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(Circulation Research. 1996;78:547-552.)
© 1996 American Heart Association, Inc.

Articles

Vital Staining of Cardiac Myocytes During Embryonic Stem Cell Cardiogenesis In Vitro

Joseph M. Metzger, Wan-In Lin, Linda C. Samuelson

From the Department of Physiology, School of Medicine, University of Michigan, Ann Arbor.

Correspondence to Dr Joseph M. Metzger, Department of Physiology, University of Michigan, School of Medicine, 7730 Medical Science II, Ann Arbor, MI 48109-0622.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac -actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell–derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when 25% of the differentiation cultures expressed the reporter construct, with expression increasing to 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell–derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic ß-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.

Key Words: contractility • myofilaments • gene expression • development

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Spontaneously contracting cardiac myocytes derived from differentiating ES cells recapitulate in vitro the programmed expression of cardiac genes characteristic of murine cardiac development in vivo.^{1 2} Important advantages of the ES cell differentiation culture system include the relative ease of genetic modification of ES cells and their noted capacity to rapidly differentiate in vitro into cardiac myocytes.² These features make feasible the generation of ES cell stable transformants to address questions of cardiac gene regulation and function in vitro. In this approach, cardiac-restricted promoters could be used to direct the expression of genes of interest into the developing ES cell–derived myocytes.

The analysis of gene expression, channel activity, or contractile function in genetically modified ES cell cardiac myocytes is complicated because of the cellular heterogeneity of the ES cell differentiation culture system. Such studies would be greatly facilitated if it were possible to label the genetically modified ES cardiac myocytes using a nontoxic cellular marker. The primary aim of the present study was to determine the feasibility of using vital stain techniques for the real-time detection of cardiac gene expression throughout ES cell cardiac myocyte development in vitro.

We established ES cell stable transformants in which the reporter gene lacZ was specifically expressed in contracting ES cell–derived cardiac myocytes throughout their entire developmental lifetime in vitro by using the -440 to +6 human cardiac -actin promoter. Cardiac -actin is one of the earliest markers of cardiac cell development. It is expressed throughout cardiac development and transiently in embryonic skeletal muscle.³ In addition, the cardiac actin promoter has been shown to be sufficient to direct cardiac-specific gene expression in stable transformants derived from embryonal carcinoma P19 cells.⁴ In the present study, attached cultures of differentiating ES cells were established to allow direct observation of transgene expression in the contracting cardiac myocytes. Detection of lacZ transgene expression in living ES cell cardiac myocytes was accomplished by using lipophilic fluorogenic ß-galactopyranoside substrates, which are cell membrane permeable.⁵ After passage across the membrane, the substrate is hydrolyzed by ß-gal, producing a fluorescent by-product that is then retained within the cell.⁵ The present study is the first to use this approach to follow, in real time, cardiac-restricted gene activation during continuous contraction of the ES cell cardiac myocytes in vitro. Vital staining will promote the study of gene activation and function during ES cell cardiogenesis in vitro. In addition, this approach should facilitate the biochemical purification of cardiac myocytes from ES cell cultures for possible use in transplantation experiments in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pluripotential ES Cells
The mouse ES cell line D3, obtained from the inner cell mass of a mouse 129/Sv+/+ day-4 blastocyst,¹ was used in the present study. ES-D3 cells were cultured on top of a monolayer of MEF feeders at a density of 1x10⁵ cells per square centimeter. MEF feeders were prepared by pretreatment with gamma irradiation (45 Gy) and plating at a density of 5x10⁴ cells per square centimeter onto gelatin-coated tissue culture plates. The ES cell culture medium was renewed daily, and the ES cells were subcultured onto new feeders every 2 to 3 days.

Culture medium for the undifferentiated ES cells consisted of DMEM with high glucose, supplemented with 15% FCS, 0.1 mmol/L ß-mercaptoethanol, and 2% LIF-containing medium. LIF has been demonstrated to inhibit the differentiation of cultured ES cells.⁶ The LIF-containing medium was collected from cultures of Chinese hamster ovary cells, which were transformed with a LIF expression plasmid (Genetics Institute).

Plasmid Constructs
The two plasmids used in the present study are diagrammed in Fig 1. The plasmid pHCActlacZ (kind gift of M. McBurney, University of Ottawa, Canada) contains the segment of a human cardiac -actin promoter fragment (-440 to +6) that has been shown to be both necessary and sufficient to direct high levels of gene expression into contracting cardiac myocytes derived from P19 embryonal carcinoma cells.⁴ In this construct, the actin promoter is used to drive expression of the Escherichia coli lacZ reporter gene. The plasmid pPGKneo contains the neomycin phosphotransferase gene with expression driven by the PGK promoter. The PGK promoter is active in undifferentiated cell types, including pluripotential ES cells.⁷

View larger version (12K): [in this window] [in a new window]   Figure 1. Linear schematics of the pHCActlacZ and pPGKneo plasmids. pHCActlacZ contains the -440 to +6 Sal I–HindIII fragment of the human cardiac -actin promoter, the E coli lacZ gene, and the simian virus 40 (SV40) polyadenylation signal. Two SV40 poly(A) signals are positioned 5' of the promoter to prevent read-through. pPGKneo contains the PGK promoter, the neomycin gene, and the herpes simplex virus thymidine kinase (HSV-tk) polyadenylation signal. Both plasmids were linearized at unique Sca I sites within the plasmid backbone. Plasmid gene fragments are not drawn to scale.

Gene Transfer Into Undifferentiated ES Cells and Generation of Stable ES Cell Transformants
pHCActlacZ and pPGKneo were linearized at unique Sca I sites, and 1 µg of pPGKneo and 10 µg of pHCActlacZ were transferred into undifferentiated ES-D3 cells (8x10⁶ cells) by electroporation (250 µF and 0.3 kV) in 0.8 mL growth medium. The electroporated ES cells were plated onto neomycin-resistant MEFs⁸ at a density of 1.6x10⁴/cm². At 24 hours after plating, the cells were renewed with medium containing 300 µg/mL of G418. The medium was renewed every other day, and the growth of G418-resistant colonies was evaluated. At 9 days after electroporation, 48 G418-resistant colonies were isolated and plated individually onto 96-well plates containing neomycin-resistant MEF. When confluent, half of the ES cells were subcultured onto 96-well plates to evaluate transgene expression, and the remainder of the cells were frozen for storage.⁹ ß-Gal expression was assessed in differentiation cultures (described below) initiated from individual clones to identify transformants, which allowed transgene expression in the ES cell–derived cardiac myocytes.

Attached Cultures of Differentiating ES Cells
Attached cultures of differentiating ES cells were established by dissociation from MEF and the formation of ES cell aggregates in hanging drop cultures, designated as day 1 of differentiation.¹⁰ Hanging drops of 300 ES cells were cultured in differentiation medium consisting of 20% FCS, 50 U/mL penicillin+50 µg/mL streptomycin, and 0.1 mmol/L ß-mercaptoethanol in DMEM. After 2 days in differentiation culture, the embryoid bodies, having formed from aggregates of the ES cells in each hanging drop, were transferred to suspension culture in 100-mm bacterial dishes and cultured an additional 3 days. Attached cultures of differentiating ES cells were initiated by plating the embryoid bodies onto gelatin-coated glass coverslips in six-well tissue culture dishes.¹⁰ Cultures were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. The growth medium for the attached differentiation cultures was changed three times per week.

Histochemical Detection of ß-Gal Activity
Attached cultures were washed with PBS and treated with a 2.2% formaldehyde/0.2% glutaraldehyde fixation solution. Cultures were then incubated with an X-Gal–containing solution at 37°C for 6 hours. Upon cleavage of X-Gal by ß-gal, an intensely blue halogenated indoxyl derivative is produced. The attached coverslips were analyzed microscopically, scoring for the number of contracting foci that stained blue.

Vital Staining
Transgene expression was determined in living cells using Imagene green and Imagene red, which are lipophilic fluorogenic C_nFDGs (Molecular Probes, Inc) that become fluorescent upon enzymatic cleavage by ß-gal. In each experiment, sketches of the contracting foci within an attached differentiation culture were made to aid in quickly locating these areas on the imaging system. Prevital stain images of the contracting foci were obtained to determine background. The differentiation medium was then changed to include 33 µmol/L Imagene red substrate, and cultures were returned to the incubator for 1 to 48 hours. Imagene red is the 12-carbon fatty alkyl analogue of the nonfluorescent lacZ substrate resorufin ß-D-galactopyranoside. The same contracting foci were then imaged to determine the extent of fluorescence. Fluorescence was detected using an Attofluor fluorescent digital imaging system (Atto Instruments, Inc). Excitation/emission filters were 440 nm/530 nm and 560 nm/630 nm for Imagene green and Imagene red, respectively. In some experiments, cultures were subsequently histochemically stained for ß-gal and showed correspondence between blue staining and fluorescence.

Indirect Immunofluorescence
Indirect immunofluorescence microscopy was used to determine whether transgene expression is restricted to the ES cell–derived cardiac myocytes. Attached cultures were fixed using 3% paraformaldehyde for 30 minutes, washed with PBS, and treated with normal goat serum (20%) to block nonspecific binding. Cultures were incubated (in a humidity chamber) with a monoclonal anti–troponin T antibody (clone JLT-12, Sigma) for 1.5 hours, washed, blocked, and exposed to a polyclonal anti–ß-gal antibody (Chemicon International) for 1.5 hours. After blocking for nonspecific binding, cells were incubated with two secondary antibodies for 1 hour: (1) goat anti-mouse antibody conjugated to Texas red and (2) goat anti-rabbit antibody conjugated to FITC. The coverslips were mounted and examined using a Leitz Aristoplan fluorescence microscope.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transformation of Pluripotential ES Cells With pHCActlacZ
pHCActlacZ, which contains the -440 to +6 human -cardiac actin promoter to drive lacZ expression, and pPGKneo, which contains the PGK promoter and the neo gene to confer resistance to the antibiotic G418, were cotransferred into undifferentiated ES cells by electroporation. The objective was to obtain a stably transformed ES cell line in which the lacZ gene was expressed specifically in ES cell–derived cardiac myocytes over a wide range of days of development in vitro. A total of 48 G418-resistant colonies were studied. Attached differentiation cultures were initiated and tested for expression of HCActlacZ. Spontaneous contractile activity was apparent in these cultures beginning at 8 to 9 days after initiation of differentiation (see "Materials and Methods"). Individual clones were assessed for ß-gal activity by X-Gal staining in differentiation cultures exhibiting spontaneously contracting myocytes. Three different clones displayed significant ß-gal–positive staining in contracting myocytes. The clone 6-4 had the highest percentage of contracting foci that were ß-gal positive (68%) and was selected for further study.

The ß-gal staining pattern in differentiation cultures established from pHCActlacZ-transformed ES cells (clone 6-4) is shown in Fig 2. The arrowheads (Fig 2A) denote specific areas in the culture that contained spontaneously contracting myocytes before ß-gal staining. The majority of these contracting foci were shown to be ß-gal positive. In control experiments, differentiation cultures established from nontransformed ES cells did not contain any ß-gal–positive cells (data not shown). Thus, the ß-gal–positive contracting myocytes resulted from expression of the transferred gene and not activation of an endogenous ß-gal–like gene.

View larger version (75K): [in this window] [in a new window]   Figure 2. Light photomicrographs of differentiation cultures derived from HCActlacZ-transformed ES cells. Panels A and B are from the same differentiation culture taken at different magnifications. Arrowheads in panel A indicate the location of spontaneously contracting myocytes. Blue staining denotes ß-gal–positive cells. Bar=150 µm.

Onset, Stability, and Cellular Specificity of Cardiac -Actin Gene Expression in ES Cell Differentiation Cultures
To determine the onset of HCActlacZ gene expression, ES cell cultures were examined in the undifferentiated state and at various time points of differentiation. Undifferentiated ES cells growing on MEF and in the presence of LIF were ß-gal negative (Fig 3A). The earliest detection of ß-gal–positive staining was at day 6 after initiation of differentiation cultures. This is 2 to 3 days before the earliest detection of spontaneous contractile activity in these cultures.¹⁰ The number of differentiation cultures containing ß-gal–positive cells increased from 25% at day 6 to 70% at day 9 of differentiation.

View larger version (16K): [in this window] [in a new window]   Figure 3. A, Activation of the HCActlacZ transgene during the early stages of ES cell differentiation. Spontaneously contracting cardiac myocytes were first observed 8 days after initiation of differentiation. Data are expressed as the fraction of cultures that were ß-gal positive divided by the total number of cultures tested for that time point. One simple embryoid body was placed in each well. Thus, each well represents one culture. Values are mean±SEM (average of seven cultures tested per group). For day 0, undifferentiated ES cells growing on MEF were X-Gal–stained. B, Duration of HCActlacZ transgene expression in ES cell–derived spontaneously contracting cardiac myocytes. Values at each day of contraction are derived by dividing the number of ß-gal–positive contracting foci by the total number of individual contracting foci tested. Thus, in panel B, we have specifically scored individual contracting foci that were ß-gal positive as opposed to scoring whole cultures that were ß-gal positive, as done in panel A. This distinction is important, because there can be multiple contracting foci in one attached culture. Values are mean±SEM. Data are from a total sample size of 777 contracting foci tested.

In the attached ES cell differentiation cultures, it was possible to determine the onset of spontaneous contraction and track the duration of continuous contractile activity among individual foci of contracting cardiac myocytes. Results indicated that expression of the transgene was relatively stable in the ES cell–derived cardiac myocytes extending over the range from 1 day to >30 days of continuous contractile activity in vitro (Fig 3B). Overall, throughout this time period, 30% of the contracting myocytes expressed the transgene.

The localization of ß-gal–positive staining to specific regions in culture where contracting myocytes were observed is evidence that HCActlacZ gene expression was restricted to the ES cell–derived contracting cardiac myocytes (Fig 2). However, because of the multiple cellular lineages that are derived from differentiating ES cells,¹ it is possible that the ß-gal–positive cells were nonmuscle cells closely associated with the spontaneously contracting cardiac myocytes. Also, early during ES cell differentiation in vitro, at time points before the onset of spontaneous contraction in cardiac myocytes, ß-gal–positive cells were detected. For these reasons, indirect immunofluorescence was performed on ES cell differentiation cultures using an anti–ß-gal antibody and a muscle-specific anti–troponin T antibody to verify that transgene expression occurred exclusively in the myocytes. Immunostaining results demonstrated that ß-gal staining was restricted to cells that were troponin T positive (Fig 4). This is direct evidence that expression of the HCActlacZ gene is restricted to the ES cell–derived myocytes. The rationale for use of the striated muscle–specific troponin T antibody was that we wished to detect the contracting myocytes over a wide range of days of development in vitro. We have recently confirmed that the ES cell–derived contracting myocytes are of cardiac origin by using a cardiac-specific troponin I antibody.¹¹

View larger version (14K): [in this window] [in a new window]   Figure 4. Indirect immunofluorescence detection and colocalization of ß-gal (A) and troponin T (B) expression in contracting cardiac myocyte foci derived from HCActlacZ-transformed ES cells. ß-Gal was detected using a polyclonal anti–ß-gal antibody with detection by a FITC-conjugated secondary antibody. Troponin T immunostaining used a monoclonal anti–troponin T antibody (Sigma) with detection by a Texas red–conjugated secondary antibody.

Vital Staining for Detection of ES Cell–Derived Contracting Cardiac Myocytes
A primary goal of the present study was to establish whether transgene expression could be detected in living ES cell myocytes by using vital stain techniques. Because X-Gal is toxic to living cells, it is not possible to use this substrate to detect transgene expression in real time. To approach this problem, we used Imagene green and Imagene red, which are commercially available, lipophilic, fluorogenic C_nFDGs (Molecular Probes, Inc). These fluorogenic substrates become fluorescent upon enzymatic cleavage by ß-gal.⁵ The substrate Imagene green contains fluorescein as the fluorophore, whereas Imagene red contains a synthetic rhodamine derivative, resorufin. Our initial studies focused on Imagene green substrates to determine which fatty acyl chain length best facilitated membrane translocation and cellular retention within ES cell myocytes. We tested substrates with 2, 4, 8, 12, and 16 carbon atoms in the fatty acyl chain. In pilot studies, it was found that C₁₂FDG worked best, whereas C₂FDG and C₄FDG were ineffective. One significant difficulty with Imagene green substrates related to the cellular autofluorescence, which was evident at the green emission wavelength in many of the ES cell differentiation cultures tested. To overcome this limitation, we used the Imagene red C₁₂RG substrate, which, because of its longer emission wavelength, significantly reduced the background cellular autofluorescence. Thus, for the rest of our studies, the Imagene red C₁₂RG substrate was used.

We performed vital staining on 31 different ES cell differentiation cultures. In these cultures, it was possible to detect a fluorescence signal as early as 1 hour after addition of the Imagene red C₁₂RG substrate to the medium (Fig 5). At 24 hours after exposure to Imagene red, the fluorescence signal increased in intensity, and during this time, there was no discernible alteration in the spontaneous contractions of the myocytes. In other experiments, we found no effect on contractility 48 hours after application of Imagene substrates. These results provide evidence that the Imagene substrate and hydrolysis products were not toxic to the ES cell–derived cardiac myocytes. Although this study focused on vital stain detection of contracting ES cell myocytes, we also found it possible to use vital staining to detect precontractile ES cell myocytes. Vital stain detection of the precontracting cells was possible 1 to 2 hours after application of the Imagene substrate, very similar to the time of detection of the contracting myocytes.

View larger version (110K): [in this window] [in a new window]   Figure 5. Vital staining of lacZ gene expression in ES cell–derived spontaneously contracting cardiac myocytes. Panels A through D are from the same field in an ES cell differentiation culture. A, Bright-field illumination in which entire field contained spontaneously contracting cardiac myocytes. B through D, Pseudo–color fluorescent images of lacZ gene expression, where pink denotes the background and increased fluorescence is indicated according to the following scale: red (highest)>yellow>green>light blue>deep blue. B, Background autofluorescence before the addition of Imagene red C12RG substrate. C, 1.5 hours after addition of 33 µmol/L Imagene red C12RG. D, 22 hours after addition of Imagene red C12RG. Bar=100 µm. Spontaneous contractile activity continued during and after washout of the Imagene vital stain, indicating that Imagene red was nontoxic to the spontaneously contracting myocytes.

To verify that the fluorescence signal was due to expression of the transgene in these cells, some of the cultures were first vital-stained and then X-Gal–stained. Results showed a correspondence between the localization of fluorescence and X-Gal staining, indicating that the vital stain was detecting expression of HCActlacZ during ES cell differentiation (data not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates vital staining of ES cell myocytes as they are developing in vitro. ES cell stable transformants were generated using the -440 to +6 human cardiac -actin promoter to drive expression of the lacZ reporter gene into contracting myocytes in vitro. Results show lacZ expression at day 6 during cardiogenesis in vitro, at a time point before detection of spontaneous contractile activity in this culture system. The kinetics of activation of this gene during ES cell cardiogenesis appears to lag by 1 day with respect to the recently defined Csx gene, a mammalian homeobox gene whose expression appears to precede other known cardiac-specific genes during development in vivo and in vitro.¹² Because of the central role of cardiac -actin during sarcomerogenesis, it is perhaps not surprising that this gene would be activated very early during the cardiogenic program in differentiating ES cells in vitro.

ES cell differentiation cultures are heterogeneous in terms of cell lineages formed in vitro.¹ Immunofluorescence experiments were therefore performed to show that expression was restricted to the myocytes in this differentiation culture system (Fig 4). The lack of formation of skeletal lineages during ES-D3 cell differentiation has been previously shown using this same system,^{11 13 14} and it has also been shown that the developing contracting myocytes express cardiac-specific contractile and regulatory isoforms.^{2 11 14 15} Thus, expression of the HCActlacZ gene appears restricted to ES cell–derived cardiac myocytes. However, it is acknowledged that because we have used a clonal derivative of the parental ES-D3 cell line, there exists the possibility of phenotypic variability in myocytes formed from the clonal versus the parental cell line. To address this possibility, we examined several independent clones and found that they all demonstrated the same main finding of transgene expression in the spontaneously contracting myocytes. In addition, it was also evident that the time of onset of spontaneous contraction, the number of contracting foci per culture, and the rate of contraction of the ES cell–derived myocytes were not different in cultures established from the ES clones compared with the parental ES cell line. Taken together, these results give support to the idea that the spontaneously contracting myocytes in the clonal derivatives are of cardiac origin, as has been shown previously for the parental ES-D3 cell line.^{1 2 10 11 14 15}

The cardiac-restricted expression pattern of this gene should be useful in targeting expression of other genes into the spontaneously contracting myocytes over a wide range of developmental time points in vitro. We recently demonstrated the feasibility of determining the contractile function of cardiac myocytes isolated from ES cell differentiation cultures.¹⁰ Electrophysiological studies have also been performed on contracting myocytes in this system.¹⁶ Stable transformants of ES cells using cardiac-restricted promoters to drive expression of wild-type or mutant contractile or channel protein genes into these cells should provide a useful tool to pursue structure-function studies during cardiogenesis in vitro.

The ability to identify ES cell–derived cardiac myocytes using vital staining will facilitate these functional studies. Vital staining will aid in the identification and subsequent isolation of those myocytes that significantly express the delivered gene. In addition, this approach makes possible longitudinal studies of cardiac gene expression in living myocytes derived from differentiating ES cells in vitro. There is good evidence that ES cell–derived cardiac myocytes recapitulate the developmentally regulated expression of cardiac contractile isoforms characteristic of myocytes developing in vivo.^{2 10 11 14 15} Vital staining in conjunction with developmentally regulated promoters, such as the myosin heavy chain promoter,¹⁷ will permit selection of cardiac myocytes at various stages of development in vitro.

Finally, the availability of this newly described ES cell line together with the vital stain approach could be used to identify cardiac myocytes at very early time points of development in vitro. By using mechanical isolation techniques¹⁰ or cell sorting techniques, it will be possible to specifically isolate these precontractile cardiac myocytes from culture. Gene expression in the precontractile cardiac cells could then be compared with that in noncardiac cells in ES cultures with the goal of identifying the factors that underlie cellular commitment to the cardiac lineage. The isolation of precontractile and contracting myocytes from ES cultures could be examined in functional studies^{10 16} or may be used as donor myocytes for grafting into myocardium in vivo.¹⁸

	Selected Abbreviations and Acronyms

 

ß-gal = ß-galactosidase CnFDG = di-ß-D-galactopyranoside, where n denotes the number of carbon atoms in the fatty acyl chain ranging from 2 to 16 C12RG = resorufin ß-D-galactopyranoside ES = embryonic stem FITC = fluorescein isothiocyanate LIF = leukemia inhibitory factor MEF = mouse embryonic fibroblast PGK = phosphoglycerate kinase X-Gal = 5-bromo-4-chloro-3-indolyl galactopyranoside

	Acknowledgments

 
This study was supported by the American Heart Association, National Center and Michigan Affiliate, Inc, and by a U-M Rackham faculty grant. Dr Metzger is an Established Investigator of the American Heart Association. We thank Dr David Yule for assistance with imaging and Dr Michael McBurney for kindly providing pHCActlacZ.

Received November 6, 1995; accepted December 18, 1995.

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