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%.

Cardiac sodium channel inhibition by lamotrigine: In vitro characterization and clinical implications.

Lamotrigine, approved for use as an antiseizure medication as well as the treatment of bipolar disorder, inhibits sodium channels in the brain to reduce repetitive neuronal firing and pathological release of glutamate. The shared homology of sodium channels and lack of selectivity associated with channel blocking agents can cause slowing of cardiac conduction and increased proarrhythmic potential. The Vaughan-Williams classification system differentiates sodium channel blockers using biophysical properties of binding. As such, Class Ib inhibitors, including mexiletine, do not slow cardiac conduction as measured by the electrocardiogram, at therapeutically relevant exposure. Our goal was to characterize the biophysical properties of NaV 1.5 block and to support the observed clinical safety of lamotrigine. We used HEK-293 cells stably expressing the hNaV 1.5 channel and voltage clamp electrophysiology to quantify the potency (half-maximal inhibitory concentration) against peak and late channel current, on-/off-rate binding kinetics, voltage-dependence, and tonic block of the cardiac sodium channel by lamotrigine; and compared to clinically relevant Class Ia (quinidine), Ib (mexiletine), and Ic (flecainide) inhibitors. Lamotrigine blocked peak and late NaV 1.5 current at therapeutically relevant exposure, with rapid kinetics and biophysical properties similar to the class Ib inhibitor mexiletine. However, no clinically meaningful prolongation in QRS or PR interval was observed in healthy subjects in a new analysis of a previously reported thorough QT clinical trial (SCA104648). In conclusion, the weak NaV 1.5 block and rapid kinetics do not translate into clinically relevant conduction slowing at therapeutic exposure and support the clinical safety of lamotrigine in patients suffering from epilepsy and bipolar disorder.

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.

Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective.

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Publisher Correction: Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction to Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].

Cross-site and cross-platform variability of automated patch clamp assessments of drug effects on human cardiac currents in recombinant cells.

Automated patch clamp (APC) instruments enable efficient evaluation of electrophysiologic effects of drugs on human cardiac currents in heterologous expression systems. Differences in experimental protocols, instruments, and dissimilar site procedures affect the variability of IC50 values characterizing drug block potency. This impacts the utility of APC platforms for assessing a drug's cardiac safety margin. We determined variability of APC data from multiple sites that measured blocking potency of 12 blinded drugs (with different levels of proarrhythmic risk) against four human cardiac currents (hERG [IKr], hCav1.2 [L-Type ICa], peak hNav1.5, [Peak INa], late hNav1.5 [Late INa]) with recommended protocols (to minimize variance) using five APC platforms across 17 sites. IC50 variability (25/75 percentiles) differed for drugs and currents (e.g., 10.4-fold for dofetilide block of hERG current and 4-fold for mexiletine block of hNav1.5 current). Within-platform variance predominated for 4 of 12 hERG blocking drugs and 4 of 6 hNav1.5 blocking drugs. hERG and hNav1.5 block. Bland-Altman plots depicted varying agreement across APC platforms. A follow-up survey suggested multiple sources of experimental variability that could be further minimized by stricter adherence to standard protocols. Adoption of best practices would ensure less variable APC datasets and improved safety margins and proarrhythmic risk assessments.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Drug-Induced Inotropic Effects in Early Drug Development. Part 2: Designing and Fabricating Microsystems for Assaying Cardiac Contractility With Physiological Relevance Using Human iPSC-Cardiomyocytes.

Contractility of the myocardium engines the pumping function of the heart and is enabled by the collective contractile activity of its muscle cells: cardiomyocytes. The effects of drugs on the contractility of human cardiomyocytes in vitro can provide mechanistic insight that can support the prediction of clinical cardiac drug effects early in drug development. Cardiomyocytes differentiated from human-induced pluripotent stem cells have high potential for overcoming the current limitations of contractility assays because they attach easily to extracellular materials and last long in culture, while having human- and patient-specific properties. Under these conditions, contractility measurements can be non-destructive and minimally invasive, which allow assaying sub-chronic effects of drugs. For this purpose, the function of cardiomyocytes in vitro must reflect physiological settings, which is not observed in cultured cardiomyocytes derived from induced pluripotent stem cells because of the fetal-like properties of their contractile machinery. Primary cardiomyocytes or tissues of human origin fully represent physiological cellular properties, but are not easily available, do not last long in culture, and do not attach easily to force sensors or mechanical actuators. Microengineered cellular systems with a more mature contractile function have been developed in the last 5 years to overcome this limitation of stem cell-derived cardiomyocytes, while simultaneously measuring contractile endpoints with integrated force sensors/actuators and image-based techniques. Known effects of engineered microenvironments on the maturity of cardiomyocyte contractility have also been discovered in the development of these systems. Based on these discoveries, we review here design criteria of microengineered platforms of cardiomyocytes derived from pluripotent stem cells for measuring contractility with higher physiological relevance. These criteria involve the use of electromechanical, chemical and morphological cues, co-culture of different cell types, and three-dimensional cellular microenvironments. We further discuss the use and the current challenges for developing and improving these novel technologies for predicting clinical effects of drugs based on contractility measurements with cardiomyocytes differentiated from induced pluripotent stem cells. Future research should establish contexts of use in drug development for novel contractility assays with stem cell-derived cardiomyocytes.

Engineered Cardiac Tissues Generated in the Biowire II: A Platform for Human-Based Drug Discovery.

Recent advances in techniques to differentiate human induced pluripotent stem cells (hiPSCs) hold the promise of an unlimited supply of human derived cardiac cells from both healthy and disease populations. That promise has been tempered by the observation that hiPSC-derived cardiomyocytes (hiPSC-CMs) typically retain a fetal-like phenotype, raising concern about the translatability of the in vitro data obtained to drug safety, discovery, and development studies. The Biowire II platform was used to generate 3D engineered cardiac tissues (ECTs) from hiPSC-CMs and cardiac fibroblasts. Long term electrical stimulation was employed to obtain ECTs that possess a phenotype like that of adult human myocardium including a lack of spontaneous beating, the presence of a positive force-frequency response from 1 to 4 Hz and prominent postrest potentiation. Pharmacology studies were performed in the ECTs to confirm the presence and functionality of pathways that modulate cardiac contractility in humans. Canonical responses were observed for compounds that act via the ß-adrenergic/cAMP-mediated pathway, eg, isoproterenol and milrinone; the L-type calcium channel, eg, FPL64176 and nifedipine; and indirectly effect intracellular Ca2+ concentrations, eg, digoxin. Expected positive inotropic responses were observed for compounds that modulate proteins of the cardiac sarcomere, eg, omecamtiv mecarbil and levosimendan. ECTs generated in the Biowire II platform display adult-like properties and have canonical responses to cardiotherapeutic and cardiotoxic agents that affect contractility in humans via a variety of mechanisms. These data demonstrate that this human-based model can be used to assess the effects of novel compounds on contractility early in the drug discovery and development process.

Considerations for an In Vitro, Cell-Based Testing Platform for Detection of Adverse Drug-Induced Inotropic Effects in Early Drug Development. Part 1: General Considerations for Development of Novel Testing Platforms.

Drug-induced effects on cardiac contractility can be assessed through the measurement of the maximal rate of pressure increase in the left ventricle (LVdP/dtmax) in conscious animals, and such studies are often conducted at the late stage of preclinical drug development. Detection of such effects earlier in drug research using simpler, in vitro test systems would be a valuable addition to our strategies for identifying the best possible drug development candidates. Thus, testing platforms with reasonably high throughput, and affordable costs would be helpful for early screening purposes. There may also be utility for testing platforms that provide mechanistic information about how a given drug affects cardiac contractility. Finally, there could be in vitro testing platforms that could ultimately contribute to the regulatory safety package of a new drug. The characteristics needed for a successful cell or tissue-based testing platform for cardiac contractility will be dictated by its intended use. In this article, general considerations are presented with the intent of guiding the development of new testing platforms that will find utility in drug research and development. In the following article (part 2), specific aspects of using human-induced stem cell-derived cardiomyocytes for this purpose are addressed.

Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 µM).

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual KV7 isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

Cardiotoxicity screening with simultaneous optogenetic pacing, voltage imaging and calcium imaging.

INTRODUCTION: The Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative seeks an in vitro test to accurately predict clinical Torsades de Pointes (TdP). We developed a cardiotoxicity assay incorporating simultaneous measurement of the action potential (AP) waveform and Ca(2+) transient (CT) in human iPSC-derived cardiomyocytes (CMs). Concurrent optogenetic pacing provided a well-controlled electrophysiological background. METHODS: We used the Optopatch platform for all-optical electrophysiology (Hochbaum et al., 2014). In a monolayer culture, a subset of cells expressed a genetically encoded, calcium and voltage reporter, CaViar (Hou, Kralj, Douglass, Engert, & Cohen, 2014), while others expressed a channelrhodopsin variant, CheRiff. Optical pacing of CheRiff-expressing cells synchronized the syncytium. We screened 12 compounds (11 acute, 1 chronic) to identify electrophysiological (AP rise time, AP50, AP90, beat rate) and CT effects in spontaneously beating and paced cultures (1Hz, 2Hz). RESULTS: CaViar reported spontaneous and paced APs and CTs with high signal-to-noise ratio and low phototoxicity. Quinidine, flecainide, E-4031, digoxin and cisapride prolonged APs, while verapamil and nifedipine shortened APs. Early after depolarizations (EADs) were elicited by quinidine, flecainide and cisapride. All but four compounds (amiodarone, chromanol, nifedipine, verapamil) prolonged AP rise time. Nifedipine and verapamil decreased CT amplitude, while digoxin increased CT amplitude. Pentamidine prolonged APs after chronic exposure. DISCUSSION: The Optopatch platform provides a robust assay to measure APs and CTs in hiPSC-CMs. This addresses the CiPA mandate and will facilitate comparisons of cell-based assays to human clinical data.

A New Perspective in the Field of Cardiac Safety Testing through the Comprehensive In Vitro Proarrhythmia Assay Paradigm.

For the past decade, cardiac safety screening to evaluate the propensity of drugs to produce QT interval prolongation and Torsades de Pointes (TdP) arrhythmia has been conducted according to ICH S7B and ICH E14 guidelines. Central to the existing approach are hERG channel assays and in vivo QT measurements. Although effective, the present paradigm carries a risk of unnecessary compound attrition and high cost, especially when considering costly thorough QT (TQT) studies conducted later in drug development. The C: omprehensive I: n Vitro P: roarrhythmia A: ssay (CiPA) initiative is a public-private collaboration with the aim of updating the existing cardiac safety testing paradigm to better evaluate arrhythmia risk and remove the need for TQT studies. It is hoped that CiPA will produce a standardized ion channel assay approach, incorporating defined tests against major cardiac ion channels, the results of which then inform evaluation of proarrhythmic actions in silico, using human ventricular action potential reconstructions. Results are then to be confirmed using human (stem cell-derived) cardiomyocytes. This perspective article reviews the rationale, progress of, and challenges for the CiPA initiative, if this new paradigm is to replace existing practice and, in time, lead to improved and widely accepted cardiac safety testing guidelines.

Evaluation of the rubidium efflux assay for preclinical identification of HERG blockade.

Inhibition of the delayed-rectifier potassium channel current, human ether-a-go-go (hERG), by pharmaceutical agents can lead to acquired long QT syndrome and the generation of potentially lethal arrhythmias and sudden death. There remains an unmet need for higher-throughput assays to screen compounds in preclinical development for the potential to block hERG and cause QT prolongation. We evaluated the rubidium efflux assay for its ability to determine block of the hERG potassium channel. hERG-transfected human embryonic kidney-293 cells were cultured on 96-well assay plates and loaded with rubidium ion by incubating in media in which potassium was replaced by 5.4 mM Rb+. Cells were exposed to test compounds and then depolarized with a K+ channel opening buffer containing 50 mM K+. The supernatant was removed, and cells were lysed using 0.1% Triton X-100. Concentration-response curves were generated for test agents by determining the Rb+ efflux using a flame atomic absorption spectrometer. Multiple trials with cisapride yielded 50% inhibitory concentration values between 308.1 +/- 11 nM to 456.3 +/- 24 nM for inhibition of Rb+ efflux and a Z factor of 0.80 +/- 0.07 (n = 5 plates, 12 wells per plate). The values for inhibition of the hERG channel exhibited a rightward shift in potency as compared to those measured using electrophysiological techniques. In addition, we evaluated 19 blinded compounds at 10 microM in the Rb+ efflux assay, and compared results to those using patch clamp electrophysiology and the dofetilide displacement binding assay. The dofetilide displacement binding assay yielded a good correlation with electrophysiological measurements of hERG block. The rubidium efflux assay lacked sensitivity to consistently identify significant channel blockade. In conclusion, the rubidium efflux assay provides a higher-throughput means to identify potent hERG channel blocking agents, but lacks the sensitivity required to accurately determine the potency of blockade.

Embryonic stem cells in predictive cardiotoxicity: laser capture microscopy enables assay development.

Embryonic stem (ES) cells offer unprecedented opportunities for in vitro drug discovery and safety assessment of compounds. Cardiomyocytes derived from ES cells enable development of predictive cardiotoxicity models to increase the safety of novel drugs. Heterogeneity of differentiated ES cells limits the development of reliable in vitro models for compound screening. We report an innovative and robust approach to isolate ES-derived cardiomyocytes using laser microdissection and pressure catapulting (LMPC). LMPC cells were readily applied onto 96-well format in vitro pharmacology assays. The expression of developmental and functional cardiac markers, Nkx 2.5, MLC2V, GATA-4, Connexin 43, Connexin 45, Serca-2a, cardiac alpha actin, and phospholamban, among others, was confirmed in LMPC ES-derived cardiomyocytes. Functional assays exhibited cardiac-like response to increased extracellular calcium (5.4 mM extracellular Ca2+) and L-type calcium channel antagonist (1 microM nifedipine). In conclusion, laser microdissection and pressure catapulting is a robust technology to isolate homogeneous ES-derived cell types from heterogeneous populations applicable to assay development.

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.

Abnormal frequency-dependent responses represent the pathophysiologic signature of contractile failure in human myocardium.

BACKGROUND: - The normal increase in isometric developed force (DF) with faster pacing rates, known as the positive force-frequency response/relationship (FFR), is altered in failing myocardium, as shown by its negative response to increased pacing. The objective of this study was to determine if increasing Ca2+ influx with L-type Ca2+ channel (L-CaCh) agonists: BayK 8644 (BayK) and FPL 64176 (FPL) or increased extracellular Ca2+ could increase contractility and normalize the FFR in failing myocardium. METHODS: - Isometric DF was measured in right ventricular trabeculae from failing (n = 28) and non-failing (n = 12) human hearts at various stimulation frequencies (0.5-2.5 Hz) before and after bath application of BayK (250 nM), FPL (100 nM), or high Ca2+ (7.0 mM). Post-rest (PR) experiments were also conducted on several trabeculae. RESULTS: - In trabeculae from failing hearts, the DF decreased with an increase in pacing. Addition of L-CaCh agonists increased DF to similar levels in trabeculae from both failing and non-failing hearts at slow pacing rates, but did not alter the negative FFR in the failing group. During increased rest intervals, the amount of PR potentiation was diminished in trabeculae from failing hearts as compared to the non-failing preparations. CONCLUSION: - This study demonstrates that the abnormal FFR observed in trabeculae from failing hearts is a reliable physiologic signature of the cardiomyopathic state even when DF, at slow stimulation frequencies, is relatively high. These studies further demonstrate that the impaired FFR is not due to an inability to further increase contractility. Rather, our findings suggest that the abnormal FFR and blunted PR potentiation alike are a reflection of an altered functional balance between Ca2+ re-uptake and Ca2+ extrusion.