Cardiovascular Research 42 (1999) ` Marie-Claude Bourin, Françoise Samson, Eric Pussard, Jean-François Giudicelli, a d a,

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1 Crdiovsculr Reserch 42 (999) Decresed type VI denylyl cyclse mrna concentrtion nd Mg -dependent denylyl cyclse ctivities nd unchnged type V denylyl cyclse mrna concentrtion nd Mn -dependent denylyl cyclse ctivities in the left ventricle of rts with myocrdil infrction nd longstnding hert filure ` Mrie-Clude Bourin, Frnçoise Smson, Eric Pussrd, Jen-Frnçois Giudicelli, d, Jen-Bptiste Michel, Jcques Hnoune, Jen-Jcques Mercdier * b d Isbelle Espinsse, Vdim Iourgenko, Christine Richer, Michele Heimburger, Nicole Defer, d c b b Abstrct INSERM U 460, Fculte de Medecine Xvier Bicht, 6rue Henri Huchrd, F-7508 Pris, Frnce b Deprtement de Phrmcologie, Le Kremlin-Bicetre, ˆ Frnce c C.N.R.S. ERS 566, Le Plessis Robinson, Frnce d INSERM U 99, Hopitl ˆ Henri Mondor, Creteil, Frnce Received 0 Februry 998; ccepted 8 September 998 Objective: To ddress the effect of longstnding left ventriculr (LV) hypertrophy nd filure on LV denylyl cyclse (AC) gene expression, mrna concentrtions of the min crdic AC isoforms were mesured in the non-infrcted re of LV from rts with myocrdil infrction (MI), without (H) or with (F) LV filure, nd in control (C) rts. Bsl, GTP- nd forskolin-stimulted Mg - nd Mn -dependent AC ctivities were lso mesured in F nd C rts. Methods: Two- nd six months fter MI, stedy-stte AC mrna concentrtions were ssessed by Northern blot nlysis nd RNse protection ssy with isoform-specific cdna nd crna probes, respectively. AC ctivities were ssessed on LV microsoml frctions using stndrd procedures. Results: Types V nd VI, nd types IV nd VII were the mjor nd minor AC mrna isoforms in both the LVs of F nd C rts. Two months fter MI, no difference in LV type V or VI mrna to glycerldehyde-3-phosphte dehydrogense (GAPDH) mrna rtios ws observed in rts with H or F compred to C. Six months fter MI, no difference in LV type V mrna concentrtion ws observed between the three rt groups, whether this level ws normlized to GAPDH, poly-(a ) or 8S RNAs. In contrst, 35% decrese in the type VI mrna to poly-(a ) RNA rtio nd 29% decrese in the type VI mrna to 8S RNA rtio ws observed only in rts with F compred to C ( p,0.05 vs. C for the two comprisons). Two- nd six months fter MI, bsl nd forskolin-stimulted Mg -dependent AC ctivities were decresed by 30 35% in F rts compred to C ( p,0.05), wheres Mn -dependent ctivities were unchnged. Conclusion: Longstnding LV hypertrophy nd filure resulting from MI in rts is not ssocited with ltered expression of the most bundnt, type V, AC mrna isoform, wheres tht of type VI is decresed. The lck of chnge in Mn -dependent AC ctivities in the LV of F rts suggests tht this decrese hs no functionl consequence on overll AC ctivity nd tht decresed Mg -dependent ctivities re relted to ltertions occurring upstrem. 999 Published by Elsevier Science B.V. All rights reserved. Downloded from by guest on 4 December 208 Keywords: Adenylyl cyclse; Rt hert; Ventriculr hypertrophy; Hert filure. Introduction regultion by the G protein system, denylyl cyclse plys pivotl role in the regultion of myocrdil contrctility. Becuse of its function to generte camp nd its dul Ctecholmine binding to b- nd b2-drenergic receptors * Corresponding uthor. Tel.: ; fx: results in intrcellulr camp production through denylyl 657. E-mil ddress: jjmercd@prtique.fr (J.J. Mercdier) Time for primry review dys / 99/ $ see front mtter 999 Published by Elsevier Science B.V. All rights reserved. PII: S (98)00283-

2 88 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) cyclse stimultion by Gs []. This ctivtes protein filure in dogs or pigs is very peculir model of hert kinse A which, in turn, stimultes the inwrd clcium filure, chrcterized by contrctile nd biochemicl lter- current nd srcoplsmic reticulum C uptke through tions occurring s erly s the first dys of pcing, nd by phosphoryltion of the L-type clcium chnnel (or the bsence of significnt ventriculr hypertrophy closely ssocited protein) [2] nd phospholmbn [3], [3,7,8]. Importntly, it hs been widely documented respectively. Following stimultion of M2 muscrinic tht myocrdil hypertrophy, which is more commonly receptors, denylyl cyclse ctivity cn lso be inhibited observed during chronic hemodynmic overlod in exby Gi [4]. A number of studies hve shown tht perimentl models s in mn, is often ssocited with the ltertions in the b-drenergic signl trnsduction pthwy reexpression of fetl gene progrm [9,20]. Regrding re ssocited with the decresed crdic contrctility tht denylyl cyclse, this would led to decresed type V nd is chrcteristic of hert filure [5]. A fll in camp incresed type VI denylyl cyclse gene expression, s production following b-drenoceptor stimultion hs long recently hypothesized by Ishikw nd Homcy [2]. been known to occur in humn hert filure [6] nd in Therefore, the im of the present work ws to determine niml models [7], indicting ltertions t some point of denylyl cyclse gene expression in the non-infrcted left the trnsduction pthwy. A decrese in b-drenergic ventricle of rts with myocrdil infrction, well-chrreceptor density hs been demonstrted [7,8], nd this ws cterized experimentl model, providing nimls exhibconfirmed t the mrna level [9]. Results on G proteins iting either compenstory hypertrophy of the non-infrcted seem to vry ccording to the species nd cuse of hert left ventricle or hert filure [ 23]. In n ttempt to filure, but there is generl greement tht n increse in correlte gene expression with the ctivity of the enzyme, Gi mrna nd protein concentrtions occurs in the we lso mesured bsl nd stimulted Mg - nd Mn - filing humn ventricle [0]. dependent denylyl cyclse ctivities in control rts nd in Up to nine isoforms of mmmlin denylyl cyclse rts with hert filure. (types I IX) hve been reported to dte, differing in their mino cid sequence, tissue distribution, mode of ctivity regultion nd chromosoml loction of the coding genes 2. Methods []. Such diversity of single effector system probbly underlies specific signling functions of the vrious iso- 2.. Experimentl design forms expressed in given tissue or individul cell, nd points to differentil regultion of the expression of the Normotensive three-month-old mle Wistr rts (Iff vrious isoforms. Six denylyl cyclse mrna isoforms Credo), weighing g, were used. Animl cre (types II, IV to VII nd IX) hve been detected in the complied with the Principles of Lbortory Animl mmmlin hert, with types V nd VI being by fr the Cre, formulted by the Ntionl Society for Medicl most bundnt ones [ 4]. Types V nd VI pper to Reserch nd the Guide for the Cre nd Use of Lborhve similr enzymtic properties, s their ctivity is not tory Animls (NIH publiction no , revised 985, stimulted by C clmodulin nd is inhibited by sub- uthoriztion no , Ministere ` de l Agriculture, micromolr concentrtions of clcium [,2]. Tobise et Frnce). Left ventriculr infrction ws produced by l. [5] hve reported differentil expression of type V nd ligtion of the left coronry rtery under ether nesthesi, VI isoforms in the rt hert during development nd s described elsewhere []. Surviving rts were mingeing, nd we hve shown tht the overll concentrtion tined on stndrd rt chow nd wter d libitum. In our of types V nd VI denylyl cyclse mrna more thn hnds, this method results in 30 40% mortlity within doubles in the rt hert from the lte fetl stge to the first post-opertive dy, with n dditionl 0 5% dulthood, type V mrna being lmost ten times more mortlity within the first six months. Such model hs bundnt thn type VI in the dult ventricle [6]. This been shown to provide, three to four weeks fter coronry increse is ccompnied by prllel rise in bsl nd rtery occlusion, rts with left ventriculr infrctions isoproterenol- nd forskolin-stimulted denylyl cyclse rnging from 4 to 59% of the left ventriculr endocrdil ctivities, suggesting tht the incresed ctivity observed circumference, with or without hert filure, depending on during erly post-ntl rt development could due to the size of the infrction []. ccumultion of the corresponding proteins. In first set of experiments (Study ), the experimentl To our knowledge, only two studies hve ddressed period ws two months. During the week before killing, denylyl cyclse gene expression during hert filure. The ech rt ws plced in metbolic cge nd urine ws first showed prllel fll in stedy-stte mrna con- collected for 5 h for cgmp determintion. At the end of centrtions of types V nd VI denylyl cyclse in the the experimentl protocol, unnesthetised rts were killed cnine model of pcing-induced hert filure [7]. In by decpittion nd blood ws collected into prechilled contrst, in pcing-induced model of hert filure in the 0-ml heprinized tubes. Blood ws centrifuged for 0 min pig, only the type VI denylyl cyclse mrna concen- t 3000 g nd the plsm ws frozen in liquid nitrogen nd trtion ws decresed [3]. However, pcing-induced hert stored t 2808C. At the sme time, the hert ws excised Downloded from by guest on 4 December 208

3 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) nd rinsed in cold sline; the tri nd surrounding vessels mixture ws sonicted on ice twice for 5 s. Following were removed, the right ventricle ws dissected long its centrifugtion t 000 g for 5 min, ctecholmines in the septl insertion nd ech ventricle ws weighed. The superntnts were extrcted on lumin nd quntified by infrct scr ws crefully dissected under binoculr mens of high-performnce liquid chromtogrphy microscope to remove ll fibrous tissue from the djcent (HPLC) with mperometric detection, s described previleft ventricle, which ws frozen in liquid nitrogen nd ously [24]. stored t 2808C until RNA purifiction nd determintion of denylyl cyclse ctivity. In second set of experiments 2.4. Probes, RNA purifiction, Northern blot nd dot (Study 2), the experimentl period ws six months. blot nlysis Animls were killed nd herts were processed s described bove, except tht one smple of ech left ventricle Probes specific for type V nd VI denylyl cyclse ws used to determine the myocrdil ctecholmine mrnas nd GAPDH mrna were those used to trnscribe concentrtion. In ech set of experiments, n pproprite the ntisense crnas used in the RNse protection ssys number of shm-operted rts (C) underwent the entire (see below). Probes specific for type IV nd VII denylyl procedure, except for coronry rtery ligtion. Smples of cyclse mrnas were gift from R. Premont (Durhm, brin were lso tken from shm-operted rts t the time NC, USA). The probe specific for type IV denylyl cyclse of killing, to prepre control RNA. All tissues were frozen mrna ws n 800-bp cdna (BmHI ScI frgment from in liquid nitrogen immeditely fter smpling nd were published sequence, [25]), corresponding to the cyclse s stored t 2808C until use. first six membrne-spnning domins. The probe specific for type VII ws 722-bp cdna frgment (nucleotides 2.2. Identifiction of hert filure 2453 to 375 from the published sequence [26]). To normlize for the mount of RNA ctully present on ech Northern blot lne or dot, synthetic probe of 24 nucleo- In Study, three indexes were used for clssifiction of tides, complementry to sequence of rt 8S rrna, ws rts with myocrdil infrction: () the presence or bused. The GAPDH cdna is 573-bp frgment obtined sence of pleurl effusion; (2) right ventriculr weight-toby reverse trnscribed polymerse chin rection (RTbody weight rtio tht ws greter thn twice the control PCR) of rt ventriculr RNA (from nucleotides 244 nd vlue, n index tht hs been shown to testify for lrge 87 of the sequence published by Tso et l. [27]). The infrcts (.40%) ssocited with hert filure (left ven- poly-(a ) probe is synthetic oligo (dt) 5 (Promeg, WI, triculr end-distolic pressure: 2862 mmhg) [] nd (3) USA). In ddition, to further ssess the severity of left urinry cgmp tht ws greter thn twice the control ventriculr overlod nd the resulting hypertrophy, we vlue, very sensitive indictor of congestive hert filure used cdna probe of 70 bp in length tht ws specific in rts [22]. When pleurl effusion nd/or the two other for rt tril ntriuretic peptide (ANP) mrna (gift from E. criteri were present, the rt ws ssigned to the subgroup Toluny, Monsnto, St. Louis, MO, USA). of severe myocrdil infrction nd hert filure (F). The For Northern blot nlysis of denylyl cyclse mrnas rts tht did not fulfil these requirements were ssigned to in rt ventricle nd brin, given their wek concentrtion in the subgroup of moderte myocrdil infrction with the hert, we used 8 mg of poly-(a ) RNA purified from compenstory left ventriculr hypertrophy (H). In Study 2, ech tissue using the Fst Trck kit from Invitrogen s rts with myocrdil infrction rrely exhibited smll ccording to the mnufcturer s instructions. Totl RNA pleurl effusion, only the presence of the two other criteri ws purified ccording to Chomczinsky nd Scchi [28]. ws used to ssign rts to the group with hert filure. RNAs were quntified densitometriclly t nm. For Northern blot nlysis of ANP mrna nd 8S rrna, 2.3. Determintion of plsm ngiotensin-converting 0 mg of totl RNA from the relevnt tissues were used. enzyme (ACE) ctivity, urinry cgmp levels nd For dot blot nlysis, 6 mg of RNA were used nd seril myocrdil ctecholmine concentrtions dilutions (3,.5, 0.75 nd mg) were trnsferred to nylon membrne fter denturtion. cdna probes were Plsm ACE ctivity ws determined s described lbelled by rndom priming (Megprime lbelling kit, previously [23] by mesuring the hydrolysis of 00 mm 32 Amershm) with [ P]-dCTP, nd oligonucleotides with 4 [glycine-- C]hippuryl-L-histidyl-L-leucine (3 mci/ mmol, 32 [g P]-ATP using T4 kinse. Probes were dded to the 27 Amershm) in the presence or bsence of 0 M cptop- 6 prehybridiztion solution t finl concentrtion of 0 ril. Urinry cgmp levels were mesured by rdioim- cpm/ ml. After hybridiztion, membrnes were wshed t munossy using n Amershm kit (TRK 500) ccording vrious finl stringencies ccording to the probe nd to the mnufcturer s recommendtions [22]. For the utordiogrms were obtined fter 4 to 8 h of membrne determintion of myocrdil ctecholmine concentrtions, exposure, except for type IV nd VII denylyl cyclse frozen smples were crushed in liquid nitrogen nd the probes, which required longer exposure durtions (24 48 resulting powder ws mixed with perchloric cid. The h). Between ech hybridiztion step, membrnes were Downloded from by guest on 4 December 208

4 90 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) dehybridized by wshing t 958C in 0.5% sodium dodecyl speed setting of 4 5. To remove cellulr debris, the sulfte (SDS). homogentes were centrifuged t 600 g for 0 min t 48C. The superntnts were collected nd centrifuged t RNse protection ssy g for 20 min t 48C. The pellets were wshed in the sme volume of homogeniztion buffer contining mm EGTA Type V nd VI denylyl cyclse mrnas were quntified nd centrifuged under the sme conditions. The procedure in the sme tube by mens of ribonuclese protection ws performed three times. After the lst wsh, the pellets ssy (RPA), with one crna riboprobe specific for ech were resuspended in 50 mm Tris HCl, ph 7.4, t protein isoform nd one crna riboprobe specific for GAPDH concentrtion of.5 mg/ ml, s determined by the method mrna, s the internl stndrd, s described previously of Brdford [3], with bovine serum lbumin s stndrd, [6]. Briefly, to void cross-hybridiztion in the RNse nd were stored t 2808C. protection ssy, probes specific for type V nd type VI Adenylyl cyclse ctivity in prticulte membrne prepdenylyl cyclse mrnas were constructed from regions in rtions (20 30 mg of protein per ssy in finl volume which the cdnas exhibit the lowest sequence identity. of 60 ml) ws mesured in buffer consisting of (finl Type V denylyl cyclse cdna is 459-bp frgment concentrtions) 70 mm Tris HCl, ph 7.4, 4 mm phos- (mino cids 498 to 650 of the sequence published by phocretine, 32 units/ ml phosphocretine kinse, mm Premont et l. [29]) obtined by mens of PCR mplific- camp, 5 mm MgCl 2, mm ATP (disodium slt), tion. Type VI denylyl cyclse cdna is 50-bp frgment 0 cpm/ ssy [ P]ATP (tetr [triethylmmonium] slt) (mino cids 708 to 877 of the sequence published by (Amershm, UK), 0.2 mm EGTA, 0.3 mm PMSF. For Premont et l. [29]) obtined by mens of PCR from denylyl cyclse ctivity stimultion, 0 mm GTP nd/or plsmid (pgem 3Z) contining n 890-bp insert of the 0 mm forskolin (finl concentrtions) ws dded to the coding sequence of type VI denylyl cyclse [30]. The rection mixture. In ddition, becuse uncoupled forms of TM cdnas were subcloned in pcr II nd fully sequenced denylyl cyclse utilize Mn-ATP but not Mg-ATP s to check for the bsence of muttions during PCR. substrte [32], we performed experiments in which 5 mm Plsmids contining the pproprite inserts were linerized MnCl2 ws substituted for 5 mm MgCl2 in the ssy with XhoI for type V nd VI denylyl cyclse cdnas, nd medium. The rection ws strted by dding the memwith EcoNI for GAPDH cdna, resulting in probes of 459, brnes nd ws stopped fter 20 min t 308C by dding 50 nd 32 bp in length, respectively. The ntisense 200 ml of 0.5 M HCl. Tubes were then boiled nd crnas were trnscribed with SP6 polymerse using the neutrlized with 250 ml of.5 M imidzole, nd the 32 Gemini II trnscription kit (Promeg) nd [ P]-UTP. The 32 [ P]cAMP formed ws quntified s described elsewhere 32 specific ctivity of the two denylyl cyclse probes ws [33]. Results re expressed s pmol of [ P]cAMP 8 consistently cpm/mg, nd tht of the GAPDH formed per min per mg of protein nd re presented s 8 probe ws cpm/ mg. The RPA ws performed mens6sem of triplicte determintions. TM using the RPA II kit (Ambion) ccording to the mnufcturer s instructions fter checking tht the crna probes 2.7. Sttisticl nlysis were in lrge excess reltive to their respective trget mrna. Protected probe trget RNA duplexes were sep- Results re presented s mens6sem. In ech study, rted by electrophoresis in 5% polycrylmide gels t 250 significnt differences between the three rt groups were V for 6 h. Gels were exposed to Cronex film (Du Pont) for identified by using one-wy nlysis of vrince 4 h (GAPDH) nd for up to three dys (denylyl cyclse). (ANOVA). When the ANOVA reveled significnt differ- Severl exposure times were used nd the utordiogrms ence, group-to-group comprisons were performed using were scnned by the Strwise imging system for densito- the t-test for multiple comprisons. The threshold for metric nlysis (Imstr). A given RNse protection experi- sttisticl significnce ws set t p,0.05. ment lwys comprised left ventriculr smples from the three experimentl groups (hypertrophic nd filing herts, nd controls). Any experiment hd n dditionl dult rt 3. Results ventriculr RNA smple in common s n internl stndrd, which ws used to normlize the dt between the 3.. Min chrcteristics of rts in Studies nd 2 vrious experiments. Study comprised 20 rts with myocrdil infrction 2.6. Adenylyl cyclse ctivity ssy nd eight shm-operted rts. Ten out of the 20 rts with myocrdil infrction were considered to hve severe Left ventriculr smples were minced with scissors in myocrdil infrction with hert filure, bsed on the 20-fold excess (w/ v) of ice-cold homogeniztion buffer criteri described bove. The remining ten rts were (0 mm Tris HCl, ph 7.4, 5 mm MgCl 2, mm EDTA) considered to hve moderte myocrdil infrction with nd homogenized for 20 s using Polytron homogenizer t compenstory left ventriculr hypertrophy. The min Downloded from by guest on 4 December 208

5 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) Tble Min chrcteristics of rts in Study Control Hypertrophy Filure p-vlue (n58) (n50) (n50) (ANOVA) Body weight, g Pleurl effusion LV weight index, mg/ g ,0.05 RV weight index, mg/ g b ,0.00 Plsm ACE ctivity, b ,0.00 nmol/ min/ ml Urinry cgmp, nmol/ 5 h b ,0.00 LV ANP mrna,.u ,0.00 Control, shm-operted rts; Hypertrophy, rts with myocrdil infrction nd compenstory hypertrophy; Filure, rts with myocrdil infrction nd hert filure. LV weight index, left ventriculr weight-to-body weight rtio; RV weight index, right ventriculr weight-to-body weight rtio; ACE, ngiotensinconverting enzyme; LV ANP mrna, left ventriculr concentrtion of the tril ntriuretic peptide mrna;.u., rbitrry units. Vlues re expressed s the men6sem. b p,0.05 versus Control vlue; p,0.05 versus Hypertrophy vlue. chrcteristics of the three groups re listed in Tble. control vlue. The men left ventriculr weight-to-body Rts with severe myocrdil infrction nd hert filure weight rtio ws incresed in the two rt groups with exhibited significnt increses in the right ventriculr myocrdil infrction compred to the controls, demonweight-to-body weight rtio, plsm ACE ctivity nd strting mrked left ventriculr hypertrophy despite urinry cgmp, reltive to both controls nd rts with myocrdil muscle loss due to infrction. The myocrdil moderte myocrdil infrction. These rts lso exhibited norepinephrine concentrtion ws decresed only in the rt left ventriculr weight-to-body weight rtio tht ws higher group with severe myocrdil infrction nd hert filure, thn tht in controls, indicting mjor hypertrophy of the both compred to controls nd rts with compenstory non-infrcted left ventricle despite myocrdil muscle loss hypertrophy. Dot blot nlysis reveled, s in the rts in due to infrction. Northern blot nlysis of ANP mrna in Study, strong ccumultion of ANP mrna in the left the three groups reveled mrked ccumultion of this ventricles of the two infrcted rt groups reltive to species in the left ventricles of the two infrcted rt groups, controls, with three-fold increse in rts with hert filure consistent with chronic hemodynmic overlod nd hy- compred to those with compenstory hypertrophy (Tble pertrophy of the non-infrcted re of the left ventricles in 2, Fig. 4). these rts (Tble ). Study 2 comprised 6 rts with myocrdil infrction 3.2. Northern blot nlysis of denylyl cyclse isoforms nd eight shm-operted rts. The chrcteristics of these expressed in control nd filing rt ventricles nimls re listed in Tble 2. Among the 6 rts with myocrdil infrction, eight were ssigned to the group A typicl Northern blot nlysis of poly-(a ) RNA with severe myocrdil infrction nd hert filure, bsed purified from rt brin nd from the ventricles of control on right ventriculr weight-to-body weight rtio nd rts nd rts with hert filure in Study 2 (hybridized with urinry cgmp excretion tht ws more thn twice the probes specific for type IV, V, VI nd VII denylyl cyclse) Tble 2 Min chrcteristics of rts in Study 2 Control Hypertrophy Filure p-vlue (n58) (n58) (n58) (ANOVA) Body weight, g LV weight index, mg/ g ,0.0 RV weight index, mg/ g b ,0.00 Urinry cgmp, nmol/ 5 h b ,0.00 LV epinephrine, ng/ g LV norepinephrine, ng/ g b 7069,0.00 LV ANP mrna,.u b 76650,0.0 Control, shm-operted rts; Hypertrophy, rts with myocrdil infrction nd compenstory hypertrophy; Filure, rts with myocrdil infrction nd hert filure. LV weight index, left ventriculr weight-to-body weight rtio; RV weight index, right ventriculr weight-to-body weight rtio; LV epinephrine, left ventriculr epinephrine concentrtion; LV norepinephrine, left ventriculr norepinephrine concentrtion. LV ANP mrna, left ventriculr concentrtion of the tril ntriuretic peptide mrna;.u., rbitrry units. Vlues re expressed s the men6sem. b p,0.05 versus Control vlue; p,0.05 versus Hypertrophy vlue. Downloded from by guest on 4 December 208

6 92 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) Fig.. Northern blot nlysis of type IV, V, VI nd VII denylyl cyclse mrnas in rt ventricle nd brin. The five pnels show the sme blot, which hs been hybridized successively with the four denylyl cyclse cdnas nd with the GAPDH cdna s described. Eight mg of poly-(a ) RNA purified from rt brin (B), control (C) nd filing (F) ventricles ws loded on ech lne. For type V nd VI denylyl cyclse nd GAPDH probes, the finl wsh ws 0.3SSC, t 508C for 5 min. For type IV nd VII denylyl cyclse probes, the finl wsh ws 3SSC t room temperture. Exposure times were 4 nd 8 h for type V nd VI denylyl cyclse mrnas, respectively, nd 24 h for types IV nd VII. is presented in Fig.. The four mrna types were detected in the non-infrcted re of the left ventricles of rts with in ll of the smples studied. With both ventriculr nd myocrdil infrction in Study (not shown). No differbrin RNAs, type VI nd VII denylyl cyclse cdnas ence ws observed in the rw concentrtion (before hybridized s single bnds of pproximtely 6 nd 6.3 kb, normliztion to GAPDH mrna) of the two denylyl respectively, in greement with previous reports [26,29], cyclse mrna species in the left ventricles of the two wheres type V denylyl cyclse cdna hybridized s two infrcted groups compred to shm-operted controls nd shrp bnds of 7 nd 5 kb ech, lso in good greement this ws lso the cse for GAPDH mrna. As result, the with previous reports [5,29]. The type IV-specific probe men type V nd type VI mrna-to-gapdh mrna rtios hybridized with mrna of pproximtely 3.5 kb, in were similr in the two infrcted rt groups nd were not greement with Go nd Gilmn [25]. However, the different from those in controls. Results were more commrna bundnce vried mrkedly ccording to the type plex in Study 2. As in Study, no significnt ltertion in of cyclse. With type IV nd type VII, hybridiztion signls type V denylyl cyclse mrna concentrtion ws obwere observed in ll tissues fter much longer exposure served in the left ventricles of rts with myocrdil time thn tht used to visulize type V nd VI mrnas. infrction compred to controls (Fig. 2). In contrst, the With left-ventriculr mrnas nd similr exposure times, type VI mrna concentrtion decresed in rts with the hybridiztion signls obtined with the type V probe myocrdil infrction compred to controls, with n inwere much stronger thn those obtined with the type VI significnt 30% decrese in rts with myocrdil infrction probe, in complete greement with our previous determi- nd compenstory hypertrophy nd 35% decrese in rts ntion of reltive type V nd VI mrna concentrtions in with severe myocrdil infrction nd hert filure the left ventricles of dult Sprgue-Dwley rts [6]. No ( vs u, p,0.05), which ws ssocicler difference in the hybridiztion signl ws detected ted with 24% decrese in GAPDH mrna concentrtion between smples from control nd filing ventricles, ( vs u, p,0.05). As result, the except with the probe for type VI denylyl cyclse, the men type V mrna-to-gapdh mrna rtio tended to signl of which ppered to be weker in filing ventricles increse by 20% in rts with severe myocrdil infrction thn in controls. nd hert filure compred to controls, but this difference did not rech sttisticl significnce (Fig. 3A). Becuse of 3.3. Quntifiction of type V nd VI denylyl cyclse the grossly prllel decrese in type VI denylyl cyclse mrna isoforms expressed in rt ventricles nd GAPDH mrna concentrtions in rts with hert filure compred to controls, the men type VI mrna-to- As in control ventricles, type V nd VI denylyl cyclse GAPDH mrna rtio did not chnge between the two rt nd GAPDH mrnas were ll detected by mens of RPA groups (Fig. 3A). The decrese in GAPDH mrna con- Downloded from by guest on 4 December 208

7 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) Fig. 2. RNse protection ssy (RPA) of type V nd VI denylyl cyclse mrna concentrtions in the left ventricles of rts in Study 2. C nd C2, control rts; H nd H2, rts with compenstory left ventriculr hypertrophy; F nd F2, rts with hert filure. A 5-mg mount of totl RNA ws used in ech ssy. Type V nd VI mrna nd (top) GAPDH mrna hybridiztion signls re from the sme utordiogrm exposure. (Bottom) GAPDH mrna hybridiztion signls result from n exposure of shorter durtion nd were those used to clculte the type V nd VI mrna-to-gapdh mrna rtios. Lengths of the protected frgments re 459, 50 nd 32 bp for type V nd VI denylyl cyclse nd GAPDH mrnas, respectively. centrtion in rts with severe myocrdil infrction nd 3.4. Adenylyl cyclse ctivities hert filure compred to controls prompted us to perform dot blot nlysis to normlize dt to poly-(a ) nd 8S Fig. 5 summrizes the results of the determintion of RNAs (Fig. 4). This nlysis confirmed the decrese in the vrious denylyl cyclse ctivities mesured in the left GAPDH mrna concentrtion in the left ventricles of rts ventricles of rts with severe myocrdil infrction nd with severe myocrdil infrction nd hert filure com- hert filure nd control rts in the two studies. In rts in pred to controls with 20% decrese in the men Study, bsl nd GTP-stimulted Mg -dependent GAPDH mrna-to-8s RNA rtio ( vs. denylyl cyclse ctivities were decresed by 30% ( , p,0.05). When normlized to poly-(a ) or vs. 66 pmol camp min mg, p,0.05) nd 44% 8S RNA, type V denylyl cyclse levels remined un- (563 vs. 276 pmol camp min mg, p,0.05), chnged in the two infrcted rt groups compred to respectively, in the left ventricles of rts with severe controls. In contrst, we found 35% decrese in the type myocrdil infrction nd hert filure compred to con- VI mrna-to-poly-(a ) RNA rtio in rts with hert trols (Fig. 5A). In shrp contrst, no difference ws filure compred to controls ( vs , observed between the two rt groups when MnCl2 ws p,0.0; Fig. 3B). A 29% decrese ws lso observed substituted for MgCl2 in the ssy medium. Similr results when type VI denylyl cyclse mrna ws normlized to were observed when denylyl cyclse ctivities were 8S RNA ( vs , p,0.05; Fig. 3C). ssyed in the presence of 0 mm forskolin (Fig. 5B). Downloded from by guest on 4 December 208 Fig. 3. Br grph showing the men (6SEM) denylyl cyclse mrna-to-gapdh mrna rtios (A), type VI denylyl cyclse mrna-to-poly-(a ) RNA rtios (B) nd type VI denylyl cyclse mrna-to-8s RNA rtios (C) in the left ventricles of rts in Study 2. White nd drk brs denote type V nd VI denylyl cyclse mrnas, respectively. C, H nd F: sme s in Fig. 2. * p,0.05 nd ** p,0.0 vs. control vlue.

8 94 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) Fig. 4. Dot blot nlysis of left ventriculr RNAs from rts in Study 2. The sme dot blot ws successively hybridized with probes tht were specific for ANP mrna, GAPDH mrna, poly-(a ) RNA nd 8S RNA. For ech smple, 3,.5, 0.75 nd mg of totl RNA were dotted. Hybridiztion signls from two rts chrcteristic of ech group re shown (top nd bottom line of ech hybridiztion). C, H nd F: sme s in Fig. 2. Downloded from by guest on 4 December 208 Fig. 5. Br grph showing the men (6SEM) Mg - nd Mn -dependent denylyl cyclse ctivities in the left ventricles of rts in Study (A, B) nd Study 2 (C, D) in the bsence (A, C) nd presence (B, D) of 0 mm forskolin. White nd drk brs denote control rts nd rts with hert filure, respectively. * p,0.05, ** p,0.0 nd *** p,0.00 vs. control vlue.

9 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) In rts in Study 2, bsl denylyl cyclse ctivity ws precisely by RPA, s they re considered to be the min decresed by 34% (56 vs pmol camp min crdic isoforms [,2], nd becuse RPA with the mg, p,0.0) in the left ventricles of rts with severe simultneous use of three probes pplied to lrge numbers myocrdil infrction nd hert filure compred to con- of smples is lredy complex. Moreover, Northern blot trols, but the decrese in GTP-stimulted denylyl cyclse nlysis reveled tht these two mrna isoforms remined ctivity did not rech sttisticl significnce (Fig. 5C). As fr more bundnt thn the other two during hert filure. in Study, no difference ws observed between the two rt As expected, in Study, coronry ligtion resulted in rts groups when MnCl2 ws substituted for MgCl2 in the with or without hert filure. Chronic hemodynmic ssy medium. When denylyl cyclse ctivities were overlod nd hypertrophy of the non-infrcted re of the ssyed in the presence of 0 mm forskolin, bsl nd left ventricle ws reflected by n incresed left ventriculr GTP-stimulted denylyl cyclse ctivities were decresed ANP mrna concentrtion in both rt groups nd the left by 35% (22469 vs pmol camp min mg, ventriculr weight-to-body weight rtio ws incresed only p,0.00) nd 29% ( vs pmol camp in rts with hert filure. In contrst to the ltered left min mg, p,0.0), respectively, in the left ventricles ventriculr ANP mrna concentrtions, we found no of rts with severe myocrdil infrction nd hert filure chnge in type V or VI denylyl cyclse mrna rw compred to controls (Fig. 5D). Here gin, no difference concentrtions or concentrtions normlized to GAPDH ws observed between the two rt groups when MnCl2 ws mrna in the left ventricle of rts with left ventriculr substituted for MgCl2 in the ssy medium. hypertrophy or hert filure. GAPDH mrna rw concentrtion lso did not chnge between the three rt groups. Considering tht this lck of chnge in denylyl 4. Discussion cyclse gene expression might hve resulted from n insufficient durtion of left ventriculr hemodynmic over- This is the first study (i) to estblish the reltive lod [7], we performed second study in which the expression of the min crdic denylyl cyclse mrna durtion of hemodynmic overlod lsted for six months isoforms in the left ventricle of norml dult rts nd rts insted of two. At the time of killing, only few infrcted with hert filure, (ii) to show tht the mrna expression rts in Study 2 exhibited smll pleurl effusion, probbly of the mjor isoform (type V) is unltered in the left becuse of the premture deth of those rts with the most ventricles of rts with hert filure, wheres type VI severe myocrdil infrctions, nd hert filure ws sdenylyl cyclse mrna expression is decresed, (iii) two sessed on the two other criteri. As expected, only rts show tht, despite decresed bsl nd stimulted Mg - with hert filure exhibited decresed left ventriculr dependent denylyl cyclse ctivities, Mn -dependent norepinephrine concentrtion compred to controls nd, in denylyl cyclse ctivities remin unltered in the left slight contrst with filing rts in Study, rts with hert ventricles of rts with hert filure. Six denylyl cyclse filure in Study 2 lso exhibited incresed left ventriculr isoforms re currently known to be expressed in the ANP mrna concentrtions compred to rts with commmmlin hert, nmely types IV, V, VI nd VII [,2] penstory hypertrophy, confirming higher degree of left nd, more recently reported, type II, which is essentilly ventriculr hemodynmic overlod in the former. As in expressed in the centrl nervous system [3], nd type IX, Study, no ltertion in left ventriculr type V denylyl which is very minor isoform in the dult hert [4]. cyclse mrna rw concentrtion ws seen in rts with Crdic expression of type IV denylyl cyclse hs only myocrdil infrction with or without hert filure reltive been shown by PCR [25] nd tht of type VII hs only to controls. It ws lso the cse when this concentrtion been reported by Wtson et l. [26] in Northern blot ws normlized to GAPDH, poly-(a ) or 8S RNAs. In nlysis of poly-(a ) RNA. In ddition, their concen- contrst, 35% decrese in type VI denylyl cyclse trtions hve never been compred to those of other mrna rw concentrtion ws observed in rts with hert crdic denylyl cyclse mrna species. Using Northern filure compred to controls. However, becuse of 24% blot nlysis, we observed tht denylyl cyclse mrna decrese in GAPDH mrna concentrtion in rts with isoforms types IV to VII re expressed in the rt left hert filure, the men type VI mrna-to-gapdh mrna ventricle, the expression levels being s following: V. rtio did not differ between the two rt groups. This VI.IV5VII. prompted us to perform dot blot experiments in these rts We exmined ventriculr concentrtions of denylyl to llow for normlizing the two denylyl cyclse mrnas cyclse mrna isoforms in the rt model of myocrdil concentrtions to poly-(a ) nd 8S RNAs. In fct, we infrction, model tht ws chrcterized extensively by observed 35% decrese when we normlized type VI us [9,22,23] nd others [], becuse it provides rts with denylyl cyclse mrna to poly-(a ) RNA, indicting tht either compenstory hypertrophy or hert filure [ 23], the stedy stte concentrtion of this denylyl cyclse thus giving the opportunity to compre the effects of these species in totl mrna is decresed compred to other two hemodynmic conditions directly. Only type V nd VI mrnas in the left ventricles of rts with hert filure due denylyl cyclse mrna concentrtions were ssessed to decresed gene trnscription, decresed mrna stbility Downloded from by guest on 4 December 208

10 96 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) or both. We lso observed smller 29% decrese when cresed type VI denylyl cyclse mrna concentrtion is type VI denylyl cyclse mrna ws normlized to 8S reminiscent of the observtion by Tobise et l. [5] of RNA becuse of n insignificnt increse in the poly-(a ) decresed denylyl cyclse ctivities in the left ventricles RNA-to-8S RNA rtio in these rts. This might indicte of nine-month-old rts compred to young nimls, which tht the overll mrna trnscription cpcity nd/ or the occurred in prllel with decresed type VI denylyl overll mrna stbility is not ltered in this model nd t cyclse concentrtions, nother sitution in which denylyl this stge of hert filure. It lso cnnot be excluded tht cyclse ctivity ws not relted to the type V mrna n increse in the poly-(a ) RNA-to-8S RNA rtio in rts concentrtion. Assuming tht type VI denylyl cyclse with left ventriculr hypertrophy nd filure in Study mrna concentrtion reflects the concentrtion of the would hve resulted in decrese in AC mrna con- encoded protein, Tobise et l. [5] postulted tht the centrtions reltive to poly-(a ) RNA. Undoubtedly, other decrese in cyclse ctivity in the left ventricles of these studies using models of hert filure should be performed rts ws relted to the decrese in type VI denylyl cyclse to ddress this specific question. Altogether, these dt gene expression, but did not explin why the incresed indicte tht cre should be tken when using GAPDH s expression of type V denylyl cyclse did not compenste houskeeping gene to normlize other mrna concentr- for this decrese. It should be pointed out tht comprisons tions, t lest in the context of hert filure, nd they point between denylyl cyclse ctivities nd denylyl cyclse to the importnce of normlizing the concentrtion of mrna concentrtions hve been put forwrd becuse, given mrna species to totl mrna [poly-(a )] nd totl given our lck of isoform-specific denylyl cyclse nti- RNA. bodies, we, nd others, hd no wy of directly determining The specific downregultion of the type VI denylyl the ctul mount of ech cyclse type nd thus hd to rely cyclse mrna isoform in rts with hert filure is in very on ssys of totl cyclse ctivity. However, it hs been good greement with the recent findings of Ping et l. [3] extensively estblished tht totl denylyl cyclse ctivities in pig model of hert filure bsed on rpid ventriculr do not directly reflect the mount of the enzyme [37] nd pcing. It is lso reminiscent of wht hs been reported in tht even forskolin-stimulted ctivities prtly depend on the rt ventricle during geing by Tobise et l. [5], the effect of G proteins [38]. extending the list of the similrities tht exist regrding Becuse uncoupled forms of denylte cyclse utilize crdic gene expression between geing nd crdic Mn-ATP but not Mg-ATP s substrte [32], we lso hypertrophy nd filure. In contrst, Ishikw et l. [7] mesured bsl nd stimulted Mn -dependent denylyl found prllel downregultion of type V nd VI denylyl cyclse ctivities in the left ventricles of rts in the two cyclse isoforms in model of hert filure due to rpid study groups. Interestingly, nd in shrp contrst with ventriculr pcing in the dog. The reson for the dis- Mg -dependent denylyl cyclse ctivities, we found tht crepncy with regrds to the type V denylyl cyclse gene ll Mn -dependent denylyl cyclse ctivities were unexpression between these two similr models of hert chnged in filing ventricles compred to controls in the filure ssocited with decresed left ventriculr denylyl two studies, whtever the ctivity mesured (bsl or cyclse ctivities is uncler. Type V denylyl cyclse GTP-stimulted, in the bsence or presence of 0 mm downregultion in the dog model could be due to the forskolin), suggesting tht the overll mounts of denylyl severity of the model, in which mny ltertions occur s cyclse do not differ between the two hemodynmic erly s the first dy of pcing [8]. Importntly, the conditions. In this context, unchnged Mn -dependent results of the present study extend the notion of type VI denylyl cyclse ctivities in the left ventricles of rts with denylyl cyclse isoform downregultion to more clini- hert filure pper to be in discrepncy with the correclly relevnt model in which hert filure occurs pro- sponding decrese in type VI denylyl cyclse mrna gressively in prllel with the development of mrked concentrtions. However, if the protein concentrtion myocrdil hypertrophy. Therefore, the fetl/ neontl prllels tht of the corresponding mrna, it is likely tht denylyl cyclse mrna phenotype is not reexpressed the effect of moderte decrese in the protein conduring rt ventriculr hypertrophy, in contrst to wht is centrtion of the minor (type VI) of the two min crdic commonly observed with other gene fmilies, such s denylyl cyclse isoforms would be blunted t the level of myosin hevy chin, following myocrdil infrction [9], overll denylyl cyclse ctivities. In ddition, s mrna s in other models of grdul crdic hypertrophy nd nd protein expressions my be divergent, it is lso filure [20]. possible tht, despite differentil regultion of the two In rts with hert filure, our observtion of decresed cyclse isoforms in the filing rt hert, the protein Mg -dependent denylyl cyclse ctivities is in good concentrtions remin unltered becuse of different greement with mny reports [2,3,34,35], nd especilly stbilities nd/ or trnsltion efficiencies of the two those of Ymmoto et l. [36] nd Sethi et l. [7], who denylyl cyclse mrna isoforms. This point will hve to used the sme experimentl model s ours. Our observ- be clrified when isoform-specific denylyl cyclse ntition nd tht of Ping et l. [3], of decresed cyclse bodies become vilble, s will the determintion of the ctivities in filing ventricles, in prllel with the de- reltive contribution of ech isoform in the overll Downloded from by guest on 4 December 208

11 I. Espinsse et l. / Crdiovsculr Reserch 42 (999) denylyl cyclse ctivities of norml nd filing herts. tion nd selective b receptor down-regultion in hert filure. Circ Obviously, mny fctors my be involved in the decresed Res 986;59: [9] Ungerer M, Bohm M, Elce JS, Erdmn E, Lohse MJ. Altered Mg -dependent denylyl cyclse ctivities during hert expression of b-drenergic receptor kinse nd b-drenergic refilure, including decresed norepinephrine myocrdil ceptors in the filing humn hert. Circultion 993;87: concentrtions such s tht found in the present study, [0] Eschenhgen T, Mende U, Nose M, et l. Increse messenger RNA b-drenergic receptor desensitiztion nd down-regultion level of the inhibitory G protein lph subunit Gi2 in humn end [6 8], incresed Gi nd G protein receptor kinse gene stge hert filure. Circ Res 992;70: [] Hnoune J, Pouille Y, Tzvr E, et l. Adenylyl cyclses: structure, expressions [0,3,36,39], or some, s yet unidentified, regultion nd function in n enzyme superfmily. Mol Cell posttrnsltionl regultion of the enzyme. The diversity of Endocrinol 997;28: the ltertions observed ccording to the species, model [2] Ishikw Y, Homcy CJ. The denylyl cyclses s integrtors of nd stge of hert filure point to the complexity of the trnsmembrne signl trnsduction. Circ Res 997;80: mechnisms involved in the regultion of camp proprotein receptor kinse expression during development of hert [3] Ping P, Anzi T, Go M, Hmmond HK. Adenylyl cyclse nd G duction in this setting nd the need for isoform-specific filure. Am J Physiol 997;273:H707 H77. ntibodies tht will llow one to ddress the precise role of [4] Premont RT, Mtsuok I, Mttei MG, et l. Identifiction nd ech cyclse isoform in the production of camp in the chrcteriztion of widely expressed form of denylyl cyclse. J vrious subcellulr comprtments [40] of myocytes of the Biol Chem 997;27: norml, hypertrophied nd filing myocrdium. [5] Tobise K, Ishikw Y, Holmer SR, et l. Chnges in type VI denylyl cyclse isoform expression correltes with decresed cpcity for camp genertion in the ging ventricle. Circ Res 994;74: Acknowledgements [6] Espinsse I, Iourgenko V, Defer N, et l. Type V, but not type VI, denylyl cyclse mrna ccumultes in the rt hert during ontogenic development. Correltion with incresed denylyl cyclse This work ws supported in prt by grnts from the ctivity. J Mol Cell Crdiol 995;27: Federtion Frnçise de Crdiologie, the Fondtion pour l [7] Ishikw Y, Sorot S, Kiuchi K, et l. Downregultion of denylyl Recherche Medicle nd the Fondtion de Frnce. Isbelle cyclse types V nd VI mrna levels in pcing induced hert filure Espinsse ws the recipient of fellowship from the in dogs. J Clin Invest 994;93: Assocition Frnçise contre les Myopthies. The uthors [8] Vtner DE, Sto N, Kiuchi K, Shnnon RP, Vtner SF. Decrese in myocrdil rynodine receptors nd ltered excittion contrction re grteful to Ctherine Rucker-Mrtin, Mrie-Clire coupling erly in the development of hert filure. Circultion Snt-Tillous, Vlerie Domergue nd Mrie-Pscle Vincent 994;90: for excellent technicl ssistnce nd to Dvid Young for [9] Michel JB, Lttion AL, Slzmnn JL, et l. Hormonl nd crdic his help in restyling this mnuscript. effects of converting enzyme inhibition in rt myocrdil infrction. Circ Res 988;62: [20] Schwrtz K, Boheler KR, de l Bstie D, Lompre AM, Mercdier JJ. References Switches in crdic muscle gene expression s result of pressure nd volume overlod. Am J Physiol 992;262:R364 R369. [] Fletcher PJ, Pfeffer JM, Pfeffer MA, Brunwld E. Left ventriculr [] Susnni EE, Vtner DE, Homcy CJ. bet-adrenergic receptor/ distolic pressure volume reltions in rts with heled myocrdil denylyl cyclse system. In: Fozzrd HA, Hber E, Jennings RB, Ktz infrction. Circ Res 98;49: AM, Morgn HE, editors. The hert nd the crdiovsculr system. 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