Cardiovascular Research

Cardiovascular Research 2002 53(4):921-935; doi:10.1016/S0008-6363(01)00522-3
© 2002 by European Society of Cardiology

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Copyright © 2002, European Society of Cardiology

Redistribution of connexin45 in gap junctions of connexin43-deficient hearts

Carolyn M. Johnson^a, Evelyn M. Kanter^a, Karen G. Green^b, James G. Laing^c,1, Tetsuo Betsuyaku^a, Eric C. Beyer^c,2, Thomas H. Steinberg^a, Jeffrey E. Saffitz^a,b,d and Kathryn A. Yamada^a,d,*

^aDepartment of Medicine, Washington University School of Medicine, St. Louis, MO, USA
^bDepartment of Pathology, Washington University School of Medicine, St. Louis, MO, USA
^cDepartment of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA
^dCenter for Cardiovascular Research, Washington University School of Medicine, St. Louis, MO, USA

kyamada{at}im.wustl.edu

^* Corresponding author. Cardiovascular Division, Box 8086, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA. Tel.: +1-314-3628-909; fax: +1-314-3628-957

Received 22 May 2001; accepted 26 October 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
Objective: Adult ventricular myocytes express two gap junction channel proteins: connexin43 (Cx43) and connexin45 (Cx45). Cx43-deficient mice exhibit slow ventricular epicardial conduction, suggesting that Cx43 plays an important role in intercellular coupling in the ventricle. Cx45 is much less abundant than Cx43 in working ventricular myocytes. Its role in ventricular conduction has not been defined, nor is it known whether expression or distribution of Cx45 is altered in Cx43-deficient mice. The present study was undertaken to determine (1) whether expression of Cx45 is upregulated and (2) whether gap junction structure and distribution are altered in Cx43-deficient mice. Methods: Ventricular tissue from neonatal Cx43^+/+, Cx43^+/– and Cx43^–/– and adult Cx43^+/+ and Cx43^+/– mice was analyzed by immunoblotting and confocal immunofluorescence microscopy. Results: Total Cx45 protein abundance measured by immunoblotting was not different in Cx43-deficient or null hearts compared to wild-type control hearts. However, the amount and distribution of Cx45 immunoreactive signal measured by quantitative confocal analysis were markedly reduced in both Cx43^+/– and Cx43^–/– hearts. Conclusion: Although the total content of Cx45 is not upregulated in Cx43-deficient hearts, the localization of Cx45 to cardiac gap junctions depends on the expression level of Cx43 and is dramatically altered in mice that express no Cx43.

KEYWORDS Cell communication; Developmental biology; Gap junctions; Histo(patho)logy; Myocytes; Remodeling

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
Gap junction channels comprised of connexin subunits are responsible for cell-to-cell communication and intercellular propagation of electrical signals throughout the heart. At least three different connexins are expressed in cardiac myocytes. Ventricular myocytes in the adult heart express connexin43 (Cx43) and connexin45 (Cx45) whereas atrial myocytes of most species including human, dog and mouse express Cx43, Cx45 and Cx40 [1–5]. Cx43 is the predominant cardiac connexin [1,4,6–8]. Studies in genetically engineered mice indicate that Cx43 is responsible for cell-to-cell communication in the ventricles [9,10], whereas Cx40 is a major determinant of that function in the atria [10–12]. The functional role of Cx43 in development, normal conduction and arrhythmogenesis is, however, complex. Conflicting reports describe modest conduction slowing [9,10,13] or lack thereof [14] in Cx43-deficient hearts. Cx43 null myocytes survive well in culture [15,16], are well coupled based on measurements of junctional conductance [15], and exhibit synchronized, rhythmic contractions [15,16]. Nevertheless, absence of cardiac Cx43 results in ventricular arrhythmias and sudden cardiac death [17], consistent with demonstrated enhancement of ischemia-induced ventricular tachycardia in Cx43-deficient hearts [18].

Cx45 appears to be the first cardiac connexin expressed during the initial stage of cardiac contractions in the mouse and then is developmentally downregulated [19]. Original studies in adult canine hearts showed that Cx45 was expressed throughout the ventricular myocardium [1,4,20]. Subsequently, Coppen and co-workers [21,22] observed that Cx45 immunofluorescence was concentrated at the endocardial surface with a distribution pattern overlapping that of Cx40-expressing myocytes of the ventricular conduction system in the adult heart. Ventricular Cx45 protein levels were below the limit of detection on immunoblots [21]. Other investigators have since observed Cx45 in low amounts in foci dispersed in the ventricular free walls of the adult heart [19], and faint β-gal staining representing Cx45 expression in myocytes of adult working myocardium [23].

The functional role of Cx45 in cardiac conduction in adult working myocardium is entirely unknown. Compared with channels made by Cx43 and Cx40, Cx45 channels have low unitary conductances and are more voltage dependent [24–27]. Although the amount of Cx45 in ventricular myocytes may be considerably less than Cx43, substantial evidence indicates that Cx43 and Cx45 can form hybrid gap junction channels with unique biophysical properties [28–30]. Thus, the functional consequences of Cx45 expression in the ventricles may be greater than its low level of expression would suggest, especially in mice deficient in Cx43. Previously, we have used genetically altered mice deficient in Cx43 protein [31] for detailed investigations into the expression, distribution and functional role of Cx43 in the heart [9,10,32]. In a recent study investigating the relationship between the amount of connexin expressed and the structure of gap junctions, we found that the number, but not the size, of gap junctions is reduced in Cx43^+/– hearts in which 50% of the wild-type level of Cx43 was expressed [32]. In the present study, we undertook a systematic approach to determine whether compensatory upregulation of Cx45 occurs in Cx43-deficient hearts, and whether the structure or distribution of gap junctions containing Cx45 is altered in Cx43-deficient hearts. We used a new anti-Cx45 antibody produced in one of our laboratories (T.H.S.), which has been extensively characterized and found to be monospecific for Cx45.

We report two new findings. First, Cx45 immunofluorescent signal was observed in both adult and neonatal mouse ventricles albeit at lower levels than described previously [1,4,20]. Second, although total Cx45 protein levels measured in immunoblots were unchanged in Cx43^+/– and Cx43^–/– hearts compared to Cx43^+/+ control hearts, the area occupied by Cx45 immunofluorescent signal in confocal images was markedly reduced in Cx43-deficient hearts, suggesting that distribution of Cx45 in ventricular myocyte gap junctions is altered dramatically in cells lacking Cx43. Thus, Cx43 expression level is a major determinant of the distribution of connexin proteins within ventricular myocyte gap junctions.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1. Cx43 mice
We maintain a colony of Cx43^+/+ and Cx43^+/– adult mice for breeding in a standard barrier facility. Founder mice (B6,129-Gja1^tm1Kdr) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Because Cx43^–/– mice die soon after birth, pregnancies were timed by housing breeding pairs overnight only. No changes in litter size, genotype distribution, or Cx43 expression have been observed over the past 4 years. The genotypes of all mice were determined by polymerase chain reaction (PCR) as described previously [16].

2.2. Characterization of anti-Cx45 antibody
The anti-Cx45 antibody used in the present study was produced at Washington University, St. Louis, MO, USA, against a mouse 6 his-carboxy terminus (amino acids 259–396) fusion protein as described by Lecanda et al. [33]. Detailed experiments demonstrating specificity of the anti-Cx45 antibody for Cx45 and absence of crossreactivity with Cx43 have been performed by us (Lin and Steinberg, unpublished observations) and in the laboratory of Professor Habo Jongsma [34]. To further validate the specificity of the anti-Cx45 antibody, we performed immunostaining experiments on control HeLa cells and HeLa cells transfected to overexpress either mouse Cx45 (HeLa-45) or rat Cx43 (HeLa-43). Anti-Cx45 antibody staining of control HeLa cells demonstrated minimal to no background expression of Cx45 in control HeLa cells (Fig. 1A). The level of anti-Cx45 staining of HeLa-43 cells was not significantly different from control HeLa cells demonstrating that the anti-Cx45 antibody did not recognize Cx43 protein (Fig. 1A). In contrast, HeLa-45 cells exhibited intense staining with the anti-Cx45 antibody (Fig. 1A). In addition, immunoblot analysis of control HeLa, HeLa-45 and HeLa-43 cells (Fig. 1B) confirmed the immunofluorescence data, demonstrating that the anti-Cx45 antibody did not recognize Cx43 protein in the HeLa-43 cell lane while demonstrating an intense band in the HeLa-45 cell lane. Finally, we tested the specificity of the anti-Cx45 antibody on Cx45-deficient E8.5 embryos [35] obtained from Professor Yosaburo Shibata and co-workers. Our anti-Cx45 antibody clearly stained Cx45^+/+ and Cx45^+/– embryos {identified both by PCR [35] and by positive X-gal (Invitrogen Life Technologies, Carlsbad, CA, USA) staining [36]} but did not stain Cx45^–/– embryos (Fig. 1C), further demonstrating specificity of the antibody used in the present study.

View larger version (261K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 (A) Cx45 (panels on the top) and Cx43 (panels on the bottom) immunostaining in HeLa cells showing monospecificity and absence of crossreactivity of anti-Cx45 antisera used in the present study. Left: Control HeLa cells exhibit low background levels of Cx45 expression. Center: HeLa cells overexpressing Cx45 (HeLa-Cx45) exhibit intense Cx45 staining reflecting abundant Cx45 expression and background levels of Cx43. Right: HeLa cells overexpressing Cx43 (HeLa-Cx43) exhibit low levels of Cx45 expression comparable to control HeLa cells and marked overexpression of Cx43. Bar=10 µm and applies to all panels. (B) Immunoblots of HeLa, HeLa-43, and HeLa-45 cells and Cx43+/+, Cx43+/– and Cx43–/– ventricular tissue samples from left to right probed with anti-Cx45 antisera (top) and anti-Cx43 antibody (middle). Corresponding Coomassie blue-stained gel is shown below. (C) Cx45 (panels on the left) and X-gal (panels on the right) staining of frozen tissue sections of whole Cx45+/+ (top), Cx45+/– (middle) and Cx45–/– (bottom) embryos (E8.5). Specific Cx45 signal is visible as punctate bright red spots in the Cx45+/+ and Cx45+/– embryos. Cx45–/– embryo is devoid of Cx45 signal. Positive X-gal staining (dark purple) identifies tissue in Cx45+/– and Cx45–/– embryos in which nls-lacZ has replaced Cx45. Bars correspond to 50 and 100 µm as indicated.

 
2.3. Immunoblotting
Immunoblot analysis was performed on ventricular tissue from neonatal (1-day-old pups) and adult hearts. Although Cx40 expression is not normally detectable in working adult ventricular myocytes, we measured Cx40 levels in addition to Cx45 and Cx43 in neonatal and adult hearts to determine whether compensatory changes in its expression occurred in Cx43-deficient hearts. Hearts were rapidly removed from anesthetized mice and frozen in liquid nitrogen. The frozen tissue was pulverized and homogenized (20 strokesx2 in a Duall homogenizer) in a hydrogencarbonate buffer solution (mM): 1 NaHCO₃, 5 EDTA, 1 EGTA, pH 8.0, containing the following protease inhibitors: 1 µM pepstatin, 100 nM aprotinin, 1 mM benzamidine, 1 mM iodoacetamide, 1 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and sonicated (3x15 s after each homogenization cycle).

Aliquots of protein (7 µg for anti-Cx43 blots; 20 or 30 µg for anti-Cx40 and anti-Cx45 blots) were added to 4x sample buffer [0.0625 M Tris, 11.25% glycerol, 0.5% sodium dodecyl sulfate (SDS), 0.001% bromphenol blue, 1% β-mercaptoethanol) and resolved by SDS–polyacrylamide (10%) gel electrophoresis and transferred (semi-dry) to nitrocellulose membranes (Schleicher and Schuell, Keene, NH, USA). Membranes were blocked in 5% nonfat, dry milk in Tris-buffered saline–Tween 20, then incubated with rabbit anti-rat Cx43 (1:5000, Zymed Laboratories, South San Francisco, CA, USA), protein A-purified rabbit anti-mouse Cx45 (1:500, Washington University), or rabbit anti-rat Cx40 (1:500, derived in our laboratories against rat Cx40 carboxy terminus amino acids 235–355) antibodies as described previously [16]. Goat anti-rabbit antibody (1:5000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used as the secondary antibody. Immunoreactivity of blots was detected by chemiluminescence (Renaissance, NEN Life Science, Wilmington, DE, USA) and quantified by densitometric analysis with Adobe Photoshop.

Multiple bands were observed on immunoblots probed with anti-Cx43 or anti-Cx45 antibodies. To determine whether the multiple bands represented phosphorylated isoforms, ventricular homogenates were treated (3 h at 37 °C) with 1 unit of alkaline phosphatase (Roche Diagnostics, Indianapolis, IN, USA or New England BioLabs, Beverly, MA, USA) per 1–125 µg protein. A single band in the alkaline phosphatase-treated lanes of Cx43 immunoblots demonstrated that the higher molecular mass bands represented phosphorylated isoforms of Cx43, consistent with our previously published findings [37]. Cx45, on the other hand, was seen as a doublet on immunoblots that was not altered in the presence of alkaline phosphatase. Thus, the Cx45 doublet may represent proteolytic cleavage products but not different phosphorylation states.

Density measurements of individual bands on each immunoblot were quantified as described previously [9,10,16,37] by normalizing all values to the darkest Cx43^+/+ band to compare each remaining band relative to the maximal signal. In addition, to validate the relative differences obtained from samples of different genotypes using our previously described method for band normalization, individual bands on the immunoblots were normalized to their respective actin bands on the corresponding Coomassie blue-stained gel. Actin-normalized values were then either further normalized to a single Cx43^+/+ control sample that was run on each gel (procedure used for Cx45 immunoblots) or compared directly without further normalization (procedure used for Cx40 immunoblots).

2.4. Confocal immunofluorescence
Confocal immunofluorescence microscopy was performed on ventricular tissue sections from neonatal and adult hearts. Atrial tissue was removed from each heart under a dissecting microscope. Frozen tissue sections on glass slides were lightly fixed with 4% paraformaldehyde prior to anti-Cx45 (1:400), anti-Cx43 (1:200) or anti-Cx40 (1:500) staining. Our anti-Cx45 and anti-Cx40 antibodies work only on frozen sections. A few additional hearts were used exclusively for anti-Cx43 immunostaining. Those hearts were fixed in 10% formalin, embedded in paraffin, sectioned (6 µm thick), and subjected to microwave antigen recovery for anti-Cx43 (1:200) staining [38]. Sections were then incubated in Cy3-conjugated goat anti-rabbit secondary antibody (1:400, Jackson ImmunoResearch).

All hearts were sliced in transverse section approximately midway between the apex and the base. Histologic sections were subsequently prepared in the same plane from the cut surface. Immunofluorescence images were collected from sections that exhibited intact tissue structure with minimal tears, folds, gaps or artefacts. All confocal images were acquired from the left ventricular midmyocardial layer where the myocytes were longitudinally (circumferentially) oriented. Subendocardial and subepicardial layers were excluded from analysis, as were myocytes oriented in an oblique plane of sectioning. Within the midmyocardium, fields containing the most robust staining were selected for imaging so we could compare the maximal signal across genotypes. Confocal images were acquired through a Zeiss microscope using an ArKr laser (568 nm, Molecular Dynamics) as described previously [32]. Five separate images (fields) were scanned from sections of each heart. Five to 11 hearts of each genotype were analyzed.

Quantitative confocal analysis of connexin immunofluorescent signal has been validated and described in detail in a previous study [32]. Briefly, test fields were digitized into a 1024x1024 matrix (1.05x10⁶ pixels/test field). An arbitrary signal intensity threshold was identified for each set of sections stained with a given primary antibody that clearly distinguished high intensity signal concentrated in discrete spots from all other areas in the test field. Signal intensity that exceeded this threshold was assumed to represent gap junctions. A much lower threshold value was also identified below which areas of the slide not covered by cells were defined. The proportion of total tissue area occupied by gap junctions (% cell area) was defined as the number of pixels with signal intensity exceeding the high threshold divided by the total number of pixels exceeding the low threshold.

Total number and mean size of gap junctions were determined by defining an individual gap junction as 5 or more contiguous pixels exceeding the high threshold value [32]. The quantitative procedures developed previously on adult ventricular tissue sections [32] were verified in the present study on neonatal ventricular tissue sections. Fig. 2 demonstrates representative confocal images of neonatal gap junctions before and after subtraction of pixels that did not achieve the signal threshold that distinguished gap junction clusters. The digital imaging software counted the number of objects and their mean size in pixels. The mean sizes were converted to µm².

View larger version (114K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Quantitative analysis of gap junction number and size by confocal microscopy and digital image processing in neonatal ventricular tissue. (A) Representative confocal image of neonatal ventricular myocardium cut in longitudinal section. Myocytes appear grey; gap junctions are bright white spots. (B) Same image as in A after subtraction of pixels that did not achieve the signal threshold that distinguished gap junctions (clusters of pixels exhibiting specific immunoreactive signal at cell–cell junctions) from other structures. (C) High-resolution image of a cluster of Cx43 immunoreactive signal identified between the arrows in A and B. (D) Same image as in C following subtraction of pixels that did not achieve the signal threshold that distinguished gap junctions from other structures. Image processing software identified eight individual gap junctions (clusters 5 contiguous pixels). Bars correspond to 10 and 1 µm as indicated.

 
2.5. Statistical analysis
All values are expressed as mean±S.D. Two group comparisons were made using group t-tests. Three group comparisons were made using one-way analysis of variance (ANOVA, SigmaStat); tests of significance between groups were performed using Tukey's or Dunn's multiple comparisons tests. A value of P<0.05 was considered significant.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
3.1. Cx43 protein expression
Immunoblot analysis was performed on ventricular tissue homogenates from 21 neonatal and 23 adult hearts. Fig. 3 shows a representative immunoblot containing samples from Cx43^+/+, Cx43^+/– and Cx43^–/– hearts. All immunoblot data are summarized in Table 1. Cx43 protein expression in Cx43^+/– hearts was reduced by 45% (P<0.001). As expected, Cx43 expression was not detected in Cx43^–/– hearts (P<0.001 vs. Cx43^+/+ and Cx43^+/– hearts). A similar 42% reduction (P<0.001) in total Cx43 protein was observed in tissue from adult Cx43^+/– (n=12) vs. Cx43^+/+ (n=11) hearts. These data corroborate previous reports that Cx43 protein expression is reduced by 50% in Cx43^+/– ventricles [9,10,16] and define Cx43 protein levels in hearts used for analysis of Cx45 expression and distribution.

View larger version (82K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Immunoblot of neonatal Cx43+/+, Cx43+/– and Cx43–/– hearts probed with anti-Cx43 antibody (top). Corresponding Coomassie blue-stained gel is shown below. Summarized data from 21 neonatal and 23 adult hearts are presented in Table 1.

 

View this table: [in this window] [in a new window]   Table 1 Immunoblot analysis of Cx45 and Cx43 expression in Cx43 wild-type and deficient mice determined using anti-Cx45 and anti-Cx43 antibodies

 
3.2. Confocal microscopy of Cx43 immunostaining
Cx43 immunostaining was performed on neonatal ventricular tissue sections from 11 hearts of each genotype. Fig. 4 shows representative images from neonatal Cx43^+/+, Cx43^+/–, and Cx43^–/– mice. Neonatal ventricular tissue showed the characteristic pattern [39–41] of punctate spots of immunofluorescent signal distributed around the cell borders. As expected, based on immunofluorescence data reported in adult hearts [32], Cx43 signal was significantly reduced in Cx43^+/– compared to Cx43^+/+ hearts and completely absent in Cx43^–/– hearts. As shown in Table 2, the % cell area occupied by Cx43 signal in Cx43^+/– tissue was reduced to 41% of the Cx43^+/+ value. The significant reduction in % cell area in neonatal Cx43^+/– compared to Cx43^+/+ hearts was the result of reductions in both the number and size of gap junctions. No Cx43 immunostaining was detected in Cx43^–/– tissue as expected.

View larger version (47K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Representative confocal images of Cx43 immunofluorescence in neonatal Cx43+/+ (left), Cx43+/– (center) and Cx43–/– (right) ventricular tissue sections stained with anti-Cx43 antibody. Bar=10 µm and applies to all panels.

 

View this table: [in this window] [in a new window]   Table 2 Confocal analysis of Cx43 immunostaining in Cx43 wild-type and deficient mice

 
3.3. Cx45 protein expression
Immunoblot analysis was performed to determine expression levels of Cx45 in 33 neonatal and 17 adult ventricles. Fig. 5 shows a representative immunoblot demonstrating specific bands for Cx45 protein in neonatal Cx43^+/+, Cx43^+/– and Cx43^–/– ventricular tissue homogenates. Quantitative data from 11 Cx43^+/+, 11 Cx43^+/– and 11 Cx43^–/– hearts are given in Table 1 and demonstrate that the level of total Cx45 protein content was similar for all three genotypes. Table 1 includes summarized Cx45 expression data from adult hearts as well; no significant difference in total Cx45 protein content between Cx43^+/+ and Cx43^+/– hearts was observed. Cx45 immunoblots were subjected to additional analysis. Individual bands on each immunoblot were normalized to their respective actin bands on the corresponding Coomassie blue-stained gel. All bands were compared to a single adult Cx43^+/+ control or a single neonatal Cx43^+/+ control, which were given a value of 1. There was no significant difference in Cx45 protein content in adult Cx43^+/+ (1.0) vs. Cx43^+/– (1.4±0.7, n=9; P=0.728) or neonatal Cx43^+/+ (1.0) vs. Cx43^+/– (1.1±0.3, n=10) vs. Cx43^–/– (2.1±1.1, n=11; P=0.413) ventricles. These data demonstrate that the total amount of ventricular Cx45 did not change in Cx43-deficient or null hearts.

View larger version (54K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Immunoblot of Cx43+/+, Cx43+/– and Cx43–/– hearts probed with anti-Cx45 antisera (top). Corresponding Coomassie blue-stained gel is shown below. Summarized data from 33 neonatal and 17 adult hearts are presented in Table 1.

 
3.4. Confocal microscopy of Cx45 immunostaining
Fig. 6 shows representative images from neonatal Cx43^+/+, Cx43^+/– and Cx43^–/– hearts and adult Cx43^+/+ and Cx43^+/– hearts stained with anti-Cx45 antisera. Cx45 expression was not confined exclusively to the endocardium and interventricular septum in the present study. Although specific Cx45 signal was observed in the endocardium consistent with expression in the conduction system, we also observed Cx45 signal at intercellular junctions between working ventricular myocytes throughout the left ventricular myocardial free wall. Cx45 immunoreactive signal in the midmyocardium from the left ventricles of Cx43^+/+, Cx43^+/– and Cx43^–/– mice was analyzed. Although Cx45 protein expression was shown by immunoblot analysis to be unchanged, inspection of the confocal images showed an obvious decrease in the area over which specific Cx45 signal was distributed in ventricular myocardium from both Cx43^+/– and Cx43^–/– mice.

View larger version (52K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Representative confocal images of Cx45 immunofluorescence in Cx43+/+ (left), Cx43+/– (center) and Cx43–/– (right) ventricular tissue sections stained with anti-Cx45 antisera. Neonatal tissue images are shown on top. Adult tissue images are shown below. Bar=10 µm and applies to all panels.

 
Quantitative analysis by digital image processing of the immunofluorescence signals confirmed this unexpected finding. As shown in Table 3, the area occupied by Cx45 signal expressed as a % of cell area was reduced by 22% in neonatal Cx43^+/– hearts (P=0.42) and by 68% in Cx43^–/– hearts (P<0.001) compared to Cx43^+/+ hearts. The number of gap junctions containing Cx45 was reduced significantly in both neonatal Cx43^+/– (P<0.001) and Cx43^–/– (P<0.001) hearts compared to Cx43^+/+ controls (Table 3). The size of gap junctions containing Cx45 was not reduced in neonatal Cx43^+/– hearts (P=0.69), but was reduced significantly in neonatal Cx43^–/– hearts (P<0.001). Thus, in Cx43^–/– hearts with complete absence of Cx43, both number and size of Cx45 gap junctions are markedly reduced. Dramatic alterations in Cx45 gap junctions were also observed in adult Cx43^+/– hearts. Cx45 immunostaining expressed as a % of cell area was reduced in adult Cx43^+/– hearts by 69% (P=0.005), due to decreases in both number (P=0.009) and size (P<0.001) of Cx45 gap junctions in Cx43^+/– compared to Cx43^+/+ hearts.

View this table: [in this window] [in a new window]   Table 3 Confocal analysis of Cx45 immunostaining in Cx43 wild-type and deficient mice

 
Sections from Cx43^+/+ and Cx43^+/– hearts were double labeled with both anti-Cx45 and anti-Cx43 antibodies. Fig. 7 demonstrates extensive colocalization of Cx45 and Cx43 in gap junctions in the ventricular myocardium of the mouse.

View larger version (94K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Double labeling of Cx43+/+ ventricular myocardium with both anti-Cx43 (left) and anti-Cx45 (right) antibodies. Antibodies used in the double label experiments were: primary for Cx43, mouse monoclonal (1:400, Chemicon International, Temecula, CA, USA); secondary for Cx43, Cy3-conjugated goat anti-mouse (1:400, Jackson ImmunoResearch Laboratories); primary for Cx45, rabbit polyclonal (1:500, Washington University); secondary for Cx45, Alexa Fluor 488 goat anti-rabbit (1:400, Molecular Probes, Eugene, OR, USA). Extensive colocalization of Cx45 and Cx43 immunoreactive signal is clearly apparent at gap junctions. Bar=10 µm and applies to both panels.

 
3.5. Cx40 protein expression
Cx40 protein expression was assessed in ventricular tissue from 22 hearts. When homogenized atria were included on the gels as a positive control, it was obvious that the level of Cx40 expression was extremely low or absent in ventricular tissue (Fig. 8A). Expression of Cx40 has been reported to be restricted to the Purkinje system and vascular endothelium [2–5,20,42] and would not be expected to yield a strong signal, if any, on immunoblots of whole ventricular homogenates. Because atrial samples were not run on every gel, we compared all Cx40 band densities normalized to each respective actin band on Coomassie blue-stained gels. Fig. 8B summarizes Cx40 protein expression and shows that total ventricular Cx40 protein levels were virtually identical, albeit minimal, in all three genotypes. These data demonstrate that ventricular Cx40 protein expression is not upregulated in Cx43-deficient or null hearts.

View larger version (91K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 (A) Immunoblot of two Cx43+/+, two Cx43+/–, two Cx43–/– neonatal ventricular samples and one atrial (Atr) sample probed with anti-Cx40 antibody (top). Corresponding Coomassie blue-stained gel is shown below. There are no detectable Cx40 bands in any of the ventricular tissue samples. (B) Summarized immunoblot data demonstrating equal levels (P=0.763) of Cx40 expression in Cx43+/+ (n=5), Cx43+/– (n=13), and Cx43–/– (n=4) hearts. (C) Fluorescence image of atrial and ventricular tissue stained with anti-Cx40 antibody demonstrating Cx40 expression (bright red punctate staining) in atrial myocardium and vascular endothelial cells in the ventricle. Bar=100 µm.

 
3.6. Confocal microscopy of Cx40 immunostaining
As expected, immunoreactive signal in ventricular tissue stained with anti-Cx40 antibody was confined to subendocardial foci (presumably His-Purkinje staining) and intramural coronary vascular endothelial cells. No Cx40 signal was observed in ventricular myocytes. Fig. 8C shows a representative fluorescence image demonstrating expected atrial staining of Cx40 and absence of Cx40 in ventricular myocardium except in vascular endothelial cells. These results confirm the low level of Cx40 protein expression in ventricular tissue as shown by immunoblot analysis (Fig. 8A and B).

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
These data demonstrate that Cx45 is expressed in neonatal and adult ventricular myocytes of the mouse. Cx45 and Cx40 are developmentally downregulated [19,43–45] and exhibit limited expression in adult ventricles. There has been uncertainty as to the level of Cx45 expression in adult mammalian ventricular myocardium based on the finding that a commercial anti-Cx45 antibody crossreacts with Cx43 [21]. The present study was performed using new, monospecific anti-Cx45 antisera that do not crossreact with Cx43 in cells overexpressing Cx43 [34]. Using this antibody, we observed that Cx45 is present in both neonatal and adult mouse ventricles as assessed by immunoblot analysis and quantitative confocal immunofluorescence microscopy, but not at the levels reported previously in the dog [1,4,20].

The physiologic role of Cx45 in developing and mature hearts has not been fully elucidated. Cx45 expression does, however, appear to be critical during early cardiogenesis as Cx45^–/– embryos do not survive [23,35]. In the adult heart, because Cx45 is present in the conduction system [21], it may play a role in propagation from the AV node to the ventricular muscle. In working ventricular myocardium Cx45 may play a modulatory role in influencing Cx43 gap junction channel conductances. Based on a large body of evidence in cell lines [28–30] and our new data demonstrating colocalization of Cx45 and Cx43 in the mouse heart, Cx43/Cx45 hybrid channels are likely to exist and function in vivo. Thus, even if present in small amounts relative to Cx43, Cx45 could influence cell-to-cell communication in normal and diseased hearts.

Cx43^–/– myocytes are coupled throughout development. Although Cx43^–/– hearts exhibit a conotruncal malformation [31,46–48] and Cx43^–/– mice do not survive long after birth, neonatal Cx43^–/– ventricular myocytes grow well in culture and form clusters of cells that exhibit synchronized contractions [15,16]. Our data suggest that Cx45 expression is responsible, at least in part, for intercellular coupling in Cx43^–/– hearts. Cx45 protein expression is not, however, upregulated in Cx43-deficient hearts to compensate for diminished or absent Cx43 protein. Our data also suggest that Cx40 is not upregulated in Cx43-deficient hearts, although Spray et al. have reported that Cx40 and possibly Cx45 channels may be present in Cx43^–/– hearts based on characteristic properties of single channel activity recorded from those hearts [15].

The pattern of Cx43 immunostaining in Cx43^+/– hearts highlights an interesting difference in the way a limited amount of Cx43 protein is packaged into gap junctions in neonatal versus adult Cx43^+/– hearts. In the latter, gap junction number is reduced, yet size remains the same [32]. In neonatal Cx43^+/– hearts, however, both number and size are reduced. Our data suggest that during maturation of Cx43-deficient hearts, cardiac Cx43 gap junctions attain a relatively normal, consistent size in the face of reduced Cx43 protein expression, whereas immature neonatal gap junctions are smaller in both number and size. Regulatory control of connexin synthesis, packaging, degradation and turnover in the adult heart may maintain an optimal size of gap junctions for efficient coordination of structure and functional activity. Specifically, the amount of Cx43 expressed does not determine the size of cardiac gap junctions as long as some Cx43 is produced (i.e., as in the Cx43^+/– heart). Other factors such as the structural stability imposed by mechanical junctions on the sarcolemma of adjacent cells, or localized conductance requirements may determine gap junction size [41].

Data from the present study demonstrate that although total ventricular Cx45 protein content is not reduced, the % cell area over which Cx45 signal is distributed is decreased. That is, the confocal images suggest that the same amount of Cx45 protein is distributed over a smaller area in Cx43-deficient and null hearts (versus Cx43^+/+ hearts). An alternative interpretation is that there may be a pool of cellular Cx45 protein that is not detected in the confocal images from Cx43-deficient and null hearts. Our data cannot distinguish between either of these two possibilities or a combination of both. In adult Cx43^+/– hearts, both % cell area occupied by Cx45 immunoreactive signal and Cx45 gap junction number were reduced significantly (Table 3). This result is not surprising given the fact that Cx45 colocalizes with Cx43 in gap junctions, and it suggests that the Cx45 protein expressed in Cx43^+/– and Cx43^+/+ hearts is distributed to gap junctions that contain Cx43. That Cx45 gap junction size was also reduced in Cx43^+/– hearts raises the interesting possibility of altered Cx43/Cx45 stoichiometry in Cx43^+/– hearts. For example, the pathophysiologic effects of reduced expression of Cx43 in Cx43^+/– hearts [9,10,13,17,18] may result not only from a reduction in the number of homotypic or homomeric Cx43 channels, but also from an increase in the number or composition of Cx45/Cx43 hybrid channels. The latter would be expected to have lower conductances [28–30] and altered gating [49].

In summary, Cx43 expression has a marked influence on the packaging (number and size) of Cx45 gap junctions. Ventricular Cx45 protein redistributes markedly in Cx43-deficient and null hearts. It is not known what regulates Cx45 and Cx40 expression. Although there is no evidence for coordinate upregulation of Cx45 or Cx40 in Cx43-deficient hearts, this does not exclude the possibility of coordinate expression of cardiac connexins under pathophysiologic conditions such as heart failure in which Cx43 is downregulated.

Time for primary review 24 days.

	Acknowledgments

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion Acknowledgments References

 
We are grateful to Drs. Kiyomasa Nishii and Yosaburo Shibata for helpful discussions, sharing protocols and for sending us Cx45^+/+, Cx45^+/– and Cx45^–/– embryos used in characterization of the anti-Cx45 antibody used in the present study. This work was supported by NIH/NHLBI grants HL50598 and HL58507 (J.E.S.) and HL66350 (K.A.Y.).

	Notes

 
¹ Present address: Washington University School of Medicine, Infectious Diseases Division, Box 8051, St. Louis, MO 63110, USA.

² Present address: The University of Chicago, Department of Pediatrics, Section of Hematology/Oncology, Chicago, IL 60637, USA.

	References

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