ABSTRACT
Our heart is comprised of many different cell types that all contribute to cardiac function. An important step in deciphering the molecular complexity of our heart is to decipher the molecular composition of the various cardiac cell types. Here we set out to delineate a comprehensive protein expression profile of the two most prevalent cell types in the heart: cardiomyocytes and cardiac fibroblasts. To this end, we isolated cardiomyocytes and fibroblasts from rat hearts and combined state-of-the-art flow cytometry with high-resolution mass spectrometry to investigate their proteome profiles right after isolation. We measured and quantified 5240 proteins in cardiomyocytes and 6328 proteins in cardiac fibroblasts. In addition to providing a global protein profile for these cardiac cell types, we also present specific findings, such as unique expression of ion channels and transcription factors for each cell type. For instance, we show that the sodium channel Scn7a and the cation channel Trpm7 are expressed in fibroblasts but not in cardiomyocytes, which underscores the importance of investigating the endogenous cell host prior to functional studies. Our dataset represents a valuable resource on protein expression profiles in these two primary cardiac cells types.
Subject(s)
Fibroblasts/metabolism , Myocytes, Cardiac/metabolism , Proteome , Proteomics , Animals , Biomarkers , Cells, Cultured , Chromatography, Liquid , Gene Expression Profiling , Proteomics/methods , Rats , Tandem Mass Spectrometry , TranscriptomeABSTRACT
INTRODUCTION: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. METHODS: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. RESULTS: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. DISCUSSION: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.
Subject(s)
ERG1 Potassium Channel/antagonists & inhibitors , Risk Assessment/methods , Torsades de Pointes/chemically induced , Bayes Theorem , Computer Simulation , Humans , Models, Biological , Patch-Clamp Techniques , Potassium Channel Blockers/pharmacology , Safety , Torsades de Pointes/physiopathologyABSTRACT
KEY POINTS: The Ca2+ and redox-sensing enzyme Ca2+ /calmodulin-dependent kinase 2 (CaMKII) is a crucial and well-established signalling molecule in the heart and brain. In vascular smooth muscle, which controls blood flow by contracting and relaxing in response to complex Ca2+ signals and oxidative stress, surprisingly little is known about the role of CaMKII. The vasodilator-induced second messenger cAMP can relax vascular smooth muscle via its effector, exchange protein directly activated by cAMP (Epac), by activating spontaneous transient outward currents (STOCs) that hyperpolarize the cell membrane and reduce voltage-dependent Ca2+ influx. How Epac activates STOCs is unknown. In the present study, we map the pathway by which Epac increases STOC activity in contractile vascular smooth muscle and show that a critical step is the activation of CaMKII. To our knowledge, this is the first report of CaMKII activation triggering cellular activity known to induce vasorelaxation. ABSTRACT: Activation of the major cAMP effector, exchange protein directly activated by cAMP (Epac), induces vascular smooth muscle relaxation by increasing the activity of ryanodine (RyR)-sensitive release channels on the peripheral sarcoplasmic reticulum. Resultant Ca2+ sparks activate plasma membrane Ca2+ -activated K+ (BKCa ) channels, evoking spontaneous transient outward currents (STOCs) that hyperpolarize the cell and reduce voltage-dependent Ca2+ entry. In the present study, we investigate the mechanism by which Epac increases STOC activity. We show that the selective Epac activator 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5-cyclic monophosphate-AM (8-pCPT-AM) induces autophosphorylation (activation) of calcium/calmodulin-dependent kinase 2 (CaMKII) and also that inhibition of CaMKII abolishes 8-pCPT-AM-induced increases in STOC activity. Epac-induced CaMKII activation is probably initiated by inositol 1,4,5-trisphosphate (IP3 )-mobilized Ca2+ : 8-pCPT-AM fails to induce CaMKII activation following intracellular Ca2+ store depletion and inhibition of IP3 receptors blocks both 8-pCPT-AM-mediated CaMKII phosphorylation and STOC activity. 8-pCPT-AM does not directly activate BKCa channels, but STOCs cannot be generated by 8-pCPT-AM in the presence of ryanodine. Furthermore, exposure to 8-pCPT-AM significantly slows the initial rate of [Ca2+ ]i rise induced by the RyR activator caffeine without significantly affecting the caffeine-induced Ca2+ transient amplitude, a measure of Ca2+ store content. We conclude that Epac-mediated STOC activity (i) occurs via activation of CaMKII and (ii) is driven by changes in the underlying behaviour of RyR channels. To our knowledge, this is the first report of CaMKII initiating cellular activity linked to vasorelaxation and suggests novel roles for this Ca2+ and redox-sensing enzyme in the regulation of vascular tone and blood flow.
Subject(s)
Action Potentials , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Animals , Calcium/metabolism , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Cells, Cultured , Guanine Nucleotide Exchange Factors/metabolism , Large-Conductance Calcium-Activated Potassium Channels/metabolism , Male , Mice , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/physiology , Rats , Rats, Wistar , VasodilationABSTRACT
Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.
ABSTRACT
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.
ABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
[reaction: see text] The enantioselective total synthesis of (-)-dactylolide is reported. The absolute stereochemistry of the tetrahydropyran was established by catalytic asymmetric Jacobsen hetero-Diels-Alder reaction. The remote C19 stereocenter was introduced by a sequence of chelation-controlled Grignard addition and Ireland-Claisen rearrangement.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Lactones/chemical synthesis , Bridged Bicyclo Compounds, Heterocyclic/chemistry , Catalysis , Lactones/chemistry , Molecular Structure , StereoisomerismABSTRACT
Potassium (K(+)) channels, with their diversity, often tissue-defined distribution, and critical role in controlling cellular excitability, have long held promise of being important drug targets for the treatment of dysrhythmias in the heart and abnormal neuronal activity within the brain. With the exception of drugs that target one particular class, ATP-sensitive K(+) (KATP) channels, very few selective K(+) channel activators or inhibitors are currently licensed for clinical use in cardiovascular and neurological disease. Here we review what a range of human genetic disorders have told us about the role of specific K(+) channel subunits, explore the potential of activators and inhibitors of specific channel populations as a therapeutic strategy, and discuss possible reasons for the difficulty in designing clinically relevant K(+) channel modulators.