Minimum Information about a Cardiac Electrophysiology Experiment (MICEE): standardised reporting for model reproducibility, interoperability, and data sharing.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work.

Heterogeneous expression of Gap junction channels in the heart leads to conduction defects and ventricular dysfunction.

BACKGROUND: - Heterogeneous remodeling of gap junctions is observed in many forms of heart disease. The consequent loss of synchronous ventricular activation has been hypothesized to result in diminished cardiac performance. To directly test this hypothesis, we designed a murine model of heterogeneous gap junction channel expression. Methods and Results-- We generated chimeric mice formed from connexin43 (Cx43)-deficient embryonic stem cells and wild-type or genetically marked ROSA26 recipient blastocysts. Chimeric mice developed normally, without histological evidence of myocardial fibrosis or hypertrophy. Heterogeneous Cx43 expression resulted in conduction defects, however, as well as markedly depressed contractile function. Optical mapping of chimeric hearts by use of voltage-sensitive dyes revealed highly irregular epicardial conduction patterns, quantified as significantly greater negative curvature of the activation wave front (-1.86+/-0.40 mm in chimeric mice versus -0.86+/-0.098 mm in controls; P<0.01; n=6 for each group). Echocardiographic studies demonstrated significantly reduced fractional shortening in chimeric mice (26.6+/-2.3% versus 36.5+/-1.6% in age-matched 129/SvxC57BL/6F1 wild-type controls; P<0.05). CONCLUSIONS: - These data suggest that heterogeneous Cx43 expression, by perturbing the normal pattern of coordinated myocardial excitation, may directly depress cardiac performance.

Null mutation of connexin43 causes slow propagation of ventricular activation in the late stages of mouse embryonic development.

Connexin43 (Cx43) is the principal connexin isoform in the mouse ventricle, where it is thought to provide electrical coupling between cells. Knocking out this gene results in anatomic malformations that nevertheless allow for survival through early neonatal life. We examined electrical wave propagation in the left (LV) and right (RV) ventricles of isolated Cx43 null mutated (Cx43(-/-)), heterozygous (Cx43(+/)(-)), and wild-type (WT) embryos using high-resolution mapping of voltage-sensitive dye fluorescence. Consistent with the compensating presence of the other connexins, no reduction in propagation velocity was seen in Cx43(-/-) ventricles at postcoital day (dpc) 12.5 compared with WT or Cx43(+/)(-) ventricles. A gross reduction in conduction velocity was seen in the RV at 15.5 dpc (in cm/second, mean [1 SE confidence interval], WT 9.9 [8.7 to 11.2], Cx43(+/)(-) 9.9 [9.0 to 10.9], and Cx43(-/-) 2.2 [1.8 to 2.7; P<0.005]) and in both ventricles at 17.5 dpc (in RV, WT 8.4 [7.6 to 9.3], Cx43(+/)(-) 8.7 [8.1 to 9.3], and Cx43(-/-) 1.1 [0.1 to 1.3; P<0.005]; in LV, WT 10.1 [9.4 to 10.7], Cx43(+/)(-) 8.3 [7.8 to 8.9], and Cx43(-/-) 1.7 [1.3 to 2.1; P<0.005]) corresponding with the downregulation of Cx40. Cx40 and Cx45 mRNAs were detectable in ventricular homogenates even at 17.5 dpc, probably accounting for the residual conduction function. Neonatal knockout hearts were arrhythmic in vivo as well as ex vivo. This study demonstrates the contribution of Cx43 to the electrical function of the developing mouse heart and the essential role of this gene in maintaining heart rhythm in postnatal life.

Visualization and functional characterization of the developing murine cardiac conduction system.

The cardiac conduction system is a complex network of cells that together orchestrate the rhythmic and coordinated depolarization of the heart. The molecular mechanisms regulating the specification and patterning of cells that form this conductive network are largely unknown. Studies in avian models have suggested that components of the cardiac conduction system arise from progressive recruitment of cardiomyogenic progenitors, potentially influenced by inductive effects from the neighboring coronary vasculature. However, relatively little is known about the process of conduction system development in mammalian species, especially in the mouse, where even the histological identification of the conductive network remains problematic. We have identified a line of transgenic mice where lacZ reporter gene expression delineates the developing and mature murine cardiac conduction system, extending proximally from the sinoatrial node to the distal Purkinje fibers. Optical mapping of cardiac electrical activity using a voltage-sensitive dye confirms that cells identified by the lacZ reporter gene are indeed components of the specialized conduction system. Analysis of lacZ expression during sequential stages of cardiogenesis provides a detailed view of the maturation of the conductive network and demonstrates that patterning occurs surprisingly early in embryogenesis. Moreover, optical mapping studies of embryonic hearts demonstrate that a murine His-Purkinje system is functioning well before septation has completed. Thus, these studies describe a novel marker of the murine cardiac conduction system that identifies this specialized network of cells throughout cardiac development. Analysis of lacZ expression and optical mapping data highlight important differences between murine and avian conduction system development. Finally, this line of transgenic mice provides a novel tool for exploring the molecular circuitry controlling mammalian conduction system development and should be invaluable in studies of developmental mutants with potential structural or functional conduction system defects.

Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43.

Cardiac arrhythmia is a common and often lethal manifestation of many forms of heart disease. Gap junction remodeling has been postulated to contribute to the increased propensity for arrhythmogenesis in diseased myocardium, although a causative role in vivo remains speculative. By generating mice with cardiac-restricted knockout of connexin43 (Cx43), we have circumvented the perinatal lethal developmental defect associated with germline inactivation of this gap junction channel gene and uncovered an essential role for Cx43 in the maintenance of electrical stability. Mice with cardiac-specific loss of Cx43 have normal heart structure and contractile function, and yet they uniformly (28 of 28 conditional Cx43 knockout mice observed) develop sudden cardiac death from spontaneous ventricular arrhythmias by 2 months of age. Optical mapping of the epicardial electrical activation pattern in Cx43 conditional knockout mice revealed that ventricular conduction velocity was significantly slowed by up to 55% in the transverse direction and 42% in the longitudinal direction, resulting in an increase in anisotropic ratio compared with control littermates (2.1+/-0.13 versus 1.66+/-0.06; P:<0.01). This novel genetic murine model of primary sudden cardiac death defines gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate.

Understanding conduction of electrical impulses in the mouse heart using high-resolution video imaging technology.

The conduction of electrical impulses in the heart depends on the ability to efficiently transfer excitatory current between individual myocytes. Several recent studies have focused on the use of optical mapping techniques to determine the electrophysiological consequences and the proarrhythmic effects of reducing intercellular coupling in newly developed connexin knockout mice. This work has begun to unravel important questions regarding the role of connexins in intercellular coupling and propagation of electrical impulses in the heart. The purpose of this review is to discuss the techniques and unique issues involved in imaging electrical wave propagation in the heart. In addition, we will review recent experimental studies that address the role of intercellular communication in the development of cardiac arrhythmias.

Conditional gene targeting of connexin43: exploring the consequences of gap junction remodeling in the heart.

Abnormalities in cardiac gap junction expression have been postulated to contribute to arrhythmias and ventricular dysfunction. We investigated the role of cardiac gap junctions by generating a heart-specific conditional knock-out (CKO) of connexin43 (Cx43), the major cardiac gap junction protein. While the Cx43 CKO mice have normal heart structure and contractile function, they die suddenly from spontaneous ventricular arrhythmias. Because abnormalities in gap junction expression in the diseased heart can be focal, we also generated chimeric mice formed from Cx43-null embryonic stem (ES) cells and wildtype recipient blastocysts. Heterogeneous Cx43 expression in the chimeric mice resulted in conduction defects and depressed contractile function. These novel genetic murine models of Cx43 loss of function in the adult mouse heart define gap junctional abnormalities as a key molecular feature of the arrhythmogenic substrate and an important factor in heart dysfunction.

High-resolution optical mapping of the right bundle branch in connexin40 knockout mice reveals slow conduction in the specialized conduction system.

Connexin40 (Cx40) is a major gap junction protein that is expressed in the His-Purkinje system and thought to be a critical determinant of cell-to-cell communication and conduction of electrical impulses. Video maps of the ventricular epicardium and the proximal segment of the right bundle branch (RBB) were obtained using a high-speed CCD camera while simultaneously recording volume-conducted ECGs. In Cx40(-/-) mice, the PR interval was prolonged (47.4+/-1.4 in wild-type [WT] [n=6] and 57.5+/-2.8 in Cx40(-/-) [n=6]; P<0.01). WT ventricular epicardial activation was characterized by focused breakthroughs that originated first on the right ventricle (RV) and then the left ventricle (LV). In Cx40(-/-) hearts, the RV breakthrough occurred after the LV breakthrough. Additionally, Cx40(-/-) mice showed RV breakthrough times that were significantly delayed with respect to QRS complex onset (3.7+/-0.7 ms in WT [n=6] and 6.5+/-0.7 ms in Cx40(-/-) [n=6]; P<0.01), whereas LV breakthrough times did not change. Conduction velocity measurements from optical mapping of the RBB revealed slow conduction in Cx40(-/-) mice (74.5+/-3 cm/s in WT [n=7] and 43.7+/-6 cm/s in Cx40(-/-) [n=7]; P<0.01). In addition, simultaneous ECG records demonstrated significant delays in Cx40(-/-) RBB activation time with respect to P time (P-RBB time; 41.6+/-1.9 ms in WT [n=7] and 55.1+/-1.3 ms in [n=7]; P<0.01). These data represent the first direct demonstration of conduction defects in the specialized conduction system of Cx40(-/-) mice and provide new insight into the role of gap junctions in cardiac impulse propagation.

Early onset heart failure in transgenic mice with dilated cardiomyopathy.

In children, dilated cardiomyopathy is due to a variety of etiologies and usually carries a grave prognosis. The purpose of the present study was to carefully follow the progression of events leading to cardiac dilatation and congestive heart failure in a dilated cardiomyopathy model in neonatal and juvenile mice. These initial steps are often not well characterized. Furthermore, the loss of gap junctions and reduced electrical coupling of cardiomyocytes frequently found in human cardiomyopathies are also observed in these early stages. By 2 wk of age, molecular markers associated with hypertrophy were already altered. Cardiomyocyte hypertrophy, reduced connexin43 expression, and decreased conduction velocity were apparent by 4 wk, before overt cardiac dysfunction (decreased shortening fraction and chamber remodeling) that was not present until 12 wk of age. Our results show that in this model cardiomyopathic changes are present by 2 wk after birth and progress rapidly during the subsequent 2 postnatal weeks. Combined with the observations of other models of heart disease, we suggest that the first 2 wk of postnatal life are absolutely critical for normal cardiac development, and events that perturb homeostasis during this period determine whether the heart will continue to develop normally. These animals exhibit early symptoms of disease including reduced connexin43 and conduction defects before impaired cardiac function and demonstrate for the first time a temporal association between decreased connexin43 levels and the initiation of a contractility deficit that ends in heart failure.

Characterization of conduction in the ventricles of normal and heterozygous Cx43 knockout mice using optical mapping.

INTRODUCTION: Gap junction channels are important determinants of conduction in the heart and may play a central role in the development of lethal cardiac arrhythmias. The recent development of a Cx43-deficient mouse has raised fundamental questions about the role of specific connexin isoforms in intercellular communication in the heart. Although a homozygous null mutation of the Cx43 gene (Cx43-/-) is lethal, the heterozygous (Cx43+/-) animals survive to adulthood. Reports on the cardiac electrophysiologic phenotype of the Cx43+/- mice are contradictory. Thus, the effects of a null mutation of a single Cx43 allele require reevaluation. METHODS AND RESULTS: High-resolution video mapping techniques were used to study propagation in hearts from Cx43+/- and littermate control (Cx43+/+) mice. Local conduction velocities (CVs) and conduction patterns were quantitatively measured by determining conduction vectors. We undertook the characterization of ECG parameters and epicardial CVs of normal and Cx43+/- mouse hearts. ECG measurements obtained from 12 Cx43+/+ and 6 Cx43+/- age matched mice did not show differences in any parameter, including QRS duration (14.5 +/- 0.9 and 15.7 +/- 2.3 msec for Cx43+/+ and Cx43+/-, respectively). In addition, using a sensitive method of detecting changes in local CV, video images of epicardial wave propagation revealed similar activation patterns and velocities in both groups of mice. CONCLUSION: A sensitive method that accurately measures local CVs throughout the ventricles revealed no changes in Cx43+/- mice, which is consistent with the demonstration that ECG parameter values in the heterozygous mice are the same as those in wild-type mice.

Reentry and fibrillation in the mouse heart. A challenge to the critical mass hypothesis.

The idea that fibrillation is only possible in hearts exceeding a critical size was introduced by W. Garrey >80 years ago and has since been generally accepted. In ventricular tissue, this critical size was originally estimated to be 400 mm(2). Recent estimates suggest that the critical size required for sustained reentry is approximately 100 to 200 mm(2), whereas 6 times this area is required for ventricular fibrillation. According to these estimates, fibrillation is not possible in the mouse heart, where the ventricular surface area is approximately 100 mm(2). To test whether sustained ventricular fibrillation could be induced in such an area, we used a high-speed video imaging system and a voltage-sensitive dye to quantify electrical activity on the epicardial surface of the Langendorff-perfused adult mouse heart. In 6 hearts, measurements during ventricular pacing at a basic cycle length (BCL) of 120 ms yielded maximum and minimum conduction velocities (CV(max) and CV(min)) of 0.63+/-0.04 and 0.38+/-0.02 mm/ms, respectively. At a BCL of 80 ms, CV(max) and CV(min) changed to 0.55+/-0.03 and 0. 34+/-0.02 mm/ms. Action potential durations (APDs), measured at 70% repolarization at those pacing frequencies were found to be 44.5+/-2. 9 and 40.4+/-2.6 ms, respectively. The wavelengths (CVxAPD) were calculated to be 28.6+/-3.4 mm in the CV(max) direction and 16.8+/-1. 5 mm in the CV(min) direction at BCL 120 ms. Wavelengths were significantly reduced (P<0.05) at BCL 80 ms (CV(max), 22.2+/-1.8 mm; CV(min), 13.7+/-0.9 mm). In 5 hearts, stationary vortex-like reentry organized by single rotors (4 of 5 hearts) or by pairs of rotors (1 of 5 hearts) was induced by burst pacing. In the ECG, the activity manifested as sustained monomorphic tachycardia. Detailed analysis showed that the local CVs were reduced in the vicinity of the rotor center, which allowed the reentry to take place within a smaller area than was calculated from wavelength measurements during pacing. In 4 of 7 hearts, burst pacing resulted in a polymorphic ECG pattern indistinguishable from ventricular fibrillation. These data challenge the critical mass hypothesis by demonstrating that ventricular tissue with an area as small as 100 mm(2) is capable of undergoing sustained fibrillatory activity.

Connexins and impulse propagation in the mouse heart.

Gap junction channels are essential for normal cardiac impulse propagation. Three gap junction proteins, known as connexins, are expressed in the heart: Cx40, Cx43, and Cx45. Each of these proteins forms channels with unique biophysical and electrophysiologic properties, as well as spatial distribution of expression throughout the heart. However, the specific functional role of the individual connexins in normal and abnormal propagation is unknown. The availability of genetically engineered mouse models, together with new developments in optical mapping technology, makes it possible to integrate knowledge about molecular mechanisms of intercellular communication and its regulation with our growing understanding of the microscopic and global dynamics of electrical impulse propagation during normal and abnormal cardiac rhythms. This article reviews knowledge on the mechanisms of cardiac impulse propagation, with particular focus on the role of cardiac connexins in electrical communication between cells. It summarizes results of recent studies on the electrophysiologic consequences of defects in the functional expression of specific gap junction channels in mice lacking either the Cx43 or Cx40 gene. It also reviews data obtained in a transgenic mouse model in which cell loss and remodeling of gap junction distribution leads to increased susceptibility to arrhythmias and sudden cardiac death. Overall, the results demonstrate that these are potentially powerful strategies for studying fundamental mechanisms of cardiac electrical activity and for testing the hypothesis that certain cardiac arrhythmias involve gap junction or other membrane channel dysfunction. These new approaches, which permit one to manipulate electrical wave propagation at the molecular level, should provide new insight into the detailed mechanisms of initiation, maintenance, and termination of cardiac arrhythmias, and may lead to more effective means to treat arrhythmias and prevent sudden cardiac death.

Structure of connexin43 and its regulation by pHi.

Electrical coupling in the heart provides an effective mechanism for propagating the cardiac action potential efficiently throughout the entire heart. Cells within the heart are electrically coupled through specialized membrane channels called gap junctions. Studies have shown that gap junctions are dynamic, carefully regulated channels that are important for normal cardiogenesis. We have recently been interested in the molecular mechanisms by which intracellular acidification leads to gap junction channel closure. Previous results in this lab have shown that truncation of the carboxyl terminal (CT) of connexin43 (Cx43) does not interfere with functional channel expression. Further, the pH-dependent closure of Cx43 channels is significantly impaired by removal of this region of the protein. Other studies have shown that the CT is capable of interacting with its receptor even when not covalently attached to the channel protein. From these data we have proposed a particle-receptor model to explain the pH-dependent closure of Cx43 gap junction channels. Detailed analysis of the CT has revealed interesting new information regarding its possible structure. Here we review the most recent studies that have contributed to our understanding of the molecular mechanisms of regulation of the cardiac gap protein Cx43.

PH regulation of connexin43: molecular analysis of the gating particle.

Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.

Intramolecular interactions mediate pH regulation of connexin43 channels.

We have previously proposed that acidification-induced regulation of the cardiac gap junction protein connexin43 (Cx43) may be modeled as a particle-receptor interaction between two separate domains of Cx43: the carboxyl terminal (acting as a particle), and a region including histidine 95 (acting as a receptor). Accordingly, intracellular acidification would lead to particle-receptor binding, thus closing the channel. A premise of the model is that the particle can bind its receptor, even if the particle is not covalently bound to the rest of the protein. The latter hypothesis was tested in antisense-injected Xenopus oocyte pairs coexpressing mRNA for a pH-insensitive Cx43 mutant truncated at amino acid 257 (i.e., M257) and mRNA coding for the carboxyl terminal region (residues 259-382). Intracellular pH (pHo) was recorded using the dextran form of the proton-sensitive dye seminaphthorhodafluor (SNARF). Junctional conductance (Gj) was measured with the dual voltage clamp technique. Wild-type Cx43 channels showed their characteristic pH sensitivity. M257 channels were not pH sensitive (pHo tested: 7.2 to 6.4). However, pH sensitivity was restored when the pH-insensitive channel (M257) was coexpressed with mRNA coding for the carboxyl terminal. Furthermore, coexpression of the carboxyl terminal of Cx43 enhanced the pH sensitivity of an otherwise less pH-sensitive connexin (Cx32). These data are consistent with a model of intramolecular interactions in which the carboxyl terminal acts as an independent domain that, under the appropriate conditions, binds to a separate region of the protein and closes the channel. These interactions may be direct (as in the ball-and-chain mechanism of voltage-dependent gating of potassium channels) or mediated through an intermediary molecule. The data further suggest that the region of Cx43 that acts as a receptor for the particle is conserved among connexins. A similar molecular mechanism may mediate chemical regulation of other channel proteins.

Effect of basolateral or apical hyposmolarity on apical maxi K channels of everted rat collecting tubule.

We are able to evert and perfuse rat cortical collecting tubules (CCT) at 37 degrees C. Patch-clamp techniques were used to study high-conductance potassium channels (maxi K) on the apical membrane. Under control conditions (150 mM Na+ and 5 mM K+ in pipette and bathing solutions), the slope conductance averaged 109.8 +/- 6.6 pS (12 channels), and reversal potential (expressed as pipette voltage) was +26.3 +/- 2.4 mV. The percent of time the channel spends in the open state and unitary current when voltage was clamped to 0 mV were 1.4 +/- 0.7% and 3.12 +/- 0.42 pA, respectively. In six patches voltage clamped to 0 mV, the isosmotic solution perfused through the everted tubule (basolateral surface) was exchanged for one made 70 mosmol/kgH2O hyposmotic to the control saline. Open probability increased from 0.019 to 0.258, an increase of 0.239 +/- 0.065 (P < 0.005). In four patches where a maxi K channel was evident, no increase in open probability was observed when a hyposmotic saline was placed on the apical surface. However, when vasopressin was present on the basolateral surface, apical application of hyposmotic saline resulted in a series of bursts of channel activity. The average increase in open probability during bursts was (0.055 +/- 0.017, P < 0.005). We conclude that one function of the maxi K channel located in the apical membrane of the rat CCT may be to release intracellular solute (potassium) during a volume regulatory decrease induced by placing a dilute solution on the basolateral surface or when the apical osmolarity is reduced in the presence of vasopressin. These data are consistent with the hypothesis that the physiological role of the channel is to regulate cell volume during water reabsorption.