The Phosphorylation of Connexin 43 in Arrhythmic Heart Tissue

Introduction:

Heart disease is the number one killer of humans in the USA, ending almost 1 million lives per year¹.  Arrhythmias alone kill 350,000 of the American victims¹. Thus, medical advances aimed at stopping or controlling arrhythmias have significant importance for the health of society. 

Cardiac cells transmit electrical impulses to their neighbors through gap junctions.  Gap junctions are constructed of six proteins, known as connexins, which determine the junction’s conductivity and, therefore, the communication from cell to cell.  If the connexins close the channel, uncoupling occurs, cells lose communication with each other, and electrical impulses fail to propagate, which can result in an arrhythmia.  Researchers believe the phosphorylation state of connexins determine the resistance of the junction.  Researchers also believe that if gap junctions become uncoupled and electrical impulses fail to propagate through a site of the tissue, that site provides a location of attachment for a core of reentry.  Previous studies proved that an ischemic heart failed to transmit electrical impulses due to its unphosphorylated gap junctions.² Thus, since ischemia often causes arrhythmias; this experiment investigated the relationship between a connexin found abundantly in ventricular tissue, Cx43, the phosphorylation state of this protein, and the core of a reentrant arrhythmia.

Methods:

We used confocal laser-scanning microscopy and immunohistochemistry to determine the phosphorylation state of Cx43 at sites of the core of reentrant arrhythmia. The tissue was fixed and permeated in formaldehyde and triton. The primary antibodies used included anti-unphosphorylated (Ser 368) Cx43 raised in mouse (Zymed) at a dilution of 1:200 and anti-phosphorylated (Ser 368) raised in rabbit (Chemicon) at a dilution of 1:400.  At dilutions of 1:1000, Alexa fluor 555 anti-rabbit IgG and Alexa fluor 488 anti-mouse IgG1, both raised in goat (Molecular Probes) were used as secondary antibodies.

Results:

To carry out the experiment, preliminary experiments were performed to fine tune the existing staining protocol.  Alterations included changing the fixation and permeating solution between methanol, ethanol, and formaldehyde/triton; blocking the tissue with rabbit serum instead of normal horse serum; using primary antibodies of different lots; using more concentrated secondary antibodies; and switching the secondary antibodies.  The final experiment used the method described above and tissues from three different rabbits: an older rabbit that generated arrhythmias, a 2.5 month old that generated arrhythmias, and a 2.5 months old that did not generate arrhythmias.  Confocal imaging revealed the tissue of the 2.5 month old rabbit that sustained arrhythmias had strongly dephosphorylated Cx43 at the core of reentry whereas the surrounding areas demonstrated predominant phosphorylated Cx43 signal, separating the healthy, well superfused tissue, from the ischemic areas with dephosphoylated Cx43.  In contrast, the healthy 2.5 month old rabbit displayed an increased amount of phosphorylation throughout the tissue and the expected dephosphorylation further into the midmyocardium.  The older rabbit also displayed a greater amount of dephosphorylation of Cx43 at the core; however, due to its fibrotic state, additional slices would be needed to analyze the results further.  For future investigation, Masson’s trichrome histology was performed on sister tissue sections to analyze the anatomic structures of the tissue with the location of the core and phosphorylation of the gap junctions.  Alpha-actinin and vimentin were also stained using immunohistochemistry to compare the myocyte and fibroblast composition in relation to the core.  These results have yet to be analyzed.

The research of dephosphorylated gap junctions at the core of reentry begins to explain the underlying causes of reentrant arrhythmias and, thus, will be a significant tool in understanding the deadly arrhythmia on a multitude of scientific levels and eventually be implemented in society to save lives.

References:

1. American Heart Association. Cardiovascular Disease Stats. Dallas, Tex.: American Heart Association; 2005.

2. Beardslee MA, et al. Dephosphorylation and Intracellular Redistribution of Ventricular Connexin 43 during Electrical Uncoupling Induced by Ischemia. Circulation Res. 2000: 656-662.