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The purpose of this review article is to summarize our current understanding of the efficacy and safety of cardiac defibrillation with nanosecond shocks. Experiments in isolated hearts, using optical mapping of the electrical activity, have demonstrated that nanosecond shocks can defibrillate with lower energies than conventional millisecond shocks. Single defibrillation strength nanosecond shocks do not cause obvious damage, but repeated stimulation leads to deterioration of the hearts. In isolated myocytes, nanosecond pulses can also stimulate at lower energies than at longer pulses and cause less electroporation (propidium uptake). The mechanism is likely electroporation of the plasma membrane. Repeated stimulation leads to distorted calcium gradients.

BACKGROUND Severe hypothermia (SH) is known to be arrhythmogenic, but the effect of therapeutic hypothermia (TH) on arrhythmias is unclear. It is hypothesized that susceptibility to Ca-mediated arrhythmia triggers would be increased only by SH.Methods and Results:Spontaneous Ca release (SCR) and resultant delayed afterdepolarizations (DADs) were evaluated by optical mapping in canine wedge preparations during normothermia (N, 36℃), TH (32℃) or SH (28℃; n=8 each). The slope (amplitude/rise time) of multicellular SCR (mSCR) events, a determinant of triggered activity, was suppressed in TH (24.4±3.4%/s vs. N: 41.5±6.0%/s), but significantly higher in SH (96.3±8.1%/s) producing higher amplitude DADs in SH (35.7±1.6%) and smaller in TH (5.3±1.0% vs. N: 10.0±1.1%, all P

Background Bisphenol S (BPS) has increasingly been used as a substitute for bisphenol A (BPA) in some “BPA-free” consumer goods and in thermal papers. Wide human exposure to BPS has been reported; however, the biological and potential toxic effects of BPS are poorly understood. Objective In this study, we sought to elucidate the sex-specific rapid effect of BPS in rat hearts and its underlying mechanism. Methods We examined the rapid effects of BPS in rat hearts using electrophysiology, confocal and conventional fluorescence imaging, and immunoblotting. Treatment was administered via acute perfusion of excised hearts or isolated cardiac myocytes. Results In female rat hearts acutely exposed to 10–9 M BPS, the heart rate was increased; in the presence of catecholamine-induced stress, the frequency of ventricular arrhythmia events was markedly increased. BPS-exposed hearts showed increased incidence of arrhythmogenic-triggered activities in female ventricular myocytes and altered myocyte Ca2+ handling, particularly spontaneous Ca2+ release from the sarcoplasmic reticulum. The dose responses of BPS actions were inverted U-shaped. The impact of BPS on myocyte Ca2+ handling was mediated by estrogen receptor β signaling and by rapid increases in the phosphorylation of key Ca2+ handling proteins, including ryanodine receptor and phospholamban. The proarrhythmic effects of BPS were female specific; male rat hearts were not affected by BPS at the organ, myocyte, or protein levels. Conclusion Rapid exposure to low-dose BPS showed proarrhythmic impact on female rat hearts; these effects at the organ, cellular, and molecular levels are remarkably similar to those reported for BPA. Evaluation of the bioactivity and safety of BPS and other BPA analogs is necessary before they are used as BPA alternatives in consumer products. Citation Gao X, Ma J, Chen Y, Wang HS. 2015. Rapid responses and mechanism of action for low-dose bisphenol S on ex vivo rat hearts and isolated myocytes: evidence of female-specific proarrhythmic effects. Environ Health Perspect 123:571–578; http://dx.doi.org/10.1289/ehp.1408679

Aim Investigate to what extent low-intensity swim training for six weeks counterbalances the adverse remodeling due to the advance of pathological hypertrophy in the left ventricle (LV) structural and mechanical properties in the early compensated phase of hypertension in male SHR. Main methods Four-month-old male SHR and Wistar rats were randomly divided into Sed (sedentary) and Ex (exercised) groups. The exercised rats were submitted to a swimming protocol (1 h/day, 5 times/week, no additional load) for six weeks. LV tissue and isolated myocytes were used to assess structural and mechanical properties. Myocytes were stimulted at frequencies (F) of 1 and 3 Hz at 37 °C. Key findings Exercised SHR showed improvement in cardiovascular parameters compared to sedentary SHR (mean arterial pressure: 13.22%; resting HR: 14.28.%). About structural and mechanical properties, swim training induced a decrease in LV myocyte thickness (10.85%), number of inflammatory cells (21.24%); collagen type III (74.23%) and type I (85.6%) fiber areas; amplitude of single myocyte shortening (47% to F1 and 28.46% to F3), timecourses of shortening (16.5% to F1 and 7.55% to F3) and relaxation (15.31% to F3) compared to sedentary SHR. Significance Six weeks of swim training attenuates the adverse remodeling of LV structural and mechanical properties in the early compensated phase of hypertension in male SHR.

Variability in the action potential of isolated myocytes and tissue samples is observed in experimental studies. Variability is manifested as both differences in the action potential (AP) morphology between cells (extrinsic variability), and also ‘intrinsic’ or beat-to-beat variability of repolarization (BVR) in the AP duration of each cell. We studied the relative contributions of experimentally recorded intrinsic and extrinsic variability to dispersion of repolarization in tissue. We developed four cell-specific parameterizations of a phenomenological stochastic differential equation AP model exhibiting intrinsic variability using APs recorded from isolated guinea pig ventricular myocytes exhibiting BVR. We performed simulations in tissue using the four different model parameterizations in the presence and the absence of both intrinsic and extrinsic variability. We altered the coupling of the tissue to determine how inter-cellular coupling affected the dispersion of the AP duration in tissue. Both intrinsic and extrinsic variability were gradually revealed by reduction of tissue coupling. However, the recorded extrinsic variability between individual myocytes produced a greater degree of dispersion in repolarization in tissue than the intrinsic variability of each myocyte., Highlights • We modelled inter-cell and beat-to-beat repolarization variability in cardiomyocytes. • We coupled the cells together into cardiac tissue. • Reducing tissue coupling increased repolarization dispersion in tissue. • Inter-cell variability had a greater effect on repolarization dispersion.

Computational electrophysiology has proven useful to investigate the mechanisms of cardiac arrhythmias at various spatial scales, from isolated myocytes to the whole heart. This article reviews how mathematical modeling has aided our understanding of human atrial myocyte electrophysiology to study the contribution of structural and electrical remodeling to human atrial fibrillation. Potential new avenues of investigation and model development are suggested.

Spontaneous transient outward currents (STOCs) have been reported in resistance and small arteries but have not yet been found in thoracic aorta. Do thoracic aorta myocytes possess cellular machinery that generates STOCs? It was found that the majority of aortic myocytes do not generate STOCs. STOCs were generated in 8.7% of freshly isolated aortic myocytes. Myocytes that did not generate STOCs we have called “silent” myocytes and myocytes with STOCs have been called “active.” STOCs recorded in active myocytes were voltage dependent and were inhibited by ryanodine, caffeine, and charybdotoxin. Forskolin was reported to increase STOCs frequency in myocytes isolated from resistance arteries. Forskolin (10 μM) triggered STOCs generation in 35.1% of silent aortic myocytes. In 36.8% percent of silent myocytes, forskolin did not trigger STOCs but increased the amplitude of charybdotoxin-sensitive outward net current to 136.1 ± 8.5% at 0 mV. Membrane-permeable 8BrcAMP triggered STOCs generation in 38.7% of silent myocytes. Forskolin- or 8BrcAMP-triggered STOCs were inhibited by charybdotoxin. 8BrcAMP also increased open probability of BKCa channels in BAPTA-AM-pretreated cells. Our data demonstrate that, in contrast to resistance arteries, STOCs are present just in the minority of myocytes in the thoracic aorta. However, cellular machinery that generates STOCs can be “switched” on by cAMP. Such an inactive cellular mechanism could modulate the contractility of the thoracic aorta in response to physiological demand.

Freshly isolated myocytes lose their viability and functionality very early, from single hours to single days. So their viability is usually tested before the experiments or monitored continuously in periodic time intervals. But simple observations using viability kits (e. g. LIVE / DEAD Cell Imaging Kit, Life Technologies) for this purpose are not sufficient because of Calcein photobleaching and furthermore repeated testing is not possible. In this paper we tested advanced methods based on Calcium degradation and fluorescence lifetime measurement or cardiomyocyte shape properties calculations.

By the mid-1990s, experts realized that drugs leading to improved ventricular remodeling were doing something remarkable in cardiac patients. The “age of cardiac remodeling” had begun. This created an experimental need for high-quality assessment of changes in cardiac tissue composition, including myocyte shape, myocardial fibrosis/collagen, and vascular remodeling. Many working in the field today have little or no training related to recognition of fixation artifacts or common errors associated with quantitative morphology. Unfortunately, such skills had become somewhat of a lost art during the ages of cardiac physiology in the mid-20th century and molecular biology, gaining prominence by the mid-1970s. Consequently, cardiac remodeling studies today are often seriously flawed to the point where data are not reproducible and subsequent researchers may be chasing the molecular basis of a nonexistent or erroneous phenotype. The current unacceptably high incidence of irreproducible data is a serious waste of time and resources as recently noted in comments by the National Institutes of Health director. The goal of this “how to” article is to share some lessons I have learned during nearly 40 years of assessing morphological changes in the heart. It is possible for any laboratory to routinely publish highly reproducible morphological data that stand the test of time and contribute to our fundamental knowledge of cardiac remodeling and the molecular mechanisms that drive it.

By means of electron probe microanalysis (EPMA), we quantified changes in total sodium [Na] and calcium [Ca] concentration owing to the following: (i) local axial stretch (LAS) of isolated rat myocytes and (ii) end-to-end stretch (ETES) of rat ventricular trabeculae. For LAS, the distance between patch pipette and a cell-attached stylus was increased by maximally 20%; this activated a nonselective cationic current ISACof approximately –0.5 nA, which was blocked by streptomycin. Trabeculae were stretched end-to-end from 85% Lmaxto Lmax. Stretch increased cytosolic [Na]totalby 34% in isolated myocytes (p < 0.001) and by 43% in trabeculae (p < 0.001). The increment in nuclear [Na]totalwas 21% in myocytes (p < 0.01) and 20% in trabeculae (p < 0.001). Stretch increased [Ca]totalin isolated myocytes, in both cytosol (from 0.63 ± 0.09 to 1.09 ± 0.20 mmol/L, p < 0.05) and nucleus (from 0.33 ± 0.05 to 0.64 ± 0.13 mmol/L, p < 0.05). In trabeculae, the stretch-induced increment of 51% in cytosolic [Ca]totalremained nonsignificant (p < 0.15). In the nucleus, [Ca]totaldid not change. We interpret the difference of stretch on nuclear calcium in myocytes vs. trabeculae with the assumption that LAS, but not ETES, produces shear-stress components that translate the mechanical stimulus deeply into the cell where it may modulate [Ca]totalby signals independent of ISAC.

Cardiac aging is associated with lengthening of the QT interval, a condition that enhances malignant ventricular arrhythmias and sudden death. The aim of this study was to establish whether ionic currents are altered in old myocytes contributing to the protracted electrical recovery of the senescent heart. Thus, mice at 3-30 months of age were studied by ECG and patch-clamp; these physiological determinations were complemented with molecular assays for the analysis of ion channel proteins. By surface ECG and telemetry system, PR, QRS and QT intervals were prolonged in mice at 25 months or older. These delays were maintained in ex-vivo Langendorff preparations. In comparison to young, epicardial monophasic action potential (AP) duration at 50% and 90% repolarization were 1.6- and 1.2-fold larger in old LV, respectively. Moreover, senescent hearts presented a 60% higher incidence of arrhythmias. In isolated myocytes, prolongation of the early (+47%), intermediate (+117%) and late (+75%) repolarization phases of the AP were identified in cells from old animals, whereas resting membrane potential, upstroke amplitude and +dV/dt were preserved. Voltage-clamp experiments were then performed to measure ionic current properties. The rapidly activating K+ current, which consists of the transient outward and ultrarapid delayed rectifier (Ito+Kur), is responsible for the early repolarization of the AP, and was significantly reduced in old myocytes. Molecular studies revealed low levels of transcripts and proteins for K+ channel subunits Kv1.4, Kv1.5 and KChiP2 in senescent cells. Also, the late Na+ current INaL, which presents slow inactivation kinetics and is operative during AP repolarization, was 1.5-fold larger in old cells. These changes were associated with alterations in gene and protein expression of Na+ channel subunits. Inhibition of INaL with mexiletine significantly shortened the intermediate and late repolarization phases of the AP in both myocytes and perfused myocardium from old mice. Importantly, INaL inhibition in vivo shortened the QT interval of senescent mice by 12%. Thus, defects in ionic current occur with aging resulting in prolongation of the AP and delays in electrical recovery which may lead to malignant ventricular arrhythmias.

Ca 2 + ions play pivotal roles in the excitation and contraction-relaxation processes of heart muscle cells. The advent of digital-imaging techniques, especially confocal laser scanning microscopy, as well as Ca 2 + -sensitive fluorescent dyes, has contributed substantially to the precise understanding of the spatiotemporal aspects of the intracellular Ca 2 + dynamics in cardiomyocytes. In this review article, we review the progress in the cytochemical and histochemical researches on the intracellular Ca 2 + concentration ([Ca 2 + ] i ) of the heart. Recent imaging technologies have revealed that subcellular [Ca 2 + ] i dynamics are spatio-temporally heterogeneous under certain pathological conditions. We focus on the Ca 2 + waves, i.e., representative of abnormal [Ca 2 + ] i dynamics that emerge under [Ca 2 + ] i -overloaded conditions, analyzed in preparations ranging from the enzymatically isolated myocytes to the whole heart. The pathophysiological significance of Ca 2 + waves in the heart is also discussed.

Single cardiac myocytes provide a model widely used to characterize the electrophysiological properties of drugs and to identify new therapeutic targets. This review focuses on isolation procedures to obtain single cardiac myocytes from several mammal species, including humans, and on patch-clamp technique as a useful method to investigate the molecular mechanism of druy actions.

Isolated adult cardiac myocytes maintained in primary culture have been used as a model of the adult myocardium for 20 years. With the recent advances and current interest in using molecular biological techniques to investigate cardiac physiology, culturing myocytes is becoming an increasingly important technique. Acutely isolated myocytes do not remain viable for the time needed for the changes in gene expression to occur, and therefore it is necessary to maintain myocytes in culture. The aims of this review are: (1) To describe a method for isolating and culturing myocytes in serum-free medium. This section is targeted at new researchers in the field, with particular emphasis on aspects of the isolation procedure which are important for optimising myocyte culture. (2) To review current knowledge of how contractile, electrophysiological and morphological properties of adult myocytes are preserved in culture. Over the past 5 to 10 years significant advances have been made in developing novel techniques which help maintain the in-vivo properties of myocytes in culture. Efficient methods for transporting exogenous genes and anti-sense oligonucleotides into adult myocytes are now available. We anticipate that in future these advances will make cultured myocytes more attractive for use in biophysical and molecular investigations of cardiac physiology.

Interest in ventricular remodeling has increased in recent years due to the development of new therapeutic agents which may produce beneficial changes in cardiac anatomy. Data collected with new techniques have provided a clear and consistent picture of myocyte remodeling in cardiac hypertrophy and failure. A maladaptive change in cardiac myocyte shape has been demonstrated in experimental animals with heart failure and in patients with congestive heart failure due to ischemic and idiopathic dilated cardiomyopathy. This article details an approach to collect comprehensive and reliable data on myocyte shape in an efficient manner using isolated myocytes. The methodology employs a Coulter Channelyzer (Model C256, Coulter Electronics, Inc., Hialeah, FL), a light microscope, and an image analysis system. Underlying problems associated with structural assessment of the heart are discussed and effective solutions are outlined.

Application of an enzymatic cell isolation technique to a turtle heart yielded mononuclear spindle-shaped myocytes. Turtle myocyte morphology revealed a long, thin, tapered cell with an average length of 221± 9.6 μm and an average width of 9.55 ± 0.87 μm. Cell volume was calculated to be 16,248 ± 24,776 μm3 and mean sarcomere length was 2.24 ± 0.09 μm. The Ca+2-tolerant myocytes shortened to a degree of 13 ± 6.1% when stimulated in an electric field. Both spontaneous and induced contractions resembled a twitch. Myocyte volume did not vary significantly with the body mass of the turtle.

Propofol is being used more often in cardiac surgery, particularly after hypothermic, hyperkalemic cardioplegic arrest (HHCA). The purpose of this study was to examine the effects of propofol on isolated myocyte contractile function under both normothermic conditions and after simulated HHCA and rewarming. Myocytes were isolated from the left ventricle of eight pigs. Myocyte contractile function was measured under both normothermic conditions and after simulated HHCA (incubation at 4 degrees C for 2 h in crystalloid cardioplegia; K+ = 24 mEq/L) using computer-assisted videomicroscopy in the presence of 2, 4, and 6 micrograms/mL propofol (11.2, 22.4, and 33.6 microM/L, respectively). Isoproterenol (25 nM) was then added and contractile function measurements repeated. Propofol caused significant dose-dependent reductions in myocyte velocity of shortening (baseline = 67 +/- 2 microns/s; propofol = 2 micrograms/mL, 45 +/- 4 microns/s; and propofol = 6 micrograms/mL, 27 +/- 3 microns/s; P < 0.05). HHCA and rewarming caused a significant reduction in myocyte velocity of shortening (29 +/- 0.9 microns/s, P < 0.05), with further significant dose-dependent reductions in contractile function after the addition of propofol. Propofol caused a decrease in beta-adrenergic responsiveness under normothermic conditions, but not after simulated HHCA. Results from the present study demonstrated for the first time that the reduction in isolated myocyte contractile function after simulated HHCA is further decreased by propofol administration.

The majority of myocardial protective techniques performed in the United States incorporate hypothermic, hyperkalemic blood or crystalloid cardioplegia. Oxygenated blood cardioplegia has not been compared with oxygenated crystalloid cardioplegia in an isolated myocyte model of hypothermic, hyperkalemic cardioplegic arrest in which direct measurements of contractile function and myocyte swelling can be made. Accordingly, isolated myocyte contractile function and myocyte profile surface area were examined after hypothermic arrest with oxygenated crystalloid or blood cardioplegia. Methods: Isolated left ventricular pig myocytes were randomly assigned to undergo cardioplegic arrest for 2 hours at 4° C. Either oxygenated crystalloid or blood cardioplegia was used. After 2 hours, myocytes were reperfused with standard cell medium at 37° C and contractile function was examined. A control group of myocytes was maintained in cell medium at 37° C for 2 hours. Myocyte velocity of shortening (micrometers per second) was examined at baseline and after β-adrenergic stimulation (isoproterenol, 25 nmol/L). Velocity of shortening declined equally from baseline control values (65 ± 2 μm/sec) in the groups subjected to oxygenated crystalloid cardioplegia and blood cardioplegia (37 ± 2 μm/sec and 42 ± 1 μm/sec, respectively; p < 0.05). Results: Although β-adrenergic stimulation caused a significant increase in velocity of shortening in all myocyte groups, the increase was less pronounced in myocytes subjected to crystalloid cardioplegia (157 ± 6 μm/sec) and blood cardioplegia (159 ± 6 μm/sec) than in normothermic control myocytes (205 ± 7 μm/sec; p < 0.05). Myocyte profile surface area, an index of cell volume, was measured in all myocyte groups. Myocyte surface area increased equally after cardioplegic arrest and rewarming in both cardioplegia groups (crystalloid 4119 ± 53 μm2; blood 3924 ± 48 μm2); surface areas in both cardioplegia groups were significantly greater than in the normothermic control group (3158 ± 39 μm2, p < 0.05). Conclusion: Equivalent effects of oxygenated crystalloid and blood cardioplegia were observed with respect to myocyte contractile function, inotropic responsiveness, and intracellular volume regulatory processes. (J THORAC CARDIOVASC SURG 1996;112:1064-72)

Background. Cardioplegic arrest with rewarming and protamine administration have been implicated in causing transient left ventricular dysfunction perioperatively. However, whether interactive effects between cardioplegic arrest and rewarming with protamine occur with respect to myocyte contractile processes remains unclear. Accordingly, using an isolated myocyte model, the present study tested the hypothesis that simulated cardioplegic arrest with rewarming and protamine would have direct and interactive effects on myocyte contractile function. Methods. Left ventricular isolated myocyte contractile function was examined using computer-aided videomicroscopy under normothermic conditions (37°C, cell medium; n=183) and after simulated hypothermic, hyperkalemic cardioplegic arrest with rewarming (4°C, 24 mEq/L K + , 2 hours; then 37°C, cell medium, 5 minutes; n=268). Myocyte function was then examined in the presence of protamine (10 to 40 μg/mL) under normothermic conditions (n = 102) and after cardioplegic arrest with rewarming (n = 175). Results. Myocyte contractile function decreased by 43% from baseline after simulated cardioplegic arrest with rewarming. Under normothermic conditions, protamine (20 μg/mL) reduced myocyte contractile function by 43.9% ± 4.3%, whereas myocyte contractile function decreased by only 31.1% ± 2.7% with protamine (20 μg/mL) after cardioplegic arrest with rewarming. Thus, the negative effects of protamine on myocyte contractility were attenuated after cardioplegic arrest when compared with normothermic conditions. Conclusions. The present study demonstrated that simulated cardioplegic arrest with rewarming and protamine have direct and interactive effects on myocyte contractile function, which are not additive or synergistic.

One proposed contributory mechanism for depressed ventricular performance after hypothermic, hyperkalemic cardioplegic arrest is a reduction in myocyte contractile function caused by alterations in intracellular calcium homeostasis. Because 2,3-butanedione monoxime decreases intracellular calcium transients, this study tested the hypothesis that 2,3-butanedione monoxime supplementation of the hyperkalemic cardioplegic solution could preserve isolated myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Myocytes were isolated from the left ventricles of six pigs. Magnitude and velocity of myocyte shortening were measured after 2 hours of incubation under normothermic conditions (37º C, standard medium), hypothermic, hyperkalemic cardioplegic arrest (4º C in Ringer's solution with 20 mEq potassium chloride), and hypothermic, hyperkalemic cardioplegic arrest with 2,3-butanedione monoxime supplementation (4º C in Ringer's solution with 20 mEq potassium chloride and 20 mmol/L 2,3-butanedione monoxime). Because β-adrenergic agonists are commonly employed after cardioplegic arrest, myocyte contractile function was examined in the presence of the β-agonist isoproterenol (25 nmol/L). Hypothermic, hyperkalemic cardioplegic arrest and rewarming reduced the velocity (32%) and percentage of myocyte shortening (27%, p < 0.05). Supplementation with 2,3-butanedione monoxime normalized myocyte contractile function after hypothermic, hyperkalemic cardioplegic arrest. Although β-adrenergic stimulation significantly increased myocyte contractile function under normothermic conditions and after hypothermic, hyperkalemic cardioplegic arrest, contractile function of myocytes exposed to β-agonist after hypothermic, hyperkalemic cardioplegic arrest remained significantly reduced relative to the normothermic control group. Supplementation with 2,3-butanedione monoxime restored β-adrenergic responsiveness of myocytes after hypothermic, hyperkalemic cardioplegic arrest. Thus, supplementation of a hyperkalemic cardioplegic solution with 2,3-butanedione monoxime had direct and beneficial effects on myocyte contractile function and β-adrenergic responsiveness after cardioplegic arrest. A potential mechanism for the effects of 2,3-butanedione monoxime includes modulation of intracellular calcium transients or alterations in sensitivity to calcium. Supplementation with 2,3-butanedione monoxime may have clinical utility in improving myocardial contractile function after hypothermic, hyperkalemic cardioplegic arrest. (J THORAC CARDIOVASC SURG 1996;111:621-9)

Delayed rectifier K+ current (IK) was studied in isolated myocytes from canine left ventricular epicardium and midmyocardium using whole cell patch-clamp techniques. IK density during activation was greater in epicardial vs. midmyocardial myocytes [1.06 +/- 0.09 vs. 0.66 +/- 0.09 pA/pF (SE); P < 0.01] measured during 3-s depolarizing pulses to +25 mV. IK density was greater in epicardial myocytes at all times examined (range 150 ms to 3 s, +25 mV) and on termination of 3-s test pulses (potentials +5 to +65 mV). Greater IK density could not be explained by differences in activation kinetics or voltage dependence of activation. Two components of IK (IK,r and IK,s) have been described in guinea pig myocytes (Sanguinetti, M. C., and N. Jurkiewicz, J. Gen. Physiol. 96: 192–214, 1990). To assess whether differences in IK density could be ascribed to IK,r or IK,s, tail currents were fit to the sum of two decaying exponentials, with each component analogous to IK,s and IK,r based on sensitivity to E-4031 and rectification properties. Greater tail current density in epicardial myocytes was due to greater IK,s with no discernible difference in IK,r. These results suggest that regional differences in IK density in left ventricular epicardium are due to a larger IK,s component, which contributes to ventricular electrical heterogeneity and may reflect the differential expression of the IsK channel.

We have developed a new method to measure [Na+]i and Ca2+ transients of a beating cardiac myocyte using a Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI) and a Ca(2+)-sensitive fluorescent probe, fluo-3. There was no interaction between two probes, and the artifact due to contraction could be eliminated in the measurement of [Na+]i. [Na+]i in guinea pig ventricular myocytes stimulated at 1 Hz was 8.0 +/- 0.7mM. Strophanthidin (10 microM), initially, increased the amplitude and the basal level of Ca2+ transients in association with an increase in [Na+]i. When arrhythmias were induced, the amplitude of Ca2+ transients decreased while [Na+]i and the basal level of Ca2 transients continued to increase. These results suggested that the diastolic [Ca2+]i was closely related to [Na+]i. However, the systolic [Ca2+]i, which could be influenced by other factors, was dissociated from [Na+]i in the condition of Ca2+ overload.

A method is presented which allows accurate automatic quantification of the protective effect of drugs in cardiomyocytes. Isolated myocytes, obtained by enzymatic digestion of heart tissue by collagenase, are kept in petri dishes which can be mounted on a microscope table. Test compounds are added to the medium. Light intensity settings and focus positions are automatically adjusted, and a number of consecutive images is acquired by scanning the stage with stepper motors, capturing frames, and storing the (pre-)images on disk. After applying a pathological stimulus to the cells, cardiomyocytes display an irreversible hypercontracture from rod- to round-shaped cells. The same fields (post-images) are captured again and also stored. After image segmentation and labeling, corresponding cells in the pre- and post-images are measured and classified into ‘rod-’ and ‘round-shaped’ groups. Finally, the protection factor is calculated as the percentage of cells remaining rod-shaped after the stimulus (survival fraction). This value is printed together with a composite image of the selected cells before and after aggression for archiving and quality control. The protection factors are evaluated to select promising cardioprotective drugs. Protection values are incorporated into a database for pharmacological information retrieval, and can be further used for determining structure–activity relationships.

It has been postulated that changes in the gross anatomic features of the heart due to various hemodynamic alterations (for example, pressure or volume overloading) are mirrored by specific changes in myocyte shape. The development of the isolated cardiac myocyte preparation has been instrumental in providing a more consistent and expeditious means for assessing changes in cellular dimensions. Data obtained from isolated myocytes collected from adult rats with various types of hemodynamic overloading confirm that cell shape changes in a consistent and predictable manner and that hypertrophy, rather than hyperplasia, is responsible for the increase in cardiac mass. The use of isolated myocytes should provide answers to many other questions regarding ventricular remodeling.

CHF patients commonly experience muscle weakness and fatigue as two major symptoms. An altered intracellular handling of ionized calcium has been suggested to play a vital role in impaired myocardial contraction.3 In isolated myocytes from patients with end stage heart failure, systolic ionized calcium levels were markedly decreased, while diastolic levels were elevated as compared to healthy controls.4 In addition, digitalis and beta-blocker medical therapy is frequently used in CHF patients and is known to increase myocardial ionized calcium levels.5

To the Editor: We read with a great interest the article by den Ruijter et al,1 which showed that acute application of ω3-polyunsaturated fatty acids (DHA and EPA) reduced action potential duration and decreased diastolic and systolic intracellular calcium in myocytes from pathological hearts explanted from rabbits and humans. Consequently, ω3-polyunsaturated fatty acids abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium levels after transients. The purpose of our …

We thank Drs Pignier and LeGrand for their interest in our work.1 In their letter, Drs Pignier and LeGrand express their belief that inhibition of the persistent component of the sodium current (INa,P) constitutes a key mechanism of the antiarrhythmic properties of ω3-polyunsaturated fatty acids in heart failure in our study,1 a …

Stretch-activated ion channels (SAC) serve as cardiac mechanotransducers. Mechanical stretch of intact tissue, isolated myocytes, or membrane patches rapidly elicits the opening of poorly selective cation, K+, and Cl− SAC. Several voltage- and ligand-gated channels also are mechanosensitive. SAC alter cardiac electrical activity and, with prolonged stretch, cause an intracellular accumulation of Ca2+ and Na+ that can serve to trigger multiple signaling cascades and ultimately may contribute to remodeling of the heart in response to hemodynamic stress. This chapter reviews the transmission of mechanical forces, the biophysical characteristics of cardiac SAC, and how SAC activity may be coupled to signaling cascades and thereby initiates the complex response of the heart to stretch.

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.

KCNH2 ( hERG1) encodes the α-subunit proteins for the rapidly activating delayed rectifier K+ current ( IKr), a major K+ current for cardiac myocyte repolarization. In isolated myocytes IKr frequently is small in amplitude or absent, yet KCNH2 channels and IKr are targets for drug block or mutations to cause long QT syndrome. We hypothesized that KCNH2 channels and IKr are uniquely sensitive to enzymatic damage. To test this hypothesis, we studied heterologously expressed K+, Na+, and L-type Ca2+ channels, and in ventricular myoctyes IKr, slowly activating delayed rectifier K+ current ( IKs), and inward rectifier K+ current ( IK1), by using electrophysiological and biochemical methods. 1) Specific exogenous serine proteases (protease XIV, XXIV, or proteinase K) selectively degraded KCNH2 current ( IKCNH2) and its mature channel protein without damaging cell integrity and with minimal effects on the other channel currents; 2) immature KCNH2 channel protein remained intact; 3) smaller molecular mass KCNH2 degradation products appeared; 4) protease XXIV selectively abolished IKr; and 5) reculturing HEK-293 cells after protease exposure resulted in the gradual recovery of IKCNH2 and its mature channel protein over several hours. Thus the channel protein for IKCNH2 and IKr is uniquely sensitive to proteolysis. Analysis of the degradation products suggests selective proteolysis within the S5-pore extracellular linker, which is structurally unique among Kv channels. These data provide 1) a new mechanism to account for low IKr density in some isolated myocytes, 2) evidence that most complexly glycosylated KCNH2 channel protein is in the plasma membrane, and 3) new insight into the rate of biogenesis of KCNH2 channel protein within cells.