Cardiovascular Research

Cardiovascular Research 1999 42(1):87-98; doi:10.1016/S0008-6363(98)00283-1
© 1999 by European Society of Cardiology

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Copyright © 1999, European Society of Cardiology

Decreased type VI adenylyl cyclase mRNA concentration and Mg²⁺-dependent adenylyl cyclase activities and unchanged type V adenylyl cyclase mRNA concentration and Mn²⁺-dependent adenylyl cyclase activities in the left ventricle of rats with myocardial infarction and longstanding heart failure

Isabelle Espinasse^a, Vadim Iourgenko^a, Christine Richer^b, Michèle Heimburger^a, Nicole Defer^d, Marie-Claude Bourin^d, Françoise Samson^c, Eric Pussard^b, Jean-François Giudicelli^b, Jean-Baptiste Michel^a, Jacques Hanoune^d and Jean-Jacques Mercadier^a,*

^aINSERM U 460, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, F-75018 Paris, France
^bDépartement de Pharmacologie, Le Krémlin-Bicêtre, France
^cC.N.R.S. ERS 566, Le Plessis Robinson, France
^dINSERM U 99, Hôpital Henri Mondor, Créteil, France

^* Corresponding author. Tel.: +33-1-4485-6158; fax: +33-1-4485-6157; e-mail: jjmercad@pratique.fr

Received 10 February 1998; accepted 8 September 1998

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: To address the effect of longstanding left ventricular (LV) hypertrophy and failure on LV adenylyl cyclase (AC) gene expression, mRNA concentrations of the main cardiac AC isoforms were measured in the non-infarcted area of LV from rats with myocardial infarction (MI), without (H) or with (F) LV failure, and in control (C) rats. Basal, GTP- and forskolin-stimulated Mg²⁺- and Mn²⁺-dependent AC activities were also measured in F and C rats. Methods: Two- and six months after MI, steady-state AC mRNA concentrations were assessed by Northern blot analysis and RNase protection assay with isoform-specific cDNA and cRNA probes, respectively. AC activities were assessed on LV microsomal fractions using standard procedures. Results: Types V and VI, and types IV and VII were the major and minor AC mRNA isoforms in both the LVs of F and C rats. Two months after MI, no difference in LV type V or VI mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA ratios was observed in rats with H or F compared to C. Six months after MI, no difference in LV type V mRNA concentration was observed between the three rat groups, whether this level was normalized to GAPDH, poly-(A⁺) or 18S RNAs. In contrast, a 35% decrease in the type VI mRNA to poly-(A⁺) RNA ratio and a 29% decrease in the type VI mRNA to 18S RNA ratio was observed only in rats with F compared to C (p<0.05 vs. C for the two comparisons). Two- and six months after MI, basal and forskolin-stimulated Mg²⁺-dependent AC activities were decreased by 30–35% in F rats compared to C (p<0.05), whereas Mn²⁺-dependent activities were unchanged. Conclusion: Longstanding LV hypertrophy and failure resulting from MI in rats is not associated with altered expression of the most abundant, type V, AC mRNA isoform, whereas that of type VI is decreased. The lack of change in Mn²⁺-dependent AC activities in the LV of F rats suggests that this decrease has no functional consequence on overall AC activity and that decreased Mg²⁺-dependent activities are related to alterations occurring upstream.

KEYWORDS Adenylyl cyclase; Rat heart; Ventricular hypertrophy; Heart failure

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Because of its function to generate cAMP and its dual regulation by the G protein system, adenylyl cyclase plays a pivotal role in the regulation of myocardial contractility. Catecholamine binding to β₁- and β₂-adrenergic receptors results in intracellular cAMP production through adenylyl cyclase stimulation by Gs [1]. This activates protein kinase A which, in turn, stimulates the inward calcium current and sarcoplasmic reticulum Ca²⁺ uptake through phosphorylation of the L-type calcium channel (or a closely associated protein) [2]and phospholamban [3], respectively. Following stimulation of M₂ muscarinic receptors, adenylyl cyclase activity can also be inhibited by Gi [4]. A number of studies have shown that alterations in the β-adrenergic signal transduction pathway are associated with the decreased cardiac contractility that is characteristic of heart failure [5]. A fall in cAMP production following β-adrenoceptor stimulation has long been known to occur in human heart failure [6]and in animal models [7], indicating alterations at some point of the transduction pathway. A decrease in β₁-adrenergic receptor density has been demonstrated [7, 8], and this was confirmed at the mRNA level [9]. Results on G proteins seem to vary according to the species and cause of heart failure, but there is general agreement that an increase in Gi mRNA and protein concentrations occurs in the failing human ventricle [10].

Up to nine isoforms of mammalian adenylyl cyclase (types I–IX) have been reported to date, differing in their amino acid sequence, tissue distribution, mode of activity regulation and chromosomal location of the coding genes [11]. Such diversity of a single effector system probably underlies specific signaling functions of the various isoforms expressed in a given tissue or individual cell, and points to differential regulation of the expression of the various isoforms. Six adenylyl cyclase mRNA isoforms (types II, IV to VII and IX) have been detected in the mammalian heart, with types V and VI being by far the most abundant ones [11–14]. Types V and VI appear to have similar enzymatic properties, as their activity is not stimulated by Ca²⁺–calmodulin and is inhibited by submicromolar concentrations of calcium [11, 12]. Tobise et al. [15]have reported differential expression of type V and VI isoforms in the rat heart during development and ageing, and we have shown that the overall concentration of types V and VI adenylyl cyclase mRNA more than doubles in the rat heart from the late fetal stage to adulthood, type V mRNA being almost ten times more abundant than type VI in the adult ventricle [16]. This increase is accompanied by a parallel rise in basal and isoproterenol- and forskolin-stimulated adenylyl cyclase activities, suggesting that the increased activity observed during early post-natal rat development could due to accumulation of the corresponding proteins.

To our knowledge, only two studies have addressed adenylyl cyclase gene expression during heart failure. The first showed a parallel fall in steady-state mRNA concentrations of types V and VI adenylyl cyclase in the canine model of pacing-induced heart failure [17]. In contrast, in a pacing-induced model of heart failure in the pig, only the type VI adenylyl cyclase mRNA concentration was decreased [13]. However, pacing-induced heart failure in dogs or pigs is a very peculiar model of heart failure, characterized by contractile and biochemical alterations occurring as early as the first days of pacing, and by the absence of significant ventricular hypertrophy [13, 17, 18]. Importantly, it has been widely documented that myocardial hypertrophy, which is more commonly observed during chronic hemodynamic overload in experimental models as in man, is often associated with the reexpression of a fetal gene program [19, 20]. Regarding adenylyl cyclase, this would lead to decreased type V and increased type VI adenylyl cyclase gene expression, as recently hypothesized by Ishikawa and Homcy [12]. Therefore, the aim of the present work was to determine adenylyl cyclase gene expression in the non-infarcted left ventricle of rats with myocardial infarction, a well-characterized experimental model, providing animals exhibiting either compensatory hypertrophy of the non-infarcted left ventricle or heart failure [21–23]. In an attempt to correlate gene expression with the activity of the enzyme, we also measured basal and stimulated Mg²⁺- and Mn²⁺-dependent adenylyl cyclase activities in control rats and in rats with heart failure.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Experimental design
Normotensive three-month-old male Wistar rats (Iffa Credo), weighing 300–320 g, were used. Animal care complied with the ‘Principles of Laboratory Animal Care’, formulated by the National Society for Medical Research and the ‘Guide for the Care and Use of Laboratory Animals’ (NIH publication no. 86-23, revised 1985, authorization no. 03237, Ministère de l’Agriculture, France). Left ventricular infarction was produced by ligation of the left coronary artery under ether anaesthesia, as described elsewhere [21]. Surviving rats were maintained on standard rat chow and water ad libitum. In our hands, this method results in a 30–40% mortality within the first post-operative day, with an additional 10–15% mortality within the first six months. Such a model has been shown to provide, three to four weeks after coronary artery occlusion, rats with left ventricular infarctions ranging from 4 to 59% of the left ventricular endocardial circumference, with or without heart failure, depending on the size of the infarction [21].

In a first set of experiments (Study 1), the experimental period was two months. During the week before killing, each rat was placed in a metabolic cage and urine was collected for 15 h for cGMP determination. At the end of the experimental protocol, unanesthetised rats were killed by decapitation and blood was collected into prechilled 10-ml heparinized tubes. Blood was centrifuged for 10 min at 3000 g and the plasma was frozen in liquid nitrogen and stored at –80°C. At the same time, the heart was excised and rinsed in cold saline; the atria and surrounding vessels were removed, the right ventricle was dissected along its septal insertion and each ventricle was weighed. The infarct scar was carefully dissected under a binocular microscope to remove all fibrous tissue from the adjacent left ventricle, which was frozen in liquid nitrogen and stored at –80°C until RNA purification and determination of adenylyl cyclase activity. In a second set of experiments (Study 2), the experimental period was six months. Animals were killed and hearts were processed as described above, except that one sample of each left ventricle was used to determine the myocardial catecholamine concentration. In each set of experiments, an appropriate number of sham-operated rats (C) underwent the entire procedure, except for coronary artery ligation. Samples of brain were also taken from sham-operated rats at the time of killing, to prepare control RNA. All tissues were frozen in liquid nitrogen immediately after sampling and were stored at –80°C until use.

2.2 Identification of heart failure
In Study 1, three indexes were used for classification of rats with myocardial infarction: (1) the presence or absence of pleural effusion; (2) a right ventricular weight-to-body weight ratio that was greater than twice the control value, an index that has been shown to testify for large infarcts (>40%) associated with heart failure (left ventricular end-diastolic pressure: 28±2 mmHg) [21]and (3) a urinary cGMP that was greater than twice the control value, a very sensitive indicator of congestive heart failure in rats [22]. When pleural effusion and/or the two other criteria were present, the rat was assigned to the subgroup of severe myocardial infarction and heart failure (F). The rats that did not fulfil these requirements were assigned to the subgroup of moderate myocardial infarction with compensatory left ventricular hypertrophy (H). In Study 2, as rats with myocardial infarction rarely exhibited a small pleural effusion, only the presence of the two other criteria was used to assign rats to the group with heart failure.

2.3 Determination of plasma angiotensin-converting enzyme (ACE) activity, urinary cGMP levels and myocardial catecholamine concentrations
Plasma ACE activity was determined as described previously [23]by measuring the hydrolysis of 100 µM [glycine-1-¹⁴C]hippuryl-L-histidyl-L-leucine (3 mCi/mmol, Amersham) in the presence or absence of 10^–7 M captopril. Urinary cGMP levels were measured by radioimmunoassay using an Amersham kit (TRK 500) according to the manufacturer’s recommendations [22]. For the determination of myocardial catecholamine concentrations, frozen samples were crushed in liquid nitrogen and the resulting powder was mixed with perchloric acid. The mixture was sonicated on ice twice for 15 s. Following centrifugation at 1000 g for 5 min, catecholamines in the supernatants were extracted on alumina and quantified by means of high-performance liquid chromatography (HPLC) with amperometric detection, as described previously [24].

2.4 Probes, RNA purification, Northern blot and dot blot analysis
Probes specific for type V and VI adenylyl cyclase mRNAs and GAPDH mRNA were those used to transcribe the antisense cRNAs used in the RNase protection assays (see below). Probes specific for type IV and VII adenylyl cyclase mRNAs were a gift from R. Premont (Durham, NC, USA). The probe specific for type IV adenylyl cyclase mRNA was an 800-bp cDNA (BamHI–SacI fragment from published sequence, [25]), corresponding to the cyclase’s first six membrane-spanning domains. The probe specific for type VII was a 722-bp cDNA fragment (nucleotides 2453 to 3175 from the published sequence [26]). To normalize for the amount of RNA actually present on each Northern blot lane or dot, a synthetic probe of 24 nucleotides, complementary to a sequence of rat 18S rRNA, was used. The GAPDH cDNA is a 573-bp fragment obtained by reverse transcribed polymerase chain reaction (RT-PCR) of rat ventricular RNA (from nucleotides 244 and 817 of the sequence published by Tso et al. [27]). The poly-(A⁺) probe is a synthetic oligo (dT)₁₅ (Promega, WI, USA). In addition, to further assess the severity of left ventricular overload and the resulting hypertrophy, we used a cDNA probe of 710 bp in length that was specific for rat atrial natriuretic peptide (ANP) mRNA (gift from E. Tolunay, Monsanto, St. Louis, MO, USA).

For Northern blot analysis of adenylyl cyclase mRNAs in rat ventricle and brain, given their weak concentration in the heart, we used 8 µg of poly-(A⁺) RNA purified from each tissue using the Fast Track kit from Invitrogen according to the manufacturer’s instructions. Total RNA was purified according to Chomczinsky and Sacchi [28]. RNAs were quantified densitometrically at 260–280 nm. For Northern blot analysis of ANP mRNA and 18S rRNA, 10 µg of total RNA from the relevant tissues were used. For dot blot analysis, 6 µg of RNA were used and serial dilutions (3, 1.5, 0.75 and 0.375 µg) were transferred to nylon membrane after denaturation. cDNA probes were labelled by random priming (Megaprime labelling kit, Amersham) with [³²P]-dCTP, and oligonucleotides with [³²P]-ATP using T4 kinase. Probes were added to the prehybridization solution at a final concentration of 10⁶ cpm/ml. After hybridization, membranes were washed at various final stringencies according to the probe and autoradiograms were obtained after 4 to 8 h of membrane exposure, except for type IV and VII adenylyl cyclase probes, which required longer exposure durations (24–48 h). Between each hybridization step, membranes were dehybridized by washing at 95°C in 0.5% sodium dodecyl sulfate (SDS).

2.5 RNase protection assay
Type V and VI adenylyl cyclase mRNAs were quantified in the same tube by means of a ribonuclease protection assay (RPA), with one cRNA riboprobe specific for each isoform and one cRNA riboprobe specific for GAPDH mRNA, as the internal standard, as described previously [16]. Briefly, to avoid cross-hybridization in the RNase protection assay, probes specific for type V and type VI adenylyl cyclase mRNAs were constructed from regions in which the cDNAs exhibit the lowest sequence identity. Type V adenylyl cyclase cDNA is a 459-bp fragment (amino acids 498 to 650 of the sequence published by Premont et al. [29]) obtained by means of PCR amplification. Type VI adenylyl cyclase cDNA is a 510-bp fragment (amino acids 708 to 877 of the sequence published by Premont et al. [29]) obtained by means of PCR from a plasmid (pGEM 3Z) containing an 890-bp insert of the coding sequence of type VI adenylyl cyclase [30]. The cDNAs were subcloned in pCR II^TM and fully sequenced to check for the absence of mutations during PCR. Plasmids containing the appropriate inserts were linearized with XhoI for type V and VI adenylyl cyclase cDNAs, and with EcoNI for GAPDH cDNA, resulting in probes of 459, 510 and 312 bp in length, respectively. The antisense cRNAs were transcribed with SP6 polymerase using the Gemini II transcription kit (Promega) and [³²P]-UTP. The specific activity of the two adenylyl cyclase probes was consistently 2–4x10⁸ cpm/µg, and that of the GAPDH probe was 0.5–1x10⁸ cpm/µg. The RPA was performed using the RPA II^TM kit (Ambion) according to the manufacturer’s instructions after checking that the cRNA probes were in large excess relative to their respective target mRNA. Protected probe–target RNA duplexes were separated by electrophoresis in 5% polyacrylamide gels at 250 V for 6 h. Gels were exposed to Cronex film (Du Pont) for 4 h (GAPDH) and for up to three days (adenylyl cyclase). Several exposure times were used and the autoradiograms were scanned by the Starwise imaging system for densitometric analysis (Imstar). A given RNase protection experiment always comprised left ventricular samples from the three experimental groups (hypertrophic and failing hearts, and controls). Any experiment had an additional adult rat ventricular RNA sample in common as an internal standard, which was used to normalize the data between the various experiments.

2.6 Adenylyl cyclase activity assay
Left ventricular samples were minced with scissors in a 20-fold excess (w/v) of ice-cold homogenization buffer (10 mM Tris–HCl, pH 7.4, 5 mM MgCl₂, 1 mM EDTA) and homogenized for 20 s using a Polytron homogenizer at a speed setting of 4–5. To remove cellular debris, the homogenates were centrifuged at 600 g for 10 min at 4°C. The supernatants were collected and centrifuged at 20 000 g for 20 min at 4°C. The pellets were washed in the same volume of homogenization buffer containing 1 mM EGTA and centrifuged under the same conditions. The procedure was performed three times. After the last wash, the pellets were resuspended in 50 mM Tris–HCl, pH 7.4, at a protein concentration of 1.5 mg/ml, as determined by the method of Bradford [31], with bovine serum albumin as standard, and were stored at –80°C.

Adenylyl cyclase activity in particulate membrane preparations (20–30 µg of protein per assay in a final volume of 60 µl) was measured in a buffer consisting of (final concentrations) 70 mM Tris–HCl, pH 7.4, 4 mM phosphocreatine, 32 units/ml phosphocreatine kinase, 1 mM cAMP, 5 mM MgCl₂, 1 mM ATP (disodium salt), 2–4x10⁶ cpm/assay [³²P]ATP (tetra [triethylammonium] salt) (Amersham, UK), 0.2 mM EGTA, 0.3 mM PMSF. For adenylyl cyclase activity stimulation, 10 µM GTP and/or 10 µM forskolin (final concentrations) was added to the reaction mixture. In addition, because uncoupled forms of adenylyl cyclase utilize Mn-ATP but not Mg-ATP as substrate [32], we performed experiments in which 5 mM MnCl₂ was substituted for 5 mM MgCl₂ in the assay medium. The reaction was started by adding the membranes and was stopped after 20 min at 30°C by adding 200 µl of 0.5 M HCl. Tubes were then boiled and neutralized with 250 µl of 1.5 M imidazole, and the [³²P]cAMP formed was quantified as described elsewhere [33]. Results are expressed as pmol of [³²P]cAMP formed per min per mg of protein and are presented as means±SEM of triplicate determinations.

2.7 Statistical analysis
Results are presented as means±SEM. In each study, significant differences between the three rat groups were identified by using one-way analysis of variance (ANOVA). When the ANOVA revealed a significant difference, group-to-group comparisons were performed using the t-test for multiple comparisons. The threshold for statistical significance was set at p<0.05.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Main characteristics of rats in Studies 1 and 2
Study 1 comprised 20 rats with myocardial infarction and eight sham-operated rats. Ten out of the 20 rats with myocardial infarction were considered to have severe myocardial infarction with heart failure, based on the criteria described above. The remaining ten rats were considered to have moderate myocardial infarction with compensatory left ventricular hypertrophy. The main characteristics of the three groups are listed in Table 1. Rats with severe myocardial infarction and heart failure exhibited significant increases in the right ventricular weight-to-body weight ratio, plasma ACE activity and urinary cGMP, relative to both controls and rats with moderate myocardial infarction. These rats also exhibited a left ventricular weight-to-body weight ratio that was higher than that in controls, indicating major hypertrophy of the non-infarcted left ventricle despite myocardial muscle loss due to infarction. Northern blot analysis of ANP mRNA in the three groups revealed a marked accumulation of this species in the left ventricles of the two infarcted rat groups, consistent with chronic hemodynamic overload and hypertrophy of the non-infarcted area of the left ventricles in these rats (Table 1).

View this table: [in this window] [in a new window]   Table 1 Main characteristics of rats in Study 1

 
Study 2 comprised 16 rats with myocardial infarction and eight sham-operated rats. The characteristics of these animals are listed in Table 2. Among the 16 rats with myocardial infarction, eight were assigned to the group with severe myocardial infarction and heart failure, based on a right ventricular weight-to-body weight ratio and urinary cGMP excretion that was more than twice the control value. The mean left ventricular weight-to-body weight ratio was increased in the two rat groups with myocardial infarction compared to the controls, demonstrating marked left ventricular hypertrophy despite myocardial muscle loss due to infarction. The myocardial norepinephrine concentration was decreased only in the rat group with severe myocardial infarction and heart failure, both compared to controls and rats with compensatory hypertrophy. Dot blot analysis revealed, as in the rats in Study 1, a strong accumulation of ANP mRNA in the left ventricles of the two infarcted rat groups relative to controls, with a three-fold increase in rats with heart failure compared to those with compensatory hypertrophy (Table 2, Fig. 4).

View this table: [in this window] [in a new window]   Table 2 Main characteristics of rats in Study 2

 

View larger version (66K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Dot blot analysis of left ventricular RNAs from rats in Study 2. The same dot blot was successively hybridized with probes that were specific for ANP mRNA, GAPDH mRNA, poly-(A+) RNA and 18S RNA. For each sample, 3, 1.5, 0.75 and 0.375 µg of total RNA were dotted. Hybridization signals from two rats characteristic of each group are shown (top and bottom line of each hybridization). C, H and F: same as in Fig. 2.

 
3.2 Northern blot analysis of adenylyl cyclase isoforms expressed in control and failing rat ventricles
A typical Northern blot analysis of poly-(A⁺) RNA purified from rat brain and from the ventricles of control rats and rats with heart failure in Study 2 (hybridized with probes specific for type IV, V, VI and VII adenylyl cyclase) is presented in Fig. 1. The four mRNA types were detected in all of the samples studied. With both ventricular and brain RNAs, type VI and VII adenylyl cyclase cDNAs hybridized as single bands of approximately 6 and 6.3 kb, respectively, in agreement with previous reports [26, 29], whereas type V adenylyl cyclase cDNA hybridized as two sharp bands of 7 and 5 kb each, also in good agreement with previous reports [15, 29]. The type IV-specific probe hybridized with a mRNA of approximately 3.5 kb, in agreement with Gao and Gilman [25]. However, the mRNA abundance varied markedly according to the type of cyclase. With type IV and type VII, hybridization signals were observed in all tissues after a much longer exposure time than that used to visualize type V and VI mRNAs. With left-ventricular mRNAs and similar exposure times, the hybridization signals obtained with the type V probe were much stronger than those obtained with the type VI probe, in complete agreement with our previous determination of relative type V and VI mRNA concentrations in the left ventricles of adult Sprague-Dawley rats [16]. No clear difference in the hybridization signal was detected between samples from control and failing ventricles, except with the probe for type VI adenylyl cyclase, the signal of which appeared to be weaker in failing ventricles than in controls.

View larger version (45K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Northern blot analysis of type IV, V, VI and VII adenylyl cyclase mRNAs in rat ventricle and brain. The five panels show the same blot, which has been hybridized successively with the four adenylyl cyclase cDNAs and with the GAPDH cDNA as described. Eight µg of poly-(A+) RNA purified from rat brain (B), control (C) and failing (F) ventricles was loaded on each lane. For type V and VI adenylyl cyclase and GAPDH probes, the final wash was 0.1xSSC, at 50°C for 15 min. For type IV and VII adenylyl cyclase probes, the final wash was 1xSSC at room temperature. Exposure times were 4 and 8 h for type V and VI adenylyl cyclase mRNAs, respectively, and 24 h for types IV and VII.

 
3.3 Quantification of type V and VI adenylyl cyclase mRNA isoforms expressed in rat ventricles
As in control ventricles, type V and VI adenylyl cyclase and GAPDH mRNAs were all detected by means of RPA in the non-infarcted area of the left ventricles of rats with myocardial infarction in Study 1 (not shown). No difference was observed in the raw concentration (before normalization to GAPDH mRNA) of the two adenylyl cyclase mRNA species in the left ventricles of the two infarcted groups compared to sham-operated controls and this was also the case for GAPDH mRNA. As a result, the mean type V and type VI mRNA-to-GAPDH mRNA ratios were similar in the two infarcted rat groups and were not different from those in controls. Results were more complex in Study 2. As in Study 1, no significant alteration in type V adenylyl cyclase mRNA concentration was observed in the left ventricles of rats with myocardial infarction compared to controls (Fig. 2). In contrast, the type VI mRNA concentration decreased in rats with myocardial infarction compared to controls, with an insignificant 30% decrease in rats with myocardial infarction and compensatory hypertrophy and a 35% decrease in rats with severe myocardial infarction and heart failure (0.59±0.07 vs. 0.91±0.10 au, p<0.05), which was associated with a 24% decrease in GAPDH mRNA concentration (0.72±0.07 vs. 0.95±0.05 au, p<0.05). As a result, the mean type V mRNA-to-GAPDH mRNA ratio tended to increase by 20% in rats with severe myocardial infarction and heart failure compared to controls, but this difference did not reach statistical significance (Fig. 3A). Because of the grossly parallel decrease in type VI adenylyl cyclase and GAPDH mRNA concentrations in rats with heart failure compared to controls, the mean type VI mRNA-to-GAPDH mRNA ratio did not change between the two rat groups (Fig. 3A). The decrease in GAPDH mRNA concentration in rats with severe myocardial infarction and heart failure compared to controls prompted us to perform dot blot analysis to normalize data to poly-(A⁺) and 18S RNAs (Fig. 4). This analysis confirmed the decrease in GAPDH mRNA concentration in the left ventricles of rats with severe myocardial infarction and heart failure compared to controls with a 20% decrease in the mean GAPDH mRNA-to-18S RNA ratio (0.76±0.03 vs. 0.95±0.04, p<0.05). When normalized to poly-(A⁺) or 18S RNA, type V adenylyl cyclase levels remained unchanged in the two infarcted rat groups compared to controls. In contrast, we found a 35% decrease in the type VI mRNA-to-poly-(A⁺) RNA ratio in rats with heart failure compared to controls (0.65±0.05 vs. 1.01±0.09, p<0.01; Fig. 3B). A 29% decrease was also observed when type VI adenylyl cyclase mRNA was normalized to 18S RNA (0.63±0.09 vs. 0.88±0.06, p<0.05; Fig. 3C).

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 RNase protection assay (RPA) of type V and VI adenylyl cyclase mRNA concentrations in the left ventricles of rats in Study 2. C1 and C2, control rats; H1 and H2, rats with compensatory left ventricular hypertrophy; F1 and F2, rats with heart failure. A 15-µg amount of total RNA was used in each assay. Type V and VI mRNA and (top) GAPDH mRNA hybridization signals are from the same autoradiogram exposure. (Bottom) GAPDH mRNA hybridization signals result from an exposure of shorter duration and were those used to calculate the type V and VI mRNA-to-GAPDH mRNA ratios. Lengths of the protected fragments are 459, 510 and 312 bp for type V and VI adenylyl cyclase and GAPDH mRNAs, respectively.

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Bar graph showing the mean (±SEM) adenylyl cyclase mRNA-to-GAPDH mRNA ratios (A), type VI adenylyl cyclase mRNA-to-poly-(A+) RNA ratios (B) and type VI adenylyl cyclase mRNA-to-18S RNA ratios (C) in the left ventricles of rats in Study 2. White and dark bars denote type V and VI adenylyl cyclase mRNAs, respectively. C, H and F: same as in Fig. 2. *p<0.05 and **p<0.01 vs. control value.

 
3.4 Adenylyl cyclase activities
Fig. 5 summarizes the results of the determination of the various adenylyl cyclase activities measured in the left ventricles of rats with severe myocardial infarction and heart failure and control rats in the two studies. In rats in Study 1, basal and GTP-stimulated Mg²⁺-dependent adenylyl cyclase activities were decreased by 30% (11±2 vs. 16±1 pmol cAMP min^–1 mg^–1, p<0.05) and 44% (15±3 vs. 27±1 pmol cAMP min^–1 mg^–1, p<0.05), respectively, in the left ventricles of rats with severe myocardial infarction and heart failure compared to controls (Fig. 5A). In sharp contrast, no difference was observed between the two rat groups when MnCl₂ was substituted for MgCl₂ in the assay medium. Similar results were observed when adenylyl cyclase activities were assayed in the presence of 10 µM forskolin (Fig. 5B).

View larger version (37K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Bar graph showing the mean (±SEM) Mg2+- and Mn2+-dependent adenylyl cyclase activities in the left ventricles of rats in Study 1 (A, B) and Study 2 (C, D) in the absence (A, C) and presence (B, D) of 10 µM forskolin. White and dark bars denote control rats and rats with heart failure, respectively. *p<0.05, **p<0.01 and ***p<0.001 vs. control value.

 
In rats in Study 2, basal adenylyl cyclase activity was decreased by 34% (15±1 vs. 23±2 pmol cAMP min^–1 mg^–1, p<0.01) in the left ventricles of rats with severe myocardial infarction and heart failure compared to controls, but the decrease in GTP-stimulated adenylyl cyclase activity did not reach statistical significance (Fig. 5C). As in Study 1, no difference was observed between the two rat groups when MnCl₂ was substituted for MgCl₂ in the assay medium. When adenylyl cyclase activities were assayed in the presence of 10 µM forskolin, basal and GTP-stimulated adenylyl cyclase activities were decreased by 35% (224±19 vs. 345±10 pmol cAMP min^–1 mg^–1, p<0.001) and 29% (279±26 vs. 391±14 pmol cAMP min^–1 mg^–1, p<0.01), respectively, in the left ventricles of rats with severe myocardial infarction and heart failure compared to controls (Fig. 5D). Here again, no difference was observed between the two rat groups when MnCl₂ was substituted for MgCl₂ in the assay medium.

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
This is the first study (i) to establish the relative expression of the main cardiac adenylyl cyclase mRNA isoforms in the left ventricle of normal adult rats and rats with heart failure, (ii) to show that the mRNA expression of the major isoform (type V) is unaltered in the left ventricles of rats with heart failure, whereas type VI adenylyl cyclase mRNA expression is decreased, (iii) two show that, despite decreased basal and stimulated Mg²⁺-dependent adenylyl cyclase activities, Mn²⁺-dependent adenylyl cyclase activities remain unaltered in the left ventricles of rats with heart failure. Six adenylyl cyclase isoforms are currently known to be expressed in the mammalian heart, namely types IV, V, VI and VII [11, 12]and, more recently reported, type II, which is essentially expressed in the central nervous system [13], and type IX, which is a very minor isoform in the adult heart [14]. Cardiac expression of type IV adenylyl cyclase has only been shown by PCR [25]and that of type VII has only been reported by Watson et al. [26]in a Northern blot analysis of poly-(A⁺) RNA. In addition, their concentrations have never been compared to those of other cardiac adenylyl cyclase mRNA species. Using Northern blot analysis, we observed that adenylyl cyclase mRNA isoforms types IV to VII are expressed in the rat left ventricle, the expression levels being as following: V > VI > IV=VII.

We examined ventricular concentrations of adenylyl cyclase mRNA isoforms in the rat model of myocardial infarction, a model that was characterized extensively by us [19, 22, 23]and others [21], because it provides rats with either compensatory hypertrophy or heart failure [21–23], thus giving the opportunity to compare the effects of these two hemodynamic conditions directly. Only type V and VI adenylyl cyclase mRNA concentrations were assessed precisely by RPA, as they are considered to be the main cardiac isoforms [11, 12], and because RPA with the simultaneous use of three probes applied to large numbers of samples is already complex. Moreover, Northern blot analysis revealed that these two mRNA isoforms remained far more abundant than the other two during heart failure. As expected, in Study 1, coronary ligation resulted in rats with or without heart failure. Chronic hemodynamic overload and hypertrophy of the non-infarcted area of the left ventricle was reflected by an increased left ventricular ANP mRNA concentration in both rat groups and the left ventricular weight-to-body weight ratio was increased only in rats with heart failure. In contrast to the altered left ventricular ANP mRNA concentrations, we found no change in type V or VI adenylyl cyclase mRNA raw concentrations or concentrations normalized to GAPDH mRNA in the left ventricle of rats with left ventricular hypertrophy or heart failure. GAPDH mRNA raw concentration also did not change between the three rat groups. Considering that this lack of change in adenylyl cyclase gene expression might have resulted from an insufficient duration of left ventricular hemodynamic overload [7], we performed a second study in which the duration of hemodynamic overload lasted for six months instead of two. At the time of killing, only a few infarcted rats in Study 2 exhibited a small pleural effusion, probably because of the premature death of those rats with the most severe myocardial infarctions, and heart failure was assessed on the two other criteria. As expected, only rats with heart failure exhibited a decreased left ventricular norepinephrine concentration compared to controls and, in slight contrast with failing rats in Study 1, rats with heart failure in Study 2 also exhibited increased left ventricular ANP mRNA concentrations compared to rats with compensatory hypertrophy, confirming a higher degree of left ventricular hemodynamic overload in the former. As in Study 1, no alteration in left ventricular type V adenylyl cyclase mRNA raw concentration was seen in rats with myocardial infarction with or without heart failure relative to controls. It was also the case when this concentration was normalized to GAPDH, poly-(A⁺) or 18S RNAs. In contrast, a 35% decrease in type VI adenylyl cyclase mRNA raw concentration was observed in rats with heart failure compared to controls. However, because of a 24% decrease in GAPDH mRNA concentration in rats with heart failure, the mean type VI mRNA-to-GAPDH mRNA ratio did not differ between the two rat groups. This prompted us to perform dot blot experiments in these rats to allow for normalizing the two adenylyl cyclase mRNAs concentrations to poly-(A⁺) and 18S RNAs. In fact, we observed a 35% decrease when we normalized type VI adenylyl cyclase mRNA to poly-(A⁺) RNA, indicating that the steady state concentration of this adenylyl cyclase species in total mRNA is decreased compared to other mRNAs in the left ventricles of rats with heart failure due to decreased gene transcription, decreased mRNA stability or both. We also observed a smaller 29% decrease when type VI adenylyl cyclase mRNA was normalized to 18S RNA because of an insignificant increase in the poly-(A⁺) RNA-to-18S RNA ratio in these rats. This might indicate that the overall mRNA transcription capacity and/or the overall mRNA stability is not altered in this model and at this stage of heart failure. It also cannot be excluded that an increase in the poly-(A⁺) RNA-to-18S RNA ratio in rats with left ventricular hypertrophy and failure in Study 1 would have resulted in a decrease in AC mRNA concentrations relative to poly-(A⁺) RNA. Undoubtedly, other studies using models of heart failure should be performed to address this specific question. Altogether, these data indicate that care should be taken when using GAPDH as a houskeeping gene to normalize other mRNA concentrations, at least in the context of heart failure, and they point to the importance of normalizing the concentration of a given mRNA species to total mRNA [poly-(A⁺)] and total RNA.

The specific downregulation of the type VI adenylyl cyclase mRNA isoform in rats with heart failure is in very good agreement with the recent findings of Ping et al. [13]in a pig model of heart failure based on rapid ventricular pacing. It is also reminiscent of what has been reported in the rat ventricle during ageing by Tobise et al. [15], extending the list of the similarities that exist regarding cardiac gene expression between ageing and cardiac hypertrophy and failure. In contrast, Ishikawa et al. [17]found a parallel downregulation of type V and VI adenylyl cyclase isoforms in a model of heart failure due to rapid ventricular pacing in the dog. The reason for the discrepancy with regards to the type V adenylyl cyclase gene expression between these two similar models of heart failure associated with decreased left ventricular adenylyl cyclase activities is unclear. Type V adenylyl cyclase downregulation in the dog model could be due to the severity of the model, in which many alterations occur as early as the first day of pacing [18]. Importantly, the results of the present study extend the notion of type VI adenylyl cyclase isoform downregulation to a more clinically relevant model in which heart failure occurs progressively in parallel with the development of a marked myocardial hypertrophy. Therefore, the fetal/neonatal adenylyl cyclase mRNA phenotype is not reexpressed during rat ventricular hypertrophy, in contrast to what is commonly observed with other gene families, such as myosin heavy chain, following myocardial infarction [19], as in other models of gradual cardiac hypertrophy and failure [20].

In rats with heart failure, our observation of decreased Mg²⁺-dependent adenylyl cyclase activities is in good agreement with many reports [12, 13, 34, 35], and especially those of Yamamoto et al. [36]and Sethi et al. [7], who used the same experimental model as ours. Our observation and that of Ping et al. [13], of decreased cyclase activities in failing ventricles, in parallel with the decreased type VI adenylyl cyclase mRNA concentration is reminiscent of the observation by Tobise et al. [15]of decreased adenylyl cyclase activities in the left ventricles of nine-month-old rats compared to young animals, which occurred in parallel with decreased type VI adenylyl cyclase concentrations, another situation in which adenylyl cyclase activity was not related to the type V mRNA concentration. Assuming that type VI adenylyl cyclase mRNA concentration reflects the concentration of the encoded protein, Tobise et al. [15]postulated that the decrease in cyclase activity in the left ventricles of these rats was related to the decrease in type VI adenylyl cyclase gene expression, but did not explain why the increased expression of type V adenylyl cyclase did not compensate for this decrease. It should be pointed out that comparisons between adenylyl cyclase activities and adenylyl cyclase mRNA concentrations have been put forward because, given our lack of isoform-specific adenylyl cyclase antibodies, we, and others, had no way of directly determining the actual amount of each cyclase type and thus had to rely on assays of total cyclase activity. However, it has been extensively established that total adenylyl cyclase activities do not directly reflect the amount of the enzyme [37]and that even forskolin-stimulated activities partly depend on the effect of G proteins [38].

Because uncoupled forms of adenylate cyclase utilize Mn-ATP but not Mg-ATP as substrate [32], we also measured basal and stimulated Mn²⁺-dependent adenylyl cyclase activities in the left ventricles of rats in the two study groups. Interestingly, and in sharp contrast with Mg²⁺-dependent adenylyl cyclase activities, we found that all Mn²⁺-dependent adenylyl cyclase activities were unchanged in failing ventricles compared to controls in the two studies, whatever the activity measured (basal or GTP-stimulated, in the absence or presence of 10 µM forskolin), suggesting that the overall amounts of adenylyl cyclase do not differ between the two hemodynamic conditions. In this context, unchanged Mn²⁺-dependent adenylyl cyclase activities in the left ventricles of rats with heart failure appear to be in discrepancy with the corresponding decrease in type VI adenylyl cyclase mRNA concentrations. However, if the protein concentration parallels that of the corresponding mRNA, it is likely that the effect of a moderate decrease in the protein concentration of the minor (type VI) of the two main cardiac adenylyl cyclase isoforms would be blunted at the level of overall adenylyl cyclase activities. In addition, as mRNA and protein expressions may be divergent, it is also possible that, despite differential regulation of the two cyclase isoforms in the failing rat heart, the protein concentrations remain unaltered because of different stabilities and/or translation efficiencies of the two adenylyl cyclase mRNA isoforms. This point will have to be clarified when isoform-specific adenylyl cyclase antibodies become available, as will the determination of the relative contribution of each isoform in the overall adenylyl cyclase activities of normal and failing hearts. Obviously, many factors may be involved in the decreased Mg²⁺-dependent adenylyl cyclase activities during heart failure, including decreased norepinephrine myocardial concentrations such as that found in the present study, β-adrenergic receptor desensitization and down-regulation [6–8], increased Gi and G protein receptor kinase gene expressions [10, 13, 36, 39], or some, as yet unidentified, posttranslational regulation of the enzyme. The diversity of the alterations observed according to the species, model and stage of heart failure point to the complexity of the mechanisms involved in the regulation of cAMP production in this setting and the need for isoform-specific antibodies that will allow one to address the precise role of each cyclase isoform in the production of cAMP in the various subcellular compartments [40]of myocytes of the normal, hypertrophied and failing myocardium.

Time for primary review 21 days.

	Acknowledgements

 
This work was supported in part by grants from the Fédération Française de Cardiologie, the Fondation pour la Recherche Médicale and the Fondation de France. Isabelle Espinasse was the recipient of a fellowship from the Association Française contre les Myopathies. The authors are grateful to Catherine Rücker-Martin, Marie-Claire Sant-Tillous, Valérie Domergue and Marie-Pascale Vincent for excellent technical assistance and to David Young for his help in restyling this manuscript.

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Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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