THE CARDIOVASCULAR RESPONSES OF THE RED-EARED SLIDER (TRACHEMYS SCRIPTA) ACCLIMATED TO EITHER 22 OR 5 C

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1 The Journl of Experimentl Biology, () Printed in Gret Britin The Compny of Biologists Limited JEB THE CARDIOVASCULAR RESPONSES OF THE RED-EARED SLIDER (TRACHEMYS SCRIPTA) ACCLIMATED TO EITHER OR 5 C I EFFECTS OF ANOXIC EXPOSURE ON IN VIVO CARDIAC PERFORMANCE J M T HICKS* AND A P FARRELL Deprtment of Biologil Sienes, Simon Frser University, Burny, British Columi, Cnd V5A S6 *Present ddress: Deprtment of Animl Siene, 57 Min Mll, Vnouver, British Columi, Cnd V6T Z4 Author for orrespondene (e-mil: frrell@sfu) Aepted 5 Septemer; pulished on WWW 4 Novemer The extreme noxi-tolerne of freshwter turtles under old onditions is well doumented, ut little is known out their rdi performne in suh situtions Using hroni theteriztion tehniques, we mesured systemi rdi power output ( ), systemi rdi output (Q sys), hert rte (fh), systemi stroke volume ( ), systemi resistne ( ) nd men rteril pressure (P sys ) in red-ered sliders (Trhemys sript) The effets of old limtion nd noxi exposure were studied Turtles were limted to either C or 5 C, nd the noxi exposure ws either ute (6 h) t C or hroni ( weeks) t 5 C Cold limtion lone deresed y 5-fold, representing Q of 88 In ddition, fh nd deresed signifintly, while inresed nd moderted the rteril hypotension Aute nd hroni noxi exposures signifintly deresed,, fh nd P sys nd inresed But the hnges were qulittively muh lrger with hroni noxi For exmple, ute noxi in C-limted turtles deresed y 66-fold, wheres hroni noxi in 5 C-limted turtles deresed y -fold The remrkle rdiovsulr down-regultion tht Summry ompnies long periods of old noxi in these turtles ws hrterized y ompring rdiovsulr sttus during hroni noxi t 5 C with tht during normoxi t C Crdi ws redued -fold, through dereses in Q sys (-fold), fh (4-fold), (57-fold) nd P sys (-fold), while ws inresed 646-fold We lso ompred rdi glyolyti rtes y ssuming tht ws proportionl to ATP supply nd tht glyolysis yielded 8 times less ATP per mole of gluose thn oxidtive metolism At C, the 66-fold derese in with noxi suggests tht Psteur effet ws needed in rdi tissues during ute noxi However, this would not e so with hroni noxi t 5 C euse of the -fold derese in We propose tht the suppression of the Psteur effet nd the lrge Q vlues for old limtion would onserve gluose stores nd enle turtles to withstnd noxi muh longer under old thn under wrm onditions Key words: turtle, Trhemys sript, hert, hert rte, stroke volume, rdi output, rdi power output, temperture, noxi Introdution Freshwter turtles n withstnd 4 months of noxi t C (Ultsh nd Jkson, 98), nd this remrkle noxitolerne is ompnied y five- to tenfold redution in metoli rte (Herert nd Jkson, 985; Jkson, 968) Beyond this, detils of this metoli depression re limited lrgely to iohemil dpttions (Storey, 996), whih provide lrgely qulittive view of energy supply nd demnd An importnt quntittive insight into the prolem of energy supply nd demnd during noxi n e provided y mesuring work performed y the hert musle euse rdi power output n e expressed s n ATP demnd, nd this ATP demnd n e relted to rtes of glyolysis However, in vivo rdi power output hs not een mesured in old-limted turtles hronilly exposed to noxi; informtion exists only for hert rte nd lood pressure Turtles (Chrysemys pit elli) exposed to noxi t C for 9 dys experiened 76-fold derese in hert rte nd 56-fold drop in men rteril lood pressure ompred with mesurements mde in normoxi nimls t C (Herert nd Jkson, 985) These oservtions, omined with mesured redutions in stroke volume in noxi perfused turtle herts, were used to predit tht rdi power output would e pproximtely 5-fold lower in old noxi turtles thn in wrm normoxi ones (Frrell et l, 994) The purpose of the present study ws to quntify diretly the degree of rdi down-regultion tht ours in vivo in turtles with old limtion nd with noxi exposure Our hroni theteriztion tehniques were sed on those pioneered y White nd Ross (966) nd perfeted y Hiks nd

2 766 J M T HICKS AND A P FARRELL Cnnul L thyroid rtery R nterior ven v L sulvin rtery L orti rh Doppler flow proe L nterior ven v R urile L pulmonry rtery L pulmonry vein Fig Ventrl view of the surgil re inluding the sites of nnultion nd flow proe plement L, left; R, right R pulmonry vein Left urile Ventrile Wng (998) when they suessfully mesured rdiovsulr sttus in Trhemys sript during h exposure to noxi t 5 C Hiks nd Wng (998) reported twofold redution in systemi rdi power output ( ) for this short noxi exposure We ntiipted finding muh lrger dereses in old-limted turtles exposed to hroni noxi To ssess rdiovsulr sttus, we mesured, systemi rdi output (Q sys), hert rte (fh), systemi stroke volume ( ), systemi resistne ( ) nd men rteril pressure (P sys ) in T sript To disset out the effets of temperture limtion from those of noxi exposure, we used turtles limted for weeks to either C or 5 C A su-group of turtles ws then sujeted to either n ute, 6 h exposure to noxi t C or to hroni, -week exposure to noxi t 5 C Mterils nd methods Experimentl nimls Red-ered sliders (Trhemys sript Gry) (ody mss 74±7 g, men ± SEM, N=) were otined from ommeril supplier (Crolin Biologil Supply Co, Burlington, NC, USA) nd held indoors in stnding wter in polypropylene ontiners with ess to sking pltforms s well s deep wter Room nd sking lights were set for h: h L:D photoperiod The turtles were fed on ommeril pellets (Wrdley Lortories In, Seuus, NJ, USA), smll piees of fish nd lettue t lest three times per week Turtles were fsted for week prior to surgery Experiments with 5 Climted turtles were onduted from Septemer 995 to My 996, while experiments with C-limted turtles were onduted from June to Septemer 996 No signifint differene ws found etween the men ody mss of the four experimentl groups Surgil preprtion Turtles were nesthetized with 4 % Hlothne vporized in 95 % O, 5 % CO nd then intuted nd ventilted with % Hlothne during surgery (Hiks nd Wng, 998) Hlothne levels were ontrolled using Hlothne vporizer (Fluote Mrk, Cyprn Ltd, UK) An eletri one sw (Mope, Detroit, MI, USA) ws used to remove 4 m 5 m piee of the plstron, therey exposing the hert nd systemi vessels A lunt proe ws used to gently remove ny musle tthed to the plstron, nd ny minor leeding ws stopped y uteriztion A PE5 polyethylene nnul, filled with heprinized sline (5 iu ml ), ws inserted olusively into the left sulvin rtery vi the thyroid rtery nd dvned towrds the hert (pproximtely 5 m) efore eing seured in ple with - guge silk thred The nnul ws led out of the shell through tror inserted t the se of the left foreleg nd seured to the skin with purse-string suture A Doppler flow proe (Iow Doppler Produts, Iow City, IA, USA) ws pled round portion of the left ort tht hd een freed of onnetive tissue (see Fig ) Leds from the proe were rried out through the hole in the plstron nd seured longside the nnul onto the rpe with ynorylte nd Flexryl (Lng Dentl Mnufturing Co, Wheeling, IL, USA) Prior to the plstron eing losed, peniillin/streptomyin/neomyin solution (Sigm Chemil Co, St Louis, MO, USA) ws spryed over the surgil field The plstron piee ws reseled in ple with lyer of Gelfom sponge (Upjohn Co, Klmzoo, MI, USA) nd seled with one dust nd ynorylte The entire re ws then overed with Flexryl Following surgery, turtles were ventilted with ir until they ould tively withdrw their lims The nimls were then llowed to reover in individul 4 l glss quri for 48 h prior to ny rdiovsulr mesurements Hemtorit ws monitored for the first week following surgery nd rnged from 4 to 6 % Turtles with levels elow % were killed y depittion A numer of turtles were not used for the experiments either euse the rteril nnul did not remin ptent or euse the Doppler flow proe filed All proedures

3 In vivo rdi performne of old-limted noxi turtles 767 were pproved y the Animl Cre Committee t Simon Frser University Instrumenttion nd terminology Systemi rteril lood pressure (P sys ) ws reorded with pressure trnsduer (Nro LDI-5, Nro, TX, USA) onneted to the nnul The pressure trnsduer ws lirted ginst stti wter olumn efore eh reording, nd pressures were regulrly referened to the wter level of the tnk in whih the turtle ws sumerged The flow proe ws onneted to Doppler flow meter (Iow Doppler Produts, Iow City, IA, USA) nd the rnge mnipulted for mximum signl strength This rnge ws reorded nd used for ll susequent mesurements Eh flow proe ws individully lirted in situ t the termintion of the experiment y delivering known volumes of diluted lood through the nnulted left ort The orti outflow ws onneted to pressure hed so tht lirtions ould e mde t physiologilly representtive pressures Clirtion urves were liner, nd r ws lwys greter thn 87 (typilly greter thn 9) Pressure nd flow signls were premplified nd displyed ontinuously on Gould hrt reorder (model 4, Clevelnd, OH, USA) Hert rte (fh) ws mesured y ounting the numer of systoli peks over min period in the C groups nd over 4 min period t 5 C Mesurements were tken from post-reth portions of the tres in the normoxi turtles Men systemi rteril pressure (P sys ), systemi rdi output (Q sys), systemi stroke volume ( ), systemi power output ( ) nd systemi resistne ( ) were lulted s follows: P sys = (systoli pressure + distoli pressure)/, () Q sys = 5Q LAo, () =(Q sys/fh)/m, () = (Q sys P sys α)/m v, (4) = P sys /Q sys, (5) where Q LAo is left orti lood flow (ml min kg ), fh is hert rte (ets min ), M is ody mss (kg), M v is ventriulr mss (g) nd α is 67 min s, onversion ftor to mw P sys ws mesured from pressure tres y lulting the men of systoli nd distoli pressure Q sys ws estimted from 5Q LAo to pproximte the ontriutions of the right ort, rotid nd sulvin rteries to totl systemi rdi output (Wng nd Hiks, 996; Comeu nd Hiks, 994) It is possile tht this estimte, whih ws determined for wrm normoxi turtles, does not hold either for noxi onditions or fter old limtion Hiks nd Wng (998) implnted the three flow proes required to mesure systemi lood flow diretly during muh shorter noxi exposures, ut reported only totl systemi flow nd not the individul rteril lood flows T Wng (personl ommunition) hs found tht rteril lood flow distriution did not vry mrkedly during short-term noxi, so our ssumption is proly vlid for our ute noxi exposures For the hroni studies, we were willing to ept the unertinty of our ssumption rther thn inrese the surgil proedures sustntilly (implnting three flow proes rther thn one) prior to hroni noxi exposure Experimentl protool During experiments, individul turtles were llowed to move freely inside wter-filled qurium ( m m 6 m) overed with lk plsti to minimize visul disturne Continuous rdiovsulr reordings were tken for periods of min without the experimenter in the room For turtles limted to C, rdiovsulr sttus ws ssessed dily during the 8-dy post-surgery reovery period, strting on the seond dy After the 8-dy reovery period, the 6 h noxi exposure ws strted for one group of C-limted turtles y denying them ess to surfe ir y fitting Plexigls over over plsti mesh grte to the top of the tnk nd y equilirting the wter with N Turtles tht were susequently limted to 5 C were lso llowed n 8-dy reovery period fter surgery, ut during this reovery period they were progressively exposed to 5 C The protool ws s follows: 6 h t 5 C on dy ; h t 5 C on dy 4; 8 h t 5 C on dy 5; no 5 C exposure on dy 6; 4 h t 5 C on dy 7; no 5 C exposure on dy 8; held t 5 C on dy 9 Agin, rdiovsulr sttus ws reorded t the end of eh re-wrming period prior to the turtles eing moved into the 5 C room Following the reovery period, the normoxi group of turtles ws llowed to limte to 5 C for 5 weeks nd rdiovsulr sttus ws mesured every seond dy The noxi group of turtles ws first held t 5 C under normoxi onditions for 4 dys efore eing sumerged for weeks in wter uled with N, s desried ove Crdiovsulr sttus ws mesured every seond dy All 5 C experiments were onduted in old room held t this temperture Sttistil nlyses Crdiovsulr vriles were determined y verging three rndom min setions of the min reording period In most ses, men vlues ± SEM for six nimls re presented (N=5 for the 5 C noxi group) Differenes etween mens of experimentl groups were determined using either one-wy or two-wy nlyses of vrine (ANOVAs), where pproprite, while multiple omprisons were performed using Student Newmn Keuls tests P<5 ws used s the level of signifine Within-group omprisons used repetedmesures ANOVA Results Eight-dy reovery period from surgery The rdiovsulr hnges ompnying the 8-dy reovery period from surgery re summrized in Figs nd Although there were signifint dy-to-dy hnges for some of the vriles, there were no onsistent trends ross ll four test groups From the perspetive of reovery, it is importnt to note tht only one of the 4 possile vrile/tretment omintions ws signifintly different etween the sixth nd eighth dys of

4 768 J M T HICKS AND A P FARRELL Hert rte, fh (ets min - ) Systemi resistne, (kp ml - min kg) Systemi stroke volume, (ml kg - ) A, P sys ,,,, fh Systemi rdi output, (ml min - kg - ) Systemi lood pressure, P sys (kp) Systemi power output, (mw g - ) Hert rte (ets min - ) Systemi stroke volume (ml kg - ) Systemi resistne (kp ml - min kg) B Time following surgery (dys) fh P sys Systemi lood pressure (kp) Systemi rdi output (ml min - kg - ) Systemi power output (mw g - ) Fig Post-surgery rdiovsulr vriles during reovery nd mesured under normoxi onditions t C for the groups of Climted turtles used susequently for the normoxi (A) nd noxi (B) experiments (N=6) Signifint differenes (P<5) etween vlues re indited y dissimilr letters Vlues re mens ± SEM reovery In ddition, the 5 C-limted groups (Fig ) showed less dy-to-dy vriility thn the C-limted groups The routine rdiovsulr vriles for the four experimentl groups of turtles mesured on the eighth dy postsurgery under normoxi onditions t C re ompred in Tle Arteril lood pressure nd were signifintly higher in the C noxi group of nimls Some of this vriility my hve refleted differenes in the winter versus summer timing of the 5 C nd C experiments, s well s the progressive exposure to 5 C for the old-limted turtles Wrm-limted turtles with nd without n ute exposure (6 h) to noxi The progressive rdiovsulr hnges tht ompnied

5 In vivo rdi performne of old-limted noxi turtles 769 Systemi stroke volume, (ml kg - ) Hert rte, fh (ets min - ) Systemi resistne, (kp ml - min kg) A fh P sys P sys , 5 Systemi rdi output, (ml min - kg - ) Systemi lood pressure, P sys (kp) Systemi power output, (kp) Time following surgery (dys) Hert rte (ets min - ) Systemi stroke volume (ml kg - ) Systemi resistne (kp ml - min kg) B,,, fh Systemi rdi output (ml min - kg - ) Systemi lood pressure (kp) Systemi power output (kp) Fig Post-surgery rdiovsulr vriles during reovery nd mesured under normoxi onditions t C for the groups of turtles susequently limted to 5 C nd used for the normoxi (A, N=6) nd noxi (B, N=5) experiments Signifint differenes (P<5) etween vlues re indited y dissimilr letters Vlues re mens ± SEM the ute exposure to noxi t C re summrized in Fig 4A Most of the rdiovsulr djustments ourred during the first h of the noxi exposure, nd none of the vriles hnged signifintly fter this Tle ompres the rdiovsulr hnges fter 6 h of noxi with the normoxi ontrol vlues The pproximtely twofold derese in oth fh nd ontriuted to the 45- fold derese in Q sys The derese in Q sys produed signifint rteril hypotension ( % derese in P sys ) even though more thn tripled As result of the derese in

6 77 J M T HICKS AND A P FARRELL Tle In vivo rdiovsulr sttus for the four experimentl groups of turtles mesured t C under normoxi onditions following the 8-dy post-surgery reovery period Systemi Systemi Men Systemi Hert rte stroke volume rdi output rteril pressure power output Systemi resistne Group (ets min ) (ml kg ) (ml min kg ) (kp) (mw g ) (kp ml min kg) C normoxi 4± 49±5 6±5 6± 66± 6±5 C noxi 6±5 ± 7±6 ±5 66±5 ±5 5 C normoxi 8±4 7±8 8±5 ± 66±6 75±5 5 C noxi 86±8 49± 77±9 8±7 5±6 8±7 Vlues re mens ± SEM for ll experimentl groups (N=6, exept 5 C noxi where N=5) Signifint differenes (P<5) etween groups re indited y dissimilr letters Tle In vivo rdiovsulr sttus for C-limted nd 5 C-limted turtles exposed to either normoxi onditions or ute noxi (6 h) t C or hroni noxi ( dys) t 5 C Systemi Systemi Men Systemi Hert rte stroke volume rdi output rteril pressure power output Systemi resistne Group (ets min ) (ml kg ) (ml min kg ) (kp) (mw g ) (kp ml min kg) C normoxi 4± 49±5 6±5 6± 66±98 6±5 C noxi ±9 78± 8± 59±6 ± ±4 5 C normoxi 5±5 77± 4±9 ±, 44±68 ±54 5 C noxi ± d 6±6 ± d ±5 ± d 88±45 Vlues re mens ± SEM for ll experimentl groups (N=6, exept 5 C noxi where N=5) Signifint differenes (P<5) etween groups re indited y dissimilr letters Q sys nd the rteril hypotension, ws 66 times lower following 6 h of noxi exposure (Tle ) thn in the normoxi group of turtles t C Cold-limted turtles under normoxi onditions The progressive rdiovsulr hnges tht ompnied the 6-dy limtion to 5 C re summrized in Fig 4B Crdiovsulr sttus ws ltered signifintly nd ppreily during the first 6 dys of limtion; very few signifint hnges ourred fter dy This rpid old limtion my reflet, in prt, the progressive introdution to 5 C tht ourred during the post-surgery reovery period Tle ompres the rdiovsulr vriles t dy 6 of 5 C limtion with those of the C-limted normoxi group Cold limtion resulted in signifint dereses in ll rdiovsulr vriles exept for, whih inresed signifintly The 47-fold derese in fh to 5 ets min, whih represented Q of 7, ontriuted signifintly to the ninefold derese in Q sys to 4 ml min kg Despite the lrge derese in Q sys, rteril hypotension ws limited to less thn twofold derese in P sys euse of 55-fold inrese in Nonetheless, the 5-fold derese in rdi to 44 mw g with 5 C limtion represented Q of 88 Cold-limted turtles exposed to hroni (-dy) noxi The progressive rdiovsulr hnges ompnying the -dy hroni exposure to noxi t 5 C re summrized in Fig 4C The rdiovsulr hnges rehed n pprent stedy stte fter dys of noxi, ie none of the rdiovsulr vriles hnged signifintly etween dy nd dy 8 of the experiment Compred with normoxi 5 Climted turtles, hroni noxi exposure resulted in signifint dereses in (-fold), Q sys (-fold), fh (5-fold) nd (-fold) nd signifint inrese (8- fold) in eyond the hnges produed y 5 C limtion lone (Tle ) These hnges were qulittively the sme s, ut quntittively muh greter thn, those produed y ute noxi t C Compred with normoxi C-limted turtles, hroni noxi exposure used profound depression of rdiovsulr sttus in 5 C-limted turtles (Tle ) There ws -fold derese in Q sys, s result of 4- fold derese in fh nd 57-fold derese in Arteril hypotension ws moderted (P sys deresed y only -fold) y 646-fold inrese in ws redued -fold to mw g from 66 mw g Of this -fold redution in, -fold derese ould e ttriuted to the effet of hroni noxi t 5 C, while 5-fold derese ould e ttriuted to old limtion Chroni noxi exposure nd old limtion ontriuted similrly to the depression of fh (5-fold versus 46-fold, respetively) nd Q sys (-fold versus ninefold, respetively) (Tle ) However, hroni noxi exposure ontriuted muh more to the inrese in (8- fold) thn old limtion (55-fold) Crdi depression nd inresed vsulr resistne re lerly signifint fetures tht ompny hroni noxi exposure in old-limted turtles

7 In vivo rdi performne of old-limted noxi turtles 77 Disussion Crdi performne under normoxi onditions In the present study, C-limted Trhemys sript were llowed n 8-dy post-surgery reovery period under normoxi onditions Given tht rdiovsulr sttus hd stilized y the eighth dy nd tht there ws resonle greement in rdiovsulr sttus mong the four groups of turtles, despite some sesonl nd proedurl differenes, we re onfident in our ontrol vlues s presented in Tle We lso followed rdiovsulr sttus during 6-dy period of limtion to 5 C nd found tht the rdiovsulr hnges ssoited with old-limtion were lrgely ompleted y the tenth dy Therefore, we re onfident tht our mesurements re inditive of old-limtion Furthermore, where omprisons re possile, rdiovsulr sttus for wrmlimted nd old-limted turtles in this study ompres fvourly with tht of previous studies For exmple, fh vlues of 4 nd 5 ets min for C nd 5 C, respetively, re omprle with those reported y Herert nd Jkson (985) for Chrysemys sript under normoxi onditions t C ( ets min ) nd fter months t C (8 ets min ) Hiks nd Wng (998) reported fh of 78 ets min for normoxi T sript t 5 C However, fh vlues reported for T sript t C y Hiks (994) nd Comeu nd Hiks (994) were 8 nd 4 ets min, respetively Some of these differenes proly represent hnges in ventiltory stte when smpling ourred For instne, towrds the end of pnoe t 9 C, turtles limted to this temperture hd hert rte of ets min tht inresed shrply to ets min with ventiltion (Shelton nd Burggren, 976) By omprison, the intrinsi fh vlues for n in situ hert preprtion for C sript, devoid of neurl ontrol nd utely exposed to 5 C nd 5 C, were 4 nd 8 ets min, respetively (Frrell et l, 994) Given tht the in situ fh t 5 C is higher thn tht in vivo for old-limted turtles, oldlimtion pprently permits the expression of physiologil hnges tht depress fh Indeed, isolted perfused herts of frogs Rn temporri when limted to low temperture hve lower ontrtion frequenies when tested over wide rnge of test temperture thn wrm-limted herts (Hrri nd To, 975) Possile mehnisms for these hnges inlude the expression of myosin isoforms with lower ATPse tivities (Vornnen, 994) nd/or lengthening of the tivtion nd relxtion phses of rdi ontrtion Autonomi modultion of the hert ould lso e involved in old limtion of turtles; these effets re onsidered in the ompnying report (Hiks nd Frrell, ) There ws good greement etween the men systemi rteril pressures reported here ( kp) nd in previous studies with turtles For exmple, P sys rnged from to 4 kp t C (Comeu nd Hiks, 994; Hiks, 994; Herert nd Jkson, 985), while Hiks nd Wng (998) reported kp for T sript t 5 C Herert nd Jkson (985) reported P sys of 9 kp t C, wheres our vlue ws kp t 5 C There ws lso good greement with previous vlues of In the present study, ws 49 ml kg t C, while Shelton nd Burggren (976) nd Hiks (994) reported vlues of 4 nd 8 ml kg for turtles t C, respetively The good greement with the vlue of 56 ml kg reported y Hiks nd Wng (998) for turtles t 5 C is espeilly importnt euse they diretly mesured lmost the entire systemi flow with three flow proes nd used only one minor ssumption This greement dds redene to the ssumption we used when we estimted Q sys from mesurement of left orti flow Unfortuntely, we hve no mens of ssessing whether our ssumption ws eqully stisftory for old-limted turtles Given the good greement etween the present nd previous studies for fh, nd P sys in wrm-limted turtles, it should not e surprising tht Q sys nd were lso omprle In the present study, Q sys ws etween 7 nd 6 ml min kg, while ws etween 5 nd 66 mw g t C Both Comeu nd Hiks (994) nd Hiks (994) reported similr levels of Q sys (9 ml min kg nd 4 ml min kg, respetively) nd (4 mw g nd 44 mw g, respetively) for turtles t C Hiks nd Wng (998) reported somewht higher vlues for Q sys (4 ml min kg ) nd ( mw g ) for T sript t 5 C Assuming tht Q sys is etween 7 nd 8 times pulmonry rdi output (Wng nd Hiks, 996; Hiks nd Wng, 998), then totl rdi power output in our study would hve rnged from 9 to 4 mw g By omprison, mximum rdi power output hs een mesured in turtle herts in situ t 56 mw g (Frrell et l, 994) nd in vivo t 46 mw g (Shelton nd Burggren, 976; Comeu nd Hiks, 996) Beyond the generl greement of rdiovsulr vriles for studies with turtles held t tempertures round C, the present study provides novel informtion on the extent nd speed with whih Q sys,, nd re djusted t 5 C Previous studies hve only mesured fh nd P sys The djustments in rdiovsulr sttus were ompleted within dys nd, y nd lrge, the mjor hnges ourred within the first dys If further djustments do our eyond the 6-dy limtion period used here, then their rtes of hnge would e low Cold limtion produed negtive hronotropi nd negtive inotropi effets sine there were sizele dereses in oth fh nd The ft tht deresed y 5-fold with Q of pproximtely 9 demonstrtes tht old-limtion involved n tive depression of rdi tivity A similr onlusion n e rehed for the 47-fold derese in fh This 5-fold redution in rdi metoli rte undoutedly reflets prllel redutions in the ATP requirements of other tissues nd, s suh, there would e sizele derese in the rte of fuel utiliztion nd wste umultion in old-limted turtles The sustntil inrese in with old limtion proly reflets prioritiztion of regionl lood flow distriution Systemi vsoonstrition, pssive ollpse of vessels due to the lower P sys or some omintion of these ould hve rought out the inrese in The involvement of drenergi nd holinergi regultion of the rdiovsulr system of the turtle with old limtion is investigted in ompnion report (Hiks nd Frrell, )

8 77 J M T HICKS AND A P FARRELL Crdi performne under noxi onditions We were impressed t the noxi-tolerne of old-limted turtles Severl turtles were llowed to reover under normoxi onditions t the end of noxi exposure (dt not shown) nd were found to e in good ondition Blood ph ws 77 in our noxi group (see Hiks nd Frrell, ) Herert nd Jkson (985) previously found tht when lood ph fell elow 7 during prolonged noxi, reovery of turtles ws impired The present study is the first to mesure systemi in old-limted turtles during hroni noxi exposure The 5 C-limted turtles under noxi hd -fold lower thn the C-limted turtles under normoxi ( mw g versus 66 mw g ) fh nd P sys hve een mesured previously under old noxi (Herert nd Jkson, l985) nd, using these vlues, totl rdi power output ws estimted to derese from 7 mw g under C normoxi to mw g under C noxi, ie 5-fold differene (Frrell et l, 994) This predition is very muh in line with the present results if we ount for the 7 8 % differene (noted ove) etween nd totl rdi power output tht reflets the rdi work ssoited with pulmonry perfusion under wrm, normoxi onditions Thus, if we use the estimted rnge for totl rdi work in normoxi Climted turtles (9 4 mw g, presented ove) nd ssume tht there ws no pulmonry flow in noxi 5 Climted turtles, then the redution in totl rdi power output would e etween 45-fold nd 7-fold [The ssumption tht pulmonry lood flow stops during hroni noxi nd tht the noxi hert works only to perfuse the systemi irultion ppers to e vlid sine Hiks nd Wng (998) found tht pulmonry lood flow stopped fter only h of noxi] The generl greement etween the two estimtes of the redution in rdi power output is prtiulrly enourging euse it lends indiret support to our ssumption tht the distriution of lood flow etween the mjor systemi vessels ws not hnged in mjor wy y old limtion nd y noxi However, given the lrge ompnying hnges in, it is possile tht the distriution of lood flow did shift somewht, nd so we urge ution in using our vlues eyond generl pplitions One possiility is tht the ererl irultion ws spred t the expense of the orgns perfused y the left ort In this se, we would hve overestimted the derese in Q sys with our ssumption Further experiments re needed to exmine regionl lood flow distriution in old-limted nd hronilly noxi turtles efore this point n e resolved Hiks nd Wng (998) reported in vivo rdiovsulr sttus for T sript t 5 C fter shorter ( h) noxi period In generl, their findings were qulittively similr to ours, ut quntittively the hnges were smller They reported 5 % derese in, 6 % derese in P sys, % derese in Q sys nd 4 % derese in fh This quntittive differene no dout reflets differene in the durtion of the ute noxi period etween the two studies In ddition, the post-surgery reovery period ws longer in our experiments, nd this my hve ontriuted to the differene Hert rte, fh (ets min - ) Systemi resistne, (kp ml - min kg) Systemi stroke volume, (ml kg - ) fh ,,, Anoxi exposure (h) There re no omprle in vivo rdiovsulr mesurements for 5 C-limted turtles However, totl,,,, P sys A Fig 4 Crdiovsulr vriles of turtles: (A) during n ute, 6 h noxi exposure t C (N=6); (B) during limtion to 5 C under normoxi onditions (N=6); (C) during limtion to 5 C nd hroni exposure to noxi onditions eginning on the seventh dy of old limtion (N=5) Signifint differenes (P<5) etween vlues re indited y dissimilr letters Vlues re mens ± SEM,, Systemi rdi output, (ml min - kg - ) Systemi lood pressure, P sys (kp) Systemi power output, (mw g - )

9 In vivo rdi performne of old-limted noxi turtles 77 Systemi stroke volume (ml kg - ) Hert rte (ets min - ) ,,,,,,,,,, B,,,,,,,,,,,,,,,, ,, fḥ,,,,,,,,,,,,,,, P sys Systemi rdi output (ml min - kg - ) Systemi lood pressure (kp) Systemi stroke volume (ml kg - ) Hert rte (ets min - ) fḥ C,,,,,,,d,,d,d d d d d d d d V s,sys P sys,,,,,,,,,, Systemi rdi output (ml min - kg - ) Systemi lood pressure (kp) Systemi resistne (kp ml - min kg) R 5 sys PO sys 4,,, 5,,,,,,,,,,,,,,,,,,,,,,, 5,,,d,,,,d,e,,d d,e d,e d,e d,e e e e Period of limtion (dys) Systemi power output (mw g - ) Systemi resistne (kp ml - min kg) Fig 4 Continued Systemi power output (mw g - ) rdi power output hs een mesured in isolted nd in situ noxi turtle hert preprtions t low tempertures On the sis of the dt of Reeves (96), Frrell et l (994) estimted tht the est isolted hert preprtion t C hd mximum noxi totl power output of 87 mw g Similrly, totl power output ws 77 mw g under noxi t 5 C for in situ perfused herts nd deresed to 7 mw g t 5 C (Frrell et l, 994) By omprison, it is ler tht rdi performne in vivo during ute noxi under wrm onditions (58 mw g fter h, Hiks nd Wng, 998; mw g fter 6 h, present study) n e well elow the intrinsi pilities of the hert Furthermore, this differene eomes two orders of mgnitude for 5 C-limted turtles hronilly exposed to noxi ( ws mw g ) The

10 774 J M T HICKS AND A P FARRELL underlying mehnisms for this hroni depression of rdi tivity in vivo wit further study Anoxi depression of the rdiovsulr system in 5 Climted turtles ws lerly quntittively greter thn in C-limted turtles For exmple, t C, the 6 h noxi exposure produed rdyrdi (-fold derese in fh), redued inotropy (9-fold derese in ) nd systemi vsoonstrition (-fold inrese in ) However, these hnges were s muh s four times lrger fter the -dy noxi exposure t 5 C These differenes ould ome out through drenergi nd holinergi regultory mehnisms Autonomi regultion of the rdiovsulr system is onsidered further in n ompnying report (Hiks nd Frrell, ) Alterntively, they ould reflet intrinsi responses to the noxi nd idoti onditions tht previl In ddition, the rteril hypotension under old noxi onditions ( -fold redution in rteril lood pressure) ould diretly ffet vsulr resistne nd modulte lood flow distriution When trnsmurl vsulr pressure flls to ritil pressure nd relxes vsulr elstin fires, the vessel ollpses nd lood flow stops (Burton, 97) Thus, if ritil losing pressures vry etween vsulr eds, then peripherl lood flow distriution ould vry s funtion of the ritil losing pressure when rteril lood pressure dereses Presumly, ritil orgns suh s the rin would hve the lowest ritil losing pressure if this were the se By mesuring for the noxi turtle hert in vivo we n ssess the extent to whih the glyolyti rte ws djusted in rdi tissues Here, we ssume tht is proportionl to ATP utilistion nd tht glyolysis supplies 8 times less ATP per mole of gluose thn oxidtive metolism In Climted turtles, ute noxi used only 66-fold derese in, so Psteur effet would e needed for the hert to ompenste for the 8-fold redution in ATP yield per mole of gluose during the ute noxi exposure In ontrst, hroni noxi used -fold derese in for 5 C-limted turtles, so tht Psteur effet would not e needed to mintin the rdi ATP supply Suppression of the Psteur effet would onserve gluose stores nd this, omined with the temperture-medited depression of rdi performne (Q =9), is proly n importnt reson why turtles n tolerte noxi muh etter under old thn under wrm onditions Our old-limtion experiments were performed over the winter months euse other etotherms show importnt physiologil modifitions on sesonl sis For exmple, the tortoise Kinixys speki hs redued metoli rte when held t 5 C in Novemer, ut not in June or August when they re normlly tive (Hiley nd Loveridge, 997) When kept under onstnt onditions, Gopherus gssizii show redued tivity nd food onsumption in the utumn (Jkson et l, 976) Whether sesonl plstiity in physiologil stte plys n importnt role in the noxi tolerne of turtles remins to e determined This work ws supported y Nturl Sienes nd Engineering Reserh Counil of Cnd reserh grnt to APF nd Simon Frser University Grdute Fellowship wrded to JMTH Referenes Burton, A C (97) Physiology nd Biophysis of the Cirultion Chigo: Yer Book Medil Pulishers In Comeu, S nd Hiks, J W (994) Regultion of entrl vsulr lood flow in the turtle Am J Physiol 67, R569 R578 Frrell, A P, Frnklin, C E, Arthur, P G, Thorrensen, H nd Cousins, K L (994) Mehnil performne of n in situ perfused hert from the turtle Chrysemys sript during normoxi nd noxi t 5 C nd 5 C J Exp Biol 9, 7 9 Hiley, A nd Loveridge, J P (997) Metoli depression during dormny in the Afrin tortoise Kinixys spekii Cn J Zool 75, 8 5 Hrri, M N E nd Tlo, A (975) Effet of seson nd temperture limtion on the hert rte temperture reltionship in the isolted frog s hert (Rn temporri) Comp Biohem Physiol 5A, 49 4 Herert, C V nd Jkson, D C (985) Temperture effets on the responses to prolonged sumergene in the turtle Chrysemys pit elli I Blood id se nd ioni hnges during nd following noxi sumergene Physiol Zool 58, Herert, C V nd Jkson, D C (985) Temperture effets on the responses to prolonged sumergene in the turtle Chrysemys pit elli II Metoli rte, lood id se nd ioni hnges nd rdiovsulr funtion in erted nd noxi wter Physiol Zool 58, Hiks, J M T nd Frrell, A P () The rdiovsulr responses of the red-ered slider (Trhemys sript) limted to either or 5 C II Effets of noxi on drenergi nd holinergi ontrols J Exp Biol, Hiks, J W (994) Adrenergi nd holinergi regultion of intrrdi shunting Physiol Zool 67, 5 46 Hiks, J W nd Wng, T (998) Crdiovsulr regultion during noxi in the turtle: n in vivo study Physiol Zool 7, 4 Jkson, C G, Trotter, J A, Trotter, T H nd Trotter, M W (976) Aelerted growth rte nd erly mturity in Gopherus gssizii (Reptil: Testudines) Herpetologi, 9 45 Jkson, D C (968) Metoli depression nd oxygen depletion in the diving turtle J Appl Physiol 4, 5 59 Reeves, R B (96) Control of glyogen utiliztion nd gluose uptke in the neroi turtle hert Am J Physiol 5, 9 Shelton, G nd Burggren, W (976) Crdiovsulr dynmis of the Cheloni during pnoe nd lung ventiltion J Exp Biol 64, 4 Storey, K B (996) Metoli dpttions supporting noxi tolerne in reptiles: reent dvnes Comp Biohem Physiol 57, Ultsh, G R nd Jkson, D C (98) Long-term sumergene t C of the turtle, Chrysemys pit elli, in normoxi nd severely hypoxi wter II Extrellulr ioni responses to extreme lti idosis J Exp Biol 96, 9 4 Vornnen, M (994) Sesonl nd temperture-indued hnges in myosin hevy hin omposition of ruin rp herts Am J Physiol 67, R567 R57 Wng, T nd Hiks, J W (996) Crdiorespirtory synhrony in turtles J Exp Biol 99, 79 8 White, F N nd Ross, G (966) Cirultory hnges during experimentl diving in the turtle Am J Physiol, 5 8