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Available online at www.sciencedirect.com


Editorial Commentary

Mouse models of cardiac conduction system markers: Revealing the past, present, and future of pacemaking and conduction Crystal M. Ripplinger, PhDn Department of Pharmacology, University of California Davis School of Medicine, Davis, CA

The cardiac conduction system (CCS) is a highly specialized and functionally distinct component of the heart, responsible for initiating and maintaining pacemaking and conduction from the early stages of development onward. Failure or dysfunction of any part of the CCS, including the sino-atrial node (SAN), atrio-ventricular node (AVN), or His–Purkinje system can result in bradycardia, heart block, arrhythmias, and even sudden death. The development of the multi-functional CCS is an extraordinarily complex, yet elegant dance of precisely timed expression of transcription factors and gene programs. Fortunately, recent years have seen an explosion of genetically modified mouse models that have greatly aided our ability to visualize and characterize the CCS. Therefore, much of our present knowledge on the detailed mechanisms of CCS patterning and development has been a result of transgenic and knock-in mouse models with reporter genes specifically or predominantly expressed in the CCS. A plethora of CCSreporter mouse lines have been developed over the past two decades (see Table in Ref. [1] for complete list) and include markers of ion channels and subunits (such as HCN4, Cx40, Cx45, and MinK), markers of transcription factors (including Gata6, Shox2, Tbx18, and Isl1), and even the useful CCS–LacZ strain that arose, somewhat by accident, from a complex genomic rearrangement resulting in CCS expression [2]. These reporter mouse lines can delineate the entire CCS, as is the case with HCN4 and CCS–LacZ [3–6], or expression profiles may be limited to specific CCS components, including the SAN (e.g., Tbx18, Shox2 [7,8]), or AVN (e.g., Tbx2 [9]). Mouse models of CCS markers are not only useful for

characterizing the specific spatio-temporal development of the CCS, but gene profiling of reporter-positive cells has also led to the discovery of additional, novel markers of the CCS [10]. Thus, mouse models of CCS markers have become an invaluable tool for studying the complex development and function of the CCS. In this issue of Trends in Cardiovascular Medicine, Liang et al. [1] provide an excellent overview of a particularly useful and robust CCS marker, HCN4. hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the “funny current,” If, that is activated during diastole in pacemaker cells and contributes to diastolic depolarization. HCN channels are expressed throughout the adult CCS, including the bundle branches and Purkinje fibers and a number of HCN4 knock-in mouse lines have been developed, including HCN4–nLacZ, –H2BGFP, and –CreERT2 [3–5]. By mouse embryonic day 16.5 (E16.5), all parts of the CCS, including SAN, AVN, and His–Purkinje system robustly express HCN4, which continues into adulthood, making the HCN4-reporter lines extremely useful for identifying and characterizing all components of the CCS. What is perhaps more intriguing, however, is the early up- and down-regulation of HCN4 that occurs in both the CCS and working myocardium throughout development. In a series of “pulse-chase” experiments, tamoxifen inductions in HCN4CreERT2 mice were performed at distinct time points from E7.5 onward and embryos were harvested at E16.5 [4]. Although the allocation of each temporally distinct subset of HCN4-expressing cells to the different components of the CCS is detailed nicely in the original report and review

CMR is supported by the American Heart Association, United States (12SDG9010015) and the US National Institutes of Health, United States (R01 HL111600). n Corresponding author. E-mail address: [email protected] (C.M. Ripplinger) http://dx.doi.org/10.1016/j.tcm.2014.09.007 1050-1738/& 2015 Elsevier Inc. All rights reserved.





by Liang et al. [1,4], a key observation emerging from these studies is that HCN4 gene expression is turned on, then off, then on again in several different precursor populations prior to complete CCS expression by E16.5. For example, inductions at E7.5 and E8.5 appear to label progressively larger populations of CCS cells, including parts of the SAN, AVN, left bundle branch, and Purkinje fibers. Of note, areas of the atrial and ventricular myocardium are also labeled with these inductions, indicating early, transient expression of HCN4 in the working myocardium. Inductions at E9.5–E12.5, however, label more restricted cell populations, with few or no labeled cells observed in the bundle branches or Purkinje fibers following E12.5 induction. As expected, inductions at E16.5 label the entire CCS, including those components not labeled at E12.5, indicating de novo HCN4 expression during the later stages of development [4]. The dynamic expression profile of HCN4 raises some interesting questions: What are the transcriptional regulatory mechanisms responsible for dynamic HCN4 expression? What, if any, is the functional significance of early, transient HCN4 expression followed by later, sustained expression? Does this paradigm offer any insight into the differentiation and maturation of distinct CCS cell phenotypes? And, perhaps most importantly, how do we translate these and other key developmental insights from mouse models into effective regenerative therapies? These questions are paramount at a time when novel stem cell differentiation approaches and cellular reprogramming techniques are rapidly expanding and providing us with a glimpse of the potential regenerative therapies that might now be possible. One exciting area of regenerative medicine related to the CCS is, of course, the development of the biological pacemaker. Recent interest has centered on the use of humaninduced pluripotent stem cells (iPSCs) as a cell-based biological pacemaker [11], and more recently, on direct reprogramming of adult ventricular myocytes into pacemakers via transfection of the transcription factor Tbx18 [12,13]. Although these approaches are still in their infancy, it is exciting to ponder whether detailed knowledge of the transcriptional regulation underlying development of the CCS could be exploited to differentiate or reprogram iPSCs or adult myocytes into distinct CCS phenotypes for use in biological pacing and beyond. Perhaps more robust pacemakers or even fast-conducting Purkinje fibers could be engineered by not only expressing the correct transcription factor or factors but also through the precise temporal control of transcription factor expression, the template for which would likely come from detailed developmental studies. Despite these exciting possibilities and the unprecedented spatial, temporal, and genetic information provided by



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CCS-reporter mouse models, there is much left to do to achieve the eventual clinical goal of regenerative medicine or biological pacemaking. Yet the detailed developmental studies reviewed here by Liang et al. may provide the best possible roadmap going forward and calls to mind the truthful adage, “You can’t know where you’re going until you know where you’ve been…”

refere nces














Liang X, Evans SM, Sun Y. Insights into cardiac conduction system formation provided by HCN4 expression. Trends Cardiovasc Med 2014 [In press]. Stroud DM, Darrow BJ, Kim SD, Zhang J, Jongbloed MRM, Rentschler S, et al. Complex genomic rearrangement in CCS-LacZ transgenic mice. Genesis 2007;45:76–82. Hoesl E, Stieber J, Herrmann S, Feil S, Tybl E, Hofmann F, et al. Tamoxifen-inducible gene deletion in the cardiac conduction system. J Mol Cell Cardiol 2008;45:62–9. Liang X, Wang G, Lin L, Lowe J, Zhang Q, Bu L, et al. HCN4 dynamically marks the first heart field and conduction system precursors. Circ Res 2013;113:399–407. Später D, Abramczuk MK, Buac K, Zangi L, Stachel MW, Clarke J, et al. A HCN4þ cardiomyogenic progenitor derived from the first heart field and human pluripotent stem cells. Nat Cell Biol 2013;15:1098–106. Rentschler S, Vaidya DM, Tamaddon H, Degenhardt K, Sassoon D, Morley GE, et al. Visualization and functional characterization of the developing murine cardiac conduction system. Development 2001;128:1785–92. Aanhaanen WTJ, Mommersteeg MTM, Norden J, Wakker V, de Gier-de Vries C, Anderson RH, et al. Developmental origin, growth, and three-dimensional architecture of the atrioventricular conduction axis of the mouse heart. Circ Res 2010;107:728–36. Sun C, Zhang T, Liu C, Gu S, Chen Y. Generation of Shox2Cre allele for tissue specific manipulation of genes in the developing heart, palate, and limb. Genesis 2013;51:515–22. Aanhaanen WTJ, Brons JF, Domínguez JN, Rana MS, Norden J, Airik R, et al. The Tbx2þ primary myocardium of the atrioventricular canal forms the atrioventricular node and the base of the left ventricle. Circ Res 2009;104:1267–74. Pallante BA, Giovannone S, Fang-Yu L, Zhang J, Liu N, Kang G, et al. Contactin-2 expression in the cardiac Purkinje fiber network. Circ Arrhythm Electrophysiol 2010;3:186–94. Mandel Y, Weissman A, Schick R, Barad L, Novak A, Meiry G, et al. Human embryonic and induced pluripotent stem cellderived cardiomyocytes exhibit beat rate variability and power-law behavior. Circulation 2012;125:883–93. Hu Y-F, Dawkins JF, Cho HC, Marbán E, Cingolani E. Biological pacemaker created by minimally invasive somatic reprogramming in pigs with complete heart block. Sci Transl Med 2014;6:245ra94. Kapoor N, Liang W, Marbán E, Cho HC. Direct conversion of quiescent cardiomyocytes to pacemaker cells by expression of Tbx18. Nat Biotechnol 2013;31:54–62.

Mouse models of cardiac conduction system markers: revealing the past, present, and future of pacemaking and conduction.

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