Background It has been found that gap junction-associated intracellular Ca2+ [Ca2+]i disturbance contributes to the arrhythmogenesis and hyperconstriction in diseased heart. However, whether functional gaps are also involved in the regulation of normal Ca2+ signaling, in particular the basal [Ca2+]i activities, is unclear. Methods and Results Global and local Ca2+ signaling and gap permeability were monitored in cultured neonatal rat ventricular myocytes (NRVMs) and freshly isolated mouse ventricular myocytes by Fluo4/AM and Lucifer yellow (LY), respectively. The results showed that inhibition of gap communication by heptanol, Gap 27 and flufenamic acid or interference of connexin 43 (Cx43) with siRNA led to a significant suppression of LY uptake and, importantly, attenuations of global Ca2+ transients and local Ca2+ sparks in monolayer NRVMs and Ca2+ sparks in adult ventricular myocytes. In contrast, overexpression of rat-Cx43 in NRVMs induced enhancements in the above measurements, and so did in HEK293 cells expressing rat Cx43. Additionally, membrane-permeable inositol 1,4,5-trisphosphate (IP3 butyryloxymethyl ester) and phenylephrine, an agonist of adrenergic receptor, could relieve the inhibited Ca2+ signal and LY uptake by gap uncouplers, whereas blockade of IP3 receptor with xestospongin C or 2-aminoethoxydiphenylborate mimicked the effects of gap inhibitors. More importantly, all these gap-associated effects on Ca2+ signaling were also found in single NRVMs that only have hemichannels instead of gap junctions. Further immunostaining/immunoblotting single myocytes with antibody against Cx43 demonstrated apparent increases in membrane labeling of Cx43 and non-junctional Cx43 in overexpressed cells, suggesting functional hemichannels exist and also contribute to the Ca2+ signaling regulation in cardiomyocytes. Conclusions These data demonstrate that Cx43-associated gap coupling plays a role in the regulation of resting Ca2+ signaling in normal ventricular myocytes, in which IP3/IP3 receptor coupling is involved. This finding may provide a novel regulatory pathway for mediation of spontaneous global and local Ca2+ activities in cardiomyocytes.
References
[1]
Stout CE, Costantin JL, Naus CC, Charles AC (2002) Intercellular calcium signaling in astrocytes via ATP release through connexin hemichannels. J Biol Chem 277: 10482–10488.
[2]
Cotrina ML, Lin JH, Alves-Rodrigues A, Liu S, Li J, et al. (1998) Connexins regulate calcium signaling by controlling ATP release. Proc Natl Acad Sci U S A 95: 15735–15740.
[3]
Retamal MA, Schalper KA, Shoji KF, Bennett MV, Saez JC (2007) Opening of connexin 43 hemichannels is increased by lowering intracellular redox potential. Proc Natl Acad Sci U S A 104: 8322–8327.
[4]
Bao X, Reuss L, Altenberg GA (2004) Regulation of purified and reconstituted connexin 43 hemichannels by protein kinase C-mediated phosphorylation of Serine 368. J Biol Chem 279: 20058–20066.
[5]
John SA, Kondo R, Wang SY, Goldhaber JI, Weiss JN (1999) Connexin-43 hemichannels opened by metabolic inhibition. J Biol Chem 274: 236–240.
[6]
Kondo RP, Wang SY, John SA, Weiss JN, Goldhaber JI (2000) Metabolic inhibition activates a non-selective current through connexin hemichannels in isolated ventricular myocytes. J Mol Cell Cardiol 32: 1859–1872.
[7]
Kang J, Kang N, Lovatt D, Torres A, Zhao Z, et al. (2008) Connexin 43 hemichannels are permeable to ATP. J Neurosci 28: 4702–4711.
[8]
Bruzzone S, Guida L, Zocchi E, Franco L, De Flora A (2001) Connexin 43 hemichannels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells. FASEB J 15: 10–12.
[9]
Li F, Sugishita K, Su Z, Ueda I, Barry WH (2001) Activation of connexin-43 hemichannels can elevate [Ca2+]i and [Na+]i in rabbit ventricular myocytes during metabolic inhibition. J Mol Cell Cardiol 33: 2145–2155.
[10]
Shintani-Ishida K, Uemura K, Yoshida K (2007) Hemichannels in cardiomyocytes open transiently during ischemia and contribute to reperfusion injury following brief ischemia. Am J Physiol Heart Circ Physiol 293: H1714–1720.
[11]
Garcia-Dorado D, Inserte J, Ruiz-Meana M, Gonzalez MA, Solares J, et al. (1997) Gap junction uncoupler heptanol prevents cell-to-cell progression of hypercontracture and limits necrosis during myocardial reperfusion. Circ 96: 3579–3586.
[12]
Wier WG, Balke CW (1999) Ca2+ release mechanisms, Ca2+ sparks, and local control of excitation-contraction coupling in normal heart muscle. Circ Res 85: 770–776.
[13]
Spach MS, Boineau JP (1997) Microfibrosis produces electrical load variations due to loss of side-to-side cell connections: a major mechanism of structural heart disease arrhythmias. Pacing Clin Electrophysiol 20: 397–413.
[14]
Hayashi H (2010) Does remodeling of gap junctions and connexin expression contribute to arrhythmogenesis? Study in an immobilization rat model. Circ J 74: 2558–2559.
[15]
Remo BF, Qu J, Volpicelli FM, Giovannone S, Shin D, et al. (2011) Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias. Circ Res 108: 1459–1466.
[16]
Ruiz-Meana M, Garcia-Dorado D, Hofstaetter B, Piper HM, Soler-Soler J (1999) Propagation of cardiomyocyte hypercontracture by passage of Na+ through gap junctions. Circ Res 85: 280–287.
[17]
Li W, Schultz C, Llopis J, Tsien RY (1997) Membrane-permeant esters of inositol polyphosphates, chemical syntheses and biological applications. Tetrahedron; 53: 12017–12040.
[18]
Luo D, Yang D, Lan X, Li K, Li X, et al. (2008) Nuclear Ca2+ sparks and waves mediated by inositol 1,4,5-trisphosphate receptors in neonatal rat cardiomyocytes. Cell Calcium 43: 165–174.
[19]
Chen L, Meng Q, Jing X, Xu P, Luo D (2010) A role for protein kinase C in the regulation of membrane fluidity and Ca2+ flux at the endoplasmic reticulum and plasma membranes of HEK293 and Jurkat cells. Cell Signal 23: 497–505.
[20]
VanSlyke JK, Musil LS (2000) Analysis of connexin intracellular transport and assembly. Methods 20: 156–164.
[21]
Bruce AF, Rothery S, Dupont E, Severs NJ (2008) Gap junction remodelling in human heart failure is associated with increased interaction of connexin43 with ZO-1. Cardiovasc Res 77: 757–765.
[22]
Opsahl H, Rivedal E (2000) Quantitative determination of gap junction intercellular communication by scrape loading and image analysis. Cell Adhes Commun 7: 367–375.
[23]
Watts SW, Tsai ML, Loch-Caruso R, Webb RC (1994) Gap junctional communication and vascular smooth muscle reactivity: use of tetraethylammonium chloride. J Vasc Res 31: 307–313.
[24]
Evans WH, Leybaert L (2007) Mimetic peptides as blockers of connexin channel-facilitated intercellular communication. Cell Commun Adhes 14: 265–273.
[25]
Gomes P, Srinivas SP, Van Driessche W, Vereecke J, Himpens B (2005) ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 46: 1208–1218.
[26]
Tong D, Gittens JE, Kidder GM, Bai D (2006) Patch-clamp study reveals that the importance of connexin43-mediated gap junctional communication for ovarian folliculogenesis is strain specific in the mouse. Am J Physiol Cell Physiol 290: C290–297.
[27]
Tilley DG (2011) G protein-dependent and G protein-independent signaling pathways and their impact on cardiac function. Circ Res 109: 217–230.
[28]
Anselmi F, Hernandez VH, Crispino G, Seydel A, Ortolano S, et al. (2008) ATP release through connexin hemichannels and gap junction transfer of second messengers propagate Ca2+ signals across the inner ear. Proc Natl Acad Sci U S A 105: 18770–18775.
[29]
Gossman DG, Zhao HB (2008) Hemichannel-mediated inositol 1,4,5-trisphosphate (IP3) release in the cochlea: a novel mechanism of IP3 intercellular signaling. Cell Commun Adhes 15: 305–315.
[30]
Evans WH, De Vuyst E, Leybaert L (2006) The gap junction cellular internet: connexin hemichannels enter the signalling limelight. Biochem J 397: 1–14.
[31]
Goodenough DA, Paul DL (2003) Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol 4: 285–294.
[32]
Naus CC, Zhu D, Todd SD, Kidder GM (1992) Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol 12: 163–175.
[33]
Zhu D, Caveney S, Kidder GM, Naus CC (1991) Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc Natl Acad Sci U S A 88: 1883–1887.
[34]
Fry T, Evans JH, Sanderson MJ (2001) Propagation of intercellular calcium waves in C6 glioma cells transfected with connexins 43 or 32. Microsc Res Tech 52: 289–300.
[35]
Oyamada Y, Zhou W, Oyamada H, Takamatsu T, Oyamada M (2002) Dominant-negative connexin43-EGFP inhibits calcium-transient synchronization of primary neonatal rat cardiomyocytes. Exp Cell Res 273: 85–94.
Kostin S, Rieger M, Dammer S, Hein S, Richter M, et al. (2003) Gap junction remodeling and altered connexin43 expression in the failing human heart. Mol Cell Biochem 242: 135–144.
[38]
Ai X, Zhao W, Pogwizd SM (2010) Connexin43 knockdown or overexpression modulates cell coupling in control and failing rabbit left ventricular myocytes. Cardiovasc Res 85: 751–762.
[39]
Hoeker GS, Katra RP, Wilson LD, Plummer BN, Laurita KR (2009) Spontaneous calcium release in tissue from the failing canine heart. Am J Physiol Heart Circ Physiol 297: H1235–1242.
[40]
Beuckelmann DJ, Nabauer M, Erdmann E (1992) Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circ 85: 1046–1055.
