GATA6 reporter gene reveals myocardial phenotypic heterogeneity ...

Am J Physiol Heart Circ Physiol 301: H1952–H1964, 2011. First published September 9, 2011; doi:10.1152/ajpheart.00635.2011.

GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling Mathieu C. Rémond,1* Grazia Iaffaldano,1* Michael P. O’Quinn,2 Nadejda V. Mezentseva,1 Victor Garcia,1 Brett S. Harris,2 Robert G. Gourdie,2 Carol A. Eisenberg,1 and Leonard M. Eisenberg1 1

New York Medical College/Westchester Medical Center Stem Cell Laboratory, Departments of Physiology and Medicine, New York Medical College, Valhalla, New York; and 2Department of Regenerative Medicine and Cell Biology, Medical University of South Carolina, Charleston, South Carolina Submitted 24 June 2011; accepted in final form 7 September 2011

Rémond MC, Iaffaldano G, O’Quinn MP, Mezentseva NV, Garcia V, Harris BS, Gourdie RG, Eisenberg CA, Eisenberg LM. GATA6 reporter gene reveals myocardial phenotypic heterogeneity that is related to variations in gap junction coupling. Am J Physiol Heart Circ Physiol 301: H1952–H1964, 2011. First published September 9, 2011; doi:10.1152/ajpheart.00635.2011.—This study examined transgenic mice whose expression of a ␤-galactosidase (lacZ) reporter is driven by a GATA6 gene enhancer. Previous investigations established that transcription of the transgene was associated with precardiac mesoderm and primary heart tube myocardium, which decreased progressively, so that its expression was no longer observed within ventricular myocardium by midgestation. Expression of this reporter in the adult was investigated for insights into myocyte homeostasis and cardiovascular biology. Morphometric analysis determined that ⬍1% of myocytes, often found in small clusters, express this GATA6-associated reporter in the adult heart. LacZ expression was also found in the ascending aorta. Myocardial expression of the transgene was not associated with a proliferative phenotype or new myocyte formation, as lacZ-positive myocytes neither labeled with cell division markers nor following 5-bromodeoxyuridine pulse-chase experimentation. Despite exhibiting normal adherens junctions, these myocytes appeared to exhibit decreased connexin 43 gap junctions. Treatment with the gap junctional blocker heptanol both in vivo and in culture elevated myocardial ␤-galactosidase activity, suggesting that deficient gap junctional communication underlies expression of the transgenic reporter. LacZ expression within the myocardium was also enhanced in response to cryoinjury and isoproterenol-induced hypertrophy. These results reveal a previously uncharacterized phenotypic heterogeneity in the myocardium and suggest that decreased gap junctional coupling leads to induction of a signaling pathway that utilizes a unique GATA6 enhancer. Upregulation of lacZ reporter gene expression following cardiac injury indicates this transgenic mouse may serve as a model for examining the transition of the heart from healthy to pathological states. myocardium; connexin 43; ␤-galactosidase transgene DESPITE BEING COMPRISED OF the most physically energetic cells in the body, the conception of the cardiac muscle is of a rather staid tissue. The bulk of the myocardium is made up of the working myocytes of the ventricles and atria, and these two cell types are often portrayed as having uniform phenotypes. The exception to this uniformity was thought to be exhibited in periods of severe disease or stress when adult cardiomyocytes begin to exhibit properties characteristic of embryonic pheno-

* M. C. Rémond and G. Iaffaldano contributed equally to this work. Address for reprint requests and other correspondence: L. M. Eisenberg, New York Medical College/Westchester Medical Center Stem Cell Laboratory, Departments of Physiology and Medicine, Vosburgh Pavilion Rm. 303, New York Medical College, Valhalla, NY (e-mail: [email protected]). H1952

types (47). Another longstanding paradigm of cardiovascular biology was that the adult myocardium is postmitotic (6, 49). Myocytes of the adult heart have been characterized as being terminally differentiated cells that have lost their ability to proliferate. It is now accepted that at least a limited amount of cardiomyocyte regeneration occurs during adulthood (4, 40). The primary mechanism of cardiomyocyte regeneration in the adult probably results from differentiation of endogenous cardiac stem cells (7, 32). Thus the myocardium may be marked by small numbers of newly generated cycling myocytes. Transgenic mice containing a regulated reporter gene under control of cis-acting genomic sequences have provided valuable model systems for studying cardiac embryology (15, 33, 43). The expression pattern of the reporter gene, usually, lacZ (␤-galactosidase) or green fluorescent protein, in these phenotypically normal genetically engineered animals allows for identification of transcriptional control elements that confer cell phenotype and temporally regulated gene expression. A surprising finding brought forth by these animal models was that the expression patterns of cardiac genes, such as Nkx2.5 and GATA6, which are uniformly exhibited throughout the developing myocardium, were actually composites of multiple distinct transcriptional programs. The 5=-flanking sequence of the Nkx2.5 gene contains several cis regulatory regions, which together control cardiac Nkx2.5 expression in its entirety but individually consign this gene’s expression in a regionally and temporally specific manner (11, 36). For example, expression of Nkx2.5 within the right ventricle, interventricular septum, and atrioventricular canal is controlled by enhancer modules that are distinct from those that direct expression in the atria (11). Likewise, enhancer sequences in the GATA6 locus that drive that gene’s expression in conduction tissue differ from those responsible for expression within working myocardium (1, 14). In these cases, it is important to realize that the expression profile of a reporter transgene under control of individual enhancer modules does not necessarily correspond to domains exhibiting higher levels of expression of the endogenous gene. Instead, the pattern of expression for the reporter gene identifies domains of expression that are regulated by distinct transcriptional programs, which comprise individual elements of the entirety of an endogenous gene’s expression profile. The transgenic tgG6/lacZ mice used in this study were generated by genomic insertion of a lacZ transgene, whose expression is driven by a 1,300-bp enhancer-containing region from the GATA6 gene (15, 29). This genetic region contains consensus binding sites for several heart-associated factors, including Nkx2.5, MEF2C, SRF, GATA-4/5/6, HAND1, and

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HETEROGENEITY OF MYOCYTE PHENOTYPE IN ADULT HEART

Hand2 (15), whose combination may account for the differential activity of this enhancer (1, 15). Previous studies (15) examining development of these mice demonstrated that lacZ expression driven by this GATA6 distal enhancer is associated with the developing myocardium. In the tgG6/lacZ mouse embryo, lacZ expression is first exhibited within precardiac mesoderm and then displayed subsequently throughout the primary heart tube myocardium. At later embryonic stages, the transgene is inactivated sequentially along the posterior (venous) to anterior (arterial) axis during formation of the multichambered heart, although a band of lacZ expression persists in the outflow tract until day 13 of embryogenesis (15). That these displays of reporter gene expression were due to regulation from the isolated GATA6 enhancer in the transgene and not from regulatory sequences affiliated with genomic insertion sites was verified by comparing multiple independently generated transgenic lines containing the same construct, which exhibited the same temporal and spatial pattern of lacZ expression during development (15, 29). It should be noted that the downregulation of this transgene that occurs during midgesta-

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tion is not symptomatic of a similar reduction of endogenous GATA6 levels at these stages of development but instead indicates that different enhancer modules maintain GATA6 expression. Based on the embryonic lacZ expression within precardiac tissue and early stage myocardium, we wondered whether these transgenic mice may also be useful for detecting myocardial progenitors and newly formed cardiomyocytes in adults. Accordingly, we examined adult hearts from these mice for expression of the lacZ reporter. Here we report that indeed the adult heart is marked by small numbers of lacZ-positive myocytes that are sprinkled throughout the myocardium. Surprisingly, lacZ-positive myocytes are not in cell cycle, as may be expected if these cells were newly generated. Instead, the lacZ label appears to be associated with myocytes that are poorly coupled to surrounding myocardial tissue and suggests that a previously undescribed heterogeneity exists in the phenotypic composition of the myocardium. In addition, mice subjected to various cardiac insults showed increased numbers of lacZ-positive myocytes, which indicate that this transgenic

Fig. 1. Expression of lacZ in the tgG6/lacZ adult mouse heart. A: dorsal view of transgenic heart after 5-bromo-4-chloro-3indolyl-␤-D-galactopyranoside (X-Gal) staining, which identifies small numbers of lacZ-positive cells dispersed within the myocardium. B: same heart as A after optically clearing with Murray’s clear, which revealed the presence of lacZ-expressing cells throughout the myocardial interior. This heart represents the predominant strain of tgG6/lacZ mice used in this study, which is the TG1852 strain. C: as a comparison, X-Gal staining of the TG2838 strain, which was independently derived using the same transgene, showed a similar lacZ distribution in the adult heart, thus confirming the lacZ response is due to the GATA6 enhancer. Moreover, previous reports (15) showed these and other sister transgenic lines generated with the same construct also exhibited identical lacZ distribution patterns in the embryo. D: parallel processing of control wild-type hearts, which remained unlabeled by this procedure, demonstrated that X-Gal staining was specific for expression of the transgenic reporter. Scale bar ⫽ 1 mm.

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model may prove to be a valuable model for understanding how pathologies develop in the heart at the tissue and cellular level. MATERIALS AND METHODS

Animals and processing of the hearts. The tgG6/lacZ mice used in this study, which have been described in previous reports (15, 18, 29), were generated using an enhancer from the GATA6 gene that drives the myocardial-associated expression of the lacZ reporter. Transgenic tgG6/lacZ and wild-type C57BL/6 mice were euthanized by cervical dislocation, and their hearts were harvested by thoracotomy. After several washes in ice-cold PBS, hearts were fixed for 1 h in ice-cold 10% neutral-buffered formalin. Hearts were then PBS washed and processed for paraffin or cryoembedding. For paraffin embedding, hearts were taken through progressive dehydration steps in gradually increasing ethanol concentrations (70, 80, 95, and 100%), followed by xylene and finally paraffin. For cryoembedding, hearts were soaked in increasing concentrations (10, 15, 20, and 30%) of sucrose in PBS and finally embedded at ⫺80°C in optimum cutting temperature embedding medium. Sections of 8- or 10-␮m thickness were produced with a microtome or cryotome, respectively, and mounted on positively charged slides. All experimentation was approved by the Institutional Animal Care and Use Committee at New York Medical College. Detection of the lacZ reporter gene. Detection of the lacZ reporter by 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) staining was carried out as described previously (18). Hearts were briefly fixed with 10% formalin and then rinsed several times with 100 mM sodium phosphate (pH 7.3), 2 mM MgCl2, 0.01 mM sodium deoxycholate, and 0.02% NP-40. Staining reactions were performed at 37°C with 5 mM potassium ferrocyanide and 1 mg/ml X-Gal overnight, rinsed in PBS, and postfixed in 10% neutral-buffered formalin. Some of the hearts were cleared according to the protocol of Miller et al. (44) for visualizing interior staining of the tissue. Hearts were pro-

gressively dehydrated in graded ethanol solutions and then placed overnight in a mixture of equal parts benzyl alcohol and benzyl benzoate (BABB or Murray’s clear). The remaining noncleared hearts were subsequently embedded in paraffin and sectioned. Quantification of ␤-galactosidase. Hearts were quickly excised to prevent degradation by proteases. The atria, left ventricle, and septum were separated from the rest of the heart and placed in ice-cold PBS. From each individual heart, pooled atria and left ventricle plus septum were minced and transferred to red blood cell lysis buffer (Biolegend). After 15 min, the reaction was stopped by dilution in excess PBS, and the samples were centrifuged at 350 g for 10 min. Pellets were resuspended and homogenized in ice-cold RIPA buffer containing 25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, and Complete protease and PhosSTOP phosphatase inhibitors (Roche) and then centrifuged at 14,000 g for 10 min to remove cellular debris. ␤-Galactosidase activity of the resulting protein samples was measured according to their hydrolysis of the fluorogenic substrate 4-methylumbelliferyl ␤-D- galactopyranoside (MUG), using the SensoLyte MUG ␤-galactosidase assay kit (Ana Spec). Immunofluorescent staining. Immunofluorescent labeling was performed on sectioned tissue as described previously (18 –20). Specificity of the antibody against ␤-galactosidase was verified by examining the patterns of immunoreactivity with parallel X-Gal stained sister sections. Detection of specific proteins was accomplished using antibodies against ␤-galactosidase (5 Prime¡3 Prime), connexin 43 (Cx43; Invitrogen), Ki67 (Abcam), phosphorylated (Ser10)-histone H3, cleaved caspase-3 (Cell Signaling Technology), sarcomeric ␣-actinin, and pan-cadherin (Sigma). To decrease fluorescent background, sections were incubated overnight at 4°C in a blocking solution consisting of PBS, 1% BSA, 0.3 M glycine, and 0.02% NaN3. For pan-cadherin and ␣-actinin staining, sections were incubated overnight at 4°C in a solution of PBS, 1% BSA, and primary antibody.

Fig. 2. Myocytes are the predominant cell type expressing the lacZ reporter gene. A: surface view of the left ventricle of an X-Gal stained adult tgG6/lacZ mouse heart showing blue stained cells within the myocardium. Note the absence of X-Gal staining in the left anterior descending coronary artery (arrow). B: closer examination of left ventricle showing that blue cells have the rod-like phenotype of differentiated myocytes. C: X-Gal staining revealed a similar distribution of lacZpositive cells within the atria. D: X-Gal stained tgG6/lacZ mouse heart sectioned in the transverse plane and processed for electron microscopy. X-Gal histochemistry produces an electron dense crystalloid precipitate (arrows) in lacZ-positive cells (*). Electron microscope verifies that lacZ-positive cells were cardiomyocytes, as indicated by presence of myofibrils in cells that display the X-Gal-derived precipitate. Scale bars in A–C ⫽ 500 ␮m and in D ⫽ 10 ␮m.


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Sections stained with ␤-galactosidase antibody were incubated for 5 h at 37°C in PBS, 1% BSA. After several PBS washes, fluorescentlabeled secondary antibody, diluted in PBS-BSA, was applied in the dark for 1 h at room temperature. Staining for Cx43 and Ki67 was performed in a similar fashion, except for an additional preincubation step with triton-lysine buffer to enhance antibody penetration. To visualize nuclei, sections were counterstained with either DAPI or TOTO-3 (Invitrogen). To avoid confusion, both fluorescent nuclear dyes were imaged as blue by the Zeiss LSM 710 laser scanning confocal microscope, which was used to visualize all immunofluorescent-stained sections. Projection of confocal z-series was achieved using the Java image analysis and processing software Image J (http://rsbweb.nih.gov/ij/) and the DeconvolutionLab plugin (Biomedical Imaging Group, EPFL). 5-Bromodeoxyuridine labeling. To study whether lacZ-positive cells were dividing, four mice were injected intraperitoneally twice a day with 50 mg/kg 5-bromodeoxyuridine (BrdU) for 3 days and euthanized after 3 or 7 days. The hearts were harvested and processed for cryoembedding. For localizing BrdU-stained cells, frozen sections were treated with 2 N HCl for 30 min and washed three times for 5 min in PBS. Primary antibody from Abcam (Ab6326) was diluted 1:100 in PBS-BSA and incubated overnight at 4°C. Goat anti-rat antibody labeled with DyLight488 was used as a secondary antibody. In addition, sections were immunostained for sarcomeric ␣-actinin to identify myocytes and counterstained with DAPI for labeling nuclei.

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Surgical and pharmacological treatments. Two different methods were undertaken to investigate the response of the heart to injury. Cardiac hypertrophy was induced with isoproterenol (17, 39; Calbiochem), which was dissolved in 0.9% NaCl before being given to mice subcutaneously at 100 mg/kg body wt. Two groups of four mice were injected with either isoproterenol or PBS (as control) for 5 days before euthanasia. To verify the effects of isoproterenol, heart weights and body weights were measured, with statistical analysis (t-test) displaying significant differences (P ⬍ 0.05) in the heart weight-to-body weight ratios of treated mice compared with control. Cryosurgery was used as a surgical method for introducing injury to the heart (27, 41). Mice were placed under anesthesia (100 mg/kg ketamine and 3 mg/kg acepromezine ip) and then positioned face up before insertion of a 22-gauge catheter into the trachea. The animals were ventilated with 2% isoflurane in O2 with a 250-␮l tidal volume at 150 cycles/min. A left thoracotomy was performed in the fourth intercostal space, with the muscles overlying the intercostal space dissected free and retracted with 5– 0 silk threads. After the pericardium was opened, a prechilled 1-mm flat surface cryoprobe (CRYAC-3: Brymill Cryogenics) was placed against the epicardial surface of the heart for 1 s. Following removal of the cryoprobe, the incision was closed in layers using 6 – 0 silk, and the skin was sealed with Nexabond. After extubation, mice were placed on ambient oxygen and a warming blanket. Following their revival, animals received two carprofen injections over a 48-h period. At later times, animals were

Fig. 3. Distribution of lacZ-positive myocytes. A and B: lacZ reporter is exhibited by small clusters of myocytes. High magnification of the surface of the heart revealed lacZ-expressing myocytes are frequently contained within groups of 2– 4 cells (arrows), as shown in surface views of the ventricle and atria, respectively. C and D: small clusters of lacZ-positive myocytes (arrows) are further displayed in sectioned tissue of the wall and base, respectively, of the left ventricle. Scale bars ⫽ 50 ␮m. E and F: morphometric analysis-of sectioned heart tissue. E: myocyte densities were determined by 2 methods based on counting 1) total nuclei and converting to myocyte numbers, and 2) myocytes directly by outlining the cell membrane by wheat germ agglutinin (WGA) staining. Myocyte densities were based on the ratio of the area counted compared with the total area of the sections. Myocyte densities were converted to average number of myocytes/section based on average area per transverse section that was tabulated in this study (33 sections total), which included those assayed for DAPI, WGA, and X-Gal staining. The 2 methods yielded an average of 25,598 and 31,882 myocytes per section, respectively, ⫾SD, as shown. F: percentage of myocytes in transgenic hearts that expressed lacZ. From X-Gal stained sections, 162, 210, and 214 lacZ-positive ventricular cells were counted from each of 3 adult hearts, with the percentage of myocytes expressing the transgene based on the entire tissue area of all tabulated sections and myocyte densities determined by both methods discussed in E. As indicated by values on individual bars, which also display SD, both tabulation methods indicated that almost 1 myocyte per 1,000 expressed lacZ. AJP-Heart Circ Physiol • VOL

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euthanized, with the hearts removed, stained for X-Gal, and sectioned as described above. For interfering with gap junction activity, mice were subjected to heptanol treatments (27, 37). For this study, nine mice were injected with 100 ␮l of 1 mM heptanol iv, which was administered for 3 consecutive days. In parallel, aged and litter-matched animals were either injected with 100 ␮l PBS, administered with the same protocol as for heptanol, or left undisturbed. All mice were euthanized after 3 h from the last injection by cervical dislocation, with hearts harvested

by thoracotomy and processed for the detection of the lacZ reporter gene. Quantitative analyses of Cx43 expression and size of ventricular myocytes. Longitudinal sections of the heart were labeled with antiCx43 and anti-␤-galactosidase antibodies as described above. An assessment of Cx43 content was obtained from confocal images, using quantitation methods validated in previous investigations (23, 30, 55). Cell membrane-associated Cx43 expression by individual ventricular myocytes was measured by determining total pixel intensity of Cx43

Fig. 4. Nonmyocyte expression of the lacZ transgene. X-Gal staining of the heart and surrounding tissues indicated that the lacZ reporter was highly exhibited within the aorta, as shown in A a whole heart view following clearing of the tissue. B: a higher magnification view of the aorta suggested that the X-Gal stained cells were smooth muscle. Subsequent sectioning of the tissue confirmed that the lacZ-positive aortic cells (arrows) were distributed in the tunica media (C) and stained positive for smooth muscle markers (D), as indicated by their subsequent labeling for smooth muscle actin (arrows). E and F: In contrast, blood vessels within the heart displayed few lacZ-positive cells. E: transverse view of the ventricle at low magnification showing lacZ-negative blood vessels (arrows) and many X-Gal-stained myocytes (blue). F: higher magnification view of the boxed area in D, showing a lacZ-negative blood vessel (*) that is adjacent to multiple lacZ-positive cells within the myocardium. G: less frequently, sectioned heart tissue would reveal a blood vessel exhibiting X-Gal stained cells (arrow), although the vast majority of intracardiac blood vessels were lacZ-negative. Scale bars in A, B, and E ⫽ 500 ␮m and in C, D, F, and G ⫽ 50 ␮m. AJP-Heart Circ Physiol • VOL

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immunofluorescence along the cell border using ImageJ to select and analyze regions of interest. From 25 visualized fields, Cx43 expression was measured from a total of 33 ␤-galactosidase-positive myocytes, as well as the surrounding ␤-galactosidase-negative myocytes from each imaged field. For each field imaged by confocal microscopy, Cx43-content of ␤-galactosidase-positive myocytes was normalized to the average values obtained for ␤-galactosidase-negative myocytes. The comparative size of ␤-galactosidase positive and negative myocytes was assessed by measuring the diameter of the short axis (25, 30, 42). These data were obtained from sections in which myocytes were longitudinally or obliquely cut and where the nucleus was centered within the cell to ensure that the short axis was in the middle plane of the myocyte. From 29 visualized fields, short axis diameter was measured from a total of 43 ␤-galactosidase-positive myocytes, as well as a total of 155 surrounding ␤-galactosidasenegative myocytes from the imaged fields. Significance of Cx43 expression and short axis diameter was determined by the t-test, as calculated using the InStat statistical application (GraphPad Software). Cardiac tissue and myocyte isolation. Hearts isolated from 1-dayold neonates were rinsed once in sterile, calcium- and magnesium-free HBSS, pH 7.4, before digestion. Cell isolation was completed by two-step tissue digestion, beginning with an overnight trypsin (500 ␮g/ml in HBSS) treatment at 4°C on a rocking platform. Following addition of soybean trypsin inhibitor, the tissue was treated with collagenase type II (1 mg/ml; Worthington) for 1 h at 37°C. The tissue was triturated with a sterile serological pipette every 30 min to assist with cell dispersion. Following a 10-min sedimentation at 200 g, dispersed tissue was plated in DMEM/F12 (Hyclone) with 5% FBS. To uncouple gap junctions, cells were treated overnight in medium plus heptanol (1 mM) before X-Gal staining. Retention of the cardiomyocyte cell architecture was accomplished by plating cells in fibrinogen gels as previously described (8).

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Cell counts and morphometric analysis. The fraction of ventricular myocytes that expressed the lacZ reporter was determined by morphometric analysis of transverse sections of the adult mouse heart (2). The densities of total myocytes within the sectioned heart tissue were discerned by two separate methods. The first involved the manual counting of nuclei on DAPI-stained transverse sections (ⱖ2,000 nuclei/section) using Image J software and the Cell Counter plug-in (Kurt De Vos, University of Sheffield; [email protected]). To determine density of nuclei per area of transverse section, all nuclei were counted within a defined area in each of four ventricular sections, with the total number of nuclei per section computed by the ratio of the total area of the section divided by area that contained the counted nuclei. Area of the sections was computed from microscopic images into pixel densities using Adobe Photoshop. The myocyte densities of each section were calculated from the number of nuclei according to previously determined values of the relative proportion of cardiac cells that are myocytes and the ratio of multinucleated to mononucleated cardiomyocytes in the adult mouse heart (3, 60). The conversion of nuclei to myocyte numbers was according to the following equation: N/[(1/a) ⫹ b] ⫽ M, where N and M are total number of nuclei and myocytes per section, respectively, a is the fraction of cardiac cells in the mouse heart that are myocytes (0.56), and b is the fraction of myocytes that are binucleated (0.915). The second method for ascertaining myocyte numbers in transverse sections involved staining tissue with fluorescent-tagged wheat germ agglutinin (Vector Laboratories), which labels cell membranes (48, 66). This enabled individual myocytes to be identified within each of the sections and counted using Image J. After myocytes numbers were compiled from two fields on each of four sections (⬎600 cells counted per section), myocyte densities were determined based on the ratio of the area counted compared with the total area of the sections. LacZ-positive myocytes were measured by counting X-Gal-stained cells from multiple transverse sections from three mouse hearts, with the number of X-Gal-stained cells counted within ventricular myo-

Fig. 5. Immunofluorescent analysis of ␤-galactosidase-labeled cells. Parallel labeling of sister sections with X-Gal (A) and fluorescent-labeled ␤-galactosidase antibody (B), which showed identical staining patterns, verified the antibody specificity for the reporter protein. C and D: Triple labeling of the sectioned heart for ␤-galactosidase (green), sarcomeric ␣-actinin (grey), and nuclei (DAPI; blue). Myocyte phenotype of lacZ-positive cells (arrows) was confirmed by their striated pattern of ␣-actinin reactivity. Expression of the lacZ reporter was not associated with a replicative phenotype, as reflected by absence of costaining with cell cycle markers. In contrast, antibodies labeling for phospho-histone H3 (E), Ki67 (F), and 5-bromodeoxyuridine (BrdU; G) (arrows) were observed in a fraction of lacZ-negative myocytes. H: LacZ-positive cells isolated from tgG6/lacZ heart, as shown by dual staining for ␤-galactosidase (green) and ␣-actinin (red), displayed the morphology of terminally differentiated myocytes. Scale bars ⫽ 20 ␮m. AJP-Heart Circ Physiol • VOL

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cardium for each heart being ⬎160. To ensure that each X-Gal-stained cell was distinct, examined sections were separated by ⱖ200 ␮m. The fraction of ventricular myocytes that were lacZ-positive for each heart was then determined by measuring the total pixel area of every section used to count X-Gal stained cells and then converting those values to total myocyte numbers based on the two methods of cell counting described above. RESULTS

To investigate lacZ expression in adult tgG6/lacZ mice, we isolated hearts from multiple animals and stained them with the ␤-galactosidase substrate X-Gal, which turns blue in presence of the enzyme. This histochemical labeling unveiled small numbers of blue-stained cells randomly scattered about the myocardium (Fig. 1A). Optical clearing of the tissue disclosed that lacZ-positive cells were distributed throughout the entire myocardial wall (Fig. 1B). X-Gal staining of independently generated sister transgenic strains showed similar lacZ distributions in the adult heart (Fig. 1C), thus confirming the lacZ response was due to the GATA6 enhancer. Parallel processing of wild-type hearts, which remained unlabeled by this procedure, demonstrated that X-Gal staining was specific for expression of the transgenic reporter (Fig. 1D). Higher magnification views of transgenic hearts revealed that X-Gal-labeled cells exhibited the rod-like morphology of differentiated myocytes, as was observed in both ventricular (Fig. 2, A and B) and atrial (Fig. 2C) myocardium. The myocyte phenotype of lacZ-positive cells was confirmed by electron microscopy, as dark crystals typical of X-Gal histochemistry (61) were displayed by

cells containing myofibrils (Fig. 2D). An interesting feature of labeled myocytes was their frequent appearance in small clusters of two to four lacZ-positive cells (Fig. 3, A–D). The proportion of cardiac cells that were genetically tagged was determined by morphometric analysis, which indicated that almost 1 per 1,000 myocytes (⬃0.08%) in the adult heart were lacZ positive (Fig. 3, E and F). In addition to myocyte expression of lacZ, the reporter gene was also prominently exhibited by smooth muscle cells within the ascending aorta and arch (Fig. 1B and Fig. 4, A–D). Expression of lacZ was much less prominent in the pulmonary artery. In contrast to the aorta, blood vessels within the heart were mostly devoid of lacZ-expressing cells (Fig. 4, E and F), although occasionally ␤-galactosidase was displayed by smooth muscle cells of intracardiac blood vessels (Fig. 4G). The numbers of lacZ-positive cells within intracardiac blood vessels was far less than observed in the myocardium, and thus the proportion of cardiac smooth muscle cells expressing the transgenic reporter could not be determined statistically. The phenotype of lacZ-positive cells within the heart was characterized by immunostaining against ␤-galactosidase. Parallel staining of sister sections with X-Gal (Fig. 5A) and immunofluorescent ␤-galactosidase (Fig. 5B) verified the specificity of antibody staining. ␤-Galactosidase expressing cells within the myocardium exhibited sarcomeres, as shown by the striated staining against sarcomeric ␣-actinin (Fig. 5, C and D). Immunostaining was used to determine if lacZ expression was associated with cell cycle or apoptosis. Although myocytes

Fig. 6. Disrupted connexin 43 (Cx43) expression in lacZ-positive myocytes. A–G: confocal z-series projections of sectioned heart tissue triple labeled for ␤-galactosidase (green), Cx43 (red), and nuclei (DAPI; blue). A and B: identical field of heart tissue that is displayed with and without ␤-galactosidase staining, respectively. Arrows in A and B indicate position of lacZ-positive myocytes that were Cx43 negative and not coupled to surrounding myocardium. C and D: another cardiac field shown with and without the green channel, respectively, that marks the position of lacZ-positive myocytes. Again, note absence of Cx43 at the termini (arrows) of lacZ expressing myocytes. As shown in E–G, Cx43 expression was not absent from all myocytes that exhibited ␤-galactosidase. E and F: identical field of heart tissue, displayed with and without the ␤-galactosidase staining, containing lacZ-positive myocytes that exhibited Cx43 at one termini (arrow), although most Cx43 was localized to cytoplasm (arrowhead). G: example of lacZ-positive myocytes exhibiting Cx43 at cell membrane (arrows). H: sectioned cardiac tissue triple stained for ␤-galactosidase (green), ␤-catenin (red), and nuclei (TOTO3; blue). Staining with ␤-catenin or other adherens junction markers indicated that all lacZ-positive myocytes displayed normal intercalated discs (arrowheads), despite differences in Cx43 expression. Scale bars ⫽ 20 ␮m. AJP-Heart Circ Physiol • VOL

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were found that exhibited staining for the mitotic markers phospho-histone H3 (Fig. 5E) and Ki67 (Fig. 5F), these cells were lacZ negative, as correspondence between these cell cycle indicators and lacZ expression was not observed. Moreover, BrdU labeling of the mice failed to show correlation with reporter gene expression, as only lacZ-negative myocytes were observed with ongoing DNA synthesis (Fig. 5G). Neither was there a correlative staining with ␤-galactosidase and the apoptotic marker caspase-3 (not shown). Isolation of tissue from transgenic hearts indicated that lacZ-positive cells displayed the morphology of terminally differentiated myocytes (Fig. 5H). The most striking disparity in the phenotype of lacZ-positive ventricular myocytes, compared with the bulk of the surrounding myocardium, was their conspicuously low expression of cell membrane-associated Cx43 (Fig. 6). Some of these genetically labeled cells displayed a complete lack of Cx43-mediated coupling with the adjacent myocardium (Fig. 6, A–D), as the normal distribution of this gap junction protein at the myocyte termini was absent in lacZ-positive cells. Other ␤-galactosidase-labeled cells exhibited incomplete Cx43 coupling at the cell membrane (Fig. 6, E and F), while a fraction of these cells displayed Cx43 at the cell border (Fig. 6G). This reduction and/or absence of Cx43 in lacZ-positive cells neither appeared to be due to aberrant intercalated disc formation nor a general decrease in junction formation at myocyte termini, as these cells exhibited normal fascia adherens junctions, as indicated by immunostaining for ␤-catenin (Fig. 6H) and pan-cadherin (not shown). Thus, despite general similarities that lacZ-positive cells shared with the predominant lacZnegative myocyte population, such as sarcomeric organization, intercalated disc configuration, and size (Fig. 7A), ventricular myocytes expressing ␤-galactosidase appeared to be distinguished by an overall decrease in Cx43 gap junctions (Fig. 7B). The relationship between gap junctional function and display of the reporter gene was examined further by treatments with heptanol, which is an inhibitor of gap junctional communication. Hearts isolated from mice subjected to tail vein injections with heptanol for three days showed a marked increase in lacZ-positive myocytes (Fig. 8, A–C), with heptanol promoting a 4.8-fold greater ␤-galactosidase activity in left ventricular myocardium than displayed by PBS-injected tgG6/ lacZ mice (Fig. 8, D and E). Moreover, ventricular tissue harvested from transgenic mice exhibited increased ␤-galactosidase activity when exposed to heptanol in culture (Fig. 8E). Upregulation of the lacZ reporter gene was also observed in response to cardiac impairment (Fig. 9). Isoproterenol-induced cardiac injury resulted in enhanced numbers of lacZ-positive myocytes and also produced an expansion of vascular lacZ expression within the coronary arteries (Fig. 9, A and B). Most strikingly, isoproterenol augmented the display of the transgene in the atria (Fig. 9, B and C), with ␤-galactosidase activity being amplified ⬎33-fold (Fig. 9D). Direct insult to the ventricular sidewall using a cryoprobe increased distribution of lacZ-positive cells within the tissue bordering the damaged area (Fig. 9E). Sectioning of these cryoinjured hearts suggested most ␤-galactosidase-expressing cells in the border zones were myocytes (Fig. 9F). In summary, these data show that the tgG6/lacZ reporter gene identifies a subpopulation of cardiomyocytes with a variant phenotype that correlates to decreased gap junctional coupling with the neighboring myocardium. We AJP-Heart Circ Physiol • VOL

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Fig. 7. Comparison of ␤-galactosidase-positive and negative myocytes. A: plot of short axis diameters of ␤-galactosidase-positive and negative ventricular myocytes, with n values of 43 and 155 cells, respectively. No significant differences were observed in the size of the myocytes distinguished by the ␤-galactosidase marker. B: Cx43 densities of ␤-galactosidase-positive and negative ventricular myocytes, as determined by fluorescent intensities along the myocyte border of confocal microscopic images of Cx43 immunolabeled sectioned hearts. Compared with the ␤-galactosidase-negative myocytes, whose mean fluorescent intensity was defined as 100 units, ␤-galactosidasenegative myocytes displayed a Cx43 content reduced on average by 34.5% (P ⬍ 0.001; n ⫽ 33).

show as well that cardiac injury leads to increased acquisition of this variant phenotype. DISCUSSION

This study examined the hearts of adult transgenic mice whose expression of a lacZ-reporter in the embryo was shown previously to identify cardiomyocytes in the primary heart tube (15). During the remodeling of the embryonic heart into a four-chambered structure, the widespread expression of the reporter gene decreases progressively, so that its expression was no longer observed within the ventricular myocardium by embryonic day 13. We picked up the story in the adult and observed small numbers of myocytes that were randomly scattered throughout the myocardium. Morphometric analysis indicated that ⬍1 per 1,000 myocytes expressed the lacZ marker in the adult heart. Our initial thinking, based on the pattern of lacZ expression in the embryonic heart, was that the transgene may tag newly generated cardiomyocytes in the adult. Yet surprisingly, these lacZ-positive myocytes were not

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Fig. 8. Heptanol treatment upregulates lacZ expression. A: dorsal view of hearts obtained from adult tgG6/lacZ mice that were treated with PBS as control (left) or with heptanol (right) for three days prior to euthanasia. Control (B) and heptanol-treated (C) hearts after optically clearing. Note the increase in lacZ-positive cells in response to heptanol, which was verified (D) by measuring ␤-galactosidase activity in left ventricular myocardial tissue from multiple control (n1–3) and heptanol-treated (H1– 4) tgG6/lacZ mice. E: summary of these results in vivo is displayed in conjunction with the experimental outcome in vitro. For these latter experiments, neonatal hearts were harvested, and ventricular tissue dissociated to generate tissue fragments. Cultured tissue was then incubated overnight in absence or presence of 1 mM heptanol before protein was isolated and ␤-galactosidase activity was measured. Both in vivo and in vitro, ␤-galactosidase activity was increased in response to this gap junctional inhibitor, with heptanol treatment eliciting, respectively, a 4.8- and-3.3 fold enhancement in the activity of the reporter protein compared with control tgG6/lacZ heart tissue. Scale bars in A–C ⫽ 500 ␮m. Error bars in E correspond to SE.

in cell cycle, which would be expected in newly generated myocytes, as they were negative for markers of cell division and proliferation (34, 63) and did not label with BrdU. By general criteria (e.g., size, shape, sarcomeric organization, and intercalated disc configuration), lacZ-positive myocytes appear to be similar to the surrounding lacZ-negative myocyte population. The only differences we discerned were a correlation between the expression of the reporter molecule driven by a GATA6 gene enhancer and deficient expression of Cx43, which is the major gap junctional protein of working myocytes (23, 59). Whether lacZ-positive ventricular myocytes exhibit other connexin isoforms has not been fully determined, although these cells do not express connexin 40 (Cx40; data not shown), which indicates they do not possess a conduction tissue phenotype (24, 59). The hypothesis that deficient gap junctional communication underlies expression of the transgenic reporter was bolstered by experiments showing that AJP-Heart Circ Physiol • VOL

administration of the gap junction inhibitor heptanol increased the numbers of lacZ-positive myocytes and ␤-galactosidase activity in the myocardium. Thus myocytes tagged by the reporter transgene would be impaired, at best, in their electrical coupling to their neighbors. Although the pedigree of the lacZ marked myocytes is yet unclear, their existence indicates the adult myocardium may exhibit a previously unknown and unsuspected phenotypic heterogeneity. The prevalence of lacZ-positive cardiomyocytes was increased in mice subjected to cardiac injury either directly via cryoprobe or by hypertensive stimulation with isoproterenol. Cardiac injuries from an infarction or cryoinjury has been shown to promote gap junctional loss at intercalated discs of myocytes bordering the injury (10, 46), which is the area where we observed increased numbers of lacZ-positive cells. Remodeling of Cx43 gap junction at the injury border zone is thought to contribute to reentrant arrhythmia following myocardial

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Fig. 9. Enhancement of lacZ reporter gene expression in response to injury. Ventral view of hearts obtained from adult tgG6/lacZ mice that were injected with either (A) PBS or (B) isoproterenol. Exposure to isoproterenol upregulated lacZ expression in the aorta and ventricular myocardium, induced ␤-galactosidase within the coronary arteries, and generated a dramatic enhancement of the reporter gene in the atria. C: sectioned view of an isoproterenol-treated heart showing high numbers of atrial myocytes that have become lacZ positive. D: increase in lacZ-positive myocytes in response to these treatments was verified by measuring ␤-galactosidase activity in cardiac tissue from isoproterenol-treated and control tgG6/lacZ mice, with isoproterenol enhancing the activity of the reporter protein by 5.2- and 33.4-fold in left ventricular and atrial myocardium, respectively. Error bars correspond to SE for values from 4 isoproterenol-treated hearts. E: left ventricular surface near the apex of tgG6/lacZ mouse hearts exposed to cryoprobe. Heart that was marked sequentially with 2 cryolesions (*) and harvested 5 days later for X-Gal staining. F: longitudinal section of a day 5 cryoinjured heart displaying X-Gal-stained cells adjacent to areas of tissue damage (*). Scale bars ⫽ 250 ␮m.

infarction (59). The localized upregulation of LacZ following injury may provide a convenient approach for marking and isolating border zone tissues/cells for subsequent analysis. Decrease in Cx43 gap junctions within ventricular muscle can also result from remodeling events that occur during the hypertrophic response to pressure overload (9, 21, 50, 52). Isoproterenol-induced hypertrophy provokes interstitial fibrosis (31, 51), which can lead to discontinuities in propagating signals via gap junctions (56). Atrial function following isoproterenol-induced hypertrophy has been less well studied, although fibrosis and Cx40 gap junctional remodeling within the atria are symptomatic of hemodynamic overload and heart failure (22, 53). The dramatic increase in atrial lacZ expression AJP-Heart Circ Physiol • VOL

that was observed in response to isoproterenol administration may be indicative of a significant functional impairment of this tissue that could result from reduced signal propagation via Cx40 gap junctions, which triggers a transcriptional program whose readout is the lacZ transgenic reporter. It should be noted that ␤-galactosidase activity detected in these mice is not related to nonspecific increase in senescent cells upon chemical or physical stresses, as endogenous ␤-galactosidase has an acidic pH optima and the histochemical and fluorogenic assays used in this study were performed under alkaline conditions (58). Accordingly, wild-type mice subjected to these treatments did not exhibit X-Gal staining. Moreover, all fluorogenic assays of ␤-galactosidase activity

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included samples from wild-type hearts, whose low background fluorescence was subtracted from values obtained from transgenic mice. In addition, the background fluorescence assayed from wild-type cardiac protein extracts did not increase upon treatment with heptanol or isoproterenol under these alkaline conditions. Thus detection of ␤-galactosidase is related to expression of a transgene that appears to label a variant phenotype within the myocardium, whose prevalence increases in response to insult or disease. In addition to its myocardial expression, the lacZ reporter was also highly exhibited in smooth muscle cells of the aorta. What is the significance of aortic lacZ expression and is it analogous to the cardiomyocyte display of ␤-galactosidase? A comparative analysis that may be informative is between the aorta and primary heart tube. One hallmark of the primary heart tube myocardium is its expression of proteins associated with differentiated smooth muscle cells, such as calponin, SM-22␣, ␣-smooth muscle actin, and smoothelin (16, 38, 54, 65), which are downregulated in embryonic myocytes as the heart more fully develops. Thus there may be a shared genetic program, involving GATA6 regulation, between primary heart tube myocardium and aortic smooth muscle. Another way the primary heart tube and aorta are similar is that they both are large blood vessels exposed to pulsatile flow. This suggests there may be a functional component regulating this shared genetic program. Accordingly, functional regulation of lacZ expression in adult cardiomyocytes may also come into play, with decreased coupling via Cx43 gap junctions leading to the upregulation of a signaling pathway that utilizes a unique GATA6 enhancer. Why would the ventricular myocardium contain small clusters of myocytes that are not coupled to the bulk of the myocardium via Cx43 gap junctions? One possible explanation is that the reporter gene provides a snapshot of cells undergoing gap junctional turnover. Gap junctions are dynamic structures, whose presence in the cell membrane is characterized by cycles of degradation and reassembly (5, 57). Studies (12, 26, 62) have shown that a delicate balance between connexin synthesis and degradation must exist for proper heart function. This process may leave small numbers of myocytes unconnected to the larger myocardial tissue, and as a consequence, these Cx43-deficient myocytes would not contribute to the propagation of electrical signal in the heart. The low number of such cells would preclude any disruptions to the electrical conductivity of the heart, which is a premise that could also underlie an alternative basis for the existence of Cx43-deficient myocytes. It is possible, due to the ability of the myocardium to tolerate low numbers of nonelectrically coupled cells (13, 45), that there is no selective pressure to promote uniformity in myocardial phenotype. In either case, the presence of these cells, which are marked by lacZ expression in the transgenic animals, would be nondisruptive to the activity of the heart. Yet, large scale reductions in the expression and/or distribution of Cx43 within the myocardium are associated with various disease states (28, 35, 59). This includes the localized Cx43 decrease at injury border zone tissues (46, 59), marked by the GATA6 enhancer in the tgG6/lacZ mice, that may be part of an adaptive mechanism for isolating these electrically unstable cells from surrounding uninjured myocardial tissues. Less is known about the relationship between GATA6 and heart disease, although a recent study (64) indicated that this transcripAJP-Heart Circ Physiol • VOL

tion factor plays a key role in assisting the maladaptive hypertrophic response. In this regard, the combination of our injury and heptanol results may suggest a pathway whereby Cx43 disruption (due to injury or disease) or inhibition (heptanol) leads to downstream activation of a particular GATA6 enhancer. Elucidating the biology of myocytes that are Cx43 deficient and under control of a GATA6 enhancer may then be informative for understanding the transition from the healthy to pathological state, with the identification and characterization of myocytes by lacZ labeling providing the means for following disease progression. Thus this lacZ reporter provides the advantage that small numbers of cells can be analyzed for phenotypic differences that could not be discerned by conventional means, such as Western blotting. Future studies will test this hypothesis and examine whether the novel myocardial cell phenotype identified in this study is a contributing factor to decreases in cardiac health. ACKNOWLEDGMENTS We thank John B. E. Burch for generosity in providing the transgenic mice. GRANTS This work was supported by the National Heart, Lung, and Blood Institute Grants HL-086815 and HL-073190. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS M.C.R., G.I., R.G.G., C.A.E., and L.M.E. conception and design of research; M.C.R., G.I., M.P.O., N.V.M., V.G., C.A.E., and L.M.E. performed experiments; M.C.R., G.I., B.S.H., R.G.G., C.A.E., and L.M.E. analyzed data; M.C.R., G.I., R.G.G., C.A.E., and L.M.E. interpreted results of experiments; M.C.R., G.I., and L.M.E. prepared figures; M.C.R., G.I., and L.M.E. drafted manuscript; M.C.R., G.I., N.V.M., B.S.H., R.G.G., C.A.E., and L.M.E. edited and revised manuscript; L.M.E. approved final version of manuscript. REFERENCES 1. Adamo RF, Guay CL, Edwards AV, Wessels A, Burch JB. GATA-6 gene enhancer contains nested regulatory modules for primary myocardium and the embedded nascent atrioventricular conduction system. Anat Rec 280: 1062–1071, 2004. 2. Anversa P, Olivetti G. Cellular basis of physiological and pathological myocardial growth. In: Handbook of Physiology. Section 2: The Cardiovascular System, edited by Page E, Fozzard HA, Solar RJ. New York: Oxford Univ. Press, 2002, p. 75–144. 3. Banerjee I, Fuseler JW, Price RL, Borg TK, Baudino TA. Determination of cell types and numbers during cardiac development in the neonatal and adult rat and mouse. Am J Physiol Heart Circ Physiol 293: H1883– H1891, 2007. 4. Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabe-Heider F, Walsh S, Zupicich J, Alkass K, Buchholz BA, Druid H, Jovinge S, Frisen J. Evidence for cardiomyocyte renewal in humans. Science 324: 98 –102, 2009. 5. Berthoud VM, Minogue PJ, Laing JG, Beyer EC. Pathways for degradation of connexins and gap junctions. Cardiovasc Res 62: 256 –267, 2004. 6. Bicknell KA, Coxon CH, Brooks G. Can the cardiomyocyte cell cycle be reprogrammed? J Mol Cell Cardiol 42: 706 –721, 2007. 7. Bollini S, Smart N, Riley PR. Resident cardiac progenitor cells: at the heart of regeneration. J Mol Cell Cardiol 50: 296 –303. 2010. 8. Brandon C, Eisenberg LM, Eisenberg CA. WNT signaling modulates the diversification of hematopoietic cells. Blood 96: 4132–4141, 2000. 9. Bupha-Intr T, Haizlip KM, Janssen PM. Temporal changes in expression of connexin 43 after load-induced hypertrophy in vitro. Am J Physiol Heart Circ Physiol 296: H806 –H814, 2009. 10. Cabo C, Yao J, Boyden PA, Chen S, Hussain W, Duffy HS, Ciaccio EJ, Peters NS, Wit AL. Heterogeneous gap junction remodeling in

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