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(Circulation. 1995;92:918-925.)
© 1995 American Heart Association, Inc.

Articles

Chronic Hypoxia Differentially Regulates ₁-Adrenergic Receptor Subtype mRNAs and Inhibits ₁-Adrenergic Receptor–Stimulated Cardiac Hypertrophy and Signaling

Hong-Tai Li, MD; Carlin S. Long, MD; D. Gregg Rokosh, PhD; Norman Y. Honbo, MA; Joel S. Karliner, MD

From 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.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background After myocardial ischemia and/or infarction, surviving cardiac myocytes in and around the injured zone develop hypertrophy to compensate for the loss of contractile units due to myocyte injury and death. One of the factors that may be involved in the development of hypertrophy after ischemic injury is norepinephrine (NE), an agent that induces hypertrophy of cardiac myocytes through the ₁-adrenergic receptor (AR). It is not known, however, whether hypoxia, a major component of ischemia, has any direct effect on NE-stimulated hypertrophy. Therefore, we sought to determine whether chronic hypoxia could alter NE-stimulated hypertrophy and if so, whether this alteration was related to ₁-AR–mediated signaling and ₁-AR changes at both the protein and mRNA levels.

Methods and Results We developed a model of chronic hypoxia in cultured neonatal rat cardiac myocytes in which myocytes were exposed to 1% oxygen for 72 hours. Initially, we observed that chronic hypoxia inhibited NE-stimulated hypertrophy, as reflected by decreases in both new protein synthesis and total protein content during chronic hypoxia. Then we found that chronic hypoxia also inhibited ₁-AR–transduced phosphatidylinositol hydrolysis, as indicated by a reduction in ₁-AR–stimulated inositol phosphate production in hypoxic cells. These observations suggested that the inhibition of NE-stimulated hypertrophy seen during chronic hypoxia was due to impairment of ₁-AR–mediated signaling and could result from changes in ₁-AR numbers and/or subtype distribution. To address this issue, we determined ₁-AR density and subtype distribution by radioligand binding and ₁-AR subtype mRNAs, including _1A/D-, _1B-, and _1C-ARs, by RNase protection assays. We found that chronic hypoxia differentially regulated both the pharmacologically defined ₁-AR subtypes and the mRNAs for the ₁-AR subtypes. Thus, hypoxia for 72 hours coordinately downregulated both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level. During normoxia, NE increased the pharmacologically defined _1A-AR density and the _1C-AR mRNA level, but hypoxia for 72 hours prevented these NE-mediated changes.

Conclusions Chronic hypoxia (1) inhibits ₁-AR–mediated hypertrophy of cardiac myocytes and ₁-AR–transduced phosphatidylinositol hydrolysis and (2) downregulates both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level.

Key Words: receptors, adrenergic, alpha • hypoxia • hypertrophy • myocytes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Myocardial ischemia or infarction leads to cardiac myocyte injury and loss. In an attempt to maintain cardiac pump function, surviving myocytes in and around the injured zone display hypertrophic growth.^{1 2} However, this hypertrophy may be inadequate to compensate for the loss of contractile units, thus leading to cardiac failure. One of the factors that may be involved in the development of hypertrophy after ischemic injury is the naturally occurring neurotransmitter norepinephrine (NE), an agent that has been shown to induce hypertrophy of cardiac myocytes both in vivo and in culture.³ Previous studies in neonatal rat cardiac myocytes have revealed that NE induces hypertrophy through the ₁-adrenergic receptor (AR), an effect that may be mediated by downstream elements in the signaling pathway activated by phosphatidylinositol (PI) hydrolysis.^{4 5 6 7} A major factor influencing the extent of cell injury that occurs both during and after myocardial ischemia and infarction is hypoxia, defined as a low circulating PO₂. Although short-term hypoxia has been reported to enhance ₁-AR–mediated responses,^{8 9 10} it is not clear whether chronic hypoxia alters NE-stimulated hypertrophy of cardiac myocytes, and if so, whether this effect is related to an alteration in ₁-AR–mediated signaling.

Changes in NE-stimulated hypertrophy and ₁-AR–mediated signaling seen with hypoxia could result from alterations in ₁-AR numbers and/or subtype distribution. Recently, three ₁-AR subtypes have been cloned, including the _1A/D-, _1B-, and _1C-ARs.^{11 12 13 14 15 16 17 18} Therefore, the previous pharmacological classification of only two subtypes (_1A and _1B) fails to describe all of the ₁-AR subtypes in tissues. Both _1A/D- and _1B-AR mRNAs have been detected in rat heart by Northern blotting with cDNA probes.¹⁵ More recently, all three subtype mRNAs have been demonstrated in both adult and neonatal rat cardiac myocytes by a sensitive RNase protection assay.^{18 19} However, it is not known whether chronic hypoxia regulates these ₁-AR subtype mRNAs.

The present study was designed to determine, first, whether chronic hypoxia alters NE-stimulated hypertrophy and if so, whether this alteration is related to ₁-AR–mediated signaling; and second, whether chronic hypoxia regulates ₁-AR density, subtype distribution, and levels of ₁-AR subtype mRNAs. To answer these questions, we developed a model of chronic hypoxia in cultured neonatal rat cardiac myocytes, in which cells were exposed to a 1% oxygen environment for 72 hours. Using this well-characterized model, which permits the study of cell growth and signaling in a homogeneous myocyte population not exposed to exogenous catecholamines and other modulations that may alter in vivo responses,^{4 5 6 8 20 21} we determined the effects of chronic hypoxia on (1) ₁-AR–stimulated hypertrophy and signaling and (2) ₁-AR density, subtype distribution, and levels of ₁-AR subtype mRNAs. Our data indicate that in neonatal rat cardiac myocytes, chronic hypoxia (1) inhibits ₁-AR–mediated hypertrophy and ₁-AR–transduced PI hydrolysis and (2) selectively downregulates both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
Primary cultures were composed of single isolated ventricular myocytes prepared from the hearts of 1-day-old rats as previously described.²⁰ Cells were plated onto 60-mm glass dishes for measurements of cell growth and inositol phosphates (IPs) or 100-mm glass dishes for radioligand binding and RNase protection assays. Cells attached at a final density of 100 to 150 cells/mm² after overnight incubation. The medium was supplemented with 1.5 µmol/L vitamin B₁₂ and 50 U/mL penicillin. The medium was changed routinely on culture day 1 to serum-free medium containing 10 µg/mL insulin and 10 µg/mL transferrin. Through culture day 3, the medium also contained 0.1 mmol/L bromodeoxyuridine to prevent nonmyocardial cell proliferation, as previously reported.²⁰

Cell yield was 5 to 7 million cells per heart, of which 90% were viable. All cultures were kept at 37°C in humidified air with 1% CO₂ to maintain pH 7.4. The cultures contained >90% myocardial cells, and cell numbers were constant over time.

Induction of Hypoxia
To produce chronic hypoxia, cells were maintained in an airtight humidified Plexiglas gas perfusion chamber gassed with 98% N₂/1% CO₂/1% O₂ for 72 hours. Gas samples were obtained via an outlet port directly into a Fyrite Gas Analyzer (United Technologies). Oxygen concentration was routinely monitored during incubation to verify that the oxygen remained between 1.0% and 1.4%, corresponding to ambient PO₂ of 7.1 to 10.0 mm Hg. 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.

To evaluate the characteristics of this chronic hypoxia model, a number of variables were measured, including cell number, intracellular ATP content, medium LDH activity, medium glucose content, and pH as previously described.²¹

Growth Measurement
Protein was measured by two methods, as previously described.^{5 6} For the first method, asymptotic labeling with [¹⁴C]phenylalanine was used to determine new protein synthesis. [¹⁴C]Phenylalanine (0.1 µCi/mL) and experimental agents or vehicle were added to the cells immediately before exposure to the normoxic or hypoxic environment as described above. At the end of the experiment, cell protein, defined as material that was trichloroacetic acid–insoluble and SDS-soluble, was taken for liquid scintillation counting. For the second method, the spectrophotometric method of Bradford was used to determine total cell protein content.²² Cells were rinsed with PBS and dissolved in 0.1% SDS at 37°C. Duplicate aliquots were taken for assay, with BSA as standard.

Determination of PI Hydrolysis
Total IPs were measured by anion exchange chromatography as previously described in our laboratory.^{6 8} In summary, cells were labeled with 1 µCi/mL [³H]myoinositol in either the presence or absence of 1 µmol/L NE just before placement in the hypoxia chamber or in the normoxia incubator. After 72 hours of normoxia or hypoxia, cells were rinsed twice with serum-free medium to remove NE and incubated in the same medium supplemented with 20 mmol/L LiCl for 10 minutes under normoxic conditions. NE with 1 µmol/L (-)-propranolol, serum, carbachol, or vehicle was then added and incubated for 1 hour. The incubation was terminated by aspiration of the medium and addition of 1 mL of 10% trichloroacetic acid with 1 mmol/L EDTA and 1 mmol/L unlabeled inositol at 4°C. The contents were collected by scraping with a rubber spatula and sonicated for 15 seconds. After centrifugation at 2000g for 10 minutes, the trichloroacetic acid was extracted by five washes with ether. [³H]IPs were eluted with 1 mol/L formic acid plus 1 mol/L ammonium formate in Dowex 1x8 anion-exchange columns (formate form). Preliminary experiments showed that [³H]myoinositol equilibrated at 48 hours and maintained a steady level at 72 hours under both normoxic and hypoxic conditions.

Radioligand Binding Study
Saturation binding studies were performed as previously described in our laboratory.⁸ After incubation under normoxic or hypoxic conditions in the presence or absence of 1 µmol/L NE, dishes were washed with minimal essential medium (MEM) with Hanks' balanced salt solution (BSS) and incubated in this medium for 10 minutes, then washed three times with ice-cold MEM with Hanks' BSS and one time with PBS and scraped with harvesting buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, and 5 mmol/L EDTA, pH 7.5). The cell mixture was centrifuged at 100 000g for 1 hour at 4°C. The pellet was resuspended in harvesting buffer and sonicated for 15 seconds. The membrane preparation then was incubated in triplicate with varying concentrations of 2-[ß-(4-hydroxy-3-[¹²⁵I]iodophenyl)-ethylaminomethyl] tetralone (¹²⁵I-HEAT) (10 to 700 pmol/L) in a total volume of 110 µL containing 70 000 to 100 000 cells at 37°C for 30 minutes. Preliminary experiments indicated that the reaction reached equilibrium by this time point. Radioactivity was determined in a gamma counter at a counting efficiency of 73%. Nonspecific binding was determined in the presence of the nonselective ₁-AR antagonist prazosin (1 µmol/L). Specific binding was defined as the difference between total binding and nonspecific binding and ranged between 60% and 90%. The maximum number of binding sites (B_max) and equilibrium binding constant (K_d) were determined by least-squares linear regression analysis using the method of Scatchard.²³ Preliminary experiments showed that <2% of binding sites were retained in the supernatant from a 100 000g pellet.

Displacement binding experiments using ¹²⁵I-HEAT and various concentrations of the _1A subtype–selective antagonist 5-methylurapidil were performed to determine ₁-AR subtype distribution using a protocol for the saturation binding reaction identical to that described above. The best two-site fit for each binding curve was calculated by minimization of the sum of squares of the errors by nonlinear regression analysis.²⁴ Two-site and one-site models were compared to determine whether the increase in the goodness of 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
Total cellular RNA was extracted from cells under normoxic or hypoxic conditions by the guanidinium thiocyanate method.²⁵ 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 RNA polymerase by use of the Maxiscript kit (Ambion, Inc) and gel-isolated after denaturing polyacrylamide electrophoresis. The DNA templates for antisense RNA probes were as follows: for _1A/D, a fragment of rat brain _1A/D-cDNA, including nucleotides 2025 to 2241¹⁵ ; for _1B, a fragment of an _1B-cDNA obtained from a rat thyroid epithelial cell line (FRTL) cDNA library,¹⁹ corresponding to nucleotides 1002 to 1259¹³ ; and for _1C, a fragment of rat cardiac _1C-cDNA,¹⁹ corresponding to nucleotides 770 to 1084 of the human _1C-subtype.¹⁷ Sizes of the probes/protected fragments were as follows: _1A/D, 267/217; _1B, 321/257; and _1C, 408/305. A GAPDH antisense RNA probe (Ambion, Inc) was used to assess RNA loading and quality. The sizes of probe and protected fragment of GAPDH were 208 and 150 bp, respectively.

The RPAII kit (Ambion, Inc) was used for both hybridization and RNase digestion. Labeled probes (5x10⁵ to 10x10⁵ cpm) were hybridized in solution with 20 to 40 µg of total RNA for 12 to 16 hours (42°C to 45°C). 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. In all experiments, in vitro–transcribed ₁-AR subtype sense cRNAs served as a positive control and tRNA as a negative control. The specificity of the ₁-AR antisense probes was verified by demonstration that no hybridization occurred between each ₁-AR probe and the sense cRNAs from the other ₁-AR subtypes. The hybridization signal for protected RNA fragments was quantified by counting of the excised gel band in a scintillation counter.

Chemicals
[³H]Myoinositol (16.5 mCi/mmol), [¹⁴C]phenylalanine (464 mCi/mmol), ¹²⁵I-HEAT (2200 Ci/mmol), and [-³²P]UTP (800 Ci/mmol) were obtained from Amersham. Prazosin, 5-methyl-urapidil, and (-)-propranolol were from RBI. (-)-NE, carbachol, and all other reagents were from Sigma Chemical Co.

Statistical Analysis
All data are expressed as mean±SEM. Comparison of numerical data was by the Student's t test for paired observations between two groups and by ANOVA followed by the Dunnett test when more than two groups were analyzed. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Characteristics of the Chronic Hypoxia Model
To determine whether chronic hypoxia in this model adversely affected cardiac myocyte survival, we made a number of observations indicating that the myocytes tolerated chronic exposure to the 1% O₂ environment. After 72 hours of hypoxia, except for a modest decline in medium glucose and pH (in the presence of NE), there were no significant changes in cell counts, LDH activity in the medium, and intracellular ATP content (Table 1).

View this table: [in this window] [in a new window]   Table 1. Characteristics of the Chronic Hypoxia Model

Influence of Chronic Hypoxia on (-)-NE–Stimulated Hypertrophy of Cardiac Myocytes
We next asked whether chronic hypoxia would alter ₁-AR–stimulated hypertrophy. We chose to examine the effects of the naturally occurring neurotransmitter NE. Previous work has shown that NE stimulates hypertrophy of neonatal rat cardiac myocytes through an ₁-AR.^{4 5} This hypertrophy was attenuated during 72 hours of hypoxia, with a 25% reduction in [¹⁴C]phenylalanine incorporation into cardiac myocytes stimulated by 1 µmol/L NE (Fig 1). Basal levels of [¹⁴C]phenylalanine in normoxic and hypoxic cells did not differ (6661±492 versus 6890±508 cpm/60-mm dish). In cells incubated with NE, total cell protein content as measured by the Bradford method was also reduced by chronic hypoxia (treated/control [T/C] ratio, 1.62±0.12 versus 1.23±0.02; n=8, P=.01). In contrast, serum-stimulated hypertrophy was unchanged during 72 hours of hypoxia, with no alteration in [¹⁴C]phenylalanine incorporation into cardiac myocytes stimulated by 1% bovine calf serum (equipotent to 1 µmol/L NE, Fig 1). In addition, total protein content in cells incubated with serum was also unchanged by 72 hours of hypoxia (T/C ratio, 1.64±0.14 versus 1.72±0.08, n=6).

View larger version (42K): [in this window] [in a new window]   Figure 1. Bar graph showing that chronic hypoxia blunts norepinephrine (NE)-stimulated cardiac myocyte hypertrophy. Data shown are fold increases in [14C]phenylalanine incorporation stimulated by NE (1 µmol/L) or serum (1% bovine calf serum) under normoxic (open bars) and hypoxic (stippled bars) conditions for 72 hours. Data are expressed as mean±SEM. n indicates number of experiments from separate cultures, each performed in triplicate. *P<.01 vs normoxia.

Influence of Chronic Hypoxia on ₁-AR–Mediated PI Hydrolysis
Total IPs were used as a marker for PI hydrolysis. Previous studies in our laboratory using anion-exchange chromatography have shown that IPs are increased through the ₁-AR in response to NE treatment.⁶ Regardless of whether cells were incubated with or without NE (1 µmol/L), after 72 hours of hypoxia, ₁-AR–stimulated IP production was reduced (31% in cells incubated with NE and 25% in cells without NE, Fig 2). The basal levels of IPs did not differ in normoxic and hypoxic cells incubated with (107.4±17.6 versus 102.4±9.3 dpm/µg protein) or without (117.4±13.7 versus 118.0±18.3 dpm/µg protein) NE.

View larger version (48K): [in this window] [in a new window]   Figure 2. Bar graph showing effect of chronic hypoxia on agonist-stimulated inositol phosphate generation. Cells were incubated with norepinephrine (NE) (+NE 72 h) or without NE (-NE 72 h) for 72 hours under normoxic (open bars) and hypoxic (stippled bars) conditions. Medium was then changed, and cells were incubated with agonist (NE, carbachol, or serum) for 1 hour. Total inositol phosphates were extracted by anion exchange chromatography (described under "Methods"). Data are expressed as mean±SEM. n indicates number of experiments from separate cultures, each performed in duplicate. *P<.01 vs cells without NE incubation under normoxic conditions; P<.01 vs cells with NE incubation under normoxic conditions.

To determine whether the decrease in IP generation stimulated by NE after chronic hypoxia was attributable to substrate depletion or to other nonspecific effects, we also compared the production of IPs stimulated by carbachol or serum between normoxic and hypoxic cells. Carbachol, which does not induce cell growth in our neonatal heart cell cultures (data not shown), stimulates PI hydrolysis via a muscarinic cholinergic receptor.²⁶ Regardless of whether cells were incubated with or without NE (1 µmol/L, 72 hours), there was no change in carbachol-stimulated IP accumulation after chronic hypoxia (Fig 2). Serum, which contains many undefined growth factors, also stimulates PI hydrolysis in our cell system. Similar to the results seen with carbachol, there was no significant change in 10% bovine calf serum–stimulated IP accumulation (equipotent to 1 µmol/L NE) after chronic hypoxia compared with normoxia (Fig 2). Taken together, these observations suggest that chronic hypoxia depresses ₁-AR–mediated PI hydrolysis.

Effects of Chronic Hypoxia on ₁-AR Density, Affinity, and Subtype Distribution
Since the decrease in NE-stimulated IP generation after chronic hypoxia could involve either a receptor or postreceptor mechanism, we next examined ₁-AR density, affinity, and subtype distribution in our system. To determine whether chronic hypoxia altered ₁-AR density and affinity, we performed equilibrium radioligand binding experiments. Four groups of preparations were used, two with NE incubation (1 µmol/L) and two without NE incubation (control cells) under either normoxic or hypoxic conditions for 72 hours. In control cells, after 72 hours of hypoxia, there was no change in either ₁-AR density or antagonist affinity (Table 2). During normoxia, NE incubation (1 µmol/L, 72 hours) had no effect on either ₁-AR density or antagonist affinity. However, during hypoxia, NE incubation decreased ₁-AR density without changing antagonist affinity (Table 2).

View this table: [in this window] [in a new window]   Table 2. Comparison of Total or Subtype 1-ARs Between Normoxic and Hypoxic Cells With or Without NE Incubation

To determine whether chronic hypoxia altered ₁-AR subtype distribution, we performed five separate displacement experiments using a highly selective _1A-AR antagonist, 5-methylurapidil.²⁷ In normoxic cardiac myocytes, a relative subtype distribution of 32.6±4.0% _1A and 67.4±4.0% _1B was calculated from the ratio of high/low affinity sites for 5-methylurapidil. This ratio is similar to that found in whole adult or neonatal rat myocardium.^{27 28 29} In control cells, after 72 hours of hypoxia, the _1A-AR proportion decreased (to 25.8±4.7%, P<.05 versus normoxia) and the _1B-AR proportion increased reciprocally. Using the high/low affinity site proportions, we calculated B_max values for _1A and _1B subtypes by multiplying the percentage of each site by the total B_max for ₁-AR measured in the equilibrium binding studies (Table 2). In control cells, after 72 hours of hypoxia, _1A-AR density was reduced by 18%, while _1B-AR density was increased by 14% (both P<.05). During normoxia, NE (1 µmol/L, 72 hours) raised _1A-AR density markedly, by 62%, while _1B-AR density was decreased by 44% (both P<.05). However, during hypoxia, NE failed to raise _1A-AR density but was still able to cause a decrease in _1B-AR density.

Effects of Chronic Hypoxia on ₁-AR Subtype mRNA Levels
The changes in ₁-AR density and subtype distribution produced by chronic hypoxia described above could result from alterations at either a transcriptional or posttranscriptional level or both. However, in contrast to the two pharmacologically defined ₁-AR subtypes, three ₁-AR subtype mRNAs have been described in rat cardiac myocytes.^{17 18} Therefore, we next asked whether ₁-AR subtype mRNA levels could be regulated by chronic hypoxia. We used RNase protection assays to measure ₁-AR subtype mRNA levels in total RNA extracted from normoxic or hypoxic cells incubated with or without NE for 72 hours. Representative autoradiograms from RNase protection assays using _1C, _1A/D, and GAPDH probes or _1B and GAPDH probes in the same reaction are shown in Fig 3. In control cells (without NE), after 72 hours of hypoxia, _1C-AR mRNA level was reduced by 37%, while _1B and _1A/D mRNA levels were unchanged (Fig 4). During normoxia, NE (1 µmol/L, 72 hours) increased the _1C level by 80% and decreased the _1B level by 57% and the _1A/D level by 56%. However, during hypoxia, although NE was able to decrease _1B and _1A/D mRNA levels to the same levels as those during normoxia, it failed to increase _1C-mRNA level (Fig 4).

View larger version (40K): [in this window] [in a new window]   Figure 3. Representative autoradiograms of RNase protection assays. Total RNA (20 µg) from a cardiac myocyte culture under various experimental conditions was hybridized with [-32P]UTP–labeled antisense 1C, 1A/D, and GAPDH probes in the same reaction (A) and 1B and GAPDH in the same reaction (B). Nx indicates 72 hours of normoxia; Hx, 72 hours of hypoxia; Nx+NE, norepinephrine (NE) incubation (1 µmol/L) during 72 hours of normoxia; and Hx+NE, NE incubation (1 µmol/L) during 72 hours of hypoxia. As shown in the lanes marked "Control" at the top, only one band was visualized when RNA was hybridized with a single probe. The 1A/D signal in lane Nx+NE was overshadowed by the background from the strong 1C signal. In separate experiments (n=4), the 1A/D signal was decreased by NE during normoxia when RNA was hybridized only with 1A/D and GAPDH probes (see Fig 4 for summary). Exposure time was 12 hours for 1C–, 1A/D–, and 1B–adrenergic receptor signals and 1 hour for GAPDH.

View larger version (23K): [in this window] [in a new window]   Figure 4. Bar graph showing regulation of 1–adrenergic receptor (AR) subtype mRNA levels by chronic hypoxia. 1-AR subtype mRNA levels were measured by RNase protection assays in neonatal rat cardiac myocytes cultured for 72 hours under the following conditions: normoxia (Nx); hypoxia (Hx); normoxia with 1 µmol/L norepinephrine (NE) (Nx+NE); and hypoxia with 1 µmol/L NE (Hx+NE). GAPDH was used to normalize 1-AR subtype mRNA signals in all assays. Data are shown as the mRNA signals for each subtype quantified by counting the excised gel band in a scintillation counter, normalized to GAPDH, and expressed as a percentage of the level in normoxic cells. The data depicted are the mean±SEM from seven or eight experiments. *P<.05 vs normoxia; P<.05 vs normoxia with 1 µmol/L NE.

Since NE is a mixed agonist that binds to both ₁- and ß-ARs, its regulation of ₁-AR subtype mRNAs could result from stimulation of either ₁-AR, ß-AR, or both. To answer this question, we examined the effects of prazosin, an ₁-AR antagonist, and (-)-propranolol, a ß-AR antagonist, on the regulation of ₁-AR subtype mRNAs by NE. After 72 hours, both the increase in _1C and the decrease in _1B and _1A/D caused by NE were blocked by 1 µmol/L prazosin but were not altered by 1 µmol/L (-)-propranolol (Fig 5). These observations indicate that NE regulates ₁-AR subtype mRNA levels through ₁-ARs.

View larger version (30K): [in this window] [in a new window]   Figure 5. Bar graph showing regulation of 1-AR subtype mRNA levels by norepinephrine (NE). 1-AR subtype mRNA levels were measured by RNase protection assays in neonatal rat cardiac myocytes cultured for 72 hours under the following conditions: control; NE (1 µmol/L); NE with propranolol (Prop, 1 µmol/L); and NE with prazosin (Praz, 1 µmol/L). Data are shown as the mRNA signals for each subtype quantified by counting the excised gel band in a scintillation counter, normalized to GAPDH, and expressed as a percentage of the level in control cells. The data depicted are the mean±SEM from three experiments. *P<.05 vs control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We used a new model of chronic hypoxia to determine the effects of this intervention on the ₁-AR system in cultured neonatal rat cardiac myocytes. The major findings of our study indicate that chronic hypoxia (1) inhibits ₁-AR–mediated hypertrophy of cardiac myocytes and ₁-AR–transduced PI hydrolysis and (2) selectively downregulates both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level.

Stimulation of cardiac ₁-ARs in vivo induces both positive inotropic and electrophysiological changes.^{30 31} Because of these important physiological effects, the role and regulation of ₁-ARs in acute myocardial ischemia or hypoxia have been extensively studied. Increased ₁-ARs on the cell surface enhance ₁-AR–mediated responses in ischemic myocardium and may lead to decreased electrophysiological stability of the heart. Thus, ₁-AR antagonists have been found to reduce the incidence of ventricular arrhythmias in animal models of ischemia.^{31 32 33} In addition, chronic stimulation of cardiac ₁-ARs leads to cardiac hypertrophy in vivo and to cardiac myocyte enlargement in culture.³ Conversely, long-term treatment with ₁-AR antagonists leads to regression of cardiac hypertrophy in guinea pig hearts with pressure overload, cardiomyopathic hamsters, and spontaneously hypertensive rats.^{34 35 36} However, there is little information on the role and regulation of ₁-ARs in myocardial hypertrophy during chronic myocardial ischemia/hypoxia. In an in vivo rat model, chronic coronary arterial stenosis was found to inhibit ₁-AR–mediated signaling and myocardial growth.³⁷ However, these results may reflect the combined influences of ischemia, decreased catecholamine levels, and cardiac sympathetic stimulation.

In this study, we determined the direct effects of chronic hypoxia, a major element of ischemia, on ₁-AR–mediated hypertrophy of cardiac myocytes in a relatively homogeneous cell population. The decreased hypertrophic response of cardiac myocytes to ₁-AR stimulation during chronic hypoxia in this model is consistent with the results reported in an in vivo model of chronic myocardial ischemia in the rat.³⁷ However, the reduced hypertrophic response seen in our culture system appears to be specific to the ₁-AR. In contrast, several other models of myocyte hypertrophy in culture such as serum, angiotensin II,³⁸ and nonmyocyte conditioned medium (C.S.L. and J.S.K., unpublished data) are not altered by chronic hypoxia. These findings suggest that ₁-AR–mediated hypertrophy may be selectively impaired by chronic hypoxia and lead to the speculation that a reduction in the ₁-AR–mediated hypertrophic response may play a critical role in the myocardial dysfunction seen after myocardial ischemia/hypoxia.

In this study, we also found that the production of IPs stimulated by ₁-AR was reduced after chronic hypoxia. This observation is similar to the report that a decrease in NE-stimulated PI hydrolysis occurs in a chronically ischemic rat model^{37 39} and suggests that the decreased ₁-AR–mediated hypertrophy may result from a decrease in ₁-AR–mediated PI hydrolysis. Several observations support the hypothesis that ₁-AR–induced PI hydrolysis is closely related to cardiac myocyte growth. First, the activation of phospholipase C is not sensitive to inhibition by pertussis toxin, although a pertussis toxin substrate is abundant in our cells.⁶ Similarly, ₁-AR–induced hypertrophy is not inhibited by pertussis toxin.⁶ Second, ₁-AR can activate protein kinase C by acting on phospholipase C, as measured by both the translocation of protein kinase C activity and immunoreactivity.^{40 41} Third, selective _1A-AR antagonists such as WB-4101, 5-methylurapidil, and (+)-niguldipine, which can block ₁-AR agonist–induced PI hydrolysis,^{7 42 43} also block ₁-AR agonist–induced hypertrophy of cardiac myocytes,^{7 29} whereas the _1B-AR antagonist chloroethylclonidine does not affect either ₁-AR–mediated PI hydrolysis or hypertrophy.^{7 29}

Of note, the observations described in this report using a chronic hypoxia model contrast with our previous report using more severe acute hypoxia, in which ₁-AR–stimulated PI hydrolysis was augmented after 1 hour but returned to control by 2 hours of hypoxia.⁸ Steinberg and Alter¹⁰ noted in a neonatal rat ventricular myocyte preparation similar to ours that augmented ₁-AR–stimulated IP generation occurred at 0.5 hour and still persisted at 6 hours of hypoxia. Similarly, Heathers et al⁹ reported an enhanced inositol triphosphate response to ₁-AR stimulation in adult canine heart cells exposed to hypoxia for 10 minutes. However, none of these studies exposed cardiac myocytes to hypoxia for longer than 6 hours. To the best of our knowledge, this study is the first to examine ₁-AR–mediated PI hydrolysis in cardiac myocytes exposed to hypoxia for 72 hours. The disparate effects of acute and chronic hypoxia on ₁-AR–mediated PI hydrolysis may indicate a change in cellular responses during chronic hypoxia, although differences in experimental models may also be responsible for the different results.

The relation between the cloned ₁-AR cDNAs and pharmacologically defined ₁-AR subtypes is not yet completely understood. The biochemical, signaling, and functional properties of expressed _1B-AR are identical to its pharmacologically defined counterpart found in various tissues.¹¹ Two virtually identical cDNA clones that encode another rat ₁-AR subtype have been independently isolated from rat cerebral cortex and rat hippocampus and designated _1A- and _1D-ARs, respectively.^{14 15} The term _1A/D has been designated to represent this subtype.⁴⁴ In contrast to the findings with the _1B-AR, the properties of the expressed receptor encoded by the cloned _1A/D-AR differ from the pharmacologically defined _1A-AR.^{14 15} However, the antagonist affinities of the cloned _1C-AR match very well with those expected for the pharmacologically defined _1A-AR,^{45 46 47 48 49 50} and the distribution of _1C mRNA in rat tissue corresponds to the distribution of pharmacologically defined _1A-AR.^{19 50 51 52} These observations suggest that the cloned _1C-AR corresponds to the pharmacologically defined _1A-AR. In further support of this possibility, we found that both NE and chronic hypoxia had a similar influence on the _1C-AR mRNA and on the pharmacologically defined _1A-AR. Specifically, NE increased both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level, whereas chronic hypoxia decreased both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level and prevented NE-stimulated increases in both pharmacologically defined _1A-AR density and the _1C-AR mRNA level. The data regarding the effects of chronic hypoxia on ₁-AR subtypes at both mRNA and protein levels are summarized in Table 3.

View this table: [in this window] [in a new window]   Table 3. Differential Regulation of 1-AR Subtypes at Both mRNA and Receptor1 Levels

In contrast to parallel decreases between the _1C-AR mRNA level and the corresponding pharmacologically defined _1A-AR density after chronic hypoxia, the modest (14%) but significant increase in the pharmacologically defined _1B-AR density after chronic hypoxia was not accompanied by an increase in the _1B-AR mRNA level. It should be recognized that the available pharmacological antagonists are not sufficiently selective to identify the three distinct cloned receptor subtypes. For example, 5-methylurapidil can differentiate the cloned _1C-AR from the cloned _1B- and _1A/D-ARs but cannot differentiate between the latter two cloned subtypes.^{49 51 52} Thus, the _1B-AR density determined by 5-methylurapidil displacement experiments in this study may represent not only the cloned _1B-AR subtype but also the cloned _1A/D-AR subtype, which was also not significantly changed at the mRNA level during chronic hypoxia. Also, we cannot exclude the possibility that hypoxia may modulate posttranscriptional regulation of _1B- and/or _1A/D-AR genes.

Since the pharmacologically defined _1A-AR but not the _1B-AR is believed to mediate hypertrophy and PI hydrolysis in cardiac myocytes,^{7 29} it is possible that the cloned _1C-AR is the subtype mediating ₁-AR–stimulated hypertrophy and PI hydrolysis in cardiac myocytes. Our novel findings that chronic hypoxia selectively downregulated the pharmacologically defined _1A-AR density and the _1C-AR mRNA level suggest that chronic hypoxia may inhibit ₁-AR–stimulated hypertrophy and signaling, at least in part, by reducing the pharmacologically defined _1A-AR density and the _1C-AR mRNA level in cardiac myocytes.

As mentioned earlier, impairment of ₁-AR–stimulated hypertrophy and PI hydrolysis produced by chronic hypoxia in cultured neonatal rat cardiac myocytes is consistent with the observations in an in vivo rat model with coronary arterial stenosis (7 days and 6 months).^{37 39} In addition, similar to our results that total ₁-AR density was decreased in hypoxic cells incubated with NE, total ₁-AR density was decreased after either 7 days or 6 months of coronary arterial stenosis.^{37 39} In contrast to our study, in which mRNA levels for _1A/D- and _1B-ARs were unchanged by chronic hypoxia, after 7 days of coronary arterial stenosis, _1A/D- and _1B-AR mRNA expression was decreased (_1C-AR mRNA was not examined).³⁹ It has been shown that ischemia may induce a local release of endogenous catecholamines, mainly NE.^{53 54} It is possible that decreases in _1A/D- and _1B-AR mRNA expression in the ischemic rat model result from the increased catecholamine level caused by ischemia.

In summary, the present study demonstrates, for the first time, that in cultured neonatal rat cardiac myocytes, chronic hypoxia selectively downregulates both the pharmacologically defined _1A-AR density and the _1C-AR mRNA level, which may contribute to the impaired ₁-AR–mediated hypertrophy and PI hydrolysis seen during chronic hypoxia. Further clarification of the role and regulation of ₁-AR subtypes should shed further light on ₁-AR–mediated signaling pathways and their relation to hypertrophy in cardiac myocytes under both physiological and pathological circumstances.

	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 Paul Simpson for providing the ₁-AR cDNA clones and critical comments.

Received December 19, 1994; revision received February 19, 1995; accepted February 22, 1995.

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