Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Abstract

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.

Figure 1

Representative quantitative immunoblots of a membrane probed first with anti-Cx45 antiserum (top), stripped, then reprobed with antiCx43 antibody (bottom). The gel was loaded with increasing amounts of Cx45 fusion protein (fp, five lanes on the left), ventricular homogenates from Cx45OE and wild-type (WT) mice (15 µg protein, four lanes in the middle), and increasing amounts of GST-Cx43 fusion protein (fp, five lanes on the right). The anti-Cx45 antiserum recognized only the Cx45 fusion protein at ∼16 kD (top gel, left lanes) and Cx45 in the ventricular homogenates (top gel, center lanes), but not the Cx43 fusion protein (top gel, right lanes). The anti-Cx43 antibody, on the other hand, recognized only the GST-Cx43 fusion protein at ∼43 kD (bottom gel, right lanes) and Cx43 in the ventricular homogenates (bottom gel, center lanes), but not the Cx45 fusion protein (bottom gel, left lanes).

Figure 2

Immunogold electron microscopy of cardiac connexins in hybrid (heteromeric and/or heterotypic) gap junctions. (A) Electron micrographic images of gap junctions decorated with 10 nm gold particles conjugated with anti-Cx43 antiserum (left), and a gap junction containing a 20 nm gold particle conjugated to anti-Cx45 antiserum (inset, arrow) among numerous 10 nm gold particles conjugated to anti-Cx43 antibody (right). Bar = 100 nm. (B) Proportions of Cx43-bound gold particles and Cx45-bound gold particles as a percent of total gap junctions in gap junction-enriched membrane preparations isolated from wild-type (WT) and Cx45OE hearts. WT hearts exhibited a larger percentage (87%) of homomeric/homotypic Cx43 gap junctions. In contrast, the percentages of Cx43 only and heteromeric gap junctions in Cx45OE hearts were roughly equivalent. Cx45 homomeric/homotypic gap junctions were not found in either WT or Cx45OE hearts. (C) The percentage of Cx43 and Cx45 in heteromeric gap junction channels was comparable in WT and Cx45OE hearts, despite the fact that there was a greater proportion of heteromeric gap junction channels in Cx45OE hearts. (D) Relative proportions of Cx43 homomeric/homotypic and Cx43/Cx45 heteromeric gap junctions by gross regions of the hearts. More heteromeric gap junctions were observed in the apex/septum compared to the LV free wall consistent with known expression of Cx45 in the cardiac conduction system which is concentrated in the subendocardial interventricular septum and in the distal Purkinje system.

Figure 3

Co-immunoprecipitation of Cx43 and Cx45 from murine ventricular myocardium. Cx45 (upper left, lane 5) or Cx43 (upper right, lane 3) were immunoprecipitated (IP) using mouse monoclonal anti-Cx45 or anti-Cx43 antibodies bound to protein G sepharose beads, respectively. The proteins in the immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis and subjected to immunoblotting (IB) with rabbit polyclonal anti-Cx43 and anti-Cx45 antibodies to reveal Cx43 (square) in Cx45-associated proteins (lower left, lane 5) and Cx45 (square) in Cx43-associated proteins (lower right, lane 3). Neither Cx43 nor Cx45 were observed in the negative controls in which mouse IgG1 was bound to protein G sepharose beads (IgG). In, supernatant of ventricular homogenates (input).

Figure 4

Cardiac-restricted ablation of Cx45 was accomplished by crossing Cx45 floxed mice with α-MHC-Cre⁺ mice. (A) Agarose gel showing banding patterns of the PCR products used to genotype cardiac-restricted ablation of Cx45. DNA samples from 6 different mice were run on this gel. Two PCR reactions were run for each mouse to determine whether they carried the Cre transgene (lanes 1, 3, 5, 7, 9 and 11) and whether they carried two wild-type, a wild-type and a floxed, or two floxed alleles (lanes 2, 4, 6, 8, 10 and 12). The sizes of the bands are as follows: Cx45 wild-type allele, 380 kb; Cx45 floxed allele, 500 kb; Cre transgene, 292 kb. (B) Immunoblot of cardiac Cx45-deficient (Cre⁺;Cx45^fl/fl) whole ventricular homogenates (F; lanes 2, 4 and 6) and controls (C; lanes 1, 3 and 5) showing markedly reduced Cx45 in Cre⁺;Cx45^fl/fl ventricles. Lane 7 shows a strong Cx45 band in atrial homogenate from Cx40KO/Cx45 knockin (KI) compared to the weaker Cx45 band in wild-type atrial homogenate (WT) shown in lane 8. The membrane was stripped and reprobed with anti-actin antibodies to show equal protein (20 µg) loading. (C) Histograms show summarized data of the confocal analysis of ventricular tissue sections stained with anti-Cx45, anti-Cx43 and anti-Cx40 antibodies. Values represent connexin immunoreactive signal as a percent of total tissue area (mean ± SD). Cx45 was significantly reduced in cardiac Cx45-deficient hearts. *p = 0.016.

Figure 5

Representative confocal images from a control heart (upper three parts) and a Cre⁺;Cx45^fl/fl heart (lower three parts) showing lack of Cx45 staining in the ventricle of the cardiac-restricted Cx45-deficient heart (lower left part). Adjacent tissue sections were stained for Cx45, Cx43 and Cx40, and every effort was made to select the same region in each heart for comparison. The ventricular chamber is at the bottom of each field. The subendocardial conduction system can be seen most clearly in the images stained for Cx40 (right parts). Cx43 and Cx45 are each expressed in this same region as well as in the ventricular myocardium beyond the subendocardial layer in the control heart (upper middle and left parts, respectively). Cx43 immunostaining is comparable in the control and Cre⁺;Cx45^fl/fl hearts (upper and lower middle parts, respectively). Bars = 10 µm.

Figure 6

Sequence of mouse Cx45 (NCBI: X63100). Amino acid residues in black and red bold font indicate the sequence of the C-terminus included in the six His-Cx45 CT fusion protein analyzed by mass spectrometry. We obtained 89% coverage of this sequence (red). Seven Ser and one Thr residues (highlighted, red underscored) were phosphorylated by CaMKII in vitro as identified by tandem mass spectrometry. Spectra and tables of fragmentation ions are given in the Supplemental Content.