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Endocrinology Vol. 144, No. 3 1053-1061
Copyright © 2003 by The Endocrine Society

ARTICLE

Midgestational Lethality in Mice Lacking the Parathyroid Hormone (PTH)/PTH-Related Peptide Receptor Is Associated with Abrupt Cardiomyocyte Death

Jin Qian1, Melissa C. Colbert1, David Witte, Chia-Yi Kuan, Eric Gruenstein, Hanna Osinska, Beate Lanske, Henry M. Kronenberg and Thomas L. Clemens

Departments of Medicine (J.Q., T.L.C.) and Molecular Genetics (E.G.), University of Cincinnati, Cincinnati, Ohio 45267; Division of Molecular Cardiovascular Biology (M.C.C., H.O.), Department of Pathology (D.W.), and Department of Molecular Developmental Biology (C.-Y.K.), Children’s Hospital Medical Center, Cincinnati, Ohio 45229; Department of Oral Pathology (B.L.), The Forsythe Institute and Harvard Dental School, Boston, Massachusetts 02115; and Endocrine Unit (H.M.K.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Thomas L. Clemens, Ph.D., Division of Endocrinology and Metabolism, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267-0547. E-mail: clementl{at}uc.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PTHrP is a key developmental regulatory protein and a potent vasoactive agent. Previous studies have shown that mice lacking either the Pthrp or the PTH type 1 receptor (Pth1r) gene exhibit severe chondrodysplasia. In addition, in most genetic backgrounds, the receptor null mice die prenatally at midgestation, but the cause of death remains elusive. Here we show the loss of the Pth1r gene in C57BL6 mice leads to massive, abrupt cardiomyocyte death and embryonic lethality between embryonic days (E) E11.5 and E12.5. PTH1R mRNA was abundantly expressed in the developing wild-type mouse heart and cardiomyocytes from E11.5 embryos demonstrated acute increases in cAMP and increased Ca2+oscillations in response to PTHrP-(1–34)NH2. Analyses of more than 300 embryos (E8–E14.5) from C57BL6/PTH1R +/- matings showed that PTH1R-/- mice survived until E11 with no obvious defects in any tissue. By E12, only 10% of the PTH1R-/- embryos survived and all PTH1R null mice were dead by E13. Ultrastructural and histological analysis revealed striking mitochondrial abnormalities at E11.5 and precipitous cardiomyocyte death between E12.0 and E12.5, followed by degenerative changes in the liver and massive necrosis of other tissues. No abnormalities were observed in the yolk sac or placenta implicating the heart degeneration as the primary cause of death. Taken together, these findings indicate that the PTH1R is required for the development of normal cardiomyocyte function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PTHrP WAS FIRST identified as the factor responsible for the paraneoplastic syndrome termed humoral hypercalcemia of malignancy (1, 2). In contrast to PTH, which is produced mainly in the parathyroid gland, PTHrP is expressed in a variety of normal fetal and adult tissues. PTH and PTHrP signal through a seven-transmembrane-spanning G protein-coupled receptor known as the type I PTH/PTHrP receptor (PTH1R) (3). PTH1R subserves the calcium-regulating functions of PTH and the actions of PTHrP but, like PTHrP, is also widely expressed in tissues not involved in calcium homeostasis (4). The PTH1R is frequently expressed in the same cells that produce PTHrP or in cells immediately adjacent to them (2). This spatial proximity of PTHrP and its receptor, together with the fact that little, if any, PTHrP circulates under normal physiological conditions (5), suggests that PTHrP functions in an autocrine/paracrine fashion.

In the mature animal, PTHrP and the PTH1R are abundantly expressed throughout the cardiovascular system, including vascular smooth muscle, endothelial cells, and heart (2). However, the precise function of PTHrP in the heart and vasculature is not entirely clear. Its marked induction by vasoconstrictors (6), together with its ability to relax smooth muscle (7), suggests that PTHrP acts as a local smooth muscle compliance factor to accommodate flow or response to contractile stimuli. Transgenic mice with targeted overexpression of PTHrP (7) or the PTH1R (8) in vascular smooth muscle develop hypotension consistent with the predicted role of this protein as a local vasodilator. In the adult heart, PTHrP exerts both chronotropic and inotropic actions. Infusion of PTHrP accelerates heart rate in the isolated perfused heart and directly influences pacemaker activity of cells in the sinus node (9, 10). PTHrP colocalizes with ANF in secretory granules in the atria (11), suggesting that it might act on cardiomyocytes in an autocrine/paracrine mode. The inotropic effect of PTHrP on the intact heart is due in part to the vasodilatory effects of this ligand on coronary vasculature (12). However, PTHrP also directly increases basal contractility of ventricular cardiomyocytes (13). Recent studies suggest that the inotropic stimulation of cardiomyocytes may be mediated by PTHrP produced in the coronary endothelium (13). All of these actions are mediated by PTH1R, most likely via changes in intracellular Ca2+ (14).

Studies in genetically altered mouse models have defined a number of developmental processes that require effective PTHrP signaling. For example, targeted disruption of the Pthrp gene results in death in the early postnatal period associated with premature terminal differentiation and calcification of chondrocytes (15). Rescue of the PTHrP null mice by directing PTHrP expression to the growth plate revealed that PTHrP was also required for mammary gland development and for tooth eruption (16). Given the actions of PTHrP on cardiac tissue, together with its established role in the development of several other tissues, we speculated that the protein might also function during the development of the cardiovascular system. In support of this theory, transgenic mice expressing PTHrP and its receptor in the embryonic heart, created by crossing the ligand and receptor-overexpressing mice, die between embryonic days (E)9.5 and E10.5 with severe thinning of the ventricle and disruption of ventricular trabeculae (8). Although the cardiovascular system appears to develop normally in the PTHrP knockout mouse, deletion of the Pth1r gene in either MF-1 outbred or C57BL6 inbred backgrounds results in midgestational death (17). PTH1R knockout embryos were reported to be smaller than normal as early as E9.5. Histologic evaluation at this time revealed a diminution of organ size but no other gross developmental defects. The number of (-/-) fetuses appeared to meet Mendelian expectations at E9.5, but all were reported dead by E14.5. PTH1R knockout embryos on a Black Swiss background survive to term yet die perinatally (17). A recent attempt to rescue the lethal phenotype using a constitutively active receptor targeted to bone chondrocytes prevented accelerated chondrogenesis; however, it did not rescue the perinatal lethality (18). Thus, the loss of PTH1R is incompatible with life irrespective of genetic background.

In the current study, we demonstrate the functional expression of the PTH1R in the developing mouse heart. We show that C57BL6 mice lacking the PTH1R die abruptly at the transition from an embryonic to a fetal heart phenotype with precipitous cardiomyocyte death. Our results suggest that effective signaling mediated by the PTH1R is required for normal heart development and/or maintenance of adequate cardiac performance. The marked swelling in the mitochondria and endoplasmic reticulum, frequently associated with ionic imbalance, indicate a potential role for PTH1R in regulating Ca2+ levels during midgestation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PTH1R null embryos
The generation of the PTH1R knockout mice has been described previously (17). Heterozygous mice in the C57BL/6–129/SvJ mixed background were backcrossed with wild-type C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME) for seven generations. Embryos homozygous for the PTH1R gene were obtained by crossing heterozygous animals. Male and female mice were placed together overnight and then separated. Pregnant mice were killed at various times during gestation and embryos removed and genotyped by PCR. All studies were approved by the local Institutional Animal Care and Use Committee.

In situ hybridization
In situ hybridization was performed as previously described (19). For generation of the antisense PTH1R riboprobe, a PvuII to SacI fragment of the mouse PTH1R cDNA (20) was cloned into pBluescript SK(+) plasmid, linearized with EcoRI and transcribed with T3 RNA polymerase. The sections were hybridized overnight at 42 C, treated with 50 µg/ml ribonuclease A (Sigma, St. Louis, MO) and 100 U/ml of ribonuclease T1 (Roche Molecular Biochemicals, Indianapolis, IN) for 30 min at 37 C, and washed to a final stringency of 0.1x standard citrate saline at 50 C. Sections were photographed under dark-field illumination.

Preparation of embryonic cardiomyocytes
Embryonic cardiomyocytes were prepared from hearts of E12.5 wild-type embryos as described (21) with the following modifications. Embryonic ventricles were minced and digested at 37 C in 0.1% collagenase (type I, Worthington) for 45 min. An equal volume of DMEM with 10% serum and antibiotics was added to the heart fragments. They were triturated, the large debris allowed to settle, and the overlying cell suspension centrifuged for 1 min at 1000 rpm. The resulting pellet was washed three times with media and the cells resuspended in 100 µl media. The cells were plated on glass coverslips in specially prepared chambers for analysis and incubated overnight at 37 C in 5% CO2.

Measurement of cAMP
Accumulation of cAMP was assayed in confluent monolayers of cells grown in 24-well plates. Cells were incubated with PTHrP-(1–34)NH2 or vehicle for 5 min in 0.5 ml of medium at 37 C in the presence of 0.2 mM 3-isobutyl-1-methylxanthine. The reaction was stopped by aspirating the medium and adding 0.2 ml of 1 N HCl. Dried extract was kept at -70 C until assay, at which time it was reconstituted with sodium acetate buffer, pH 6.2. Assay of cAMP was carried out as described previously (22).

Cytoplasmic calcium measurements
The kinetics of cytoplasmic calcium were measured as previously described (23) except that microphotometry was used in place of video image analysis to increase the time resolution. Briefly, cells were plated onto coverslips that had been treated with fibronectin to increase cell adherence. Cells were plated at a low enough density to provide areas containing single cells or very small clusters of cells. Immediately before the start of an experiment, cells were loaded with 3 µM fluo-3-acetoxymethylester (fluo-3AM) (Molecular Probes, Inc., Eugene, OR) for 20 min at 37 C in DMEM. At the end of the load period, cells were washed with DMEM without fluo-3AM and postincubated for 20 min at 37 C. The medium was replaced by prewarmed loading buffer in a microincubator on the microscope stage. A rectangular aperture in the microphotometer unit was used to isolate the field of view to a single contracting cell or a small cluster of cells that contained one or more synchronously oscillating cells for measurement. Changes in cytoplasmic calcium expressed as changes in fluorescence intensity were measured using the InCyt-Pm1 microphotometry system from Intracellular Imaging, Inc. (Cincinnati, OH). Cells were illuminated at 485 nm and fluo-3 fluorescence was detected at 530 nm. Measurements were taken at a rate of 20/sec.

Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay
Embryos were harvested at E11.5, E12, E12.25, and E12.5, fixed overnight in Bouin’s solution, and infiltrated with sucrose before cryosectioning for TUNEL analysis. Sections were permeabalized with 0.2% Triton X-100 and labeled with digoxygenin-11-deoxyuridine triphosphate and terminal transferase (Roche) at 37 C for 1 h. After blocking in horse serum (5%), the slides were incubated with mouse antidigoxygenin (1:250) overnight at 4 C. Following extensive washing, the sections were incubated in biotinylated secondary antibody followed by Texas red-labeled streptavidin for 1 h at room temperature. All sections were counterstained with the nucleic acid dye Hoechst 33342.

Electron microscopic analysis
For ultrastructure examination, embryos were initially fixed in utero with 3.5% glutaraldehyde in cardioplegic buffer (5% dextrose, 25 mM KCl in PBS) using gravity perfusion through uterine vasculature. The embryos were then excised and further fixed in 3.5% glutaraldehyde in 0.1 M cacodylate buffer, for 1–2 h at 4 C. The embryonic hearts were trimmed and fixed overnight in the same fixative at 4 C, osmicated, and processed for embedding in epoxy resin (Embed 812). Cardiac ventricles were first identified on 1-µm sections by light microscopy, and thin sections prepared which were examined using a Carl Zeiss (Thornwood, NY) 912 Omega electron microscope at accelerating voltage 100 kV. Photographs were taken under magnifications 5000 and 8000. At least 25 micrographs were taken for each analysis: those shown are representative.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A functional PTH1R is expressed in the developing mouse heart
The disturbances in cardiac development seen in double transgenic mice overexpressing both PTHrP and PTH1R (7) suggested that early activation of PTH1R signaling interfered with either heart development or function. Because the timing and location of PTH1R in the embryonic mouse heart has not been previously reported, we examined the expression of native PTH1R mRNA by in situ hybridization. In general, our results are in agreement with the distribution in tissues other than the heart as described by Karperien et al. (24). As early as E8 and clearly by E9.5 (Fig. 1AGo), the extraembryonic fetal tissues such as yolk sac and the parietal endoderm lining Reichart’s membrane showed strong expression. No expression in the embryo proper could be detected until E10, where signal was observed in many mesodermal structures including the heart (not shown). By E11.5, PTH1R mRNA was found throughout the heart muscle, in the endocardial cushions, and the pericardial sac surrounding the heart (Fig. 1BGo). In the heart, mRNA levels diminished by E14 (not shown). Diffuse expression was observed in the mesenchymal tissue of the developing lung, the mesonephric tubules, and the epithelium lining the ureteral buds as described by others (24). Robust expression was noted in endochondral bone (Fig. 1CGo).



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  		Figure 1. PTH1R mRNA expression during development. A, A section through an E9.5 embryo. There is strong expression in the extraembryonic tissues (bright white grains) including the parietal and visceral endodermal layers of the yolk sac (arrows). There is no expression in the embryo (*) or myometrium (m). B, A sagittal section through the heart in the E11.5 embryo hybridized with the antisense probe. Expression is greatest in the endocardial cushions (arrowheads), with lower expression in the ventricular trabeculae (v), the atria (a), and pericardium (p). D, The heart hybridized with the control sense probe. C, A section through the chest wall of an E16 embryo. Strong expression is present in the developing rib particularly in the growth plate (arrows). There is also a layer of signal in the dermis (arrowheads). Magnification, x100, dark-field illumination.

 
To examine the signaling properties of PTH1R in the embryonic heart, cardiomyocytes were cultured from wild-type embryos harvested at E11.5. The PTH1R signals primarily by coupling to either Gs or Gq (3). In vascular tissues, PTH1R couples preferentially to Gs with activation of adenylate cyclase (25). Therefore, embryonic cardiomyocytes were assayed for their cAMP responses to PTHrP. Exposure of cardiomyocytes to PTHrP-(1–34)NH2 (Bachem, Torrance, CA) significantly increased cAMP accumulation in a dose-dependent manner (Fig. 2AGo). In general, elevation of intracellular cAMP in cardiomyocytes activates L-type Ca2+ channels and modulates Ca2+ levels (26). To determine if PTHrP-(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34)NH2 altered Ca2+ fluxes in the embryonic heart, cardiomyocytes were loaded with Fluo-3 and stimulated with either PTHrP-(1–34)NH2 or dibutyryl cAMP (Sigma). Both agents increased the amplitude without changing the frequency of spontaneous Ca2+ oscillations in embryonic cardiomyocytes as measured by photometry, but the response to PTHrP was greater than to cAMP (Fig. 2BGo).



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  		Figure 2. Functional coupling of the PTH1R in the normal developing mouse heart. Primary cardiomyocytes were cultured from E11.5 wild-type embryos as described in Materials and Methods. A, Cells were exposed to increasing concentrations of PTHrP-(1–34)NH2 or left untreated and cellular cAMP was determined as described in Materials and Methods. Asterisks denote a significant increase in cAMP compared with controls (P < 0.05). B, Cells were loaded with Fluo 3 and changes in cytoplasmic calcium expressed as changes in fluorescence intensity were measured using microphotometry (see Materials and Methods). Addition of 10 nM PTHrP (1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 )NH2 or 1 mM dibutyryl cAMP increased the amplitude of spontaneous Ca2+ oscillations. The bottom right panel illustrates the mean data collected from four independent experiments. Asterisks denote a significant difference from control (untreated) cells (P < 0.001). The results illustrated are representative of multiple (three or more) experiments.

 
PTH1R null mice die at mid-gestation
As previously reported (17), matings of heterozygous C57Bl6 PTH1-R (-/+) mice resulted in no live homozygous null mice, but the expected ratios of heterozygous and wild-type mice. Gross examination of embryos from litters at E11.5–12.5 showed that the growth and development of some, but not all, null embryos were clearly retarded (Fig. 3Go, top right), and pericardial edema and pooling of blood were often observed. These defects likely contribute to the developmental asynchrony observed. When embryos were genotyped, 80% of the expected Mendelian ratio of homozygous null mutants was detected up to E10.5 (Fig. 3Go, bottom). The early loss of homozygous nulls could be the consequence of effects on parietal endoderm formation, which is dependent upon PTHrP, and may contribute to their overall smaller size (27). Between E11 and E12, there was a precipitous drop in the number of surviving null embryos, and by E13 none were found in any of the litters examined. Therefore, the PTH1R null embryos die abruptly at midgestation.



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  		Figure 3. Midgestational death of PTH1R null mice. Litters from heterozygote intercrosses were collected and analyzed. Top panel compares the normal gross morphological appearance of an E12 heterozygous wild-type embryo (left) with that of an E12-/- littermate (right). The null embryo (right) exhibits a delay in overall development with prominent pericardial pooling of blood (arrow). Forelimb and hindlimb buds are indicated (L). Bottom panel, Kaplan-Meier survival plot of PTH1R (-/-), (-/+), and wild-type controls from a total of 334 embryos genotyped.

 
Midgestational lethality is associated with massive and rapid cardiomyocyte cell death
Mouse embryos were harvested at gestational ages ranging from E9.5–E12.5 and examined histologically. No structural malformations or cellular alterations in any organs of the null embryos were seen before E11.0. In particular, the structure and vascularization of the yolk sac and placenta were unremarkable (not shown). The earliest pathological findings were observed in the E11.5 embryos. Sagittal sections revealed the beginnings of separation of the endocardial lining from the trabeculae in the developing heart (Fig. 4Go, compare A with B); however, no abnormalities of the underlying myocardial tissue were observed. At E12.0, the first signs of heart failure were clearly present with dilation and occasional effusion in the pericardial sac (Fig. 4CGo). The endocardial separation progressed and became more widespread throughout the heart (Fig. 4DGo) but was never observed in the wild-type or heterozygous littermates at any age (see Fig. 4Go, A and E). Evidence of myocardial necrosis was variable, characterized by microscopic foci of myocardial cells that showed loss of cytoplasmic detail, and increased cytoplasmic eosinophilic staining with nuclear pyknosis and fragmentation. Importantly, extensive histologic analysis of multiple null embryos over this time showed no evidence of abnormalities or degeneration in any other tissue. In general, cardiac necrosis became much more extensive and severe by E12.5 (Fig. 4FGo). There was, however, a range of severity of the lesions with some mice exhibiting very early focal areas of necrosis and others with total cardiac necrosis and loss of structural integrity. By this stage, necrosis was not limited to the heart although the earliest lesions were generally identified in this tissue. Thus, within a 12-h window, the heart underwent abrupt and massive degeneration followed by progressive necrotic changes in other tissues.



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  		Figure 4. Myocardial lesions in PTH1R null embryos. A, A section through the heart of a wild-type E11.5 embryo. Arrows indicate the endothelial lining, which is tightly apposed to the ventricular trabeculae (t). B, A section through a (-/-) littermate of the embryo shown in panel A. In contrast to A, there is evidence of endocardial separation (arrows) from the underlying trabeculae (t), although the heart has developed normally. C, A sagittal section through an E12.0 embryo (-/-). Note the dilated pericardial sac (*). No abnormalities were observed elsewhere in the embryo and no degenerative changes could be detected in the surrounding tissues; a, atria; v, ventricle. D, Higher magnification of the ventricle from embryo section shown in panel C. There is further marked separation of the endocardial layer (arrowheads), but no light microscopic changes in the underlying myocardium. E, An E12.5 wild-type heart. Arrowheads indicate the endothelial layer tightly attached to the trabeculae (t) with healthy underlying cardiomyocytes. F, A null embryo at E12.5. There is evidence of marked endocardial layer separation (arrows) and the underlying myocardium now shows signs of extensive widespread necrosis and loss of structural integrity. The myocardial fibers show increased eosinophilic staining, loss of cytoplasmic detail, and nuclear pyknosis. (All panels: bright-field illumination, hematoxylin and eosin; C, x40; D, x100; and A, B, E and F, x200.) The histologic findings illustrated are representative of those observed in numerous sections from multiple embryos.

 
Apoptosis does not account for myocardial cell death
The rapidity and extent of myocardial cell degeneration and the known role of PTH1R in regulating programmed cell death (28) prompted us to examine apoptosis in the hearts of null embryos. Embryos were isolated at E11.5, E12.0, E12.25, and E12.5 to cover the full range over which lethality occurred, and cell death was examined by TUNEL assays. As a positive control, sections were also taken from the lamina terminalis, an area in which caspase-dependent apoptosis occurs normally during brain development (29). As can be seen in Fig. 5Go, many positively labeled cells were found in the lamina terminalis of comparably staged wild-type (A) and null (D) embryos. In contrast, no positive nuclei were identified in either wild-type or null hearts at E11.5 (not shown) or E12 (Fig. 5Go, B and E) and only rarely seen in hearts at E12.5 (Fig. 5Go, C and F). The scarcity of TUNEL positive cells is in stark contrast to the degree of cardiomyocyte degeneration observed in null embryos. These results indicate the loss of PTH1R is not associated with increased programmed cell death, which therefore cannot account for massive cardiomyocyte degeneration.



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  		Figure 5. Assessment of cardiomyocyte apoptosis. TUNEL staining was performed on sections from wild-type (A–C) and null (D–F) embryos at E12 (A, B, D, and E) and E12.5 (C and F). A and D, The lamina terminalis. Multiple TUNEL positive nuclei (arrows) can be seen in both wild-type and null embryos and are characteristic of this region of the brain in embryos at this age. In contrast, TUNEL staining of embryonic heart sections show no or a rare positive cell(s) (arrows) in either wild-type or null mouse hearts at E12 and E12.5. A, Atria; t, trabeculae; vw, ventricular wall. The findings illustrated are representative of those observed in numerous sections from multiple embryos.

 
Ultrastructural abnormalities in PTH1R null mouse hearts
In an attempt to detect abnormalities that might contribute to cellular degeneration in the null hearts, cardiomyocytes were examined by transmission electron microscopy. Because the first subtle signs of cardiac degeneration were detected in null hearts at E11.5, litters were examined at this stage. In wild-type hearts, ultrastructural analysis showed that the sarcomeres were well organized with normally spaced Z-bands. Mitochondria were small, abundantly interspersed among the developing sarcomeres, and the cristae were generally parallel and tightly packed. The perinuclear membrane was tightly apposed, and chromatin arrangement was normal (Fig. 6Go, A and C). In contrast, the ultrastructure in null hearts exhibited clearly abnormal changes (Fig. 6Go, B and D). Myofibrils were occasionally disarrayed with Z-band irregularities and reduced Z band distances suggestive of contracture. Mitochondria appeared larger; many were round or swollen and irregularly shaped. The cristae were disordered and distorted with occasional inclusions, whereas the endoplasmic reticulum appeared distended. In isolated cells that were clearly degenerating, the mitochondria were grossly swollen with complete loss of cristae. Enlarged perinuclear space was observed in the null cardiomyocytes, whereas the chromatin organization was identical to the wild-type hearts (compare nuclei in Fig. 6Go, A and B). No typical apoptotic nuclei were seen in more than 50 nuclei/cells examined by electron microscopy. These ultrastructural changes, swelling of the mitochondria and endoplasmic reticulum, are consistent with defects of maintaining ionic homeostasis and imminent cell death.



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  		Figure 6. Ultrastructural analysis of PTH1R null hearts. Cardiomyocytes from wild-type (A and C) and PTH1R null hearts (B and D) were examined from embryos at E11.5. Ventricular myocytes in wild-type hearts (A) have nuclei with normal chromatin arrangement and a regular double profile of nuclear lamina. The null cardiomyocytes (B) also have normal chromatin arrangement but exhibit nuclei with lamina separated from the sarcoplasm (thick arrow). Bar, 2 µm. C, Wild-type cardiomyocytes exhibit mitochondria with mostly small, regularly arranged cristae (arrow). Profiles of the sarcoplasmic reticulum cisternae are flattened with numerous ribosomes on the outer surface (arrowheads). The corresponding magnification of null PTH1R cardiomyocytes (D) show myofibrils with fewer sarcomeres, and the mitochondria are larger and swollen with abnormal cristae (arrows). The sarcoplasmic reticulum cisternae, some decorated with ribosomes, are grossly enlarged and swollen (arrowheads). Bar, 1 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
It has become increasingly clear that PTHrP and PTH1R signaling play a role in many developing tissues. Although ablation of either PTHrP or the PTH1R results in chondrodysplasia and perinatal lethality, receptor null animals die before birth (17). The exact cause of death in the PTH1R mutants has been puzzling, particularly because the successful rescue of the bone phenotype fails to prevent neonatal death (18). In this report, we describe a previously unrecognized developmental role for the PTH1R. Our data demonstrate that this receptor is abundantly expressed and functional in the embryonic heart at this critical time. The absence of receptor signaling in homozygous null embryos precipitates abrupt cardiomyocyte death at E11.5-E12.5 presaged by ultrastructural changes that include mitochondrial dysmorphology.

The presence of a functional PTH1R throughout the normal developing murine heart at midgestation, together with the precipitous cardiomyocyte death in its absence, clearly indicates that signaling through this G protein-coupled receptor is required for normal cardiomyocyte function. Previous studies have shown that PTHrP and PTH impact apoptotic and antiapoptotic signaling pathways in a number of cell types including chondrocytes (28), intestinal cells (30), prostate cancer cells (31), and 293 cells transfected with the PTH1R (32). We initially suspected that the heart defects in the PTH1R null mice resulted from cardiomyocyte apoptosis. However, our results, both TUNEL and electron microscopic data, show no evidence of apoptosis or apoptotic nuclear changes. In general, condensation of chromatin is followed by atrophy or involution of apoptotic cell bodies, separating them from their neighbors. During the atrophic process, the subcellular organelles tend to shrink (32A 32B ). In contrast, swollen mitochondria and abnormalities in the endoplasmic reticulum were observed in the cardiomyocytes of null embryos. These changes would be consistent with Ca2+ dysregulation. Increased mitochondrial-free Ca2+ triggers opening of the mitochondrial permeability transition pore, a causative factor in collapse of the inner transmembrane potential, an increase in reactive oxygen species, and decreased ATP production (33), which could lead to cardiomyocyte death and necrotic changes. Therefore, our data demonstrate that apoptosis is not a major contributor to myocardial cell death in the null embryos.

Activation of the embryonic murine cardiomyocyte PTH1R was associated with increased accumulation of cAMP and the amplitude of Ca2+ oscillations as determined by microphotometry. This finding is consistent with a potential role in maintaining cardiomyocyte function or regulating embryonic Ca2+. Previous work using neonatal and adult cardiomyocytes suggests that PTH1R modulates Ca2+ influx via L-type Ca channels (14). However, whereas spontaneous activity of early embryonic cardiomyocytes depends upon sodium-calcium exchange, it is apparently independent of the known L-type Ca2+ channels before E13 (34, 35, 36, 37). ß-Adrenergic receptors, present in the early heart, stimulate cAMP production similar to PTH1R but do not activate L-type Ca2+ currents in response to isoproterenol stimulation. Effective coupling of adenylate cyclase to the L-type Ca channel does not occur until the late fetal stage (38). Preliminary results suggest a similar uncoupling of PTH1R to L-type Ca2+ channel activation at this stage, in spite of cAMP production in cardiomyocytes in response to ligand (Colbert, M. C., and T. L. Clemens, unpublished). Thus, even in wild-type cardiomyocytes PTH1R signals coupled to Gs may not act via the L-type Ca2+channel. This raises the possibility that the PTH1R might influence Ca2+ by activation of additional signaling pathways such as PLC. Studies are currently in progress to further elucidate these potential signaling pathways. Nevertheless, the early midgestational lethality of PTH1R mutants indicates the receptor is necessary during the transition from an embryonic to fetal heart. Further evidence supporting the critical nature of the PTH1R to heart development can be inferred by the ability of transgenic overexpression of the receptor in the heart using a smooth muscle {alpha} actin promoter (8) to rescue midgestational cardiac lethality in the C57Bl6 embryos (39).

As indicated above, the phenotype and severity of developmental abnormalities in mice lacking the PTH1R is highly dependent on the genetic background. For example, Black Swiss mice homozygous for the disrupted pthr1 allele survived until later in gestation (E17-P0) and exhibited chondrodysplasia. However, targeted overexpression of the PTH1R to chondrocytes in the PTH1R knockout mice (Black Swiss) normalized the growth plate defects but did not prevent the prenatal lethality suggesting PTH1R signaling is critical in tissues other than bone. The possibility that cardiac defects contribute to the perinatal demise of the Black Swiss PTH1R null mice is currently under investigation. Interestingly, mice lacking the Pthrp gene also exhibit chondrodysplasia but survive until birth (15). There are a number of possible explanations for this apparent paradox. First, PTHrP is produced by decidual tissues (40), and thus transfer of PTHrP from these or other maternal sources to the fetus could rescue the ligand knockout mice. Alternatively, it is possible that there are additional (yet to be identified) ligand(s) that are capable of activating the PTH1R and thereby could substitute for PTHrP. The recently identified ligand for the PTH2R, termed TIP39, is an unlikely candidate because it does not activate the PTH1R (41). Another possibility is that PTHrP, which is known to be elevated in the plasma (and possibly other tissues, e.g. heart) of the PTH1R null embryos (42), could activate receptors or signaling pathways distinct from the PTH1R. For example, several different cell types produce mid-region PTHrP fragments capable of raising intracellular free calcium (43), suggesting the existence of novel receptors for these posttranslationally derived PTHrP products. It is unknown whether such fragments or receptors exist in murine fetal tissues, but if they did, they could in theory activate novel signaling pathways to produce the defects observed in the heart of the PTH1R null embryos.

Whether PTH1R signaling is essential for normal cardiac function in humans as it is in mice remains to be established. Circumstantial evidence supporting such a role is evident from the abnormalities seen in the rare fatal Blomstrand’s chondrodysplasia caused by an inactivating mutation of the PTH1R (44). These infants also die before birth with coarctation of the aorta and hydrops fetalis, the latter condition typically caused by high output heart failure. Furthermore, the extreme rarity of Blomstrand’s fetuses also indicates that the lack of a functional PTH1R is usually lethal early in gestation.

In summary, our studies demonstrate that mice lacking the PTH1R die abruptly at midgestation with evidence of precipitous cardiomyocyte cell death. We conclude that a functional PTH1R is required for establishment and/or maintenance of normal cardiac performance at the midgestational transition from embryonic to fetal heart.

These results implicate a critical role of PTH1R in embryonic cardiomyocyte development.


   Acknowledgments
 
We thank Pam Groen, Kathy Saalfeld, Lisa McMillin, and William Stuart for technical assistance; and Chris Woods for photography assistance.


   Footnotes
 
This work was supported by NIH Grants HL-47811 (to T.L.C.), HL-36059 (to M.C.C.), and DK-56246 (to H.M.K.).

1 J.Q. and M.C.C. contributed equally to this work. Back

Abbreviations: E, Embryonic day; Pth1r, PTH type 1 receptor; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling.

Received October 2, 2002.

Accepted for publication November 7, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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