Circulation Research May 11, 2012 Journal Club Localization of Islet-1–Positive Cells in the Healthy and Infarcted Adult Murine Heart Florian Weinberger, Dennis Mehrkens, Felix W. Friedrich, Mandy Stubbendorff, Xiaoqin Hua, Jana Christina Müller, Sonja Schrepfer, Sylvia M. Evans, Lucie Carrier, Thomas Eschenhagen Circ Res. 2012;110: PDF [with Online Supplement]: Related Editorial by Mark Sussman [PDF]: Myocardial Isl + land: A Place With Lots of Rhythm, but No BeatMyocardial Isl + land: A Place With Lots of Rhythm, but No Beat Included in the Journal Club pack: Abstract, Novelty & Significance section, and all figures.

Abstract Rationale: The transcription factor Islet-1 is a marker of cardiovascular progenitors during embryogenesis. The isolation of Islet-1–positive (Islet-1+) cells from early postnatal hearts suggested that Islet-1 also marks cardiac progenitors in adult life. Objective: We investigated the distribution and identity of Islet-1+ cells in adult murine heart and evaluated whether their number or distribution change with age or after myocardial infarction. Methods and Results: Distribution of Islet-1+ cells in adult heart was investigated using gene targeted mice with nuclear β-galactosidase inserted into the Islet-1 locus. nLacZ-positive cells were only present in 3 regions of the adult heart: clusters in the interatrial septum and around the pulmonary veins, scattered within the wall of the great vessels, and a strictly delimited cluster between the right atrium and superior vena cava. Islet-1+ cells in the first type of clusters coexpressed markers for parasympathetic neurons. Positive cells in the great arteries coexpressed smooth muscle actin and myosin heavy chain, indicating a smooth muscle cell identity. Very few Islet-1+ cells within the outflow tract expressed the cardiomyocyte marker α-actinin. Islet-1+ cells in the right atrium coexpressed the sinoatrial node pacemaker cell marker HCN4. Cell number and localization remained unchanged between 1 to 18 months of age. Consistently Islet-1 mRNA was detected in human sinoatrial node. Islet-1+ cells could not be detected in the infarct zone 2 to 28 days after myocardial infarction, aside from 10 questionable cells in 1/13 hearts. Conclusions: Our results identify Islet-1 as a novel marker of the adult sinoatrial node and do not provide evidence for Islet-1+ cells to serve as cardiac progenitors. Localization of Islet-1–Positive Cells in the Healthy and Infarcted Adult Murine Heart

Novelty and Significance What Is Known? In early cardiac development, cells expressing the transcription factor Islet-1 form the right ventricle, the outflow tract, parts of the atria (the second heart field) and the sinoatrial node (SAN) and atrioventricular node (AVN). Islet-1 + cells from fetal hearts or embryonic stem cells form cardiac myocytes, smooth muscle and endothelial cells when placed in cell culture, suggesting that Islet-1 + cells may also serve a cardiovascular progenitor role in the adult heart. In the adult mouse heart, Islet-1 positivity was detected by antibody staining in distinct clusters in the heart basis whose identity remained unclear. What New Information Does This Article Contribute? Genetic labeling experiments showed that Islet-1 in the adult mouse heart is expressed in HCN4+ pacemaker cells of the SAN, but not the AVN, parasympathetic nervous ganglia, smooth muscle cells of the proximal part of the great arteries, and few cardiac myocytes of the outflow tract area. The expression pattern was highly reproducible and stable for up to 18 months. No Islet-1+ cells were found in healthy ventricular myocardium. These cells were found only in an atypical form in 10/ cells in >4000 sections of hearts 2 to 28 days after myocardial infarction. Islet-1 is an established marker of early cardiovascular development; however, its role in the adult heart, specifically its progenitor role, remains obscure. Given the technical difficulties in studying Islet-1 distribution in the heart by immunologic means, we used a genetic approach, that is, a mouse in which Islet-1 expression drives a nuclearly localized β-galactosidase. This technique allows not only histological identification of Islet-1+ cells with high sensitivity and specificity but even the macroscopic identification in the whole heart, facilitating the analysis of regional distribution. Islet-1+ cells were undetectable in >5000 sections from normal or injured myocardium. This is important because it provides strong evidence against the idea that Islet-1+ cells could serve a progenitor role in the normal or injured postnatal heart. Stable Islet-1+ expression in very distinct and functionally specialized cells suggest that Islet-1+ expression in the postnatal heart is not a spurious left-over from development but serves an important role that warrants further studies. The findings of this study also establish Islet-1 as a new and, compared with HCN4, a specific marker of a subpopulation of SAN pacemaker cells

nLacZ+ cells in an Isl-1–nLacZ adult mouse heart. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

Characterization of nLacZ+ cells in the interatrial septum and around the pulmonary veins. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

Characterization of nLacZ+ cells in the proximal aorta and pulmonary artery. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

nLacZ+ cells in the outflow tract and the aortic valve. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

nLacZ+ cells in the sinoatrial node. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

Isolated sinoatrial node cells after X-gal staining. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

Islet-1 mRNA in murine and human sinoatrial node tissue. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association

Isl-1–nLacZ-hearts after myocardial infarction. Weinberger F et al. Circulation Research 2012;110: Copyright © American Heart Association