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Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

Kang M, Lin N, Li C, Meng Q, Zheng Y, Yan X, Deng J, Ou Y, Zhang C, He J, Luo D - Cell Commun. Signal (2014)

Bottom Line: Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes.Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.

Results: In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.

Conclusions: These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

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Related in: MedlinePlus

IP3R-associated regulation of electronic spreading among monolayer neonatal ventricular myocytes. Coordinated spontaneous Ca2+ oscillations were measured by confocal microscopy in cultured NRVMs loaded with fluo-4. Representative images and traces illustrate the global Ca2+ transients prior to and after 2-APB (3 μM, 10 minutes) or heptanol (1 mM, 2 minutes) treatment followed by addition of IP3/BM (20 μM, 6 minutes) or ATP (5 μM, 3 minutes) (A). Ca2+ transient uncoupling, represented by the percentage of dysynchronous transients in four to five connected cells indicated with circles in control image, were found in heptanol, 2-APB (B and C), or pan-IP3R shRNA (D) treated cells. IP3/BM and ATP could rescue the uncoupled transients induced by heptanol but not by 2-APB or pan-IP3R shRNA. Nifedipine (0.3 μM, 10 minutes), PMA (1 μM, 20 minutes) or isoprenaline (Iso, 0.1 μM, 2 minutes) did not mimic the effect of 2-APB or IP3/BM on coupled spontaneous Ca2+ oscillations (E). **P <0.01 vs. the cells treated with vehicle or scramble shRNA; ##P <0.01 vs. heptanol alone, n = 10–23 independent determinations for each bar.
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Fig3: IP3R-associated regulation of electronic spreading among monolayer neonatal ventricular myocytes. Coordinated spontaneous Ca2+ oscillations were measured by confocal microscopy in cultured NRVMs loaded with fluo-4. Representative images and traces illustrate the global Ca2+ transients prior to and after 2-APB (3 μM, 10 minutes) or heptanol (1 mM, 2 minutes) treatment followed by addition of IP3/BM (20 μM, 6 minutes) or ATP (5 μM, 3 minutes) (A). Ca2+ transient uncoupling, represented by the percentage of dysynchronous transients in four to five connected cells indicated with circles in control image, were found in heptanol, 2-APB (B and C), or pan-IP3R shRNA (D) treated cells. IP3/BM and ATP could rescue the uncoupled transients induced by heptanol but not by 2-APB or pan-IP3R shRNA. Nifedipine (0.3 μM, 10 minutes), PMA (1 μM, 20 minutes) or isoprenaline (Iso, 0.1 μM, 2 minutes) did not mimic the effect of 2-APB or IP3/BM on coupled spontaneous Ca2+ oscillations (E). **P <0.01 vs. the cells treated with vehicle or scramble shRNA; ##P <0.01 vs. heptanol alone, n = 10–23 independent determinations for each bar.

Mentions: Next, the electrical spreading between adjacent cells was determined by assessing the coordination of spontaneous Ca2+ transients in monolayer NRVMs, because a direct linkage between GJs and coordinate Ca2+ transients have been identified [28,29]. Normally, all connected NRVMs oscillate simultaneously because of direct ion propagation through GJs, but this coupling property can be disrupted by GJ inhibition, causing desynchronized Ca2+ transients. Thus, the asynchronous Ca2+ oscillations among four to five adjacent cells represent a non-invasive interruption of electrical propagation through GJs, which is expressed as a percentage of the total transients during a 2-minute recording. As shown in Figure 3A-C, coordinated Ca2+ transients were discontinued by addition of the gap uncoupler, heptanol or Gap 27, and changed to asynchronous Ca2+ spiking. 2-APB (3 μM) that inhibited the 6-CFDA diffusion (Figure 2B) also interfered with the coordination rhythm of Ca2+ transients. Similarly, knocking down pan-IP3R expression, but not any IP3R isoform, with shRNA caused partial desynchronization of the rhythmic Ca2+ oscillations. Moreover, consistent with the FRAP assay results, IP3/BM and ATP resynchronized these distorted Ca2+ transients in heptanol- or Gap 27-treated cells (Figure 3B and Additional file 2: Video 1), but not in 2-APB- (Figure 3C and Additional file 3: Video 2) or pan-IP3R shRNA-treated cells (Figure 3D). Neither isoprenaline, which stimulates the β1-adrenergic receptor, nor phorbol myristate acetate (PMA), which activates PKC, mimicked this recovery effect of IP3/BM. Furthermore, nifedipine, which inhibits L-type Ca2+ channels, did not elicit any uncoupling effect on Ca2+ oscillations (Figure 3B,C and E), although all of these reagents did affect the Ca2+ signaling rate and amplitude (Figure 3E, data not completely shown).Figure 3


Cx43 phosphorylation on S279/282 and intercellular communication are regulated by IP3/IP3 receptor signaling.

Kang M, Lin N, Li C, Meng Q, Zheng Y, Yan X, Deng J, Ou Y, Zhang C, He J, Luo D - Cell Commun. Signal (2014)

IP3R-associated regulation of electronic spreading among monolayer neonatal ventricular myocytes. Coordinated spontaneous Ca2+ oscillations were measured by confocal microscopy in cultured NRVMs loaded with fluo-4. Representative images and traces illustrate the global Ca2+ transients prior to and after 2-APB (3 μM, 10 minutes) or heptanol (1 mM, 2 minutes) treatment followed by addition of IP3/BM (20 μM, 6 minutes) or ATP (5 μM, 3 minutes) (A). Ca2+ transient uncoupling, represented by the percentage of dysynchronous transients in four to five connected cells indicated with circles in control image, were found in heptanol, 2-APB (B and C), or pan-IP3R shRNA (D) treated cells. IP3/BM and ATP could rescue the uncoupled transients induced by heptanol but not by 2-APB or pan-IP3R shRNA. Nifedipine (0.3 μM, 10 minutes), PMA (1 μM, 20 minutes) or isoprenaline (Iso, 0.1 μM, 2 minutes) did not mimic the effect of 2-APB or IP3/BM on coupled spontaneous Ca2+ oscillations (E). **P <0.01 vs. the cells treated with vehicle or scramble shRNA; ##P <0.01 vs. heptanol alone, n = 10–23 independent determinations for each bar.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig3: IP3R-associated regulation of electronic spreading among monolayer neonatal ventricular myocytes. Coordinated spontaneous Ca2+ oscillations were measured by confocal microscopy in cultured NRVMs loaded with fluo-4. Representative images and traces illustrate the global Ca2+ transients prior to and after 2-APB (3 μM, 10 minutes) or heptanol (1 mM, 2 minutes) treatment followed by addition of IP3/BM (20 μM, 6 minutes) or ATP (5 μM, 3 minutes) (A). Ca2+ transient uncoupling, represented by the percentage of dysynchronous transients in four to five connected cells indicated with circles in control image, were found in heptanol, 2-APB (B and C), or pan-IP3R shRNA (D) treated cells. IP3/BM and ATP could rescue the uncoupled transients induced by heptanol but not by 2-APB or pan-IP3R shRNA. Nifedipine (0.3 μM, 10 minutes), PMA (1 μM, 20 minutes) or isoprenaline (Iso, 0.1 μM, 2 minutes) did not mimic the effect of 2-APB or IP3/BM on coupled spontaneous Ca2+ oscillations (E). **P <0.01 vs. the cells treated with vehicle or scramble shRNA; ##P <0.01 vs. heptanol alone, n = 10–23 independent determinations for each bar.
Mentions: Next, the electrical spreading between adjacent cells was determined by assessing the coordination of spontaneous Ca2+ transients in monolayer NRVMs, because a direct linkage between GJs and coordinate Ca2+ transients have been identified [28,29]. Normally, all connected NRVMs oscillate simultaneously because of direct ion propagation through GJs, but this coupling property can be disrupted by GJ inhibition, causing desynchronized Ca2+ transients. Thus, the asynchronous Ca2+ oscillations among four to five adjacent cells represent a non-invasive interruption of electrical propagation through GJs, which is expressed as a percentage of the total transients during a 2-minute recording. As shown in Figure 3A-C, coordinated Ca2+ transients were discontinued by addition of the gap uncoupler, heptanol or Gap 27, and changed to asynchronous Ca2+ spiking. 2-APB (3 μM) that inhibited the 6-CFDA diffusion (Figure 2B) also interfered with the coordination rhythm of Ca2+ transients. Similarly, knocking down pan-IP3R expression, but not any IP3R isoform, with shRNA caused partial desynchronization of the rhythmic Ca2+ oscillations. Moreover, consistent with the FRAP assay results, IP3/BM and ATP resynchronized these distorted Ca2+ transients in heptanol- or Gap 27-treated cells (Figure 3B and Additional file 2: Video 1), but not in 2-APB- (Figure 3C and Additional file 3: Video 2) or pan-IP3R shRNA-treated cells (Figure 3D). Neither isoprenaline, which stimulates the β1-adrenergic receptor, nor phorbol myristate acetate (PMA), which activates PKC, mimicked this recovery effect of IP3/BM. Furthermore, nifedipine, which inhibits L-type Ca2+ channels, did not elicit any uncoupling effect on Ca2+ oscillations (Figure 3B,C and E), although all of these reagents did affect the Ca2+ signaling rate and amplitude (Figure 3E, data not completely shown).Figure 3

Bottom Line: Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes.Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Inositol 1,4,5-trisphosphate receptor (IP3R) plays a pivotal role in the Ca2+ release process in a variety of cell types. Additionally, IP3R is distributed in ventricular intercalated discs, but its function(s) in this particular site remains unknown. Connexin (Cx43), the predominant gap junction (GJ) protein in ventricular myocardium, is linked to several signaling pathways that regulate Cx43 properties by (de)phosphorylation on multiple residues. Here, we investigated the regulatory role of IP3R in cell-cell communication and the mechanism(s) underlying this effect.

Results: In neonatal rat and adult mouse ventricular myocytes IP3R co-localized and co-immunoprecipitated with Cx43 in GJ plaques detected by immunostaining and western blot assays. Blocking IP3R with antagonists or silencing pan-IP3R expression with shRNA hindered the 6-carboxyfluorescein (6-CFDA) diffusion through GJs and desynchronized Ca2+ transients among confluent neonatal myocytes in culture, whereas stimulation of IP3R with IP3 ester or ATP exerted the opposite effect. Likewise, 6-CFDA propagation through GJs was modulated by IP3R activation or inhibition in cell pairs of isolated adult cardiomyocytes. Furthermore, IP3R activation or IP3R suppression promoted or suppressed, respectively, Cx43 phosphorylation on S279/282. Site-directed mutagenesis indicated that expression of a mutant Cx43-S282A (alanine) inhibited S279/282 phosphorylation and GJ permeability, while the S279A mutant showed the opposite effect in ventricular myocytes. Expression of these mutants in HEK293 cells revealed that cells with a dual S279/282 mutation failed to express exogenous Cx43, whereas cells with a single S279 or S282 mutation displayed Cx43 overexpression with increased phosphorylation of S279/282 and promotion of intercellular communication.

Conclusions: These results demonstrated, for the first time, that IP3R physically interacts with Cx43 and participates in the regulation of Cx43 phosphorylation on S279/282, thereby affecting GJ intercellular communication in ventricular myocytes.

Show MeSH
Related in: MedlinePlus