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Pathophysiological heterogeneity in cardiac tissue is related to the occurrence of arrhythmias. Of importance are regions of slowed conduction, which have been implicated in the formation of conduction block and reentry. Experimentally, it has been a challenge to produce local heterogeneity in a manner that is both reversible and well controlled. Consequently, we developed a dual-zone superfusion chamber that can dynamically create a small (5 mm) central island of heterogeneity in cultured cardiac cell monolayers. Three different conditions were studied to explore the effect of regionally slowed conduction on wave propagation and reentry: depolarization by elevated extracellular potassium, sodium channel inhibition with lidocaine, and cell-cell decoupling with palmitoleic acid. Using optical mapping of transmembrane voltage, we found that the central region of slowed conduction always served as the core region around which a spiral wave formed and then revolved following a period of rapid pacing. Because of the localized slowing in the core region, we observed experimentally for the first time an S shape of the spiral wave front near its tip. These results indicate that a small region of slowed conduction can play a crucial role in the formation, anchoring, and modulation of reentrant spiral waves.

Electrophysiological variability in cardiomyocytes derived from pluripotent stem cells continues to be an impediment for their scientific and translational applications. We studied the variability of action potentials (APs) recorded from clusters of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) using high-resolution optical mapping. Over 23,000 APs were analyzed through four parameters: APD30, APD80, triangulation and fractional repolarization. Although measures were taken to reduce variability due to cell culture conditions and rate-dependency of APs, we still observed significant variability in APs among and within the clusters. However, similar APs were found in spatial locations with close proximity and in some clusters formed distinct regions having different AP characteristics that were reflected as separate peaks in the AP parameter distributions, suggesting multiple electrophysiological phenotypes. Using a recently developed automated method to group cells based on their entire AP shape, we identified distinct regions of different phenotypes within single clusters and common phenotypes across different clusters when separating APs into 2 or 3 subpopulations. The systematic analysis of the heterogeneity and potential phenotypes of large populations of hESC-CMs can be used to evaluate strategies to improve the quality of pluripotent stem cell-derived cardiomyocytes for use in diagnostic and therapeutic applications and in drug screening.

Background Sheets of cultured cardiac cells constitute an emerging experimental model of cardiac tissue electrophysiology and arrhythmia. It has been used to study fundamental properties of reentrant (spiral) waves and electric field interactions with tissue structure and thus constitutes a valuable model for investigations of fibrillation and defibrillation. Methods Optical mapping can be used to visualize action potential propagation or calcium waves in confluent monolayers of neonatal rat ventricular cells. Spiral waves can be initiated by burst pacing and terminated by an electric field shock. Results Work from this laboratory and others is reviewed regarding the behavior of single and multiple spiral waves in this model system as they pertain to fibrillation (dynamic instabilities) and defibrillation (interaction of electric fields with reentrant waves). Conclusions Subject to limitations, the cultured cell monolayer is a controlled experimental model that will be useful for further study of basic aspects of fibrillation and defibrillations.

Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene modification of NRVM monolayers by two vectors: one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, inward rectifier K+channel (Kir) 2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan blue exclusion, annexin V binding followed by flow cytometry (FCM), and terminal transferase dUTP nick-end labeling assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, small interfering RNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of inward rectifier K+current in cardiac arrhythmias.

The goal of this study was to engineer cardiac tissue constructs with uniformly anisotropic architecture, and to evaluate their electrical function using multi-site optical mapping of cell membrane potentials. Anisotropic polymer scaffolds made by leaching of aligned sucrose templates were seeded with neonatal rat cardiac cells and cultured in rotating bioreactors for 6-14 days. Cells aligned and interconnected inside the scaffolds and when stimulated by a point electrode, supported macroscopically continuous, anisotropic impulse propagation. By culture day 14, the ratio of conduction velocities along vs. across cardiac fibers reached a value of 2, similar to that in native neonatal ventricles, while action potential duration and maximum capture rate, respectively, decreased to 120ms and increased to approximately 5Hz. The shorter culture time and larger scaffold thickness were associated with increased incidence of sustained reentrant arrhythmias. In summary, this study is the first successful attempt to engineer a cm(2)-size, functional anisotropic cardiac tissue patch.

Background— Functional reentry in the heart takes the form of spiral waves. Drifting spiral waves can become pinned to anatomic obstacles and thus attain stability and persistence. Lidocaine is an antiarrhythmic agent commonly used to treat ventricular tachycardia clinically. We examined the ability of small obstacles to anchor spiral waves and the effect of lidocaine on their attachment. Methods and Results— Spiral waves were electrically induced in confluent monolayers of cultured, neonatal rat cardiomyocytes. Small, circular anatomic obstacles (0.6 to 2.6 mm in diameter) were situated in the center of the monolayers to provide an anchoring site. Eighty reentry episodes consisting of at least 4 revolutions were studied. In 36 episodes, the spiral wave attached to the obstacle and became stationary and sustained, with a shorter reentry cycle length and higher rate. Spiral waves could attach to obstacles as small as 0.6 mm, with a likelihood for attachment that increased with obstacle size. After attachment, both conduction velocity of the wave-front tip and wavelength near the obstacle adapted from their pre-reentry values and increased linearly with obstacle size. In contrast, reentry cycle length did not correlate significantly with obstacle size. Addition of lidocaine 90 μmol/L depressed conduction velocity, increased reentry cycle length, and caused attached spiral waves to become quasi- attached to the obstacle or terminate. Conclusions— Anchored spiral waves exhibit properties of both unattached spiral waves and anatomic reentry. Their behavior may be representative of functional reentry dynamics in cardiac tissue, particularly in the setting of monomorphic tachyarrhythmias.

Objective: Attempts to cardiovert tachycardia by rapid point pacing can sometimes result in transient or stable increase of the heart rate (acceleration), changed ECG morphology, and/or fibrillation. The goal of this study was to investigate the effect of rapid pacing on the dynamics of functional reentry in monolayer cultures of cardiac cells. Methods: Fully confluent, uniformly anisotropic monolayers of neonatal rat ventricular myocytes were prepared using methods of microabrasion. Cells were paced by a point electrode at rest and during functional reentry, and membrane voltages were optically mapped. Results: Point pacing readily induced single loop anisotropic functional reentry with monomorphic optical pseudo-ECG (pECG) and average rotation period of 193 ± 52 ms ( n =71 monolayers). Attempts to cardiovert reentry by rapid pacing at rates 10–50% faster than the reentry rate were successful in 57/71 monolayers. In 14/71 monolayers, the number of rotating waves was stably increased by 1 to 4, yielding a 10–70% acceleration of pECG rate and change to a different monomorphic or polymorphic pECG. The resulting multi-wave functional reentries were classified based on the number and direction of their rotating waves. The higher the number of waves in the multi-wave reentry, the more accelerated was the rate of cell firing in the monolayer. Importantly, stable acceleration was only inducible in monolayers with relatively deep and broad conduction velocity restitution relationships. Reapplication of point pacing further accelerated, decelerated, or eventually terminated the reentrant activity. Conclusions: These results suggest that stable multiplication of rotating waves in conjunction with a deep and broad conduction velocity restitution relationship is a possible mechanism for stable acceleration of functional reentry by rapid pacing.

Transmembrane potential responses of single cardiac cells stimulated at rest were studied with uniform rectangular field pulses having durations of 0.5-10 ms. Cells were enzymatically isolated from guinea pig ventricles, stained with voltage sensitive dye di-8-ANEPPS, and stimulated along their long axes. Fluorescence signals were recorded with spatial resolution of 17 microm for up to 11 sites along the cell. With 5 and 10 ms pulses, all cells (n = 10) fired an action potential over a broad range of field amplitudes (approximately 3-65 V/cm). With 0.5 and 1 ms pulses, all cells (n = 7) fired an action potential for field amplitudes ranging from the threshold value (approximately 4-8 V/cm) to 50-60 V/cm. However, when the field amplitude was further increased, five of seven cells failed to fire an action potential. We postulated that this paradoxical loss of excitation for higher amplitude field pulses is the result of nonuniform polarization of the cell membrane under conditions of electric field stimulation, and a counterbalancing interplay between sodium current and inwardly rectifying potassium current with increasing field strength. This hypothesis was verified using computer simulations of a field-stimulated guinea pig ventricular cell. In conclusion, we show that for stimulation with short-duration pulses, cells can be excited for fields ranging between a low amplitude excitation threshold and a high amplitude threshold above which the excitation is suppressed. These results can have implications for the mechanistic understanding of defibrillation outcome, especially in the setting of diseased myocardium.

Mechanoelectric coupling (MEC) has been studied extensively in the heart at the tissue and organ levels, but to only a limited extent in single cells because of the technical challenges. New results are presented in which MEC was studied in 57 single frog ventricular myocytes that were held on both ends by glass holding pipettes. Axial stretch was applied either by displacement of the pipettes, or by a glass fiber around which the cell was wrapped, that was displaced in a pulsatile or sinusoidal fashion. Electrical activity of the cell was monitored either by active contraction, by intracellular action potentials, or by focal extracellular potentials. Of more than 350 stretches applied to 57 cells with amplitudes ranging from 3% to 35%, only 4 cases of mechanically induced stimulation were observed. In 252 stretches applied to 32 cells in which action potential duration (APD) was measured, no change >20% was observed, except in 3 cells in which APD increased by >100%, and in 2 cells with extended triggered activity. Thus, in contrast to studies in intact tissue, single frog ventricular myocytes are generally insensitive to direct axial stretch. However, robust mechanosensitive responses were observed in 7 of 57 (∼12%) cells. The results of other single cell studies are reviewed, and the significance of differences in tissue-level and single cell results is discussed.

Describes experiments that determine how the inter-pulse interval between rectangular pulses in a train of pulses alters the threshold of electroporation of 1-pamitoyl 2-oleoyl phosphatidycholine bilayer lipid membranes. The bilayers were exposed to a train of sixteen 100-/spl mu/s duration pulses. Threshold voltage and the sequence number of the pulse in the train, where onset of the electroporation occurred, were recorded for six inter-pulse intervals (/spl infin/, 1000 /spl mu/s, 100 /spl mu/s, 10 /spl mu/s, 1 /spl mu/s, 0 /spl mu/s). The threshold voltage of the pulse train decreased linearly with the logarithm of the inter-pulse interval. When the inter-pulse interval was 1 /spl mu/m, electroporation threshold dropped to that of a single pulse with duration 1600 /spl mu/s (equal to the sum of all pulse durations in the train). In this case, the occurrence of bilayer rupture was almost equally frequent for all pulses in the train. When the inter-pulse interval between the pulses exceeded 1 /spl mu/s, the influence of the previous pulse on the response to the following pulse declined. It became more likely that the bilayer ruptured during the first half of the train. These experimental observations suggest that a train of pulses applied with short inter-pulse interval (less than 1 ms) can lower the electroporation threshold of bilayer lipid membranes.

Changes in transmembrane voltage (V(m)) of cardiac cells during electric field stimulation have a complex spatial- and time-dependent behaviour that differs significantly from electrical stimulation of space-clamped membranes by current pulses. A multisite optical mapping system was used to obtain 17 or 25 microm resolution maps of V(m) along the long axis of guinea-pig ventricular cells (n = 57) stained with voltage-sensitive dye (di-8-ANEPPS) and stimulated longitudinally with uniform electric field (2, 5 or 10 ms, 3-62 V cm(-1)) pulses (n = 201). The initial polarizations of V(m) responses (V(mr)) varied linearly along the cell length and reversed symmetrically upon field reversal. The remainder of the V(m) responses had parallel time courses among the recording sites, revealing a common time-varying signal component (V(ms)). V(ms) was depolarizing for pulses during rest and hyperpolarizing for pulses during the early plateau phase. V(ms) varied in amplitude and time course with increasing pulse amplitude. Four types of plateau response were observed, with transition points between the different responses occurring when the maximum polarization at the ends of the cell reached values estimated as 60, 110 and 220 mV. Among the cells that had a polarization change of > 200 mV at their ends (for fields > 45 V cm(-1)), some (n = 17/25) had non-parallel time courses among V(m) recordings of the various sites. This implied development of an intracellular field (E(i)) that was found to increase exponentially with time (tau = 7.2 +/- 3.2 ms). Theoretical considerations suggest that V(ms) represents the intracellular potential (phi(i)) as well as the average polarization of the cell, and that V(mr) is the manifestation of the extracellular potential gradient resulting from the field stimulus. For cells undergoing field stimulation, phi(i) acts as the cellular physiological state variable and substitutes for V(m), which is the customary variable for space-clamped membranes.

Although intracellular calcium ([Ca2+]i) transients in cardiac cells have been well studied in the uniformly polarized cell membrane, how these transients are modified during field stimulation when the cell membrane is nonuniformly polarized has not been investigated. In this study we characterized the effects of uniform field stimuli on [Ca2+]itransients in isolated guinea pig cardiac cells. Single guinea pig cells were enzymatically isolated, loaded with the [Ca2+]ifluorescent indicator fluo-3, and stimulated along their longitudinal axes with S1 or S1-S2 (S1-S2 = 50 ms) pulses. The fluorescence signals were recorded simultaneously from up to 12 sites along the cell length using a multisite mapping system. S1 pulse, applied during the resting phase of the action potential, induced [Ca2+]itransients that had an earlier onset at the anodal-facing end, suggesting that [Ca2+]igradients (∇[Ca2+]i) develop during the rising phase of the [Ca2+]itransients. With the assumption that the peak change in [Ca2+]iis 980 nM, ∇[Ca2+]iwas estimated to be ∼3.4 nM/μm in the anodal half of the cell for a nominal 10 V/cm field and negligible in the cathodal half. The S2 pulse that was applied during the plateau of the action potential also perturbed the [Ca2+]itransients and produced [Ca2+]igradients directed from the center to either end of the cell. Mean ∇[Ca2+]iin the anodal half of the cell (∼4.2 nM/μm) was found to be statistically higher than in the cathodal half (∼2.8 nM/μm).

Type 2 long QT (LQT2) syndrome is a cardiac disorder associated with hERG channel mutation that may lead to tachyarrhythmia and sudden death. We developed a syncytial model of cardiomyocytes (CMs) differentiated from iPS cells derived from an LQT2 patient harboring an A422T mutation. The A422T mutation has been shown to cause a marked decrease in IKr current mainly due to a trafficking defect in the hERG channel. Immunostaining of cTnI revealed that CMs were isotropically distributed with well-formed sarcomeres. Both wild type (WT) and LQT2 monolayers were stable in culture for at least 60 days with spontaneous activity, and could be paced at cycle lengths (CL) from 2000 down to 300 ms. After staining the monolayers with di-4-ANEPPS, optical mapping showed that all monolayers formed a functional syncytium that supported propagating action potentials. LQT2 monolayers had longer APD80s than WT monolayers (332±43 ms, n=6, vs. 267±46 ms, n=18, mean±SD, CL = 700 ms, p Application of 0.2μM E-4031 (IKr blocker) slowed repolarization and prolonged APD80 in both WT and LQT2 monolayers, and slowed conduction velocity in WT monolayers (4.7±1.0 cm/s, n=6 vs. 10.4±1.4 cm/s, n=11 control, CL = 1000 ms). The slowing effect may reflect a role of IKr in setting the maximum diastolic potential in these CMs where IK1 is weakly expressed, as has been recently suggested. Application of 10μM ML-T531 (IKr activator) shortened APD80 in LQT2 monolayers (313±36 ms vs. 162±19 ms, n=4, p

Introduction: Nav1.5, the main voltage-gated Na+ channel in the heart, has been shown to be involved in many cardiac diseases such as long QT syndrome, Brugada syndrome, and heart failure. Na+ channels are importantly regulated by Ca2+ calmodulin (CaM) mediated signaling: however, a fundamental understanding and physiological significance of CaM regulation of Na+ channel is incomplete. Here, we have created a transgenic mouse that harbors a mutated CaM binding motif (NaV1.5-IQ/AA), which is critical for Na+ channel regulation by Ca2+-CaM. Methods: Ventricle mass and function were analyzed with electrocardiogram, echocardiogram, and detailed invasive pressure-volume analysis. Additionally, single ventricular myocytes were obtained. Whole cell patch clamp was used to record membrane ionic currents, including sodium current, Ca2+ current, K+ currents and NCX current. Results: Homozygous mice are embryonic lethal and IQ/AA+/- mice exhibit a dramatic phenotype consisting of dilated cardiomyopathy (DCM) at 4-6 months of age with prolongation of QT. The Na+ current in IQ mice exhibits an enhanced slowly inactivating late component (INa,L) with concomitant up regulation of Na+/Ca2+ exchanger currents. Consistent with other models of DCM and heart failure, DCM in IQ/AA+/- mice was associated with a down regulation of transient outward K+ currents (Ito) and an increase in T-type Ca2+ currents. Chronic treatment with ranolazine designed to block INa,L prevented electrical remodeling of the hearts including an increase in INa,L and a down regulation of Ito. Consistent with the changes in INa,L and Ito, in ranolazine-fed IQ/AA+/- mice, the QT interval was decreased compared to vehicle (p Conclusions: The data suggest that loss of CaM-mediated regulation of Na+ channel induces dilated cardiomyopathy by enhancing late Na+ current. Taken together, our data demonstrate a dynamic interplay for Ca2+ and Na+ signaling via the CaM binding motif of Na+ channels and highlight the critical importance of late Na+ currents to myopathy and arrhythmia.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are the most promising source of cardiomyocytes (CMs) for experimental and clinical applications, but their use is largely limited by a structurally and functionally immature phenotype that most closely resembles embryonic or fetal heart cells. The application of physical stimuli to influence hPSC-CMs through mechanical and bioelectrical transduction offers a powerful strategy for promoting more developmentally mature CMs. Here we summarize the major events associated with in vivo heart maturation and structural development. We then review the developmental state of in vitro derived hPSC-CMs, while focusing on physical (electrical and mechanical) stimuli and contributory (metabolic and hypertrophic) factors that are actively involved in structural and functional adaptations of hPSC-CMs. Finally, we highlight areas for possible future investigation that should provide a better understanding of how physical stimuli may promote in vitro development and lead to mechanistic insights. Advances in the use of physical stimuli to promote developmental maturation will be required to overcome current limitations and significantly advance research of hPSC-CMs for cardiac disease modeling, in vitro drug screening, cardiotoxicity analysis and therapeutic applications., published_or_final_version

Light-mediated silencing and stimulation of cardiac excitability, an important complement to electrical stimulation, promises important discoveries and therapies. To date, cardiac optogenetics has been studied with patch-clamp, multielectrode arrays, video microscopy, and an all-optical system measuring calcium transients. The future lies in achieving simultaneous optical acquisition of excitability signals and optogenetic control, both with high spatio-temporal resolution. Here, we make progress by combining optical mapping of action potentials with concurrent activation of channelrhodopsin-2 (ChR2) or halorhodopsin (eNpHR3.0), via an all-optical system applied to monolayers of neonatal rat ventricular myocytes (NRVM). Additionally, we explore the capability of ChR2 and eNpHR3.0 to shape action-potential waveforms, potentially aiding the study of short/long QT syndromes that result from abnormal changes in action potential duration (APD). These results show the promise of an all-optical system to acquire action potentials with precise temporal optogenetics control, achieving a long-sought flexibility beyond the means of conventional electrical stimulation.

The use of human embryonic stem cell cardiomyocytes (hESC-CMs) in tissue transplantation and repair has led to major recent advances in cardiac regenerative medicine. However, to avoid potential arrhythmias, it is critical that hESC-CMs used in replacement therapy be electrophysiologically compatible with the adult atrial, ventricular, and nodal phenotypes. The current method for classifying the electrophysiology of hESC-CMs relies mainly on the shape of the cell's action potential (AP), which each expert subjectively decides if it is nodal- like, atrial-like or ventricular-like. However, the classification is difficult because the shape of the AP of an hESC-CMs may not coincide with that of a mature cell. In this paper, we propose to use a metamorphosis distance for comparing the AP of an hESC-CMs to that of an adult cell model. This involves constructing a family of APs corresponding to different stages of the maturation process, and measuring the amount of deformation between APs. Experiments show that the proposed distance leads to better interpolation and classification results.

Introduction The question of how a defibrillation shock affects the myocardium far (> approximately 1 mm; the space constant of continuum tissue models) from the electrode is not fully understood. According to a long-standing, yet to be verified, hypothesis, the relatively high-resistance intercellular gap junctions may help in coupling the shock effect to the distant myocardium by redistributing the defibrillation current and creating a sawtooth pattern of polarization in which every cell undergoes hyperpolarization and depolarization. The goal of this study was to conduct an in-depth theoretical and experimental investigation of the sawtooth effect in the simplest coupled system, that of an isolated cell-pair. Methods and results Theoretically, we present a relationship between sawtooth amplitude (STA) and junctional resistance (Rj), and show that, in a cell-pair with two cells of different lengths, the sawtooth effect may not necessarily appear as a reversal in polarization across the junction when Rj is below a critical value. Experimentally, we optically mapped transmembrane potential responses along the lengths of enzymatically isolated guinea pig cell-pairs at 10- or 17-microm resolution, and estimated STA as the magnitude of discontinuity in responses at the intercellular junction. From 14 cell-pairs, STA was estimated to be approximately 11 mV for a nominal 10 V/cm field. Based on our theoretical results, this value corresponds to an Rj of approximately 18 Mohms. Conclusion The intercellular junction induces a measurable sawtooth effect in the simplest system of an isolated cell-pair. An accounting for the sawtooth effect might be essential for understanding field-tissue interaction far from the electrode and to accurately predict tissue response during field stimulation.

Transmembrane potential ( V m) responses in cardiac strands with different curvature were characterized during uniform electric-field stimulation with the use of modeling and experimental approaches. Linear and U-shaped strands (width 100–150 μm) were stained with voltage-sensitive dye. V m was measured by optical mapping across the width and at sites of beginning curvature. Field pulses were applied transverse to the strands during the action-potential plateau. For linear strands, V mcontained 1) a rapid passive component ( V m ar) nearly linear and symmetric across the width, 2) a slower hyperpolarizing component ( V m as) greater and faster on the anodal side, and 3) at high field strengths a delayed depolarizing component ( V m ad) greater on the anodal side. For U-shaped strands, V m at sites of beginning curvature also contained rapid and slow components ( V m br and V m bs, respectively) that included contributions from the linear strand response and from the fiber curvature. V m ar, V m br, and part of V m bs could be attributed to passive behavior that was modeled, and V m as, V m ad, and part of V m bs could be attributed to active membrane currents. Thus curved strands exhibit field responses separable into components with characteristic amplitude, spatial, and temporal signatures.

The focal extracellular potential (FEP) described in this study is an electrophysiological signal related to the transmembrane potential ( V m) of cardiac myocytes that avoids the mechanical fragility, interference with contraction, and intracellular contact associated with conventional whole cell recording. One end of a frog ventricular myocyte was secured into a glass holding pipette. The FEP was measured differentially between this pipette and a bath pipette while the cell was voltage- or current-clamped by a third whole cell pipette. The FEP appeared as an amplitude-truncated action potential, while FEP duration accurately reflected the action potential duration (APD) at 90% repolarization (APD90). FEP magnitude increased as the holding pipette K+ concentration ([K+]) was increased. The FEP-voltage relation was quasi-linear at negative V m with a slope that increased with elevated holding pipette [K+]. Increasing the membrane conductance inside the holding pipette by adding amphotericin B or cromakalim linearized the FEP-voltage relation across all V m. The FEP accurately reported electrical activation and APD90 during changes of stimulation frequency and episodes of cellular stretch.

Because stretch-induced activation may be important in generating clinically relevant arrhythmias in the heart, we delineated the ability of different types of stretches to activate ventricular tissue. Geometrically simple sheets of frog ( Rana catesbeiana) ventricular tissue were mounted to allow stretches to be applied perpendicular to one edge. Every heart could be activated by a stretch pulse ( n = 25), and several parameters were varied to determine their effects on mechanical activation threshold. At shorter coupling intervals, a larger stretch was needed to excite the tissue, and activation-recovery intervals were shorter, similar to previously published electrically probed strength-interval and restitution relations. Additionally, the tissue became easier to activate as the speed of the stretch increased from 0.09 to 2.6% length/ms. The increment in stretch needed for activation decreased as the baseline stretch increased from 0 to 6% length. Thus we show that mechanical activation is similar to electrical activation and that increasing uniquely mechanical parameters such as the speed of the applied stretch or baseline level of stretch can decrease the mechanical activation threshold.

Action Potential Change During RF Ablation. introduction: Although radiofrequency (RF) catheter ablation is used to treat certain cardiac arrhythmias, little is known regarding transient changes in cellular electrophysiology during and following RF delivery. Optical recordings of action potential (OAP) with voltage-sensitive dyes allow immunity from electrical noise during RF delivery. The purpose of this study was to clarify the possible synergistic effects of both the thermal and electrotonic components of RF ablation. Methods and Results: In this study, OAPs were recorded on the epicardium of 16 isolated Langendorff-perfused rabbit hearts within or adjacent to lesions made by RF catheters. Hearts were perfused at room temperature with Tyrode's solution containing 2,3-butanedione monoxime and stained by the voltage-sensitive dye di-4-ANEPPS. OAPs were recorded before, during, and after RF pulses. Within the lesion, the action potential duration at 80% repolarization (APD80) of OAP decreased rapidly during the RF pulse, without recovery following the pulse. In the border zone surrounding the lesion, the RF energy resulted in a rapid decrease in APD80, which recovered promptly after the pulse (recovery time constant: 82 ± 37 sec). APD80 was nonlinearly related to temperature during the RF ablation and responded faster to RF ablation than to purely thermal injury. Conclusion: The application of RF energy results in significant changes in myocardial cellular electrophysiologic properties. The RF energy bas a combination of thermal and electrotonic effects on the myocardial tissue. The results of this in vitro study may illustrate the cellular basis for commonly observed phenomena in clinical practice.

The effects of the channel-forming peptide gramicidin D (gD) on the conductance and electroporation thresholds of planar bilayer lipid membranes, made of the synthetic lipid 1-palmitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 200-900 mV) rectangular voltage pulses of 15 ms duration were used to perturb the bilayers and monitor the transmembrane conductance. Electroporation voltage thresholds were found, and conductance was recorded before and after electroporation. Gramicidin was added to the system in peptide/lipid ratios of 1:10, 000, 1:500, and 1:15. The addition of gD in a ratio of 1:10,000 had no effect on electroporation, but ratios of 1:500 and 1:15 significantly increased the thresholds by 16% (p < 0.0001) and 40% (p < 0.0001), respectively. Membrane conductance before electroporation was measurable only after the addition of gD and increased monotonically as the peptide/lipid ratio increased. The effect of gD on the membrane area expansivity modulus (K) was tested using giant unilamellar vesicles (GUVs). When gD was incorporated into the vesicles in a 1:15 ratio, K increased by 110%, consistent with the increase in thresholds predicted by an electromechanical model. These findings suggest that the presence of membrane proteins may affect the electroporation of lipid bilayers by changing their mechanical properties.

Source Separation and Field Stimulation. Introduction: While it is now understood that the tissue geometry and the electric field distribution are important in generating virtual electrodes, the effects of interaction between a collection of electrodes have not been examined. To develop a basis for understanding such interactions, we have studied a single pair of oppositely polarized virtual sources. Although such oppositely polarized pairs of virtual electrodes can be generated by a variety of field distributions and tissue geometries, we examine one simple system that incorporates the salient features of source interaction. Methods and Results: Our model system is a homogeneous tissue strip stimulated by a uniform extracellular field. To clarify virtual source interaction, we show that field stimulated tissue can be equivalently polarized by a set of intracellular current sources with magnitude and distribution defined by the generalized activating function. In our model system, an intracellular current source is produced at one edge of the tissue and an Intracellular current sink at the other. Therefore, the tissue length acts to modulate the overlap, or interaction, between the polarizations arising from each source. To quantify the effects of source interaction, the chronaxie and rheobase values of the strength-duration relation were determined for source separations varying between 1.0 cm and 100 μm (active membrane dynamics were modeled with the Luo-Rudy phase I formulation). At all separations > 3.0 mm, the chronaxie was constant at 3.09 msec and the rheobase was 0.38 V/cm. Under 0.2 mm, the chronaxie decreased to 0.55 msec while the rheobase increased linearly with the inverse of source separation. The dependence of these parameters on separation primarily reflects passive electrotonic interactions between the two virtual electrodes. However, the exact values are strongly dependent upon active tissue properties—largely the inward rectifier potassium channel and activation of the sodium current. Conclusion: Tissue excitation in response to field stimulation is strongly modulated by the proximity of, and therefore the interaction between, oppositely polarized virtual electrode sources.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.

Cardiac tissue dysfunction can result from high-intensity electrical shocks and is manifested as changes in transmembrane potential ( V m). Ten-millisecond shock pulses (SPs) of varying intensity and polarity were applied to frog ventricle in diastole, and V m was quantified directly under the stimulating electrode by an optical method using voltage-sensitive dye. As SP intensities were increased, the shock-induced action potential (AP) plateau and AP amplitude (APAs) decreased sigmoidally toward 75–85% of the control AP amplitude (APAc) and zero, respectively. APAswas shifted toward lower current densities for anodal compared with cathodal SPs (half-maximal values 185 and 238 mA/cm2, respectively; P = 0.02). Recovery of APAs was marginally significant 1 s after SP delivery ( P = 0.063). The peak change in V mduring SP (across all intensity levels) was −200% APAc for anodal and +125% APAc for cathodal pulses. In conclusion, we show that SP reduces APA in a sigmoidal fashion at strengths >10–20 × diastolic threshold and is more deleterious for anodal polarities.

To fully understand the mechanisms of defibrillation, it is critical to know how a given electrical stimulus causes membrane polarizations in cardiac tissue. We have extended the concept of the activating function, originally used to describe neuronal stimulation, to derive a new expression that identifies the sources that drive changes in transmembrane potential. Source terms, or virtual electrodes, consist of either second derivatives of extracellular potential weighted by intracellular conductivity or extracellular potential gradients weighted by derivatives of intracellular conductivity. The full response of passive tissue can be considered, in simple cases, to be a convolution of this "generalized activating function" with the impulse response of the tissue. Computer simulations of a two-dimensional sheet of passive myocardium under steady-state conditions demonstrate that this source term is useful for estimating the effects of applied electrical stimuli. The generalized activating function predicts oppositely polarized regions of tissue when unequally anisotropic tissue is point stimulated and a monopolar response when a point stimulus is applied to isotropic tissue. In the bulk of the myocardium, this new expression is helpful for understanding mechanisms by which virtual electrodes can be produced, such as the hypothetical "sawtooth" pattern of polarization, as well as polarization owing to regions of depressed conductivity, missing cells or clefts, changes in fiber diameter, or fiber curvature. In comparing solutions obtained with an assumed extracellular potential distribution to those with fully coupled intra- and extracellular domains, we find that the former provides a reliable estimate of the total solution. Thus the generalized activating function that we have derived provides a useful way of understanding virtual electrode effects in cardiac tissue.

Cultured heart cells have long been valuable for characterizing biological mechanism and disease pathogenesis. However, these preparations have limitations, relating to immaturity in key properties like excitation-contraction coupling and β-adrenergic stimulation. Progressive attenuation of the latter is intimately related to pathogenesis and therapy in heart failure. Highly valuable would be a long-term culture system that emulates the structural and functional changes that accompany disease and development, while concurrently permitting ready access to underlying molecular events. Accordingly, we here produce functional monolayers of adult guinea-pig ventricular myocytes (aGPVMs) that can be maintained in long-term culture for several weeks. At baseline, these monolayers exhibit considerable myofibrillar organization and a significant contribution of sarcoplasmic reticular (SR) Ca(2+) release to global Ca(2+) transients. In terms of electrical signaling, these monolayers support propagated electrical activity and manifest monophasic restitution of action-potential duration and conduction velocity. Intriguingly, β-adrenergic stimulation increases chronotropy but not inotropy, indicating selective maintenance of β-adrenergic signaling. It is interesting that this overall phenotypic profile is not fixed, but can be readily enhanced by chronic electrical stimulation of cultures. This simple environmental cue significantly enhances myofibrillar organization as well as β-adrenergic sensitivity. In particular, the chronotropic response increases, and an inotropic effect now emerges, mimicking a reversal of the progression seen in heart failure. Thus, these aGPVM monolayer cultures offer a valuable platform for clarifying long elusive features of β-adrenergic signaling and its plasticity.

Field-Induced Excitatory Responses Across Propagating Waves. Introduction: Biphasic (BP) defibrillation waveforms have been shown to be significantly more efficacious than equivalent monophasic (MP) waveforms. However, when defibrillation falls, it tends to do so first in distal regions of the heart where induced field gradient magnitudes are lowest. We tested the hypothesis that the improved efficacy of BP waveforms results from their enhanced ability to prevent the initiation of new postshock activation fronts behind preexisting; wavetails, rather than from any significantly improved ability to terminate preexisting wavefronts. Methods and Results: An idealized computer model of a one-dimensional cardiac strand was used to investigate the spatial and temporal interactions between an underlying propagation front (or tail) and uniform MP or BP field stimuli of various intensities. Axial discontinuities from intercellular junctions induced sawtooth patterns of polarization during such field stimuli, enabling the shocks to interact directly with all cells. MP and BP diastolic thresholds were essentially equal. All suprathreshold MP and BP field stimuli successfully terminated preexisting wavefronts by directly depolarizing tissue ahead of those fronts, thus blocking their continued progression. However, the postshock response at the wavetail was significantly dependent on the shape and strength of the administered field. Low-strength MP stimuli induced an all-or-none excitation response across the wavetail, producing a sharp spatial transmembrane voltage gradient from which a new sustained anterogradely propagating wavefront was initiated. In contrast, low-strength BP field stimuli induced a spatially graded excitatory response whose voltage gradient was insufficient to initiate such a wavefront. Higher-strength MP and BP stimuli both produced graded excitatory responses with no subsequent propagation. Conclusions: Shock-induced spatial “all-or-none” excitatory responses facilitate, and graded excitatory responses prevent, the postshock initiation of new propagating wavefronts. Moreover, BP field stimuli can induce such graded excitatory responses at significantly lower stimulus strengths than otherwise equivalent MP stimuli. Therefore, these results support an alternative “graded excitatory response” mechanism for the improved efficacy of BP over MP field stimuli in low gradient regions.

Recent theoretical models of cardiac electrical stimulation or defibrillation predict a complex spatial pattern of transmembrane potential (Vm) around a stimulating electrode, resulting from the formation of virtual electrodes of reversed polarity. The pattern of membrane polarization has been attributed to the anisotropic structure of the tissue. To verify such model predictions experimentally, an optical technique using a fluorescent voltage-sensitive dye was used to map the spatial distribution of Vm around a 150-microns-radius extracellular unipolar electrode. An S1-S2 stimulation protocol was used, and vm was measured during an S2 pulse having an intensity equal to 10x the cathodal diastolic threshold of excitation. The recordings were obtained on the endocardial surface of bullfrog atrium in directions parallel and perpendicular to the cardiac fibers. In the longitudinal fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) but only in a region within 445 +/- 112 microns (and 616 +/- 78 microns for anodal pulses) from the center of the electrode (n = 9). Outside this region, vm reversed polarity and reached a local maximum at 922 +/- 136 microns (and 988 +/- 117 microns for anodal pulses) (n = 9). Beyond this point vm decayed to zero over a distance of 1.5-2 mm. In the transverse fiber direction, the membrane depolarized for cathodal pulses (and hyperpolarized for anodal pulses) at all distances from the electrode. The amplitude of the response decreased with distance from the electrode with an exponential decay constant of 343 +/- 110 microns for cathodal pulses and 253 +/- 91 microns for anodal pulses (n = 7). The results were qualitatively similar in both fiber directions when the atrium was bathed in a solution containing ionic channel blockers. A two-dimensional computer model was formulated for the case of highly anisotropic cardiac tissue and qualitatively accounts for nearly all the observed spatial and temporal behavior of vm in the two fiber directions. The relationships between vm and both the "activating function" and extracellular potential gradient are discussed.

The excitation threshold of enzymatically dissociated single frog ventricular myocytes was measured as a function of cell length or sarcomere length. Field stimulation with 3 ms duration rectangular current pulses was applied via two platinum wire electrodes oriented either parallel or perpendicular to the long axis of the cell. Excitation threshold was measured as the amplitude of applied current, with an error of less than 1%. Single cells were held isometrically by two glass pipettes and wrapped around an optical fibre, which was used to control cell length. Sarcomere length was measured using video microscopy and a phase-locked loop system. Changes in cell length and sarcomere length were induced by mechanical stretch and release of the whole cell. The excitation threshold decreased with increasing resting length (preload), and increased with decrease of the cell length back to its original length, regardless of the orientation of the electric field. The measured change in excitation threshold with stretch was of the order of 1%/% strain. These results suggest that stretch affects the excitation threshold of cardiac cells by factors other than simple shape changes in the cell, perhaps through the activity of mechanosensitive ionic channels.

Experimental and clinical observations confirm that certain biphasic (BP) defibrillation shocks are significantly more efficacious than equivalent monophasic (MP) shocks, yet the mechanisms underlying these improvements are still not well understood. The authors used two separate, but related, computer models to investigate in detail the excitation responses of active cardiac cells and tissue to idealized premature extracellular MP and BP field stimuli. The results revealed a large disparity in MP and BP excitation responses to low-strength, but not high-strength, fields. In particular, at these low-strength levels, the polarity reversal within BP shocks effectively extends excitability to earlier cellular refractory states than can be achieved with simple MP shocks. Moreover, whereas low-strength MP shocks induce a distinct all-or-none excitatory response to varying shock prematurities, the excitatory response to equivalent BP shocks remains highly graded. In tissue simulations where such field stimuli intersected propagating wave fronts, the all-or-none excitatory response elicited by low-strength MP shocks created a postshock discontinuity in the spatial transmembrane voltage profile, which initiated a new propagation wave front. In contrast, the graded excitatory response elicited by BP waveforms effectively prevented the formation of postshock wave fronts. High-strength MP and BP stimuli prevented renewed propagation equally well. In conclusion, these results suggest a new mechanism for BP defibrillation superiority over MP waveforms: that the graded excitatory response to BP stimuli at low-field strengths effectively prevents the formation of large spatial transmembrane voltage gradients, which can lead to renewal of propagated wave fronts

Human embryonic stem cells have emerged as the prototypical source from which cardiomyocytes can be derived for use in drug discovery and cell therapy. However, such applications require that these cardiomyocytes (hESC-CMs) faithfully recapitulate the physiology of adult cells, especially in relation to their electrophysiological and contractile function. We review what is known about the electrophysiology of hESC-CMs in terms of beating rate, action potential characteristics, ionic currents, and cellular coupling as well as their contractility in terms of calcium cycling and contraction. We also discuss the heterogeneity in cellular phenotypes that arises from variability in cardiac differentiation, maturation, and culture conditions, and summarize present strategies that have been implemented to reduce this heterogeneity. Finally, we present original electrophysiological data from optical maps of hESC-CM clusters.

In this work, we utilize a CMOS active pixel sensor in a fluorescence imaging setup. The ability to sense small light intensity changes on top of a large baseline with spatial resolution at the subcellular scale is required in fluorescence imaging. The CMOS imager presented in [1] is perfect for this application with the ability to resolve fine features coupled with high dynamic range. By using a custom imager with a relay lens we are able to realize a dramatic decrease in device size, cost and complexity of the whole system.

Background The production of cardiomyocytes from human induced pluripotent stem cells (hiPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling, and cardiac regeneration. However, existing protocols for the differentiation of hiPSC to the cardiac lineage are inefficient and highly variable. We describe a highly efficient system for differentiation of human embryonic stem cells (hESC) and hiPSC to the cardiac lineage. This system eliminated the variability in cardiac differentiation capacity of a variety of human pluripotent stem cells (hPSC), including hiPSC generated from CD34+ cord blood using non-viral, non-integrating methods. Methodology/Principal Findings We systematically and rigorously optimized >45 experimental variables to develop a universal cardiac differentiation system that produced contracting human embryoid bodies (hEB) with an improved efficiency of 94.7±2.4% in an accelerated nine days from four hESC and seven hiPSC lines tested, including hiPSC derived from neonatal CD34+ cord blood and adult fibroblasts using non-integrating episomal plasmids. This cost-effective differentiation method employed forced aggregation hEB formation in a chemically defined medium, along with staged exposure to physiological (5%) oxygen, and optimized concentrations of mesodermal morphogens BMP4 and FGF2, polyvinyl alcohol, serum, and insulin. The contracting hEB derived using these methods were composed of high percentages (64–89%) of cardiac troponin I+ cells that displayed ultrastructural properties of functional cardiomyocytes and uniform electrophysiological profiles responsive to cardioactive drugs. Conclusion/Significance This efficient and cost-effective universal system for cardiac differentiation of hiPSC allows a potentially unlimited production of functional cardiomyocytes suitable for application to hPSC-based drug development, cardiac disease modeling, and the future generation of clinically-safe nonviral human cardiac cells for regenerative medicine.

The use of stem cells for cardiac regeneration is a revolutionary, emerging research area. For proper function as replacement tissue, stem cell-derived cardiomyocytes (SC-CMs) must electrically couple with the host cardiac tissue. Electrophysiological mapping techniques, including microelectrode array (MEA) and optical mapping, have been developed to study cardiomyocytes and cardiac cell monolayers, and these can be applied to study stem cells and SC-CMs. MEA recordings take extracellular measurements at numerous points across a small area of cell cultures and are used to assess electrical propagation during cell culture. Optical mapping uses fluorescent dyes to monitor electrophysiological changes in cells, most commonly transmembrane potential and intracellular calcium, and can be easily scaled to areas of different sizes. The materials and methods for MEA and optical mapping are presented here, together with detailed notes on their use, design, and fabrication. We also provide examples of voltage and calcium maps of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs), obtained in our laboratory using optical mapping techniques.

The use of stem cells for cardiac regeneration is a revolutionary, emerging research area. For proper function as replacement tissue, stem cell-derived cardiomyocytes (SC-CMs) must electrically couple with the host cardiac tissue. Electrophysiological mapping techniques, including microelectrode array (MEA) and optical mapping, have been developed to study cardiomyocytes and cardiac cell monolayers, and these can be applied to study stem cells and SC-CMs. MEA recordings take extracellular measurements at numerous points across a small area of cell cultures and are used to assess electrical propagation during cell culture. Optical mapping uses fluorescent dyes to monitor electrophysiological changes in cells, most commonly transmembrane potential and intracellular calcium, and can be easily scaled to areas of different sizes. The materials and methods for MEA and optical mapping are presented here, together with detailed notes on their use, design, and fabrication. We also provide examples of voltage and calcium maps of mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs), obtained in our laboratory using optical mapping techniques.

Electroporation of the cardiac cell membrane may result from intense electric fields applied to cardiac muscle, associated for example with defibrillation and cardioversion. We analyzed the distribution of voltage levels sufficient to cause electroporation in enzymatically isolated frog cardiac cells, using the cell-attached patch-clamp technique with rectangular pulses similar to those used in experimental studies of cardiac defibrillation. Five-millisecond monophasic or ten-millisecond biphasic symmetric (1/1) and asymmetric (1/0.5) rectangular pulses of either polarity were applied to the cell membrane in 100-mV steps from 0.2 to 0.8 V. The membrane conductance was continuously monitored by a low-voltage pulse train. In a total of 77 cells, we observed a step increase in conductance, occurring in 21% of cells at a transmembrane potential of 0.3 V, 52% at 0.4 V, 14% at 0.5 V, and 13% at 0.6-0.8 V. Electroporation occurred with this voltage distribution regardless of pulse shape, polarity, or the presence of all of the following ionic channel blockers: tetrodotoxin, barium, tetraethylammonium, 4-aminopyridine, cadmium, nickel, and gadolinium. The time course of membrane recovery was highly variable. The maintenance of a high membrane conductance after the shock pulse was associated with irreversible cell contracture provided that Ca2+ was included in the patch-pipette solution. However, with biphasic asymmetric pulses, the conductance recovered very quickly (< or = 37 ms).(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical stimulation of cardiac cells by imposed extracellular electric fields results in a transmembrane potential which is highly nonuniform, with one end of the cell depolarized and the other end hyperpolarized along the field direction. To date, the implications of the close proximity of oppositely polarized membranes on excitability have not been explored. In this work we compare the biophysical basis for field stimulation of cells at rest with that for intracellular current injection, using three Luo-Rudy type membrane patches coupled together as a lumped model to represent the cell membrane. Our model shows that cell excitation is a function of the temporal and spatial distribution of ionic currents and transmembrane potential. The extracellular and intracellular forms of stimulation were compared in greater detail for monophasic and symmetric biphasic rectangular pulses, with duration ranging from 0.5 to 10 ms. Strength-duration curves derived for field stimulation show that over a wide range of pulse durations, biphasic waveforms can recruit and activate membrane patches about as effectively as can monophasic waveforms having the same total pulse duration. We find that excitation with biphasic stimulation results from a synergistic, temporal summation of inward currents through the sodium channel in membrane patches at opposite ends of the cell. Furthermore, with both waveform types, a net inward current through the inwardly rectifying potassium channel contributes to initial membrane depolarization. In contrast, models of stimulation by intracellular current injection do not account for the nonuniformity of transmembrane potential and produce substantially different (even contradictory) results for the case of stimulation from rest.

An optical method was used to measure action potentials from frog ventricle, in vitro, under normal physiological conditions with 0.5-1 mM Ca2+ Ringer's solution. The approach presented in this paper involves a portable fluorimeter coupled to a multimode optical fiber running into a glass pipette ("optrode") to carry both excitation light to and fluorescence from the ventricle stained with the voltage sensitive dye di-4-ANEPPS. A suction technique was used to stabilize the optrode-tissue interface, significantly reducing motion artifacts from the beating ventricle. The typical fractional change in fluorescence intensity for an action potential was -9%. The optical recordings faithfully reproduced membrane action potentials as measured with microelectrode recordings. To confirm further the validity of our method we studied the effect of an increasing stimulation rate on the optical action potential. The amplitude of the action potential did not increase, and the change in action potential duration was similar to published results obtained with microelectrode recordings, suggesting that our optical action potentials are relatively free of motion artifacts. Finally, our optical recordings suggest that during anodal and cathodal point stimulation, the time course of membrane potential differs from that predicted simply by a passive cable model.

This review focuses on mechanoelectric feedback and mechanotransduction in cardiac cells and stem-cell derived cardiomyocyte progenitor cells. Topics include: methods to apply mechanical stimuli to isolated cells and tissues; methods for patterned growth of cells; effects of stretch and shear stress on cellular function and tissue electrophysiology; regulation of structural and junctional proteins by stretch; the role of the cytoskeleton in mechanotransduction and heart failure; signaling pathways involved in mechanotransduction and load-induced hypertrophic responses; and the role of substrate stiffness in stem cell differentiation and maturation of excitation-contraction coupling.

Previous studies have postulated an important role for the inwardly rectifying potassium current ( I K1 ) in controlling the dynamics of electrophysiological spiral waves responsible for ventricular tachycardia and fibrillation. In this study, we developed a novel tissue model of cultured neonatal rat ventricular myocytes (NRVMs) with uniform or heterogeneous Kir2.1expression achieved by lentiviral transfer to elucidate the role of I K1 in cardiac arrhythmogenesis. Kir2.1-overexpressed NRVMs showed increased I K1 density, hyperpolarized resting membrane potential, and increased action potential upstroke velocity compared with green fluorescent protein–transduced NRVMs. Opposite results were observed in Kir2.1-suppressed NRVMs. Optical mapping of uniformly Kir2.1 gene-modified monolayers showed altered conduction velocity and action potential duration compared with nontransduced and empty vector-transduced monolayers, but functional reentrant waves could not be induced. In monolayers with an island of altered Kir2.1 expression, conduction velocity and action potential duration of the locally transduced and nontransduced regions were similar to those of the uniformly transduced and nontransduced monolayers, respectively, and functional reentrant waves could be induced. The waves were anchored to islands of Kir2.1 overexpression and remained stable but dropped in frequency and meandered away from islands of Kir2.1 suppression. In monolayers with an inverse pattern of I K1 heterogeneity, stable high frequency spiral waves were present with I K1 overexpression, whereas lower frequency, meandering spiral waves were observed with I K1 suppression. Our study provides direct evidence for the contribution of I K1 heterogeneity and level to the genesis and stability of spiral waves and highlights the potential importance of I K1 as an antiarrhythmia target.

During defibrillation, cardioversion, and electrocution trauma, heart cells are exposed to potential gradients that increase the transmembrane potential (Vm). At sufficiently high Vm, pathological increases in cell permeability can occur. With enzymatically isolated frog heart cells (n = 29) we investigated the voltage and time sufficient for electroporation or cardiac cell membranes with rectangular voltage pulses, particularly with 5-msec monophasic, and 5- or 10-msec biphasic pulses. The rectangular voltage pulse (monophasic 0.1-1.5 V, 0.1-100 msec or symmetric biphasic 0.1-1 V, 0.4-10 msec [total duration]) was applied to the cell membrane using the cell-attached patch clamp technique, and a low voltage pulse train was added so that membrane conductance could be monitored continuously. Step increases in membrane conductance (breakdown) were observed, indicative of electroporation, and occurred with different combinations of pulse amplitude and duration; for example, for monophasic square pulses: (1 V, 0.2 msec) or (0.5 V, 0.5 msec), and for biphasic pulses: (1 V, 0.4 msec total duration) or (0.5 V, 0.8 msec). Using 5- or 10-msec rectangular pulses, breakdown occurred at a voltage around 0.4 V independent of polarity or waveform. The recovery of the permeabilized cell membrane after the voltage pulse was highly variable, in some cases not recovering at all while in other cases recovering after a lapse of seconds to minutes. These results suggest that monophasic and biphasic pulses of approximately 1 V, 0.2-0.4 msec and approximately 0.4 V, 5 msec can permeabilize the heart cell membrane even for minutes, time enough to cause an alteration in the cellular ionic composition leading to depressed or unexcitable tissue, a precursor for cardiac arrhythmia.

Orthogonal sequential shock can defibrillate the heart with greater efficacy compared with single shock defibrillation. In this study we tested the hypothesis that cardiac cells have a preferred orientation in their response to excitatory extracellular electric fields, so that orthogonal shocks may stimulate distinct populations of cells. A micropaddle electrode system was used to deliver rectangular pulses for extracellular field stimulation of individual heart cells. We found that single frog and guinea pig ventricular myocytes are excitable with rectangular pulse field stimulation over a wide range of pulse durations, ranging from 10 msec to as little as 20 microseconds. The excitation field strength varies inversely with pulse duration as described by the Weiss-Lapicque equation, although the frog myocytes show a significant "notch" at pulse durations of approximately 1-2 msec, and the guinea pig myocytes are more excitable than predicted for pulse durations of less than 0.2 msec. Every myocyte tested was more excitable when the long axis of the cell was oriented parallel to the stimulating field than when perpendicular to the field. For 2-msec pulses, the difference in field strength was a factor of 5.8 +/- 2.0 (n = 30) for frog and 2.6 +/- 0.5 (n = 23) for guinea pig myocytes. Complete excitation strength-duration curves were obtained in seven frog and 14 guinea pig cells for both parallel and perpendicular cell orientations.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential gradients imposed across cell or lipid membranes break down the insulating properties of these barriers if an intensity and time-dependent threshold is exceeded. Potential gradients of this magnitude may occur throughout the body, and in particular in cardiac tissue, during clinical defibrillation, ablation, and electrocution trauma. To study the dynamics of membrane electropermeabilization a cell-attached patch clamp technique was used to directly control the potential across membrane patches of single ventricular cells enzymatically isolated from frog (Rana pipiens) hearts. Ramp waveshapes were used to reveal rapid membrane conductance changes that may have otherwise been obscured using rectangular waveshapes. We observed a step increase (delta t less than 30 microseconds) or breakdown in membrane conductance at transmembrane potential thresholds of 0.6-1.1 V in response to 0.1-1.0 kV/s voltage ramps. Conductance kinetics on a sub-millisecond time scale indicate that breakdown is preceded by a period of instability during which the noise and amplitude of the membrane conductance begin to increase. In some cells membrane breakdown was observed to be fully reversible when using an intershock interval of 1 min (20-23 degrees C). These findings support energetic models of membrane electropermeabilization which describe the formation of membrane pores (or growth of existing pores) to a conducting state (instability), followed by a rapid expansion of these pores when the energy barrier for the formation of hydrophilic pores is overcome (breakdown).

Electrical field stimulation of the heart occurs during pacing and defibrillation. In cardiac tissue, the generalized activating function is the mathematical forcing function that drives changes in cellular transmembrane potential in response to field stimulation. It is the underlying principle giving rise to virtual electrodes near a physical electrode or throughout the tissue. The pattern and strength of the virtual electrodes is the result of a complex interaction between the spatial patterns of local electric field and local tissue properties of conductivity. This review defines the generalized activating function and illustrates its utility in a number of examples.

Morphogenesis is often considered a function of transcriptional synchrony and the spatial limits of diffusing mitogens; however, physical constrainment by the cell microenvironment represents an additional mechanism for regulating self-assembly of subcellular structures. We asked whether myocyte shape is a distinct signal that potentiates the organization of myofibrillar arrays in cardiac muscle myocytes. We engineered the shape of neonatal rat ventricular myocytes by culturing them on microfabricated fibronectin islands, where they spread and assumed the shape of the island. Myofibrillogenesis followed, both spatially and temporally, the assembly of unique actin networks whose architecture was predictable given the shape of the island. Subsequently, the z lines of the sarcomeres aligned and registered in distinct patterns in different regions of the myocytes in such a way that orthogonal axes of contraction could be distinctly engineered. These data suggest that physical constrainment of muscle cells by extracellular matrix may be an important regulator of myofibrillar organization.

The electrocardiogram (ECG) is a measure of the collective electrical behavior of the heart based on body surface measurements. With computational models or tissue preparations, various methods have been used to compute the pseudo-ECG (pECG) of bipolar and unipolar leads that can be given clinical interpretation. When spatial maps of transmembrane potential (Vm) are available, pECG can be derived from a weighted sum of the spatial gradients of Vm. The concept of a lead field can be used to define sensitivity curves for different bipolar and unipolar leads and to determine an effective operating height for the bipolar lead position for a two-dimensional sheet of heart cells. The pseudo-vectorcardiogram (pVCG) is computed from orthogonal bipolar lead voltages, which are derived in this study from optical voltage maps of cultured monolayers of cardiac cells. Rate and propagation direction for paced activity, rotation frequency for reentrant activity, direction of the common pathway for figure-eight reentry, and transitions from paced activity to reentry can all be distinguished using the pVCG. In contrast, the unipolar pECG does not clearly distinguish among many of the different types of electrical activity. We also show that pECG can be rapidly computed by two geometrically weighted sums of Vm, one that is summed over the area of the cell sheet and the other over the perimeter of the cell sheet. Our results are compared with those of an ad hoc difference method used in the past that consists of a simple difference of the sum of transmembrane potentials on one side of a tissue sheet and that of the other.