Analysis of beta-adrenergic receptor mRNA levels in human ventricular biopsy specimens by quantitative polymerase chain reactions: Progressive reduction of beta1-adrenergic receptor mRNA in heart failure

146

JACC Vol. 27, No. 1 January 1996:146-54

Analysis of Beta-Adrenergic Receptor mRNA Levels in Human Ventricular Biopsy Specimens by Quantitative Polymerase Chain Reactions: Progressive Reduction of Betal-Adrenergic Receptor mRNA in Heart Failure S T E F A N E N G E L H A R D T , M I C H A E L B O H M , MD,* E R L A N D E R D M A N N , MD,* M A R T I N J. L O H S E , M D

Martinsried, Wiirzburg and Cologne, Germany

Objectives. This study investigated the relation between the severity of heart failure and the extent of the reduction of betal-adrenergic receptor messenger ribonucleic acid (mRNA) levels in biopsy specimens from the ventricular septum obtained during cardiac catheterization of patients with various degrees of heart failure. Background. Heart failure is accompanied by desensitization of the beta.adrenergic receptor system, which is in part due to downregulation of betal-adrenergic receptors. Downregulation of betat-adrenergic receptors has been suggested to be caused by reductions in mRNA levels. Methods. Because biopsy specimens were small and receptor mRNAs not abundant, mRNA levels were determined by quantitative reverse transcription/polymerase chain reactions. This method was validated by measuring synthetic ribonucleic acid (RNA) standards and samples from explanted hearts by solution hybridization assays. Both methods yielded similar results, but the polymerase chain reaction method was -1,000-fold more

sensitive. Sources of variations in the polymerase chain reaction were quantitated and found to be best controlled for by determination of the glyceraldehyde phosphate dehydrogenase mRNA as an endogenous control. Results. Betal-adrenergic receptor mRNA levels in the biopsy specimens were decreased by 7% in mild (New York Heart Association functional class II), 26% in moderate (functional class III) and >50% in severe heart failure (functional class IV). There was a good correlation between hemodynamic indicators of heart failure and betal-adrenergic receptor mRNA levels. In contrast, beta2-adrenergic receptor mRNA levels were apparently unaffected by heart failure. Conclusions. Reduced betal-adrenergic receptor mRNA levels occur early in heart failure and can be detected in septal biopsy specimens during right heart catheterization. The reduction in betat-adrenergic receptor expression may contribute to further loss of cardiac function. (J Am Coil Cardiol 1996;27:146-54)

The failing human heart shows a decreased responsiveness to beta-adrenergic receptor agonists, which may contribute to the loss of cardiac contractility (1-5). This decrease in responsiveness of the cardiac beta-adrenergic receptor system appears to be due in part to a reduction in the number of beta 1-adrenergic receptors, the predominant subtype in the human heart (1-6). The sympathetic nervous system responds to heart failure with an increased activity (7-9), resulting in increased levels of catecholamines at postsynaptic sites as well as in the systemic circulation and, thus, in enhanced stimulation of adrenergic receptors. The reduction in cardiac betal-adrenergic receptor

levels may therefore be a consequence of the increased stimulation of these receptors by norepinephrine released from the sympathetic nerves. In agreement with this hypothesis, beta-adrenergic receptor downregulation can be induced by chronic volume overload, which is also accompanied by increased sympathetic activity (10). A variety of mechanisms regulate the function and number of beta-adrenergic receptors (reviewed in Lohse [11]). Probably the most important mechanism causing long-term agonistinduced desensitization is the reduction of receptor number ("downregulation") caused by reduced levels of the corresponding messenger ribonucleic acid (mRNA) (11). This mechanism has been studied in quite some detail for the beta2-adrenergic receptor and has been attributed to a reduced mRNA stability (12-16). We have recently begun to investigate (17,18) whether such alterations in mRNA levels might be the basis of the reduced levels of betal-adrenergic receptors in heart failure. These studies were done on explanted hearts whose function was either severely impaired (New York Heart Association class IV) or was essentially normal (donor hearts). In severe heart

From the Laboratorium ftir Molekulare Biologic der Universitfit Mflnchen, M~x-Planck-Institut flit Biochemie, Martinsried and Institut fiir Pharmakologie und Yoxikologie der Universitfit Wfirzburg, Wtirzburg; and *Klinik III ftir Innere Medizin der Universit~t K61n, Cologne, Germany. This study was supported by grants from the Deutsche Forschungsgemeinschaft, Bonn, Germany (Drs. Lohse and B6hm) and the Fonds der Chemischen Industrie, Frankfurt, Germany (Dr. Lohse). Manuscript received January 17, 1995; revised manuscript received May 26, 1995, accepted August 10, 1995. Address for correspondence: Dr. Martin J. Lohse, Institut ffir Pharmakologie und Yoxikologie, Versbacher Strasse 9, 97078 Wiirzburg, Germany. 01996 by the American Collegc of Cardiology

0735-1097/96/$15.00 I)735-1097(95)00425-4

JACC Vol. 27, No. 1 January. 1996:146-54

E N G E L H A R D T Err AL. B E T A - A D R E N E R G I C R E C E P T O R MRNA IN V E N T R I C U L A R BIOPSY SPECIMENS

failure the betal-adrenergic receptor mRNA levels were decreased by -50%, whereas betaz-adrenergic receptor mRNA levels were unaltered (17). These findings closely paralleled the expression of the two receptors as measured by radioligand binding. Our data were confirmed by Bristow et al. (19), who in addition determined the absolute levels of beta-adrenergic receptor mRNAs. There are several reasons why it appears important to extend such studies to biopsy specimens from patients. 1) Trauma and complex treatment may affect the adrenergic receptor system in both failing and donor heart explants (20,21); 2) There is a significant interval between explantation and freezing, particularly for donor hearts; and 3) studies on explants show results at the two extremes of cardiac function: presumably healthy donor hearts and severely failing recipient hearts. However, the intermediate stages of heart failure appear equally important for drawing conclusions about the relations between the observed effects and the development of cardiac dysfunction. Recent studies in pacing-induced heart failure in dogs (22) suggested that the downregulation of betal-adrenergic receptors might be an early event in the development of heart failure, and that, therefore, this downregulation might be a causal factor in the further deterioration of cardiac function. In humans, the degree of beta-adrenergic receptor downregulation correlates with the degree of heart failure in patients with idiopathic dilated cardiomyopathy or mitral valve disease (23,24). Single myocardial biopsy specimens are too small to study beta-adrenergic receptors by radioligand binding or their mRNA levels by conventional techniques. The only technique available to investigate changes in a number of proteins in a single biopsy specimen is the determination of the respective mRNA levels by reverse transcription/polymerase chain reactions (25). This exquisitely sensitive method is prone to several artifacts and problems of standardization (17,25-30). Therefore, we set out to validate and standardize the polymerase chain reaction method by the parallel use of solution hybridization assays and attempted to use these methods to measure the mRNA levels of the beta1- and betaz-adrenergic receptor in biopsy specimens obtained during cardiac catheterization of patients with various degrees of contractile dysfunction.

Methods Patients and biopsy procurement. Biopsy specimens from the interventricular septum were obtained by right heart catheterization from a series of patients with different stages of heart failure New York Heart Association class 0 to IV. The studies had been approved by the ethical committee of the medical faculty, and all patients gave written informed consent before undergoing cardiac catheterization. Because of ethical considerations, catheterization was performed only in patients with suspected myocarditis as the clinical indication for this procedure. Several biopsy specimens were examined histopatbologically, and if no evidence for myocarditis was found,

147

a single specimen held at -80°C as a reserve was used for the present study. The clinical data of these patients are given in Table 1. Their overall cardiac function ranged from normal to severe impairment due to dilated cardiomyopathy or other causes. The clinical classification according to New York Heart Association criteria was made the day before catheterization independently by two experienced staff cardiologists. Determination of functional capacity by treadmill exercise was contraindicated in all patients in view of suspected myocarditis. Hemodynamic data obtained during cardiac catheterization are given in Table 1 for all patients whose left ventrieular function was assessed. In addition to these data, the diagnoses were based on clinical examinations, chest X-ray films and echocardiography. No patient received catecholamines or beta-adrenergic receptor antagonists. The biopsy specimens (-3 to 5 mg wet weight) were placed in liquid nitrogen immediately after removal and kept at -80°C. Two experienced physicians (M.B., E.E.) performed the cardiac catheterization in all patients; the method of tissue procurement was the same for all biopsies. Ribonucleic acid preparation and reverse transcription. Ribonucleic acid (RNA) from the frozen tissue samples was prepared by a shortened version of the protocol of Chomczinski and Sacchi (31) essentially as described (17,18). The final RNA pellet was dried and then dissolved in 10/~1 water. The purity, checked by measuring the ratio of the absorbance at 260 and 280 nm, was 1.8 to 2.0 in all cases. The amount of RNA obtained varied between 0.7 and 5 ~g/biopsy. Five hundred milligrams of this RNA was reverse transcribed into complementary deoxyribonucleic acid (cDNA) by using random hexamers and Superscript II reverse transcriptase (Gibeo) as described (17,18). In all experiments, separate reactions containing no reverse transcriptase were done as controls. Quantitative polymerase chain reactions. Sense and antisense oligonucleotide primer pairs were synthesized to match the sequences of the human beta 1- (32) and beta 2- (33) adrenergic receptors and human glyceraldehyde phosphate dehydrogenase (34). Details about the primers and the expected polymerase chain reaction products are given in Table 2. Polymerase chain reactions were done with the transcript obtained from 50 ng RNA (i.e., 10% of the reverse transcribed cDNA). The assay contained 0.5 ~mol/liter of the respective primers, 1.25 U Thermus aquaticus polymerase (Boehringer), 200 ~mol/liter deoxynucleotides plus 0.3 tzCi of [alpha-32p]deoxycytidine triphosphate (Amersham), 1.5 retool/ liter magnesium chloride, 50 retool/liter potassium chloride, 10 retool/liter Tris-hydrochloride, pH 8.3, in a volume of 50 ~1. Amplifications were done in a Perkin-Elmer model 480 thermal cycler with denaturation at 94°C for 1 rain (3 rain in the first cycle), annealing for 1 rain at the temperatures indicated in Table 2 and an extension at 72°C for 1 rain (10 min in the last cycle). Multiple samples from different biopsy specimens were assayed for each gene by using a single master reaction mixture. The reverse transcription/polymerase chain reaction products were isolated by vertical agarose gel electrophoresis;

148

ENGELHARDT ET AL. BETA-ADRENERGIC RECEPTOR MRNAIN VENTRICULAR BIOPSY SPECIMENS

JACC Vol. 27, No. 1 January 1996:I46-54

Table 1. Clinical Data From Cardiac Catheterization of Patients With Different Stages of Heart Failure Pt No. NYHA 0-I 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean SEM NYHA 11 14 15 16 17

18 19 20 Mean SEM NYHA Ill 21 22 23 24 25 Mean SEM NYHA IV 26 27 28 29 Mean SEM

Age (yr)/ Gender

LVEDP (ram Hg)

LVEDV (ml)

EF (%)

33/F 47/F 32/F 53/M 23/M 33/M 19/M 37/F 47/M 21/F 19/F 47/F 34/M 32 4.(1

14

136

11

148

44/F 65/M 53/M

Diagnosis

10

125

6 10 16 20

272* 200* 110 150

16

123

72 68 72 80 70 80 70 53 49

--

--

--

--

--

--

NF

--

--

--

NF

--

NF

7

--

202*

--

NF NF Beginning DCM NF Beginning DCM Beginning DCM NF Beginning DCM NF NF

12.2

162.9

1.5

17.4

68.2 3.6

57/M 21/F 46/M 49 5.4

14 ll 8 8 15 --11.2 1.5

282 301 200 300 100 --236.6 38.9

45 59 50 30 75* --51.8 7.5

DCM DCM DCM DCM Restrictive cardiomyopatby DCM DCM

45/M 25/M 20/F 57/M 48/M 39 7.1

18 24 -24 4 17.5 4.7

201 366 -289 300 289 33.9

32 48 -26 36 35.5 4.6

DCM DCM DCM DCM DCM

49/M 39/F 39/M 17/F 36 6.8

25 25 8 -19.3 5.7

300 52 257 -203 76.5

10 66* 75 20 42.8 16.3

DCM Restrictive cardiomyopatby Mitral valve regurgitation DCM

57/M

*Postextrasystolic beats. DCM = dilated cardiomyopathy; EF = ejection fraction; F = female; LVEDP = left ventricular end-diastolic pressure; LVEDV = left ventricular end-diastolic volume; M = male; NF = nonfailing; NYHA = New York Heart Association functional class; -- = data not available.

after excision their phosphorus-32 content was determined by Cerenkov counting. Background radioactivity present in empty lanes was subtracted from all data. In all experiments, a template-free control R N A template, not subjected to reverse transcription, and known amounts of plasmids containing the respective sequences were amplified to monitor the accuracy and efficiency of the polymerase chain reaction method. Synthesis of RNA standards. Sense and antisense R N A for the human beta t- and beta2-adrenergic receptor was transcribed from vectors that were generated by cloning the full length c D N A of the human beta t- (32) or beta2-adrenergic

receptor (33) (sense R N A ) or a 1,140-base pair SacI-EcoRV (beta2) or a 627-base pair NotI-ApaI (betal) fragment (radiolabeled antisense R N A ) behind the T7 p r o m o t e r in the vector pGEM-9Zf (Promega). Unlabeled R N A was transcribed from 1 /~g of appropriately linearized vector with 10 U of T 7 - R N A polymerase (Promega), 30 U of RNAsin (Promega), 1 retool/liter nucleotides, 10 mmol/liter DTT, 2 retool/liter spermidine, 6 mmol/liter magnesium chloride, 10 retool/liter sodium chloride in 50/zl of 40 mmol/liter Tris-hydrochloride, p H 7.9, during 1 h at 37°C. Subsequently the deoxyribonucleic acid ( D N A ) was digested with 1 U of R Q 1 - D N a s e at 37°C for 30 rain. For the generation of radiolabeled antisense R N A , the

JACC Vol. 27, No. 1 January' 1996:146-54

ENGELHARDT ET AL. BETA-ADRENERGIC RECEPTOR MRNA IN VENTRICULAR BIOPSY SPECIMENS

149

Table 2. Sequences of Forward and Reverse OligonuclcotidePrimers Specificfor Human Sequences of Betaj- and Beta2-Adrenergic Receptors and Glyceraldehyde-3-PhosphatcDehydrogenase

mRNA /3~AR /32AR GAPDH

Primer 5' 5' 5' 5' 5' 5'

CTC A c e AAC CTC TTC ATe ATG 3' GAA ACG GCG CTC GCA G e T G 3' CCT CCT AAA "ITG GAT AGG 3' AGT CTG T I T AGT GTT CTG 3' GeT T I T AAC TCT GGT AAA GTG G 3' T e A CGC CAC AGT TTC CCG GAG G 3'

Position in Coding Sequence (base pairs)

Orientation

272 794 925 1,295 63 592

Forward Reverse Forward Reverse Forward Reverse

Annealing Temp.

Distance (base pairs)

(oc)

522

58

370

50

529

58

Annealing Temp. = temperature of the annealing step in the respective polymerase chain reaction process;/31AR, ,82AR - beta 1- and beta2-adrenergic receptors; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; mRNA = messenger ribonueleic acid. The positions of the primers in the coding sequence of the respective messenger ribonucleic acids, forward or reverse orientation and the length of the generated polymerase chain reaction products (Distance) are indicated. Template sequences were from Ref. 32 to 34.

transcription was done in the presence of 50/.tCi of [alpha32p]cytidine triphosphate and 0.25 retool/liter each of adenosine triphosphate, guanosine triphosphate and uridine triphosphate. The RNA was then isolated by gel filtration on push columns followed by polyacrylamide gel electrophoresis according to standard methods (35). The RNA concentration was determined photometrically and from the incorporated radioactivity. Solution hybridization assays. Levels of mRNA were quantitated by a solution hybridization/RNase protection method adapted from Hellmann et al. (36). In brief, 50/xg of total RNA prepared from human heart tissue or known amounts of beta l- or betaz-adrenergic receptor mRNA were incubated with 10 pg of labeled antisense RNA (-20,000 cpm) in 40/zl of 40% formamide, 1 mmol/liter ethylenediaminetetraacetic acid, 40 retool/liter Pipes, pH 6.7, and 400 mmol/liter sodium chloride. The probe was allowed to hybridize at 68°C for 18 h. Unhybridized single-stranded probe was then digested by incubation with RNase A (40 tzg/ml) and RNase T1 (2 t~g/ml) for 1 h at 30°C. After the addition of denatured salmon sperm DNA (50 ttg/ml), RNA-RNA hybrids were precipitated by the addition of 10% trichloroacetic acid, recovered by filtration over Whatman GF/C filters, washed with 30 ml of 7.5% trichloroacetic acid and then quantitated by Cerenkov counting. Statistical analysis. Data are expressed as mean value _+ SEM. Comparison between different groups was performed by one-way analysis of variance followed by the Scheff6 procedure for multiple comparisons. In addition, these data were analyzed for linear trends by using the InStat program.

transcriptase (Fig. 1) or the RNA samples (not shown). This observation is important because the genes of both receptors contain no introns, and thus contamination of the RNA preparations with genomic DNA would give the same polymerase chain reaction products. Such controls were done with all polymerase chain reactions reported in this study. Figure 2 shows the amplification characteristics that were obtained after reverse transcription of the mRNAs for the betal-adrenergic receptor (Fig. 2A) and glyceraldehyde phosphate dehydrogenase (Fig. 2B). The amount of polymerase chain reaction product increased exponentially up to 28 cycles in the case of glyceraldehyde phosphate dehydrogenase, and up to 36 cycles in the case of the betal-adrenergic receptor. The efficiency of amplification was calculated from the slope of the respective curves and was 48.7% (glyceraldehyde phosphate dehydrogenase) or 47.3% (betal-adrenergic receptor). This similarity in amplification efficiency is a prerequisite for the use of glyceraldehyde phosphate dehydrogenase as an endogenous standard. In analogous experiments, the amplification efficiency for the beta2-adrenergic receptor polymerase

Figure 1. Reversetranscriptiou/polymerasechainreaction productsgenerated from total heart ribonucleicacidfor the betal-adrenergicreceptor (/3jAR), glyceraldehydephosphate dehydrogenase (GAPDH), and beta2-adrenergic receptor (/32AR).To the right of each specificlane is the respective control (C) in which the reverse transcriptase was omitted. Molecularweight(MW) markerswere loaded on the left and right sides of the gel, and the sizes of the relevant markers are indicated. The polymerase chain reaction products were of the expected size (see Table 2). bp = base pairs.

Results Quantification of RNA. In an initial set of experiments, the preparation of RNA and the reverse transcription/polymerase chain reactions were optimized to allow the quantitation of multiple RNAs in a single biopsy specimen. Figure 1 shows that each of the potymerase chain reactions yielded a single specific product of the expected size, and that no such product was obtained in control samples lacking either the reverse

653 bp X_

J

653 bp

517 bp - ~ 453 bp

517 bp ~ 453 bp ~ MW

B1AR C B2AR C GAPDH C

MW

150

E N G E L H A R D T ET AL. B E T A - A D R E N E R G I C R E C E P T O R MRNA IN V E N T R I C U L A R BIOPSY SPECIMENS

JACC Vol. 27, No. 1 January 1996:146-54

Figure 2. Amplification of the

A E"

polymerase chain reaction (PCR) product for the betal-adrenergic receptor (/31AR) (A) and glyceraldehyde phosphate dehydrogenase (GADPH) (B). After reverse transcription of 500 ng of total heart ribonucleic acid, the polymerase chain reactions were done with 10% of the resulting cDNA for the indicated number of cycles. The polymerase chain reaction products were resolved by agarose gel electrophoresis (left panels), and the incorporated radioactivity was quantitated by excision of the bands and Cerenkov counting (right). Amplification dficiency (E) was calculated according to Chelly et al. (30) and was 47.3% for the betal-adrenergic receptor product and 48.7% for the glyceraldehyde phosphate dehydrogenase product, respectively. Data are expressed as mean value + SEM (not shown when smaller than symbol size) from three independent experiments, bp = base pairs.

10000

_ f 6 5 3 bp ~517

RIAR , ~ (522 bp)

bp

-~453 bp

~

~

~ 30

32

34

36

lOOO.

MW

3'o

amplification cycles

3'2

3'6

PCR cycles

B O.

u 653 bp J m 517 bp - ~ 453 bp

GAPDH-]~. (529 bp)

10000-~

0-.I '10

o

lO0O.

,,r

16

19

22

25

28

MW

a.

,~

amplification cycles

100-

18

1'9 2'2 2's 2'8 PCR cycles

chain reaction was determined at 48.3% (not shown). On the basis of these experiments, we routinely used 22 cycles for the determination of glyceraldehyde phosphate dehydrogenase mRNA levels and 32 cycles for the beta l- and betaz-adrenergic receptor mRNAs. Because of its high sensitivity, the quantitative reverse transcription/polymerase chain reaction method is prone to artifacts, and it seemed necessary to compare the results obtained this way with those of a more conventional technique. We chose solution hybridization (12,13,36) as the reference method. Figure 3 compares the results of the two methods for the betal-adrenergic receptor mRNA. With the use of various amounts of synthetic full-length betal-adrenergic receptor mRNA as standards, the polymerase chain reaction method was about three orders of magnitude more sensitive than the solution hybridization assay. Furthermore, the linear range of the polymerase chain reaction method was larger, extending over more than two orders of magnitude. However, both methods yielded similar values for the levels of beta]adrenergic receptor mRNA in human heart: The polymerase chain reaction method detected 6 _+ 0.9 × 10 20 mol of beta~adrenergic receptor mRNA in 50 ng of total RNA prepared from the free left ventricular wall, whereas 5 2 1.1 × 10 ]7 mol of receptor mRNA in 50/xg total RNA was measured by solution hybridization. This corresponds to 1.2 or 1.0 amol beta~-adrenergic receptor mRNA/gg total RNA, respectively. These results underline the validity of the polymerase chain reaction method for the quantitation of rare mRNAs, and its ability to determine their absolute levels. Similar polymerase chain reaction experiments gave a value of 0.4 _+ 0.05 amol//xg total RNA for the beta2-adrenergic receptor mRNA.

Differences in tissue procurement, RNA quality, efficiency of reverse transcription and polymerase chain reaction and, finally, product quantitation contribute to variations encountered in the quantitative polymerase chain reaction method.

Figure 3. Quantitation of betal-adrenergic receptor messenger ribonucleic acid (mRNA) in human heart by reverse transcription/ polymerase chain reaction (RT-PCR) and solution hybridization. Known amounts of beta:receptor mRNA synthesized with T7-RNA polymerase or 50 ng (polymerase chain reaction) or 50 ~g (solution hybridization) of total RNA from left ventricular myocardium were quantitated as described under Methods. The radioactivity signal obtained by the two methods is shown. Arrows indicate the signal generated from 50 ng (polymerase chain reaction) or 50/xg (solution hybridization) of total heart RNA. The results read from the standard curve are 6 x 10-20 tool of betaL-receptor RNA/50 ng of total heart RNA (polymerase chain reaction) or 5 × 10-17 mol of betas-receptor RNA/50 ~g of total heart RNA (solution hybridization). Values are expressed as mean value _+SEM (not shown when smaller than symbol size) from three independent experiments.

~

108

10 4

10 4-

cpm

-103cpm

10 3 -

d

10 2

i

i

i

I

HYBRIDIZATION

J

J

J

1 0 .20 1 0 -19 10-18 10 "17 10 -16 10-15 1 0 -14

Bl-adrenergic receptor RNA (mol)

10 2

JACC Vol. 27, No. l January 1996:146-54

ENGELHARDT ET AL. BETA-ADRENERGIC RECEPTOR MRNA IN VENTRICULAR BIOPSY SPECIMENS

Because a variety of standardization methods have been advocated to counteract such problems, we wondered about the origin of the major variations in quantitative polymerase chain reaction reactions. To do this, we quantitated the polymerase chain reaction products for the betal-adrenergic receptor mRNA starting from different steps of the procedure (Fig. 4A). When multiple samples were excised from adjacent sites of the left ventricular free wall of a human heart explant and then processed separately, the coefficient of variation of the resultant quantitation of betaa-adrenergic receptor mRNA was -0.6. Starting at the next defined step and using multiple aliquots of an RNA preparation from a single sample, the coefficient of variation was already reduced to almost half. Starting later in the procedure, the variation decreased progressively. Similar data were obtained for the glyceraldehyde phosphate dehydrogenase mRNA, although probably because of its higher mRNA levels and the lower number of polymerase chain reaction cycles required, the variations were smaller at all steps (legend to Fig. 4). These results indicate that the major sources of variation are located in the treatment of tissue and the RNA preparation; that is, steps that occur before the addition of external RNA standards. Therefore, it appeared more reasonable to normalize to an internal standard such as glyceraldehyde phosphate dehydrogenase, which does not appear to be subject to variations in heart failure (17). Indeed, normalization of the values obtained for the betalreceptor mRNA with the respective glyceraldehyde phosphate dehydrogenase mRNA values resulted in a significant reduction of the coefficient of variation (Fig. 4B). This finding underlines the validity of the normalization to an endogenous standard. As a consequence, the levels of glyceraldehyde phosphate dehydrogenase mRNA were determined in all samples and were used for normalization. Beta-adrenergie receptor mRNA in myocardial biopsy specimens. We then used these methods to quantitate the mRNAs for the beta1- and beta2-adrenergic receptors in cardiac biopsy specimens. The specimens were taken from the septum during right ventricular catheterization and were from a total of 29 patients with different degrees of cardiac dysfunction. The clinical and hemodynamic data of these patients are given in Table 1 and in the Methods section. The absolute values of the expression of glyceraldehyde phosphate dehydrogenase in these biopsy specimens were very similar in all groups (Table 3). In agreement with our earlier data on explanted hearts (17,18), this finding indicates that this mRNA is not affected by cardiac failure and appears to be a valid control. The levels of beta2-adrenergic receptor mRNA were very similar in all four groups and thus apparently unaffected by cardiac failure (Fig. 5). In contrast, the levels of betal-adrenergic receptor mRNA were significantly lower in failing hearts than in the control hearts in functional classes 0 and I (Fig. 6). Reductions were already apparent in biopsy specimens obtained from hearts with modest (functional class I1 -7%) and moderate (class III -26%) heart failure, but the most pronounced alterations were seen in severely failing hearts (class IV), where the reduction amounted to >50%. The inverse relation of betas-

A

151

0.8

p.

.9

0.6

z. m >

~

0.4

._~

o

0.2

O

tissue

RNA

cDNA

PCR product

starting point in RT/PCR procedure

B

0.8

i..

.2 ,¢.,

0.6

o~ >

"6

0.4

t" Q} N.-

o

0.2

o

/'Jl AR

BIAR/GAPDH

Figure 4. Sourcesof variation in the quantitationof betal-adrenergic receptor (/31AR) messenger RNA (mRNA) levels in human heart by reverse transcription/polymerasechain reaction (RT/PCR). Multiple samples were taken from adjacentsites in the free wallof an explanted nonfailinghuman left ventricle and were processed as describedunder Methods. At each individualstep, multiple aliquots were taken and then processed and quantitated separately. Panel A shows the coefficient of variation (SD/mean) as a function of the starting point of independent processingof individualsamples. Similarexperimentsfor the mRNA of glyceraldehydephosphate dehydrogenase (GAPDH) gave the followingcoefficientsof variations for the same four steps: 0.47, 0.08, 0.05, 0.01. Panel B showsthe coefficientof variation of the beta~-adrenergic receptor mRNA values from multiple independent samples without (left column) or with (right column) normalization with the respectivevalues for the mRNA of glyceraldehydephosphate dehydrogenase, which was determined in parallel, but in a separate reaction. Data are from five independent determinations, eDNA = complementarydeoxyribonucleicacid.

adrenergic receptor mRNA and functional class was highly significant, as shown by testing for a linear trend (p < 0.001). The decline in betal-adrenergic receptor mRNA levels in the biopsy specimens correlated best with the functional class of these patients (r = -0.61, p < 0.001) and was similar for right atrial pressure (r = -0.55, p < 0.01) and left ventricular end-diastolic pressure (r = -0.50, p < 0.05) as indicators of

152

ENGELHARDT ET AL. BETA-ADRENERGIC RECEPTOR MRNA IN VENTRICULAR BIOPSY SPECIMENS

JACC Vol. 27, No. 1 January 1996:146-54

Table 3. Levels of Glyceraldehyde-3-PhosphateDehydrogenase Messenger Ribonucleic Acid in Different Stages of Heart Failure PCR product (cpm)

NYHA I

NYHA II

NYHA III

NYHA IV

1,044 +- 61

986 -+ 68

1,141 _+ 16

1,032+ 82

Levels of glyceraldehyde-3-phosphate dehydrogenase messenger ribonucleic acid in biopsy specimens taken from the right septum of patients with different degrees of heart failure. The specimens from the patients listed in Table 1 were analyzed by quantitative reverse transcription/polymerase chain reaction (PCR), and the results (mean value _+ SEM) are given in cpm of incorporated radioactivity. NYHA = New York Heart Association functional class.

z

n - , -<,

Ez

,_n" O--

1000-

o O

"

~E

500 -

¢,-.right or left ventricular dysfunction, respectively. The correlation with ejection fraction was not significant (r = 0.37). When only patients with dilated cardiomyopathy were analyzed, the correlation coefficient was 0.58 (p < 0.01) for ejection fraction and -0.56 (p < 0.01) for left ventricular end-diastolic pressure. There was no correlation between left ventricular end-diastolic volume and mRNA levels (r = -0.03). These data indicate that betat-adrenergic receptor mRNA levels are linked to cardiac function and that these levels are progressively reduced in heart failure. Discussion

The loss of beta-adrenergic receptor responsiveness in heart failure has been demonstrated in several independent studies (1-6). It appears to have two components: reduction in Figure 5. Levels of beta2-adrenergic receptor messenger ribonucleic acid (mRNA) in biopsy specimens taken from the right septum of patients with different degrees of heart failure. The specimens from the patients listed in Table 1 were analyzed by quantitative polymerase chain reaction, were normalized with the respective values of the glyceraldehyde phosphate dehydrogenase mRNA as described in Figure 4, and the levels of receptor mRNA were read from a standard curve as shown in Figure 3. Data are expressed as mean value +_SEM. NYHA = New York Heart Association functional classification; RNA = ribonucleic acid.

ooj

z

A

4001

T T

Ez 0 --

300'

0

~E

200"

e- ¢N

100" i

0"

0-1

II

III

NYHA-Classification

IV

O-

0-1

II

III

IV

NYHA-Classification Figure 6. Levels of betal-adrenergic receptor messenger ribonucleic acid (mRNA) in biopsy specimens taken from the right septum of patients with different degrees of heart failure. The specimens from the patients listed in Table 1 were analyzed by quantitative polymerase chain reaction, were normalized with the respective values of the glyceraldehyde phosphate dehydrogenase mRNA as described in Figure 4, and the levels of receptor mRNA were read from a standard curve as shown in Figure 3. Data are expressed as mean value _ SEM. One-way analysis of variance (ANOVA) followed by testing for a linear trend showed a significant inverse relation between functional class and betal-adrenergic receptor mRNA (r = -0.61, p < 0.001). In class IV the betal-adrenergic receptor mRNA levels were significantly lower than values in class 0-I (p < 0.01 [ANOVA followed by Scheff6 test]). Abbreviations as in Figure 5. receptor numbers, largely confined to the betal-subtype, and impaired function of the remaining receptors. These alterations have been proposed to play a role in the deterioration of cardiac function, as the inability to enhance contractility in response to catecholamines may further compromise the heart's ability to meet the demand. Beta-adrenergic receptor mRNA quantification. Reduced expression of betal-adrenergic receptors in the failing human heart has been attributed to a concomitant loss of the corresponding mRNA (17,19). We sought to complement those studies, which were performed on specimens from explanted hearts, by developing techniques that would enable us to investigate beta-adrenergic receptor expression in single endomyocardial biopsy specimens. Because of the small size of the biopsy specimens and the need to measure the expression of several proteins, determination of mRNA levels by quantitative reverse transcription/polymerase chain reaction method appeared to be the only possible technique. Optimization of the RNA preparation resulted in yields of >1/~g of total RNA from a single biopsy specimen (2 to 5 rag), which was free from genomic DNA. Normalization is a critical problem in the quantitative polymerase chain reaction method. Basically, there are two different approaches: Either an unrelated, nonregulated mRNA is quantitated in parallel or various kinds of RNA standards are added to the RNA preparation. Although the latter method, often used as competitive polymerase chain

JACC Vol. 27, No. 1 January 1996:146-54

E N G E L H A R D T ET AL. B E T A - A D R E N E R G I C R E C E P T O R MRNA IN V E N T R I C U L A R BIOPSY SPECIMENS

reaction (27,28), has recently gained considerable popularity, it controls only for variations encountered after the preparation of total RNA from the samples. However, our data show that the major sources of variation are proximal to the possible addition of exogenous RNA standards, and that the steps that can be controlled by exogenous RNA standards (reverse transcription/polymerase chain reaction and product quantitation) are only minor sources of variation. Consequently, normalization to the glyceraldehyde phosphate dehydrogenase mRNA (which is unaltered in heart failure) appeared the best way to control for variations in biopsy quality, RNA preparation and efficiency of the polymerase chain reactions. The glyceraldehyde phosphate dehydrogenase standards were determined in parallel but in separate reaction tubes to avoid competition between the two different polymerase chain reaction products, which has been shown to be a problem in single-tube reactions (17,37). The normalized values were then analyzed by a standard curve obtained with known amounts of synthetic receptor mRNA. This was possible because the amplification efficiency was similar for pure synthetic receptor mRNA and for receptor mRNA amplified from total RNA preparations from heart. This method of quantitation gave results in good agreement with simultaneous determination by solution hybridization but was considerably more sensitive. The absolute values of beta-adrenergic receptor mRNA levels in nonfailing left ventricular myocardium were - 1 amol//zg total RNA for the beta 1- and -0.4 amol//zg total RNA for the beta2-subtype. The values reported recently by Bristow and co-workers (19) correspond to - 2 amol//zg total RNA for the betal-subtype (assuming that polyA+-RNA constitutes -3% of total RNA [38]) and to -37 amol//xg total RNA for the beta2subtype. Thus, our data are similar for the betal-adrenergic receptor but for unknown reasons are quite dissimilar for the betaz-adrenergic receptor. Beta-adrenergic receptor mRNA in diseased myocardium. The quantitation of beta-adrenergic receptor mRNAs in cardiac biopsy specimens with these methods gave two main results: 1) a reduction of betal-adrenergic receptor mRNA levels in heart failure but no change for the beta2-subtype, and 2) a good correlation between the degree of heart failure and the reduction of betal-adrenergic receptor mRNA levels. A reduction of betal-adrenergic receptors in the failing human heart with dilated cardiomyopathy has been observed by most investigators (1-6), whereas, with few exceptions (4,17,39), betaz-receptor levels have been found unaltered in most forms of heart failure. It is not clear how such a selective reduction of only the betal-SUbtype might occur. In fact, when expressed in Chinese hamster ovary cells, betaz-adrenergic receptors are more readily downregulated after agonist stimulation than is the betas-subtype (40). This betal-selective downregulation may be due to the presence of the two receptor subtypes on different cells or to a cyclic adenosine monophosphate-independent mechanism--possibly specific for the heart--leading to downregulation of betal-adrenergic receptors. Alternatively, betal-adrenergic receptor downregulation in heart failure may not simply be a consequence of

153

increased catecholamine levels. These issues will require investigations in better defined systems. Our data show that the decrease in cardiac function and the loss of betal-adrenergic receptor mRNA occur in a roughly parallel manner. The correlation with the mRNA levels was better for functional class than for hemodynamic indexes of cardiac function. In particular, in high output failure such as mitral valve disease or restrictive heart disease the receptor mRNA levels were reduced according to functional class, even though the ejection fraction was maintained. This finding agrees with the observation that radioligand binding to betaadrenergic receptors was reduced in mitral valve disease (24). Together with the observation of an early decline of beta ladrenergic receptors in pacing-induced heart failure in dogs (22), these data suggest a possible causal role of beta ladrenergic receptor downregulation in the development of heart failure. The biopsy specimens studied here were taken from the septum during right ventricular catheterization; that is, from a site that may be regarded functionally as the outside of the left ventricle. Recent studies (41,42) suggest that local rather than systemic changes are responsible for the downregulation of beta l.adrenergic receptors in heart failure. In our patients with global heart failure the decline in betal-adrenergic receptor mRNA levels in the biopsy specimens correlated equally well with hemodynamic indicators of left (left ventricular enddiastolic pressure) or right (right atrial pressure) heart dysfunction. Furthermore, the reduction in betaa-adrenergic receptor mRNA levels (-51%) found in the biopsy specimens of patients (in functional class IV) matched very closely the reduction (-50%) reported earlier (17) on specimens excised from the left ventricles of explanted severely failing hearts. This observation suggests that the behavior of the septum in global heart failure is similar to that of the left ventricle. It is remarkable, that in heart failure the betal-adrenergic receptor mRNA levels are reduced by the same extent as the levels of the receptors themselves (i.e., by -50% in functional class IV [17]). This finding suggests that other mechanisms of agonist-induced receptor downregulation do not play a major role in heart failure. A good correlation between beta 1adrenergic receptor levels and their mRNA levels in cardiac explants supports this notion (19). Conclusions. We developed a methodology to determine the levels of multiple low abundance mRNAs in cardiac biopsy specimens. This allowed us to observe a reduction of the mRNA levels for beta 1- but not for beta2-adrenergic receptors in failing hearts, and a good correlation between the reduction in betax-adrenergic receptor mRNA and the degree of heart failure. It will be interesting to investigate whether there is a similar correlation in the temporal development of heart failure in individual patients and whether the variations in the betal-adrenergic receptor mRNA levels (particularly in functional class 0 to I) will be reflected in the future development of cardiac function in these patients.

154

ENGELHARDT ET AL. BETA-ADRENERGIC RECEPTOR MRNA IN VENTRICULAR BIOPSY SPECIMENS

References 1. Bristow MR, Ginsburg R, Minobe W, et al. Decreased catecholamine sensitivity and beta-adrenergic receptor density in failing human hearts. N Engl J Med 1982;307:205-11. 2. Homcy C J, Vatner SF, Vatner DE./3-Adrenergic receptor regulation in the heart in pathophysiologic states: abnormal adrenergic responsiveness in cardiac disease. Annu Rev Physiol 1991;53:137-59. 3. B6hm M, Gierschik P, Jakobs KI-I, et al. Increase of G~, in human hearts with dilated but not ischemic cardiomyopathy. Circulation 1990;82:1249-65. 4. Bristow MR, Anderson FL, Port DP, et al. Differences in /3-adrenergic neuroeffector mechanisms in ischemic versus idiopathic dilated cardiomyopathy. Circulation 1991;84:1024-39. 5. Brodde O-E. Beta-adrenoceptors in cardiac disease. Pharmacol Ther 1993; 60:405-30. 6. Steinfath M, Geertz B, Schmitz W, et al. Distinct down-regulation of cardiac /3~- and /32-adrenoceptors in different human heart diseases. Naunyn Schmiedeberg's Arch Pharmacol 1991;343:217-20. 7. Cohn JN. Abnormalities of peripheral sympathetic nervous system control in congestive heart failure, Circulation 1990;82 Suppl I:59-67. 8. Packer M. Neurohormonal interactions and adaptations in congestive heart failure. Circulation 1988;77:721-30. 9. Swedberg K, Eneroth P, Kjekshus J, Wilhelmsen L. Hormones regulating cardiovascular function in patients with severe congestive heart failure and their relation to mortality. Circulation 1990;82:1730-6. 10. Hammond HK, Roth DA, Insel PA, et al. Myocardial beta-adrenergicreceptor expression and signal transduction after chronic volume-overload hypertrophy and circulatory congestion. Circulation 1992;85:269-80. 11. Lohse MJ. Molecular mechanisms of membrane-receptor desensitization. Biochim Biophys Acta 1993;1179:171-88. 12. Hadcock JR, Malbon CC. Down-regulation of /3-adrenergic receptors: agonist-induced reduction in receptor mRNA levels. Proc Natl Acad Sci USA 1988;85:502l-5. 13. Hadcock JR, Wang H, Malbon CC. Agonist-induced destabilization of /3-adrenergic receptor mRNA: attenuation of glucocorticoid-induced upregulation of/3-adrenergic receptors. J Biol Chem 1989;264:19928-33. 14. Bouvier M, Collins S, O'Dowd BF, et al. Two distinct pathways for cAMP-mediated down-regulation of the/3z-adrenergic receptor: phosphorylation of the receptor and regulation of its mRNA level. J Biol Chem 1989;264:16786-92. 15. Collins S, Bouvier M, Bolanowski MA, Caron MG, Lefkowitz RH. Cyclic AMP stimulates transcription of the/32-adrenergic receptor gene in response to short term agonist exposure. Proc Natl Acad Sci USA 1989;86:4853-7. 16. Port JD, Huang LY, Malbon CC. Beta-adrenergic agonists that downregulate receptor messenger-RNA up-regulate a M(r) 35,000 protein(s) that selectively binds to beta-adrenergic-receptor messenger-RNAs. J Biol Chem 1992;267:4103-8. 17. Ungerer M, B6hm M, Elce JS, Erdmann E, Lohse MJ. Altered expression of /3-adrenergic-receptor kinase and /3j-adrenergic receptors in the failing human heart. Circulation 1993;87:454-63. 18. Ungerer M, Parruti G, B0hm M, et al. Expression of /3-arrestins and /3-adrenergic receptor kinases in the failing human heart. Circ Res 1994;74: 206 -13. 19. Bristow MR, Minobe WA, Raynolds MV, et al. Reduced beta(l) receptor messenger-RNA abundance in the failing human heart. J Clin Invest 1993;92:2737- 45. 20. Hamill RW, Wolf PD, McDonald JV, Lee LA, Kelly M. Catecholamines predict outcome in traumatic brain injury. Ann Neurol 1987;21:438-43. 21. Shivalkar B, Loon JV, Wieland W, et al. Variable effects of explosive or gradual increase in intracranial pressure on myocardial structure and function. Circulation 1993;87:230-9. 22. Kiuchi K, Shannon RP, Komamura K, et al. Myocardial /3-adrenergic

JACC Vol. 27, No. 1 January 1996:146-54

receptor function during the development of pacing-induced heart failure. J Clin Invest 1993;91:907-14. 23. Fowler MB, Laser JA, Hopkins GL, Minobe W, Bristow MR. Assessment of the /3-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response. Circulation 1986;74:1290-302. 24. Brodde OE, Zerkowski HR, Doetsch N, et al. Myocardial beta-adrenoceptor changes in heart failure: concomitant reduction in betal- and beta2adrenoceptor function related to the degree of heart failure in patients with mitral valve disease. J Am Coil Cardiol 1989;14:323-31. 25. Wang AM, Doyle MV, Mark DF. Quantitation of mRNA by the polymerase chain reaction. Proc Natl Acad Sci USA 1989;86:9717-21. 26. Feldman AM, Ray PE, Silan CM, ct al. Selective gene expression in failing human heart. Circulation 1991;83:1866-72. 27. Gilliland G, Perrin S, Blanchard K, Bunn HF. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci USA 1990;87:2725-9. 28. Piatak MJ, Sagg MS, Yang LC, et al. High levels of HIV-1 in plasma during all stages of infection determined by competitive PCR. Science 1993;259: 1749-54. 29. Lu W, Han D-S, Yuan J, Andrieu J-M. Multi-target PCR analysis by capillary electrophoresis and laser-induced fluorescence. Nature 1994;368: 269-7l. 30. Chelly J, Kaplan JC, Maire P, Gautron S, Kahn A. Transcription of the dystrophin gene in human muscle and non-muscle tissues. Nature 1988;333: 858-60. 31. Chomczinski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162:156-9. 32. Frielle T, Collins S, Daniel KW, et al. Cloning of the cDNA for the human /31-adrenergic receptor. Proc Natl Acad Sci USA 1987;84:7920-4. 33. Kobilka BK, Dixon RAF, Frielle T, et al. cDNA for the human/32-adrenergic receptor: a protein with multiple membrane-spanning domains and encoded by a gene whose chromosomal location is shared with that of the receptor for platelet-derived growth factor. Proc Natl Acad Sci USA 1987;84:46-50. 34. Hanauer A, Mendel JL. The glyceraldehyde 3 phosphate dehydrogenase geue family: structure of a human cDNA and of an X chromosome linked pseudogene; amazing complexity of the gene family in mouse. EMBO J. 1984;3:2627-33. 35, Ausubel FM, Brent R, Kingston RE, et al. Current Protocols in Molecular Biology. New York: Current Protocols Inc., 1987-95. 36, Hellmann W, Suzuki F, Ohakubo H, et al. Angiotensinogen in extrahepatic rat tissues: application of a solution hybridization assay. Naunyn Schmiedeberg's Arch Pharmacol 1988;338:327-31. 37. Murphy LD, Herzog CE, Rudick JB, Fojo AT, Bates SE. Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry 1990;29:10351-6. 38. Brandhorst BP, McConkey EH. Stability of nuclear RNA in mammalian cells. J Mol Biol 1974;85:451-63. 39. Brodde O-E, Zerkowski HR, Borst HG, Maier W, Michel MC. Drug- and disease-induced changes of human cardiac/3 l- and/32-adrenoceptors. Eur Heart J 1989;10 Suppl B:38-44. 40. Suzuki T, Nguyen HCT, Nantel LF, et al. Distinct regulation of/31- and /32-adrenergic receptors in chinese hamster fibroblasts. Mol Pharmacol 1992;41:542-8. 41. Bristow MR, Minobe W, Rassmussen R, et al./3-adrenergic neuroeffector abnormalities in the failing human heart are produced by local rather than systemic mechanisms. J Clin Invest 1992;89:803-15. 42. Yoshikawa T, Handa S, Suzuki M, Nagami K. Abnormalities in sympathoneuronal regulation are localized to failing myocardium in rabbit heart. J Am Coil Cardiol 1994;24:210-5.