Deletion of the last five C-terminal amino acid residues of connexin43 leads to lethal ventricular arrhythmias in mice without affecting coupling via gap junction channels

Abstract

The cardiac intercalated disc harbors mechanical and electrical junctions as well as ion channel complexes mediating propagation of electrical impulses. Cardiac connexin43 (Cx43) co-localizes and interacts with several of the proteins located at intercalated discs in the ventricular myocardium. We have generated conditional Cx43D378stop mice lacking the last five C-terminal amino acid residues, representing a binding motif for zonula occludens protein-1 (ZO-1), and investigated the functional consequences of this mutation on cardiac physiology and morphology. Newborn and adult homozygous Cx43D378stop mice displayed markedly impaired and heterogeneous cardiac electrical activation properties and died from severe ventricular arrhythmias. Cx43 and ZO-1 were co-localized at intercalated discs in Cx43D378stop hearts, and the Cx43D378stop gap junction channels showed normal coupling properties. Patch clamp analyses of isolated adult Cx43D378stop cardiomyocytes revealed a significant decrease in sodium and potassium current densities. Furthermore, we also observed a significant loss of Nav1.5 protein from intercalated discs in Cx43D378stop hearts. The phenotypic lethality of the Cx43D378stop mutation was very similar to the one previously reported for adult Cx43 deficient (Cx43KO) mice. Yet, in contrast to Cx43KO mice, the Cx43 gap junction channel was still functional in the Cx43D378stop mutant. We conclude that the lethality of Cx43D378stop mice is independent of the loss of gap junctional intercellular communication, but most likely results from impaired cardiac sodium and potassium currents. The Cx43D378stop mice reveal for the first time that Cx43 dependent arrhythmias can develop by mechanisms other than impairment of gap junction channel function.

Fig. 1

Lethality and impaired cardiac conduction in neonatal Cx43D378stop mice. a Homozygous Cx43D378stop:αMyHC-Cre mice die within the first day after birth. In contrast, control littermates (heterozygous Cx43D378stop, heterozygous or homozygous Cx43floxD378stop mice) showed a normal life expectancy after birth. n > 10 per group. b In contrast to controls (left panel) newborn Cx43D378stop hearts (right panel) display irregular thickened trabeculae with abnormal pouch formation (asterisk) at the subpulmonary outflow tract but in contrast to Cx43KO mice the pulmonary outflow tract is still open. PV, pulmonary vein; RV, right ventricle. n > 7 per group. c Representative examples of surface-ECG recordings of a control (left) and a Cx43D378stop (right) fetus (ED 16.5). d Statistical analysis reveal significantly 3.3-fold prolonged QRS intervals in mutant mice compared to controls. n = 6 for Cx43D378stop and n = 7 for control mice

Fig. 2

Lethal ventricular arrhythmias in adult Cx43D378stop mice. a Tamoxifen-injected adult (3-month-old) homozygous Cx43D378stop:αMyHC-Cre-ER(T2) mice die between day 16 and 21 after last tamoxifen injection. In contrast, oil-injected control littermates (heterozygous or homozygous Cx43floxD378stop mice) survived normally. n > 10 per group. b Statistical evaluation of the QRS interval in long-tem ECG recordings revealed a 2.1-fold prolongation of ventricular depolarization in comparison to control mice. n = 4 per group. c Northern blot analysis verified expression of the Cx43D378stop mRNA and absence of Cx43 mRNA in adult Cx43D378stop mice 12 days after last tamoxifen injection. d–g Representative long-term ECG recordings of a Cx43D378stop mutated mouse recorded until its death. d Normal sinus rhythm at the start of recording on day 10 after last tamoxifen injection. e First occurrence of non-sustained monomorphic tachycardia on day 15. f Polymorphic tachycardia on day 17. g Polymorphic tachycardia, degenerating in ventricular fibrillation, causing death on day 18. h QRS prolongation and bundle branch block deformation on day 15

Fig. 3

Epicardial mapping of Langendorff-perfused hearts from control and Cx43D378stop mice. Representative examples of spontaneous conduction in the left ventricle of a control animal (a) and a Cx43D378stop mouse (b) during sinus rhythm. Isochronal maps show a broad conduction delay in mutant mice. Isochronal lines are shown at distances of 1 ms. n = 3 per group

Fig. 4

Coupling analysis of Cx43D378stop gap junction channels. Injection of neurobiotin into one cell at the periphery of beating cardiomyocyte clusters leads to intercellular dye transfer between neighboring cells in control (a, left panel) and Cx43D378stop:αMyHC-Cre (a, right panel) cardiomyocytes indicated by blue color. For better evaluation, the blue colored area was bordered by a black line. Statistical analysis of the coupling experiments revealed no significant difference in coupling among Cx43D378stop:αMyHC-Cre compared to control cardiomyocytes (b). Data are mean ± SEM of ≥20 injections in each cell line. Bar: 20 μm. c Application of octanol (2 mM) produced full uncoupling of Cx43D378stop homotypic junctions within ~3 s. Repeated ramps of voltage (V_j) were used to measure junctional current (I_j). Junctional conductance was calculated from the ratio, I_j/V_j. d Single channel events measured in HeLaCx43D378stop cell pair during an exposure to CO₂ that reduced cell–cell coupling initially to three and at the end of the recording to one functional channel with conductance of ~105 pS. n = 27 for junctional conductance and n = 129 for single channel events

Fig. 5

Sodium current and depolarization-activated outward potassium current properties recorded from adult cardiac myocytes isolated from Cx43D378stop hearts. a Average peak sodium current density as a function of voltage command. Peak current amplitude at –40 mV: Control: –32.0 ± 1.5 pA/pF; n = 13. Cx43D378stop: –18.7 ± 2.1 pA/pF; n = 14. P < 0.001. b Voltage-dependence of I_Na steady-state inactivation curves. Voltage for half-maximal inactivation (V_1/2):–80.2 ± 1.2 mV for control and 78.7 ± 1.4 mV for Cx43D378stop (pNS). c Time course of I_Na recovery from inactivation. Time constant (one exponential function): 5.1 ± 0.3 ms for control and 5.3 ± 0.4 ms for Cx43D378stop (pNS). d Inactivation time course (one exponential function) of I_Na: 2.6 ± 0.1 ms for control and 3.2 ± 0.2 ms for Cx43D378stop, P < 0.04. e Average peak potassium current density as a function of voltage command. Peak current amplitude at +40 mV: Control: +38.7 ± 3.4 pA/pF; n = 14. Cx43D378stop: +29.1 ± 2.1 pA/pF; n = 15, P < 0.03. f Current densities of three different outward current components at +40 mV. A1 (I_{to, f}): 23.5 ± 3.3 pA/pF for control and 15.5 ± 1.2 pA/pF for Cx43D378stop, P < 0.04; A2 (I_k, _slow): 11.1 ± 0.9 pA/pF for control and 10.9 ± 1.6 pA/pF for Cx43D378stop (pNS); A0 (I_ss): 6.0 ± 0.5 pA/pF for control and 5.0 ± 0.6 pA/pF for Cx43D378stop (pNS)

Fig. 6

Localization of Na_v1.5 and N-Cadherin in adult Cx43D378stop hearts. a Immunological staining of Na_v1.5 (green), N-Cadherin (red) and nuclei (blue) shows co-localization of Na_v1.5 and N-Cadherin at intercalated discs between ventricular cardiomyocytes in sections of control and Cx43D378stop:αMyHC-Cre-ER(T2) hearts (merged magnifications, 63×). Bar: 20 μm. b Statistical evaluation (Pearson's coefficient) reveals a significantly reduced co-localization (~33 %) of Na_v1.5 and N-Cadherin at intercalated discs of Cx43D378stop hearts. Data are mean ± SEM of 158 intercalated discs analyzed for control and 393 for Cx43D378stop hearts

Fig. 7

Expression and localization of mutated Cx43D378stop protein, ZO-1 and Na_v1.5 in the heart. Immunological staining of Cx43 (red), ZO-1 (green) and nuclei (blue) in sections of control (a) and Cx43D378stop hearts (b) verifies expression and membrane insertion of mutated Cx43D378stop protein. No obvious alterations in gap junction plaque formation or size were found for the Cx43D378stop protein. Cx43 and Cx43D378stop proteins both co-localized with ZO-1 at intercalated discs between ventricular cardiomyocytes (merged magnifications, 100×). Bar 20 μm. c Statistical evaluation (Pearson's coefficient) revealed no significant difference in the colocalization of Cx43 and ZO-1 between control and Cx43D378stop hearts. Data are mean ± SEM of 147 intercalated discs analyzed for control and 165 for Cx43D378stop hearts. Western blot expression analyses showed no significant changes in Cx43 protein expression in newborn (d) and adult (e) Cx43D378stop hearts compared to controls. Equal levels of ZO-1 and Na_v1.5 protein in neonatal (f) and adult (g) Cx43D378stop hearts were detected in both groups. Blots were reprobed with actin- or GAPDH-antibodies to verify equal loading. n = 4 per group