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(Circulation Research. 2000;86:723.)
© 2000 American Heart Association, Inc.

MiniReview

Connexin Expression and Turnover

Implications for Cardiac Excitability

Jeffrey E. Saffitz, James G. Laing, Kathryn A. Yamada

From the Departments of Pathology and Medicine and the Center for Cardiovascular Research, Washington University, St. Louis, Mo.

Correspondence to Jeffrey E. Saffitz, Department of Pathology, Box 8118, Washington University School of Medicine, 660 S Euclid Ave, St. Louis, MO 63110. E-mail saffitz{at}pathbox.wustl.edu

	Abstract

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
Abstract—Electrical activation of the heart requires current transfer from one cell to another via gap junctions, arrays of densely packed intercellular channels. The extent to which cardiac myocytes are coupled is determined by multiple mechanisms, including tissue-specific patterns of expression of diverse gap junction channel proteins (connexins), and regulatory pathways that control connexin synthesis, intracellular trafficking, assembly into channels, and degradation. Many connexins, including those expressed in the heart, have been found to turn over rapidly. Recent studies in the intact adult heart suggest that connexin43, the principal cardiac connexin, is surprisingly short-lived (half-life 1.3 hours). Both the proteasome and the lysosome participate in connexin43 degradation. Other ion channel proteins, such as those forming selected voltage-gated K⁺ channels, may also exhibit rapid turnover kinetics. Regulation of connexin degradation may be an important mechanism for adjusting intercellular coupling in the heart under normal and pathophysiological conditions.

Key Words: gap junctions • connexins • proteolysis • proteasome • lysosome

	Introduction

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
Gap junctions couple cardiac myocytes electrically and metabolically by facilitating the intercellular exchange of ions, signaling molecules (<1 kDa in size), and other molecular information.¹ At least 4 different members of the connexin family of gap junction channel proteins are expressed in the heart: connexin (Cx) 43, Cx45, Cx40, and Cx37.^{2 3 4 5 6} These proteins are expressed in different amounts and combinations in different cell types and regions of the heart. Each protein forms channels with distinct biophysical properties, including unitary conductances and voltage sensitivities.^{7 8 9 10} A particular pattern of connexin expression may, therefore, confer specific conduction properties on a given cardiac tissue. Results of recent studies have implicated other potential determinants of intercellular coupling, including mechanisms regulating connexin synthesis, assembly, and degradation. This review focuses on connexin turnover kinetics and explores the hypothesis that regulation of connexin degradation may modulate cell-cell coupling of cardiac myocytes and thereby affect impulse propagation, especially in response to pathophysiological stimuli.

	Rapid Turnover of Connexins

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
Electron microscopic images of the intercalated disk create the impression that gap junctions are static structures. In fact, the opposite is true. Many connexins, including those expressed in the heart (eg, Cx43, Cx45, and Cx37) and those expressed abundantly in the liver and elsewhere (eg, Cx32 and Cx26), have half-lives of 1 to 5 hours.^{11 12 13 14 15 16 17} Although not all connexins are short-lived (Cx46 in the lens has an apparent half-life of >1 day,¹⁸ which may reflect the unusual metabolic conditions in the lens), dynamic turnover of connexins in the heart may be an important mechanism for modulating intercellular coupling and impulse propagation.

The first evidence that gap junction proteins turn over rapidly in cardiac myocytes came from studies by Laird et al,¹⁴ who showed that newly synthesized Cx43 disappeared with a half-life of 2 hours in cultured neonatal rat ventricular myocytes. The discovery of the rapid turnover of Cx43 raised questions about the intracellular pathways responsible for degradation of gap junction proteins. Insights into this issue were first revealed in studies of Cx43 turnover in E36 Chinese hamster ovary cells and ts20 cells, a mutant line of E36 cells, which carry a temperature-sensitive mutation in the E1 ubiquitin-activating protein and, therefore, fail to degrade proteins dependent on ubiquitin pathways when exposed to elevated temperatures.¹⁹ Metabolic labeling and pulse-chase studies in control E36 cells revealed a Cx43 half-life of 2 hours, whereas Cx43 accumulated in ts20 cells when the ubiquitin-conjugation system was inactivated at the restrictive temperature.¹⁹

	Connexin Degradation by Proteasomal and Lysosomal Pathways

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
The major organelles of intracellular proteolysis are the proteasome and the lysosome.^{20 21} Pathways responsible for degradation of proteins can be elucidated by incubating cells with specific and selective inhibitors of the proteasome (peptide aldehydes such as acetyl-leucyl-leucyl-norleucinal [ALLN] or carboxybenzoyl-leucyl-leucyl-leucinal [MG132]²² or the fungal metabolite lactacystin²³ ) or the lysosome (lysosomotropic amines such as primaquine or chloroquine, weak bases such as ammonium chloride,²⁴ or cathepsin inhibitors such as leupeptin or E-64²⁵ ). By using this approach, treatment of BWEM cells (a smooth muscle-like cell line originally derived from embryonic rat heart) with inhibitors of either pathway resulted in accumulation of Cx43,²⁶ indicating that both the lysosome and proteasome participate in Cx43 degradation. Confocal microscopy of BWEM cells treated with the proteasomal inhibitor lactacystin revealed accumulation of Cx43 signal at or near appositional membranes, whereas intracellular vesicular staining was more abundant in cells treated with the lysosomal inhibitor E-64.²⁶ Thus, proteasomal degradation may involve initial targeting of Cx43 at the junctional membrane, causing protein to accumulate there when proteasomal proteolysis is inhibited. Conversely, internalization of Cx43 leading to its accumulation in intracellular vesicles may occur when lysosomal proteolysis is inhibited.

To further test the hypothesis that rapid degradation of Cx43 involves protein present in cell-surface gap junctions, studies have been performed in BWEM cells treated with brefeldin A (BFA),²⁶ a compound that disrupts delivery of newly synthesized proteins to the cell surface. Cells exposed to BFA exhibited dramatic loss of Cx43 membrane staining that coincided with a decrease in the total cellular content of Cx43 assessed by immunoblotting.²⁶ BFA-induced loss of Cx43 was prevented by coincubating cells with proteasomal or lysosomal inhibitors.²⁶ However, when cells were treated simultaneously with BFA and a proteasomal inhibitor (either MG132 or lactacystin), plasma membrane staining of Cx43 was comparable to that in untreated cells.²⁶ These results provide further evidence that Cx43 already assembled within gap junctional plaques undergoes proteasomal degradation.

Intracellular sites and pathways of connexin degradation vary in different cell types, and results observed in established cell lines may not be applicable to cardiac myocytes. Accordingly, additional studies have been performed using proteolysis inhibitors in primary cultures of neonatal rat ventricular cardiac myocytes. As observed in previous studies of BWEM cells,²⁶ Cx43 accumulated in myocytes incubated with either lysosomal or proteasomal inhibitors,²⁷ indicating that both pathways degrade Cx43 in cardiac myocytes. In contrast to what was observed in established cell lines, however, lysosomal inhibition caused Cx43 to accumulate in appositional membranes rather than in intracellular vesicles.²⁷ In addition, electron microscopy revealed increased length of gap junction profiles in cells treated with either chloroquine or lactacystin.²⁷ Thus, both proteasomal and lysosomal pathways are involved in the degradation of Cx43 that resides within the intercalated disk in cardiac myocytes.

An important consideration in analysis of connexin turnover in cultured cardiac myocytes concerns the effects of cell isolation. Freshly disaggregated adult myocytes contain intact gap junctions on their surfaces,^{28 29} indicating the presence of junctional membranes of former neighbors. These gap junctions become internalized and disappear rapidly.^{28 29} Because disaggregation followed by active reestablishment of cell junctions in culture could increase connexin synthesis and degradation rates compared with those in the intact heart, studies of connexin turnover dynamics in intact myocardium are of particular interest. Metabolic labeling and pulse-chase studies have been performed in isolated, perfused adult rat hearts.¹⁷ Radioactive Cx43 disappeared in a monoexponential fashion with a calculated half-life of 1.3 hours,¹⁷ a rate similar to that seen in cultured myocytes.^{14 16} In contrast, the amount of radioactivity in actin did not change measurably, consistent with its reported half-life of 11 days. Thus, Cx43 turns over as rapidly in the intact adult heart as in cultured myocytes.

Perfusion of isolated adult rat hearts with either proteasomal or lysosomal inhibitors produced a marked increase in C43 immunofluorescence at intercalated disks in the known distribution of gap junctions.¹⁷ The intracellular pattern of vesicular Cx43 staining reported in previous studies of established cell lines treated with lysosomal inhibitors²⁶ was not, however, observed in ventricular myocytes of intact rat hearts perfused with ammonium chloride.¹⁷ Furthermore, immunoblots prepared from ventricular homogenates revealed that nonphosphorylated Cx43 accumulated in ALLN-treated hearts, whereas inhibition of lysosomal proteolysis with leupeptin or ammonium chloride causes marked accumulation of phosphorylated Cx43.¹⁷ It is not known how changes in phosphorylation state contribute to protein stability or whether dephosphorylated Cx43 may be a target for degradation. It is also not known whether protein that accumulates in response to inhibition of lysosomal or proteasomal degradation is present in functional channels that could alter intercellular coupling. These questions have important implications for potential therapeutic strategies designed to interfere with connexin degradation and thereby reduce the incidence of arrhythmias dependent on impaired coupling.

All of the cardiac connexins are subject to phosphorylation^{14 15 16 30 31} involving, in most cases, serine residues. Phosphorylation of Cx43 has been characterized most extensively. Progressive phosphorylation of newly synthesized Cx43 occurs over time and is associated with its assembly into channels and transport to the cell surface.¹⁵ Phosphorylated isoforms of Cx43, identified by their altered mobility on SDS-polyacrylamide gels, are resistant to extraction in 1% Triton X-100, suggesting that they are associated with the cortical cytoskeleton at the plasma membrane.¹⁵ Cx43 can be phosphorylated by protein kinase C, mitogen-activated protein kinase, and the src protein kinase.^{32 33 34} Actions of these kinases usually lead to diminished single-channel or whole-cell conductances or diminished dye coupling, but the responsible mechanisms are poorly defined.^{35 36} A recent study showed that src-dependent phosphorylation of Cx43, but not Cx45, occurs in cardiomyopathic hamster hearts.³⁷ The exact sites and the biological consequences of phosphorylation of the cardiac connexins have not been elucidated.

Increasing evidence has focused attention on the role of phosphorylation in the regulation of connexin stability. Results of recent studies suggest that phosphorylation of specific serine residues on Cx43 and Cx45 alters connexin degradation^{38 39} but by complex, disparate mechanisms. For example, phosphorylation of Cx43 on ser255 by p34^cdc2 kinase in Rat1 cells in the G₂/M phase of the cell cycle promotes endocytosis and degradation of Cx43.³⁸ In contrast, phosphorylation of serine residues in the carboxyl terminal of Cx45 dramatically stabilizes the protein in transfected HeLa cells.³⁹ Thus, phosphorylation of serine residues may alter connexin stability and/or target the protein for degradation.

It might be argued that previous studies involving metabolic labeling and pulse-chase strategies have not formally ruled out the possibility that rapid turnover of connexins occurs selectively in an intracellular pool of protein, whereas some or all of the protein in gap junctions is sufficiently long-lived that it does not become labeled during relatively brief pulse intervals. However, results of recent studies in which Cx43 has been visualized in living cells previously transfected to express Cx43 tagged on its carboxyl terminal with green fluorescent protein (Cx43-GFP) have provided dramatic evidence of the dynamic nature of Cx43 in cell-surface gap junctions.⁴⁰ Time-lapse movies have shown continuous transport of apparently newly synthesized Cx43-GFP to the plasma membrane of MDCK cells where discrete patches of fluorescent signal (presumed gap junctional plaques) were seen to oscillate and occasionally to coalesce. Cx43-GFP was removed from the plasma membrane by budding and internalization and usually formed distinct endocytic vesicles of two different sizes. The smaller of these vesicles appeared to deliver Cx43-GFP back to the cell surface.⁴⁰ Although the fate of Cx43-GFP in living cells has not been followed for more than 40 minutes (due to technical limitations), future studies may be able to directly visualize turnover of Cx43 in gap junction plaques.

	Stress- or Injury-Induced Acceleration of Cx43 Degradation

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
Electron microscopy has revealed intracellular membrane-bound vesicular structures containing morphologically identifiable gap junctions referred to as annular gap junctions. Annular gap junctions occur in many cell types including cardiac myocytes.^{28 29 41 42} They are especially abundant in recently disaggregated adult myocytes.^{28 29} Their presence suggests that arrays of channels at the cell surface can be internalized and possibly degraded by lysosomes. It is currently not known whether degradation of gap junctions may contribute to abnormal electrical remodeling in the heart and arrhythmogenesis in the setting of hypertrophy, ischemia, or infarction.

Recent studies in which cultured neonatal cardiac myocytes have been subjected to heat stress bear on the relation between myocyte injury and Cx43 degradation.²⁷ Exposure of cultured myocytes to heat stress (43.5°C for 30 minutes) resulted in dramatic loss of Cx43 protein content (assessed by immunoblotting or immunohistochemistry). Heat stress also resulted in accumulation of the heat shock protein HSP70. Degradation of Cx43 was prevented during an interval of heat stress by simultaneously incubating cells with proteasomal or lysosomal inhibitors, suggesting that both pathways participate in heat-induced proteolysis of Cx43. Furthermore, when heat-stressed cells were allowed to reaccumulate Cx43 during a 3-hour recovery interval, a subsequent interval of heat shock failed to cause degradation of Cx43. These results suggest that a factor induced during an initial interval of heat stress (possibly HSP70) may have protected against subsequent Cx43 degradation in response to additional injury.²⁷

The wider implications of these results with respect to changes in gap junction structure and function during ischemic preconditioning have not been explored in detail. Enhanced degradation and turnover of connexins could reduce cell-cell coupling, slow conduction, and promote reentrant arrhythmias. Differential targeting of Cx43 to ubiquitin-dependent or -independent proteolysis pathways could provide mechanisms by which cardiac myocytes regulate "normal" and "pathophysiologic" degradation responses. Furthermore, disparate ubiquitin-dependent pathways may result in either protein degradation or stabilization. Phosphorylation at PEST motifs (regions rich in pro, glu, ser, and thr sequences) has been identified as a general mechanism for targeting proteins for rapid degradation.^{43 44} Conversely, phosphorylation of c-Mos, c-Fos, and c-Jun by mitogen-activated protein kinases suppresses their ubiquitination and degradation.⁴⁵ Little is known about the specific signaling pathways and enzymes responsible for targeting connexins and other proteins for degradation in the heart, much less the wide range of physiological and pathophysiological conditions in which these reactions occur.

	Degradation of Other Channel Proteins

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
The discovery that connexins turn over rapidly raises questions about whether other proteins involved in cardiac excitability and conduction are similarly regulated. This issue has not been studied systematically. Of the voltage-gated channels studied thus far, Kv1.5 proteins are among those reported to have the shortest half-lives (4 hours) based on disappearance of protein in cells treated with the protein synthesis inhibitor cycloheximide.⁴⁶ Levitan and Takimoto⁴⁷ have suggested that rapid upregulation of voltage-gated K⁺ channels and concomitant regulation of changes in excitability may be mediated by rapid turnover of Kv channel proteins. Voltage-gated Na⁺ and Ca²⁺ channels have been reported to have half-lives of 15 to 26 hours.^{48 49} Thus, turnover of K⁺ channel and gap junction proteins but apparently not Na⁺ or Ca²⁺ channel proteins may influence minute-to-minute changes in excitability. Other membrane-bound receptors have been reported to turn over with half-lives of 6 to 8 hours (A₁ adenosine receptors,⁵⁰ IP₃ receptors^{51 52} ), days (fetal acetylcholine receptors⁵³ ), or weeks (adult acetylcholine receptors,⁵³ sarcoplasmic reticulum Ca²⁺-ATPase pump⁵² ).

Intracellular pathways responsible for degrading voltage-gated ion channel proteins have not been elucidated in detail. Of the various integral plasma membrane proteins in animal cells found to undergo ubiquitination, the only ion channel is the epithelial Na⁺ channel (ENaC), a short-lived protein with a half-life of 1 hour.⁵⁴ Mutations in the ENaC PPxY motif, which interacts with WW domains of the ubiquitin-protein ligase Nedd4, disrupt ubiquitin-mediated degradation of ENaC and result in increased ENaC activity and Liddle’s syndrome.⁵⁵ Other channels, receptors, and transporters may be degraded by ubiquitin-mediated pathways. A proteolytic cleavage product of the skeletal muscle ryanodine receptor bears homology to S5a, a proteasome subunit that targets polyubiquitinated proteins to the 26S proteasome.⁵⁶ Agonist stimulation may accelerate protein turnover. For example, the muscarinic agonist, carbachol, increases the rate of degradation of the IP₃ type I receptor by a Ca²⁺-dependent mechanism.⁵¹ Diminished Ca²⁺ entry may initiate acetylcholine receptor loss at the neuromuscular junction.⁵⁷ Blockade of acetylcholine receptors by -bungarotoxin results in a shift in the half-life of the protein from 14 days to 1 day.⁵⁸ A number of growth factor receptors have also been found to undergo ubiquitin-mediated lysosomal degradation in response to agonist stimulation.²⁴

	Summary of Intracellular Pathways of Cx43 Degradation

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
The Figure shows a proposed model of the degradation of gap junction proteins based on current knowledge. The connexin polypeptide is synthesized on endoplasmic reticulum (ER)–associated ribosomes and inserted into the ER membrane. Some unoligomerized connexin polypeptides may be degraded in the ER in the same manner as other misfolded or improperly oligomerized secretory and membrane proteins. Excess, defective, or unused protein could undergo ubiquitination and be exported from the ER to the cytosol where it becomes degraded by the proteasome. Most newly synthesized connexin polypeptides in the ER are subsequently oligomerized to form hemichannels either in the ER or the Golgi. The hemichannels are transported to the sarcolemma where they may dock with hemichannels of neighboring cells to form complete gap junction channels. Individual channels then aggregate to form gap junctional plaques. Degradation of gap junctional plaques requires actions of both the proteasome and the lysosome, which may occur in parallel or sequentially. Gap junction channels may be internalized within intracellular vesicles either as channel arrays (annular gap junctions) or as channels disrupted from the plaques by partial proteasomal proteolysis.⁵⁹ Either form may undergo lysosomal degradation.

View larger version (70K): [in this window] [in a new window]   Figure 1. Proposed model of intracellular pathways of connexin degradation. Refer to text for explanation and references.

	Implications for Cardiac Excitability

Top Abstract Introduction Rapid Turnover of Connexins Connexin Degradation by... Stress- or Injury-Induced... Degradation of Other Channel... Summary of Intracellular... Implications for Cardiac... References

 
Because gap junctions play a fundamental role in cell-cell communication and impulse propagation, it seems likely that cardiac myocytes have evolved multiple regulatory mechanisms to control the level of intercellular communication in the heart. The fact that Cx43 turns over so rapidly in the intact heart suggests that one mechanism controlling coupling is rapid adjustment in the number of channels between cells. Undoubtedly, other mechanisms controlling connexin synthesis, assembly into channels, and single-channel properties, including unitary conductances and open-probability times, also play crucial roles in regulating the extent to which cells are coupled to one another, especially under pathophysiological conditions. Many of these regulatory mechanisms may be mediated by changes in connexin phosphorylation. Cardiac myocytes may uncouple rapidly and completely, as occurs in the setting of acute, severe ischemia. In the face of sublethal injury, however, cardiac myocytes may uncouple partially as part of a more generalized cellular response to injury involving complex structural and functional adaptations. Alterations in intercellular coupling in viable but structurally remodeled myocardium undoubtedly change the conduction properties of the tissue and, in some settings, may create anatomic substrates of arrhythmias dependent on derangements in conduction. Alterations in connexin expression and spatial remodeling of gap junctions in regions bordering healing infarcts have been strongly implicated in the development of slow, heterogeneous conduction and conduction block critical in reentrant arrhythmogenesis.⁶⁰ Downregulation of Cx43 expression and loss of the largest gap junctions have been described in hibernating myocardium in patients with chronic heart failure,⁶¹ although the contributions of these changes to electrical and contractile dysfunction have not been elucidated. The extent to which regulation of connexin turnover dynamics is used by cells to control levels of intercellular coupling is not known, nor is it clear whether alterations in connexin expression in diseased myocardium are mediated by changes in the delicate balance between connexin synthesis and connexin degradation. More detailed understanding of mechanisms underlying cellular responses to injury will not only provide novel insights into fundamental disease processes that promote arrhythmogenesis but may also lead to novel therapies to prevent lethal rhythm disturbances in patients with heart disease.

Received January 28, 2000; accepted February 10, 2000.

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