Abstract-The epicardial border zone (EBZ) of canine infarcts has increased anisotropy because of transverse conduction slowing. It remains unknown whether changes in gap junctional conductance (G j ) accompany the increased anisotropy. Ventricular cell pairs were isolated from EBZ and normal hearts (NZ). Dual patch clamp was used to quantify G j . At a transjunctional voltage (V j ) of ϩ10 mV, side-to-side G j of EBZ pairs (9.2Ϯ3.4 nS, nϭ16) was reduced compared with NZ side-to-side G j (109.4Ϯ23.6 nS, nϭ14, PϽ0.001). Key Words: gap junction Ⅲ myocardial infarction Ⅲ arrhythmias A fter coronary occlusion, a border zone of myocytes survives on the epicardial surface of healing canine infarcts, the epicardial border zone (EBZ). 1,2 The EBZ is characterized by reduced conduction velocity and increased anisotropy 1,2 associated with the occurrence of reentrant circuits and ventricular tachycardia. 1 Reduced sodium current 3,4 in EBZ myocytes may contribute to decreased conduction velocity. We studied gap junctional conductance in pairs of EBZ myocytes to determine if alterations occur and, therefore, might also contribute to changes in conduction and anisotropy. Connexin43 (Cx43) was quantified to determine if conductance changes were related to alterations in quantity of this gap junctional protein. Materials and Methods Preparation of Myocyte PairsCell pairs were obtained from EBZ of infarcted canine left ventricle, 5 days after coronary occlusion. Surgical methods for occlusion 1,2 and enzymatic techniques for cell disaggregation 4 have been described. EBZ tissue was removed from a region between the LAD and first circumflex branch that was visibly identified as infarct by its pale appearance (Figure 1), similar to the region sampled in previous studies of EBZ cells. 3,4 Because tachycardia was not induced nor reentry mapped, tissues did not come specifically from the central common pathway of reentrant circuits where we previously described redistribution of Cx43 gap junctions 5 (Figure 1). For normal pairs (NZ), tissue from a similar region in noninfarcted hearts was used.Studies were performed on both end-to-end-and side-to-sidecoupled EBZ and NZ myocyte pairs, 2 to 8 hours after isolation. The morphological criterion for side-to-side coupling was greater than 50% contact of cell lengths. The criterion for end-to-end coupling was contact of more than 60% of the end-to-end surfaces between each of two paired cells and less than 10% contact of side-to-side cell surfaces. 6 According to these criteria, about 60% of all cell pairs isolated were end-to-end coupled, and 40% were side-to-side coupled. End-to-end coupled pairs were easily identified under the optical microscope, because intercalated disks between the two cells could be seen clearly. It was sometimes difficult to identify a side-to-side coupled pair under the microscope because, on occasion, either a single large myocyte, or three myocytes coupled with each other without clear borders, resembled a cell pair. To verify that currents were recorded from a...

Gap junction conductance and distribution is heterogeneous in different regions of reentrant circuits. Lateralization of Cx43 gap junctions in CCP of reentrant circuits is associated with normal transverse conductance between cell pairs. In contrast, absence of lateralization in OP is associated with reduced transverse conductance. Despite normal anisotropic ratio, conduction velocity in CCP region remains slower than normal. This suggests that the effects of Cx43 remodeling in the infarcted heart should be interpreted in conjunction with other types of remodeling occurring in the EBZ (i.e. sarcolemmal ion channels).

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Abstract-Mice with cardiac-restricted inactivation of the connexin43 gene (CKO mice) have moderate slowing of ventricular conduction and lethal arrhythmias. Mechanisms through which propagation is maintained in the absence of Cx43 are unknown. We evaluated gap junctional conductance in CKO ventricular pairs using dual patch clamp methods. Junctional coupling was reduced to 4Ϯ2 nS (side-to-side) and 11Ϯ2 nS (end-to-end), including 21% of cell-pairs with no detectable coupling, compared with 588Ϯ104 nS (side-to-side) and 558Ϯ92 nS (end-to-end) in control cell-pairs. Key Words: gap junctions Ⅲ connexin Ⅲ remodeling Ⅲ arrhythmias C hanges in gap junction structure and function 1 in pathological conditions (gap junction remodeling) may contribute to arrhythmogenesis. 2-9 Gap junction protein quantity is reduced in heart failure, cardiomyopathy, and ischemia 2,3,5 and is redistributed around the cell perimeter in infarct border zones. 3,4 Junctional conductance is decreased in ischemia and ventricular hypertrophy. 6 -9 These changes are associated with propagation slowing and sometimes arrhythmias, although other pathological changes in diseased myocardium are also likely to play a role. Recently, genetic strategies have been used to specifically examine relationships between cardiac gap junction expression and function. 10 -14 Using a conditional gene-targeting strategy, we generated mice deficient in myocardial Cx43 (CKO mice) and demonstrated that such mice succumb to lethal ventricular tachyarrhythmias. 10 Interestingly, despite the virtual loss of the principal connexin responsible for ventricular cell coupling, effects on conduction velocity were relatively modest, with about 50% slowing. 10 In this study, we directly measured transjunctional conductance in ventricular myocyte pairs from CKO mice. We found that conductance is markedly reduced to 1% to 2% of controls. This level of conductance is predicted by theoretical studies to have a much greater conduction slowing effect [15][16][17] than observed in vivo in this model, 10 suggesting that additional mechanisms support impulse propagation when gap junctional conductance is severely reduced. 18 Materials and Methods Murine ModelGap junctional conductance was investigated in ventricular myocyte cell pairs obtained from a gene-targeted murine model in which the Cx43 gene was conditionally inactivated exclusively in cardiomyocytes by use of the Cre/loxP system (CKO mice). 10 Immunohistochemical studies and Western blot analysis have demonstrated that as many as 95% of myocytes from these CKO mice fail to express Cx43. 10 For controls, we used littermates that are homozygous "floxed" but do not express Cre recombinase and express wild-type levels of Cx43. 19 We also studied a heterozygous group that has a Ϸ50% reduction in Cx43 in the heart (unpublished data, 2003). Preparation of Ventricular MyocytesAnimals were cared for according to the guiding principles in DHEW (NIH) No. 85-23. Protocols were approved by IACUC at New York University and Columbia. CKO and con...

Objective: The rapid (I ) and slow (I ) components of delayed rectifier currents play an important role in determining the cardiac adjacent to the region where myocytes were isolated. mRNA levels of all three subunits were reduced 2 days after LAD occlusion (by 4869%, 6865%, and 4564% for dERG, dIsK and dKvLQT1, respectively, n58 each). By day 5, the dKvLQT1 message returned to control while those of dERG and dIsK remained reduced (by 5267% and 7666%, respectively

It has been shown for a Shaker channel (H-4) that its NHz-terminal cytoplasmic domain may form a "ball and chain" structure, with the "chain" tethering the "ball" to the channel while the "ball" capable of binding to the channel in its open state and causing inactivation. Equivalent structures have not been identified in mammalian Shaker homologues. We studied the functional role of the NH2-terminal region of a fast-inactivating mammalian K channel, RHK1 (Kvl.4), by deleting different domains in this region and examining the resultant changes in channel properties at whole cell and single channel levels. Deleting the NH2-terminal hydrophobic domain (domain A) or the subsequent positive charges (domain I) from RHKI greatly slowed the decay of whole cell currents, suggesting the existence of a ball-like structure in RHK1 similar to that in the Shaker channel. The function of the ball appeared to be abolished by deleting domain A, while modified but maintained by deleting domain I. In the latter case, the data suggest that the positive charges needed for the function of the ball can be replaced by amino acids from a following region (domain III) that has a high positive charge density. Deleting multiple domains from the NHz terminus of RHK1 corresponding to the chain in Shaker H-4 did not induce expected changes in channel properties that might result from a shortening of a chain. A comparison of single channel kinetics of selected mutant channels with those of the wild-type channel indicated that these deletion mutations slowed whole cell currents by prolonging burst durations and by increasing the probability of reopening during depolarization. There were no changes in single channel current amplitude or latency to first opening. In conclusion, our observations indicate that the inactivation mechanism of RHK1 is similar to that of Shaker H-4 in that a positively charged cytoplasmic domain is important for such a process. The NHz-terminal domain is not involved in channel activation or ion permeation process.

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We studied the effects of cell swelling on membrane currents of canine ventricular myocytes using the whole-cell patch-clamp method. Cell swelling was induced by lowering the osmolarity of the bath solution to 60% of control. Cell width and currents were measured simultaneously. Cell swelling induced little or no change in the L-type Ca, the inward rectifier, and the transient outward currents, but a marked increase in the slow delayed rectifier current (IKs) was seen. We further examined the role of protein kinase activities in IKs modulation by cell swelling. This modulation was not affected by inhibiting serine/threonine kinases using H-8. On the other hand, the modulation was inhibited by genistein (a protein tyrosine kinase inhibitor) although not by daidzein (an inactive analogue of genistein). Our data suggest that in canine ventricle cell swelling can increase protein tyrosine kinase activity, which can augment IKs and contribute to changes in membrane electrical activity observed under these conditions.