Prapa Kanagaratnam, Vias Markides, Dominic W.Lamb, Richard J.Schilling, D. Wyn Davies, Anthony W.C. Chow, Pravina Patel, Nicholas S. Peters.
St Mary’s Hospital and Imperial College School of Medicine, London, UK
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At the cellular level, membrane channel properties, gap-junctional
coupling and local cellular architecture determine cardiac conduction1,2. We have used epicardial
multi-electrode mapping combined with immunoconfocal quantification of gap-junctional proteins and interstitial
connective tissue to understand the relationship between structure, ultrastructure and conduction properties at
the tissue level. Gap junctions contain channels consisting of two hemi-channels, termed connexons2,3. Each
connexon is constructed from six connexin proteins. Connexins are a multigene family of proteins, which have
a high degree of molecular homology, but form gap junctions with different functional properties. In the human
atrium, the predominant connexins are connexin43 (Cx43) and connexin40 (Cx40). Connexin45 (Cx45) has also
been shown to be present at low levels. Gap junctions are considered to present rate-determining resistive
discontinuities to propagation and the functional properties conferred by the relative levels of the connexins
expressed are, therefore, thought to determine the conduction properties of myocardium. Extrapolating in-vitro
cell pair or two-dimensional multicellular data to predict the role of ultrastructural determinants of conduction in
the intact tissue is difficult because the properties of the tissue will be determined by not only the mode of
intercellular communication and intracellular function, but also by the properties of the extracellular space and
of the functional effects of the anatomical structures formed by tissue reorientation, folding and thickenening
(such as at the crista terminalis, triangle of Koch).
In order to understand the role of gross structure and ultrastructure in conduction in the human right atrium, we
have studied patients undergoing cardiac surgery. Epicardial mapping was performed on the intact right atrial
free wall during sinus rhythm and pacing prior to commencing cardiopulmonary bypass, and a biopsy was
excised from the mapped region. Careful positioning of the array with respect to the sulcus terminalis ensured
a consistent position with the anterior 56 electrodes covering the trabeculated portion of the free wall.
Electrograms were acquired on-line during sinus rhythm and (having inspected activation maps during sinus
rhythm) pacing at 5 mA at a 500 ms interval from a site that closely simulated the direction of activation during
sinus rhythm. Following mapping, an excision biopsy was taken from the mapped region of the trabeculated
right atrial free wall before cardiopulmonary bypass was established. Three randomly selected consecutive sinus
beats and three consecutive paced beats reproducing the direction and pattern of sinus propagation, were
selected for off-line analysis. For each selected cycle, an activation time was assigned to each of the 56
electrodes overlying the trabeculated right atrium, at the maximum negative dV/dt deflection of the unipolar
electrogram. The activation time of each electrode and those of the immediate neighbouring electrodes were
used to calculate the local conduction velocities over the entire array by the method of triangulation.
The atrial myocardial biopsy was then prepared for immunolabeling of both Cx43 and Cx40. Connexin signal
quantity was expressed per unit area of myocyte. The role of endomysial connective tissue (between individual
myocytes) and total connective tissue (endomysial and perimysial-between muscle bundles) in determining
conduction velocity was also investigated by quantifying endomysial connective tissue in subselected fields and
represented as area of endomysial tissue per unit area of myocardial field.
We have found using these techniques that although there is no apparent relationship between conduction
velocity and the quantity of Cx43 signal or of total connexin signal (Cx40+Cx43), an increase in the quantity of
Cx40 signal was associated with a reduction in conduction velocity during sinus rhythm (p=0.036). There was a
stronger correlation between the relative quantities of immunolabel (Cx40/Cx40+Cx43) and conduction velocity
during sinus rhythm (p<0.005), such that as the proportion of Cx40 immunolabel increased the conduction velocity
decreased4. Conduction velocity during pacing at 500 ms intervals did not correlate with the quantity of Cx40,
Cx43, total connexin signal or the relative immunofluorescence of the two connexins. However, the relative
signal quantity of immunolabeled Cx40 to total connexin (Cx40/Cx40+Cx43) correlated with the change in
conduction velocity during pacing (mean paced conduction velocity – mean sinus conduction velocity) such that
a lower proportion of Cx40 was associated with a decrease in conduction velocity at a shorter cycle length
(p<0.02). The quantities of Cx40, Cx43 and total connexin signals did not correlate with the changes seen during
pacing. Neither the mean total connective tissue autofluorescence (endomysial+perimysial connective tissue)
nor endomysial autofluorescence alone correlated with sinus conduction velocity, paced conduction velocity or
change in conduction velocity on pacing.
The results of the study, therefore, indicate that the relationship between Cx40 immunofluorescence and
conduction velocity is not simple. The findings suggest either that Cx40 and Cx43 form heteromeric connexons
in such a manner that an increasing proportion of Cx40 reduces coupling or that Cx40 somehow inhibits the
formation of functional homomeric Cx43 connexons thereby reducing coupling. In addition, at the whole tissue
level, it appears that the connective tissue content has little impact on conduction properties.
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