Connexins, Conduction, and Atrial Fibrillation
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《新英格兰医药杂志》
Atrial fibrillation has always been the little sister to its ventricular counterpart — but during the past decade, it has captured our attention, mainly because it is so common. Atrial fibrillation affects nearly 2.5 million Americans, and this number could double over the next 50 years as the population ages.1 Although not the killer that ventricular fibrillation is, atrial fibrillation is by no means benign. Roughly 15 percent of strokes in the United States have been attributed to this arrhythmia.2
The pathogenesis of atrial fibrillation is complex, even as compared with the other tachyarrhythmias. Atrial fibrillation arises from dynamic interactions among an unusually wide range of structural, electrophysiological, inflammatory, and genetic factors — and once it develops, it has the curious property of inducing further changes that promote its likelihood to recur and its resistance to antiarrhythmia therapies.3 Most patients with atrial fibrillation have associated cardiovascular diseases such as diabetes, hypertension, heart failure, valvular dysfunction, hyperthyroidism, and myocardial infarction. These patients usually have abnormal atria characterized by dilatation, fibrosis, and degenerative changes in atrial myocytes. These structural alterations create an anatomical substrate that slows impulse propagation and alters atrial refractoriness, which are properties that promote reentry.
However, not all patients with atrial fibrillation have such structural alterations or identifiable underlying diseases. The analysis of patients with lone or idiopathic atrial fibrillation has yielded important mechanistic insights. For example, in some persons, foci within the pulmonary veins discharge either continuously to drive atrial fibrillation or in short bursts that can cause atrial fibrillation if there is an underlying functional abnormality in atrial conduction or refractoriness.
The study of patients with lone atrial fibrillation has also brought into sharper focus another important insight about pathogenesis: atrial fibrillation can be inherited. A recent study suggests that perhaps 30 percent of patients with atrial fibrillation (with or without structural heart disease) have a family history of the disease.4 Thus, it is becoming increasingly apparent that, even in patients with anatomical substrates such as atrial dilatation and fibrosis, genetic factors may determine whether atrial fibrillation develops or becomes sustained. Similarly, patients with structurally normal atria may have a so-called genetic substrate, in which atrial fibrillation arises from mutations in critical genes that affect atrial conduction, refractoriness, or both.
Previous studies in patients with familial atrial fibrillation have implicated dominant mutations in the genes encoding cardiac potassium-channel proteins (reviewed by Roberts5). These mutations cause a gain of function that shortens the duration of action potentials and the effective refractory period — changes that increase the likelihood of reentry. Now, a study by Gollob et al.,6 described in this issue of the Journal, has implicated mutations in a gene encoding a gap-junction–channel protein in the pathogenesis of atrial fibrillation. Because gap junctions are responsible for the intercellular transfer of electrical current (Figure 1), a mutation in a gap-junction–channel protein (referred to as a "connexin") could impair conduction and thereby promote atrial fibrillation.
Figure 1. Structure of a Gap Junction, a Densely Packed Array of Channels in the Plasma Membranes of Neighboring Cells.
Hemichannels, each composed of six connexin subunits, dock in the extracellular space to create aqueous pores that link the cytoplasmic compartments of adjacent cells and facilitate intercellular communication. Adapted from Makowski et al.7 with the permission of the publisher.
Gollob et al. sequenced GJA5, the gene for the gap-junction protein connexin 40 (the number indicates the predicted molecular weight in kilodaltons), in 15 patients with idiopathic atrial fibrillation. They identified four novel heterozygous missense mutations in four patients. They then characterized the effects of these mutations on the ability of connexin 40 to form gap junctions and to conduct current from one cell to another.
A Pro88Ser substitution, identified in two patients, was detected in DNA from atrial muscle but not in DNA from lymphocytes. Cells expressing this mutant form of connexin 40 did not form visible gap junctions; instead, the protein seemed to accumulate inside the cells, suggesting a transport defect that prevents the protein from reaching the cell surface, from assembling into gap junctions, or both. Not surprisingly, current did not pass between cells expressing this mutant. When connexin 40 expression was examined in atrial tissue harboring the Pro88Ser mutation, some cells showed fewer gap junctions than normal and exhibited intracellular accumulation of connexin 40, whereas others appeared normal. This mosaic pattern suggests the presence of a somatic mutation in an early myocardial progenitor cell. Interestingly, an identical dominant Pro88Ser mutation in connexin 32 (a connexin expressed in Schwann cells) and in connexin 50 (expressed in lens epithelium) has been implicated in Charcot–Marie–Tooth disease8 and cataracts,9 respectively.
One patient exhibited two nonallelic mutations involving the Gly38Asp and Met163Val substitutions. Like the Pro88Ser mutation, these mutations were found in DNA from atrial tissue but not from lymphocytes, again suggesting a somatic source. When expressed in cultured cells, the Gly38Asp mutant made only sparse gap junctions, whereas most of the protein was intracellular. Electrical coupling in cells expressing this mutant connexin 40 was significantly lower than in cells expressing wild-type connexin 40.
In contrast, the Met163Val mutant formed apparently normal gap junctions (although some protein was seen inside the cells) and the level of coupling was similar to that seen in cells expressing wild-type connexin 40. This substitution may therefore be a benign polymorphism. However, a different picture emerged from the analysis of the fourth mutation, a germ-line Ala96Ser substitution identified in one patient. This mutant made apparently normal junctions, but their coupling was markedly lower than that in wild-type cells. This observation implicates a dominant negative mechanism rather than a transport defect. This hypothesis was supported by additional experiments showing that electrical coupling was considerably lower in cells that coexpressed mutant forms of connexin 40 along with wild-type connexin 40 or wild-type connexin 43 (another atrial gap-junction protein) than in cells expressing wild-type connexin 40 alone. Thus, connexin 40 mutations may reduce coupling by impairing the transport or assembly of connexins into gap junctions or by inhibiting channel function.
Because the myocardium is composed of discrete cells, each surrounded by an insulating lipid bilayer, the electrical activation of the heart requires the intercellular transfer of current through gap junctions. Normal cardiac myocytes are extensively coupled so that impulses propagate rapidly, as though the tissue were a uniform conductive medium. The results of Gollob et al. add to the growing recognition that alterations in the expression, distribution, or function of gap junctions may lead to conduction disturbances that can promote arrhythmogenesis.10
Gap junctions are densely packed arrays of intercellular channels made up of members of the large family of connexin proteins. Each cell contributes a hemichannel comprising six connexin molecules; these hemichannels dock in the extracellular space to form an aqueous pore that connects the cytoplasmic compartments of neighboring cells (Figure 1). Ions, small molecules, and even small peptides can pass through the junction, allowing cells to conduct current and exchange chemical signals. The human genome contains 21 connexin genes, and most cells express more than one type of connexin. Atrial muscle, for example, expresses both connexin 40 and connexin 43 and lesser amounts of connexin 45. The biologic functions fulfilled by the expression of multiple connexins in the heart are now being elucidated. For example, the knockout of connexin 40 in mice prolongs the P wave, the PQ interval, and the QRS interval, a finding consistent with diminished atrial conduction velocity.11 It also leads to spontaneous atrial arrhythmias, clearly showing that connexin 40 plays an important role in atrial electrophysiology. It is likely that individual atrial channels can consist of more than one connexin, but the diversity of their composition and their biophysical properties are poorly understood.
Finally, it should be emphasized that altered electrical coupling may contribute to atrial fibrillation in patients without mutant connexin 40. Gap-junction remodeling and changes in the expression levels of connexin 40 and connexin 43 have been described in the atria of patients with chronic atrial fibrillation, although the results have been inconsistent and their mechanistic significance is uncertain. The potential interactions between genetic factors and acquired changes in gap junctions remain to be elucidated. The further study of patients with lone atrial fibrillation and atrial fibrillation related to underlying cardiac disease may shed light on these important questions.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School — both in Boston.
References
Go AS, Hylek EM, Phillips KA, et al. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) study. JAMA 2001;285:2370-2375.
Rockson SG, Albers GW. Comparing the guidelines: anticoagulation therapy to optimize stroke prevention in patients with atrial fibrillation. J Am Coll Cardiol 2004;43:929-935.
Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation 1995;92:1954-1968.
Fox CS, Parise H, D'Agostino RB Sr, et al. Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring. JAMA 2004;291:2851-2855.
Roberts R. Genomics and cardiac arrhythmias. J Am Coll Cardiol 2006;47:9-21.
Gollob MH, Jones DL, Krahn AD, et al. Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med 2006;354:2677-2688.
Makowski L, Caspar DLD, Phillips WC, Goodenough DA. Gap junction structures: II. Analysis of the x-ray diffraction data. J Cell Biol 1977;74:629-645.
Nelis E, Simokovic S, Timmerman V, et al. Mutation analyses of the connexin 32 (Cx32) gene in Charcot-Marie-Tooth neuropathy type 1: identification of five new mutations. Hum Mutat 1997;9:47-52.
Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonula pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998;62:526-532.
Kanno S, Saffitz JE. The role of gap junctions in electrical conduction and arrhythmogenesis in the heart. Cardiovasc Pathol 2001;10:169-177.
Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K. Conduction disturbances and increased atrial vulnerability in connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 1999;99:1508-1515.(Jeffrey E. Saffitz, M.D.,)
The pathogenesis of atrial fibrillation is complex, even as compared with the other tachyarrhythmias. Atrial fibrillation arises from dynamic interactions among an unusually wide range of structural, electrophysiological, inflammatory, and genetic factors — and once it develops, it has the curious property of inducing further changes that promote its likelihood to recur and its resistance to antiarrhythmia therapies.3 Most patients with atrial fibrillation have associated cardiovascular diseases such as diabetes, hypertension, heart failure, valvular dysfunction, hyperthyroidism, and myocardial infarction. These patients usually have abnormal atria characterized by dilatation, fibrosis, and degenerative changes in atrial myocytes. These structural alterations create an anatomical substrate that slows impulse propagation and alters atrial refractoriness, which are properties that promote reentry.
However, not all patients with atrial fibrillation have such structural alterations or identifiable underlying diseases. The analysis of patients with lone or idiopathic atrial fibrillation has yielded important mechanistic insights. For example, in some persons, foci within the pulmonary veins discharge either continuously to drive atrial fibrillation or in short bursts that can cause atrial fibrillation if there is an underlying functional abnormality in atrial conduction or refractoriness.
The study of patients with lone atrial fibrillation has also brought into sharper focus another important insight about pathogenesis: atrial fibrillation can be inherited. A recent study suggests that perhaps 30 percent of patients with atrial fibrillation (with or without structural heart disease) have a family history of the disease.4 Thus, it is becoming increasingly apparent that, even in patients with anatomical substrates such as atrial dilatation and fibrosis, genetic factors may determine whether atrial fibrillation develops or becomes sustained. Similarly, patients with structurally normal atria may have a so-called genetic substrate, in which atrial fibrillation arises from mutations in critical genes that affect atrial conduction, refractoriness, or both.
Previous studies in patients with familial atrial fibrillation have implicated dominant mutations in the genes encoding cardiac potassium-channel proteins (reviewed by Roberts5). These mutations cause a gain of function that shortens the duration of action potentials and the effective refractory period — changes that increase the likelihood of reentry. Now, a study by Gollob et al.,6 described in this issue of the Journal, has implicated mutations in a gene encoding a gap-junction–channel protein in the pathogenesis of atrial fibrillation. Because gap junctions are responsible for the intercellular transfer of electrical current (Figure 1), a mutation in a gap-junction–channel protein (referred to as a "connexin") could impair conduction and thereby promote atrial fibrillation.
Figure 1. Structure of a Gap Junction, a Densely Packed Array of Channels in the Plasma Membranes of Neighboring Cells.
Hemichannels, each composed of six connexin subunits, dock in the extracellular space to create aqueous pores that link the cytoplasmic compartments of adjacent cells and facilitate intercellular communication. Adapted from Makowski et al.7 with the permission of the publisher.
Gollob et al. sequenced GJA5, the gene for the gap-junction protein connexin 40 (the number indicates the predicted molecular weight in kilodaltons), in 15 patients with idiopathic atrial fibrillation. They identified four novel heterozygous missense mutations in four patients. They then characterized the effects of these mutations on the ability of connexin 40 to form gap junctions and to conduct current from one cell to another.
A Pro88Ser substitution, identified in two patients, was detected in DNA from atrial muscle but not in DNA from lymphocytes. Cells expressing this mutant form of connexin 40 did not form visible gap junctions; instead, the protein seemed to accumulate inside the cells, suggesting a transport defect that prevents the protein from reaching the cell surface, from assembling into gap junctions, or both. Not surprisingly, current did not pass between cells expressing this mutant. When connexin 40 expression was examined in atrial tissue harboring the Pro88Ser mutation, some cells showed fewer gap junctions than normal and exhibited intracellular accumulation of connexin 40, whereas others appeared normal. This mosaic pattern suggests the presence of a somatic mutation in an early myocardial progenitor cell. Interestingly, an identical dominant Pro88Ser mutation in connexin 32 (a connexin expressed in Schwann cells) and in connexin 50 (expressed in lens epithelium) has been implicated in Charcot–Marie–Tooth disease8 and cataracts,9 respectively.
One patient exhibited two nonallelic mutations involving the Gly38Asp and Met163Val substitutions. Like the Pro88Ser mutation, these mutations were found in DNA from atrial tissue but not from lymphocytes, again suggesting a somatic source. When expressed in cultured cells, the Gly38Asp mutant made only sparse gap junctions, whereas most of the protein was intracellular. Electrical coupling in cells expressing this mutant connexin 40 was significantly lower than in cells expressing wild-type connexin 40.
In contrast, the Met163Val mutant formed apparently normal gap junctions (although some protein was seen inside the cells) and the level of coupling was similar to that seen in cells expressing wild-type connexin 40. This substitution may therefore be a benign polymorphism. However, a different picture emerged from the analysis of the fourth mutation, a germ-line Ala96Ser substitution identified in one patient. This mutant made apparently normal junctions, but their coupling was markedly lower than that in wild-type cells. This observation implicates a dominant negative mechanism rather than a transport defect. This hypothesis was supported by additional experiments showing that electrical coupling was considerably lower in cells that coexpressed mutant forms of connexin 40 along with wild-type connexin 40 or wild-type connexin 43 (another atrial gap-junction protein) than in cells expressing wild-type connexin 40 alone. Thus, connexin 40 mutations may reduce coupling by impairing the transport or assembly of connexins into gap junctions or by inhibiting channel function.
Because the myocardium is composed of discrete cells, each surrounded by an insulating lipid bilayer, the electrical activation of the heart requires the intercellular transfer of current through gap junctions. Normal cardiac myocytes are extensively coupled so that impulses propagate rapidly, as though the tissue were a uniform conductive medium. The results of Gollob et al. add to the growing recognition that alterations in the expression, distribution, or function of gap junctions may lead to conduction disturbances that can promote arrhythmogenesis.10
Gap junctions are densely packed arrays of intercellular channels made up of members of the large family of connexin proteins. Each cell contributes a hemichannel comprising six connexin molecules; these hemichannels dock in the extracellular space to form an aqueous pore that connects the cytoplasmic compartments of neighboring cells (Figure 1). Ions, small molecules, and even small peptides can pass through the junction, allowing cells to conduct current and exchange chemical signals. The human genome contains 21 connexin genes, and most cells express more than one type of connexin. Atrial muscle, for example, expresses both connexin 40 and connexin 43 and lesser amounts of connexin 45. The biologic functions fulfilled by the expression of multiple connexins in the heart are now being elucidated. For example, the knockout of connexin 40 in mice prolongs the P wave, the PQ interval, and the QRS interval, a finding consistent with diminished atrial conduction velocity.11 It also leads to spontaneous atrial arrhythmias, clearly showing that connexin 40 plays an important role in atrial electrophysiology. It is likely that individual atrial channels can consist of more than one connexin, but the diversity of their composition and their biophysical properties are poorly understood.
Finally, it should be emphasized that altered electrical coupling may contribute to atrial fibrillation in patients without mutant connexin 40. Gap-junction remodeling and changes in the expression levels of connexin 40 and connexin 43 have been described in the atria of patients with chronic atrial fibrillation, although the results have been inconsistent and their mechanistic significance is uncertain. The potential interactions between genetic factors and acquired changes in gap junctions remain to be elucidated. The further study of patients with lone atrial fibrillation and atrial fibrillation related to underlying cardiac disease may shed light on these important questions.
No potential conflict of interest relevant to this article was reported.
Source Information
From the Department of Pathology, Beth Israel Deaconess Medical Center and Harvard Medical School — both in Boston.
References
Go AS, Hylek EM, Phillips KA, et al. Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) study. JAMA 2001;285:2370-2375.
Rockson SG, Albers GW. Comparing the guidelines: anticoagulation therapy to optimize stroke prevention in patients with atrial fibrillation. J Am Coll Cardiol 2004;43:929-935.
Wijffels MC, Kirchhof CJ, Dorland R, Allessie MA. Atrial fibrillation begets atrial fibrillation: a study in awake chronically instrumented goats. Circulation 1995;92:1954-1968.
Fox CS, Parise H, D'Agostino RB Sr, et al. Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring. JAMA 2004;291:2851-2855.
Roberts R. Genomics and cardiac arrhythmias. J Am Coll Cardiol 2006;47:9-21.
Gollob MH, Jones DL, Krahn AD, et al. Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation. N Engl J Med 2006;354:2677-2688.
Makowski L, Caspar DLD, Phillips WC, Goodenough DA. Gap junction structures: II. Analysis of the x-ray diffraction data. J Cell Biol 1977;74:629-645.
Nelis E, Simokovic S, Timmerman V, et al. Mutation analyses of the connexin 32 (Cx32) gene in Charcot-Marie-Tooth neuropathy type 1: identification of five new mutations. Hum Mutat 1997;9:47-52.
Shiels A, Mackay D, Ionides A, Berry V, Moore A, Bhattacharya S. A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant "zonula pulverulent" cataract, on chromosome 1q. Am J Hum Genet 1998;62:526-532.
Kanno S, Saffitz JE. The role of gap junctions in electrical conduction and arrhythmogenesis in the heart. Cardiovasc Pathol 2001;10:169-177.
Hagendorff A, Schumacher B, Kirchhoff S, Luderitz B, Willecke K. Conduction disturbances and increased atrial vulnerability in connexin40-deficient mice analyzed by transesophageal stimulation. Circulation 1999;99:1508-1515.(Jeffrey E. Saffitz, M.D.,)