Cellular Biology

Localization of Islet-1–Positive Cells in the Healthy and Infarcted Adult Murine HeartNovelty and Significance

Florian Weinberger, Dennis Mehrkens, Felix W. Friedrich, Mandy Stubbendorff, Xiaoqin Hua, Jana Christina Müller, Sonja Schrepfer, Sylvia M. Evans, Lucie Carrier, Thomas Eschenhagen
https://doi.org/10.1161/CIRCRESAHA.111.259630
Circulation Research. 2012;110:1303-1310
Originally published May 10, 2012

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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.

Introduction

Myocardial damage after myocardial infarction or other cardiac diseases results in loss of working myocardium and subsequent heart failure. Within the past years, improved revascularization strategies have enabled more patients to survive myocardial infarction, thereby increasing the incidence of heart failure.1 Despite new pharmacological and device-based therapies prognosis for heart failure remains poor, and new therapeutic options are urgently needed. In this context, regenerative approaches have gained increasing attention. Adult cardiac myocytes were classically regarded as withdrawn from the cell cycle. This dogma has been challenged by recent findings suggesting that a very small fraction of adult cardiomyocytes in human heart is regenerated.2 Fate-mapping experiments in mice provided evidence that cells from an unknown source differentiate into cardiomyocytes after myocardial injury, but not in healthy hearts.3 A subsequent study showed that this effect can be stimulated by bone marrow cell therapy, probably due to stimulation of endogenous cardiac progenitors.4 A study using a feline model also suggested cardiac regeneration by endogenous progenitor cells.5 These data lend support to the concept of resident cardiac stem or progenitor cells as proposed earlier.6 These cell types have been characterized by the expression of general (c-kit,7 and sca-18) or more restricted (Islet-19) stem or progenitor cell marker proteins. Their stimulation might be a particularly attractive therapeutic strategy as it avoids immunologic problems and could be achieved pharmacologically. Yet, the existence, relevance and character of resident cardiac progenitors remain ambiguous.10

Editorial, see p 1267

In This Issue, see p 1265

Cells that express the transcription factor Islet-1 are attractive candidates as resident cardiac progenitors because the role of Islet-1 in early cardiac development has been unambiguously demonstrated. Islet-1 is a transcription factor of the LIM-homeodomain family, which was first identified in the β-cells of the pancreas as an enhancer of the insulin gene.11 It has been extensively studied in the nervous system, where it is essential for the differentiation of motor neurons.12,13 Islet-1 became popular in the field of cardiovascular research after the discovery of severe cardiac malformation in Islet-1–deficient mice. These animals lack the outflow tract, the right ventricle, and most of the atria. Lineage tracing studies revealed that these structures originate from Islet-1 positive (Islet-1+) precursors.14 Islet-1+ cells do not only differentiate into cardiac myocytes but also contribute to the formation of the sinoatrial node (SAN), the atrioventricular node (AVN), and smooth muscle cells of the coronary arteries.15 Importantly, murine Islet-1+ cells retain this wide cardiovascular differential potential in vitro when isolated from embryonic and neonatal hearts16,17 or from murine or human embryonic stem cells.9,16 Islet-1+ cells from embryonic and neonatal hearts can therefore be regarded as real cardiac progenitors. Recently, small numbers of Islet-1+ cells were identified in adult heart.18,19 Khattar et al differentiated 2 clusters of Islet-1+ cells in adult murine hearts,18 and Genead et al detected Islet-1+ cells in hearts of young adult rats.19 Both studies relied on immunofluorescent methods, which are subject to ambiguity because of the extremely low expression level of Islet-1. Furthermore, they are not well suited to analyze the exact anatomic localization of scattered cells in 3D. The present study used a genetic approach to improve the sensitivity and specificity of detection of Islet-1+ cells and evaluated their localization and number throughout adult life and after myocardial infarction.

Methods

An expanded Methods section is available in the Online Data Supplement.

Animals

The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the NIH (Publication No. 85–23, revised 1985). Isl-1–nLacZ-mice were created by targeted insertion of a DNA cassette containing nLacZ in exon 1 of the Islet-1 gene.13 Final investigations were done in heterozygous Isl-1–nLacZ-mice on a mixed Blackswiss and C57BL/6J background, and wild-type littermates were used as controls.

Bromo-Chloro-Indolyl-Galactopyranoside Staining

Hearts were perfusion-fixed with 4% paraformaldehyde, washed in 0.9% NaCl, and afterward incubated in a staining solution containing potassium ferrocyanide (5 mmol/L), potassium ferricyanide (5 mmol/L), MgCl2 (2 mmol/L), 0.02% NP-40, 0.01% sodium deoxycholate, Tris pH 7.4 (20 mmol/L), and 1 mg/mL bromo-chloro-indolyl-galactopyranoside (X-Gal) in phosphate-buffered saline for 2 hours at 37°C. Cells were fixed in 0.05% glutaraldehyde for 10 minutes at 4°C and afterward incubated in the staining solution over night. Islet-1+ cells could be visualized by a blue nuclear signal.

Histology

Serial cryosections (10 μm) were either performed in a 4-chamber view or a transversal short-axis view. All sections were collected. Sections were postfixed in 4% paraformaldehyde for 10 minutes at room temperature and eosin-counterstained to allow for an overview. Consecutive sections were used for immunofluorescence, immunohistochemistry, and staining for cholinesterase activity.

Isolation of Smooth Muscle Cells

The ascending aorta and the pulmonary artery were excised, cleared of connective tissue, and longitudinally opened. Aorta and pulmonary artery were cut into 5 to 10 pieces, which were transferred onto 35-mm cell culture plates. These pieces were cultured in DMEM containing 10% FCS, and 1% penicillin/streptomycin and immunofluorescence was performed before the cells formed a confluent monolayer.

Preparation of mRNA From Murine and Human SAN Tissue

Mice were euthanized by cervical dislocation. Hearts were quickly removed and placed in modified Tyrode solution (NaCl 119.8 mmol/L, KCl 5.4 mmol/L, CaCl2 0.2 mmol/L, MgCl2 1.05 mmol/L, NaHCO3 22.6 mmol/L, NaH2PO4 0.42 mmol/L, glucose 5.05 mmol/L, Na2EDTA 0.05 mmol/L, ascorbic acid 0.56 mmol/L). The SAN region (approximately 1×1 mm) was carefully dissected using a Stemi SV6 stereo microscope (Zeiss). We obtained human SAN tissue, which was routinely prepared from postmortem human hearts, as described by Hudson,20 during scientific autopsy (between 1–3 days postmortem). RNA was isolated using the High-Capacity cDNA RT-Kit (Applied Biosystem).

Isolation of Mouse SAN Cells

Isolation of SAN cells was performed according to Mangoni et al.21 The SAN region was dissected as described above and isolation of SAN cells was achieved by digestion using Liberase TH (Roche Applied Science) and manual agitation.

Results

The distribution of Islet-1+ cells was investigated in 30 heterozygous Isl-1–LacZ mice at different time points after birth (10 weeks to 18 months). After light perfusion-fixation and X-gal staining, nLacZ+ cells were easily recognizable by eye or stereo microscopy when present in clusters. Microscopic identification of even single isolated nLacZ+ cells was unambiguous due to the nuclear restriction of the blue signal and immunohistology showed that nLacZ+ cells expressed Islet-1 (Online Figure I and Sun et al15). Hearts from 6 nontransgenic littermates that were subjected to identical fixation and staining procedures did not show any comparable signal (Online Figure II).

nLacZ+ cells were not randomly distributed within the heart but were strictly confined to certain anatomic structures. There was no macroscopic or microscopic evidence for nLacZ+ cells in the muscular layer of the left or right ventricular walls (Online Figure III; n=20, ≈500–600 slices per heart). In contrast, we consistently identified three different regions of nLacZ+ cells in adult heart (Figure 1A through 1C). Two regions were visible in an anterior view of the heart (Figure 1A) and a third region was located at the back of the heart (Figure 1B). A cluster of nLacZ+ cells was located in the border zone between right atrium and the superior vena cava. nLacZ+ cells were also scattered around the proximal part of the great vessels (aorta and pulmonary artery). The additional clusters on the back of the heart contained only few nLacZ+ cells (2–50 cells). These small clusters were located around the pulmonary veins and inside the interatrial septum. Very few single nLacZ+ cells were localized along the wall of the pulmonary veins. We identified positive cells in these 3 anatomic regions in all transgenic animals that were analyzed and did not observe alterations of localization or cell number with age.

Figure 1.
Figure 1.

nLacZ+ cells in an Isl-1–nLacZ adult mouse heart. A, Anterior view, and B, posterior view of a 20-week-old Isl-1–nLacZ-mouse heart after X-gal staining. C, Magnification of the nLacZ+ structures marked with asterisks in A and B. AO indicates aorta; CG, cardiac ganglion; PA, pulmonary artery; RA, right atrium; RV, right ventricle; and SVC, superior vena cava.

The clusters of nLacZ+ cells on the posterior side of the heart consisted of round, large cells. Further characterization revealed that these were cardiac ganglia, as shown by the coexpression of acetylcholinesterase (AChE; Figure 2A through 2C). Coexpression of β-galactosidase (β-gal) and the parasympathetic marker choline acetyltransferase (ChAT) characterized them as parasympathetic ganglia (Figure 2D). Very few single nLacZ+ neurons were located in the nervous plexus surrounding the pulmonary veins (Figure 2C).

Figure 2.
Figure 2.

Characterization of nLacZ+ cells in the interatrial septum and around the pulmonary veins. AChE+ (brown) and nLacZ+ cells (blue nucleus) in a cardiac parasympathetic ganglion in A, low magnification, and B, high magnification. C, Single nLacZ+ cell around a pulmonary vein. Asterisks in A mark structures showed in higher magnification in B and C. D, Brightfield and confocal microscopy showing coexpression of ChAT (green) and β-gal in a parasympathetic ganglion. Nuclei are stained red (DRAQ5). CG indicates cardiac ganglion; PV, pulmonary vein. Scale bars: 500 μm (A), 20 μm (B), and 10 μm (C and D).

nLacZ+ cells were also detected in the proximal part of the aorta and the trunk of the pulmonary artery (Figure 1 and Figure 3A through 3C). The absolute majority of these cells were located in the muscular layer above the aortic and pulmonary valve. We isolated these cells for further characterization. They coexpressed β-gal and the smooth muscle markers smooth muscle actin and smooth muscle myosin heavy chain (Figure 3D and 3E). Very few single nLacZ+ cells were present in the aortic valve leaflets (Figure 4A and 4C). A third type of nLacZ+ cells existed in the left ventricular outflow tract region (Figure 4A through 4E), where a small number of nLacZ+ cells coexpressed β-gal and the cardiomyocyte marker α-actinin, indicating that these were cardiomyocytes.

Figure 3.
Figure 3.

Characterization of nLacZ+ cells in the proximal aorta and pulmonary artery. A through C, nLacZ+ cells in the muscular layer of the proximal aorta after X-gal staining and eosin counterstaining. D and E, nLacZ+ cells cultured after isolation from the proximal pulmonary artery, stained with X-gal and antibodies against smooth muscle α-actin (D, green) and smooth muscle myosin heavy chain (E, green). Nuclei are stained red (DRAQ5). Visualization by differential interference contrast confocal microscopy (DIC) and confocal fluorescent microscopy. Note the black nucleus in DIC as sign of nLacZ positivity. AO indicates aorta; LV, left ventricle; RA, right atrium; RV, right ventricle; and SVC, superior vena cava. Scale bars: 500 μm (A), 100 μm (B), and 10 μm (C through E).

Figure 4.
Figure 4.

nLacZ+ cells in the outflow tract and the aortic valve. A, Histological overview, showing the localization of nLacZ+ cells in the muscular layer of the outflow tract, and B, high magnification after X-gal staining. C, Asterisk marks a single nLacZ+ cell in the aortic valve in high magnification. D, Brightfield visualization of nLacZ+ cells scattered in the myocardium of the outflow tract and the muscular layer of the proximal aorta. Corresponding staining for α-actinin (E, green), showing the typical cross-striation in a nLacZ+ cell in the myocardium of the outflow tract. AO indicates aorta; AV, aortic valve; LV, left ventricle; RA, right atrium; RV, right ventricle; and SAN, sinoatrial node. Scale bars: 500 μm (A), 100 μm (B), and 10 μm (C).

A fourth type of nLacZ+ cells was located in the muscular wall of the right atrium. These cells were aligned along the border zone between the right atrium and the vena cava superior (Figure 1 and Online Figure IV) and expressed the cardiomyocyte marker α-actinin (Figure 5A and 5D). Because of the peculiar localization between right atrium and vena cava superior and the typical “comma-shaped” form, we performed staining for the SAN marker hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4). Costaining for HCN4 and X-gal indicated that these cells were SAN cells (Figure 5B, 5C, 5E, and 5F). Interestingly, only a subpopulation (approximately 50%) of HCN4-positive cells stained positive for nLacZ. The AChE-positive and HCN4-positive AVN was consistently negative for nLacZ+ cells (Online Figure V). Isolation of cells from the SAN region resulted in single cells showing typical heterogeneous morphology (spindle-shaped, elongated spindle-shaped, atrial-like). Compared with whole-mount histologies, a much smaller subpopulation (0.1% to 5%) of the isolated cells stained positive for β-gal (Figure 6).

Figure 5.
Figure 5.

nLacZ+ cells in the sinoatrial node. A, Differential interference contrast confocal microscopy, and B and C, brightfield microscopy. D through F, Corresponding immunofluorescence staining for α-actinin (D, green) and HCN4 (E and F, green). Nuclei are stained red (DRAQ5). Scale bars: 20 μm (A and B) and 10 μm (C).

Figure 6.
Figure 6.

Isolated sinoatrial node cells after X-gal staining. A through E, Isolated cells from murine SAN with heterogeneous cellular morphology. Brightfield microscopy of elongated spindle-shaped cells (A and D), spindle-shaped cells (B and E), and atrial-like–shaped cells (C and F) showing nLacZ+ (A through C) and nLAcZ− cells (D through F), respectively. Scale bars: 5 μm.

To investigate whether Islet-1 is also expressed in murine wild-type and human SAN, we isolated RNA from murine and postmortem human SAN tissue. Islet-1 mRNA was detectable in all tissue samples containing the SAN in C57BL/6J from 12 to 44 weeks of age (n=31), but not from the corresponding atrial or ventricular myocardium (Figure 7A). Islet-1 mRNA was also detectable in human postmortem samples containing the SAN, but not from the corresponding ventricular myocardium (n=4; Figure 7B).

Figure 7.
Figure 7.

Islet-1 mRNA in murine and human sinoatrial node tissue. A, Qualitative detection of Islet-1 mRNA by RT-PCR in murine SAN tissue from four C57/BL6J wild-type animals (12 and 32 weeks), and B, human SAN tissue samples from 4 different patients. LV indicates left ventricular tissue; RV, right ventricular tissue; LA, left atrial tissue; RA, right atrial tissue; and SAN, sinoatrial node tissue.

To investigate the role of lslet-1+ cells after myocardial injury, we subjected a group of Isl-1–LacZ mice to myocardial infarction and examined the hearts at different time points after ischemic injury (2, 7, 14, and 28 days after left anterior descending artery ligation, n=3–4; Figure 8A through 8D). nLacZ+ cells could still be detected in the SAN, the trunk of the great vessels, and cardiac ganglia (Online VI, A through C) but were not present in the infarct region in 12 of 13 examined hearts 2, 14, and 28 days after myocardial infarction (in >4000 analyzed sections). However, in 1 heart examined 7 days after myocardial infarction, we observed 10 questionable cells in the infarct area showing atypical but clearly nuclear LacZ staining (Online Figure VII, A).

Figure 8.
Figure 8.

Isl-1–nLacZ-hearts after myocardial infarction. Histological overview, showing nLacZ-hearts 2 days (A) and 28 days (C) after left anterior descending artery ligation. Insets mark the regions shown in higher magnification (B and D). Examples of the infarct border zone 2 days (B) (hematoxylin and eosin staining) and 28 days (D) after myocardial infarction (sirius red staining). Scale bars: 50 μm.

Discussion

The transcription factor Islet-1 marks a population of cardiovascular progenitors of the second heart field in early murine development.14 The observation that few Islet-1+ cells are also present in the early postnatal heart and can differentiate into cells of all 3 cardiovascular cell types in vitro16,17 led to the idea that Islet-1+ cells represent a resident cardiac progenitor cell population with therapeutic potential.22 By using gene targeted Islet-1 nLacZ knock-in mice we provide evidence that the postnatal (1–18 months) murine heart is essentially devoid of Islet-1+ cells except well defined and consistent populations in the SAN, a fraction of cardiac ganglia, the media of the proximal aspects of the aorta, and pulmonary artery and very few single cells in the outflow tract area. Given the high sensitivity and specificity of nLacZ staining and the consistency of the staining pattern we believe that the findings are unambiguous.

Our results are mainly consistent with 2 recent findings and significantly expand them.18,19 Khattar et al described Islet-1–expressing cells in 2 types of clusters in close proximity to the heart basis. One consisted of neuronal cells and a second one was formed by cardiomyocytes. In a longitudinal study, Genead et al identified single Islet-1+ cells in the outflow tract of young adult rats that expressed the cardiomyocyte marker troponin T. Both studies mainly relied on classic immunohistological techniques. The difference between these findings may indicate species differences. On the other hand they might also be due to technical limitations. In the light of the present findings, the first cluster most likely represented parasympathetic ganglia, the second one the SAN and the troponin T+/Islet-1+ cells in the outflow tract of rats correspond to the few nLacZ+ cardiomyocytes in our model.

The first, also macroscopically visible clusters of nLacZ+ cells, were located in the posterior aspects of the atria and around the pulmonary veins. These cells were marked as parasympathetic neurons by their coexpression of AChE and ChAT. AChE marks neuronal structures in general, whereas ChAT almost exclusively marks parasympathetic neurons.23,24 The finding of Islet-1 expression in differentiated neurons was not surprising since it was known from chicken embryos that Islet-1 expression persists in neurons after leaving the cell cycle.25 Additionally, recent studies by Sun et al have demonstrated a key role for Islet-1 in sensory neuron development.26 Interestingly, not all cardiac ganglia contained nLacZ+ cells. Rysevaite et al described about twenty AChE-positive cardiac ganglia in the adult murine heart.27 Similar to their results, we observed cardiac ganglia of different size and cell number (2–50 cells), most of them being localized around the pulmonary veins. These nLacZ+ ganglia were detectable in up to 1.5-year-old mice, which had a mixed genetic background of Blackswiss and C57BL/6J. In this respect, our findings are identical to the clusters of Islet-1+ cells observed in 129SvJ×C57BL/6J and in 129SvJ mice by Khattar et al.18

The second cluster of Islet-1+ cells clearly marks the SAN. Approximately 50% of the HCN4 and α-actinin–positive cells of the SAN were also nLacZ+, suggesting the existence of at least 2 populations of pacemaker cells in the adult murine SAN, an Islet-1+ and an Islet-1− subpopulation. In all likelihood, the Islet-1+ subpopulation of the SAN is identical to the second cluster of cells described by Khattar et al.18 The authors described this cluster to be located in the right atrium and the interatrial septum. We were not able to detect any nLacZ+ cardiomyocytes in the interatrial septum. The difference might be species- or even strain-dependent, as Khattar et al showed differences between mouse strains. Alternatively, it may be a question of labeling techniques. In any case, the existence of a robust Islet-1+ SAN subpopulation is in agreement with recent data from Mommersteg et al28 showing that the SAN develops partly from cells that coexpress Islet-1 and Tbx18. This is a very small population, resulting from an overlap of Islet-1+ progenitors from the second heart field and Tbx18+ sinus venosus progenitors. These cells maintain Islet-1 positivity after myocardial differentiation during embryonic development. Furthermore, it fits to the data by Sun et al,15 who showed that even though the Islet-1 expression in the right atria becomes more and more locally confined during embryonic development, it persists in the SAN up to 3 days postnatally (the latest time point evaluated in their study). Sun et al15 described attenuation of Islet-1 expression during this process, suggesting that Islet-1 expression would diminish within weeks. Our data now show that Islet-1 positivity of the SAN remains unaltered for at least 18 months, indicating that it serves a continuous, as yet unidentified, function in pacemaker cells. After isolation of SAN cells we observed that only a small subpopulation of sinoatrial node cells stained positive for β-gal. nLacZ+ cells were not restricted to one of the previously described SAN cell types.21 The proportion of nLacZ+ cells varied markedly between the different preparations (0.1% to 5%) but was always much lower than that observed in histology. The low proportion of nLacZ+ cells probably is due to the technical difficulties in obtaining isolated single SAN cells and suggests that nLacZ+ SAN cells were more susceptible to the digestion process. In any case, it confirms the existence of a subset of Islet-1+ SAN pacemaker cells. These results are in accordance with previous findings of Sun et al,15 who observed colocalization of Islet-1 and GFP in a HCN4-H2B-GFP knock-in mouse model during cardiac development. Of note, Islet-1 was also expressed in AVN during cardiac development, whereas we could not detect nLacZ+ cells within the AVN of adult animals. The mRNA data from human heart samples suggest that the murine findings extend to the human SAN as well.

Besides the 2 compact clusters of Islet-1+ cells, we identified significant numbers of more disseminated nLacZ+ smooth muscle cells in the proximal aorta and the trunk of the pulmonary artery. Since Islet-1 progenitors can give rise to smooth muscle cells14–15 and Sun et al showed persistent Islet-1 expression in this particular region of the heart on the third postnatal day,15 we were not surprised by this finding. Again, the specific function of Islet-1+ compared with the majority of Islet-1− smooth muscle cells in the media of the great vessels remains unknown at present.

In contrast to adult newt hearts, which contain Islet-1–expressing cardiomyocytes,29 Islet-1 expression was considered to be downregulated during murine cardiac differentiation.12,13,16,21 Therefore, we were surprised by the finding that a small number of ventricular cardiomyocytes exhibit Islet-1 positivity, all of them located in the outflow tract area. This could suggest that these cells are cardiomyogenic progenitors. Indeed, Smart et al showed that by priming mice with thymosin β4, cardiac progenitors marked by the expression of Wilms tumor 1 gain Islet-1 expression during cardiac differentiation30 after myocardial injury. However, although our study cannot exclude the possibility that Islet-1 plays a role during cardiac regeneration, several findings argue against the proposed role of Islet-1+ cardiovascular progenitors remaining in the adult heart. Recent data in mice, in contrast to zebrafish,31 support the hypothesis that cardiac regeneration occurs from an undifferentiated precursor rather than from cardiac myocytes.3 We found different cell types that expressed Islet-1, all of them highly differentiated. Furthermore, the localization of these cells and especially the absence of Islet-1+ cells from the working myocardium of the left and right ventricle (except for the very few cells in the outflow tract) make it unlikely that these cells play a significant role for myocardial regeneration. When analyzing >4000 sections (and thereby more than 100 Mio cells) we found that 12 of 13 hearts did not contain any nLAcZ+ cells after myocardial infarction. In 1 heart with a very large myocardial infarction, we detected 10 cells that showed an atypical but nuclear LacZ staining in the subendocardial infarct region 7 days after myocardial infarction. Whether the staining in these cells really reflects Islet-1 is unknown at present, but the frequency would then account for 10 of >25 Mio analyzed cells at this time point and therefore is unlikely to be of biological relevance.

Limitations

Genetic models such as the one used in our study have the inherent limitation that the expression of the marker is never present in 100% of the cells intended for transgene expression and that the expression pattern may vary between different lines of mice. Moreover, we had to use heterozygous Isl-1–LacZ-mice because insertion of nLacZ into the Islet-1 gene locus caused disruption of the endogenous Islet-1 gene and homozygous knockout of Islet-1 is lethal. Therefore, the mice were functionally heterozygous Islet-1 knockout mice. This was not associated with apparent abnormalities, but unrecognized pathologies cannot be excluded completely. On the other hand, partial allele usage may have caused underestimation of the number of Islet-1+ cells. The fact that we found very consistent distribution of nLacZ+ cells in 25 animals of different ages suggests that such error, if existing, was minor. In addition, costaining with an Islet-1 antibody confirmed that nLacZ+ cells were indeed Islet-1 positive (Online Figure I). Staining for β-gal can also give rise to nonspecific positivity, but nontransgenic littermate controls were consistently negative and the strict nuclear localization of the signal facilitated discrimination between a true signal and noise in almost all cases. Nevertheless, we observed 10 cells in the subendocardial infarct region of 1 heart 7 days after myocardial infarction that showed an atypical but nuclear LacZ staining (Online Figure VII, A). We could not definitely discriminate between a true positive signal and false positivity (eg, caused by macrophages that are able to catalyze X-gal32). Although it might be possible that Islet-1 was expressed between the analyzed time points, the long half-life of β-gal protein of ≈30 hours33 render it unlikely that such transient expression would go unnoticed.

In conclusion, our results identify Islet-1 as a novel marker of a subpopulation of SAN cells, parasympathetic neurons, a subpopulation of smooth muscle cells in the proximal aspects of the great arteries and few cardiac myocytes in the outflow tract of the adult heart. The role of Islet-1 in these distinct heart regions deserves further studies.

Sources of Funding

This study was supported by the LeDucq Foundation. D.M. was supported by the Werner Otto Stiftung.

Disclosures

None.

Acknowledgments

We thank the Mouse Pathology Core Facility (University Medical Center of Hamburg-Eppendorf) for immunohistochemical staining for Islet-1, K. Püschel (University Medical Center of Hamburg-Eppendorf) for providing the human SAN samples, and J. Starbatty for excellent technical assistance.

Footnotes

  • In February 2012, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.77 days.

  • The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.111.259630/-/DC1.

  • Non-standard Abbreviations and Acronyms

    AChE
    acetylcholinesterase
    AVN
    atrioventricular node
    ChAT
    choline acetyltranferase
    HCN4
    potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4
    SAN
    sinoatrial node
    X-gal
    bromo-chloro-indolyl-galactopyranoside

  • Received October 25, 2011.
  • Revision received March 7, 2012.
  • Accepted March 8, 2012.

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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/≈100 000 000 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.

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  • Localization of Islet-1–Positive Cells in the Healthy and Infarcted Adult Murine HeartNovelty and Significance
    Florian Weinberger, Dennis Mehrkens, Felix W. Friedrich, Mandy Stubbendorff, Xiaoqin Hua, Jana Christina Müller, Sonja Schrepfer, Sylvia M. Evans, Lucie Carrier and Thomas Eschenhagen
    Circulation Research. 2012;110:1303-1310, originally published May 10, 2012
    https://doi.org/10.1161/CIRCRESAHA.111.259630
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