Assessment of sarcomere shortening and calcium transient in primary human and dog ventricular myocytes.

Understanding translation from preclinical observations to clinical findings is important for evaluating the efficacy and safety of novel compounds. Of relevance to cardiac safety is profiling drug effects on cardiomyocyte (CM) sarcomere shortening and intracellular Ca dynamics. Although CM from different animal species have been used to assess such effects, primary human CM isolated from human organ donor heart represent an ideal non-animal alternative approach.

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect).

Validation of using cardioplegic solutions for preserving cardiac function in isolated rabbit heart assays.

Introduction: Cardioplegic solutions were first developed to preserve heart function during cardiac surgeries and heart transplants but have application in the nonclinical setting. Due to lack of lab space in the vivarium, cardioplegic solution was used to conserve cardiac function for ex-vivo studies performed in a separate building. All studies in this report were conducted with isolated female rabbit hearts (IRHs) via retrograde perfusion using the Langendorff apparatus to investigate if cardioplegia usage affects cardiac function.

Comprehensive in vitro pro-arrhythmic assays demonstrate that omecamtiv mecarbil has low pro-arrhythmic risk.

Omecamtiv mecarbil (OM) is a myosin activator (myotrope), developed as a potential therapeutic agent for heart failure with reduced ejection fraction. To characterize the potential pro-arrhythmic risk of this novel sarcomere activator, we evaluated OM in a series of International Conference on Harmonization S7B core and follow-up assays, including an in silico action potential (AP) model. OM was tested in: (i) hERG, Nav1.

Effects of omecamtiv mecarbil on calcium-transients and contractility in a translational canine myocyte model.

Omecamtiv mecarbil (OM) is a selective cardiac myosin activator (myotrope), currently in Phase 3 clinical investigation as a novel treatment for heart failure with reduced ejection fraction. OM increases cardiac contractility by enhancing interaction between myosin and actin in a calcium-independent fashion. This study aims to characterize the mechanism of action by evaluating its simultaneous effect on myocyte contractility and calcium-transients (CTs) in healthy canine ventricular myocytes.

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF.

Decreased contractility and altered responses to inotropic agents in myocytes from tachypacing-induced heart failure canines.

Contractility measurements using primary isolated cardiac myocytes (CM) have commonly been used in understanding the physiology and pharmacology of cellular mechanics. In the majority of studies, CM from healthy animals were used, and fewer studies were performed with CM from diseased hearts. To better understand the translational value of contractility on the cellular level of a diseased animal model, myocytes were isolated from left ventricles of a tachypacing-induced heart failure (HF) canine model, and their contractility was measured by recording sarcomere shortening using an image-based IonOptix video system.

Oxytocin does not directly alter cardiac repolarization in rabbit or human cardiac myocytes.

Oxytocin, a nine amino acid peptide, is highly conserved in placental mammals, including humans. Oxytocin has a physiological role in parturition and parenteral administration of the synthetic peptide is used to induce labor and control postpartum hemorrhage. Endogenous levels of oxytocin before labor are ∼20 pg/mL, but pharmacological administration of the peptide can achieve levels of 110 pg/mL (0.

Human embryonic stem cell derived cardiac myocytes detect hERG-mediated repolarization effects, but not Nav1.5 induced depolarization delay.

Introduction: Cardiac safety is of paramount importance in contemporary drug development. Efficient and sensitive evaluation of cardiac safety in the research and development of new molecular agents begins with preclinical in-vitro models. A new model that is currently under evaluation is the human embryonic stem-cell derived cardiac myocytes (hESC-CM) (Peng, Lacerda, Kirsch, Brown, & Bruening-Wright, 2010).

Itraconazole decreases left ventricular contractility in isolated rabbit heart: mechanism of action.

Itraconazole (ITZ) is an approved antifungal agent that carries a "black box warning" in its label regarding a risk of negative cardiac inotropy based on clinical findings. Since the mechanism of the negative inotropic effect is unknown, we performed a variety of preclinical and mechanistic studies to explore the pharmacological profile of ITZ and understand the negative inotropic mechanism. ITZ was evaluated in: (1) an isolated rabbit heart (IRH) preparation using Langendorff retrograde perfusion; (2) ion channel studies; (3) a rat heart mitochondrial function profiling screen; (4) a mitochondrial membrane potential (MMP) assay; (5) in vitro pharmacology profiling assays (148 receptors, ion channels, transporters, and enzymes); and (6) a kinase selectivity panel (451 kinases).

BeKm-1, a peptide inhibitor of human ether-a-go-go-related gene potassium currents, prolongs QTc intervals in isolated rabbit heart.

Drug-induced cardiac arrhythmia, specifically Torsades de pointes, is associated with QT/QTc interval prolongation, thus prolongation of the QT interval is considered as a biomarker for Torsades de pointes risk (N Engl J Med 350:1013-1022, 2004). Specific inhibition of human ether-a-go-go-related gene (hERG) potassium channels has been recognized as the main mechanism for QT prolongation (Cardiovasc Res 58:32-45, 2003). This mechanism has been demonstrated for a variety of small-molecule agents, which access the inner pore of the hERG channel preferentially from inside the cell.

Pharmacological effects of nonselective and subtype-selective nicotinic acetylcholine receptor agonists in animal models of persistent pain.

Nicotinic acetylcholine receptors (nAChRs) are longstanding targets for a next generation of pain therapeutics, but the nAChR subtypes that govern analgesia remain unknown. We tested a series of nicotinic agonists, including many molecules used or tried clinically, on a panel of cloned neuronal nAChRs for potency and selectivity using patch-clamp electrophysiology and a live cell-based fluorescence assay. Nonselective nicotinic agonists as well as compounds selective either for alpha4beta2 or for alpha7 nAChRs were then tested in the formalin and complete Freund's adjuvant models of pain.

Species-specific pharmacology of Trichloro(sulfanyl)ethyl benzamides as transient receptor potential ankyrin 1 (TRPA1) antagonists.

Agonists of TRPA1 such as mustard oil and its key component AITC cause pain and neurogenic inflammation in humans and pain behavior in rodents. TRPA1 is activated by numerous reactive compounds making it a sensor for reactive compounds in the body. Failure of AITC, formalin and other reactive compounds to trigger pain behavior in TRPA1 knockout mice, as well as the ability of TRPA1 antisense to alleviate cold hyperalgesia after spinal nerve ligation, suggest that TRPA1 is a potential target for novel analgesic agents.

Ethanol dual modulatory actions on spontaneous postsynaptic currents in spinal motoneurons.

Recently we have shown that acute ethanol (EtOH) exposure suppresses dorsal root-evoked synaptic potentials in spinal motoneurons. To examine the synaptic mechanisms underlying the reduced excitatory activity, EtOH actions on properties of action potential-independent miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs) were studied in spinal motoneurons of newborn rats. Properties of mEPSCs generated by activation of N-methyl-D-aspartate receptors (NMDARs) and non-NMDA receptors and of mIPSCs mediated by glycine and gamma-aminobutyric acid-A receptors (GlyR and GABA(A)R) were examined during acute exposure to 70 and 200 mM EtOH.

Interactions between multiple rhythm generators produce complex patterns of oscillation in the developing rat spinal cord.

Neural networks capable of generating coordinated rhythmic activity form at early stages of development in the spinal cord. In this study, voltage-imaging techniques were used to examine the spatiotemporal pattern of rhythmic activity in transverse slices of lumbar spinal cord from embryonic and neonatal rats. Real-time images were recorded in slices stained with the voltage-sensitive fluorescent dye RH414 using a 464-element photodiode array.