Calcium Transient Duration Research Articles

Dexmedetomidine inhibited arrhythmia susceptibility to adrenergic stress in RyR2R2474S mice through regulating the coupling of membrane potential and intracellular calcium

BackgroundDexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist, can decrease the incidence of arrhythmias, such as catecholaminergic polymorphic ventricular tachycardia (CPVT). However, the underlying mechanisms by which DEX affects cardiac electrophysiological function remain unclear. MethodsRyanodine receptor (RyR2) heterozygous R2474S mice were used as a model for CPVT. WT and RyR2R2474S/+ mice were treated with isoproterenol (ISO) and DEX, and electrocardiograms were continuously monitored during both in vivo and ex vivo experiments. Dual-dye optical mapping was used to explore the anti-arrhythmic mechanism of DEX. ResultsDEX significantly reduced the occurrence and duration of ISO-induced of VT/VF in RyR2R2474S/+ mice in vivo and ex vivo. DEX remarkably prolonged action potential duration (APD80) and calcium transient duration (CaTD80) in both RyR2R2474S/+ and WT hearts, whereas it reduced APD heterogeneity and CaT alternans in RyR2R2474S/+ hearts. DEX inhibited ectopy and reentry formation, and stabilized voltage-calcium latency. ConclusionDEX exhibited an antiarrhythmic effect through stabilizing membrane voltage and intracellular Ca2+. DEX can be used as a beneficial perioperative anesthetic for patients with CPVT or other tachy-arrhythmias.

Defining cardiac stability during Langendorff perfusion

The Langendorff heart preparation is a valuable ex vivo technique for studying cardiac electrophysiology. For the past century it has been used in toxicology studies, drug testing, and disease modeling and has contributed significantly to understanding cardiac physiology, development, and pathological states. This study aimed to investigate the stability and reproducibility of the modern Langendorff heart preparation under various experimental conditions and to establish cardiac metric standards with corresponding exclusion criteria. Isolated hearts were extracted from adult Sprague Dawley rats and retrograde perfused with a modified Krebs-Henseleit buffer. A cohort of isolated hearts were used to establish a control baseline of multiple physiological parameters measured over time (e.g., electrocardiogram, flow rate, electrophysiology, electrical and calcium dynamics). These control baseline values were then compared to hearts exposed to the most common media additives in Langendorff studies (e.g., blebbistatin(−/−), bovine serum albumin, and voltage and calcium indicator dye). Over a three-hour period, cardiac metrics were monitored in stable Langendorff-perfused hearts. The addition of blebbistatin, DMSO or dyes did not change EP or ECG measurements as compared to control baseline. The most significant drift in all the EP values was ventricular effective refractory period (VERP). With or without blebbistatin, VERP increased from 40 to 60ms over the course of three hours. In that time heart rate and flow also decreased slightly. Blebbistatin was associated with an increased action potential duration but did not alter calcium transient duration. It was also observed that 1% circulating bovine serum albumin quenched the rh237 voltage dye signal 80% as measured by signal to noise. Constant pressure Langendorff-perfusion with a crystalloid buffer provides a reliable and reproducible model for cardiac electrophysiology research with a few caveats. The total length of study should be carefully considered to account for adaptations in cardiac physiology during longer perfusion times. As an excitation-contraction uncoupler, blebbistatin reduces motion artifact but delays action potential duration. Although BSA is used to minimize cardiac edema, it interferes with membrane bound voltage dyes. This study sets physiological parameters as a useful guide for future cardiac studies that use the isolated heart as a research model. American Physiological Society, R01HD108839 to N.G.P. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Comparative cardiotoxicity assessment of bisphenol chemicals and estradiol using human induced pluripotent stem cell-derived cardiomyocytes.

Bisphenol A (BPA) is commonly used to manufacture consumer and medical-grade plastics. Due to health concerns, BPA substitutes are being incorporated-including bisphenol S (BPS) and bisphenol F (BPF)-without a comprehensive understanding of their toxicological profile. Previous studies suggest that bisphenol chemicals perturb cardiac electrophysiology in a manner that is similar to 17β-estradiol (E2). We aimed to compare the effects of E2 with BPA, BPF, and BPS using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Cardiac parameters were evaluated using microelectrode array (MEA) technology and live-cell fluorescent imaging. Cardiac metrics remained relatively stable after exposure to nanomolar concentrations (1-1000 nM) of E2, BPA, BPF, or BPS. At higher micromolar concentrations, chemical exposures decreased the depolarization spike amplitude, and shortened the field potential, action potential duration, and calcium transient duration (E2 ≥ BPA ≥ BPF ≫ BPS). Cardiomyocyte physiology was largely undisturbed by BPS. BPA-induced effects were exaggerated when coadministered with an L-type calcium channel (LTCC) antagonist or E2, and reduced when coadministered with an LTCC agonist or an estrogen receptor alpha antagonist. E2-induced effects were not exaggerated by coadministration with an LTCC antagonist. Although the observed cardiac effects of E2 and BPA were similar, a few distinct differences suggest that these chemicals may act (in part) through different mechanisms. hiPSC-CM are a useful model for screening cardiotoxic chemicals, nevertheless, the described findings should be validated using a more complex ex vivo and/or in vivo model.

Up-regulated small-conductance calcium-activated potassium currents contribute to atrial arrhythmogenesis in high-fat feeding mice.

Metabolic syndrome (MetS) is associated with arrhythmias and cardiovascular mortality. Arrhythmogenesis in MetS results from atrial structural and electrical remodelling. The small-conductance Ca2+-activated K+ (SK) currents modulate atrial repolarization and may influence atrial arrhythmogenicity. This study investigated the regulation of SK current perturbed by a high-fat diet (HFD) to mimic MetS. Thirty mice were divided into two groups that were fed with normal chow (CTL) and HFD for 4 months. Electrocardiography and echocardiography were used to detect cardiac electrical and structure remodelling. Atrial action potential duration (APD) and calcium transient duration (CaTD) were measured by optical mapping of Langendorff-perfused mice hearts. Atrial fibrillation (AF) inducibility and duration were assessed by burst pacing. Whole-cell patch clamp was performed in primarily isolated atrial myocytes for SK current density. The SK current density is higher in atrial myocytes from HFD than in CTL mice (P ≤ 0.037). The RNA and protein expression of SK channels are increased in HFD mice (P ≤ 0.041 and P ≤ 0.011, respectively). Action potential duration is shortened in HFD compared with CTL (P ≤ 0.015). The shortening of the atrial APD in HFD is reversed by the application of 100 nM apamin (P ≤ 0.043). Compared with CTL, CaTD is greater in HFD atria (P ≤ 0.029). Calcium transient decay (Tau) is significantly higher in HFD than in CTL (P = 0.001). Both APD and CaTD alternans thresholds were higher in HFD (P ≤ 0.043), along with higher inducibility and longer duration of AF in HFD (P ≤ 0.023). Up-regulation of apamin-sensitive SK currents plays a partial role in the atrial arrhythmogenicity of HFD mice.

Evidence for the cardiodepressive effects of the plasticizer di-2-ethylhexyl phthalate.

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 μg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 μg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 μg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 μg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials.

KairoSight-3.0: A validated optical mapping software to characterize cardiac electrophysiology, excitation-contraction coupling, and alternans.

Cardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical imaging of voltage-sensitive and calcium-sensitive probes allows for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance the characterization of cardiac parameters using optical signals. To test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop the KairoSight-3.0 software. Cardiac maps were validated with a user-specified manual mapping approach. Manual maps of action potential duration (30 or 80 % repolarization), calcium transient duration (30 or 80 % reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97 % of manual and software values falling within 10 ms of each other and >75 % within 5 ms for action potential duration and calcium transient duration measurements (n = 1000-2000 pixels). Further, our software package includes additional measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps. KairoSight-3.0 has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, alternans, and the excitation-contraction coupling with satisfactory accuracy.

Ryanodine receptor inhibition with acute dantrolene treatment reduces arrhythmia susceptibility in human hearts.

Ryanodine receptor 2 (RyR2) hyperactivity is observed in structural heart diseases that are a result of ischemia or heart failure. It causes abnormal calcium handling and calcium leaks that cause metabolic, electrical, and mechanical dysfunction, which can trigger arrhythmias. Here, we tested the antiarrhythmic potential of dantrolene (RyR inhibitor) in human hearts. Human hearts not used in transplantation were obtained, and right ventricular outflow tract (RVOT) wedges and left ventricular (LV) slices were prepared. Pseudo-ECGs were recorded to determine premature ventricular contraction (PVC) incidences. Optical mapping was performed to determine arrhythmogenic substrates. After baseline optical recordings, tissues were treated with 1) isoproterenol (250 nM), 2) caffeine (200 mM), and 3) dantrolene (2 or 10 mM). Optical recordings were obtained after each treatment. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Isoproterenol shortened action potential duration (APD) in the RV, RVOT, and LV regions and shortened calcium transient duration (CaTD) in the LV. Caffeine further shortened APD in the RV, did not modulate APD in the RVOT, and prolonged APD in the LV. In addition, in the LV, CaTD prolongation was also observed. More importantly, adding dantrolene did not alter APD in the RV or RVOT regions but produced a trend toward APD prolongation and significant CaTD prolongation in the LV, restoring these parameters to baseline values. In conclusions, dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates in the human heart and could be a novel antiarrhythmic therapy in patients with structural heart disease.NEW & NOTEWORTHY Ryanodine receptor 2 hyperactivity is observed in structural heart diseases caused by ischemia or heart failure. It causes abnormal calcium leaks, which can trigger arrhythmias. To prevent arrhythmias, we applied dantrolene in human hearts ex vivo. Isoproterenol and caffeine treatment increased PVC incidence, whereas dantrolene reduced the PVC burden. Dantrolene treatment suppresses triggers and reverses arrhythmogenic substrates and could be a novel antiarrhythmic therapy in patients with structural heart disease.

Abstract GS.11: Exercise Therapy During Doxorubicin Treatment Provides Sex-specific Cardioprotection

Moderate-intensity exercise is routinely prescribed to patients with cardiovascular diseases to improve cardiac function. It has also been recommended in patients undergoing anti-cancer chemotherapy to attenuate cardiotoxic effects. We recently identified that moderate endurance exercise in healthy mice causes the remodeling of cardiac physiology in a sex-specific manner. Therefore, we hypothesized that exercise therapy applied during doxorubicin chemotherapy would produce sex-specific cardioprotection. Male and female mice were divided into three groups, 1) sedentary control, 2) DOX - doxorubicin treated at 5 mg/kg/week for 6 weeks, and 3) EXDOX - 2 weeks of forced treadmill running for 2 weeks before DOX + exercise for an additional 6 weeks. Mouse health was tracked daily. Total body weight, echocardiography, and electrocardiography were assessed every 2 weeks. At the end of the protocol, triple parametric optical mapping was performed to determine the remodeling of cardiac metabolism-excitation-contraction coupling (MECC). DOX treatment increased morbidity and reduced total body weight in both males and females. DOX-induced action potential duration (APD) prolongation in both sexes and, additionally, in males alone, increased anisotropic ratio of conduction (index for arrhythmogenicity) and rise time of calcium transients (CaRT). In EXDOX mice, the onset of morbidity was delayed in both males and females compared to DOX mice. Total body weight in male EXDOX mice, but not females, was further reduced compared to DOX treatment alone. Echocardiography revealed increased LV wall thickness in male and female EXDOX mice vs DOX, while stroke volume was reduced in females alone. Next, DOX-induced APD prolongation was prevented in EXDOX females but not males. Furthermore, DOX-induced increase in CaRT was further increased in EXDOX males, in addition to increased calcium transient duration and reduced calcium decay constant. Taken together, these data suggest significant sex dimorphism in cardiac adaptation to DOX treatment and exercise therapy during DOX treatment. Female mice undergoing exercise therapy during DOX treatment demonstrated greater cardioprotection and attenuation of MECC remodeling.

In vivo KCNQ1-suppression-replacement gene therapy in transgenic rabbits with type 1 long QT syndrome

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): Pfizer Background Type 1 long QT syndrome (LQT1) is a genetic channelopathy characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs. Patients with LQT1 may manifest QT prolongation and ventricular arrhythmias that can culminate in sudden cardiac death. Purpose With this project, we aim to investigate whether our mutation independent, KCNQ1-specific suppression-replacement (SupRep) gene therapy can restore the deficient K+-channel and thereby restore the healthy phenotype. Methods Our proprietary dual component SupRep gene therapy was created by combining a custom-designed KCNQ1 shRNA and a shRNA-immune (shIMM) KCNQ1 cDNA into AAV9. In vivo AAV9-mediated KCNQ1SupRep gene therapy was performed by targeted intra-aortic root construct injection (1E12 vg/kg body weight) during balloon occlusion using a Swan Ganz catheter. 2 weeks after in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 (KCNQ1-Y315S, loss-of IKs) and wild-type (WT) rabbits to determine the effect of SupRep therapy on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes to evaluate the effect of SupRep on the cardiomyocyte’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) both, in vitro – after plasmid transfection, and in vivo – after AAV9-mediated gene therapy. AAV9-KCNQ1-construct expression was verified with immunohistochemistry targeting the GFP-tag. Results After injection of 3 LQT1 rabbits so far, no significant changes were observed in the LQT1 rabbits’ QTc before and after in vivo AAV9-mediated gene therapy. However, at the cellular level, LQT1 cardiomyocytes transfected in vitro with the SupRep-plasmid demonstrated a significant reduction of APD90 (ms, 1Hz stimulation at 37°) compared to LQT1 control cells (SupRep-in-vitro, 358±21 vs. control 447±22, p<0.05). No differences were observed between sham-plasmid transfected cardiomyocytes and LQT1 control cells after one day of culturing. Similarly, cardiomyocytes isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). Conclusion In vivo KCNQ1-suppression-replacement gene therapy normalizes cellular action potential and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the cardiomyocyte level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.

Sustained high-fat diet increases ventricular susceptibility to arrhythmias and impairs myocardial calcium cycling

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Medizinische Fakultät, Heinrich Heine Universität Düsseldorf Background – Obesity is one of the most relevant factors for cardiovascular disease in western societies. Independent of coronary heart disease, obesity promotes a multimodal arrhythmic substrate and increases the susceptibility to ventricular arrhythmias (VA) eventually leading to sudden cardiac death. Purpose – Aim of the present study was to develop an ex vivo mouse model to characterize obesity-mediated VAs and left ventricular (LV) myocardial disruption of calcium homeostasis. Methods – All experiments were performed ex vivo in retrogradely perfused hearts from 32 – 36 week-old C57Bl/6J mice (n=14). Five mice received a normal chow diet (NCD), 9 mice were fed a sustained high-fat diet (HFD) over a 6-month period to generate diet-induced-obesity (DIO). We combined an endocardial catheter-based technique to assess VA susceptibility with an epicardial dual fluorescence imaging approach employing a potentiometric (Rh237) and a calcium sensitive (Rhod2) dye to assess action potential and calcium cycling characteristics. Optical mapping data was analysed using a MATLAB-based algorithm. VA susceptibility was quantified by an established scoring system. Results – Mice receiving a sustained HFD were significantly heavier than those receiving NCD (NCD: 34.8±2.1g, HFD: 53.3±3.2g, P<0.0001). The number of induced VAs and VA susceptibility were increased in HFD compared to NCD (number of induced VAs: NCD: 1.4±0.7, HFD: 6.3±0.8, P=0.005, Figure 1A; VA susceptibility: NCD: 1.4±0.7, HFD: 18.1±2.3, P=0.001, Figure 1B). Ventricular tachycardias (VT) were only inducible in HFD, additionally, those hearts presented mostly polymorphic VTs (VT inducibility: NCD: 0.0 %, HFD: 88.9 %, P=0.003, Figure 1C; VT morphology: monomorphic VT: 25 %, polymorphic VT: 75 %). Epicardial optical mapping of the anterior LV wall showed a prolonged action potential duration (APD) and prolonged calcium transient duration (CaTD) in HFD (APD90 at CL 100 ms: NCD: 45.1±1.5 ms, HFD: 50.8±1.8 ms, P=0.03; CaTD90 at 100 ms pacing cycle length (PCL): NCD: 61.0±2.3 ms, HFD: 66.6±0.6 ms, P=0.02, Figure 2A). Furthermore, CaT alternans (ALT) threshold defined as amplitude alternans > 10 % of two consecutive beats was decreased (PCL ALT threshold: NCD: 60.0±1.6 ms, DIO: 67.8±1.2 ms, P=0.006, Figure 2B), with mean ALT being increased in HFD (mean ATL at 60 ms PCL: NCD: 15.4±2.4 %, HFD: 25.7±3.0 %, P=0.04). Conclusions – Our findings show that sustained HFD in mice increases ventricular susceptibility to VTs, especially polymorphic VTs indicating heterogeneity in wave propagation. DIO leads to disruption of LV myocardial calcium homeostasis as demonstrated by a decreased ALT threshold and increased mean ALT. Our data suggest that LV calcium mishandling may be a driver for arrhythmogenesis in DIO and modulation may present a perspective therapeutical approach. Further experiments will address molecular mechanisms and identify potential targets of modulation.

PO-02-210 AAV9-MEDIATED KCNQ1-SUPPRESSION-REPLACEMENT GENE THERAPY IN TRANSGENIC RABBITS WITH TYPE 1 LONG QT SYNDROME

Type 1 long QT syndrome (LQT1) is characterized by both haploinsufficient and dominant-negative loss-of-function pathogenic variants in the KCNQ1-encoded Kv7.1 K+ channels conducting IKs current, which contributes to the cardiac action potential. No current therapies target the molecular cause of LQT1. To rescue the pathologic phenotype in the transgenic KCNQ1-Y315S LQT1 rabbit model using our proprietary KCNQ1-specific suppression-replacement (SupRep) gene therapy. Our dual component SupRep gene therapy was created by combining into a single construct a custom-designed KCNQ1 shRNA (suppression) and a cardiac-specific troponin C (cTnC) promoter driven shRNA-immune KCNQ1 cDNA (replacement). The KCNQ1-SupRep construct was packaged into AAV9 (AAV9-KCNQ1-SupRep), a nominally immunogenic and cardiotropic viral vector with limited off-target delivery. AAV9-KCNQ1-SupRep was injected in vivo via a targeted intra-aortic root injection (1E12 vg/kg bodyweight) during balloon occlusion using a Swan Ganz catheter. Two weeks post in vivo gene therapy, 12-lead ECGs were assessed in adult transgenic LQT1 and wildtype (WT) rabbits to ascertain the effect of SupRep on the rabbit’s QTc. Patch-clamp and calcium transient measurements were performed in isolated ventricular cardiomyocytes (CMs) to evaluate the effect of SupRep on the CM’s action potential duration (APD90) and Ca2+ transient duration (Ca2+90) post in vivo gene therapy. AAV9-KCNQ1-SupRep expression was verified with immunohistochemistry. After injection of 3 LQT1 rabbits so far, no statistically significant changes were observed in the LQT1 rabbits’ QTc pre/post in vivo AAV9-KCNQ1-SupRep. However, CMs isolated from the 3 LQT1 rabbits that underwent in vivo SupRep gene therapy demonstrated pronounced shortening (ms) of both APD90 and Ca2+90 compared to LQT1 controls, leading to levels similar to WT controls (APD90, 1Hz, 37°: LQT1: 530±18, WT: 445±18, LQT1-SupRep: 375±25, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT) (Ca2+90: LQT1: 487±23, WT: 346±16, LQT1-SupRep: 393±19, p<0.0001 LQT1 vs. LQT1-SupRep, p=ns LQT1-SupRep vs. WT). In vivo AAV9-KCNQ1-SupRep gene therapy normalizes cellular AP and calcium transient duration in transgenic LQT1 rabbits. Further in vivo experiments will be conducted to evaluate whether this therapeutic correction at the CM level will translate into normalization of the LQT1 rabbits’ QTc both at rest and during stress testing.

PO-02-241 CARDIAC SYMPATHETIC REMODELING AND SK CHANNELS UPREGULATION, THE POSSIBLE MECHANISMS OF NOISE EXPOSURE ASSOCIATED ATRIAL ARRHYTHMOGENESIS

Environmental noise exposure may increase sympathetic tone and lead to atrial fibrillation (AF) and mortality. The small conductance Ca2+- activated K+ (SK) channels play important roles in the repolarization of atria. The regulation of SK channels might be involved in modulating cardiac electrophysiology via the autonomic nervous system (ANS). Apamin blocks SK channels specifically and is used for evaluating the roles of SK channels regulation related to noise exposure. To determine how ANS regulates SK channels under noise exposure. We used a noise exposure (NOE) mice model with broad-band noise 20-20k Hz for 28 days to induce atrial myopathy. A total of 12 NOE and 9 control mice were used in the study. Dual optical mapping was performed via Langendorff perfused intact hearts to measure the baseline and post-apamin atrial action potential duration (APD), calcium transient duration (CaTD) and conduction velocity (CV) in the NOE and control mice. The expression of SK channel proteins (SK1-3), tyrosine hydroxylase (TH) and growth-associated protein 43 (GAP43) was detected by immunohistochemistry. The degree of myocardial fibrosis in the atria was detected by Masson's trichrome staining. Optical mapping study showed no APD difference (26.26±3.69 vs 27.76±2.36 ms, p=0.69) but prolonged CaTD (52.67±1.91 vs 49.65±3.4 ms, p=0.03) in NOE compared with control mice atria at baseline. The ratio of ΔAPD (the difference in the APD after and before apamin) and baseline was higher in NOE than in control mice (0.221±0.231 vs 0.011±0.52, p=0.015), meaning that noise exposure contributed to the upregulation of SK channels. Atrial CV was higher in NOE than in control mice (44.9±4 vs 39.2±3.5 cm/s, p=0.019); after apamin administration, it was significantly lower in NOE than in control mice (30.9±4.8 vs 34.2±3.5 cm/s, p=0.045). IHC study showed SK1-3, TH, GAP43 and trichrome staining were significantly higher in NOE than in control atria (18445.3±2527.9 vs 11228.4±2276.9 μm2/ mm2, 16063.6±1974 vs 9760.17±2576.8 μm2/mm2, 9304.3±633.1 vs 5994.7±1131.8 μm2/ mm2, 1019.9±189.8 vs 622.6±252.3 μm2/ mm2, 733.5±449.1 vs 117.4±53.4 μm2/ mm2, 7449.9±4274.7 vs 492.6±353.9 μm2/ mm2, all p≤0.01). Additionally, AF can be induced in 17% (2/12) of NOE but never in control mice (p=0.14). Noise exposure induces cardiac ANS remodeling, fibrosis and SK currents upregulation. It partially explains the mechanism of noise-induced atrial arrhythmia.

BS-452758-1 SUPPRESSION-REPLACEMENT GENE THERAPY REVERSE REMODELS THE INTRACELLULAR CALCIUM HANDLING DISEASE PHENOTYPE OF TNNT2-MEDIATED HYPERTROPHIC CARDIOMYOPATHY

Hypertrophic cardiomyopathy (HCM), characterized by otherwise unexplained left ventricular hypertrophy, has a prevalence of 1:500. Pathogenic variants in TNNT2-encoded cardiac troponin T causes a thin filament subtype of sarcomeric HCM. A key cellular feature of the pathological remodeling in TNNT2-mediated HCM involves dysregulation of intracellular calcium handling in ventricular cardiomyocytes (CMs). To develop a TNNT2 “suppression-replacement (SupRep)” gene therapy to reverse the intracellular calcium perturbations associated with thin filament, sarcomeric HCM using induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) derived from a patient with TNNT2-R288P-mediated HCM. Custom-designed TNNT2 targeting shRNAs were tested for knockdown (KD) efficiency using TSA201 cells and RT-qPCR. A dual-component SupRep TNNT2 gene therapy was created by cloning into a single construct a custom-designed TNNT2 shRNA that produced greatest KD (suppression) and a “shRNA-immune” (shIMM) TNNT2 cDNA (replacement). Patient-specific TNNT2-R288P and CRISPR/Cas9 variant-corrected isogenic control (IC) iPSC-CMs were generated from a patient with HCM. The intracellular calcium indicator, Fluo-4, was used to detect intracellular free calcium. Our lead TNNT2-shRNA had an 80% KD efficiency of TNNT2. TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep showed a similar KD efficiency. Compared to the IC iPSC-CMs, the TNNT2-R288P iPSC-CMs showed increased irregular events (30.4% vs. 10.9%, p<0.0001), prolonged calcium transient duration 90 (CTD90, 1.01 ± 0.03 s vs. 0.89 ± 0.02 s, p<0.0001) and prolonged peak to 90% decay time (0.57 ± 0.02 s vs. 0.49 ± 0.01 s, p=0.0012). Compared to untreated cells, transfection with TNNT2-SupRep gene therapy, the TNNT2-R288P iPSC-CMs showed a decrease in irregular events (30.4% vs. 14%, p<0.0001), shortened CTD90 (1.01 ± 0.03 s vs. 0.91 ± 0.01 s, p=0.003), and shortened peak to 90% decay time (0.57 ± 0.02 s vs. 0.50 ± 0.02 s, p=0.02). There was no significant difference in irregular events, CTD90, or peak to 90% decay time between TNNT2-R288P iPSC-CMs treated with TNNT2-SupRep and IC iPSC-CMs. Here, we provide the first proof-of-principle SupRep gene therapy for TNNT2-mediated HCM. TNNT2-SupRep gene therapy almost fully restored the impaired intracellular calcium handling properties of TNNT2-R288P patient-derived iPSC-CMs back to the normal state achieved by the CRISPR/Cas9 gene-edited cure.

Regulation of oxytocin-induced calcium transients and gene expression in engineered myometrial tissues by tissue architecture and matrix rigidity

The uterus is susceptible to benign tumors known as fibroids, which have been associated with many pregnancy complications, including preterm labor. However, the impact of fibrotic tissue remodeling on the physiology of the myometrium, the smooth muscle layer of the uterus, is poorly understood, in large part due to a lack of model systems. In this study, we engineered healthy-like and fibrotic-like myometrium by culturing human myometrial smooth muscle cells on polyacrylamide hydrogels micropatterned with fibronectin to independently tune matrix rigidity and tissue alignment, respectively. We then evaluated calcium transients in response to oxytocin stimulation. Isotropic myometrial tissues on stiff substrates (representing fibrotic myometrium) had shorter calcium transients due to shorter decay time compared to aligned myometrial tissues on soft substrates (representing healthy myometrium). Calcium transients in aligned tissues had longer response times and longer decay times than isotropic tissues, irrespective of substrate stiffness. The amplitude of calcium transients was also higher on soft substrates compared to stiff substrates, irrespective of tissue alignment. We also performed RNA sequencing to detect differentially expressed genes between healthy- and fibrotic-like tissues, which revealed that a bitter taste receptor shown to induce smooth muscle relaxation, TAS2R31, was down-regulated in fibrotic-like tissues. Finally, we measured oxytocin-induced calcium transients in response to pre-treatment with progesterone, caffeine, thrombin, and nifedipine to demonstrate applications for our model system in drug screening. Both progesterone and caffeine caused a decrease in calcium transient duration, as expected, while thrombin and nifedipine had less impact. Collectively, our engineered model of the myometrium enables new insights into myometrial mechanobiology and can be extended to identify or screen novel drug targets.

Increased vulnerability to atrial and ventricular arrhythmias caused by different types of inhaled tobacco or marijuana products

The emergence of a plethora of new tobacco products marketed as being less harmful than smoking, such as electronic cigarettes and heated tobacco products, and the increased popularity of recreational marijuana have raised concerns about the potential cardiovascular risk associated with their use. The purpose of this study was to investigate whether the use of novel tobacco products or marijuana can cause the development of proarrhythmic substrate and eventually lead to arrhythmias. Rats were exposed to smoke from tobacco, marijuana, or cannabinoid-depleted marijuana, to aerosol from electronic cigarettes or heated tobacco products, or to clean air once per day for 8 weeks, following by assays for blood pressure, cardiac function, ex vivo electrophysiology, and histochemistry. The rats exposed to tobacco or marijuana products exhibited progressively increased systolic blood pressure, decreased cardiac systolic function with chamber dilation, and reduced overall heart rate variability, relative to the clean air negative control group. Atrial fibrillation and ventricular tachycardia testing by ex vivo optical mapping revealed a significantly higher susceptibility to each, with a shortened effective refractory period and prolonged calcium transient duration. Histological analysis indicated that in all exposure conditions except for air, exposure to smoke or aerosol from tobacco or marijuana products caused severe fibrosis with decreased microvessel density and higher level of sympathetic nerve innervation. These pathophysiological results indicate that tobacco and marijuana products can induce arrhythmogenic substrates involved in cardiac electrical, structural, and neural remodeling, facilitating the development of arrhythmias.

Abstract 15591: Acute Dantrolene Treatment Reverses Arrhythmogenic Substrate and Reduces Triggers Induced by Ryanodine Receptor 2 Hyperactivity in Human Hearts

Introduction: Ryanodine receptor 2 (RyR2) hyperactivity is commonly observed in structural heart diseases that are a result of ischemia or heart failure. It results in abnormal calcium handling and calcium leak that can trigger arrhythmias. Dantrolene, a RyR inhibitor, is used to prevent calcium leak from RyR1 in skeletal muscles. Hypothesis: Suppressing RyR2 hyperactivity with acute dantrolene treatment can reduce arrhythmia burden in human hearts. Methods: Human donor hearts that were not used in transplantation were obtained and 1 cm 3 cubes were isolated from the left ventricle. Slices at 400 μm thickness were generated from these cubes, pinned and perfused with Tyrode’s solution in a recording tissue chamber. Pseudo-ECGs were recorded with ECG electrodes and the slices were optically mapped to measure conduction velocity (CV), action potential duration (APD) and calcium transient duration (CaTD). After an initial 30 min equilibration period, baseline optical recordings were obtained. Slices were then treated with 1) Isoproterenol (Iso, 250 nM) to stimulate β-adrenergic receptors, 2) Caffeine (200 μM) for RyR2 receptor activation and 3) Dantrolene (10 μM), sequentially. Dual optical recordings, Vm and Ca, were obtained after each treatment. Results: Substrate: Iso reduced APD (425±36 vs 394±43, p=0.03), as expected, and addition of Caffeine restored APD to baseline. More importantly, addition of Dantrolene prolonged APD even further (460±90, p=0.04). CaTD was also similarly reduced by Iso (445±48 vs 387±25, p=0.04) and restored to baseline by Caffeine+Iso. No significant changes in CV were measured under any condition. Trigger: In slices where PVCs were inducible, Caffeine+Iso increased occurrence of PVCs (20 vs 53 PVCs/min) while addition of Dantrolene reduced PVC incidence (18 PVCs/min). Conclusions: Dantrolene treatment, acutely reversed proarrhythmogenic substrate and reduced triggers and could therefore be a suitable approach for anti-arrhythmic therapy in patients with structural heart disease. Additionally, human LV slice preparation is useful in antiarrhythmic drug studies.

Abstract P1145: Hydrogel Matrix Support Of Suspension-differentiated Hipscs Impacts Resulting In Vitro Cardiac Tissue Functionality

Functional in vitro cardiac tissues are needed to provide insight into the cellular level of disease. Previously, we demonstrated hydrogel-encapsulated hiPSCs undergo cardiac differentiation and achieve cardiomyocyte (CM) maturation including T-tubule formation. To investigate the effect of encapsulation on differentiation and cardiac tissue functionality, here we compared PEG-fibrinogen (PF) microsphere (MS)-supported differentiation approach to self-aggregated embryoid body (EB) differentiation. Briefly, hiPSCs were encapsulated or formed into EBs; cardiac differentiation was initiated by Wnt activation and subsequent inhibition. CM content, contractile functionality, drug response, and electrophysiological properties were determined by flow cytometry, microscopy, and optical mapping, respectively.Initial diameter was 673±22 μm (coefficient of variance, COV=0.03) for MS and145±34 μm (COV=0.24) for EBs. The number of CMs produced (differentiation day 10) per initial hiPSC was significantly higher for MS (2.8 ± 1.8) than EBs (0.6±0.12). MS also yielded a higher percentage of CMs; MS-encapsulated hiPSC differentiation resulted in 74.7±13.8% CMs while for EBs it was 56±16.1 % (cTnT+, MF20+, n=14 and 5 batches, respectively, p&lt;0.05). Myocardial contractility was also enhanced in cardiac MS as compared to EBs (similar CM content, day 10); maximum contraction velocity was four times higher for MS than EBs (139±17.5 μm/s versus 33.4±5.1 μm/s) and maximum relaxation velocity was fourteen times higher (97±7.2 μm/s versus 7.4±0.4 μm/s).Furthermore, cardiac MS responded more vigorously to isoproterenol (IP) and propranolol (P) (frequency +124% (IP), - 61% (P), versus +49% (IP) and -27%(P) for EB), suggesting that MS-CM β-adrenergic signaling may be more mature. Calcium transient propagation was more uniform in MS-CMs compared to EB-CMs; calcium transient duration (CTD) at 50% and 80% repolarization and calcium transient velocity were similar. In conclusion, MS supported cardiac differentiation supported higher CM content, faster contractility, and improved drug response in comparison to similarly differentiated EBs, demonstrating the potential of encapsulation to enhance cardiac tissue functionality for disease modeling.

Calcium signaling in individual APP/PS1 mouse dentate gyrus astrocytes increases ex vivo with Aβ pathology and age without affecting astrocyte network activity.

Astrocytes are critical for healthy brain function. In Alzheimer’s disease, astrocytes become reactive, which affects their signaling properties. Here, we measured spontaneous calcium transients ex vivo in hippocampal astrocytes in brain slices containing the dentate gyrus of 6‐ (6M) and 9‐month‐old (9M) APPswe/PSEN1dE9 (APP/PS1) mice. We investigated the frequency and duration of calcium transients in relation to aging, amyloid‐β (Aβ) pathology, and the proximity of the astrocyte to Aβ plaques. The 6M APP/PS1 astrocytes showed no change in spontaneous calcium‐transient properties compared to wild‐type (WT) astrocytes. 9M APP/PS1 astrocytes, however, showed more hyperactivity compared to WT, characterized by increased spontaneous calcium transients that were longer in duration. Our data also revealed an effect of aging, as 9M astrocytes overall showed an increase in calcium activity compared to 6M astrocytes. Subsequent calcium‐wave analysis showed an increase in sequential calcium transients (i.e., calcium waves) in 9M astrocytes, suggesting increased network activity ex vivo. Further analysis using null models revealed that this network effect is caused by chance, due to the increased number of spontaneous transients. Our findings show that alterations in calcium signaling in individual hippocampal astrocytes of APP/PS1 mice are subject to both aging and Aβ pathology but these do not lead to a change in astrocyte network activity. These alterations in calcium dynamics of astrocytes may help to understand changes in neuronal physiology leading to cognitive decline and ultimately dementia.

Mechanisms of dysfunction in patient-derived cardiomyocytes with the hypertrophic cardiomyopathy-myosin binding protein-C e258K mutation

Mutations in cardiac myosin binding protein-C (cMyBP-C) are the most common cause of hypertrophic cardiomyopathy (HCM). We recently described the highly penetrant MYBPC3-c.772G>A (p.E258K) mutation with founder effect in Tuscany, Italy. Isolated myofibrils from patient myectomies indicated accelerated cross-bridge kinetics and higher energy cost of tension. Concurrently, prolonged action potentials and calcium transient duration preserves twitch contraction amplitude and duration in intact cells and trabeculae (Pioner et al., Biophys J, 2020;118:35a).

B-PO05-014 A RETROSPECTIVE STUDY OF SEASONAL VARIABILITY OF CARDIAC ELECTROPHYSIOLOGY IN THE RABBIT HEART

Rabbits are an important animal model for the study of cardiac electrophysiology and are suitable for analyzing mechanisms of cardiac arrhythmias. Rabbits are known to have seasonal variation in activity, behavior, and breeding; however, there are few data related to seasonal variation in electrophysiological parameters in the rabbit heart. To determine if there is significant seasonal variation in cardiac electrophysiology of the New Zealand White (NZW) rabbit heart. Male NZW rabbit hearts (N = 55) were purchased from an approved vendor and housed on a 12:12 hr light:dark cycle. Hearts were Langendorff-perfused for simultaneous optical mapping of Vm and Ca2+ with voltage-sensitive (RH237) and calcium-sensitive (Rhod2-AM) indicators, respectively. Data were collected from 2012-2016 and retrospectively analyzed. We defined the seasons meteorologically: December-February as winter, March-May as spring, June-August as summer and September-November as autumn. All data were collected while pacing at a constant cycle length of 300 ms. Retrospective data revealed seasonal variability in the action potential duration (APD) and in the calcium transient duration (CaTD). For summer compared to spring and autumn, APD50 (121.1 ± 11 vs. 137 ± 19.8 and vs. 135.3 ± 11.3 ms, p < 0.01 for both) and APD80 (151.1 ± 11.7 vs. 166.4 ± 17.6 and vs. 163.5 ± 11.9 ms, p < 0.01 for both) were significantly shorter, but not different from winter. For summer compared to spring, CaTD50 (123.8 ± 5.5 vs. 137.7 ± 13.2 ms, p < 0.01) and CaTD80 (162.8 ± 6.1 vs. 178.4 ± 16.9 ms, p = 0.11) were shorter. There was not significance difference in Vm-Ca2+ coupling (i.e., CaTD - APD) between seasons. APD and CaTD are significantly shorter in the summer months of isolated rabbit hearts. These data may provide insight into seasonal patterns of arrhythmia in humans, but should also be considered with regard to experimental rigor and reproducibility.