Response to Letters Regarding Article, “Abnormal Conduction and Morphology in the Atrioventricular Node of Mice with Atrioventricular Canal Targeted Deletion of Alk3/Bmpr1a Receptor”
We would like to thank Drs Gourdie and Sedmera for their comments about our recent study on the role of bone morphogenic protein (BMP) signaling in atrioventricular (AV) node function.1 We agree that the mechanism responsible for the “split” or “twin” AV nodes we observed in Class IV hearts is as yet unknown. Our data suggest that disruption of BMP signaling affects normal fibrotic deposition, but it was not our contention that this was the primary means of abnormal AV node morphology. In the Discussion section we presented 2 alternative mechanisms (outlined on pages 2541 to 2542), “… BMP signaling is required to maintain proper continuity between the AV node and His bundle or the His bundle and the bundle branches” and “… disrupted BMP signaling could affect patterning of the AV canal, thereby impacting formation of the entire central conduction system.” We agree that the hypothesis of disrupted fusion of conduction system modules2,3 is a possibility and should have been included in the Discussion. We thank Drs Gourdie and Sedmera for pointing out this important oversight.
We also thank Anderson et al for reading our study with interest. In response to their comments, we would like to stress the extent of histological analysis that was undertaken. Please note that histological analysis was done blinded from the optical mapping results. At least 300 sections were generated for each heart at 6-μm intervals, following a coronal plane of sectioning and starting from a posterior position and moving anteriorly. One of every 30 sections was stained with Masson’s trichrome and observed under a microscope to narrow down the sections going through the AV conduction system. Within that region, 1 of every 4 to 10 sections was further stained and analyzed. Analysis included identification of the His bundle and tracing of the conduction tissue in more dorsal sections. In none of the slides observed was the “AVN” connected to atrial myocardium. Sections through mutant hearts were compared with similar sections through hearts from CCS-LacZ mice that were whole-mount stained for β-galactosidase in order to identify the cardiac conduction system (not shown). We agree that immunostaining with other markers of the conduction system would have positively identified the AV structure within the mutant sections themselves.
However, we disagree that our central finding—that altered BMP signaling disrupts AV junction formation and AV node function—was primarily based on histological evidence. Our study defined mutants on the basis of detailed electrophysiological examination, which included pharmacological agents that target the AV node, volume-conducted ECGs, and optical mapping of ventricular activation patterns obtained during spontaneous activity. Specifically, pharmacological treatment of Class III mutants (hearts where ventricular activation primarily occurred via bypass conduction) with high doses of procainamide resulted in third-degree heart block. These data strongly indicated severe conduction defects within the AV junction. We agree it is possible that other regions of the central conduction system may also have been affected; however, no cGATA-6 Cre–mediated recombination has been detected in the bundle branches or Purkinje fibers.4 The latter finding argues for a central role of alterations within the node. Finally, we did not define a specific mechanism, nor did we state that BMP signaling is limited to the AV node; rather, we concluded that disruption of Alk3 function broadly affects the AV junction. We agree that more detailed analysis of cardiac conduction system morphology is required.
Stroud DM, Gaussin V, Burch JB, Yu C, Mishina Y, Schneider MD, Fishman GI, Morley GE. Abnormal conduction and morphology in the atrioventricular node of mice with atrioventricular canal–targeted deletion of Alk3/Bmpr1a receptor. Circulation. 2007; 116: 2535–2543.
Gourdie RG, Mima T, Thompson RP, Mikawa T. Terminal diversification of the myocyte lineage generates Purkinje fibers of the cardiac conduction system. Development. 1995; 121: 1423–1431.
Chuck ET, Freeman DM, Watanabe M, Rosenbaum DS. Changing activation sequence in the embryonic chick heart. Implications for the development of the His-Purkinje system. Circ Res. 1997; 81: 470–476.