The results presented here demonstrate that Notch can modify the transcriptome and cellular electrophysiology of cardiac myocytes to resemble cells of the specialized conduction system. These findings are relevant to the better understanding of how to control cardiovascular progenitor cell differentiation and how to engineer regenerative cardiac tissues.
Various approaches have been suggested for the generation of a biological pacemaker. These have included the manipulation ex-vivo
of human embryonic stem cell or induced pluripotent stem cells to become pacemaker cells, followed by implantation into diseased hearts. However, many important barriers remain to be overcome with a cell therapy approach, including cell-cell coupling between injected cells and native myocardium. A second approach involves gene therapy. For example, adenoviral-mediated delivery of Hcn2 channels, which contribute to the “funny current”, has been performed in dogs, and this type of biological pacemaker compared favorably with that of electronic units in the same animal (25–27
and reviewed in 1
). A third approach, supported by the results reported in this manuscript, is to directly reprogram existing cardiomyocytes in vivo
to a conduction-like phenotype. Results from Cx40-Cre
lineage analysis suggest that the differentiation step to the ventricular conduction system has definitively occurred by 16.5 dpc in the mouse4
. However, it is encouraging that our findings indicate that plasticity of mature myocytes to adopt a conduction-like phenotype persists later in life than previously appreciated. We have clearly demonstrated the ability to reprogram a small percentage of cells in vivo to Purkinje-like cells using a single factor. Thus, although further characterization of the effects of Notch will need to be undertaken prior to meeting a bar for convincing therapeutic value, this study suggests the possibility that cellular reprogramming strategies may be a viable strategy. It will be interesting to determine whether adenovirally-delivered Notch, either alone or in combination with other factors, may enhance the development of conduction tissue from adult myocardium and whether this effect is stable once the Notch signal is turned off.
Amongst the genes up-regulated by Notch activation in vivo are Nkx2-5
, which have been previously implicated in development and function of the conduction system.11, 28–30,31
Like Notch, Nkx2-5 and Tbx5 each function to regulate cardiac morphogenesis and cellular electrophysiology. Our ex-vivo
experiments using myocytes isolated at various ages indicate that Notch can activate Nkx2-5
at all times tested, while up-regulation of Tbx5
was restricted to prenatal stages. Future studies will address the mechanism for differential responsiveness under specific developmental contexts.
The development of cardiac preexcitation resembling Wolff-Parkinson-White syndrome in Notch activated mice15
is likely to result from the combined effects of several distinct Notch functions, including effects on morphogenesis and cellular electrophysiology. Mispatterning of the AV boundary likely contributes to the abnormal presence of myocardium traversing between atrium and ventricle. However, the ability of Notch to alter cellular electrophysiology, and specifically to promote a Purkinje fiber-like phenotype in ventricular cardiomyocytes, may be equally significant for altering the electrical conducting properties of accessory pathway tissue, which is required for a bypass tract to become functional and for the animal to manifest preexcitation. In Notch activated newborn hearts, and in neonatal human hearts32
, isolated strands of cardiomyocytes are observed in the absence of electrical preexcitaton; these remnants of AV canal myocardium are not electrically active. In Notch activated mice, as in some humans, preexcitation emerges only later in life, perhaps due to Notch-mediated changes in cellular electrophysiology of bypass myocardium. Tbx2
, genes implicated in preexcitation syndromes, also regulate the expression and/or function of ion channel genes such as Scn5a
Thus, changes in cellular electrophysiology may play an integral role in the development of preexcitation syndromes, antegrade conduction properties of accessory pathways, and risk of sudden cardiac death. Future studies will aim to determine the individual currents regulated by Notch, and to clarify the degree to which the role of specific currents and ion channel functions are conserved between mouse and human in specialized conduction tissue.
Microdeletions of Bone Morphogenetic Protein-2
) have recently been associated with a syndrome of WPW together with Alagille syndrome.35
, known to be implicated in Alagille syndrome, is located 3.8 MB centromeric from BMP-2
, raising the question of whether altered Notch signaling contributes to the observed WPW phenotype in this human syndrome. Almost one-third of WPW patients develop atrial fibrillation at a young age, which often resolves with ablation of the bypass tracts. An intriguing hypothesis is whether Notch pathway induced alterations in ion channel expression within the atrium, or near the atrial insertion of the accessory pathway, may play a role in the genesis of atrial fibrillation in humans.
While our in vitro studies demonstrate an effect of activating Notch on a large percentage of infected cardiomyocytes, the effect in vivo is more modest. In ventricular myocardium, there are cells that shift their phenotype to become similar to Purkinje cells as evidenced by upregulation of both Cntn2 and Cx40 and a characteristic action potential morphology, though there are also cells that can best be described as transitioning towards a Purkinje phenotype in response to Notch activation, as well as non-responders (Supplemental Figure 4
). The identification of additional signals that augment or restrict the degree of conversion will require further study. In Notch-activated hearts, we observed spatially-restricted non-homogenous up-regulation of Cntn2 and phenotypic variability in cellular electrophysiology. Possible explanations include additional combinatorial signals, either cooperative or inhibitory, which contribute to the competency of ventricular cardiomyocytes to respond to activated Notch in vivo. Alternatively, there may be a stronger bias against conduction system reprogramming inherent in a subset of cardiomyocytes, which is overcome by higher amounts of activated Notch provided by adenoviral delivery in the in vitro system. One future approach to address these possibilities might be to inject activated Notch into neonatal rodent ventricles, an approach that has been employed previously (Palatinus et al. Am J Phys
., 2011). Whether Notch is also required during conduction system development, apart from its previously demonstrated effects on the AV node15
or whether alternative signaling pathways can instruct cardiomyocytes to become conduction cells in the absence of Notch signaling, is not entirely clear. However, Notch-regulated genes appear to be important for patterning the ventricular conduction system as evidenced by postnatal patterning defects observed with CCS-LacZ
Notch signaling controls many facets of development, postnatal homeostasis and disease in many organ systems. The results presented here reveal a novel function for Notch signaling to influence cardiomyocyte cell fate decisions, and future investigations will further probe the interactions between Notch and other signaling pathways known to orchestrate conduction system lineage decisions in the heart.