Regulation of intercellular coupling in acute and chronic heart disease

Effective pump function of the heart depends on the precise control of spatial and temporal patterns of electrical activation. Accordingly, the distribution and function of gap junction channels are important determinants of the conduction properties of myocardium and undoubtedly play other roles in intercellular communication crucial to normal cardiac function. Recent advances have begun to elucidate mechanisms by which the heart regulates intercellular electrical coupling at gap junctions in response to stress or injury. Although responses to increased load or injury are generally adaptive in nature, remodeling of intercellular junctions under conditions of severe stress creates anatomic substrates conducive to the development of lethal ventricular arrhythmias. Potential mechanisms controlling the level of intercellular communication in the heart include regulation of connexin turnover dynamics and phosphorylation.

gap junctions; cardiac hypertrophy; heart failure; arrhythmias

Authorship

J.E. Saffitz

Washington University School of Medicine, Departments of Pathology and Medicine, and the Center for Cardiovascular Research, St. Louis, MO, USAWashington University School of MedicineUSASt. Louis, MO, USAWashington University School of Medicine, Departments of Pathology and Medicine, and the Center for Cardiovascular Research, St. Louis, MO, USA

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Figures

Figures (4)

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Figure 1
- The rate of connexin43 (Cx43) synthesis increases in cultured neonatal rat ventricular myocytes incubated with angiotensin II (A-II) but not cAMP. The upper panel shows an immunoprecipitation assay of radiolabeled Cx43 isolated from cultures incubated with [³⁵S]-methionine during the final 2 h of a 6- or 24-h treatment interval with A-II or dibutyryl cAMP (db-cAMP). As shown in the lower panel, the amount of radioactivity incorporated into Cx43 increased significantly in cultures treated with A-II compared with control cultures (means ± SD; *P<0.03) which were incubated with [³⁵S]-methionine in the absence of A-II or db-cAMP. The effects of A-II were blocked by the AT-1 receptor blocker losartan (Los). In contrast, there was no increase in radioactivity incorporated into Cx43 in cultures exposed to db-cAMP for 6 or 24 h. Thus, accumulation of Cx43 induced by cAMP appears to be due to diminished degradation rather than enhanced synthesis. Reprinted from Ref. 10 with permission.

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Figure 2
- Connexin43 (Cx43) and actin decay curves from pulse-chase experiments in adult rat hearts. Hearts were metabolically labeled with perfusate containing [³⁵S]-methionine, followed by chase periods of 0, 60, 120 and 240 min (N = 4 at each time point for Cx43). Radiolabeled Cx43 or actin immunoprecipitated from heart homogenates was separated on 12.5% SDS-PAGE gels and detected by autoradiography. Representative immunoprecipitation assays are shown. Actin was immunoprecipitated as a band at ~43 kDa (arrow). Cx43 and actin signals were quantified by densitometry and normalized to unchased values (given a value of 1.0). Background gray scale densitometry values were subtracted from each specific band to quantify signal intensity at each time point. A greater background level in the 0-min lane in the actin preparation accounted for the appearance of a greater signal intensity in the actin band at 0 min compared with 120- and 240-min time points. The decay constant, k (derived from the equation y = e^-kt), was used to calculate Cx43 half-life. Reprinted from Ref. 24 with permission.

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Figure 3
- Immunoblot analysis of Cx43 content in hearts perfused for 4 h with either buffer alone (Con) or protease inhibitors including leupeptin (Leu), ALLN, or NH₄Cl. Aliquots of total protein (30 µg) were separated by SDS-PAGE and probed with a rabbit polyclonal anti-Cx43 antibody. The total amount of Cx43 signal was increased in hearts perfused with all protease inhibitors. However, in hearts treated with the lysosomal inhibitors (leupeptin or NH₄Cl), there was selective accumulation of phosphorylated isoforms of Cx43 which migrate at apparent molecular mass of 44-46 kDa. In contrast, perfusion of hearts with the proteasomal inhibitor ALLN caused accumulation of nonphosphorylated Cx43 which migrates at 41 kDa. Reprinted from Ref. 24 with permission.

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Figure 4
- Representative laser scanning confocal images of Cx43 immunofluorescence in a control heart (A) or in hearts perfused for 4 h with the proteasomal inhibitor ALLN (B) or the lysosomal inhibitor NH₄Cl (C). Increased signal is apparent at sites of intercellular apposition. Reprinted from Ref. 24 with permission.

Figure 1 - The rate of connexin43 (Cx43) synthesis increases in cultured neonatal rat ventricular myocytes incubated with angiotensin II (A-II) but not cAMP. The upper panel shows an immunoprecipitation assay of radiolabeled Cx43 isolated from cultures incubated with [³⁵S]-methionine during the final 2 h of a 6- or 24-h treatment interval with A-II or dibutyryl cAMP (db-cAMP). As shown in the lower panel, the amount of radioactivity incorporated into Cx43 increased significantly in cultures treated with A-II compared with control cultures (means ± SD; *P<0.03) which were incubated with [³⁵S]-methionine in the absence of A-II or db-cAMP. The effects of A-II were blocked by the AT-1 receptor blocker losartan (Los). In contrast, there was no increase in radioactivity incorporated into Cx43 in cultures exposed to db-cAMP for 6 or 24 h. Thus, accumulation of Cx43 induced by cAMP appears to be due to diminished degradation rather than enhanced synthesis. Reprinted from Ref. 10 with permission.

Figure 2 - Connexin43 (Cx43) and actin decay curves from pulse-chase experiments in adult rat hearts. Hearts were metabolically labeled with perfusate containing [³⁵S]-methionine, followed by chase periods of 0, 60, 120 and 240 min (N = 4 at each time point for Cx43). Radiolabeled Cx43 or actin immunoprecipitated from heart homogenates was separated on 12.5% SDS-PAGE gels and detected by autoradiography. Representative immunoprecipitation assays are shown. Actin was immunoprecipitated as a band at ~43 kDa (arrow). Cx43 and actin signals were quantified by densitometry and normalized to unchased values (given a value of 1.0). Background gray scale densitometry values were subtracted from each specific band to quantify signal intensity at each time point. A greater background level in the 0-min lane in the actin preparation accounted for the appearance of a greater signal intensity in the actin band at 0 min compared with 120- and 240-min time points. The decay constant, k (derived from the equation y = e^-kt), was used to calculate Cx43 half-life. Reprinted from Ref. 24 with permission.

Figure 3 - Immunoblot analysis of Cx43 content in hearts perfused for 4 h with either buffer alone (Con) or protease inhibitors including leupeptin (Leu), ALLN, or NH₄Cl. Aliquots of total protein (30 µg) were separated by SDS-PAGE and probed with a rabbit polyclonal anti-Cx43 antibody. The total amount of Cx43 signal was increased in hearts perfused with all protease inhibitors. However, in hearts treated with the lysosomal inhibitors (leupeptin or NH₄Cl), there was selective accumulation of phosphorylated isoforms of Cx43 which migrate at apparent molecular mass of 44-46 kDa. In contrast, perfusion of hearts with the proteasomal inhibitor ALLN caused accumulation of nonphosphorylated Cx43 which migrates at 41 kDa. Reprinted from Ref. 24 with permission.

Figure 4 - Representative laser scanning confocal images of Cx43 immunofluorescence in a control heart (A) or in hearts perfused for 4 h with the proteasomal inhibitor ALLN (B) or the lysosomal inhibitor NH₄Cl (C). Increased signal is apparent at sites of intercellular apposition. Reprinted from Ref. 24 with permission.

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Regulation of intercellular coupling in acute and chronic heart disease

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