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ß-Adrenergic receptors (ßAR) regulate cardiomyocyte proliferation during early postnatal life

Department of Pediatrics,
^* Rhode Island Hospital, Women and Infants’ Hospital of Rhode Island, Brown Medical School, Providence, Rhode Island 02905, USA

¹Correspondence: Department of Pediatrics, Women & Infants’ Hospital of RI, 101 Dudley St., Providence, RI 02905, USA. E-mail: Yi-Tang_Tseng{at}brown.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cardiomyocyte development switches from hyperplasmic to hypertrophic growth between postnatal days 3 and 4 in rats. The mechanisms responsible for this transition have been controversial. ß-Adrenergic receptor (ßAR) activation of mitogenic responses in vitro has been reported. We hypothesized that tonic activation of the ßAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of the current study was to elucidate the roles of ßAR in rat cardiomyocyte growth in vivo. We demonstrated that ßAR blockade induced a significant reduction in cardiomyocyte proliferation as measured by the BrdU labeling index. Blockade of ßAR did not affect p38 or p44/42 MAPK activities. We further demonstrated that ßAR blockade induced a prompt deactivation of the p70 ribosomal protein S6 kinase (p70 S6K). To confirm these results, we measured p70 S6K activity directly. Basal activity of p70 S6K in neonatal cardiomyocytes was fourfold higher than that of insulin-treated adult rat liver. The activity of p70 S6K was reduced by 60% within 1 min after ßAR blockade. We conclude that the ßAR are involved in regulation of neonatal cardiomyocyte proliferation and that this mitogenic control may be mediated via the p70 S6K pathway.—Tseng, Y.-T., Kopel, R., Stabila, J. P., McGonnigal, B. G., Nguyen, T. T., Gruppuso, P. A., Padbury, J. F. ß-Adrenergic receptors (ßAR) regulate cardiomyocyte proliferation during early postnatal life.

Key Words: MAPK • p70 S6K • propranolol

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
CARDIOMYOCYTES DISPLAY TWO developmentally regulated modes of growth. During fetal and early postnatal life in rats, cardiomyocytes are actively proliferative. After the third to fourth day of life, cardiomyocytes are arrested in the G₁ phase of the cell cycle and mitosis is irreversibly blocked (1 , 2) . Growth of cardiomyocytes in later phases of cardiac development is solely via hypertrophy. The molecular mechanisms responsible for the switch from hyperplasia to hypertrophy are not understood completely (3 , 4) .

Redundant mechanisms for the regulation of cardiomyocyte cell division and growth converge on one or several serine/threonine kinases, including the mitogen-activated protein kinase (MAPK) pathways and the pathway leading to phosphorylation of ribosomal protein S6. Many G-protein-coupled receptors are also able to activate these signaling cascades and induce changes in cell proliferation and growth (5) . The best studied among these is activation of MAPK by the ß2-adrenergic receptor. Other kinase cascades activated by ß-adrenergic receptor (ßAR) include c-Jun amino-terminal kinase, phosphatidylinositol 3-kinase (PI3K), p70 ribosomal protein S6 kinase (p70 S6K), and p38 MAPK (6 7 8 9) . Although ßAR activation of the PI3K/p70 S6K pathway has been demonstrated, the role of ßAR during the proliferative period of cardiac development and regulation of signaling kinases in this role have not been examined.

Whereas mitogenic pathways have been examined in isolated neonatal rat cardiomyocytes, the relative role of each pathway was not assessed nor was the relationship to in vivo regulation of proliferation or growth. We hypothesized that tonic activation of the ßAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. The purpose of this study was to elucidate the role of ßAR in rat cardiomyocyte cell growth and proliferation. These initial studies focused on demonstration of the role for ßAR in vivo and on identification of signaling kinases that might be important in that role.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Materials and animal
All chemicals were purchased from Sigma Chemical Co. (St Louis, MO) and all antibodies were purchased from New England Biolabs (Beverly, MA) unless specified otherwise. Timed pregnant Sprague-Dawley rats (Harlan Laboratories, Cambridge, MA) were housed in cages with free access to food and water and monitored for date of delivery of pups. Experiments were conducted on day 3 after birth. Pups were weighed and randomly assigned to either the control or treatment group. The pups in the treatment group were injected intraperitoneally (i.p.) with S (-) propranolol (10 mg/kg), the biologically active form, whereas the control pups were injected i.p. with PBS vehicle. After injection, pups were immediately returned to their cage with the dam. In an experiment involving the 5-bromo-2'deoxyuridine (BrdU) labeling index, the pups were injected i.p. with BrdU (50 mg/kg) 1 h after PBS or propranolol injection and immediately returned to their cage. Pups were decapitated 1 h after BrdU injection and hearts were removed rapidly. The hearts were placed in 10% formalin overnight, rinsed, and stored in 70% alcohol at room temperature. The preserved hearts were embedded in paraffin and horizontal sections (5 µm) of the ventricles were mounted onto slides. In an experiment involving p44/42 MAPK, p38 MAPK, and p70 S6K Western blots and the p70 S6K activity assays, pups were injected i.p. with PBS or propranolol for 1 to 30 min. After injection, hearts were removed rapidly, placed in liquid nitrogen, and stored at -80°C.

Immunohistochemistry
The heart sections were first deparaffinized and rehydrated. Immunohistochemical detection of BrdU-labeled nuclei was carried out as described (10) . The sections were treated with 3% H₂O₂, digested with 0.1% protease XIV, denatured in 2N HCl, and washed in 0.1M Borax (pH 8.5). The sections were then incubated with a mouse monoclonal anti-BrdU antibody (Vector Laboratories, Burlingame, CA) overnight at 4°C in a humidified chamber. A mouse IgG was used as a negative control. The sections were then incubated with a biotinylated anti-mouse IgG (H+L) secondary antibody (Vector Laboratories) for 30 min at room temperature. Bound antibody was detected using the Vectastain^® ABC-peroxidase kit (Vector Laboratories), developed in liquid 3,3'-diaminobenzidine chromogen (BioGenex, San Ramon, CA) for 2 to 5 min, and lightly counterstained with Harris modified hematoxylin (Fisher Scientific, Fair Lawn, NJ). Permount was used as the mounting media and sections were coverslipped. The immunohistochemical studies were repeated three or four times on samples prepared from different animals.

Image analysis
Images were first acquired with a Nikon Eclipse E800 bright-field microscope (Bay Shore, NY) with a Plan Apo 40x lens. At least three images from three different zones (inner, middle, and outer segments) of the ventricular myocardium were acquired for a total of nine images from each heart. Heart sections from three to four different animals each from both the control and the propranolol-treated groups were analyzed. Images were analyzed using the NIH Image Analysis program (Scion Corp., Frederick, MD). The BrdU labeling index of each heart section was calculated according to the following formula: 100 x (the number of BrdU-positive nuclei/total number of nuclei). Each image was analyzed twice to obtain an average labeling index.

Western blotting
The activity of the signaling kinases potentially regulated by ßAR stimulation (p44/42 MAPK, p38 MAPK, p70 S6K) was measured initially by Western blotting. The whole hearts were pulverized in a glass homogenizer with cold lysis buffer containing 0.2% Triton X-100 and proteins were prepared as described (11) . Protein concentrations were determined using the bicinchoninic acid assay (12) . A total of 25 µg heart lysates from each sample were resolved in 10% SDS-PAGE gels, electrotransferred onto PVDF membranes, and blocked in 5% nonfat dry milk overnight at 4°C. Membranes were incubated with either an anti-phospho-p44/42 MAPK antibody, an anti-phospho-p38 MAPK antibody, or an anti-p70 S6K antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After washing, membranes were incubated with an HRP-conjugated secondary antibody (Amersham Pharmacia Biotech, Piscataway, NJ) for 1 h at room temperature. Membranes were then developed using a Western blot chemiluminescence reagent (NEN Life Science, Boston, MA) and exposed to X-ray films. Membranes were stripped and reprobed with an anti-p44/42 MAPK antibody and an anti-p38 MAPK antibody to determine total p44/42 MAPK activity and total p38 MAPK activity, respectively. Western blotting was repeated three times using different animals for each kinase.

Measurement of the p70 S6 kinase activity
A total of 500 µg proteins from each sample was immunoprecipitated with an anti-p70 S6K antibody on a rotator overnight at 4°C. Normal rabbit serum was used as a negative control. Insulin-treated adult liver lysates were used as a positive control. p70 S6 kinase assays were carried out using commercially available reagents to determine the in vitro phosphorylation of p70 S6K (Upstate Biotechnology, Lake Placid, NY). Incubations were at 30°C for 90 min. Samples from vehicle control and propranolol-treated (1 to 30 min) animals were analyzed simultaneously.

Statistics
All results were shown as the mean ± SE. For the BrdU labeling index, statistical significance of the difference between the control and propranolol-treated groups were measured with the paired Student’s t test. For p70 S6K activity studies, statistical significance was determined by one-way analysis of variance, followed by a Dunnett test. A probability of P < 0.01 was considered to represent a significant difference.

	RESULTS

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The effect of ßAR blockade on neonatal cardiomyocyte proliferation
Effects of ßAR blockade on cardiomyocyte proliferation in neonatal rats were determined by the BrdU labeling index (Fig. 1A , B ). BrdU was injected 1 h after injection of vehicle or propranolol to ensure ßAR blockade. We examined the labeling index in three regions of the heart ventricle: subepicardium, myocardium, and subendocardium. The mouse IgG negative control did not detect any labeling (not shown). High labeling indexes were seen in the control animals, with the highest seen in the subepicardium (45.7±3.0%), followed by the subendocardium (35.4±7.7%) and the myocardium (19.4±7.8%) (Fig. 1B ). This pattern of higher BrdU labeling in the outer (subepicardium) and inner (subendocardium) walls of the heart was observed consistently in hearts from multiple litters, in multiple stained slides, and with experimental study design modifications. Labeling indexes in all three regions were significantly reduced (P<0.01, subepicardium=15.4±1.7%, subendocardium=11.6±2.1%, and myocardium=7.9±2.1%) by propranolol treatment (Fig. 1B ). These results indicate that blockade of the ßAR signaling inhibits the high proliferation rate of neonatal cardiomyocytes.

View larger version (115K): [in this window] [in a new window]   Figure 1. A) Immunohistochemical staining of formalin-fixed and paraffin-embedded 3-day-old rat heart sections using anti-BrdU antibody (brown). Shown is a representative from multiple experiments. Nuclei are counterstained using hematoxylin (blue). Animals were treated with vehicle control (upper panels) or propranolol (lower panels) before injection with BrdU as described in Materials and Methods. Inner (subendocardium), middle (myocardium), and outer (subepicardium) segments of the ventricular myocardium are shown. B) Densitometric analysis of BrdU staining results shown in panel A. Data are calculated as described in Materials and Methods and expressed as mean ± SE. Significance of difference from the vehicle control value, *P < 0.01.

Blockade of ßAR affects signaling kinase activity
One of the goals of our studies was to identify whether and which one of the major signaling kinases was involved in the regulation of ßAR-mediated cardiomyocyte proliferation. The p44 and p42 MAPK, also known as extracellular signal-regulated kinase 1 (ERK1) and ERK2 (13) , are among the most studied MAPK in the heart. When activated through phosphorylation at threonine and tyrosine residues, they may play a role in cardiomyocyte hypertrophy (14 , 15) . Therefore, Western blotting with anti-dual phosphorylated p44/42 MAPK antibodies was used to detect active ERK1 and ERK2 in the neonatal rat heart. We first examined the activities of p44/p42 MAPK 1 h after ßAR blockade. Both phosphorylated and total p44/42 MAPK were detected in all of the neonatal hearts (Fig. 2A ). Propranolol treatment did not affect the phosphorylated p44/42 MAPK levels and total p44/42 MAPK levels remained unchanged (Fig. 2B ). Because activation of MAPK and other signaling kinases can be rapid, occurring in minutes rather than hours, we also examined phosphorylated and total p44/42 MAPK at 1, 2, 5, 10, and 20 min after propranolol administration (data not shown). Similar results were observed. At no time did ßAR blockade result in a reduction in p44/42 MAPK phosphorylation.

View larger version (27K): [in this window] [in a new window]   Figure 2. Effect of ßAR blockade on p44/42 MAPK in neonatal rat heart. A) Three-day-old rats were treated with PBS or propranolol for 1 h. Heart lysates were resolved by SDS-PAGE and analyzed by Western blotting for phosphorylated p44/42 MAPK (P-MAPK) and total p44/42 MAPK (total MAPK). The triplicate samples in each group are heart samples from 3 different rats. B) Densitometric analysis of the p44/42 MAPK Western blot results shown in panel A. Data are expressed as the activity of phosphorylated p44/42 MAPK as % of the total p44/42 MAPK and are presented as mean ± SE.

P38 MAPK is among the other signaling kinases expressed in the heart that may play a role in regulation of cardiomyocyte proliferation. We examined the effects of propranolol administration on myocardial p38 MAPK activity. Moderate levels of total p38 MAPK were detected (not shown). ßAR blockade did not alter the levels of total p38 MAPK. No phosphorylated p38 MAPK was detected at times ranging from 1 to 20 min after propranolol injection.

As described above, PI3K and p70 S6K have been ascribed important roles in the regulation of early cardiac growth (7) . Since p70 S6K is downstream from PI3K, we examined the effects of ßAR blockade on p70 S6K. The anti-p70 S6K antibody recognized both the active (phosphorylated p70 S6K) and inactive (dephosphorylated p70 S6K) forms of p70 S6K. A downward shift in gel running reflects the faster migration of the dephosphorylated form of the enzyme (Fig. 3 ). In vehicle control (lane 1), there was a high level of expression of the phosphorylated-active form of p70 S6K. Propranolol injection resulted in a prompt and substantial reduction in active p70 S6K (downward shift). We also measured kinase activity of p70 S6K directly (Fig. 4 ). All data were calculated by subtracting the activity of normal rabbit serum (the negative control). There was a high basal level of p70 S6K activity in neonatal heart (vehicle control, open bar) fourfold greater than that of insulin-treated adult rat liver positive control (shaded bar). Blockade of ßAR induced a prompt and significant reduction (F=24.6, P<0.001) of p70 S6 kinase activity (solid bars). The p70 S6K activity was reduced by 60% 1 min after propranolol injection and was still down by 40% 30 min later. These results suggest that the p70 S6K is involved in the ßAR-mediated regulation of cardiomyocyte proliferation during early postnatal development.

View larger version (23K): [in this window] [in a new window]   Figure 3. Effect of ßAR blockade on p70 S6K in neonatal rat heart. Shown is a representative blot of 3 experiments with similar results. Three-day-old rats were treated with propranolol for one to 20 min as indicated on top of each lane. C, Control treated with vehicle for 1 min. Heart lysates were resolved by SDS-PAGE and analyzed by Western blotting using anti-p70 S6K antibodies. The position of active (P-p70 S6K) and inactive p70 S6K are shown by the upper and lower arrow, respectively.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of ßAR blockade on p70 S6 kinase activity in neonatal rat heart. Shown is a representative of 3 experiments with similar results. Three-day-old rats were treated with propranolol for 1 to 30 min as indicated (solid bars). Vehicle control animals were treated with PBS for 1 to 5 min (open bar). Liver from adult rat treated with insulin was used as a positive control (shaded bar). Data are presented as percent of the vehicle control and are mean ± SE from 2 to 4 different samples. Significance of difference from the vehicle control value, *P < 0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The purpose of this study was to elucidate the role of ßAR in rat cardiomyocyte cell growth and development. The immunohistochemical studies demonstrated that the ßAR play a critical role in cardiomyocyte proliferation during early postnatal development. The p38 MAPK and the ERK1/ERK2 pathways were not affected by ßAR blockade. However, p70 S6K activity was promptly reduced after ßAR blockade. The data suggest that regulation of neonatal cardiomyocyte cell growth is mediated in part by ßAR and downstream p70 S6K.

Several signal transduction pathways regulate cardiac growth and/or proliferation. These redundant mechanisms for the regulation of cardiomyocyte cell division and growth converge on one or several serine/threonine kinases. Several G-protein-coupled receptors (AR, ßAR, angiotensin II, and endothelin-1) are able to activate these signaling cascades and induce changes in cell proliferation and growth (5) . The relative importance of each of pathway is likely to depend on the developmental stage and/or pathobiological setting in which it is activated. We hypothesized that tonic activation of the ßAR signaling regulates cell division in neonatal cardiomyocytes via effects on signaling kinases known to be important in cell cycle regulation. We did not observe a reduction in the levels of phosphorylated p44/42 MAPK after ßAR blockade. This dissociation of MAPK activity from cell proliferation is striking and is reminiscent of a similar dissociation in regulation of fetal rat hepatocyte proliferation (16) . It has been reported that norepinephrine induces cardiomyocyte hypertrophy by activating the raf-1/MAPK cascade through both 1AR and ßAR (15) . One plausible explanation for the lack of reduction in phosphorylated p44/42 MAPK in neonatal rat hearts after ßAR blockade could be a change in the activity of one or several of the MAPK phosphatases (MKPs) recently identified to be important in MAPK signaling (17) . MKPs play important roles in cellular functions by inhibiting the MAPK signaling cascade. Whether ßAR blockade induces changes in the activity of MKPs, however, remains to be investigated.

We saw no effect of ßAR blockade on the levels of total or phosphorylated p38 MAPK. Whereas p38 MAPK may be involved in cardiogenesis, its role may be most important in cardiomyocyte hypertrophy and ischemic preconditioning protection (18 , 19) . Activation of p38 MAPK in neonatal cardiomyocytes by AR and endothelin-1 via a G-protein-dependent mechanism has been demonstrated but would not account for our observations on the effect of ßAR blockade on BrdU incorporation (20) .

We observed a high basal expression of p70 S6K activity. Activity in neonatal heart samples was fourfold greater than that of samples extracted from adult rat liver after insulin treatment. Treatment with propranolol resulted in a 60% reduction in activity within 1 min after ßAR blockade. We cannot completely rule out the possibility that reduction in p70 S6K is simply a by-product of cardiomyocyte growth arrest and may not be directly mediated by ßAR blockade. However, this is consistent with the demonstration that ßAR stimulation of cardiomyocytes with isoproterenol results in a significant increase in p70 S6K activity (8) . This result was observed in isolated adult cardiomyocytes and only after acquisition of the capacity for hypertrophic responses. ßAR stimulation, which increased p70 S6K activity, had no effect on phosphorylated p44/42 MAPK in the same experiment (8) . p70 S6K is a downstream target of PI3K (21) . Recent studies suggest a greater importance for the PI3K/p70 S6K pathway than p44/42 or p38 MAPK in mediating cardiac effects (7 , 8 , 22) . Recently, a central role for PI3K in regulation of early cardiac growth has been suggested by studies in transgenic mice overexpressing PI3K in the heart (23) . Cardiac-specific expression of a constitutively active PI3K resulted in increased cardiac growth and activation of both p70 S6K and PKB. Reduced cardiac growth was seen in animals expressing a dominant negative PI3K. Levels of ERK1 were not affected in these animals. Likewise, cardiomyocyte differentiation of embryonic stem cells is completely blocked by inhibition of PI3K (24) . Our results confirm and extend these results suggesting importance for the PI3K/p70 S6K pathway in early neonatal cardiomyocyte proliferation.

The regulation of ßAR expression is unique during fetal and early postnatal life. The high secretion rate of catecholamines is associated with a high degree ligand occupancy of the ßAR in the heart. Disruption of the ß1AR leads to a high proportion of embryonic death (25) whereas no prenatal lethality is associated with ß2AR knockout (26) . Disruption of both ß1 and ß2AR did not significantly affect basal physiology (27) . Since these double knockout animals were developed on a mixed strain background, they do not display increased embryonic lethality as ß1AR knockout animals do. Although litter size appeared to be normal, detailed cardiac morphological characterization has yet to be examined. It would be interesting to compare cardiac BrdU labeling indices between wild-type and ß1AR/ß2AR double knockout animals. In contrast, overexpression of ß1AR or ß2AR lead to cardiomyopathy (28 , 29) . Multiple studies suggest a role for sympathetic stimulation in hypertrophic cardiac growth during congestive heart failure and other pathological states (30) . This is corroborated experimentally where chronic, subhypertensive doses of norepinephrine or isoproterenol result in significant cardiac hypertrophy. Whereas adrenergic signaling via both - and ß-adrenergic pathways induces hypertrophy, the effects after infusion of isoproterenol confirm the role of ßAR stimulation in this mechanism. In pregnant rats, propranolol results in significant impairment of fetal somatic and heart growth and a delay in cardiac cellular development (31) . Likewise, sympathetic denervation is associated with decreased cardiac DNA synthesis in the first week of life and impaired sympathetic regulation of cardiac growth in older animals (32) . Our results suggest that this may result from blockade of the major role of ßAR stimulation in regulating cardiac growth in late fetal and early postnatal life.

	ACKNOWLEDGMENTS

 
The authors thank Joan Boylan, Hiroko Sekimoto, Marija Hleb, and Surendra Sharma for technical assistance. This work was supported by NICHD 2PO1 HD11343.

Received for publication February 27, 2001. Revision received May 7, 2001.

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