Gap junction remodeling and spironolactone-dependent reverse remodeling in the hypertrophied heart

Abstract

Pressure overload is a common pathological insult to the heart and the resulting hypertrophy is an independent risk factor for sudden cardiac death. Gap junction remodeling (GJR) has been described in hypertrophied hearts; however, a detailed understanding of the remodeling process and its effects on impulse propagation is lacking. Moreover, there has been little progress developing therapeutic strategies to diminish GJR. Accordingly, transverse aortic banding (TAC) was performed in mice to determine the effects of progressive pathological hypertrophy on connexin (Cx)43 expression, posttranslational phosphorylation, gap junction assembly, and impulse propagation. Within 2 weeks after TAC, total and phospho-Cx43 abundance was reduced and incorporation of Cx43 into gap junctional plaques was markedly diminished. These molecular changes were associated with progressive slowing of impulse propagation, as determined by optical mapping with voltage-sensitive dyes. Treatment with the aldosterone receptor antagonist spironolactone, which has been shown to diminish sudden arrhythmic death in clinical trials, was examined for its effects on GJR. We found that spironolactone blunted the development of GJR and also potently reversed established GJR, both at the molecular and functional levels, without diminishing the extent of hypertrophy. These data suggest a potential mechanism for some of the salutary electrophysiological and clinical effects of mineralocorticoid antagonists in myopathic hearts.

Figure 1

Molecular and functional GJR in TAC mice. A, Representative cross-sections of hearts showing progressive hypertrophy. B, Western blot analysis demonstrating GJR. Hearts were harvested at the indicated times post-TAC and either total cellular lysates (T) or Triton X-100 insoluble pellet fractions (P) were prepared. Antibodies were directed toward the Cx43 C terminus (Cx43-C); Cx43 N terminus (Cx43-N); vinculin (Vinc); S365-phosphoCx43 (pS365); or S325/328/330-phosphoCx43 (pS325). C, Immunofluorescent staining shows progressive loss of junctional Cx43. Sections were stained with a pan-Cx43 antibody. D, Representative optical maps demonstrate progressive slowing of CV. Perfusion of 4-week post-TAC hearts with αGA resulted in additional slowing of CV. Time points include sham (S), 2 weeks (2W), 4 weeks (4W), and 8 weeks (8W) post-TAC.

Figure 2

Aberrant phosphorylation of Cx43 in TAC mice. Sections were prepared at the indicated time points and stained with antibodies recognizing all forms of Cx43 (Total Cx43), S365-phosphoCx43 (pS365), or S325/328/330-phosphoCx43 (pS325). Diminution of both phosphorylated forms of Cx43 is evident as early as 1 week post-TAC. Scale bar=50 μm.

Figure 3

Inhibition of GJR with spironolactone. Groups of mice were subjected to either sham operation (S) or TAC (T) and fed either normal chow (−SPI) or chow supplemented with spironolactone (+SPI). Analyses were performed 4 weeks after surgery. A, Northern blot analysis demonstrates no changes in Cx43 mRNA abundance. B, Western blot analysis with a panCx43 antibody demonstrates increased slow-mobility Cx43 in both S+SPI and T+SPI hearts. C, Representative immunofluorescent staining demonstrates loss of Cx43 gap junction plaques in TAC−SPI hearts but substantial improvement in mice treated with spironolactone (TAC+SPI). D, Representative optical maps from each of the 4 groups. E, Representative immunofluorescent staining in reversal experiment demonstrates improvement after 2 weeks of treatment with spironolactone. F, Representative optimal maps from reversal experiment.

Figure 4

Enhanced Cx43 phosphorylation with spironolactone. Sections were prepared from each of the experimental groups 4 weeks after randomization and stained with antibodies recognizing all forms of Cx43 (Total Cx43), S365-phosphoCx43 (pS365), or S325/328/330-phosphoCx43 (pS325). Representative images are shown. Reduced immunoreactive Cx43 is seen in the TAC−SPI hearts with all 3 antibodies. Spironolactone treatment partially restores the appearance of the gap junctional plaques. Scale bar=50 μm.