Cardiovascular Research 39 (1998) Giovanni Battista Ambrosio, Luciano Dalla Libera

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1 Crdiovsulr Reserh 39 (1998) Skeletl musle myosin hevy hin expression in rts with monorotlineindued rdi hypertrophy nd filure. Reltion to blood flow nd degree of musle trophy, b b b Giorgio Vesovo *, Cludio Ceoni, Plmir Bernohi, Roberto Ferrri, Ugo Crrro, Giovnni Bttist Ambrosio, Luino Dll Liber First Division of Internl Mediine, Venie City Hospitl, Cstello, Venie, Itly b Centro di Fisioptologi Crdiovsolre, Fondzione Mugeri, Gussgo, Bresi, Itly CNR Unit for Musle Biology nd Pthophysiology, University of Pdu, Pdu, Itly Reeived 11 September 1997; epted 28 Jnury 1998 Abstrt Bkground: In ongestive hert filure (CHF) the skeletl musle of the lower limbs develops myopthy hrterised by trophy nd shift from the slow to the fst type fibres. The mehnisms responsible for these hnges re not ler yet. Objetives: We investigted the influene of blood flow nd degree of musle trophy on the myosin hevy hins (MHC) omposition of the soleus nd extensor digitorum longus (EDL) of rts with right ventrile hypertrophy nd filure. Methods: CHF ws indued in 16 rts by injeting 30 mg/ kg monorotline. Eight nimls hd the sme dose of monorotline but resulting in ompensted right ventrile hypertrophy. Two ge- nd diet-mthed groups of ontrol nimls (nine nd five respetively) were lso studied. The reltive perentge of MHC1 (slow isoform), MHC2 (fst oxidtive) nd MHC2b (fst glyolyti) ws determined by densitometri sn fter eletrophoreti seprtion. The reltive weights of soleus nd EDL (musle weight/ body weight) were tken s n index of musle trophy. Skeletl musle blood flow ws mesured by injeting fluoresent mirospheres. Results: CHF nd Control (Con) rts showed similr degree of trophy both in soleus ( vs p5ns), nd EDL ( vs , p50.09). In CHF rts these two musles showed sttistilly signifint MHCs redistribution towrd the fst type isozymes. In ft in EDL of CHF rts MHC2 ws % vs % of the Con ( p,0.05). MHC2b ws however higher ( % vs %, p50.017). In the soleus of CHF rts MHC1 ws deresed ( % vs %, p50.02), while MHC2 ws inresed ( % vs %; p50.028). Similr hnges were not found in the musles of the ompensted hypertrophy nimls. No orreltion ws found between MHC pttern nd the reltive musle weight in the CHF nimls. Soleus blood flow in CHF rts ws signifintly lower thn tht of Con ( ml/ min/ g vs p,0.05), while no differenes were found in EDL ( ml/ min/ g vs , p5ns). Conlusions: In rts with CHF skeletl musle myopthy hrterised by shift of the MHCs towrd the fst type isoforms ours. The mgnitude of the shift orreltes neither with the degree of trophy, nor with the skeletl musle blood flow, suggesting tht these two ftors do not ply pivotl role in the pthogenesis of the myopthy Elsevier Siene B.V. All rights reserved. Keywords: Hert filure; Myosin hevy hins; Skeletl musle; Monorotline 1. Introdution hve been desribed. These involve loss of musle bulk [3], hnges in musle fibres with redution of the type 1 Congestive hert filure (CHF) is hrterised by nd shift towrd the fst type 2b [3 6], mitohondril disordered skeletl musle performne tht n in prt bnormlities [7], ltered musle metbolism [8,9]. explin ftigue nd shortness of breth [1,2]. A series of Whether musle trophy due to deonditioning plys role bnormlities possibly ontributing to exertionl ftigue in the genesis of this myopthy is not ler yet. We hve reently suggested tht the CHF myopthy is speifi being * Corresponding uthor. Tel.: 139 (41) ; Fx: 139 (41) ; E-mil: ldl@iv.bio.unipd.it Time for primry review 30 dys / 98/ $ Elsevier Siene B.V. All rights reserved. PII: S (98)

2 234 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) not relted to detrining. The gstronemius of ptients n index of musle trophy. Right ventrile weight/ body with profound disuse trophy shows in ft preferentil weight (RVW/ BW) ws lulted nd tken s n index synthesis of the slow myosin hevy hin (MHC) 1 [10], of right ventrile hypertrophy. Before srifie blood but the debte on the possible uses of this myopthy is smple ws lso drwn for determintion of b-anp (Atril still open. It hs been hypothesised tht ytokine tivtion Ntriureti Peptide). The ssy ws performed with the nd loss of nboli funtion [11], ergo metboloreeptors method previously desribed by Comini et l. [17]. dysfuntion [12], or hnges in blood flow [13,9] my be Experiments were pproved by the Ethil Committee of of importne. This ltter point is still debted nd there the Interdeprtmentl Biologil Building of the University re onfliting reports on the role of blood flow. Wilson et of Pdu, nd were rried out ording to the Itlin l. [13] hve in ft reported n impired nutritive flow to lws. skeletl musle, while Mssie et l. [9] showed no reltion between severity of CHF nd blood flow. The present study ws undertken in the ttempt to shed some light on 2.2. Effet of monorotline on the skeletl musle the pthophysiologil role of musle trophy nd nutritive Although seletive trget orgns for monorotline re blood flow on skeletl musle ltertions. We used well liver nd lung [18,19], we looked t MHCs hnges in the hrterised nd lrgely epted niml model of hert EDL nd Sol of four rts, 2 weeks fter the injetion of 30 filure [14,15]: the monorotline-treted rt. Mono- mg/ kg monorotline. Four ge- nd diet-mthed ontrols rotline is pyrrolizidine lkloid tht indues pulmonry were lso killed. RVW/BW, sol/bw nd EDL/BW were vsulr disese with severe hypertension, right ventrile mesured. hypertrophy nd in some nimls overt CHF hert filure [16]. We lso ompred nimls with overt hert filure to rts with ompensted right ventrile hypertrophy Eletrophoreti seprtion of MHCs 2. Methods 2.1. Experimentl model Four groups of nimls were studied: 16 nimls with CHF (CHF) 8 nimls with right ventrile hypertrophy (Hyp) 9 sline-injeted ontrols for the CHF group (Con) 5 sline-injeted ontrols for the Hyp group (ConHyp) Sprgue Dwley rts of weight 75 to 100 g were injeted intrperitonelly with monorotline (30 mg/ kg). Treted rts were llowed to et freely from supply of stndrd rt ubes. The ontrol rts were diet-mthed to the treted rts by llowing them only the quntity of food onsumed on the previous dy by the treted rts. The CHF rts re those nimls tht 4 weeks fter monorotline injetion develop pleurl, perirdil nd peritonel effusions, - ompnied by severe right ventrile hypertrophy nd dilttion. Rts tht do not develop signs of deompenstion t 4 weeks (Hyp) [16], re kept live for n extr week. They develop onentri right ventrile hypertrophy, but not CHF. They hve ge- nd diet-mthed ontrols sine they grow slightly more, rehing higher body weight. Rts were kept in m ges. After 4 nd 5 weeks respetively, nimls were killed, body weight (BW), right ventrile weight (RVW), lung weight mesured, together with pleurl nd peritonel effusions. soleus (Sol) nd extensor digitorum longus (EDL) were exised nd weighed. The rtio Sol/ BW nd EDL/ BW (reltive musle weight) ws then lulted nd tken s The method is n improvement of tht published by Crrro nd Ctni [20] nd desribed in detil by Vesovo et l. [10]. Musles were homogenised nd solubilised in 2.3% sodium dodeyl sulphte (SDS), 10% glyerol, 0.5% 2-merptoethnol, nd 6.25 mm Tris HCl, ph 6.8. The running buffer ws dded to the smple in rtio of 1:1 (v/ v). Anlytil SDS-polyrylmide gel eletrophoresis of MHC ws performed on 7.5% polyrylmide slbs with 37.5% (v/v) glyerol. The stking gel ws omposed of 37.5% glyerol, 4% T rylmide Bis (36.5:1), 12.5 mm Tris HCl (ph 6.8), nd 0.1% SDS. Running buffer ws mde of 50 mm Tris, 384 mm glyine, ph 8.3, nd 0.2% SDS. Seprtion of MHC ws hieved using onstnt urrent t 4 ma. After 12 h the running buffer ws hnged nd eletrophoresis restrted with the sme prmeters for nother 24 h. Gel ontining pproximtely 0.2 mg of protein per bnd ws stined with 0.1% Coomssie brillint blue in 5% eti id 40% methnol nd destined with 40% methnol 7% eti id. Identifi- tion of individul MHCs ws performed in seprte series of experiments by immunoblotting the gel bnds with pnel of monolonl ntibodies [10] Assessment of MHCs ontent The perent distribution of MHC1, MHC2 nd MHC2b ws determined by densitometri sn of the stined slb gels. A GS300 trnsmittne refletne snning densitometer (Hoefer Sientifi Instruments) onneted to MIntosh SE Apple omputer ws used. Dt were pro- essed with GS370 densitometry softwre. A liner re- sponse is obtined on densitometry when mg of individul MHC is nlysed. Quntittive densitometry

3 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) ws performed using internl MHC stndrds eletrophoresed in the sme gel s desribed by Sndri et l. [21]. higher thn tht of other previously employed methods in whih rdiotive nd oloured mirospheres were used Skeletl musle blood flow determintion 2.6. Histology nd morphometri nlysis Skeletl musle blood flow ws determined ording to On eh musle ross-setion ws tken for histologithe Glenny et l. method [22] in nother group of nimls: l exmintion. Setions were stined with Azn Mllory. six CHF, ten Con, eight Hyp nd six ConHyp rts were The ross-setionl re of fibres nd the volume frtion studied. The rts were nesthetised with pentobrbitl (45 of extrellulr spe, whih is omposed by interstitium mg/kg body weight) nd nnul ws introdued into the plus fibrosis, were lulted by mens of omputerised rotid rtery for the injetion of fluoresent mirospheres. intertive method using Zeiss Ibs 2000 instrument, s Red mirospheres [FluoSpheres polystyrene, 15mm, red desribed by Angelini et l. [23]. fluoresent (580/ 605)] for blood flow determintion were supplied s suspension in 10 ml of 0.15 M NCl with 0.02% Tween 20 nd 0.02% thimerosl. The suspension 2.7. Sttistil nlysis 6 ontining 1.0?10 mirospheres/ ml (Moleulr Probes Student s t-test for unpired dt nd liner regression Europe) ws vigorously vortexed for 3 5 min nd ulws used when pproprite. Men6S.D. re reported. A trsonited. Vi the rotid nnul it ws injeted into the 5 5% differene ws onsidered sttistilly signifint. left ventrile over 10 s in volume of 400 ml (4?10 mirospheres). The nnul ws then flushed with 0.5 ml of heprinised sline. Strting 10 s before the injetion of the fluorospheres, referene blood smple ws olleted 3. Results for 90 s from the femorl rtery nnul t onstnt rte of 0.51 ml/min. The skeletl musles (Sol nd EDL) were 3.1. Postmortem exmintion (Tble 1) exised, weighed nd digested overnight in 4 ml of 4 M KOH. The digested tissue piees were then individully A postmortem exmintion of the rts ws rried out in filtered through 10 mm pore polyrbonte filters (Poretis, order to ssess the presene or bsene of CHF nd of Livermore, CA, USA) by use of negtive pressure. The ompensted rdi hypertrophy in the different groups of tubes nd filtering burettes (Millipore) were wshed twie nimls ording to the riteri listed in Setion 2. The with 2.0% Tween, nd the olleted fluid ws filtered to presene of CHF ws onfirmed in 16 rts (CHF group). pik up residul mirospheres. Filters were pled in Conentri ompensted right ventrile hypertrophy ws polypropylene test tubes nd the fluoresent dye eluted deteted in eight rts (Hyp group). No ltertions were from the retined mirospheres by 1 ml of Cellosolve found in the ontrol rts. The ourrene of CHF ws lso ette. Fluoresene ws determined by spetroflthe presene of sites nd pleurl effusions nd by the onfirmed by the RVW/BW, by the lung weight/bw, by uorophotometer. Tissue blood flow ould then be lub-anp ssy whih showed signifintly higher levels of lted with respet to the referene blood smple, using the following eqution: this peptide in the deompensted nimls (see Tble 1). flow to smple (ml/min) 5 (fluoresene intensity of smple) 3 (referene withdrwl rte ml/ min) ]]]]]]]]]]]]]]]] fluoresene intensity in referene blood smple In our series of experiments the reltionship between number of mirospheres nd fluoresene intensity ws liner (r.0.99) nd the estimted number of mirospheres per smple ws between 100 nd 200. The detetion limit of the fluoresent mirospheres method is two- to fourfold 3.2. Body weight in CHF, Hyp nd ontrol rts The Hyp rts nd their reltive ontrols showed similr BW ( g vs respetively; p5ns), whih were both signifintly ( p50.001) higher thn those of the CHF nimls nd their reltive ontrols. Between these ltter groups BW lso differed signifintly ( vs ; p50.005). Tble 1 Right ventrile weight/ body weight (RVW/ BW), lung weight/ body weight, sites, pleurl effusions nd b-anp in the Con, CHF, ConHyp nd Hyp rts Con CHF ConHyp Hyp RVW/ BW b b Lung weight/ BW (mg/ g) Asites (ml) Pleurl effusions (ml) Plsm b-anp (fmol/ ml) b p,0.01, p,0.01, p,0.001.

4 236 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) Tble 2 EDL nd Sol weight in CHF nd Con rts, Hyp nd ConHyp rts CHF Con Hyp ConHyp Musle weight (mg) EDL b (n516) (n59) b (n58) (n55) Musle weight (mg) Sol d (n516) d (n59) (n58) (n55) Reltive musle weight EDL (n516) (n59) (n58) (n55) Reltive musle weight Sol (n516) (n59) e (n58) e (n55) Reltive musle weight (musle weight/ body weight) in the sme four groups of nimls. b d e p,0.002, p,0.0001, p,0.001, p,0.002, p, Sol nd EDL weight (Tble 2) similr in the four groups of nimls for both EDL nd Sol. Sol weight ws signifintly higher in Hyp rts thn in CHF ( p,0.001). No sttistilly signifint differenes 3.6. MHCs eletrophoreti pttern were found between Hyp nd ConHyp ( p5ns), while CHF rts showed lower Sol weight when ompred to MHC isoforms were seprted s desribed in Setion 2 Con ( p,0.002). Similrly EDL weight ws identil in nd re presented in Fig. 1. In rt skeletl musle three the Hyp nd ConHyp groups ( p5ns), though signifint MHC isoforms (MHC1, MHC2b nd MHC2) were sepdifferene ould be noted between CHF nd Con rts ( p,0.002). As expeted CHF rts showed muh lower EDL weight thn Hyp rts ( p,0.0001). rted on the bsis of their reltive mobility EDL [Fig. 2( nd b)] MHC2 in Con rts ws % vs of the 3.4. Reltive musle weights (Tble 2) CHF ( p,0.05). MHC2b ws signifintly lower in the Con thn in the CHF rts ( vs , p5 The Sol weight/bw ws similr in CHF nd Con rts 0.017). A similr shift towrd the fst type isoforms ws ( p5ns). In the Hyp the Sol reltive musle weight ws not present in the Hyp rts tht showed perentge of signifintly higher thn in the ConHyp ( p,0.009). EDL MHC2 nd MHC2b similr to tht of the ConHyp weight/ BW of CHF rts ws slightly though not sig- ( vs , p5ns nd vs. nifintly lower thn tht of Con ( p50.09). In the Hyp p5ns respetively). nd ConHyp groups the rtio ws lmost identil ( p5 NS) Sol [Fig. 3( nd b)] The Sol showed hnges in the isozyme pttern similr 3.5. Histology nd morphometri nlysis to those seen in EDL with shift towrd the fst MHCs. CHF nimls showed in ft signifintly lower per- There were no differenes in fibres ross-setionl re entge of MHC1 when ompred to Con ( vs. between CHF nd Hyp nimls nd their reltive ontrols , p50.02), while MHC2 ws signifintly high- 2 ( m in CHF vs in Con, p50.2; er ( vs , p50.028). In the Hyp group in Hyp vs in Con Hyp, p5ns). no hnges were deteted when ompred to ConHyp The fibrosis nd interstitil volume frtions, mesured either for MHC1 ( vs , p5ns) or ltogether, rnged from 3.8 to 4.2%, nd were therefore MHC2 ( vs , p5ns). Fig. 1. Eletrophoreti seprtion on polyrylmide SDS slb-gel of myosin hevy hins (MHC) (rrows indite the three isoforms: MHC1 slow type, MHC2 fst oxidtive nd MHC2b fst glyolyti ) seprted on the bsis of their reltive mobility in order from fstest to slowest. () Extensor digitorum longus (EDL) from Con rt. (b) EDL from CHF rt. () Sol from Con rt. (d) Sol from CHF rt.

5 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) Fig. 2. () Perent distribution on myosin hevy hin (MHC) 2 nd 2b in the extensor digitorum longus (EDL) in the CHF nd Con rts, showing n 1 inresed expression of the fst glyolyti isoform MHC2b in CHF. * p,0.05, p (b) Perent distribution of myosin hevy hin (MHC) 2 nd 2b in the extensor digitorum longus (EDL) in the Hyp nd ConHyp rts, showing no hnges in MHCs pttern with ompensted hypertrophy. * p5ns, 1 p5ns. There ws signifint orreltion between MHC1 nd 3.8. Correltion between degree of musle trophy nd b-anp in the CHF group (r50.64, p,0.01) nd MHC omposition borderline signifine for RVW/ BW (r50.5, p50.05). There ws no orreltion in the Hyp group between these The degree of musle trophy ws orrelted with the prmeters. perent distribution of MHCs within the CHF nd Hyp groups nlysed seprtely Effet of 2 weeks monorotline-tretment on skeletl musle CHF group In the Sol of the 15 CHF rts mentioned in Setion 2.1, Two weeks of tretment with monorotline did not no orreltion ws found either for MHC1 or MHC2 ffet either Sol (MHC vs , p5ns), (r50.15 p5ns, r50.10 p5ns respetively) (Fig. 4( or EDL MHC omposition (MHC vs. nd b)). The sme ws true for EDL degree of trophy nd , p5ns). The degree of trophy ws similr in both MHC2 (r50.14, p5ns) nd MHC2b (r50.25, the monorotline nd ontrols both in EDL ( in p5ns) (Fig. 5 nd 5b). the monorotline vs in ontrols, p5ns) nd Sol ( in the monorotline vs in Hyp group [Fig. 6( nd b)] ontrols, p5ns). Right ventrile hypertrophy ws not In the nimls mentioned in Setion 2.1, the degree of present in the monorotline-treted rts s shown by the trophy of Sol ws signifintly orrelted with both RVW/ BW whih ws identil to tht of the ontrols MHC2 (r50.6, p50.028) nd MHC1 (r50.6, p5 ( vs , p5ns) ). Similr results were obtined for EDL, being Fig. 3. () Perent distribution of myosin hevy hin (MHC) 1 nd 2 in the Sol of CHF nd Con rts, showing n inresed expression of the fst 1 oxidtive MHC2 in CHF. * p50.028, p (b) Perent distribution of myosin hevy hin 1 nd 2 in the Sol of Hyp nd ConHyp rts, showing 1 no hnges in MHCs pttern with ompensted hypertrophy. * p5ns, p5ns.

6 238 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) Fig. 4. () Liner regression between degree of Sol trophy (Sol weight/ body weight) nd perentge of myosin hevy hin 1 (%MHC1), showing no orreltion between these two prmeters in the CHF group (n515). (b) Liner regression between Sol degree of trophy (Sol weight/ body weight) nd perentge of myosin hevy hin 2 (MHC2), showing no orreltion between these two prmeters in the CHF group (n515). Fig. 5. () Liner regression between EDL degree of trophy (EDL weight/ body weight) nd perentge of myosin hevy hin 2 (MHC2), showing no orreltion between these two prmeters in the CHF group (n515). (b) Liner regression between EDL degree of trophy (EDL weight/ body weight) nd perentge of myosin hevy hin 2b (MHC2b), showing no orreltion between these two prmeters in the CHF group (n515). r50.64 ( p50.015) for MHC2 nd r50.65 ( p50.015) ml/ min/ g of tissue. This ws signifintly for MHC2b (Fig. 7( nd b). lower ( p,0.05) thn tht of Con rts ( ). The blood flow of EDL ws not signifintly different from tht 3.9. Skeletl musle blood flow (Tble 3) of Con rts ( vs , p5ns) CHF group Hyp group The skeletl musle blood flow of the Sol ws The Sol blood flow in Hyp rts ws This Fig. 6. () Liner regression between Sol degree of trophy (Sol weight/ body weight) nd perentge of myosin hevy hin 1 (MHC1), showing in the Hyp nd ConHyp groups signifint orreltion between these two prmeters (n513). (b) Liner regression between Sol degree of trophy (Sol weight/ body weight) nd perentge of myosin hevy hin 2 (MHC2), showing in the Hyp nd ConHyp groups signifint orreltion between these two prmeters (n513).

7 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) Fig. 7. () Liner regression between EDL degree of musle trophy (EDL weight/ body weight) nd perentge of myosin hevy hin 2 (MHC2), showing in the Hyp nd ConHyp groups signifint orreltion between these two prmeters (n513). (b) Liner regression between EDL degree of trophy (EDL weight/ body weight) nd perentge of myosin hevy hin 2b (MHC2b), showing in the Hyp nd ConHyp groups signifint orreltion between these two prmeters (n513). ws not sttistilly different from tht mesured in the EDL. It is widely epted tht CHF is hrterised by ConHyp rts ( , p5ns). In the EDL there ws the ourrene of speifi myopthy with intrinsi slight, but not signifint, redution in blood flow when bnormlities of musle fibres [1,24,25]. This seems to be ompred to the ConHyp rts ( vs , true espeilly in the leg musles, but lso ours in other p5ns). res [3,26,27]. In our CHF rts the ourrene of skeletl musle myopthy is doumented by the speifi hnges in MHCs pttern tht reflet hnges in fibre type. 4. Disussion These re similr to those reported by Simonini et l. [28] in rt model of CHF due to MI (myordil infrtion). In this pper we desribe, for the first time, biohemil However wht triggers the myopthy is still debted. The nd struturl modifitions ourring in the skeletl present dt do not support previously published hypothmusle of well estblished model of CHF [14,15], eses in whih detrining nd the onsequent trophy hd together with mesurements of blood flow. We hve in ft been dvoted s the possible use of the CHF myopthy found n inresed expression of more ftiguble MHCs [29,30]. This is in keeping with previous report of our isoforms in musles with different funtion nd bioener- group [10] tht showed in disuse trophy in mn the geti hrteristis. In the fst twith EDL the fst preferentil synthesis of the slow MHC1 nd with those of glyolyti MHC2b ws inresed s ws the fst oxidtive Simonini et l. [31] who showed no explntion in the MHC2 in the slow twith Sol. We hve lso reported redued tivity for the hnges ourring in MHCs on hnges in musle trophism in CHF nd Hyp nimls. expression in CHF rts. Though we tried to limit physil CHF rts hve in ft loss of EDL musle bulk s tivity in the Hyp nd Con rts by restriting them in indited by EDL weight/ BW, while Sol, whih is prini- smll ges, rts with severe CHF might hve exerised plly involved in mintining posture, showed no sign of less, but it should not hve influened skeletl musle trophy. We ould detet neither hnges in fibres ross- myosin gene expression. It hs been in ft shown by setionl re, nor replement fibrosis. Moreover no Simonini et l. by monitoring loomotor tivity of CHF orreltion ws found between musle hnges in terms of rts, tht different levels of physil tivity do not MHCs omposition, tht n be onsidered speifi influene MHCs gene expression [31]. It hs been lso biohemil mrker of the myopthy, nd the ourrene debted for long time whether hnges in nutritive blood of trophy. We hve lso shown tht ompensted hy- flow ould limit exerise tolerne nd ply n importnt pertrophy is ompnied by none of the bove mentioned role in the development of this myopthy [13,32,33]. As biohemil, trophi, or morphologi hnges. In the sme previously suggested by Mssie et l. in mn [9] our dt musles we hve mesured nutritive resting blood flow do not support this hypothesis for two resons: first of ll whih ws only slightly redued in the Sol, but not in beuse the resting blood flow in EDL ws unhnged, Tble 3 Skeletl musle blood flow in CHF, Con, Hyp nd ConHyp rts Sol CHF Sol Con EDL CHF EDL Con Sol Hyp Sol ConHyp EDL Hyp EDL ConHyp Blood flow * * (ml/ min/ g) (n56) (n510) (n56) (n510) (n58) (n56) (n58) (n56) p,0.05.

8 240 G. Vesovo et l. / Crdiovsulr Reserh 39 (1998) seondly beuse the slight redution found in the Sol of of skeletl musle in ptients with hroni hert filure. Int J Crdiol CHF rts do not explin hnges in MHCs omposition of 1988;18: [4] Mnini DM, Coyle E, Coggn A, et l. Contribution of intrinsi the sme order of mgnitude of those ourring in the skeletl musle hnges to 31P NMR skeletl bnormlities in EDL. In this se blood flow redution ould simply ptients with hroni hert filure. Cirultion 1992;85: represent flow redistribution towrd the more oxygen [5] Sullivn MJ, Green HJ, Cobbe FR. Skeletl musle biohemistry demnding, fst musles, in order to preserve preise nd histology in mbultory ptients with long term hert filure. movements. Cirultion 1990;81: [6] Drexler H, Reide U, Munzel T, Konig H, Funke E, Just H. Mesurement of blood flow with fluoresent miro- Altertions of skeletl musle in hroni hert filure. Cirultion spheres is n extremely relible method, ertinly superior 1992;85: to rdiotive or oloured mirospheres [34]. The repro- [7] Drexler H. Skeletl musle filure in hert filure. Cirultion duibility of the method is firly high both in terms of 1992;85: bsolute number of mirospheres per smple, nd linerity [8] Wilson JR, Mnini DM, Dunkmnn WB. Exertionl ftigue due to skeletl musle dysfuntion in ptients with hert filure. Cirulbetween number of mirospheres nd fluoresene intensition 1993;87: ty. [9] Mssie BM, Conwy M, Yonge R, et l. Skeletl musle metbolism We n exlude diret effet of monorotline on the in ptients with ongestive hert filure: reltion to linil severity skeletl musle beyond the toxi effet, tht is known to be nd blood flow. Cirultion 1987;76: seletive, on the two speifi trget orgns, nmely liver [10] Vesovo G, Serfini F, Fhin L, et l. Speifi hnges in skeletl musle myosin hevy hin omposition in rdi filure: differnd lung [14,15]. Hyp nimls hd in ft the sme dose of enes ompred with disuse trophy s ssessed on mirobiopsies by lkloid s CHF rts, without showing ny histologi high resolution eletrophoresis. Hert 1996;76: dmge or, more importntly, ny sign of MHCs redistri- [11] MMurry J, Abdullh I, Drgie HJ, Shpiro D. Inresed onbution. Moreover the speifilly designed experiment entrtions of tumor nerosis ftor in heti ptients with with 2 weeks monorotline-tretment showed no hnges severe hroni hert filure. Br Hert J 1991;66: in MHCs pttern, when the hert showed no sign of [12] Cots AJS, Clrk AL, Piepoli M, Volterrni M, Poole-Wilson PA. Symptoms nd qulity of life in hert filure. The musle hypothhypertrophy or filure, nd the skeletl musle no trophi esis. Br Hert J 1994;72: hnges. The monorotline-treted rt is n unique model [13] Wilson JR, Mrtin JL, Shwrtz D, Ferrro N. Exerise intolerne for studying the hert filure syndrome [16,35,36]. The in ptients with hroni hert filure: role of impired nutritive flow dignosis of overt hert filure vs. ompensted right to skeletl musle. Cirultion 1984;69: ventrile hypertrophy is in ft well estblished nd esily [14] Reindel JF, Gney PE, Wgner JG, Sloombe RF, Roth RA. Development of morphologi, hemodynmi, nd biohemil done llowing omprison between these two hnges in lungs of rts given monorotline pyrrole. Toxiol Appl pthophysiologil onditions [16], lthough the our- Phrmol 1990;106: rene of CHF in our nimls ws strengthened by the [15] Wilson DW, Segl HJ, Pn LC, Lme MW, Estep JE, Morin D. mesurement of b-anp. Mehnisms nd pthology of monorotline pulmonry toxiity. In onlusion our study suggests tht musle bnor- Crit Rev Toxiol 1992;22: [16] Vesovo G, Jones SM, Hrding SE, Poole-Wilson PA. Isoproterenol mlities of the CHF syndrome re neither due to detrining sensitivity of isolted rdi myoytes from rts with monotrophy, nor to blood flow redution. It my well be tht rotline-indued right-sided hypertrophy nd rdi filure. J Mol ytokines (TNF), insulin resistne or other neuro hor- Cell Crdiol 1989;21: monl mehnisms my ontribute to its development [17] Comini L, Agnoletti G, Pnzli A, et l. Ativtion of ANP [11,37]. We n lso speulte on the pthophysiology of synthesis during ongestive hert filure in rts treted with monorotline. Am J Physiol 1995;268:H this myopthy sine it hs been reently demonstrted in [18] Pn LC, Lme MW, Morin D, Wilson DW, Segll HJ. Red blood the rt with CHF tht mrna levels enoding b-mhc re ells ugment trnsport of retive metbolites of monorotline depressed, wheres those enoding MHC2b re inresed. from liver to lung in isolted nd tndem liver nd lung preprtions. It suggests tht the redistribution in MHC omposition is Toxiol Appl Phrmol 1991;110: not due to seletive myosin or fibre trophy, but rther to [19] Roth RA, Reindel JF. Lung vsulr injury from monorotline pyrrole, puttive hepti metbolite. Adv Exp Med Biol speifi shift in isomyosin synthesis [28]. At the sme 1991;283: time musle wste n our beuse of nonseletive fibre [20] Crrro U, Ctni C. A sensitive SDS-PAGE method seprting loss, possibly medited by poptosis [38]. myosin hevy hin isoforms of rt skeletl musle revels the heterogeneous nture of the embryoni myosin. Biohim Biophys Res Commun 1983;116: [21] Sndri M, Rizzi C, Ctni C, Crrro U. Smll nd lrge sle Referenes preprtive purifition of myosin light nd hevy hins by seletive KDS preipittion of myosin subunits: yield by SDS-PAGE [1] Minotti JR, Pilly P, Ok R, Wells L. Skeletl musle size: nd quntittive orthogonl densitometry. Bsi Appl Myology reltionship to musle funtion in hert filure. J Appl Physiol 1992;2: ;75: [22] Glenny RW, Bernrd S, Brinkley M. Vlidtion of fluoresent- [2] Wilson JR, Mnini DM, Dunkmn WW. Exertionl ftigue due to lbelled mirospheres for mesurement of regionl orgn perfusion. skeletl musle dysfuntion in ptients with hert filure. Cirul- J Appl Physiol 1993;74: tion 1993;87: [23] Angelini A, Vesovo G, Clliri I, Vlente ML, Thiene G. Correl- [3] Lipkin DP, Jones DA, Round JM, Poole-Wilson PA. Abnormlities tion between endomyordil biopsies nd ventrile full thikness

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