Single Heart Cells Research Articles

A simple antegrade perfusion method for isolating viable single cardiomyocytes from neonatal to aged mice.

The aim of this study was to establish a simple and reproducible antegrade perfusion method for isolating single viable mouse heart cells and to determine the standard practical protocols that are appropriate for mice of various ages. Antegrade perfusion was performed by injecting perfusate from near the apex of the left ventricle of the excised heart, the aorta of which was clamped, using an infusion pump. This could thoroughly perfuse the myocardium through the coronary circulation. All procedures were carried out on a prewarmed heater mat under a microscope, which allows for the processes of injection and perfusion to be monitored. With appropriate adjustment of the size of the injection needle, the composition and amount of enzyme solution and the perfusion flow rate, this antegrade perfusion method could be applied to the hearts of neonatal to aged mice. We examined the morphological characteristics and electrophysiological properties of the isolated ventricular and atrial myocytes and found that these cells were mostly identical to those obtained with the traditional Langendorff‐based retrograde perfusion method. Interstitial nonmyocytes, such as cardiac progenitor cells, were also isolated simultaneously from the supernatant fraction of the centrifugation, similar to the retrograde perfusion method. The results suggest that single heart cells can be well isolated with high degree of quality by the present antegrade perfusion method, regardless of the age of the mouse.

In This Issue

HomeCirculation ResearchVol. 122, No. 3In This Issue Free AccessIn BriefPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessIn BriefPDF/EPUBIn This Issue Ruth Williams Ruth WilliamsRuth Williams Search for more papers by this author Originally published2 Feb 2018https://doi.org/10.1161/RES.0000000000000197Circulation Research. 2018;122:385Open Software to Quantify Cardiac Contraction (p e5)Sala et al create MUSCLEMOTION, an open-source software for quantifying contractions in both heart muscle cells and tissues.Download figureDownload PowerPointMeasurement and analysis of cardiac contractility are of high importance in studying muscle function, assessing disease phenotypes, and evaluating drug toxicity. Unlike electrophysiological measurements, for which there are well-established and standardized techniques applicable to a range of samples, the techniques for measuring myocardial contractions tend to be sample-specific, not easily comparable, and require specialized expertise. Sala and colleagues have now developed a novel software that can quantify contractions in movies of specimens as diverse as single heart cells, cultured cardiac myocytes, 3D cardiac organoids, and even living Zebrafish hearts. They tested the ability of their software, called MUSCLEMOTION, to detect drug-induced and disease-related contraction differences in a variety of samples and showed that the performance of the software was comparable to various gold-standard techniques tailored to those specific samples. By making MUSCLEMOTION user-friendly and freely accessible, the authors hope to facilitate comparisons of contraction data, not just between samples, but between laboratories as well, thereby facilitating data replication and reproducibility.64 Novel Genetic Loci for Coronary Artery Disease (p 433)Van der Harst and Verweij provide evidence for an omnigenic model of coronary artery disease.Download figureDownload PowerPointCoronary artery disease (CAD) is a multifactorial condition with contributions from both genetic and environmental factors. It has been associated with a number of genetic loci, yet linking these to potential pathological pathways remains difficult. Van der Harst and Verweij have now carried out the largest GWAS (genome-wide association study) to date. Examining nearly 8 million single nucleotide polymorphisms (SNPs) in a total of 122 733 CAD patient samples, they identified 64 novel CAD-associated loci. These candidate genes, however, are involved in such broad biological functions and expressed in such diverse tissues that the contribution of genetic factors appears to be even more complicated than first thought, as many loci have no obvious link to CAD. Based on these findings, the authors suggest that the model of CAD as a polygenic disease—one caused by multiple genes affecting a handful of key pathways—may be incorrect. Instead, they surmise that the results point to an omnigenic model—whereby the link between many SNPs and CAD may be one of interconnectedness of gene-regulatory mechanisms. If so, determining cell-specific networks of gene expression may be the best route to understanding CAD pathology.Long-Term MRI Follow-Up of MVO After Cell Therapy (p 479)Traverse et al report the final 2-year results of the stem cell TIME trial.Download figureDownload PowerPointStem cell therapies are under investigation for their potential to promote cardiac tissue repair after myocardial infarction (MI). Because changes to the myocardium following reperfusion could influence cell engraftment and survival, the timing of cell delivery was examined in the TIME trial (Timing In Myocardial Infarction Evaluation), which compared delivery of cells on day 3 with day 7 following MI in 120 patients. At 6 months, the results indicated that cells delivered at either time point offered no benefits to patients. And 2 years later, these results remain the same. However, the study has also provided one of the largest MRI datasets of patients following MI. By analyzing these data, Traverse and colleagues show that patients who had microvascular obstructions at baseline have poorer outcomes—larger infarct size, more adverse left ventricle remodeling, reduced recovery of ventricle function, and greater likelihood of requiring an implantation device. From these findings, the team suggests that the presence of microvascular obstruction may be a powerful predictor of subsequent left ventricle failure, and that microvascular dysfunction identifies the most at-risk patients. Previous Back to top Next FiguresReferencesRelatedDetails February 2, 2018Vol 122, Issue 3 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000197 Originally publishedFebruary 2, 2018 PDF download Advertisement

Abstract 239: Transcriptomic Profiling Maps Anatomically Patterned Subpopulations Among Single Embryonic Cardiac Cells

Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genomewide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0% ± 3.2% and 91.2% ± 2.8% accuracy, respectively (99.4% ± 1.0% and 99.1% ± 1.1% if a ±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5/ cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.

Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells

Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0%± 3.2% and 91.2%± 2.8% accuracy, respectively (99.4%± 1.0% and 99.1%± 1.1% if a±1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5-/- cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.

Ros in Cardiac Calcium Signaling

Reactive oxygen species (ROS) are thought to play an important role in the pathophysiology of diverse cells including those in the heart. In addition, recent work by many investigators suggests that there is a significant dynamic signaling role of ROS in many cell types, including cardiomyocytes. Using isolated single heart cells from rat and mouse, confocal imaging and cellular electrophysiology, we investigated the roles of transient elevations of ROS by using a rapid superfusion system to exogenously apply H2O2 (100 [[Unsupported Character - Symbol Font μ]]M). We examined Ca2+ sparks, [Ca2+]i transients and membrane currents. We also investigated the roles of the secondary messengers PKA and CaMKII in the ROS-dependent signaling that we examined. Clear alterations of some Ca2+ signals were observed with complex contributions from PKA- and CaMKII dependent signals. The roles of mitochondria and NADPH oxidases in this process are discussed.

Parameters in Dynamic Models of Complex Traits are Containers of Missing Heritability

Polymorphisms identified in genome-wide association studies of human traits rarely explain more than a small proportion of the heritable variation, and improving this situation within the current paradigm appears daunting. Given a well-validated dynamic model of a complex physiological trait, a substantial part of the underlying genetic variation must manifest as variation in model parameters. These parameters are themselves phenotypic traits. By linking whole-cell phenotypic variation to genetic variation in a computational model of a single heart cell, incorporating genotype-to-parameter maps, we show that genome-wide association studies on parameters reveal much more genetic variation than when using higher-level cellular phenotypes. The results suggest that letting such studies be guided by computational physiology may facilitate a causal understanding of the genotype-to-phenotype map of complex traits, with strong implications for the development of phenomics technology.

Calcium ion spiral wave induced by random calcium ion sparks in single heart cell

Calcium ion spiral wave induced by random released calcium ion sparks in single heart cell is studied using the improved discrete fire-diffuse-fire model and phase analysis method. The results show that calcium ion spiral wave can be excited in resting heart cell by calcium ion sparks at an appropriate release frequency. The key of the generation of calcium ion spiral wave is the emergence of the phase singularity caused by the calcium sparks in the different time and space sequences. The number of phase singularity is exponentially linked with calcium ion spark frequency, and has the limit in a fixed system.

Noise-induced synchronous stochastic oscillations in small scale cultured heart-cell networks

This paper reports that the synchronous integer multiple oscillations of heart-cell networks or clusters are observed in the biology experiment. The behaviour of the integer multiple rhythm is a transition between super- and subthreshold oscillations, the stochastic mechanism of the transition is identified. The similar synchronized oscillations are theoretically reproduced in the stochastic network composed of heterogeneous cells whose behaviours are chosen as excitable or oscillatory states near a Hopf bifurcation point. The parameter regions of coupling strength and noise density that the complex oscillatory rhythms can be simulated are identified. The results show that the rhythm results from a simple stochastic alternating process between super- and sub-threshold oscillations. Studies on single heart cells forming these clusters reveal excitable or oscillatory state nearby a Hopf bifurcation point underpinning the stochastic alternation. In discussion, the results are related to some abnormal heartbeat rhythms such as the sinus arrest.

A cellular automata-based model for simulating restitution property in a single heart cell

Ventricular fibrillation is the cause of the most sudden mortalities. Restitution is one of the specific properties of ventricular cell. The recent findings have clearly proved the correlation between the slope of restitution curve with ventricular fibrillation. This; therefore, mandates the modeling of cellular restitution to gain high importance. A cellular automaton is a powerful tool for simulating complex phenomena in a simple language. A cellular automaton is a lattice of cells where the behavior of each cell is determined by the behavior of its neighboring cells as well as the automata rule. In this paper, a simple model is depicted for the simulation of the property of restitution in a single cardiac cell using cellular automata. At first, two state variables; action potential and recovery are introduced in the automata model. In second, automata rule is determined and then recovery variable is defined in such a way so that the restitution is developed. In order to evaluate the proposed model, the generated restitution curve in our study is compared with the restitution curves from the experimental findings of valid sources. Our findings indicate that the presented model is not only capable of simulating restitution in cardiac cell, but also possesses the capability of regulating the restitution curve.

Solute Transport in the Transverse Tubules of Cardiac Ventricular Myocytes

Electrical excitation in mammalian cardiac ventricular myocytes underlies the activation of cell-wide Ca2+ release and hence contraction. This process can occur rapidly in relatively large myocytes because transverse tubules (TTs) penetrate deep into the cells. The TT network also permits extracellular solute to be carried into the cell volume and thereby allows for improved inflow and egress of substrates. We have studied TT morphology in relaxed and contracted cells with the aim to characterize transport function and physical properties of the TT system.Using living isolated rat ventricular myocytes, we examined the movement of substrate within the TTs under different conditions using sulforhodamine B and fluorescence imaging. In addition, we examined the TT network with respect to its size, shape and complexity using super-resolution STED (stimulated emission depletion) microscopy. The lipophilic indicator Di-8-ANEPPS was used to identify and characterize the TTs. A rapid bulk solution changer was used to measure the extracellular marker sulforhodamine B concentration changes within the T-tubule matrix at rest and during field stimulation. STED imaging of resting myocytes revealed tubules of about 250 nm in diameter (see related abstract: Wagner et al. 2010). We hypothesized that if TTs collapse and expand dynamically during contraction, solute within the TT network would exchange more rapidly during contractions than when the myocytes are quiescent. Testing this hypothesis by rapid solution change revealed that TT solute exchange was significantly faster during stimulated contraction in single heart cells. Consistent with this finding was the observation that inhibition of contraction by cytochalasin D treatment in paced myocytes reduced the rate of solute exchange. These results suggest that TT solute exchange is accelerated by the mechanical deformation of the TTs during contraction. Future work should enable us to test this hypothesis more directly.

Rem Selectively Abolishes β1-adrenergic Regulation Of CaV1.2 Channels In Heart

β-adrenergic modulation of cardiac CaV1.2 channels is critical for sympathetic regulation of the heartbeat, and its disruption is a harmful hallmark of heart failure. RGK (Rem, Rem2, Rad, Gem/Kir) GTPases potently inhibit CaV channels by interacting with their auxiliary β subunits. Intriguingly, RGK proteins are present in heart, and their levels are elevated in heart failure. We examined the impact of the RGK GTPase, Rem, on CaV1.2 channels in heart cells and assessed whether there was crosstalk with the β-adrenergic modulation of the channel. Cultured adult guinea pig ventricular myocytes expressed robust CaV1.2 currents (ICa,L) (15.08 pA/pF) and responded to β1-adrenergic activation (1 μM isoproterenol + 1 μM ICI118,551) with a sharp, three-fold increase in current density. Isochronal cardiac cells expressing YFP-Rem, achieved through adenovirus infection, displayed a markedly lower basal current density (5.85 pA/pF). Nevertheless, the effect of Rem in heart is quantitatively smaller than seen in recombinant channels expressed in HEK 293 cells, which feature a virtual ablation of ICa,L. Surprisingly, the remaining Rem-insensitive ICa,L in guinea pig heart cells was essentially unresponsive to β1-adrenergic stimulation. This was not due to disruption of the signaling pathway because isoproterenol-mediated increase in cardiac IKs remained unchanged. Intriguingly, the Rem insensitive ICa,L remained responsive to forskolin. These results reveal an unexpected crosstalk between RGK GTPases and β-adrenergic signaling pathway at the level of cardiac ICa,L, and suggests that Rem selectively inhibits spatially distinct CaV1.2 channels in single heart cells.

Total internal reflection fluorescence microscopy study of spiral Ca2+ waves in single heart cell

Spiral wave phenomena exist in many scales of nature and have attracted the attention of scientists from different fields. Although much work has been done on qualitative analysis of spiral waves, the mechanism of spiral waves' spontaneous formation and termination in living systems is still not clear. Here, by using total internal reflection fluorescence microscopy, we show the spiral waves of calcium signals in single rat cardiac myocytes and the simulation of the waves comprising calcium sparks. The mechanism of the formation and termination of spiral waves is attributable to the calcium release channels' refractory resulting from their stochastic release. We suggest that this mechanism can be adapted to other living systems.

Micro-dynamics of Ca2+ signals in single heart cells

We used a total internal reflection fluorescence microscope based on the near field optics principle to study the Ca2+ signals in single heart cells of Sprague-Dawley rat. Due to the high signal to noise ratio and high speed of data acquisition of this microscope, the Ca2+ images show complex 2-dimensional wave patterns. The local elementary Ca2+ release units play important role in the formation and transformation of Ca2+ wave. Simulations based on Fire-Diffuse-Fire model show that the Ca2+ wave, composed of Ca2+ release units, may steadily exist in single heart cell. Our results will be helpful for understanding the micro-dynamics of living excitable medium.

A Simple Technique for Isolating Healthy Heart Cells from Mouse Models

Single heart cells of mouse models provide powerful tools for heart research. However, their isolation is not easy, and it imposes a significant bottleneck on their use in cellular studies of the heart. Aiming to overcome this problem, this report introduces a novel technique that reproducibly isolates healthy heart cells from mouse models. Using simple devices that ensure easy handling and the rapid aortic cannulation of a small mouse heart, cell isolation was done under physiological conditions without using the "KB" medium or 2,3-butanedione monoxime (BDM). The isolated cells consistently had a healthy appearance and a high viability of 75 +/- 5% (mean +/- SD) in Tyrode solution containing 1.8 mM Ca2+. After 8 h of storage at 37 degrees C, they still had a viability of 45 +/- 12%. The cells showed normal contraction properties when field-stimulated, and they generated normal action potentials and membrane currents under the whole-cell clamp condition. The beta-adrenergic signal transduction of the cells was also normal when it was examined with the isoproterenol enhancement of the L-type Ca2+ current.

Metabolic monitoring of the electrically stimulated single heart cell within a microfluidic platform

A device based on five individually addressable microelectrodes, fully integrated within a microfluidic system, has been fabricated to enable the real-time measurement of ionic and metabolic fluxes from electrically active, beating single heart cells. The electrode array comprised one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an electrochemical lactate microbiosensor, that were used to measure the amounts of lactate produced by the heart cell. The device also allowed simultaneous in-situ microscopy, enabling optical measurements of cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+. Initial experiments aimed to create a metabolic profile of the beating heart cell, and results show well defined excitation-contraction (EC) coupling at different rates. Ca2+ transients and extracellular pH measurements were obtained from continually paced single myocytes, both as a function of the rate of cell contraction. Finally, the relative amounts of intra- and extra-cellular lactate produced during field stimulation were determined, using cell electroporation where necessary.

Letter Regarding the Article by Xue et al, “Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes”

To the Editor: The article by Xue et al1 on human embryonic stem cell-derived pacemakers illustrates that embryonic stem cells differentiated into spontaneously beating cardiocytes may function as biological pacemakers and mentions a potential limitation: The intrinsic pacemaker rate was slower than desirable. They suggest that incorporating HCN pacemaker channel genes might achieve more desirable rates, an idea consistent with our published results using HCN2 in gene- and adult human mesenchymal stem cell (hMSC)-based therapies.2–4 However, certain of the comments by Xue et al misinterpret our own work on HCN-loaded hMSCs. They state that “…these modified, undifferentiated, human mesenchymal stem cells are incapable of pacing quiescent cells because the former are neither electrically active nor genuine cardiac cells” (p 19). This statement suggests a misunderstanding of the rationale and underlying biophysics of the hMSC experiments. In fact, generation of pacemaker activity does not require delivery of an “excitable differentiated cardiac cell,” but only that the delivered cell (1) carry sufficient pacemaker current and (2) make gap junctions; thus, the hMSC-myocyte pair should behave as a pacemaker unit entirely equivalent to a single heart cell with substantial if. We clearly demonstrated both …

Angiotensin II-induced increase of T-type Ca 2+ current and decrease of L-type Ca 2+ current in heart cells

The effect of angiotensin II (Ang II) on the T- and L-type calcium currents ( I Ca) in single ventricular heart cells of 18-week-old fetal human and 10-day-old chick embryos was studied using the whole-cell voltage clamp technique. Our results showed that in both, human and chick cardiomyocytes, Ang II (10 −7 M) increased the T-type calcium current and decreased the L-type I Ca. The effect of Ang II on both types of currents was blocked by the AT 1 peptidic antagonist, [Sar 1, Ala 8] Ang II (2 × 10 −7 M). Protein kinase C activator, phorbol 12,13-dibutyrate, mimicked the effect of Ang II on the T- and L-type calcium currents. These results demonstrate that in fetal human and chick embryo cardiomyocytes Ang II affects the T- and L-type Ca 2+ currents differently, and this effect seems to be mediated by the PKC pathway.

Stimulation of Isolated Ventricular Myocytes Within an Open Architecture Microarray

This paper is concerned with the physiological responses of single heart cells within microfluidic chambers, in response to stimulation by integrated microelectrodes. To enable these investigations, which included the measurement of action potential duration, intracellular Ca2+ and cell shortening, a series of microfluidic chambers (50 microm wide, 180 microm long, 400 microm high, 500 microm pitch) and connecting channels (200 microm wide, 5000 microm long, 50 microm high, 500 microm pitch) were replica-moulded into the silicone elastomer, polydimethylsiloxane (PDMS). The structures were formed against a master of posts and lines, photolithograhically patterned into the high aspect ratio photoresist SU-8. The chambers within the slab of PDMS were aligned against pairs of stimulating gold microelectrodes (50 microm long, 20 microm wide, 0.1-10 microm thick, 180 microm apart) patterned on a microscope coverslip base, thus defining cavities of approximately 4 nL volume. The assembly was filled with physiological saline and single isolated rabbit ventricular myocytes were introduced by micropipetting, thus creating limited volumes of saline above individual myocytes that could be varied between 4 nL and > or = 4 microL. The application of transient current pulses to the cells via the electrodes caused transient contractions with constant amplitude (recorded as changes in sarcomere length), confirming that excitation contraction coupling (EC coupling) remained functional in these limited volumes. Continuous monitoring of the intracellular Ca2+ (using calcium sensitive dyes) showed, that in the absence of bath perfusion, the amplitude of the transients remained constant for approximately 3 min in the 4-nL volume and approximately 20 min for the 4 microL volume. Beyond this time, the cells became unexcitable until the bath was renewed. The action potential duration (APD) was recorded at stimulation frequencies of 1 Hz and 0.5 Hz using potential sensitive dyes and was prolonged at the higher pacing rate. These studies show the prolonged electrical stimulation of isolated adult cardiac myocytes in microchambers with unimpaired EC coupling as verified on optical records of the action potential, Ca2+ transients and cell shortening. The open architecture provided free (pipetting) access for drug dispensation without cross talk between neighboring microwells, and multiplexed optical detection can be realized to study EC coupling on arrays of cells under both control and experimental conditions.

Cytoskeletal structure and recovery in single human cardiac myocytes

Mechanical support of the failing human heart with a left ventricular assist device (LVAD) normalizes many components of myocyte structure and function. We hypothesized that recovery of the cytoskeleton, a major site of mechanotransduction in cardiac myocytes, is crucial for sustained improvement of myocardial function. We therefore measured the effects of LVAD support on 4 cytoskeletal proteins in single human heart cells. Myocytes were isolated from non-failing (NF), hypertrophied (H), failing (F) and LVAD-supported failing (L) human hearts. Protein quantitation was performed using Western blot analysis and cellular distribution was determined by immunolabeling and confocal microscopy. alpha-actinin did not differ in cells from H or F as compared with NF, and L had no effect. Vinculin was not quantitatively different in H or F vs NF, but localization at the intercalated disks was significantly decreased in H and absent in F, and this pattern was consistently reversed in L. Desmin protein was significantly increased in F vs NF, both in quantity and distribution, and these increases were reversed in L. beta-tubulin was increasingly polymerized in H and F, and the hyperpolymerization was reversed in L. On the level of the single cardiomyocyte, major proteins of the cytoskeleton are significantly altered in hypertrophied and failing human hearts. These alterations are reversed by mechanical unloading with an LVAD, suggesting that the cytoskeleton is not the limiting factor in determining full cardiac recovery.

Bradykinin induced a positive chronotropic effect via stimulation of T- and L-type calcium currents in heart cells.

Using Fluo-3 calcium dye confocal microscopy and spontaneously contracting embryonic chick heart cells, bradykinin (10(-10) M) was found to induce positive chronotropic effects by increasing the frequency of the transient increase of cytosolic and nuclear free Ca2+. Pretreatment of the cells with either B1 or B2 receptor antagonists (R126 and R817, respectively) completely prevented bradykinin (BK) induced positive chronotropic effects on spontaneously contracting single heart cells. Using the whole-cell voltage clamp technique and ionic substitution to separate the different ionic current species, our results showed that BK (10(-6) M) had no effect on fast Na+ inward current and delayed outward potassium current. However, both L- and T-type Ca2+ currents were found to be increased by BK in a dose-dependent manner (10(-10)-10(-7) M). The effects of BK on T- and L-type Ca2+ currents were partially blocked by the B1 receptor antagonist [Leu8]des-Arg9-BK (R592) (10(-7) M) and completely reversed by the B2 receptor antagonist D-Arg[Hyp3,D-Phe7,Leu8]BK (R-588) (10(-7) M) or pretreatment with pertussis toxin (PTX). These results demonstrate that BK induced a positive chronotropic effect via stimulation of T- and L-type Ca2+ currents in heart cells mainly via stimulation of B2 receptor coupled to PTX-sensitive G-proteins. The increase of both types of Ca2+ current by BK in heart cells may explain the positive inotropic and chronotropic effects of this hormone.