growing group of nicotine users and the cardiac effects of nicotine on this potentially vulnerable population is unknown. Thus, data from this study may have implications for tobacco products and other nicotine containing products.

COSMAS: a lightweight toolbox for cardiac optical mapping analysis

Optical mapping is widely used in experimental cardiology, as it allows visualization of cardiac membrane potential and calcium transients. However, optical mapping measurements from a single heart or cell culture can produce several gigabytes of data, warranting automated computer analysis. Here we present COSMAS, a software toolkit for automated analysis of optical mapping recordings in cardiac preparations. COSMAS generates activation and conduction velocity maps, as well as visualizations of action potential and calcium transient duration, S1-S2 protocol analysis, and alternans mapping. The software is built around our recent ‘comb’ algorithm for segmentation of action potentials and calcium transients, offering excellent performance and high resistance to noise. A core feature of our software is that it is based on scripting as opposed to relying on a graphical user interface for user input. The central role of scripts in the analysis pipeline enables batch processing and promotes reproducibility and transparency in the interpretation of large cardiac data sets. Finally, the code is designed to be easily extended, allowing researchers to add functionality if needed. COSMAS is provided in two languages, Matlab and Python, and is distributed with a user guide and sample scripts, so that accessibility to researchers is maximized.

Acute Effects of Ibrutinib on Ventricular Arrhythmia in Spontaneously Hypertensive Rats

BackgroundThe Bruton's Tyrosine Kinase Inhibitor ibrutinib is associated with ventricular arrhythmia (VA) and sudden death. However, the pro-arrhythmic electrophysiological dysregulation that results from ibrutinib with age and cardiovascular disease is unknown. ObjectivesThis study sought to investigate the acute effects of ibrutinib on left ventricular (LV) VA vulnerability, cytosolic calcium dynamics, and membrane electrophysiology in old and young spontaneous hypertensive rats (SHRs). MethodsLangendorff-perfused hearts of young (10 to 14 weeks) and old (10 to 14 months) SHRs were treated with ibrutinib (0.1 μmol/l) or vehicle for 30 min. Simultaneously, LV epicardial action potential and cytosolic calcium transients were optically mapped following an incremental pacing protocol. Calcium and action potential dynamics parameters were analyzed. VA vulnerability was assessed by electrically inducing ventricular fibrillations (VFs) in each heart. Western blot analysis was performed on LV tissues. ResultsIbrutinib treatment resulted in higher vulnerability to VF in old SHR hearts (27.5 ± 7.5% vs. 5.7 ± 3.7%; p = 0.026) but not in young SHR hearts (8.0 ± 4.9% vs. 0%; p = 0.193). In old SHR hearts, following ibrutinib treatment, action potential duration (APD) alternans (p = 0.008) and APD alternans spatial discordance (p = 0.027) were more prominent. Moreover, calcium transient duration 50 was longer (p = 0.032), calcium amplitude alternans ratio was significantly lower (p = 0.001), and time-to-peak of calcium amplitude was shorter (p = 0.037). In young SHR hearts, there were no differences in calcium and APD dynamics. ConclusionsIbrutinib-induced VA is associated with old age in SHR. Acute dysregulation of calcium and repolarization dynamics play important roles in ibrutinib-induced VF.

NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Differentiating the effects of β-adrenergic stimulation and stretch on calcium and force dynamics using a novel electromechanical cardiomyocyte model.

Cardiac electrophysiology and mechanics are strongly interconnected. Calcium is crucial in this complex interplay through its role in cellular electrophysiology and sarcomere contraction. We aim to differentiate the effects of acute β-adrenergic stimulation (β-ARS) and cardiomyocyte stretch (increased sarcomere length) on calcium-transient dynamics and force generation, using a novel computational model of cardiac electromechanics. We implemented a bidirectional coupling between the O'Hara-Rudy model of human ventricular electrophysiology and the MechChem model of sarcomere mechanics through the buffering of calcium by troponin. The coupled model was validated using experimental data from large mammals or human samples. Calcium transient and force were simulated for various degrees of β-ARS and initial sarcomere lengths. The model reproduced force-frequency, quick-release, and isotonic contraction experiments, validating the bidirectional electromechanical interactions. An increase in β-ARS increased the amplitudes of force (augmented inotropy) and calcium transient, and shortened both force and calcium-transient duration (lusitropy). An increase in sarcomere length increased force amplitude even more, but decreased calcium-transient amplitude and increased both force and calcium-transient duration. Finally, a gradient in relaxation along the thin filament may explain the nonmonotonic decay in cytosolic calcium observed with high tension. Using a novel coupled human electromechanical model, we identified differential effects of β-ARS and stretch on calcium and force. Stretch mostly contributed to increased force amplitude and β-ARS to the reduction of calcium and force duration. We showed that their combination, rather than individual contributions, is key to ensure force generation, rapid relaxation, and low diastolic calcium levels.NEW & NOTEWORTHY This work identifies the contribution of electrical and mechanical alterations to regulation of calcium and force under exercise-like conditions using a novel human electromechanical model integrating ventricular electrophysiology and sarcomere mechanics. By better understanding their individual and combined effects, this can uncover arrhythmogenic mechanisms in exercise-like situations. This publicly available model is a crucial step toward understanding the complex interplay between cardiac electrophysiology and mechanics to improve arrhythmia risk prediction and treatment.

Cardiac‐specific Deletion of Filamin C Decouples Myocyte Contractility and Calcium Handling in Adult Mice

Costameres are sub‐membranous protein complexes in striated muscle that play roles in mechanical signal transduction and force transmission between the Z‐disc of the sarcomere and the extracellular matrix. In cardiac myocytes (CMs), the cytoskeletal protein Filamin C (FLNC) interacts with multiple proteins in the Z‐disc and the costamere, suggesting that it contributes to costamere‐based mechanotransduction in the heart. Moreover, mutations in the gene that encodes FLNC have been associated with multiple forms of human cardiomyopathies. To investigate the role of FLNC in cardiac mechanosignaling and pathological remodeling, we employed a recently developed murine model of inducible and cardiac‐specific FLNC knockout (FLNC‐icKO). Previous studies with this mouse model used echocardiographic measurements of adult FLNC‐icKO mice to reveal a severe dilated cardiomyopathy (DCM) phenotype by 2 weeks post‐induction, including significantly reduced ventricular fractional shortening. Correspondingly, we found that intact papillary muscles dissected from adult FLNC‐icKO hearts have significantly reduced twitch force compared to those dissected from wild‐type (WT) hearts. Interestingly, while the fractional shortening of isolated CMs from FLNC‐icKO hearts is also significantly reduced compared to WT CMs, the calcium transient (peak amplitude and duration) during a twitch is not different between groups. These results suggest that contractility and calcium handling in FLNC‐null hearts are decoupled. Potential mechanisms that may be responsible for this apparent decoupling are a disruption in force transmission between the sarcomere and the cell membrane, an increased myofilament lattice spacing, or altered calcium sensitivity of the myofilaments upon the deletion of FLNC in CMs.Support or Funding InformationFunding: HL007444‐36 to JDP, HL144872 to JC, and HL137100 to ADM.

Pharmacological activation of IKr in models of long QT Type 2 risks overcorrection of repolarization.

Current treatment for congenital long QT syndrome Type 2 (cLQTS2), an electrical disorder that increases the risk of life-threatening cardiac arrhythmias, is aimed at reducing the incidence of arrhythmia triggers (beta-blockers) or terminating the arrhythmia after onset (implantable cardioverter-defibrillator). An alternative strategy is to target the underlying disease mechanism, which is reduced rapid delayed rectifier current (IKr) passed by Kv11.1 channels. Small molecule activators of Kv11.1 have been identified but the extent to which these can restore normal cardiac signalling in cLQTS2 backgrounds remains unclear. Here, we examined the ability of ICA-105574, an activator of Kv11.1 that impairs transition to the inactivated state, to restore function to heterozygous Kv11.1 channels containing either inactivation enhanced (T618S, N633S) or expression deficient (A422T) mutations. ICA-105574 effectively restored Kv11.1 current from heterozygous inactivation enhanced or expression defective mutant channels in heterologous expression systems. In a human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of cLQTS2 containing the expression defective Kv11.1 mutant A422T, cardiac repolarization, estimated from the duration of calcium transients in isolated cells and the rate corrected field potential duration (FPDc) in culture monolayers of cells, was significantly prolonged. The Kv11.1 activator ICA-105574 was able to reverse the prolonged repolarization in a concentration-dependent manner. However, at higher doses, ICA-105574 produced a shortening of the FPDc compared to controls. In vitro and in silico analysis suggests that this overcorrection occurs as a result of a temporal redistribution of the peak IKr to much earlier in the plateau phase of the action potential, which results in early repolarization. Kv11.1 activators, which target the primary disease mechanism, provide a possible treatment option for cLQTS2, with the caveat that there may be a risk of overcorrection that could itself be pro-arrhythmic.

P6300Endothelial cell activation by pro-inflammatory cytokines exerts novel paracrine effects on co-cultured cardiomyocytes

Abstract Background Anti-inflammatory therapies have failed to meet expectations in recent clinical trials for heart failure, despite the number of studies demonstrating pro-fibrotic, arrhythmogenic and hypertrophic effects of inflammation. To enlighten this, we sought to examine the contribution of non-myocytes in the cardiac inflammatory response. Endothelial cells (EC) can regulate cardiomyocyte (CM) function with multiple soluble factors. While many studies have shown the effects of pro-inflammatory cytokines on EC or CM separately, it is still unclear how the activation of EC can affect CM function. Purpose We studied the effect of pro-inflammatory pre-conditioning of EC on CM in indirect co-culture systems and in an ex vivo model of cardiac tissue. We hypothesised that pro-inflammatory activation of EC would alter the contractility of co-cultured CM. Methods Human cardiac microvascular EC were first pre-conditioned for 24h with Cytomix (1 ng/ml TNF-α, 1 ng/ml IL-1β, 25 ng/ml IL-6 Rα/IL-6 chimera) and co-cultured in a transwell system (pore size: 0.4μm) with adult rat ventricular CM. Co-culture supernatants were screened using a Cytokine Profiler Array. In vitro analysis of calcium handling in CM utilised the optical mapping technique and Fluo-4. Contractility of cardiac tissue was measured ex vivo using myocardial slices of 300 μm, prepared from left ventricles of adult rat and cultured for 24h with field stimulation and a fixed stretch producing a sarcomeric length of 2.2 μm. Results Treatment of EC by Cytomix prior to co-cultures induced a release of CC and CXC chemokines, G-/GM-CSF, ST2 and the adhesion molecule ICAM-1. Using published RNAseq datasets we noticed that adult CM do not constitutively express the receptors for the chemokines identified with the Profiler Array. However, duration of calcium transients in CM was significantly reduced from 454 to 322 ms in co-cultures with EC pre-conditioned by Cytomix, compared to untreated EC. Amplitude and time to peak were unchanged. In contrast, myocardial slices treated with Cytomix demonstrated a significant increase in contractility compared to control (from 3.3 to 5.3 mN/mm2) but no significant change in the duration of contraction (from 467 to 434 ms) or the rate of relaxation. Conclusions Pre-conditioned EC exert paracrine effects on the calcium handling of isolated CM, suggesting pro-inflammatory activation of proximal EC can affect CM function. In myocardial slices, pro-inflammatory stimulation provoked an inotropic response. The divergence of effect with findings in isolated cells may reflect differences in experimental design and multicellularity. Future work will aim to characterise the soluble mediators involved, and selectively target EC in slices to contextualise the effects described in co-cultures. Ultimately, this may inform the development of novel anti-inflammatory strategies for clinical use. Acknowledgement/Funding British Heart Foundation

Atrial fibrillation and its arrhythmogenesis associated with insulin resistance

BackgroundInsulin resistance (IR) is considered as a risk factor for atrial fibrillation (AF) even before diabetes develops. The pathophysiology and underlying mechanism are largely unclear.MethodsWe investigated the corresponding mechanism in two IR models of rats fed 15-week high-fat (HFa) and high-fructose/cholesterol (HFr) diets. AF was evaluated and induced by burst atrial pacing. Isolated atrial myocytes were used for whole-cell patch clamp and calcium assessment. Ex vivo whole heart was used for optical mapping. Western blot and immunofluorescence were used for quantitative protein evaluation.ResultsBoth HFa and HFr rat atria were vulnerable to AF evaluated by burst atrial pacing. Isolated atrial myocytes from HFa and HFr rats revealed significantly increased sarcoplasmic reticulum calcium content and diastolic calcium sparks. Whole-heart mapping showed prolonged calcium transient duration, conduction velocity reduction, and repetitive ectopic focal discharge in HFa and HFr atria. Protein analysis revealed increased TGF-β1 and collagen expression; increased superoxide production; abnormal upregulation of calcium-homeostasis-related proteins, including oxidized CaMKIIδ, phosphorylated-phospholamban, phosphorylated-RyR-2, and sodium-calcium exchanger; and increased Rac1 activity in both HFa and HFr atria. We observed that inhibition of CaMKII suppressed AF in both HF and HFr diet-fed rats. In vitro palmitate-induced IR neonatal cardiomyocytes and atrial fibroblasts expressed significantly more TGF-β1 than did controls, suggesting paracrine and autocrine effects on both myocytes and fibroblasts.ConclusionsIR engenders both atrial structural remodeling and abnormal intracellular calcium homeostasis, contributing to increased AF susceptibility. The inhibition of CaMKII may be a potential therapeutic target for AF in insulin resistance.

Single cell investigation of mechanically-induced arrhythmias during acute ischemia

Introduction: Ischemia-induced ventricular arrhythmias are a major cause of sudden death. We have demonstrated a contribution of altered mechanics to ischemia-induced arrhythmias, which appear to be calcium-driven and facilitated by a ‘vulnerable window’ (VW) created by spatio-temporal differences in voltage-calcium dynamics. The aim of our current work is to test the hypothesis that mechanically-induced arrhythmias during acute ischemia are indeed calcium-driven, due to distinct changes in action potential (APD) and calcium transient (CaTD) duration.

Age-associated changes in electrical function of the zebrafish heart

Age-associated changes in cardiac structure and function have been observed from the molecular to whole organ level in humans and mammalian models. Understanding the mechanisms involved is important for explaining the development of cardiac disease with age and developing novel strategies for its treatment and prevention. The zebrafish represents a powerful model for cardiovascular research, as various cardiac pathologies have been recapitulated, it is easily genetically modified, and it is a relatively low cost and high-throughput option. In aged zebrafish, myocyte hypertrophy, increased ventricular density and fibrosis, valvular lesions, and reductions in coronary vasculature have been described. The functional consequences of these structural changes however, are relatively unknown. In the current study, we investigated age-related changes in cardiac function in the isolated zebrafish heart. In older animals, heart rate was less stable, sinoatrial node recovery time was increased, and the heart rate response to vagal nerve stimulation was reduced, suggesting an age-dependent change in sinoatrial node function. These changes were accompanied by age-related differences in intracardiac innervation, with the total number and proportion of cholinergic neurons being higher in older animals. In contrast, calcium transient duration was highly variable in young animals, while baseline heart rate and rates of contraction and relaxation were also highly variable, but on average did not change with age. These results suggest that age-associated changes in both myocyte and intracardiac neuronal structure and function exist in the zebrafish heart, offering a new model for studies of cardiac ageing.

Disruption of neonatal cardiomyocyte physiology following exposure to bisphenol-a

Bisphenol chemicals are commonly used in the manufacturing of polycarbonate plastics, polyvinyl chloride plastics, resins, and thermal printing applications. Humans are inadvertently exposed to bisphenols through contact with consumer products and/or medical devices. Recent reports have shown a link between bisphenol-a (BPA) exposure and adverse cardiovascular outcomes; although these studies have been limited to adult subjects and models. Since cardiac physiology differs significantly between the developing and adult heart, we aimed to assess the impact of BPA exposure on cardiac function, using a neonatal cardiomyocyte model. Neonatal rat ventricular myocytes were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium-handling parameters in the presence of control or bisphenol-supplemented media. A range of doses were tested to mimic environmental exposure (10−9–10−8M), maximum clinical exposure (10−5M), and supraphysiological exposure levels (10−4M). Acute BPA exposure altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability. BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes, prolonged calcium transient upstroke and duration time. Alterations in calcium handling also increased the propensity for alternans and skipped beats. Notably, the effect of BPA-treatment on calcium handling was partially reversible. Our data suggest that acute BPA exposure could precipitate secondary adverse effects on contractile performance and/or electrical alternans, both of which are dependent on intracellular calcium homeostasis.

Environmental chemicals can disrupt neonatal cardiomyocyte physiology

BackgroundThe ubiquitous nature of plastics has raised concerns pertaining to continuous exposure to plastic polymers and human health risks. Of particular concern is the use of endocrine‐disrupting chemicals in plastic production, including bisphenol A (BPA). Widespread and continuous exposure to BPA can occur through dietary intake, inhalation, dermal and intravenous exposure via consumer products and medical devices. Recent studies have shown a link between BPA exposure and adverse cardiovascular outcomes. This is particularly concerning for vulnerable populations who are at risk of high BPA exposure due to medical treatment, including pediatric patients. Despite significant differences in the cardiac physiology of the developing and adult heart, our current understanding of BPA‐induced cardiac dysfunction has been limited to adult models.ObjectiveCardiac physiology differs significantly between the pediatric and adult heart. We aimed to determine the extent to which BPA exposure contributes to cardiovascular dysfunction, using a neonatal cardiomyocyte model.MethodsNeonatal rat ventricular myocytes (NRVM) were monitored to assess cell viability, spontaneous beating rate, beat rate variability, and calcium‐handling in the presence of control or bisphenol‐supplemented media. A range of environmental and clinically‐relevant exposure levels were tested [10−9 – 10−5M].ResultsAcute BPA exposure (15 – 20 min) altered cardiomyocyte functionality, resulting in a slowed spontaneous beating rate and increased beat rate variability beginning at low concentrations (10−8M BPA). BPA exposure also impaired intracellular calcium handling, resulting in diminished calcium transient amplitudes and prolonged calcium transient duration time, which replicated our previously published studies in isolated‐whole hearts. BPA‐induced changes in calcium handling increased the propensity for calcium transient alternans, beginning at low BPA concentrations (10−9M). The observed alterations in calcium kinetics following BPA exposure were not the result of cytotoxicity or altered cell viability.ConclusionOur studies show that acute BPA chemical exposure can alter neonatal cardiomyocyte physiology. Additional studies are necessary to determine the effects of bisphenol exposure on pediatric heart electrophysiology and mechanical function, as well as the impact of long‐term treatment that more closely mimics clinical exposure.Support or Funding InformationThis publication was supported by the National Institutes of Health (R00ES023477, UL1TR000075), Children's Research Institute and Children's National Heart Institute.

Exercise Training Protects Cardiomyocytes from Deleterious Effects of Palmitate.

We investigated the effects of palmitate, a high saturated fat, on Ca2+, action potential and reactive oxygen species dynamics in cardiomyocytes from untrained and trained mice. Male mice were subjected to moderate intensity exercise training on a treadmill. Cardiomyocytes of untrained and trained mice were isolated, treated for 30 min with palmitate and intracellular calcium transient and action potential duration were recorded. Additionally, we assessed reactive oxygen species generation. Treatment of cardiomyocytes from untrained mice with palmitate induced a significant decrease in Ca2+ transient magnitude by 34%. Exercise training did not change cardiomyocyte Ca2+ dynamics in the control group. However, trained cardiomyocytes were protected from deleterious effects of palmitate. Action potential duration was not altered by palmitate in either untrained or trained cardiomyocytes. Moreover, palmitate treatment increased reactive oxygen species generation in both untrained and trained cardiomyocytes. Nevertheless, the levels of reactive oxygen species in trained cardiomyocytes treated with palmitate were still 27% lower than those seen at basal conditions in untrained cardiomyocytes. Taken together, these findings indicate that exercise training protects cardiomyocytes from deleterious effects of palmitate possibly by inhibiting exacerbated ROS production.

Inwardly rectifying potassium channels influence Drosophila wing morphogenesis by regulating Dpp release.

Loss of embryonic ion channel function leads to morphological defects, but the underlying reason for these defects remains elusive. Here, we show that inwardly rectifying potassium (Irk) channels regulate release of the Drosophila bone morphogenetic protein Dpp in the developing fly wing and that this is necessary for developmental signaling. Inhibition of Irk channels decreases the incidence of distinct Dpp-GFP release events above baseline fluorescence while leading to a broader distribution of Dpp-GFP. Work by others in different cell types has shown that Irk channels regulate peptide release by modulating membrane potential and calcium levels. We found calcium transients in the developing wing, and inhibition of Irk channels reduces the duration and amplitude of calcium transients. Depolarization with high extracellular potassium evokes Dpp release. Taken together, our data implicate Irk channels as a requirement for regulated release of Dpp, highlighting the importance of the temporal pattern of Dpp presentation for morphogenesis of the wing.

Cardiac electrical defects in progeroid mice and Hutchinson–Gilford progeria syndrome patients with nuclear lamina alterations

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 β-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.

Cardiac arrhythmia in a mouse model of sodium channel SCN8A epileptic encephalopathy

Patients with early infantile epileptic encephalopathy (EIEE) are at increased risk for sudden unexpected death in epilepsy (SUDEP). De novo mutations of the sodium channel gene SCN8A, encoding the sodium channel Nav1.6, result in EIEE13 (OMIM 614558), which has a 10% risk of SUDEP. Here, we investigated the cardiac phenotype of a mouse model expressing the gain of function EIEE13 patient mutation p.Asn1768Asp in Scn8a (Nav1.6-N1768D). We tested Scn8aN1768D/+ mice for alterations in cardiac excitability. We observed prolongation of the early stages of action potential (AP) repolarization in mutant myocytes vs. controls. Scn8aN1768D/+ myocytes were hyperexcitable, with a lowered threshold for AP firing, increased incidence of delayed afterdepolarizations, increased calcium transient duration, increased incidence of diastolic calcium release, and ectopic contractility. Calcium transient duration and diastolic calcium release in the mutant myocytes were tetrodotoxin-sensitive. A selective inhibitor of reverse mode Na/Ca exchange blocked the increased incidence of diastolic calcium release in mutant cells. Scn8aN1768D/+ mice exhibited bradycardia compared with controls. This difference in heart rate dissipated after administration of norepinephrine, and there were no differences in heart rate in denervated ex vivo hearts, implicating parasympathetic hyperexcitability in the Scn8aN1768D/+ animals. When challenged with norepinephrine and caffeine to simulate a catecholaminergic surge, Scn8aN1768D/+ mice showed ventricular arrhythmias. Two of three mutant mice under continuous ECG telemetry recording experienced death, with severe bradycardia preceding asystole. Thus, in addition to central neuron hyperexcitability, Scn8aN1768D/+ mice have cardiac myoycte and parasympathetic neuron hyperexcitability. Simultaneous dysfunction in these systems may contribute to SUDEP associated with mutations of Scn8a.

Generation of Functional Cardiomyocytes from the Synoviocytes of Patients with Rheumatoid Arthritis via Induced Pluripotent Stem Cells.

Cardiovascular disease is a leading cause of morbidity in rheumatoid arthritis (RA) patients. This study aimed to generate and characterise cardiomyocytes from induced pluripotent stem cells (iPSCs) of RA patients. Fibroblast-like synoviocytes (FLSs) from patients with RA and osteoarthritis (OA) were successfully reprogrammed into RA-iPSCs and OA-iPSCs, respectively. The pluripotency of iPSCs was confirmed by quantitative reverse transcription-polymerase chain reaction and immunofluorescence staining. Established iPSCs were differentiated into cardiomyocytes using a small molecule-based monolayer differentiation protocol. Within 12 days of cardiac differentiation from patient-specific and control-iPSCs, spontaneously beating cardiomyocytes (iPSC-CMs) were observed. All iPSC-CMs exhibited a reliable sarcomeric structure stained with antibodies against cardiac markers and similar expression profiles of cardiac-specific genes. Intracellular calcium signalling was recorded to compare calcium-handling properties among cardiomyocytes differentiated from the three groups of iPSCs. RA-iPSC-CMs had a lower amplitude and a shorter duration of calcium transients than the control groups. Peak tangential stress and the maximum contractile rate were also decreased in RA-iPSC-CMs, suggesting that contractility was reduced. This study demonstrates the successful generation of functional cardiomyocytes from pathogenic synovial cells in RA patients through iPSC reprogramming. Research using RA-iPSC-CMs might provide an opportunity to investigate the pathophysiology of cardiac involvement in RA.

Calcium dynamics predict direction of synaptic plasticity in striatal spiny projection neurons

The striatum is a major site of learning and memory formation for sensorimotor and cognitive association. One of the mechanisms used by the brain for memory storage is synaptic plasticity - the long-lasting, activity-dependent change in synaptic strength. All forms of synaptic plasticity require an elevation in intracellular calcium, and a common hypothesis is that the amplitude and duration of calcium transients can determine the direction of synaptic plasticity. The utility of this hypothesis in the striatum is unclear in part because dopamine is required for striatal plasticity and in part because of the diversity in stimulation protocols. To test whether calcium can predict plasticity direction, we developed a calcium-based plasticity rule using a spiny projection neuron model with sophisticated calcium dynamics including calcium diffusion, buffering and pump extrusion. We utilized three spike timing-dependent plasticity (STDP) induction protocols, in which postsynaptic potentials are paired with precisely timed action potentials and the timing of such pairing determines whether potentiation or depression will occur. Results show that despite the variation in calcium dynamics, a single, calcium-based plasticity rule, which explicitly considers duration of calcium elevations, can explain the direction of synaptic weight change for all three STDP protocols. Additional simulations show that the plasticity rule correctly predicts the NMDA receptor dependence of long-term potentiation and the L-type channel dependence of long-term depression. By utilizing realistic calcium dynamics, the model reveals mechanisms controlling synaptic plasticity direction, and shows that the dynamics of calcium, not just calcium amplitude, are crucial for synaptic plasticity.

Characterisation of cardiomyocytes derived from human induced pluripotent stem cells as monolayers

BackgroundHuman myocardial tissue for use in physiological research is difficult to obtain, and differences between human cardiac tissue and that used in animal models preclude full understanding of mechanisms of human arrhythmia. Stem-cell-derived cardiomyocytes might offer an alternative source of tissue. Therefore we aimed to explore the physiological features of cardiac tissue produced from human induced pluripotent stem (iPS) cells in monolayer form and investigated its response to β-adrenergic modulation. MethodsCardiomyocyte monolayers were produced as per the protocol of Burridge and colleagues. Monolayers were loaded with a calcium-sensitive dye (Fluo 4-AM, 5 μM) and imaged with fluorescence microscopy. Isoprenaline was used for β agonism and propranolol for β antagonism. Images were analysed with custom-written MatLab software to calculate relevant physiological variables. Ethics approval was not required because iPS cell lines were transformed. FindingsSeven monolayers were produced. They stained positive for troponin T and exhibited spontaneous contraction. Mean cycle length and calcium transient duration were 4·04 s (SD 2·90) and 1·88 s (1·18), respectively. Both variables were shortened on addition of isoprenaline (0·3 μM): cycle length 2·27 s (2·02) (p=0·0063) and duration 1·26 s (1·05) (p=0·0041). Propranolol (50 μM) reversed the effect of isoprenaline: cycle length 6·14 s (6·14) (p=0·045) and duration 2·43 s (1·04) (p=0·0009). The calcium transient amplitude reduced with cycle length because of a rise in baseline intracellular calcium (p=0·0138). In an additional monolayer, spiral waves were seen, which provided focal sources of excitation at higher frequencies than those in monolayers driven by non re-entrant mechanisms (cycle length 1·34 s [0·49], p=0·0497). InterpretationCardiomyocytes were successfully derived from human iPS cells and showed a chronotropic response to β-adrenergic modulation. The data support the plausibility of understanding arrhythmogenesis by use of human iPS derived cardiomyocytes. The immaturity of the derived cells is a potential limitation of the study. The presence of spiral waves in one case was notable since spiral waves or rotors are thought to drive arrhythmias such as atrial fibrillation. FundingBritish Heart Foundation.