Collaborative science in action: A 20 year perspective from the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization dedicated to resolving global health challenges through collaborative scientific efforts across academia, regulatory authorities and the private sector. Collaborative science across non-clinical disciplines offers an important keystone to accelerate the development of safer and more effective medicines. HESI works to address complex challenges by leveraging diverse subject-matter expertise across sectors offering access to resources, data and shared knowledge. In 2008, the HESI Cardiac Safety Committee (CSC) was established to improve public health by reducing unanticipated cardiovascular (CV)-related adverse effects from pharmaceuticals or chemicals. The committee continues to significantly impact the field of CV safety by bringing together experts from across sectors to address challenges of detecting and predicting adverse cardiac outcomes. Committee members have collaborated on the organization, management and publication of prospective studies, retrospective analyses, workshops, and symposia resulting in 38 peer reviewed manuscripts. Without this collaboration these manuscripts would not have been published. Through their work, the CSC is actively addressing challenges and opportunities in detecting potential cardiac failure modes using in vivo, in vitro and in silico models, with the aim of facilitating drug development and improving study design. By examining past successes and future prospects of the CSC, this manuscript sheds light on how the consortium's multifaceted approach not only addresses current challenges in detecting potential cardiac failure modes but also paves the way for enhanced drug development and study design methodologies. Further, exploring future opportunities and challenges will focus on improving the translational predictability of nonclinical evaluations and reducing reliance on animal research in CV safety assessments.

A novel ryanodine receptor 2 inhibitor, M201-A, enhances natriuresis, renal function and lusi-inotropic actions: Preclinical and phase I study.

BACKGROUND AND PURPOSE: The ryanodine receptor 2 (RyR2) is present in both the heart and kidneys, and plays a crucial role in maintaining intracellular Ca2+ homeostasis in cells in these organs. This study aimed to investigate the impact of M201-A on RyR2, as well as studying its effects on cardiac and renal functions in preclinical and clinical studies. EXPERIMENTAL APPROACH: Following the administration of M201-A (1,4-benzothiazepine-1-oxide derivative), we monitored diastolic Ca2+ leak via RyR2 and intracellular Ca2+ concentration in isolated rat cardiomyocytes and in cardiac and renal function in animals. In a clinical study, M201-A was administered intravenously at doses of 0.2 and 0.4 mg·kg-1 once daily for 20 min for four consecutive days in healthy males, with the assessment of haemodynamic responses. KEY RESULTS: In rat heart cells, M201-A effectively inhibited spontaneous diastolic Ca2+ leakage through RyR2 and exhibited positive lusi-inotropic effects on the rat heart. Additionally, it enhanced natriuresis and improved renal function in dogs. In human clinical studies, when administered intravenously, M201-A demonstrated an increase in natriuresis, glomerular filtration rate and creatinine clearance, while maintaining acceptable levels of drug safety and tolerability. CONCLUSIONS AND IMPLICATIONS: The novel drug M201-A inhibited diastolic Ca2+ leak via RyR2, improved cardiac lusi-inotropic effects in rats, and enhanced natriuresis and renal function in humans. These findings suggest that this drug may offer a potential new treatment option for chronic kidney disease and heart failure.

Inherited physical capacity: Widening divergence from young to adult to old.

Cardiorespiratory performance segregates into rat strains of inherited low- and high-capacity runners (LCRs and HCRs); during adulthood, this segregation remains stable, but widens in senescence and is followed by segregated function, health, and mortality. However, this segregation has not been investigated prior to adulthood. We, therefore, assessed cardiorespiratory performance and cardiac cell (cardiomyocyte) structure-function in 1- and 4-month-old LCRs and HCRs. Maximal oxygen uptake was 23% less in LCRs at 1-month compared to HCRs at 1-month, and 72% less at 4 months. Cardiomyocyte contractility was 37-56% decreased, and Ca2+ release was 34-62% decreased, in 1- and 4-month LCRs versus HCRs. This occurred because HCRs had improved contractility and Ca2+ release during maturation, whereas LCRs did not. In quiescent cardiomyocytes, LCRs displayed 180% and 297% more Ca2+ sparks and 91% and 38% more Ca2+ waves at 1 and 4 months versus HCRs. Cell sizes were not different between LCRs and HCRs, but LCRs showed reduced transverse-tubules versus HCRs, though no discrepant transverse-tubule generation occurred during maturation. In conclusion, LCRs show reduced scores for aerobic capacity and cardiomyocyte structure-function compared to HCRs and there is a widening divergence between LCRs and HCRs during juvenile to near-adult maturation.

A novel method for the percutaneous induction of myocardial infarction by occlusion of small coronary arteries in the rabbit.

Arrhythmic sudden cardiac death (SCD) is an important cause of mortality following myocardial infarction (MI). The rabbit has similar cardiac electrophysiology to humans and is therefore an important small animal model to study post-MI arrhythmias. The established approach of surgical coronary ligation results in thoracic adhesions that impede epicardial electrophysiological studies. Adhesions are absent following a percutaneously induced MI, which is also associated with reduced surgical morbidity and so represents a clear refinement of the approach. Percutaneous procedures have previously been described in large rabbits (3.5-5.5 kg). Here, we describe a novel method of percutaneous MI induction in smaller rabbits (2.5-3.5 kg) that are readily available commercially. New Zealand White rabbits (n = 51 males, 3.1 ± 0.3 kg) were anesthetized using isoflurane (1.5-3%) and underwent either a percutaneous MI procedure involving microcatheter tip deployment (≤1.5 Fr, 5 mm), coronary ligation surgery, or a sham procedure. Electrocardiography (ECG) recordings were used to confirm ST-segment elevation indicating coronary occlusion. Blood samples (1 and 24 h) were taken for cardiac troponin I (cTnI) levels. Ejection fraction (EF) was measured at 6-8 wk. Rabbits were then euthanized (Euthatal) and hearts were processed for magnetic resonance imaging and histology. Mortality rates were similar in both groups. Scar volume, cTnI, and EF were similar between both MI groups and significantly different from their respective sham controls. Thus, percutaneous coronary occlusion by microcatheter tip deployment is feasible in rabbits (2.5-3.5 kg) and produces an MI with similar characteristics to surgical ligation with lower procedural trauma and without epicardial adhesions.NEW & NOTEWORTHY Surgical coronary ligation is the standard technique to induce myocardial infarction (MI) in rabbits but is associated with procedural trauma and the generation of thoracic adhesions. Percutaneous coronary occlusion avoids these shortcomings and is established in pigs but has only been applicable to large rabbits because of a mismatch between the equipment used and target vessel size. Here, we describe a new scalable approach to percutaneous MI induction that is safe and effective in 2.5-3.5-kg rabbits.

A comprehensive protocol combining in vivo and ex vivo electrophysiological experiments in an arrhythmogenic animal model.

Ventricular arrhythmias contribute significantly to cardiovascular mortality, with coronary artery disease as the predominant underlying cause. Understanding the mechanisms of arrhythmogenesis is essential to identify proarrhythmic factors and develop novel approaches for antiarrhythmic prophylaxis and treatment. Animal models are vital in basic research on cardiac arrhythmias, encompassing molecular, cellular, ex vivo whole heart, and in vivo models. Most studies use either in vivo protocols lacking important information on clinical relevance or exclusively ex vivo protocols, thereby missing the opportunity to explore underlying mechanisms. Consequently, interpretation may be difficult due to dissimilarities in animal models, interventions, and individual properties across animals. Moreover, proarrhythmic effects observed in vivo are often not replicated in corresponding ex vivo preparations during mechanistic studies. We have established a protocol to perform both an in vivo and ex vivo electrophysiological characterization in an arrhythmogenic rat model with heart failure following myocardial infarction. The same animal is followed throughout the experiment. In vivo methods involve intracardiac programmed electrical stimulation and external defibrillation to terminate sustained ventricular arrhythmia. Ex vivo methods conducted on the Langendorff-perfused heart include an electrophysiological study with optical mapping of regional action potentials, conduction velocities, and dispersion of electrophysiological properties. By exploring the retention of the in vivo proarrhythmic phenotype ex vivo, we aim to examine whether the subsequent ex vivo detailed measurements are relevant to in vivo pathological behavior. This protocol can enhance greater understanding of cardiac arrhythmias by providing a standardized, yet adaptable model for evaluating arrhythmogenicity or antiarrhythmic interventions in cardiac diseases.NEW & NOTEWORTHY Rodent models are widely used in arrhythmia research. However, most studies do not standardize clinically relevant in vivo and ex vivo techniques to support their conclusions. Here, we present a comprehensive electrophysiological protocol in an arrhythmogenic rat model, connecting in vivo and ex vivo programmed electrical stimulation with optical mapping. By establishing this protocol, we aim to facilitate the adoption of a standardized model for investigating arrhythmias, enhancing research rigor and comparability in this field.

Proarrhythmic changes in human cardiomyocytes during hypothermia by milrinone and isoprenaline, but not levosimendan: an experimental in vitro study.

BACKGROUND: Accidental hypothermia, recognized by core temperature below 35 °C, is a lethal condition with a mortality rate up to 25%. Hypothermia-induced cardiac dysfunction causing increased total peripheral resistance and reduced cardiac output contributes to the high mortality rate in this patient group. Recent studies, in vivo and in vitro, have suggested levosimendan, milrinone and isoprenaline as inotropic treatment strategies in this patient group. However, these drugs may pose increased risk of ventricular arrhythmias during hypothermia. Our aim was therefore to describe the effects of levosimendan, milrinone and isoprenaline on the action potential in human cardiomyocytes during hypothermia. METHODS: Using an experimental in vitro-design, levosimendan, milrinone and isoprenaline were incubated with iCell2 hiPSC-derived cardiomyocytes and cellular action potential waveforms and contraction were recorded from monolayers of cultured cells. Experiments were conducted at temperatures from 37 °C down to 26 °C. One-way repeated measures ANOVA was performed to evaluate differences from baseline recordings and one-way ANOVA was performed to evaluate differences between drugs, untreated control and between drug concentrations at the specific temperatures. RESULTS: Milrinone and isoprenaline both significantly increases action potential triangulation during hypothermia, and thereby the risk of ventricular arrhythmias. Levosimendan, however, does not increase triangulation and the contractile properties also remain preserved during hypothermia down to 26 °C. CONCLUSIONS: Levosimendan remains a promising candidate drug for inotropic treatment of hypothermic patients as it possesses ability to treat hypothermia-induced cardiac dysfunction and no increased risk of ventricular arrhythmias is detected. Milrinone and isoprenaline, on the other hand, appears more dangerous in the hypothermic setting.

Does the small conductance Ca2+-activated K+ current ISK flow under physiological conditions in rabbit and human atrial isolated cardiomyocytes?

BACKGROUND: The small conductance Ca2+-activated K+ current (ISK) is a potential therapeutic target for treating atrial fibrillation. AIM: To clarify, in rabbit and human atrial cardiomyocytes, the intracellular [Ca2+]-sensitivity of ISK, and its contribution to action potential (AP) repolarisation, under physiological conditions. METHODS: Whole-cell-patch clamp, fluorescence microscopy: to record ion currents, APs and [Ca2+]i; 35-37°C. RESULTS: In rabbit atrial myocytes, 0.5 mM Ba2+ (positive control) significantly decreased whole-cell current, from -12.8 to -4.9 pA/pF (P < 0.05, n = 17 cells, 8 rabbits). By contrast, the ISK blocker apamin (100 nM) had no effect on whole-cell current, at any set [Ca2+]i (∼100-450 nM). The ISK blocker ICAGEN (1 µM: ≥2 x IC50) also had no effect on current over this [Ca2+]i range. In human atrial myocytes, neither 1 µM ICAGEN (at [Ca2+]i âˆ¼ 100-450 nM), nor 100 nM apamin ([Ca2+]i âˆ¼ 250 nM) affected whole-cell current (5-10 cells, 3-5 patients/group). APs were significantly prolonged (at APD30 and APD70) by 2 mM 4-aminopyridine (positive control) in rabbit atrial myocytes, but 1 µM ICAGEN had no effect on APDs, versus either pre-ICAGEN or time-matched controls. High concentration (10 µM) ICAGEN (potentially ISK-non-selective) moderately increased APD70 and APD90, by 5 and 26 ms, respectively. In human atrial myocytes, 1 µM ICAGEN had no effect on APD30-90, whether stimulated at 1, 2 or 3 Hz (6-9 cells, 2-4 patients/rate). CONCLUSION: ISK does not flow in human or rabbit atrial cardiomyocytes with [Ca2+]i set within the global average diastolic-systolic range, nor during APs stimulated at physiological or supra-physiological (≤3 Hz) rates.

Electrically stimulated in vitro heart cell mimic of acute exercise reveals novel immediate cellular responses to exercise: Reduced contractility and metabolism, but maintained calcium cycling and increased myofilament calcium sensitivity.

Cardiac cellular responses to acute exercise remain undescribed. We present a model for mimicking acute aerobic endurance exercise to freshly isolated cardiomyocytes by evoking exercise-like contractions over prolonged periods of time with trains of electrical twitch stimulations. We then investigated immediate contractile, Ca2+ , and metabolic responses to acute exercise in perfused freshly isolated left ventricular rat cardiomyocytes, after a matrix-design optimized protocol and induced a mimic for acute aerobic endurance exercise by trains of prolonged field twitch stimulations. Acute exercise decreased cardiomyocyte fractional shortening 50%-80% (p < .01). This was not explained by changes to intracellular Ca2+ handling (p > .05); rather, we observed a weak insignificant Ca2+ transient increase (p = .11), while myofilament Ca2+ sensitivity increased 20%-70% (p < .05). Acidic pH 6.8 decreased fractional shortening 20%-70% (p < .05) because of 20%-30% decreased Ca2+ transients (p < .05), but no difference occurred between control and acute exercise (p > .05). Addition of 1 or 10 mM La- increased fractional shortening in control (1 mM La- : no difference, p > .05; 10 mM La- : 20%-30%, p < .05) and acute exercise (1 mM La- : 40%-90%, p < .01; 10 mM La- : 50%-100%, p < .01) and rendered acute exercise indifferent from control (p > .05). Intrinsic autofluorescence showed a resting NADstate of 0.59 ± 0.04 and FADstate of 0.17 ± 0.03, while acute exercise decreased NADH/FAD ratio 8% (p < .01), indicating intracellular oxidation. In conclusion, we show a novel approach for studying immediate acute cardiomyocyte responses to aerobic endurance exercise. We find that acute exercise in cardiomyocytes decreases contraction, but Ca2+ handling and myofilament Ca2+ sensitivity compensate for this, while acidosis and reduced energy substrate and mitochondrial ATP generation explain this.

Pro-arrhythmic effect of escitalopram and citalopram at serum concentrations commonly observed in older patients - a study based on a cohort of 19,742 patients.

BACKGROUND: For a decade, patients have been advised against using high citalopram- and escitalopram-doses due to risk for ventricular arrhythmia and cardiac arrest. Still, these drugs are widely used to treat depression and anxiety especially in older patients. It is unclear why they are cardiotoxic and at what serum concentrations patients are at risk for arrhythmias. Thus, how many patients that are at risk for iatrogenic cardiac arrest is unknown. METHODS: We studied the arrhythmogenic effects of citalopram, escitalopram and their metabolites on human cardiomyocytes. Concentrations showing pro-arrhythmic activity were compared with observed drug and metabolite serum concentrations in a cohort of 19,742 patients (age 12-105 years) using escitalopram or citalopram in Norway (2010-2019). As arrhythmia-risk is related to maximum serum concentration, this was simulated for different age-groups from the escitalopram patient material. FINDINGS: Therapeutic concentrations of both citalopram and escitalopram but not their metabolites showed pro-arrhythmic changes in the human cardiac action potential. Due to age-dependent reduction of drug clearance, the proportion of patients above threshold for arrhythmia-risk increased with age. 20% of patients >65 years were predicted to reach potentially pro-arrhythmic concentrations, following intake of 10 mg escitalopram. INTERPRETATION: All patients that are using escitalopram or citalopram and have genetic disposition for acquired long-QT syndrome, are >65 years, are using additional pro-arrhythmic drugs or have predisposition for arrhythmias, should be monitored with therapeutic drug monitoring (TDM) to avoid exposure to potentially cardiotoxic concentrations. Serum concentrations should be kept below 100 nM, to reduce arrhythmia-risk. FUNDING: This study was funded by The Research Council of Norway (project number: 324062).

Physiology of intracellular calcium buffering.

Calcium signaling underlies much of physiology. Almost all the Ca2+ in the cytoplasm is bound to buffers, with typically only ∼1% being freely ionized at resting levels in most cells. Physiological Ca2+ buffers include small molecules and proteins, and experimentally Ca2+ indicators will also buffer calcium. The chemistry of interactions between Ca2+ and buffers determines the extent and speed of Ca2+ binding. The physiological effects of Ca2+ buffers are determined by the kinetics with which they bind Ca2+ and their mobility within the cell. The degree of buffering depends on factors such as the affinity for Ca2+, the Ca2+ concentration, and whether Ca2+ ions bind cooperatively. Buffering affects both the amplitude and time course of cytoplasmic Ca2+ signals as well as changes of Ca2+ concentration in organelles. It can also facilitate Ca2+ diffusion inside the cell. Ca2+ buffering affects synaptic transmission, muscle contraction, Ca2+ transport across epithelia, and the killing of bacteria. Saturation of buffers leads to synaptic facilitation and tetanic contraction in skeletal muscle and may play a role in inotropy in the heart. This review focuses on the link between buffer chemistry and function and how Ca2+ buffering affects normal physiology and the consequences of changes in disease. As well as summarizing what is known, we point out the many areas where further work is required.

Does Enhanced Structural Maturity of hiPSC-Cardiomyocytes Better for the Detection of Drug-Induced Cardiotoxicity?

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are currently used following the Comprehensive in vitro Proarrhythmic Assay (CiPA) initiative and subsequent recommendations in the International Council for Harmonization (ICH) guidelines S7B and E14 Q&A, to detect drug-induced cardiotoxicity. Monocultures of hiPSC-CMs are immature compared to adult ventricular cardiomyocytes and might lack the native heterogeneous nature. We investigated whether hiPSC-CMs, treated to enhance structural maturity, are superior in detecting drug-induced changes in electrophysiology and contraction. This was achieved by comparing hiPSC-CMs cultured in 2D monolayers on the current standard (fibronectin matrix, FM), to monolayers on a coating known to promote structural maturity (CELLvo™ Matrix Plus, MM). Functional assessment of electrophysiology and contractility was made using a high-throughput screening approach involving the use of both voltage-sensitive fluorescent dyes for electrophysiology and video technology for contractility. Using 11 reference drugs, the response of the monolayer of hiPSC-CMs was comparable in the two experimental settings (FM and MM). The data showed no functionally relevant differences in electrophysiology between hiPSC-CMs in standard FM and MM, while contractility read-outs indicated an altered amplitude of contraction but not changes in time course. RNA profiling for cardiac proteins shows similarity of the RNA expression across the two forms of 2D culture, suggesting that cell-to-matrix adhesion differences may explain account for differences in contraction amplitude. The results support the view that hiPSC-CMs in both 2D monolayer FM and MM that promote structural maturity are equally effective in detecting drug-induced electrophysiological effects in functional safety studies.

Palmitoylation of the pore-forming subunit of Ca(v)1.2 controls channel voltage sensitivity and calcium transients in cardiac myocytes.

Mammalian voltage-activated L-type Ca2+ channels, such as Ca(v)1.2, control transmembrane Ca2+ fluxes in numerous excitable tissues. Here, we report that the pore-forming α1C subunit of Ca(v)1.2 is reversibly palmitoylated in rat, rabbit, and human ventricular myocytes. We map the palmitoylation sites to two regions of the channel: The N terminus and the linker between domains I and II. Whole-cell voltage clamping revealed a rightward shift of the Ca(v)1.2 current-voltage relationship when α1C was not palmitoylated. To examine function, we expressed dihydropyridine-resistant α1C in human induced pluripotent stem cell-derived cardiomyocytes and measured Ca2+ transients in the presence of nifedipine to block the endogenous channels. The transients generated by unpalmitoylatable channels displayed a similar activation time course but significantly reduced amplitude compared to those generated by wild-type channels. We thus conclude that palmitoylation controls the voltage sensitivity of Ca(v)1.2. Given that the identified Ca(v)1.2 palmitoylation sites are also conserved in most Ca(v)1 isoforms, we propose that palmitoylation of the pore-forming α1C subunit provides a means to regulate the voltage sensitivity of voltage-activated Ca2+ channels in excitable cells.

Phenomenological analysis of simple ion channel block in large populations of uncoupled cardiomyocytes.

Current understanding of arrhythmia mechanisms and design of anti-arrhythmic drug therapies hinges on the assumption that myocytes from the same region of a single heart have similar, if not identical, action potential waveforms and drug responses. On the contrary, recent experiments reveal significant heterogeneity in uncoupled healthy myocytes both from different hearts as well as from identical regions within a single heart. In this work, a methodology is developed for quantifying the individual electrophysiological properties of large numbers of uncoupled cardiomyocytes under ion channel block in terms of the parameters values of a conceptual fast-slow model of electrical excitability. The approach is applied to a population of nearly 500 rabbit ventricular myocytes for which action potential duration (APD) before and after the application of the drug nifedipine was experimentally measured (Lachaud et al., 2022, Cardiovasc. Res.). To this end, drug action is represented by a multiplicative factor to an effective ion conductance, a closed form asymptotic expression for APD is derived and inverted to determine model parameters as functions of APD and $\varDelta $APD (drug-induced change in APD) for each myocyte. Two free protocol-related quantities are calibrated to experiment using an adaptive-domain procedure based on an original assumption of optimal excitability. The explicit APD expression and the resulting set of model parameter values allow (a) direct evaluation of conditions necessary to maintain fixed APD or $\varDelta $APD, (b) predictions of the proportion of cells remaining excitable after drug application, (c) predictions of stimulus period dependency and (d) predictions of dose-response curves, the latter being in agreement with additional experimental data.

Initiation of ventricular arrhythmia in the acquired long QT syndrome.

AIMS: Long QT syndrome (LQTS) carries a risk of life-threatening polymorphic ventricular tachycardia (Torsades de Pointes, TdP) and is a major cause of premature sudden cardiac death. TdP is induced by R-on-T premature ventricular complexes (PVCs), thought to be generated by cellular early-afterdepolarisations (EADs). However, EADs in tissue require cellular synchronisation, and their role in TdP induction remains unclear. We aimed to determine the mechanism of TdP induction in rabbit hearts with acquired LQTS (aLQTS). METHODS AND RESULTS: Optical mapping of action potentials (APs) and intracellular Ca2+ was performed in Langendorff-perfused rabbit hearts (n = 17). TdP induced by R-on-T PVCs was observed during aLQTS (50% K+/Mg++ & E4031) conditions in all hearts (P < 0.0001 vs. control). Islands of AP prolongation bounded by steep voltage gradients (VGs) were consistently observed before arrhythmia and peak VGs were more closely related to the PVC upstroke than EADs, both temporally (7 ± 5 ms vs. 44 ± 27 ms, P < 0.0001) and spatially (1.0 ± 0.7 vs. 3.6 ± 0.9 mm, P < 0.0001). PVCs were initiated at estimated voltages of ∼ -40 mV and had upstroke dF/dtmax and Vm-Ca2+ dynamics compatible with ICaL activation. Computational simulations demonstrated that PVCs could arise directly from VGs, through electrotonic triggering of ICaL. In experiments and the model, sub-maximal L-type Ca2+ channel (LTCC) block (200 nM nifedipine and 90% gCaL, respectively) abolished both PVCs and TdP in the continued presence of aLQTS. CONCLUSION: These data demonstrate that ICaL activation at sites displaying steep VGs generates the PVCs which induce TdP, providing a mechanism and rationale for LTCC blockers as a novel therapeutic approach in LQTS.

Dynamic but discordant alterations in zDHHC5 expression and palmitoylation of its substrates in cardiac pathologies.

S-palmitoylation is an essential lipid modification catalysed by zDHHC-palmitoyl acyltransferases that regulates the localisation and activity of substrates in every class of protein and tissue investigated to date. In the heart, S-palmitoylation regulates sodium-calcium exchanger (NCX1) inactivation, phospholemman (PLM) inhibition of the Na+/K+ ATPase, Nav1.5 influence on membrane excitability and membrane localisation of heterotrimeric G-proteins. The cell surface localised enzyme zDHHC5 palmitoylates NCX1 and PLM and is implicated in injury during anoxia/reperfusion. Little is known about how palmitoylation remodels in cardiac diseases. We investigated expression of zDHHC5 in animal models of left ventricular hypertrophy (LVH) and heart failure (HF), along with HF tissue from humans. zDHHC5 expression increased rapidly during onset of LVH, whilst HF was associated with decreased zDHHC5 expression. Paradoxically, palmitoylation of the zDHHC5 substrate NCX1 was significantly reduced in LVH but increased in human HF, while palmitoylation of the zDHHC5 substrate PLM was unchanged in all settings. Overexpression of zDHHC5 in rabbit ventricular cardiomyocytes did not alter palmitoylation of its substrates or overall cardiomyocyte contractility, suggesting changes in zDHHC5 expression in disease may not be a primary driver of pathology. zDHHC5 itself is regulated by post-translational modifications, including palmitoylation in its C-terminal tail. We found that in HF palmitoylation of zDHHC5 changed in the same manner as palmitoylation of NCX1, suggesting additional regulatory mechanisms may be involved. This study provides novel evidence that palmitoylation of cardiac substrates is altered in the setting of HF, and that expression of zDHHC5 is dysregulated in both hypertrophy and HF.

Adrenoceptor sub-type involvement in Ca2+ current stimulation by noradrenaline in human and rabbit atrial myocytes.

Atrial fibrillation (AF) from elevated adrenergic activity may involve increased atrial L-type Ca2+ current (ICaL) by noradrenaline (NA). However, the contribution of the adrenoceptor (AR) sub-types to such ICaL-increase is poorly understood, particularly in human. We therefore investigated effects of various broad-action and sub-type-specific α- and ß-AR antagonists on NA-stimulated atrial ICaL. ICaL was recorded by whole-cell-patch clamp at 37 °C in myocytes isolated enzymatically from atrial tissues from consenting patients undergoing elective cardiac surgery and from rabbits. NA markedly increased human atrial ICaL, maximally by ~ 2.5-fold, with EC75 310 nM. Propranolol (ß1 + ß2-AR antagonist, 0.2 microM) substantially decreased NA (310 nM)-stimulated ICaL, in human and rabbit. Phentolamine (α1 + α2-AR antagonist, 1 microM) also decreased NA-stimulated ICaL. CGP20712A (ß1-AR antagonist, 0.3 microM) and prazosin (α1-AR antagonist, 0.5 microM) each decreased NA-stimulated ICaL in both species. ICI118551 (ß2-AR antagonist, 0.1 microM), in the presence of NA + CGP20712A, had no significant effect on ICaL in human atrial myocytes, but increased it in rabbit. Yohimbine (α2-AR antagonist, 10 microM), with NA + prazosin, had no significant effect on human or rabbit ICaL. Stimulation of atrial ICaL by NA is mediated, based on AR sub-type antagonist responses, mainly by activating ß1- and α1-ARs in both human and rabbit, with a ß2-inhibitory contribution evident in rabbit, and negligible α2 involvement in either species. This improved understanding of AR sub-type contributions to noradrenergic activation of atrial ICaL could help inform future potential optimisation of pharmacological AR-antagonism strategies for inhibiting adrenergic AF.

Metformin Reduces Potassium Currents and Prolongs Repolarization in Non-Diabetic Heart.

Metformin is the first choice drug for the treatment of type 2 diabetes due to positive results in reducing hyperglycaemia and insulin resistance. However, diabetic patients have higher risk of ventricular arrhythmia and sudden cardiac death, and metformin failed to reduce ventricular arrhythmia in clinical trials. In order to explore the mechanisms responsible for the lack of protective effect, we investigated in vivo the effect of metformin on cardiac electrical activity in non-diabetic rats; and in vitro in isolated ventricular myocytes, HEK293 cells expressing the hERG channel and human induced pluripotent stem cells derived cardiomyocytes (hIPS-CMs). Surface electrocardiograms showed that long-term metformin treatment (7 weeks) at therapeutic doses prolonged cardiac repolarization, reflected as QT and QTc interval duration, and increased ventricular arrhythmia during the caffeine/dobutamine challenge. Patch-clamp recordings in ventricular myocytes isolated from treated animals showed that the cellular mechanism is a reduction in the cardiac transient outward potassium current (Ito). In vitro, incubation with metformin for 24 h also reduced Ito, prolonged action potential duration, and increased spontaneous contractions in ventricular myocytes isolated from control rats. Metformin incubation also reduced IhERG in HEK293 cells. Finally, metformin incubation prolonged action potential duration at 30% and 90% of repolarization in hIPS-CMs, which is compatible with the reduction of Ito and IhERG. Our results show that metformin directly modifies the electrical behavior of the normal heart. The mechanism consists in the inhibition of repolarizing currents and the subsequent decrease in repolarization capacity, which prolongs AP and QTc duration.

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

Prediction of Ventricular Arrhythmias by QRS/QTc - Ratio in Citalopram or Escitalopram Intoxication.

Background: The U.S. Food and Drug Administration (FDA) has stated that citalopram and escitalopram should not be used at daily doses above 40 mg/20 mg due to risk for development of fatal ventricular arrhythmias like torsade de pointes (TdP). Yet, supratherapeutic serum concentrations of citalopram are common and predicting patients at risk for TdP is of high clinical value. Accordingly, we investigated whether QRS/QTc; developed for predicting TdP in hypothermic patients could be used in citalopram intoxication. Methods: A total of 16 publications describing patients suffering from complications due to citalopram or escitalopram treatment, or intoxication with the same substances, were included after a systematic search. The main criterion for inclusion was admission ECG, either with given QRS and QTc values or with attached ECG-files that enabled calculation. Results: QRS/QTc rather that QTc alone emerged as a marker of ventricular arrhythmia in the 16 included case reports, with highly significant (p < 0.0005) lower values in patients displaying ventricular arrhythmias. Conclusion: Citalopram and escitalopram are extensively used in treatment of depressive disorders, and a large proportion of patients have supratherapeutic serum concentrations. Calculation of QRS/QTc in available case reports show that this novel ECG-marker has potential to predict patients at risk for developing ventricular arrhythmias.