Connexin43 cardiac gap junction remodeling: lessons from genetically engineered murine models

Abstract

Sudden cardiac death is responsible for several hundred thousand deaths each year in the United States. Multiple lines of evidence suggest that perturbation of gap junction expression and function in the heart, or what has come to be known as cardiac gap junction remodeling, plays a key mechanistic role in the pathophysiology of clinically significant cardiac arrhythmias. Here we review recent studies from our laboratory using genetically engineered murine models to explore mechanisms implicated in pathologic gap junction remodeling and their proarrhythmic consequences, with a particular focus on aberrant posttranslational phosphorylation of connexin43.

Fig. 1

Aberrant posttranslational phosphorylation of Cx43 in ODDD mutant hearts. a Western blot analysis using antibodies recognizing all forms of Cx43 (panCx43) and nonphosphorylated Cx43 (np-Cx43) showing specific loss of phosphorylated Cx43. Lanes 1–4 are from wild-type hearts and lanes 5–8 are from ODDD hearts. b Western blot analysis using antibodies recognizing all forms of Cx43 (panCx43), phosphoS365-Cx43 (pS365) and phosphoS325/328/330-Cx43 (pS325), showing specific loss of PS365 and pS325. Lanes 1, 3, 5, 7 and 9 are from wild-type hearts and lanes 2, 4, 6 and 8 are from ODDD hearts. c Immunohistochemical staining of wild-type and ODDD mutant hearts with panCx43, pS325 and pS365 antibodies. Phosphorylated forms of Cx43 are virtually absent in ODDD mutant hearts. Bar 20 μm. d Representative signal-averaged surface electrocardiograms (lead II) from a wild type (WT) and an ODDD mutant mouse. Note the diminished QRS amplitude in the mutant. e Optical mapping of the left ventricular surface of a representative WT heart and 3 individual ODDD mutant hearts showing significant slowing of conduction in the mutant hearts. f Programmed electrical stimulation showing return of sinus rhythm after premature beats in a wild type heart, but induction of sustained VT in an ODDD heart (middle), and spontaneous VT in an ODDD heart (right). Adapted from Kalcheva et al. (2007)

Fig. 2

Structural, molecular and functional gap junction remodeling with pressure overload hypertrophy. a Cross-sections of hearts showing progressive hypertrophy after transverse aortic constriction (TAC). b Western blot analysis demonstrating progressive reduction in total and phosphoCx43 expression in total cellular lysates (T) or Triton X-100 insoluble pellet fractions (P). Antibodies recognized the Cx43 C-terminus (Cx43-C); Cx43 amino-terminus (Cx43-N); vinculin (Vinc); S365-phosphoCx43 (pS365) or S325/328/330-phospho-Cx43 (pS325). c Immunostaining showing progressive loss of junctional Cx43 with TAC, especially S325/328/330-pCx43. d Representative optical maps demonstrating progressive slowing of conduction velocity after TAC. e Immunostaining showing loss of Cx43 gap junction plaques in TAC mice treated with vehicle alone (T-SPI), but substantial improvement in mice treated with spironolactone (T + SPI), comparable to sham-operated mice receiving vehicle alone (S-SPI) or spironolactone (S + SPI). Scale bar 50 μm. f Representative optical maps from each of the 4 groups. Adapted from Qu et al. (2009)

Fig. 3

Molecular and functional analysis of CK1δ mutant mice. a High-resolution Western blot analysis of whole cell lysates (WCL) or Triton X-100 insoluble pellets (pellet) prepared from ventricles of mice with the indicated genotypes, probed with polyclonal panCx43 antisera. Wild type Cx43 lysate treated with calf intestine phosphatase (CIP) migrates at P0 and is shown for comparison to various major phosphorylated forms of Cx43 (P1, P2, P3). b Representative immunofluorescent staining with panCx43 (Cx43, green) and N-cadherin (N-cad, red) antibodies at baseline and 4 weeks after TAC. Scale bar 10 μm. Magnified views of individual gap junction plaques for each genotype after TAC are shown below. c Representative immunoblots of whole cell lysates at baseline (B) and after TAC (T) from each of the indicated genotypes, probed with polyclonal panCx43 and GAPDH antibodies. d Representative activation maps from each of the indicated genotypes at baseline and after TAC, showing blunting of conduction slowing in S3E mutant mice. Adapted from Remo et al. (2011)