AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H1299-H1308, 2006. First published February 24, 2006; doi:10.1152/ajpheart.00017.2006
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/3/H1299    most recent
00017.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sucharov, C. C.
Right arrow Articles by Bristow, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sucharov, C. C.
Right arrow Articles by Bristow, M. R.

A beta1-adrenergic receptor CaM kinase II-dependent pathway mediates cardiac myocyte fetal gene induction

Carmen C. Sucharov, Peter D. Mariner, Karin R. Nunley, Carlin Long, Leslie Leinwand, and Michael R. Bristow

University of Colorado Cardiovascular Institute, Denver, Aurora and Boulder, Colorado

Submitted 4 January 2006 ; accepted in final form 24 February 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
beta-Adrenergic signaling plays an important role in the natural history of dilated cardiomyopathies. Chronic activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress ultimately harms the failing heart by mechanisms that include alterations in gene expression. Here, we show that stimulation of beta-ARs with isoproterenol in neonate rat ventricular myocytes causes a "fetal" response in the relative activities of the human cardiac fetal and/or adult gene promoters that includes repression of the human and rat {alpha}-myosin heavy chain ({alpha}-MyHC) promoters with simultaneous activation of the human atrial natriuretic peptide (ANP) and rat beta-MyHC promoters. We also show that the promoter changes correlate with changes in endogenous gene expression as measured by mRNA expression. Furthermore, we show that these changes are specifically mediated by the beta1-AR, but not the beta2-AR, and are independent of {alpha}1-AR stimulation. We also demonstrate that the fetal gene response is independent of cAMP and protein kinase A, whereas inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK) pathway blocks isoproterenol-mediated fetal gene program induction. Finally, we show that induction of the fetal program is dependent on activation of the L-type Ca2+ channel. We conclude that in neonatal rat cardiac myocytes, agonist-occupied beta1-AR mobilizes Ca2+ stores to activate fetal gene induction through cAMP independent pathways that involve CaMK.

myosin; adenosine 3',5'-cyclic monophosphate

beta-ADRENERGIC SIGNALING plays an important antithetical role in the natural history of dilated cardiomyopathies (DCMs) exerting favorable compensatory effects on cardiac function and promoting the development and progression of the DCM phenotype (4, 6, 11, 13). Activation of beta-adrenergic receptors (beta1-AR and beta2-AR) during periods of cardiac stress initially results in increases in heart rate and contractility, effectively improving cardiac output, but then ultimately harms the failing heart (5, 8) by mechanisms that include alterations in gene expression (19). Unfortunately, however, the elucidation of the pathways involved in the harmful components of beta-adrenergic signaling has been hindered by a lack of a reliable experimental model.

At the cellular level, myocardial failure is characterized by changes in the expression of genes involved in many critical functions of the cardiac myocyte, including those comprising the contractile apparatus. A subset of these molecular changes has been described as a recapitulation of a "fetal" gene program because many embryonically expressed genes that are downregulated postnatally are reactivated, whereas several "adult" genes are repressed (15). Of the changes that are observed in failing hearts, increases in expression of the fetal genes skeletal {alpha}-actin and atrial natriuretic peptide (ANP) with coordinate decreases in expression of {alpha}-myosin heavy chain ({alpha}-MyHC), the ratio of {alpha}-MyHC to beta-MyHC, and sarcoplasmatic reticulum ATPase 2a (SRCA2a) are the most widely recognized.

In the intact heart, chronic beta-adrenergic stimulation of the myocardium has been shown to change gene expression patterns in a manner that also mimics the fetal gene program observed in failing hearts (3, 19, 27). Several beta-AR blocking agents have been identified that successfully treat heart failure patients with DCMs by acting to favorably alter the biology of the failing heart (7). It was recently shown that patients with DCMs who respond to beta1-adrenergic receptor blockade by an improvement in systolic function and a reversal of remodeling also have a reversal of the fetal gene expression (19). In these responder's hearts, the ratio of {alpha}-MyHC to beta-MyHC expression and total SRCA2a are increased, whereas atrial natriuretic peptide (ANP) gene expression is reduced (19). Given the evidence of the importance of beta-adrenergic signaling in heart muscle disease and heart failure, elucidation of the molecular details of how beta-adrenergic stimulation leads to fetal gene induction could lead to the identification of additional therapeutic targets.

Neonatal rat ventricular myocytes (NRVMs) have been used successfully by many investigators as a cell culture model for investigating several signaling pathways involved in heart disease. In the case of beta-adrenergic signaling, however, NRVMs have been problematic. That is, treatment of NRVMs with isoproterenol, a nonselective beta-AR agonist, induces myocyte hypertrophy similar to that observed in cardiac disease. However, this hypertrophic response does not consistently induce a fetal gene program (18, 31). As a result, little progress has been made in dissecting the effects of beta-adrenergic activation. Moreover, the question of whether beta-adrenergic stimulation can directly elicit a comprehensive fetal gene response has been raised.

In the work presented here, we demonstrate that the stimulation of NRVMs with 10 nM to 1 µM isoproterenol can consistently induce an upregulation of ANP, brain natriuretic peptide (BNP), and skeletal {alpha}-actin expression and a downregulation of {alpha}-MyHC and SERCA2a. This change is easily monitored by measuring the relative activities of the human and rat cardiac MyHC gene promoters, making this system a valuable model to experimentally examine the molecular pathways involved in beta-adrenergic activation. As a proof of principle, we go on to show that the isoproterenol-induced fetal response is specifically mediated by the beta1-AR and not the beta2-AR. Surprisingly, the beta1-AR fetal gene response is independent of cAMP, because treatment of the cells with cAMP analogs had no effect on the activity of MyHC promoters or endogenous fetal and/or adult gene expression, and pretreatment of myocytes with protein kinase A (PKA) inhibitors did not affect isoproterenol-mediated responses. Furthermore, we provide evidence that Ca2+/calmodulin kinase (CaMK) appears to play an important role in the isoproterenol-mediated fetal gene response because inhibition of CaMK resulted in an inhibition of isoproterenol-mediated effects. Finally, we show that isoproterenol-mediated induction of the FGP is dependent on activation of Ca2+ channels because inhibition of the L-type Ca2+ channel (LTCC) channels results in inhibition of the isoproterenol effects on the FGP. Our results suggest that changes in gene expression mediated by beta1-adrenergic stimulation occur through a cAMP independent pathway and are likely to occur through a Ca2+-related mechanism.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Materials. Isoproterenol (Sigma) was dissolved in 1 mmol/l ascorbic acid solution, and the concentrations used are described in RESULTS. Additional reagents were added to the cells with the following final concentrations: 1 µM 8-bromoadenosine 3',5'-cyclic monophosphate sodium salt (Sigma), 1 µM N6,2'-O-dibutytyladenosine 3',5'-cyclic monophosphate sodium salt (Sigma), 10 µM H-89 (Sigma), 1 µg/ml 14–22 amide myristoylated (Calbiochem), 100 nM prazosin (Sigma), 200 nM CGP-20712A (Tocris), 300 nM ICI-118551 (Tocris), 2 µM KN-93 (Sigma), and 1 µM nicarpidine HCl (Sigma). Except as described in the figures, cells were treated with 100 nM isoproterenol 48 h after being plated and harvested 48 h later.

Plasmid construction and preparation. The human {alpha}-MyHC promoter construct and rat {alpha}-MyHC and beta-MyHC promoter construct have been described previously (21, 32). The human ANP promoter (–2593/+18-luc) was a gift of Dr. David Gardner. Plasmid DNA was prepared using alkaline-lysis kits from Qiagen. DNA was eluted in 10 mM Tris and stored at 4°C.

Cell culture and transfections. NRVMs were cultured as previously described (18, 29, 30, 36). Briefly, cells were isolated by trypsin digestion from the ventricles of 1- to 3-day-old rats. Isolation procedures were done in a very gentle manner, and cells were plated at a concentration of 1.5 x 105 cells per well in 12-well tissue culture gelatin-coated plates in medium containing 5% bovine calf serum. After 24 h, the medium was changed to serum-free medium containing insulin, transferrin, bovine serum albumin, vitamin B12, and penicillin. Supplements are prepared monthly. Wells were precoated with a 0.1% gelatin solution and dried in a hood under UV lights. In addition, all medium solutions were buffered with HEPES (pH 7.5) to a final concentration of 20 mM. Protocol for animal work is in accordance with PHS Animal Welfare Assurance, ID A3269–01 and approved by the Institutional Animal Care and Use Committee.

Cells were transfected using Fugene 6 (Roche) with 0.25 µg DNA per well. DNA was mixed with Fugene reagent at a ratio of 3 µl Fugene to 1 µg DNA in serum-free medium. Isoproterenol and pharmacological inhibitors were added to the culture medium on the day following transfection. Transfection experiments were done in tripliclates in six or more different experiments (n > 18). In cotransfection experiments, the total DNA amount was kept constant by the addition of an empty vector.

RNase protection assay. Total cellular RNA was extracted with TRIzol (Invitrogen) for use in RNase protection assays (RPAs). RPAs were performed essentially as described (12, 24). Briefly, 5 µg of RNA were hybridized against [32P]RNA probes specific to skeletal {alpha}-actin, SRCA2A, {alpha}-MyHC, beta-MyHC, ANF, BNP, and GAPDH. RPAs were performed using the RPAII kit (Ambion) from mRNA extracted from a minimum of three different experiments.

Incorporation of [3H]leucine. General cell growth was measured by the incorporation of [3H]leucine as described previously (20). Immediately after treatment of cells with isoproterenol and/or adrenergic receptor blockers, 1 µCi of [3H]leucine was added to each well (12-well plates) of NRVMs. After 48 h, plates were washed two times with ice-cold PBS. The cells were precipitated on ice with 5% trichloroacetic acid. Proteins were then solubilized by the addition of 0.1% SDS and incubation at 37°C on a rocker for 4 h. [3H]leucine incorporation was measured by scintillation counter.

Immunofluorescence. Immunofluorescence was done according to Harrison et al. (9). Cells were washed with Tris buffered saline and 0.1% Tween-20 (TBST) and fixed with 10% formaldehyde for 20 min. Cells were again washed with TBST and incubated with 0.1% Triton-X for 30 min. Cells were then blocked with 1% BSA in TBST for 1 h followed by 1 h incubation with 1:500 dilution of the {alpha}-actinin antibody (Sigma A-7811). Cells were then washed with TBST and incubated with 1:1,000 dilution of Alexa 594 anti-mouse antibody (Molecular Probes A11032) for 1 h. Cells were washed three times with TBST and one time with water and sequentially covered with mounting solution (Southern Biotech) and glass coverslips. Images were captured at a x20 magnification with a fluorescence microscope (Nikon E800) equipped with a digital camera (Zeiss AxioCam) and Zeiss Axiovision version 3.0.6.36 [EC] imaging software.

Statistical analysis. All values are means ± SE. All analyses were performed using ANOVA with a P < 0.05 in a two-tailed distribution considered to be statistically significant. The posttest comparison was performed by the method of Bonferroni.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 
Isoproterenol induces fetal gene expression of pathological markers. Although NRVMs have been used successfully to study the molecular and cellular effects of many pathology-asociated biological substances, their use for the examination of beta-adrenergic stimulation has yielded conflicting results, making it difficult to study beta-AR-mediated signaling pathways in a cell culture model. It is possible that the lack of beta-AR response in NRVMs is related to cell density and/or hypertrophic conditions in the media. With this in mind, special care was taken during the isolation and culturing of NRVMs to prevent cell damage, reduce fibroblast contamination, minimize contact-mediated stimulation by plating the cells at low density, and remove all traces of serum after plating.

As shown in Fig. 1, we are able to consistently prepare cells that have measurable pathological responses to beta-AR stimulation. Isoproterenol treatment (100 nM) resulted in a decrease in {alpha}-MyHC and SRCA2a gene expression, and although beta-MyHC gene expression was not increased, there was a decrease in the ratio of {alpha}-MyHC to beta-MyHC mRNA, which is consistent with a pathological response. Consistent with a pathological response, skeletal {alpha}-actin, BNP, and ANP mRNA expression were upregulated (Fig. 1A). Furthermore, isoproterenol treatment causes significant increase in protein synthesis as measured by an increase in the incorporation of [3H]leucine compared with untreated control (Fig. 1B). Measurement of labeled amino acid incorporation has been previously described and validated as a marker of cardiac myocyte hypertrophy (16, 17, 29, 30). These data clearly indicate that a fetal gene response is elicited in these cells and confirm our ability to use NRVMs as a model for studying beta-AR signaling.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 1. Isoproterenol promotes cardiac hypertrophy measured by changes in gene expression and an increase in protein content. Neonatal rat ventricular myocytes (NRVMs) were treated with 100 nM isoproterenol for 48 h. A: total RNA was isolated and analyzed by RNase protection assay (RPA). {alpha}-MyHC and beta-MyHC, {alpha}- and beta-myosin heavy chain, respectively; ANP, atrial natriuretic peptide, BNP, brain natriuretic peptide; SRCA2a, sarcoplasmic reticulum ATPase 2a. B: cellular hypertrophy was measured by the increase in protein synthesis as described in the text. Results were compared with control experiments defined as 100% in all figures. C: immunofluorescence with anti-{alpha}-actinin antibody of cells treated with isoproterenol (ISO) or vehicle control.

 
Promoter studies demonstrate fetal response to isoproterenol treatment. To further demonstrate that isoproterenol elicits fetal changes in gene expression in NRVMs and provide a useful technique for measuring these changes, we transfected NRVMs with heterologous promoter constructs in which either the human or rat {alpha}-MyHC promoters and the human ANP or rat beta-MyHC promoters were driving the expression of luciferase. As shown in Fig. 2, A–D, treatment of NRVMs with 10 nM to 1 µM of isoproterenol results in a fetal response in each of these reporter constructs. More specifically, the human and rat {alpha}-MyHC-luc promoter activities are repressed by isoproterenol treatment, whereas the activities of the human ANP-luc and rat beta-MyHC-luc reporter constructs are increased. Because the beta-AR response elicits repression of {alpha}-MyHC promoter at a 10 nM to 1 µM isoproterenol range, all subsequent experiments were performed with 100 nM isoproterenol.


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 2. Human and rat {alpha}-MyHC promoters (A and C) and human ANP (B) and rat beta-MyHC (D) promoters were treated with increasing concentrations of ISO. Luciferase activity was measured 48 h after treatment. N.S. not significant.

 
Fetal gene response is mediated through beta1-AR and not through beta2-AR. To confirm that the fetal gene response observed in the promoter-reporter experiments is specifically mediated through beta-ARs, the pharmacological beta-AR antagonist propranolol was used in combination with isoproterenol treatment of NRVMs. As shown in Fig. 3, the addition of propranolol prevented the isoproterenol-mediated repression of both the human and rat {alpha}-MyHC-luc reporters as well as the activation of the beta-MyHC-luc reporter.


Figure 3
View larger version (9K):
[in this window]
[in a new window]
  		Fig. 3. Changes in promoter activity are mediated by beta1-adrenergic receptor (beta1-AR). NRVMs were treated with 1 µM of the beta-AR blocker propranolol 15 min before ISO treatment. Luciferase activity was measured 48 h later. To determine which beta-AR is involved in changes in promoter activity, 200 nM of the beta1-AR-specific inhibitor CGP-20712a and 300 nM of the beta2-AR-specific inhibitor ICI-118551 were added to the cells 15 min before treatment with 100 nM ISO. A and B: MyHC and ANF promoter response to ISO is mediated by beta1- and not beta2-AR activity. Cells were harvested, and luciferase activity was measured 48 h after ISO treatment.

 
These data led us to ask which beta-adrenergic receptor, beta1-AR or beta2-AR, is mediating the fetal gene response. To answer this question, each receptor was blocked individually by the addition of the receptor-specific blockers CGP-20712A and ICI-118551, which selectively inhibit the beta1-AR and beta2-AR, respectively. As shown in Fig. 3, CGP-20712A, but not ICI-118551, blocks the isoproterenol-mediated changes in promoter activities of our reporter constructs, indicating that the fetal gene response to isoproterenol treatment is mediated through beta1-ARs and not through beta2-ARs.

Endogenous changes are specifically mediated by beta1-AR. To confirm that our reporter studies were accurately reflecting the regulation of endogenous responses to isoproterenol through the beta1-AR, mRNA levels of the fetal gene program were measured. As shown in Fig. 4A, repression of SRCA2a and {alpha}-MyHC, and activation of BNP, ANP, and skeletal {alpha}-actin in response to isoproterenol are all blocked by CGP-20712A, but not by ICI-118551, consistent with the promoter-reporter system. Increase in protein synthesis, however, was blocked by the beta1-AR blocker, CGP-20712A and by the beta2-AR blocker ICI-118551 (Fig. 4B). Taken together, these data demonstrate that isoproterenol induces a pathological response in NRVMs that is specifically mediated by activation of the beta1-adrenergic receptor.


Figure 4
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. Changes in endogenous fetal gene expression and in cellular hypertrophy in response to ISO are mediated by beta1-AR and not beta2-AR. A: cells were treated with the specific inhibitors as described in Fig. 3. mRNA was extracted 48 h after treatment and analyzed by RPA. Bars represent a total of four individual experiments. Each mRNA was normalized to GAPDH. B: NRVMs were treated with propranolol or the beta1-AR- or beta2-AR-specific inhibitors or a combination of both followed by different concentrations of ISO as described. Cellular hypertrophy was measured by the increase in protein synthesis as described in the text.

 
Isoproterenol-mediated changes in the fetal gene program do not occur through {alpha}-AR. To rule out the possibility that the changes in promoter activity were not mediated by {alpha}1-ARs, cells were treated with 1 µM of the specific {alpha}1-AR antagonist prazosin before treatment with isoproterenol. {alpha}1-AR blockade did not affect {alpha}-MyHC, beta-MyHC, or ANP promoter activity at isoproterenol concentrations of 100 nM or 1 µM, providing further evidence that lower concentrations of isoproterenol induces the fetal gene program through a beta-adrenergic-mediated pathway (Fig. 5). However, at higher concentrations, prazosin reduced the isoproterenol effect, suggesting that at concentrations higher than 1 µM, isoproterenol activation is leaky and stimulates the {alpha}-AR pathway.


Figure 5
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 5. Prazosin does not block isoproterenol-mediated repression of the {alpha}-MyHC promoters (A and C) or activation of the ANP or beta-MyHC promoters (B and D). NRVMs were treated with 1 µM prazosin 15 min before ISO treatment. Luciferase activity was measured 48 h later.

 
Inhibition of isoproterenol-activation of the FGP is independent of PKA. In cardiac myocytes, many beta1-adrenergic responses are mediated through cAMP activation of PKA (26). To determine whether PKA is involved in isoproterenol-mediated changes in {alpha}-MyHC promoter activity, cells were treated with 8-bromo-cAMP or dibutyrl cAMP. The {alpha}-MyHC promoter-reporter system failed to respond to 8-bromo-cAMP (Fig. 6A) or dibutyrl cAMP (data not shown). We then sought to determine whether the small molecule PKA inhibitor H-89, or the PKA peptide inhibitor PKI14–22 was capable of blocking the isoproterenol-mediated effect. Neither PKI14–22 nor H-89 blocked isoproterenol-mediated repression of the human {alpha}-MyHC promoter (Fig. 6B), human ANF, and rat beta-MyHC promoters (data not shown). PKA inhibition did not prevent the downregulation of {alpha}-MyHC, although it did reduce beta-MyHC gene expression. PKA inhibition failed to prevent downregulation of SRCA2A. Although PKA inhibition reduced the degree of inhibition of ANP, BNP, and skeletal {alpha}-actin, these genes were still upregulated in response to isoproterenol (Fig. 8). To confirm the results of the pharmacological treatment, cells were transfected with the human {alpha}-MyHC-luc vector promoter and a construct that expresses a constitutively active form of PKA (caPKA). caPKA did not change the activity of the {alpha}-MyHC promoter (Fig. 6C). As a positive control for PKA-mediated effects, a synthetic promoter construct containing three copies of the cAMP response element binding site was cotransfected with caPKA in NRVMs; this construct was upregulated by the caPKA (data not shown).


Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6. Changes in endogenous gene expression and {alpha}-MyHC promoter activity in response to ISO are independent of cAMP-PKA pathway. A: {alpha}-MyHC promoter activity is not repressed in response to cAMP treatment. Cells were treated with 1 µM 8-bromoadenosine cAMP sodium salt. B: quantification of RPA. Bars represent a total of three individual experiments. Each mRNA was normalized to GAPDH. Cells were treated with 10 µM H-89 or 1 µg/ml 14–22 amide myristoylated for 1 h before treatment with 100 nM ISO. C: repression of {alpha}-MyHC promoter activity in response to ISO is not blocked by inhibition of PKA. Cells were treated with 10 µM H-89 or 1 µg/ml 14–22 amide myristoylated for 1 h before treatment with 100 nM ISO. D: a constitutively active PKA (caPKA) cDNA construct does not alter {alpha}-MyHC promoter activity. Results were compared with control experiments defined as 100%. These include vehicle-treated controls or cotransfection with empty vectors.

 

Figure 8
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. L-type Ca2+ channels (LTCCs) are involved in beta1-AR mediated fetal gene expression. NRVMs were pretreated with nicardipine for 15 min followed by ISO treatment as described in the text. RNA was extracted and analyzed by RPA. Bars represent a total of three individual experiments. Each mRNA was normalized to GAPDH.

 
Isoproterenol-mediated changes in promoter activity and mRNA expression are regulated through CaMKII. In cardiac myocytes it was recently shown that CaMKII is involved in the pathological aspect of the beta1-AR-mediated response (35, 37, 40). It has also been shown that beta-adrenergic stimulation induces expression of cardiac ankyrin repeat protein expression in NRVMs through a CaMK- and PKA-dependent pathway (41). Moreover, CaMK has been implicated in the regulation of the hypertrophic markers skeletal {alpha}-actin, BNP, ANF, and beta-MyHC (see Refs. 22 and 39 and review in Ref. 38). We next sought to determine whether CaMKII plays a role in the changes in gene expression mediated by beta1-AR in cardiac myocytes. Inhibition of CaMKII by KN-93 prevented the majority of endogenous mRNA fetal gene program induction (Fig. 7A). KN-93 pretreatment prevented isoproterenol-associated downregulation of {alpha}-MyHC or upregulation of skeletal {alpha}-actin and natriuretic peptides. However, inhibition of CaMK was not capable of preventing repression of SRCA2a, suggesting that SRCA2a gene repression is regulated by a different pathway. Surprisingly, KN-93 alone resulted in a decrease in ANP, beta-MyHC, and SRCA2a mRNA levels. These results suggest that inhibition of CaMKII not only blocks the effect of isoproterenol but results in repression of certain genes that are upregulated in NRVMs under the cell culture conditions used in this study.


Figure 7
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 7. Activation of beta1-AR-mediated fetal gene expression is dependent on Ca2+/calmodulin-dependent protein (CaMKII). NRVMs were pretreated with KN-93 for 1 h followed by ISO treatment as described in the text. RNA was extracted and analyzed by RPA. Bars represent a total of three individual experiments. Each mRNA was normalized to GAPDH.

 
Isoproterenol-activation of FGP is mediated by LTCC. The results presented above suggest that Ca2+ is an important mediator of fetal gene activation in response to beta1-AR stimulation. beta1-AR signaling has been shown to activate LTCCs through Gs{alpha} (14), and CaMKII has been shown to activate LTCCs (1), both independently of cAMP activation, making LTCCs a likely candidate for Ca2+ regulation in response to beta1-AR activation. To determine whether LTCC participates in the isoproterenol-mediated activation of the fetal gene program, NRVMs were pretreated with the LTCC inhibitor nicardipine. As shown in Fig. 8, inhibition of LTCCs with nicardipine prevented downregulation of {alpha}-MyHC and SRCA2a and upregulation of ANP, BNP, and skeletal {alpha}-actin. Interestingly, inhibition of LTCC increased upregulation of beta-MyHC in response to isoproterenol stimulation.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 DISCLOSURES
 REFERENCES
 
In this study we demonstrate that isoproterenol induces a hypertrophic response in NRVMs as measured by an induction of the fetal gene program as well as an increase in cellular incorporation of radiolabeled leucine. The changes in gene expression invoked by isoproterenol were similar to those observed in the intact hearts of adult animals and human subjects exhibiting pathological hypertrophy and myocardial failure (3, 19). This fetal gene expression response to isoproterenol is beta-AR specific and is not mediated through {alpha}1-ARs. The fetal gene response is mediated through a beta1-AR receptor pathway that is cAMP and PKA independent but dependent on CaMKII and LTCC.

Previous studies in NRVMs were unable to detect a change in endogenous MyHC levels with beta-adrenergic stimulation, suggesting that in this system beta-adrenergic pathways were not involved in the fetal gene response (31), despite the fact that beta-adrenergic stimulation has been shown to increase ANP gene expression (2, 23). This lack of a comprehensive beta-adrenergic fetal gene induction in NRVMs was in contrast to results in the adult rat intact myocardium as well as clinical data in the human heart (3, 19, 27). However, our data indicate that NRVM treatment with isoproterenol demonstrated a fetal gene response as assessed by the activities of {alpha}-MyHC, beta-MyHC, and ANP reporter constructs, as well as an increase in protein synthesis based on increased levels of [3H]leucine incorporation. As argued previously (18), the hypertrophy observed in NRVMs upon treatment with isoproterenol could be dependent on increased contractility and thus might not be associated with the changes in gene expression that ordinarily accompany pathological hypertrophy. However, we demonstrated that beta-adrenergic stimulation alters the activities of the human and rat cardiac MyHC promoters and human ANP promoter in directions consistent with the "fetal" gene response that is considered to be the molecular marker of pathological hypertrophy (33). Several important conclusions can be drawn from these data. First, the use of transiently transfected luciferase reporters can serve as a convenient assay for measuring the pathological effects of isoproterenol treatment on NRVMs. The results of the luciferase assays closely mimicked the endogenous mRNA data shown in Fig. 1, providing a useful tool for observing the fetal gene response in these cells. Second, the ability of isoproterenol to elicit a pathological response of the {alpha}-MyHC promoter falls within a defined range of isoproterenol concentrations. As can be seen in Fig. 2, A and C, there is no significant response of the {alpha}-MyHC promoter when the isoproterenol concentration is lower than 10 nM or higher than 1 µM. At lower concentrations, there is likely insufficient activation of the beta-ARs to elicit a fetal response. At higher concentrations, however, the {alpha}-AR pathway is activated that eclipse beta-adrenergic signaling, preventing the repression of the {alpha}-MyHC promoter.

Moreover, isoproterenol induced a fetal pattern of endogenous gene expression in NRVMs with repression of SRCA2a mRNA expression and activation of skeletal {alpha}-actin, BNP, and ANP mRNA expression. Importantly, the {alpha}-MyHC-to-beta-MyHC ratio is downregulated, and the alteration in this balance is likely responsible for the changes in contractile activity as seen in experiments with single cells (10). Changes in {alpha}-MyHC-to-beta-MyHC ratios without activation of beta-MyHC gene expression have also been observed in failing human hearts (19). Taken together, these data demonstrate that beta-adrenergic pathway is able to induce "pathological" hypertrophy and a fetal gene program response in NRVMs.

Inhibition of beta1-AR resulted in complete blockade of isoproterenol-mediated repression or activation of the promoters analyzed or endogenous gene expression, whereas beta2-AR blockade did not change the isoproterenol-mediated effects. This corroborates previous work in NRVMs demonstrating that isoproterenol-mediated increases in ANP gene transcription and hypertrophy are beta1-AR dependent, and data showing that the use of beta1-adrenergic blockers can reverse fetal gene expression patterns in vivo (19, 23). Increase in protein synthesis was repressed by both beta1-AR and beta2-AR blockers (Fig. 4, A and B). This is in agreement with other studies (25, 28). However, Morisco et al. (23) have shown that increase in cell size in response to 10 µM isoproterenol treatment is blocked by ICI-118551 (23). In our system, this concentration induces a {alpha}-AR response making it difficult to draw comparisons between the two studies.

Stimulation of beta-AR by catecholamines results in the activation of the beta-AR-adenylyl cyclase-PKA cascade (34), classically thought to mediate most of the biological responses to beta-adrenergic stimulation. For this reason, it was surprising that PKA inhibition by different methods was incapable of blocking isoproterenol-mediated effects on any aspect of fetal gene induction, although it reduced upregulation of ANP, BNP, and skeletal {alpha}-actin. These results suggest that cAMP-PKA pathway might be involved in a minor upregulation of the fetal gene program, but it is not responsible for the changes observed in response to isoproterenol treatment. These findings are in agreement with Bogoyevitch et al. (2), who demonstrated in NRVMs that isoproterenol-mediated hypertropy and increased ANP promoter activity utilized cAMP independent signaling.

Recent work in NRVMs has shown that CaMKII is involved in beta-AR-mediated response in both cases independent of cAMP and PKA (35, 37, 40). Here we show that the pathological changes in gene expression in response to beta1-AR stimulation are also dependent on activation of CaMKII, with the exception of SRCA2a. SRCA2a repression was not blocked by CaMKII inhibition suggesting that regulation of SRCA2a gene expression is mediated by a different pathway. Finally, activation of CaMKII is dependent on cellular Ca2+ influx, so we next determined the role of Ca2+ channels in beta1-AR response. Inhibition of LTCC resulted in blockade of beta1-AR activation of fetal gene induction, suggesting that Ca2+ plays a major role in initiation of this response.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
DISCLOSURES
 REFERENCES
 
This work was possible with the support of National Heart, Lung, and Blood Institute Grants 2R01 HL-48013, R01HL-56510, and R01 HL-48013-10S1.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
REFERENCES
 
M. R. Bristow is an Officer and has equity in Myogen Inc. and ARCA Discovery, Inc.; L. A. Leinwand has equity in Myogen, Inc.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. R. Bristow, Campus Box B130, UCHSC, Denver, CO 80262 (e-mail: michael.bristow{at}uchsc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 DISCLOSURES
 REFERENCES
 

  1. Anderson ME. Calmodulin kinase and L-type calcium channels: a recipe for arrhythmias? Trends Cardiovasc Med 14: 152–161, 2004.[CrossRef][ISI][Medline]
  2. Bogoyevitch MA, Andersson MB, Gillespie-Brown J, Clerk A, Glennon PE, Fuller SJ, and Sugden PH. Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy. Biochem J 314: 115–121, 1996.
  3. Boluyt MO, Long X, Eschenhagen T, Mende U, Schmitz W, Crow MT, and Lakatta EG. Isoproterenol infusion induces alterations in expression of hypertrophy-associated genes in rat heart. Am J Physiol Heart Circ Physiol 269: H638–H647, 1995.[Abstract/Free Full Text]
  4. Bonow RO. Aortic regurgitation. Curr Treat Options Cardiovasc Med 2: 125–132, 2000.
  5. Bristow MR. Mechanism of action of beta-blocking agents in heart failure. Am J Cardiol 80: 26L–40L, 1997.[CrossRef][Medline]
  6. Devereux RB and Roman MJ. Left ventricular hypertrophy in hypertension: stimuli, patterns, and consequences. Hypertens Res 22: 1–9, 1999.[ISI][Medline]
  7. Eichhorn EJ and Bristow MR. Medical therapy can improve the biological properties of the chronically failing heart. A new era in the treatment of heart failure. Circulation 94: 2285–2296, 1996.[Abstract/Free Full Text]
  8. Esler M, Kaye D, Lambert G, Esler D, and Jennings G. Adrenergic nervous system in heart failure. Am J Cardiol 80: 7L–14L, 1997.[CrossRef][Medline]
  9. Harrison BC, Roberts CR, Hood DB, Sweeney M, Gould JM, Bush EW, and McKinsey TA. The CRM1 nuclear export receptor controls pathological cardiac gene expression. Mol Cell Biol 24: 10636–10649, 2004.[Abstract/Free Full Text]
  10. Herron TJ, Korte FS, and McDonald KS. Loaded shortening and power output in cardiac myocytes are dependent on myosin heavy chain isoform expression. Am J Physiol Heart Circ Physiol 281: H1217–H1222, 2001.[Abstract/Free Full Text]
  11. Iwanaga Y, Kihara Y, Inagaki K, Onozawa Y, Yoneda T, Kataoka K, and Sasayama S. Differential effects of angiotensin II versus endothelin-1 inhibitions in hypertrophic left ventricular myocardium during transition to heart failure. Circulation 104: 606–612, 2001.[Abstract/Free Full Text]
  12. Kinugawa K, Yonekura K, Ribeiro RC, Eto Y, Aoyagi T, Baxter JD, Camacho SA, Bristow MR, Long CS, and Simpson PC. Regulation of thyroid hormone receptor isoforms in physiological and pathological cardiac hypertrophy. Circ Res 89: 591–598, 2001.[Abstract/Free Full Text]
  13. Kono T, Sabbah HN, Rosman H, Alam M, Jafri S, and Goldstein S. Left ventricular shape is the primary determinant of functional mitral regurgitation in heart failure. J Am Coll Cardiol 20: 1594–1598, 1992.[Abstract]
  14. Lader AS, Xiao YF, Ishikawa Y, Cui Y, Vatner DE, Vatner SF, Homcy CJ, and Cantiello HF. Cardiac Gsalpha overexpression enhances L-type calcium channels through an adenylyl cyclase independent pathway. Proc Natl Acad Sci USA 95: 9669–9674, 1998.[Abstract/Free Full Text]
  15. Lompre AM, Schwartz K, d'Albis A, Lacombe G, Van Thiem N, and Swynghedauw B. Myosin isoenzyme redistribution in chronic heart overload. Nature 282: 105–107, 1979.[CrossRef][Medline]
  16. Long CS, Hartogensis WE, and Simpson PC. Beta-adrenergic stimulation of cardiac non-myocytes augments the growth-promoting activity of non-myocyte conditioned medium. J Mol Cell Cardiol 25: 915–925, 1993.[CrossRef][ISI][Medline]
  17. Long CS, Henrich CJ, and Simpson PC. A growth factor for cardiac myocytes is produced by cardiac nonmyocytes. Cell Regul 2: 1081–1095, 1991.[ISI][Medline]
  18. Long CS, Kariya K, Karns L, and Simpson PC. Sympathetic modulation of the cardiac myocyte phenotype: studies with a cell-culture model of myocardial hypertrophy. Basic Res Cardiol 87, Suppl 2: 19–31, 1992.[CrossRef][ISI][Medline]
  19. Lowes BD, Gilbert EM, Abraham WT, Minobe WA, Larrabee P, Ferguson D, Wolfel EE, Lindenfeld J, Tsvetkova T, Robertson AD, Quaife RA, and Bristow MR. Myocardial gene expression in dilated cardiomyopathy treated with beta-blocking agents. N Engl J Med 346: 1357–1365, 2002.[Abstract/Free Full Text]
  20. Maass A, Grohe C, Kubisch C, Wollnik B, Vetter H, and Neyses L. Hormonal induction of an immediate-early gene response in myogenic cell lines–a paradigm for heart growth. Eur Heart J 16, Suppl C: 12–14, 1995.
  21. Mariner PD, Luckey SW, Long CS, Sucharov CC, and Leinwand LA. Yin Yang 1 represses alpha-myosin heavy chain gene expression in pathologic cardiac hypertrophy. Biochem Biophys Res Commun 326: 79–86, 2005.[CrossRef][ISI][Medline]
  22. McDonough PM, Stella SL, and Glembotski CC. Involvement of cytoplasmic calcium and protein kinases in the regulation of atrial natriuretic factor secretion by contraction rate and endothelin. J Biol Chem 269: 9466–9472, 1994.[Abstract/Free Full Text]
  23. Morisco C, Zebrowski DC, Vatner DE, Vatner SF, and Sadoshima J. Beta-adrenergic cardiac hypertrophy is mediated primarily by the beta(1)-subtype in the rat heart. J Mol Cell Cardiol 33: 561–573, 2001.[CrossRef][ISI][Medline]
  24. Patten M, Hartogensis WE, and Long CS. Interleukin-1beta is a negative transcriptional regulator of alpha1-adrenergic induced gene expression in cultured cardiac myocytes. J Biol Chem 271: 21134–21141, 1996.[Abstract/Free Full Text]
  25. Ponicke K, Heinroth-Hoffmann I, and Brodde OE. Role of beta 1- and beta 2-adrenoceptors in hypertrophic and apoptotic effects of noradrenaline and adrenaline in adult rat ventricular cardiomyocytes. Naunyn Schmiedebergs Arch Pharmacol 367: 592–599, 2003.[CrossRef][ISI][Medline]
  26. Rockman HA, Koch WJ, Milano CA, and Lefkowitz RJ. Myocardial beta-adrenergic receptor signaling in vivo: insights from transgenic mice. J Mol Med 74: 489–495, 1996.[CrossRef][ISI][Medline]
  27. Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P, Yang J, Antos CL, Shelton JM, Bassel-Duby R, Olson EN, and Williams RS. Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci USA 98: 3328–3333, 2001.[Abstract/Free Full Text]
  28. Schwarz B, Percy E, Gao XM, Dart AM, Richardt G, and Du XJ. Altered calcium transient and development of hypertrophy in beta2-adrenoceptor overexpressing mice with and without pressure overload. Eur J Heart Fail 5: 131–136, 2003.[CrossRef][ISI][Medline]
  29. Simpson P, McGrath A, and Savion S. Myocyte hypertrophy in neonatal rat heart cultures and its regulation by serum and by catecholamines. Circ Res 51: 787–801, 1982.[Abstract/Free Full Text]
  30. Simpson P and Savion S. Differentiation of rat myocytes in single cell cultures with and without proliferating nonmyocardial cells. Cross-striations, ultrastructure, and chronotropic response to isoproterenol. Circ Res 50: 101–116, 1982.[Free Full Text]
  31. Simpson PC, Kariya K, Karns LR, Long CS, and Karliner JS. Adrenergic hormones and control of cardiac myocyte growth. Mol Cell Biochem 104: 35–43, 1991.[ISI][Medline]
  32. Sucharov CC, Mariner P, Long C, Bristow M, and Leinwand L. Yin Yang 1 is increased in human heart failure and represses the activity of the human alpha-myosin heavy chain promoter. J Biol Chem 278: 31233–31239, 2003.[Abstract/Free Full Text]
  33. Vikstrom KL, Bohlmeyer T, Factor SM, and Leinwand LA. Hypertrophy, pathology, and molecular markers of cardiac pathogenesis. Circ Res 82: 773–778, 1998.[Abstract/Free Full Text]
  34. Wallukat G. The beta-adrenergic receptors. Herz 27: 683–690, 2002.[CrossRef][ISI][Medline]
  35. Wang W, Zhu W, Wang S, Yang D, Crow MT, Xiao RP, and Cheng H. Sustained beta1-adrenergic stimulation modulates cardiac contractility by Ca2+/calmodulin kinase signaling pathway. Circ Res 95: 798–806, 2004.[Abstract/Free Full Text]
  36. Waspe LE, Ordahl CP, and Simpson PC. The cardiac beta-myosin heavy chain isogene is induced selectively in alpha 1-adrenergic receptor-stimulated hypertrophy of cultured rat heart myocytes. J Clin Invest 85: 1206–1214, 1990.[ISI][Medline]
  37. Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE Jr,. Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, and Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nat Med 11: 409–417, 2005.[CrossRef][ISI][Medline]
  38. Zhang T and Brown JH. Role of Ca2+/calmodulin-dependent protein kinase II in cardiac hypertrophy and heart failure. Cardiovasc Res 63: 476–486, 2004.[Abstract/Free Full Text]
  39. Zhu W, Zou Y, Shiojima I, Kudoh S, Aikawa R, Hayashi D, Mizukami M, Toko H, Shibasaki F, Yazaki Y, Nagai R, and Komuro I. Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy. J Biol Chem 275: 15239–15245, 2000.[Abstract/Free Full Text]
  40. Zhu WZ, Wang SQ, Chakir K, Yang D, Zhang T, Brown JH, Devic E, Kobilka BK, Cheng H, and Xiao RP. Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. J Clin Invest 111: 617–625, 2003.[CrossRef][ISI][Medline]
  41. Zolk O, Marx M, Jackel E, El-Armouche A, and Eschenhagen T. Beta-adrenergic stimulation induces cardiac ankyrin repeat protein expression: involvement of protein kinase A and calmodulin-dependent kinase. Cardiovasc Res 59: 563–572, 2003.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Circ. Res.Home page
M. Metrich, A. Lucas, M. Gastineau, J.-L. Samuel, C. Heymes, E. Morel, and F. Lezoualc'h
Epac Mediates {beta}-Adrenergic Receptor-Induced Cardiomyocyte Hypertrophy
Circ. Res., April 25, 2008; 102(8): 959 - 965.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W. Mao, S. Fukuoka, C. Iwai, J. Liu, V. K. Sharma, S.-S. Sheu, M. Fu, and C.-s. Liang
Cardiomyocyte apoptosis in autoimmune cardiomyopathy: mediated via endoplasmic reticulum stress and exaggerated by norepinephrine
Am J Physiol Heart Circ Physiol, September 1, 2007; 293(3): H1636 - H1645.
[Abstract] [Full Text] [PDF]

Home page
 PhysiologyHome page
M. Kang, K. Y. Chung, and J. W. Walker
G-Protein Coupled Receptor Signaling in Myocardium: Not for the Faint of Heart
Physiology, June 1, 2007; 22(3): 174 - 184.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/3/H1299    most recent
00017.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sucharov, C. C.
Right arrow Articles by Bristow, M. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sucharov, C. C.
Right arrow Articles by Bristow, M. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.