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(Circulation. 1996;94:3303-3310.)
© 1996 American Heart Association, Inc.

Articles

Chronic Hypoxia Increases ß₁-Adrenergic Receptor mRNA and Density but Not Signaling in Neonatal Rat Cardiac Myocytes

Hong-Tai Li, MD; Norman Y. Honbo, MA; Joel S. Karliner, MD

the Cardiology Section, Veterans Affairs Medical Center; the Cardiovascular Research Institute; and the Department of Medicine, University of California, San Francisco.

Correspondence to Joel S. Karliner, MD, Cardiology Section (111C), Veterans Affairs Medical Center, 4150 Clement St, San Francisco, CA 94121. E-mail Karliner.Joel-S@SanFrancisco.VA.Gov.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background It is well recognized that the ß-adrenergic receptor–adenylylcyclase system is altered during myocardial ischemia/hypoxia. However, there are no data regarding either regulation of ß-adrenergic receptors, particularly at the mRNA level, or adenylylcyclase activity in isolated cardiac myocytes exposed to chronic hypoxia.

Methods and Results In a chronic hypoxia model in which neonatal rat ventricular myocytes were exposed to a 1% O₂ environment for 72 hours, we investigated (1) ß₁-mRNA and receptor expression and adenylylcyclase activity and (2) ß₁-mRNA and receptor downregulation and adenylylcyclase desensitization induced by prolonged norepinephrine incubation. We found that hypoxia for 72 hours increased myocardial membrane ß₁-adrenergic receptor density by 44%. This increase was not associated with a corresponding decrease in cytosolic ß₁-adrenergic receptors. RNase protection assays demonstrated that hypoxia increased the steady-state levels of ß₁-mRNA by 109%. Adenylylcyclase activity stimulated by isoproterenol, sodium fluoride, guanyl-5'-imidodiphosphate, and forskolin in hypoxic membranes was not altered compared with normoxic controls. Hypoxia for 72 hours also did not affect norepinephrine-induced ß₁-mRNA and receptor downregulation and adenylylcyclase desensitization in response to isoproterenol, guanyl-5'-imidodiphosphate, or forskolin.

Conclusions In neonatal rat cardiac myocytes, chronic hypoxia (1) increases ß₁-mRNA and receptor expression but does not alter adenylylcyclase activity stimulated at either the receptor or the postreceptor level and (2) does not affect agonist-induced ß₁-mRNA and receptor downregulation and desensitization of the adenylylcyclase response.

Key Words: receptors, adrenergic, beta • myocytes • hypoxia • norepinephrine • mRNA • signal transduction

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Adrenergic receptors play an important role in physiological responses related to cardiac contraction, hypertrophy, and excitability. Numerous studies in animal models have demonstrated that both ß₁- and ₁-ARs are extensively altered in myocardial ischemia.^{1 2 3 4 5 6 7 8} In intact animal models of chronic hypoxia, a decrease in myocardial ß-ARs has been reported.^{9 10 11 12 13} However, AR regulation in such models may result from both elevated endogenous catecholamine levels and ischemia/hypoxia itself. In vitro ischemia/hypoxia models with cultured cardiac myocytes have been used to examine the direct effect of ischemia/hypoxia on ARs. However, most studies focused on short-term hypoxia (<6 hours) and revealed conflicting results.^{14 15 16 17 18} To investigate whether hypoxia has any effect on catecholamine-induced cardiac hypertrophy, we recently developed an isolated chronic hypoxia model in which cultured neonatal rat cardiac myocytes are exposed to a 1% O₂ environment for 72 hours.¹⁹ We showed that NE-stimulated hypertrophy of cardiac myocytes is reduced during chronic hypoxia.¹⁹ We also found that ₁-AR subtypes are differentially regulated at both the mRNA and protein levels and that ₁-AR–mediated signaling is decreased during chronic hypoxia.¹⁹ However, it is not known whether chronic hypoxia has any effect on ß₁-mRNA and protein expression or on adenylylcyclase activity.

Downregulation of cardiac ß₁-ARs and desensitization of the adenylylcyclase response to agonists that act at both the receptor and the postreceptor levels have been consistent findings in failing human hearts (reviewed in References 20 and 21). These phenomena have been attributed to elevated circulating catecholamine levels in patients with heart failure^{20 21} and have been mimicked by in vivo isoproterenol infusion^{22 23} and prolonged incubation with NE or isoproterenol in cultured cardiac myocytes.^{24 25 26} However, it is not known whether hypoxia has any effect on catecholamine-induced downregulation of ß₁-ARs or desensitization of the adenylylcyclase response. Therefore, we designed the present study to determine first, whether chronic hypoxia regulates the expression of ß₁-mRNA and receptors or influences adenylylcyclase activity, and second, whether chronic hypoxia affects the ability of NE to downregulate the expression of ß₁-mRNA and receptors and to desensitize the adenylylcyclase response to agonist stimulation.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Primary cell cultures were composed of single isolated ventricular myocytes prepared from the hearts of 1-day-old rats as previously described.²⁷ Cells were plated onto 100-mm glass dishes at a final density of 100 to 150 cells/mm² after overnight incubation. The medium was supplemented with B₁₂ (1.5 µmol/L) and penicillin (50 U/mL). The medium was changed routinely on culture day 1 with medium containing 5% bovine calf serum and on day 4 with medium containing 10 µg insulin and 10 µg transferrin per milliliter. Through culture day 3, the medium also contained 0.1 mmol/L bromodeoxyuridine to prevent low-level nonmyocardial cell proliferation as previously reported.²⁷ Cell yield was 5 to 8 million per heart, of which 90% were viable. All cultures were kept at 37°C in humidified air with 1% CO₂ to maintain pH at 7.4. The cultures contained >90% myocardial cells, and cell numbers were constant over time.

Induction of Hypoxia
Chronic hypoxia was induced as previously described.¹⁹ Briefly, cells were maintained in an airtight, humidified, acrylic plastic perfusion chamber gassed with 98% N₂/1% CO₂/1% O₂ for 72 hours. In concurrent experiments, normoxic conditions were created by placing cultured cells in a Forma Scientific incubator gassed with 99% air/1% CO₂ at 37°C. Reoxygenation was accomplished by placing the hypoxic cells in normoxic conditions for 72 hours as described above. We have previously shown that the myocytes tolerate chronic exposure to the 1% O₂ environment without significant changes in lactate dehydrogenase release and ATP depletion.¹⁹

Radioligand Binding Assay
Saturation binding studies were performed as previously described in our laboratory.¹⁶ After incubation under normoxic, hypoxic, or reoxygenated conditions, dishes were immediately placed on ice and washed three times with 1 mmol/L Tris/2 mmol/L EGTA and lysed in situ in this hypotonic buffer for 20 minutes at 4°C. Brief exposure of hypoxic myocytes to room air when cells were taken out of the hypoxia chamber had no effect on the results. The resultant particulate preparation was harvested and centrifuged at 40 000g for 30 minutes. The pellet (membrane fraction) was resuspended in 50 mmol/L Tris HCl/1 mmol/L MgCl₂, pH 7.4, and stored at -70°C; the supernatant (cytosol fraction) of the original 40 000g spin was recentrifuged at 125 000g for 24 hours at 4°C. For assays, 80 µL of each cell fraction preparation was incubated with 10 µL of (±)¹²⁵I-ICYP at a final concentration ranging from 20 to 600 pmol/L in a total volume of 100 µL at 37°C in 12x75-mm polypropylene tubes for 30 minutes. Preliminary experiments indicated that the reaction reached equilibrium by 30 minutes at 200 pmol/L of ¹²⁵I-ICYP. Binding reactions were terminated by the addition of 5 mL of 50 mmol/L Tris HCl, pH 7.5, with subsequent filtration through glass fiber filters (Whatman GF/C). The test tubes and filters were washed with an additional 20 mL of 50 mmol/L Tris HCl. Filtering and washing required <30 seconds with use of a Brandel Harvester (Brandel Laboratories). Retained radioactivity was counted in a gamma counter at an efficiency of 73%. All determinations were performed in triplicate (membrane fractions) or duplicate (cytosolic fractions). For membranes, each tube contained 10 to 20 µg protein, and for cytosolic fractions, each tube contained 5 to 10 µg protein. Specific binding was defined as binding that could not be displaced by 1 µmol/L (-)-propranolol (final concentration) and ranged from 75% to 95%. The maximum number of ¹²⁵I-ICYP binding sites (Bmax) and the radioligand dissociation constant (K_d) were determined by Scatchard analysis.²⁸

Displacement binding experiments with ¹²⁵I-ICYP and various concentrations (100 pmol/L to 1 nmol/L) of the ß₁-AR selective antagonist betaxolol were performed to determine the relative proportion of ß₁/ß₂-ARs in our cardiac myocyte culture. Displacement binding experiments with ¹²⁵I-ICYP and various concentrations (1 nmol/L to 1 mmol/L) of (-)-isoproterenol were performed to determine the agonist affinity of ß-ARs in the presence and absence of 100 µmol/L GTP. The best two-site fit for each displacement binding curve was calculated by minimization of the sum of squares of the errors by nonlinear regression analysis.²⁹ Two-site and one-site models were compared to determine whether the increase in the goodness of the fit was significantly more than would be expected on the basis of chance alone, using a partial F test. A value of P<.05 was considered significant.

RNase Protection Assay
RNase protection assays were performed as previously described.¹⁹ Total cellular RNA was extracted from cells under normoxic, hypoxic, and reoxygenated conditions by the guanidinium thiocyanate method.³⁰ RNA concentration and purity were determined by measurement of absorbance at 260 and 280 nm with a spectrophotometer (model DU-65, Beckman Instruments, Inc).

Antisense RNA probes were labeled by incorporation of [-³²P]UTP into the RNA during transcription with T₇ or T₃ RNA polymerase by use of the Maxiscript kit (Ambion, Inc) and gel isolated after denaturing polyacrylamide electrophoresis. The DNA templates for the antisense RNA probes were as follows: for ß₁-AR, a fragment of rat ß₁-AR cDNA, including nucleotides 1952 to 2421³¹ ; for ß₂-AR, a fragment of rat ß₂-AR cDNA, including nucleotides 793 to 1052.³² Sizes of the probes/protected fragments were as follows: ß₁-AR, 534/469 bp, and ß₂-AR, 307/259 bp. The specificity of the ß₁-AR and ß₂-AR probes was verified by demonstration that no hybridization occurred between the ß₁-AR or ß₂-AR probe and the sense cRNA from ß₂-AR or ß₁-AR. A ß-actin antisense control template containing a 126-bp cDNA fragment of the rat ß-actin gene or an 18S RNA antisense control template containing 80 bp of a highly conserved region of the human ribosomal RNA gene (Ambion, Inc) was used to synthesize an antisense ß-actin or 18S RNA probe and assess RNA loading and quality. Probe sizes for ß-actin and 18S RNA were 180 and 109, respectively.

The RPAII kit (Ambion, Inc) was used for both hybridization and RNase digestion. Labeled probes (5x10⁵ cpm) were hybridized in solution with 15 to 50 µg of total RNA for 12 to 16 hours (58°C). The specific activity of probes was as follows: ß₁-AR and ß₂-AR, 1 to 2x10⁹ cpm/µg; ß-actin, 1 to 3x10⁷ cpm/µg; and 18S RNA, 4 to 8x10³ cpm/µg. Unhybridized single-stranded RNA was digested with RNase A and T₁ (1:40). Protected RNA-RNA hybrids were resolved on a 5% denaturing acrylamide gel and visualized by autoradiography. The hybridization signal for protected fragments was determined by counting the excised gel band in a scintillation counter.

Adenylylcyclase Activity
Adenylylcyclase activity was determined as previously described.¹⁶ Briefly, the assay mixture contained, in a final volume of 200 µL, 0.083 mmol/L [-³²P]ATP (1 to 2x10⁵ cpm), 1.7 mmol/L MgCl₂, 0.33 mmol/L cAMP, 0.42 mmol/L Tris-HEPES buffer (pH 7.5), 4.2 U creatine kinase, 11 mmol/L phosphocreatine, and 20 to 40 µg protein. Incubations were performed at 30°C for 30 minutes, and the results obtained were linear with protein concentration. The reaction reached a plateau by 30 minutes and was terminated by the addition of 300 µL of a solution containing 1 to 1.6x10⁴ cpm ³H-cAMP in 0.345 mol/L HCl and boiling for 4 minutes. Each sample containing 350 µL was applied to a column containing 2.6 g neutral alumina. Five milliliters of 0.1 mol/L ammonium acetate was added to each column, and then 13 mL of liquid scintillation cocktail (Universol, ICN Biomedicals) was added to each eluted sample. Recovery of added ³H-cAMP was 70% to 90%, and background counts were virtually zero.

Chemicals
¹²⁵I-ICYP (2200 mCi/mmol) was from NEN. [-³²P]ATP (3000 Ci/mmol) and [-³²P]UTP (800 Ci/mmol) were from Amersham. ³H-cAMP recovery marker (30 to 50 Ci/mmol) was from DuPont. Hexokinase and glucose-6-phosphate dehydrogenase were from Boehringer-Mannheim Corp. (-)-Propranolol was from RBI. All other reagents were from Sigma Chemical Co.

Data Analysis
All results are expressed as mean±SE and were compared by use of Student's t test for paired data between two groups and by ANOVA followed by Dunnett's test when more than two groups were analyzed. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Hypoxia Studies
ß₁-AR Density and Affinity
Neonatal rat ventricular myocytes, like adult rat myocytes, contain predominantly ß₁-ARs.^{33 34 35} To further characterize the ß₁- and ß₂-AR proportions in our myocyte cultures, we performed displacement binding experiments using ¹²⁵I-ICYP and a selective ß₁-antagonist, betaxolol. Betaxolol–¹²⁵I-ICYP displacement curves yielded a two-site fit in all three separately performed experiments. The relative proportion of high-affinity (K_d 167±3 nmol/L) and low-affinity sites (K_d 2.03±0.43 µmol/L) was 96±2% and 4±1%, respectively. Therefore, the ¹²⁵I-ICYP binding sites in the present study almost exclusively reflected ß₁-ARs. Representative equilibrium binding curves are shown in Fig 1. After 72 hours of hypoxia, myocardial membrane ß₁-AR density increased by 43% and antagonist K_d increased by 46% (Table 1). In the cytosol after chronic hypoxia, there was no significant change in either ß₁-AR density or antagonist K_d. After 72 hours of reoxygenation, myocardial membrane ß₁-AR density remained somewhat elevated, but the difference from control was no longer significant.

View larger version (19K): [in this window] [in a new window]   Figure 1. Representative equilibrium binding experiments. Data shown are equilibrium binding curves from a typical binding experiment that used125I-ICYP and membrane fractions of neonatal rat cardiac myocytes cultured under normoxic conditions () or after 72 hours of hypoxia (). Inset shows Scatchard transformation of these data.

View this table: [in this window] [in a new window]   Table 1. Effects of Chronic Hypoxia and Reoxygenation on ß1-AR Density

Displacement binding experiments with (-)-isoproterenol and ¹²⁵I-ICYP were performed to determine ß₁-AR agonist affinity. Chronic hypoxia shifted IC₅₀ rightward and induced a modest decrease in the proportion of ß₁-ARs in the high-affinity state (Table 2).

View this table: [in this window] [in a new window]   Table 2. Analysis of (-)-Isoproterenol Displacement Curves

ß₁-AR mRNA Levels
Similar to the radioligand binding results, our myocyte cultures contained predominantly ß₁-mRNA. Only a faint ß₂-mRNA signal was detected in myocyte cultures that contained 10% nonmyocytes compared with nonmyocyte cultures that contained almost exclusively ß₂-ARs and ß₂-mRNA (data not shown). Therefore, any ß₂-mRNA in our myocyte cultures was likely due to the presence of small numbers of nonmyocytes and was not studied in hypoxia experiments. The levels of ß₁-mRNA increased by 109% after 72 hours of hypoxia and tended to return to control values after 72 hours of reoxygenation (Fig 2).

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of chronic hypoxia on ß1-mRNA levels. ß1-mRNA levels were measured by RNase protection assays with total RNA from neonatal rat cardiac myocytes cultured under normoxic conditions (Nx 72 h and Nx 72 h/Rx 72 h), hypoxia (Hx 72 h), and reoxygenation (Hx 72 h/Rx 72 h). Top, Representative autoradiogram of an RNase protection assay. Shown is the gel region containing the specific protected fragments, indicated by the absence of a signal in the lane with tRNA. Sizes of the probes/protected fragments were 534/469 for ß1-AR and 109/80 bp for 18S RNA, respectively. Bottom, Summary of results of RNase protection assays. Data are shown as the ß1-mRNA signal measured by counting the excised gel band in a scintillation counter and expressed as a percentage of the level in normoxic cells (Nx 72 h). The data depicted are the mean±SE from eight separate experiments. *P<.05 vs Nx 72 h. Note that there was no significant change in the steady-state ß1-mRNA levels when cells were cultured under normoxic conditions (Nx 72 h vs Nx 72 h/Rx 72 h).

Adenylylcyclase Activity
Basal membrane adenylylcyclase activity was unchanged from control values after 72 hours of hypoxia. Isoproterenol-stimulated adenylylcyclase activity was measured in the presence of 100 µmol/L GTP and was not altered after 72 hours of hypoxia (Fig 3). Likewise, adenylylcyclase activity stimulated by agonists that act on G proteins (sodium fluoride and GppNHp) and on the catalytic subunit of adenylylcyclase (forskolin) also was not altered after 72 hours of hypoxia.

View larger version (37K): [in this window] [in a new window]   Figure 3. Effect of chronic hypoxia on adenylylcyclase activity stimulated by isoproterenol (ISO, 0.1 µmol/L), sodium fluoride (NaF, 100 µmol/L), GppNHp (100 µmol/L), and forskolin (100 µmol/L). Adenylylcyclase activity was assayed with the use of membranes from neonatal rat cardiac myocytes cultured under normoxic conditions (open bars) and after 72 hours of hypoxia (stippled bars). Isoproterenol-stimulated adenylylcyclase was determined in the presence of 100 µmol/L GTP, and data shown are the net isoproterenol responses after subtracting the activity stimulated by 100 µmol/L GTP alone. Data shown are the mean±SE for five separate experiments, each performed in triplicate. None of the changes in adenylylcyclase activity during chronic hypoxia were significant.

Hypoxia Studies in the Presence of NE
ß₁-AR Density and Affinity
We have previously shown that ß₁-ARs are downregulated by prolonged exposure to NE.²⁵ To determine whether chronic hypoxia altered ß₁-AR downregulation induced by prolonged exposure to NE, four groups of preparations were used, two with and two without NE under either normoxic or hypoxic conditions. During normoxia, NE incubation (1 µmol/L, 72 hours) decreased ß₁-AR density by 60.5±2.9% (Fig 4). During hypoxia, NE incubation induced a similar decrease (49.9±7.4%; P=NS versus normoxia). Like control cells incubated without NE, hypoxia also caused an increase in antagonist K_d in cells incubated with NE (108.7±6.0 versus 79.7±3.5 pmol/L, n=4; P<.01).

View larger version (18K): [in this window] [in a new window]   Figure 4. Effect of chronic hypoxia on downregulation of ß1-ARs after 72 hours of NE incubation. We measured ß1-ARs using 125I-ICYP in membranes from neonatal rat cardiac myocytes cultured in the presence of NE (1 µmol/L) or vehicle under normoxic (Nx) or hypoxic (Hx) conditions for 72 hours. Data shown are the mean±SE for four separate experiments, each performed in triplicate. Nx+NE indicates NE incubation during 72 hours of normoxia; Hx+NE, NE incubation during 72 hours of hypoxia. *P<.01 vs Nx; P<.01 vs Nx+NE.

ß₁-AR mRNA Levels
Because there were no previous data regarding the regulation of ß₁-mRNA levels by NE in neonatal rat cardiac myocytes, we first determined the ß₁-mRNA response to NE over time and found it to be biphasic (Fig 5). Incubation with NE for 1 hour increased the levels of ß₁-mRNA by 133%, whereas prolonged NE treatment (24 to 72 hours) caused a decrease of 31% to 38%. Both the increase (at 1 hour) and decrease (at 72 hours) in ß₁-mRNA induced by NE could be blocked by a selective ß₁-antagonist, betaxolol, but not by an ₁-antagonist, prazosin (Fig 6), suggesting that alterations in the ß₁-mRNA signal induced by NE are mediated through ß₁-ARs.

View larger version (41K): [in this window] [in a new window]   Figure 5. Effect of NE treatment on ß1-mRNA levels. ß1-mRNA levels were measured by RNase protection assays with total RNA from neonatal rat cardiac myocytes cultured in the presence of NE (1 µmol/L) or vehicle for 1 to 72 hours. Top, Representative autoradiogram of an RNase protection assay. Shown is the gel region containing the specific protected fragments, indicated by the absence of a signal in the lane with tRNA. Preliminary experiments showed that ß-actin mRNA levels were not altered by NE (1 µmol/L, 1 to 72 hours), and therefore the ß-actin probe was used as an internal control. The unequal ß-actin signal shown among different samples here was due to uneven loading of samples (see bottom graph for summary of results from four separate experiments). Sizes of the probes/protected fragments were 534/469 for ß1-AR and 180/126 bp for ß-actin, respectively. Bottom, Summary of results of RNase protection assays. Data are shown as the ß1-mRNA signal measured by counting the excised gel band in a scintillation counter and expressed as a percentage of the level in control cells under identical time points. The data depicted are the mean±SE from four separate experiments. *P<.05 vs control. There were no significant changes in ß1-mRNA levels in control cells from 1 to 72 hours.

View larger version (47K): [in this window] [in a new window]   Figure 6. Blockade of NE-induced changes in ß1-mRNA levels. Shown is a representative autoradiogram of RNase protection assay. ß1-mRNA was measured with total RNA from neonatal rat cardiac myocytes cultured for 1 or 72 hours under the following conditions: control; NE 1 µmol/L; NE with prazosin (PZN) 1 µmol/L; and NE with betaxolol (BTX) 1 µmol/L. Shown is the gel region containing the specific protected fragments, indicated by the absence of a signal in the lane with tRNA. Both the increase (1 hour) and the decrease (72 hours) in ß1-mRNA induced by NE treatment were blocked by betaxolol but not by prazosin. Similar results were obtained in two additional experiments.

To determine whether chronic hypoxia regulated the decrease in ß₁-mRNA levels induced by prolonged NE exposure, four groups of preparations were used, two with and two without NE under either normoxic or hypoxic conditions. During normoxia, NE incubation (1 µmol/L, 72 hours) decreased ß₁-mRNA levels by 36.7±3.7% (Fig 7). During hypoxia, NE incubation induced a similar decrease (43.3±5.9%).

View larger version (24K): [in this window] [in a new window]   Figure 7. Effect of chronic hypoxia on ß1-mRNA downregulation after 72 hours of NE incubation. ß1-mRNA levels were measured with the use of RNase protection assays with total RNA from neonatal rat cardiac myocytes cultured in the presence of NE (1 µmol/L) or vehicle under normoxic or hypoxic conditions for 72 hours. Top, Representative autoradiogram of an RNase protection assay. Shown is the gel region containing the specific protected fragments, indicated by the absence of a signal in the lane with tRNA. Bottom, Summary of results of RNase protection assays. Data are shown as the ß1-mRNA signal measured by counting the excised gel band in a scintillation counter and expressed as a percentage of the level in control normoxic cells (Nx). The data depicted are the mean±SE from five separate experiments. Nx indicates 72 hours of normoxia; Hx, 72 hours of hypoxia; Nx+NE, NE incubation during 72 hours of normoxia; and Hx+NE, NE incubation during 72 hours of hypoxia. *P<.01 vs Nx; P<.05 vs Nx+NE.

Desensitization of Adenylylcyclase Activity
We have previously shown that both isoproterenol- and GppNHp-stimulated adenylylcyclase activity is desensitized after prolonged incubation with NE in neonatal rat cardiac myocytes.²⁵ Similarly, as shown in Fig 8 (top), both isoproterenol- and GppNHp-stimulated adenylylcyclase activity were reduced after 72 hours of incubation with NE (1 µmol/L). In addition, forskolin-stimulated adenylylcyclase activity was desensitized after 72 hours of NE incubation. Hypoxia for 72 hours had no effect on NE-induced desensitization of isoproterenol-, GppNHp-, and forskolin-stimulated adenylylcyclase activity (Fig 8, bottom).

View larger version (34K): [in this window] [in a new window]   Figure 8. Effect of chronic hypoxia on desensitization of the adenylylcyclase response to isoproterenol (ISO, 0.1 µmol/L), GppNHp (100 µmol/L), and forskolin (100 µmol/L). Adenylylcyclase activity was assayed with the use of membranes from neonatal rat cardiac myocytes cultured in the presence of NE 1 µmol/L (stippled bars) or vehicle (open bars) under normoxic (top) and hypoxic conditions (bottom). Isoproterenol-stimulated adenylylcyclase was determined in the presence of 100 µmol/L GTP, and data shown are the net isoproterenol responses after subtracting the activity stimulated by 100 µmol/L GTP alone. Data shown are the mean±SE for nine (normoxia) and five (hypoxia) separate experiments, each performed in triplicate. *P<.05 and **P<.01 vs control membranes (without NE incubation).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The results of this study demonstrate that in neonatal rat cardiac myocytes, chronic hypoxia increases both ß₁-mRNA and receptor expression but does not affect adenylylcyclase activity stimulated at either the receptor or the postreceptor levels. Chronic hypoxia also has no influence on agonist-induced ß₁-mRNA and receptor downregulation or on desensitization of the adenylylcyclase response. These observations are in sharp contrast to the effects of chronic hypoxia on ₁-AR subtypes and signaling.¹⁹ The virtually opposite responses of ß₁- and ₁-AR systems summarized in Table 3 indicate that chronic hypoxia differentially regulates AR gene expression and signaling in neonatal rat ventricular myocytes. Our finding that the ß₁-adenylylcyclase cascade was preserved during chronic hypoxia also suggests that the ß₁-adenylylcyclase system may not mediate the reduced hypertrophic response to NE that occurs during chronic hypoxia in neonatal rat cardiac myocytes.¹⁹

View this table: [in this window] [in a new window]   Table 3. Differential Regulation of Adrenergic Subtype mRNAs, Receptors,* and Signaling by Prolonged NE Stimulation and Chronic Hypoxia

Our observations in cell culture differ from previous reports in whole-animal models.^{9 10 11 12 13} Exposure to chronic hypoxia for 2 to 5 weeks in rats decreased myocardial ß-AR density.^{9 10 11 12} In lambs subjected to 2 weeks of chronic hypoxia by means of a right-to-left shunt, left ventricular ß-ARs and ß₁-mRNA levels were reduced.^{13 36} Because these investigations were performed in intact animals, circulating catecholamine levels were elevated in response to chronic hypoxia.^{11 37} Thus, it is likely that the catecholamine response to hypoxia, rather than hypoxia per se, reduces ß-AR and mRNA levels in intact animals. Hypoxia and ischemia are related phenomena in which the energy metabolism is greatly impaired, and both play important roles in coronary heart disease. Our observation that chronic hypoxia increases ß₁-mRNA in the present study is consistent with a recent report in the isolated perfused heart³⁸ in which a rapid increase in ß₁-mRNA levels occurred during myocardial ischemia (15 to 60 minutes), an effect that was not blocked by ß-AR antagonists and may be due to ischemia itself.³⁸

In our previous report of acute hypoxia (0% O₂ for 1 to 2 hours), a decrease in membrane ß₁-AR density was associated with a concomitant rise in cytosolic ß₁-ARs.¹⁶ In the present study, we did not detect any change in the cytosolic ß₁-AR population after chronic hypoxia. We then hypothesized that the increased ß₁-ARs produced by chronic hypoxia could result from alterations at either a transcriptional or posttranscriptional level or both. Several observations suggest that ß₁-mRNA regulation plays an important role in ß₁-AR expression. First, reduced ß₁-ARs clearly correlate with reduced ß₁-mRNA levels.^{36 39 40 41 42 43} In rat C6 glioma cells, glucocorticoids downregulated both ß₁-AR density and mRNA.³⁹ In brown adipose tissue and heart of hypothyroid rats, both ß₁-AR density and mRNA were decreased.⁴⁰ Similar results were also obtained in hypoxic lamb,³⁶ failing porcine,⁴¹ and failing human ventricular myocardium.^{42 43} Second, increased ß₁-ARs correlate well with increased ß₁-mRNA levels.^{44 45} In the hearts of rats subjected to thyroxine intoxication, both ß₁-AR density and ß₁ were increased.⁴⁴ This observation was supported by the finding that in cultured neonatal rat ventricular myocytes, both ß₁-AR density and ß₁ were increased by thyroid hormone.⁴⁵ Thus, an increase in ß₁-mRNA expression may contribute to the increased membrane ß₁-AR density that occurs during chronic hypoxia.

Although myocardial membrane ß₁-AR density was increased after 72 hours of hypoxia, isoproterenol-stimulated adenylylcyclase activity was unchanged. These results suggest that ß₁-ARs are uncoupled from adenylylcyclase. Analysis of displacement binding curves showed that chronic hypoxia induced a small decrease in the proportion of ß₁-ARs in the high-affinity state. However, this decrease is so modest that it may not account for the apparent receptor-effector uncoupling after chronic hypoxia. Because adenylylcyclase activity stimulated by agonists that act on G proteins (sodium fluoride and GppNHp) and on the catalytic subunit of adenylylcyclase (forskolin) was unchanged after 72 hours of hypoxia, it seems unlikely that postreceptor alterations are responsible for the receptor-effector uncoupling. This is further supported by the observation that chronic hypoxia has no effect on agonist-induced desensitization of adenylylcyclase, a phenomenon in which inhibitory G proteins participate.^{22 23 26 46} A recent study⁴⁷ in failing porcine hearts has indicated that increased myocardial ß-AR kinase activity precedes ß-AR downregulation and correlates well with ß-AR uncoupling. It is possible that ß-AR kinases, which are increased in heart failure^{42 47 48} and which were not examined in the present study, may be increased during chronic hypoxia and contribute to ß₁-AR uncoupling.

Prolonged catecholamine incubation in neonatal rat cardiac myocytes has been proposed as an in vitro model for chronic heart failure, in which ß₁-ARs are downregulated and the adenylylcyclase response is desensitized.^{24 25 26} A recent study⁴⁹ reported that there are defects in multiple components of the membrane ß₁-adenylylcyclase system in the epicardial border zone of the 5-day infarcted canine heart. These abnormalities could result from either elevated endogenous catecholamine levels, ischemia/hypoxia, or both. Like surviving myocardial tissues in the epicardial border zone of the infarcted heart,⁴⁹ cardiac myocytes are still viable after 72 hours of hypoxia in our in vitro model.¹⁹ We reasoned that hypoxia might influence the downregulation of ß₁-ARs and desensitization of the adenylylcyclase response after prolonged incubation with NE. We found that hypoxia did not affect NE-induced ß₁-mRNA and receptor downregulation and either receptor-mediated (isoproterenol) or postreceptor-mediated (GppNHp and forskolin) desensitization of adenylylcyclase stimulation. This finding indicates that hypoxia itself has no effect on the desensitization of adenylylcyclase responses induced by prolonged NE incubation in this model.

The objective of the present study was to determine the direct effect of chronic hypoxia on the ß-AR–adenylylcyclase system in isolated neonatal rat cardiac myocytes. However, we recognize that our approach may have certain limitations, and caution should be taken in extrapolating our data to in vivo models of ischemia/hypoxia. The accumulation of metabolic products of ischemia, presynaptic innervation, and external workload on the intact heart undoubtedly influence the effects of ischemia on the individual myocyte. The use of neonatal cardiac myocytes may not reflect the situation in adult cells, although the ß-AR–adenylylcyclase system in the neonate has characteristics similar to that of the adult.³⁵ In addition, to reduce nonmyocyte contamination, we plated cells at low density. This may result in a relative loss of cell:cell contact and myocyte:nonmyocyte interaction. However, the present study demonstrates for the first time that chronic hypoxia directly alters the expression of ß₁-AR mRNA and receptors both in the presence and absence of agonist (NE) in isolated cardiac myocytes. The marked differences in mRNA, receptor, and signaling between ß₁- and ₁-ARs indicate that chronic hypoxia differentially regulates adrenergic responses in cardiac myocytes.

	Selected Abbreviations and Acronyms

 

AR = adrenergic receptor Bmax = maximum number of binding sites GppNHp = 5'-guanylylimidodiphosphate ICYP = iodocyanopindolol Kd = radioligand dissociation constant NE = norepinephrine

	Acknowledgments

 
This work was supported by program project grant HL-25847 from the National Heart, Lung, and Blood Institute and the Research Service, Department of Veterans Affairs. We are grateful to Dr Mark W. Hamblin for providing the ß₁-AR cDNA clone and Dr Claire Frazer for providing the ß₂-AR cDNA clone. We thank Dr Mary Gray, Trini Miguel, and Jim Roberts for assistance with the adenylylcyclase experiments.

	Footnotes

 
Presented in part at the 66th Scientific Sessions of the American Heart Association, Atlanta, Ga, November 7-11, 1993, and the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 11-16, 1995, and published in abstract form (Circulation. 1993;88[suppl I]:I-571; 1995;92[suppl I]:I-236).

Received June 25, 1996; accepted August 6, 1996.

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