CardioMotion: identification of functional and structural cardiotoxic liabilities in small molecules through brightfield kinetic imaging.

Cardiovascular toxicity is an important cause of drug failures in the later stages of drug development, early clinical safety assessment, and even postmarket withdrawals. Early-stage in vitro assessment of potential cardiovascular liabilities in the pharmaceutical industry involves assessment of interactions with cardiac ion channels, as well as induced pluripotent stem cell-derived cardiomyocyte-based functional assays, such as calcium flux and multielectrode-array assays. These methods are appropriate for the identification of acute functional cardiotoxicity but structural cardiotoxicity, which manifests effects after chronic exposure, is often only captured in vivo. CardioMotion is a novel, label-free, high throughput, in vitro assay and analysis pipeline which records and assesses the spontaneous beating of cardiomyocytes and identifies compounds which impact beating. This is achieved through the acquisition of brightfield images at a high framerate, combined with an optical flow-based python analysis pipeline which transforms the images into waveform data which are then parameterized. Validation of this assay with a large dataset showed that cardioactive compounds with diverse known direct functional and structural mechanisms-of-action on cardiomyocytes are identified (sensitivity = 72.9%), importantly, known structural cardiotoxins also disrupt cardiomyocyte beating (sensitivity = 86%) in this method. Furthermore, the CardioMotion method presents a high specificity of 82.5%.

Best practice considerations for nonclinical in vivo cardiovascular telemetry studies in non-rodent species: Delivering high quality QTc data to support ICH E14/S7B Q&As.

The ICH E14/S7B Questions and Answers (Q&As) guideline introduces the concept of a "double negative" nonclinical scenario (negative hERG assay and negative in vivo QTc study) to demonstrate that a drug does not produce a clinically relevant QT prolongation (i.e., no QT liability). This nonclinical "double negative" data package, along with negative Phase 1 clinical QTc data, may be sufficient to substitute for a clinical Thorough QT (TQT) study in some specific cases. While standalone GLP in vivo cardiovascular studies in non-rodent species are standard practice during nonclinical drug development for small molecule programs, a variety of approaches to the design, conduct, analysis and interpretation are utilized across pharmaceutical companies and contract research organizations (CROs) that may, in some cases, negatively impact the stringent sensitivity needed to fulfill the new Q&As. Subject matter experts from both Pharma and CROs have collaborated to recommend best practices for more robust nonclinical cardiovascular telemetry studies in non-rodent species, with input from clinical and regulatory experts. The aim was to increase consistency and harmonization across the industry and to ensure delivery of high quality nonclinical QTc data to meet the proposed sensitivities defined within the revised ICH E14/S7B Q&As guideline (Q&As 5.1 and 6.1). The detailed best practice recommendations presented here cover the design and execution of the safety pharmacology cardiovascular study, including optimal methods for acquiring, analyzing, reporting, and interpreting the resulting QTc and pharmacokinetic data to allow for direct comparison to clinical exposures and assessment of safety margin for QTc prolongation.

Improving the in Vivo QTc assay: The value of implementing best practices to support an integrated nonclinical-clinical QTc risk assessment and TQT substitute.

Recent updates and modifications to the clinical ICH E14 and nonclinical ICH S7B guidelines, which both relate to the evaluation of drug-induced delayed repolarization risk, provide an opportunity for nonclinical in vivo electrocardiographic (ECG) data to directly influence clinical strategies, interpretation, regulatory decision-making and product labeling. This opportunity can be leveraged with more robust nonclinical in vivo QTc datasets based upon consensus standardized protocols and experimental best practices that reduce variability and optimize QTc signal detection, i.e., demonstrate assay sensitivity. The immediate opportunity for such nonclinical studies is when adequate clinical exposures (e.g., supratherapeutic) cannot be safely achieved, or other factors limit the robustness of the clinical QTc evaluation, e.g., the ICH E14 Q5.1 and Q6.1 scenarios. This position paper discusses the regulatory historical evolution and processes leading to this opportunity and details the expectations of future nonclinical in vivo QTc studies of new drug candidates. The conduct of in vivo QTc assays that are consistently designed, executed and analyzed will lead to confident interpretation, and increase their value for clinical QTc risk assessment. Lastly, this paper provides the rationale and basis for our companion article which describes technical details on in vivo QTc best practices and recommendations to achieve the goals of the new ICH E14/S7B Q&As, see Rossman et al., 2023 (this journal).

The incidence of spontaneous arrhythmias in telemetered beagle dogs, Göttingen Minipigs and Cynomolgus non-human primates: A HESI consortium retrospective analysis.

INTRODUCTION: Characterization of the incidence of spontaneous arrhythmias to identify possible drug-related effects is often an important part of the analysis in safety pharmacology studies using telemetry. METHODS: A retrospective analysis in non-clinical species with and without telemetry transmitters was conducted. Electrocardiograms (24 h) from male and female beagle dogs (n = 131), Göttingen minipigs (n = 108) and cynomolgus non-human primates (NHP; n = 78) were analyzed. RESULTS: Ventricular tachycardia (VT) was observed in 3% of the dogs but was absent in minipigs and NHPs. Ventricular fibrillation (VF) was not observed in the 3 species. Ventricular premature beats (VPBs) were more frequent during daytime and atrioventricular blocks (AVBs) were more frequent at night in all species. A limited number of animals exhibited a high arrhythmia frequency and there was no correlation between animals with higher frequency of an arrhythmia type and the frequency of other arrythmias in the same animals. Clinical chemistry or hematology parameters were not different with or without telemetry devices. NHP with a transmural left ventricular pressure (LVP) catheter exhibited a greater incidence of VPBs and PJCs compared to telemetry animals without LVP. DISCUSSION: All species were similar with regards to the frequency of ventricular ectopic beats (26-46%) while the dog seemed to have more frequent junctional complexes and AVB compared to NHP and minipigs. Arrhythmia screening may be considered during pre-study evaluations, to exclude animals with abnormally high arrhythmia incidence.

A novel cardiovascular systems model to quantify drugs effects on the inter-relationship between contractility and other hemodynamic variables.

The use of systems-based pharmacological modeling approaches to characterize mode-of-action and concentration-effect relationships for drugs on specific hemodynamic variables has been demonstrated. Here, we (i) expand a previously developed hemodynamic system model through integration of cardiac output (CO) with contractility (CTR) using pressure-volume loop theory, and (ii) evaluate the contribution of CO data for identification of system-specific parameters, using atenolol as proof-of-concept drug. Previously collected experimental data was used to develop the systems model, and included measurements for heart rate (HR), CO, mean arterial pressure (MAP), and CTR after administration of atenolol (0.3-30 mg/kg) from three in vivo telemetry studies in conscious Beagle dogs. The developed cardiovascular (CVS)-contractility systems model adequately described the effect of atenolol on HR, CO, dP/dtmax, and MAP dynamics and allowed identification of both system- and drug-specific parameters with good precision. Model parameters were structurally identifiable, and the true mode of action can be identified properly. Omission of CO data did not lead to a significant change in parameter estimates compared to a model that included CO data. The newly developed CVS-contractility systems model characterizes short-term drug effects on CTR, CO, and other hemodynamic variables in an integrated and quantitative manner. When the baseline value of total peripheral resistance is predefined, CO data was not required to identify drug- and system-specific parameters. Confirmation of the consistency of system-specific parameters via inclusion of data for additional drugs and species is warranted. Ultimately, the developed model has the potential to be of relevance to support translational CVS safety studies.

Applying the CiPA approach to evaluate cardiac proarrhythmia risk of some antimalarials used off-label in the first wave of COVID-19.

We applied a set of in silico and in vitro assays, compliant with the Comprehensive In Vitro Proarrhythmia Assay (CiPA) paradigm, to assess the risk of chloroquine (CLQ) or hydroxychloroquine (OH-CLQ)-mediated QT prolongation and Torsades de Pointes (TdP), alone and combined with erythromycin (ERT) and azithromycin (AZI), drugs repurposed during the first wave of coronavirus disease 2019 (COVID-19). Each drug or drug combination was tested in patch clamp assays on seven cardiac ion channels, in in silico models of human ventricular electrophysiology (Virtual Assay) using control (healthy) or high-risk cell populations, and in human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. In each assay, concentration-response curves encompassing and exceeding therapeutic free plasma levels were generated. Both CLQ and OH-CLQ showed blocking activity against some potassium, sodium, and calcium currents. CLQ and OH-CLQ inhibited IKr (half-maximal inhibitory concentration [IC50 ]: 1 µM and 3-7 µM, respectively) and IK1 currents (IC50 : 5 and 44 µM, respectively). When combining OH-CLQ with AZI, no synergistic effects were observed. The two macrolides had no or very weak effects on the ion currents (IC50  > 300-1000 µM). Using Virtual Assay, both antimalarials affected several TdP indicators, CLQ being more potent than OH-CLQ. Effects were more pronounced in the high-risk cell population. In hiPSC-derived cardiomyocytes, all drugs showed early after-depolarizations, except AZI. Combining CLQ or OH-CLQ with a macrolide did not aggravate their effects. In conclusion, our integrated nonclinical CiPA dataset confirmed that, at therapeutic plasma concentrations relevant for malaria or off-label use in COVID-19, CLQ and OH-CLQ use is associated with a proarrhythmia risk, which is higher in populations carrying predisposing factors but not worsened with macrolide combination.

Echocardiographic and hemodynamic indices of myocardial contractility simultaneously evaluated in telemetered beagle dogs: A HESI-sponsored cross-company evaluation.

INTRODUCTION: Alterations in cardiac contractility can have significant clinical implications, highlighting the need for early detection of potential liabilities. Pre-clinical methods to assess contractility are typically invasive and their translation to human measures of cardiac function are not well defined. Clinically, cardiac function is most often measured non-invasively using echocardiography. The objective of these studies was to introduce echocardiography into standard large animal cardiovascular safety pharmacology studies and determine the feasibility of this combination. METHODS: A consortia of laboratories combined their data sets for evaluation. At each site, telemetered beagle dogs, in a 4 × 4 Latin square crossover study design (n = 4), were administered either pimobendan (positive inotrope) or atenolol (negative inotrope) orally at clinically relevant dose levels. Standard telemetry parameters were collected (heart rate, mean arterial blood pressure, etc.) continuously over 24 h, as well as derived contractility endpoints: QA interval and LV +dP/dtmax. At Tmax, echocardiography was performed in conscious dogs with minimal restraint to collect contractility parameters: ejection fraction (EF) and fractional shortening (FS). RESULTS: Correlations between telemetry and echo contractility endpoints showed that, in general, a change in LV +dP/dtmax of 1000 mmHg/s translates to a 5.2% change in EF and a 4.2% change in FS. Poor correlations were shown between QA interval derived simultaneously, to both EF and FS. DISCUSSION: Comparing data from telemetry-only groups to those that included echocardiography collections showed no effect in the ability to interpret test article-related effects, providing the foundation for the inclusion of echocardiography without compromising standard telemetry data quality.

A Proof-of-Concept Evaluation of JTPc and Tp-Tec as Proarrhythmia Biomarkers in Preclinical Species: A Retrospective Analysis by an HESI-Sponsored Consortium.

INTRODUCTION: Based on the ICH S7B and E14 guidance documents, QT interval (QTc) is used as the primary in vivo biomarker to assess the risk of drug-induced torsades de pointes (TdP). Clinical and nonclinical data suggest that drugs that prolong the corrected QTc with balanced multiple ion channel inhibition (most importantly the l-type calcium, Cav1.2, and persistent or late inward sodium current, Nav1.5, in addition to human Ether-à-go-go-Related Gene [hERG] IKr or Kv11.1) may have limited proarrhythmic liability. The heart rate-corrected J to T-peak (JTpc) measurement in particular may be considered to discriminate selective hERG blockers from multi-ion channel blockers. METHODS: Telemetry data from Beagle dogs given dofetilide (0.3 mg/kg), sotalol (32 mg/kg), and verapamil (30 mg/kg) orally and Cynomolgus monkeys given medetomidine (0.4 mg/kg) orally were retrospectively analyzed for effects on QTca, JTpca, and T-peak to T-end covariate adjusted (Tpeca) interval using individual rate correction and super intervals (calculated from 0-6, 6-12, 12-18, and 18-24 hours postdose). RESULTS: Dofetilide and cisapride (IKr or Kv11.1 blockers) were associated with significant increases in QTca and JTpca, while sotalol was associated with significant increases in QTca, JTpca, and Tpeca. Verapamil (a Kv11.1 and Cav1.2 blocker) resulted in a reduction in QTca and JTpca, however, and increased Tpeca. Medetomidine was associated with a reduction in Tpeca and increase in JTpca. DISCUSSION: Results from this limited retrospective electrocardiogram analysis suggest that JTpca and Tpeca may discriminate selective IKr blockers and multichannel blockers and could be considered in the context of an integrated comprehensive proarrhythmic risk assessment.

An evaluation of the utility of LVdP/dt40, QA interval, LVdP/dtmin and Tau as indicators of drug-induced changes in contractility and lusitropy in dogs.

INTRODUCTION: The importance of drug-induced effects on the inotropic state of the heart is well known. Unlike hemodynamic and cardiac electrophysiological methods, which have been routinely used in drug safety testing for years, the non-clinical assessment of drug effects on myocardial contractility is used less frequently with no established translation to humans. The goal of these studies was to determine whether assessment of alternate measures of cardiac inotropy could detect drug-induced changes in the contractile state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. This study also evaluated drug-induced effects on lusitropy (relaxation) parameters of the heart. METHODS: A double 4×4 Latin square study design using Beagle dogs (n=8) was conducted. Drugs were administrated orally. Arterial blood pressure (BP), left ventricular pressure (LVP) and the electrocardiogram (ECG) were assessed across different laboratories using the same protocol. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control). Animals were instrumented with an ITS telemetry system or DSI's D70-PCTP or PhysioTel™ Digital system. The data acquisition and analysis systems used were Ponemah, Notocord or EMKA. RESULTS: The derived inotropic and lusitropic parameters evaluated included peak systolic and end diastolic LVP, LVdP/dtmax, LVdP/dt40, QA interval, LVdP/dtmin and Tau. This study showed that LVdP/dt40 provided essentially identical results to LVdP/dtmax qualifying it as an index to assess drug effects on cardiac contractility. LVdP/dt40 provided an essentially identical assessment to that of LVdP/dtmax. The QA interval did not react sensitively to the drugs tested in this study; however, it did detect large effects and could be useful in early cardiovascular safety studies. The lusitropic parameter, LVdP/dtmin, was modestly decreased, and Tau was increased, by atenolol and itraconazole. At the doses tested, amrinone and pimobendan produced no changes in LVdP/dtmin while Tau was modestly increased. The drugs did not produce effects on BP, HR or the ECG at the doses tested. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. DISCUSSION: These findings indicate that this experimental model can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems. While LVdP/dt40 produced responses similar to LVdP/dtmax, the QA interval and lusitropic parameters LVdP/dtmin and Tau were not markedly changed at the dose of drugs tested. Further studies with drugs that affect early diastolic relaxation through calcium handling are needed to better evaluate drug-induced changes on lusitropic properties of the heart.

Evaluation of a method utilizing PhysioFlow®, a novel signal morphology-based form of impedance cardiography, to measure cardiac output in the conscious beagle.

INTRODUCTION: Currently, standard methods for measuring cardiac output are either invasive (i.e. flow probe) or are limited in terms of short measurement intervals and measurement variability (i.e. echocardiography). The ability to reliably measure cardiac output in a non-invasive manner in large animals would provide a valuable tool to expand functional cardiovascular endpoints in preclinical safety studies. PhysioFlow® is a novel method that uses waveform analysis of an impedance signal to measure cardiac output non-invasively. Unlike cardiac impedance techniques in the past, PhysioFlow® is not dependant on thoracic structure or basal thoracic impedance (Z0) and therefore this methodology is transferrable from human to animal models. METHODS: Three tool compounds with known effects on cardiac output were administered to conscious beagle dogs to determine if the non-invasive PhysioFlow® system could detect the expected changes in stroke volume and cardiac output as determined by literature references using the current standard methodologies (e.g. aortic blood flow and thermodilution). RESULTS: The PhysioFlow® system was able to detect increases in cardiac output when dosed with 20µg/kg of Dobutamine, a decrease in cardiac output when dosed with 0.1mg/kg of Acepromazine, and no significant change in cardiac output when dosed with 2mg/kg of Minoxidil. These results are within expected ranges based on published literature (Stepien et al., 1995; Taylor et al., 2007). DISCUSSION: PhysioFlow®, a signal morphology-based impedance cardiography, can be utilized to reliably and non-invasively measure cardiac output in beagle dogs.

The evaluation of drug-induced changes in cardiac inotropy in dogs: Results from a HESI-sponsored consortium.

INTRODUCTION: Drug-induced effects on the cardiovascular system remain a major cause of drug attrition. While hemodynamic (blood pressure (BP) and heart rate (HR)) and electrophysiological methods have been used in testing drug safety for years, animal models for assessing myocardial contractility are used less frequently and their translation to humans has not been established. The goal of these studies was to determine whether assessment of contractility and hemodynamics, when measured across different laboratories using the same protocol, could consistently detect drug-induced changes in the inotropic state of the heart using drugs known to have clinically relevant positive and negative effects on myocardial contractility. METHODS: A 4×4 double Latin square design (n=8) design using Beagle dogs was developed. Drugs were administrated orally. Arterial blood pressure, left ventricular pressure (LVP) and the electrocardiogram were assessed. Each of the six laboratories studied at least 2 drugs (one positive inotrope (pimobendan or amrinone) and one negative inotrope) (itraconazole or atenolol) at 3 doses selected to match clinical exposure data and a vehicle control. Animals were instrumented with an ITS telemetry system, DSI's D70-PCTP system or DSI's Physiotel Digital system. Data acquisition and analysis systems were Ponemah, Notocord or EMKA. RESULTS: Derived parameters included: diastolic, systolic and mean arterial BP, peak systolic LVP, HR, end-diastolic LVP, and LVdP/dtmax as the primary contractility index. Blood samples were drawn to confirm drug exposures predicted from independent pharmacokinetic studies. Across the laboratories, a consistent change in LVdP/dtmax was captured despite some differences in the absolute values of some of the hemodynamic parameters prior to treatment. DISCUSSION: These findings indicate that this experimental model, using the chronically instrumented conscious dog, can accurately and consistently detect changes in cardiac contractility, across multiple sites and instrumentation systems, and that data obtained in this model may also translate to clinical outcomes.

Effects of chronic gap junction conduction-enhancing antiarrhythmic peptide GAP-134 administration on experimental atrial fibrillation in dogs.

BACKGROUND: Abnormal intercellular communication caused by connexin dysfunction may contribute to atrial fibrillation (AF). The present study assessed the effect of the gap junction conduction-enhancing antiarrhythmic peptide GAP-134 on AF inducibility and maintenance in a dog model of atrial cardiomyopathy. METHODS AND RESULTS: Twenty-four dogs subject to simultaneous atrioventricular pacing (220 bpm for 14 days) were randomly assigned to placebo treatment (PACED-CTRL; 12 dogs) or oral GAP-134 (2.9 mg/kg BID; PACED-GAP-134; 12 dogs) starting on day 0. UNPACED-CTRL (4 dogs) and UNPACED-GAP-134 (4 dogs) served as additional control groups. Change in left atrial (LA) systolic area from baseline to 14 days was calculated using transoesophageal echocardiography. At 14 days, animals underwent an open-chest electrophysiological study. PACED-CTRL dogs (versus UNPACED-CTRL) had a shorter estimated LA wavelength (8.0+/-1.4 versus 24.4+/-2.5 cm, P<0.05) and a greater AF vulnerability (mean AF duration, 1588+/-329 versus 25+/-34 seconds, P<0.05). Oral GAP-134 had no effect on AF vulnerability in UNPACED dogs. Compared with PACED-CTRL dogs, PACED-GAP-134 dogs had a longer estimated LA wavelength (10.2+/-2.8 versus 8.0+/-1.4 cm, respectively, P<0.05). Oral GAP-134 did not significantly reduce AF inducibility or maintenance in the entire group of 24 PACED dogs; in a subgroup of dogs (n=11) with less than 100% increase in LA systolic area, oral GAP-134 reduced AF induction from 100% to 40% and mean AF duration from 1737+/-120 to 615+/-280 seconds (P<0.05). CONCLUSIONS: Oral GAP-134 reduces pacing-induced decrease in LA wavelength and appears to attenuate AF vulnerability in dogs with less atrial mechanical remodeling. Gap junction modulation may affect AF in some circumstances.

GAP-134 ([2S,4R]-1-[2-aminoacetyl]4-benzamidopyrrolidine-2-carboxylic acid) prevents spontaneous ventricular arrhythmias and reduces infarct size during myocardial ischemia/reperfusion injury in open-chest dogs.

The antiarrhythmic dipeptide, GAP-134, ([2S,4R]-1[2-aminoacetyl]-4-benzamido-pyrrolidine-2-carboxylic acid) was evaluated in canine ischemia/reperfusion model. In dogs subjected to 60-minute ischemia and 4-hour reperfusion, GAP-134 was administered 10 minutes before reperfusion as a bolus + intravenous (IV) infusion. The doses administered were 0.25 microg/kg bolus + 0.19 microg/kg per hour infusion; 2.5 microg/kg + 1.9 microg/kg per hour; 25 mg/kg + 19 mg/kg per hour; 75 mg/kg + 57 mg/kg per hour. Ventricular ectopy was quantified during reperfusion, including premature ventricular contractions (PVC) and ventricular tachycardia (VT). Total incidence of VT was reduced significantly with the 2 highest doses of GAP-134 (1.7 + 0.8; 2.2 + 1.4 events; P < .05) compared to controls (23.0 + 6.1). Total PVCs were reduced significantly from 11.1 + 1.6% in control animals to 2.0% + 0.7% and 1.8% + 0.8% after the 2 highest doses of GAP-134. Infarct size, expressed as percentage of left ventricle, was reduced significantly from 19.0% + 3.5% in controls to 7.9% + 1.5% and 7.1% + 0.8% (P < .05) at the 2 highest doses of GAP-134. GAP-134 is an effective antiarrhythmic agent with potential to reduce ischemia/reperfusion injury.

Discovery of a class of potent gap-junction modifiers as novel antiarrhythmic agents.

In an effort to discover potent, orally bioavailable compounds for the treatment of atrial fibrillation (AF) and ventricular tachycardia (VT), we developed a class of gap-junction modifiers typified by GAP-134 (1, R(1)=OH, R(2)=NH(2)), a compound currently under clinical evaluation. Selected compounds with the desired in-vitro profile demonstrated positive in vivo results in the mouse CaCl(2) arrhythmia model upon oral administration.

The gap junction modifier, GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], improves conduction and reduces atrial fibrillation/flutter in the canine sterile pericarditis model.

Gap junction uncoupling can alter conduction pathways and promote cardiac re-entry mechanisms that potentiate many supraventricular arrhythmias, such as atrial fibrillation (AF) and atrial flutter (AFL). Our objective was to determine whether GAP-134 [(2S,4R)-1-(2-aminoacetyl)-4-benzamido-pyrrolidine-2-carboxylic acid], a small dipeptide gap junction modifier, can improve conduction and ultimately prevent AF/AFL. In rat atrial strips subjected to metabolic stress, GAP-134 prevented significantly conduction velocity slowing at 10 nM compared with vehicle (p < 0.01). In the canine sterile pericarditis model, conduction time (CT; n = 5), atrial effective refractory period (AERP; n = 3), and AF/AFL duration/inducibility (n = 16) were measured 2 to 3 days postoperatively in conscious dogs. CT was significantly faster after GAP-134 infusion (average plasma concentration, 250 nM) at cycle lengths of 300 ms (66.2 +/- 1.0 versus 62.0 +/- 1.0 ms; p < 0.001) and 200 ms (64.4 +/- 0.9 versus 61.0 +/- 1.3 ms; p < 0.001). No significant changes in AERP were noted after GAP-134 infusion. The mean number of AF/AFL inductions per animal was significantly decreased after GAP-134 infusion (2.7 +/- 0.6 versus 1.6 +/- 0.8; p < 0.01), with total AF/AFL burden being decreased from 12,280 to 6063 s. Western blot experiments showed no change in connexin 43 expression. At concentrations exceeding those described in the AF/AFL experiments, GAP-134 had no effect on heart rate, blood pressure, or any electrocardiogram parameters. In conclusion, GAP-134 shows consistent efficacy on measures of conduction and AF/AFL inducibility in the canine sterile pericarditis model. These findings, along with its oral bioavailability, underscore its potential antiarrhythmic efficacy.

Discovery of (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid hydrochloride (GAP-134)13, an orally active small molecule gap-junction modifier for the treatment of atrial fibrillation.

Rotigaptide (3) is an antiarrhythmic peptide that improves cardiac conduction by modifying gap-junction communication. Small molecule gap-junction modifiers with improved physical properties were identified from a Zealand Pharma peptide library using pharmaceutical profiling, established SAR around 3, and a putative pharmacophore model for rotigaptide. Activity of the compounds was confirmed in a mouse cardiac conduction block model of arrhythmia. Dipeptide 9f (GAP-134) was identified as a potent, orally active gap-junction modifier for clinical development.

Reduced sarcoplasmic reticulum Ca(2+) load mediates impaired contractile reserve in right ventricular pressure overload.

Myocardial contractile reserve is significantly attenuated in patients with advanced heart failure. The aim of this study was to identify mechanisms of impaired contractile reserve in a large animal model that closely mimics human myocardial failure. Progressive right ventricular hypertrophy and failure were induced by banding the pulmonary artery in kittens. Isometric contractile force was measured in right ventricular trabeculae (n=115) from age-matched Control and Banded feline hearts. Rapid cooling contractures (RCC) were used to determine sarcoplasmic reticulum (SR) Ca(2+) load while assessing the ability of changes in rate, adrenergic stimulation and bath Ca(2+) to augment contractility. The positive force-frequency relationship and robust pre- and post-receptor adrenergic responses observed in Control trabeculae were closely paralleled by increases in RCC amplitude and the RCC2/RCC1 ratio. Conversely, the severely blunted force-frequency and adrenergic responses in Banded trabeculae were paralleled by an unchanged RCC amplitude and RCC2/RCC1 ratio. Likewise, supraphysiologic levels of bath Ca(2+) were associated with severely reduced contractility and RCC amplitude in Banded trabeculae compared to Controls. There were no differences in myofilament Ca(2+) sensitivity or length-dependent increases in contractility between Control and Banded trabeculae. There was a significant decrease in SR Ca(2+)-ATPase pump abundance and phosphorylation of phospholamban and ryanodine receptor in Banded trabeculae compared with Controls. A reduced ability to increase SR Ca(2+) load is the primary mechanism of reduced contractile reserve in failing feline myocardium. The similarity of impaired contractile reserve phenomenology in this feline model and transplanted hearts suggests mechanistic relevance to human myocardial failure.

Sex-based differences in myocardial contractile reserve.

Recent studies have identified sex differences in heart function that may affect the risk of developing heart failure. We hypothesized that there are fundamental differences in calcium (Ca) regulation in cardiac myocytes of males and premenopausal females. Isometric force transients (n = 45) were measured at various stimulation frequencies to define the force frequency responses (FFR) (0.5, 1.0, 1.5, and 2.0 Hz) during either changes in bath Ca ([Ca]o) (1.0, 1.75, 3.5, and 7.0 mM) or length-tension (20, 40, 60, 80, and 100% L(max)) in right ventricle trabeculae from normal male (MT) and premenopausal female (FT) cats. Force-Ca measurements were also obtained in chemically skinned trabeculae. Under basal conditions (0.5 Hz, 1.75 mM Ca, 80% L(max)) both MT and FT achieved similar developed forces (DF) (MT 11 +/- 1, FT = 10 +/- 1 mN/mm2). At low rates and lengths, there is no sex difference. At higher preloads and rates, there is a separation in DF in MT and FT. At basal [Ca]o both MT and FT exhibited positive FFR (2.0 Hz, 1.75 mM Ca: MT 38 +/- 3, FT 21 +/- 4 mN/mm2); however, at higher [Ca]o, MT achieved greater DF (2.0 Hz, 7.0 mM Ca: MT 40 +/- 3 and FT = 24 +/- 4 mN/mm2). We detected no sex difference in myofilament Ca sensitivity at a sarcomere length of 2.1 mum. However, rapid cooling contractures indicated greater sarcoplasmic reticulum (SR) Ca load in MT at higher frequencies. Despite virtually identical contractile performance under basal conditions, significant sex differences emerge under conditions of increased physiological stress. Given the lack of sex differences in myofilament Ca sensitivity, these studies suggest fundamental sex differences in cellular Ca regulation to achieve contractile reserve, with myocardium from males exhibiting higher SR Ca load.

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart.

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.