MAXIMUM CARDIAC PERFORMANCE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) AT TEMPERATURES APPROACHING THEIR UPPER LETHAL LIMIT

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1 The Journl of Experimentl Biology 199, (1996) Printed in Gret Britin The Compny of Biologists Limited 1996 JEB MAXIMUM CARDIAC PERFORMANCE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) AT TEMPERATURES APPROACHING THEIR UPPER LETHAL LIMIT A. P. FARRELL, A. K. GAMPERL, J. M. T. HICKS, H. A. SHIELS AND K. E. JAIN Deprtment of Biologicl Sciences, Simon Frser University, Burny, BC, Cnd, V5A 1S6 Accepted 8 Novemer 1995 Numerous studies hve exmined the effect of temperture on in vivo nd in situ crdiovsculr function in trout. However, little informtion exists on crdic function t tempertures ner the trout s upper lethl limit. This study mesured routine nd mximum in situ crdic performnce in rinow trout (Oncorhynchus mykiss) following cclimtion to 15, 18 nd 22 C, under conditions of tonic (30 nmol l 1 ), intermedite ( nmol l 1 ) nd mximl (200 nmol l 1 ) drenergic stimultion. Hert rte incresed significntly with oth temperture nd drenline concentrtion. The Q 10 vlues for hert rte rnged from 1.28 t 30 nmol l 1 drenline to 1.36 t 200 nmol l 1 drenline. In contrst to hert rte, mximum stroke volume declined y pproximtely 20 % (from 1.0 to 0.8 ml kg 1 ) s temperture incresed from 15 to 22 C. This decrese ws not llevited y mximlly stimulting the hert with 200 nmol l 1 drenline. Becuse of the equl nd opposite effects of incresing temperture on hert rte nd Summry stroke volume, mximum crdic output did not increse etween 15 nd 22 C. Mximum power output decresed (y pproximtely %) t ll drenline concentrtions s temperture incresed. This reduction reflected poorer pressure-generting ility t tempertures ove 15 C. These results, in comintion with erlier work, suggest (1) tht pek crdic performnce occurs round the trout s preferred temperture nd well elow its upper lethl limit; (2) tht the diminished crdic function concomitnt with cclimtion to high tempertures ws ssocited with inotropic filure; (3) tht Q 10 vlues for crdic rte functions, other thn hert rte per se, hve limited predictive vlue t tempertures ove the trout s preferred temperture; nd (4) tht hert rte is poor indictor of crdic function t tempertures ove 15 C. Key words: hert, stroke volume, hert rte, crdic output, temperture, drenline, rinow trout, Oncorhynchus mykiss. Introduction Much of our knowledge on fish crdiovsculr responses to temperture chnge is limited to informtion on hert rte. There re two resons for this. First, hert rte is the esiest crdic vrile to mesure. Second, in mmmls t lest, hert rte is relile predictor of crdic performnce. If hert rte were eqully relile s predictor of integrted crdic function in fish, this lrge dt se would hve tremendous predictive potentil for fish crdic performnce. Unfortuntely, there re mny indictions tht hert rte lone is poor predictor of integrted crdic function in fish. Among the concerns re the following: (1) stroke volume cn chnge y s much s, nd even more thn, hert rte when crdic output increses (Frrell, 1991; Frrell nd Jones, 1992); (2) the reltive contriutions of stroke volume nd hert rte to chnges in crdic output vry etween species nd s function of temperture (see Kolok nd Frrell, 1994); (3) mximum stroke volume cn decrese t high hert rtes (Frrell et l. 1989); nd (4) mximum isometric tension developed y crdic muscle decreses t high contrction frequencies (i.e. negtive stircse effect) (Ask et l. 1981; Ask, 1983; Driedzic nd Gesser, 1985, 1988). In view of these oservtions, it would e unwise to predict chnges in crdic performnce from temperture-induced chnges in hert rte lone. Indeed, temperture-induced increse in hert rte does not necessrily produce proportionl increse in crdic output (Brett, 1971; Ymmitsu nd Itzw, 19; Kolok nd Frrell, 1994). The present study, which reports the first mesurements of mximum crdic performnce in rinow trout t tempertures ner their upper lethl limit (23 25 C; Blck, 1953), extends our knowledge of how numerous vriles (hert rte, stroke volume, crdic output, power output) ffect crdic function in fish. The results support our contention tht hert rte is poor indictor of integrted crdic performnce in fish. In ddition, this novel informtion should prove vlule in predicting the effects of incresed environmentl temperture on fish performnce. Our pproch ws to use n in situ perfused hert to mesure routine nd mximum crdic performnce t 15, 18 nd 22 C fter the fish hd cclimted to these tempertures. The in situ perfused rinow trout hert

2 664 A. P. FARRELL AND OTHERS is n pproprite model for investigting the reltionship etween high environmentl temperture nd crdic performnce since it is cple of performing t work levels equl to mximum in vivo levels (Frrell et l. 1989). Mterils nd methods Experimentl nimls Rinow trout [Oncorhynchus mykiss (Wlum)] (weighing g) were otined from locl supplier (West Creek Trout Frms, Aldergrove, BC, Cnd) nd mintined in 2000 l fireglss tnk receiving dechlorinted Vncouver tpwter. Throughout the experiment only one stock of fish ws used. All fish were initilly mintined t 15 C, efore susequent exposure to 18 nd then 22 C. Fish were cclimted t ech temperture for t lest 2 weeks prior to use. Wter temperture ws regulted to within 1 C of the desired test temperture y Min-O-Cool cooling unit (Frigid Units, Blissfield, MI, USA) nd two countercurrent het exchngers of locl construction. Photoperiod ws 12 h:12 h L:D. Fish were fed commercilly prepred trout pellets dily. Perfused hert preprtions Fish were nesthetized in uffered solution of tricine methne sulphonte (0.1 g l 1 MS 222, with 0.1 g l 1 sodium icronte) nd trnsferred to n operting tle where their gills were irrigted with erted uffered nesthetic t 4 C (0.05 g l 1 MS 222 in 0.1 g l 1 sodium icronte). Fish were injected with 0.6 ml of heprinized ( i.u. ml 1 ) sline vi the cudl vessels, nd n in situ hert preprtion ws otined, s detiled in Frrell et l. (1986) nd modified y Frrell et l. (1989). Briefly, n input cnnul ws secured into the sinus venosus through heptic vein nd perfusion with sline contining 30 nmol l 1 drenline ws egun immeditely. Silk thred ws used to secure the input cnnul nd to occlude ny remining heptic veins. The output cnnul ws inserted into the ventrl ort t point confluent with the ulus rteriosus nd tied firmly in plce. Finlly, silk ligtures were tied round ech ductus Cuvier to occlude these veins nd to crush the crdic rnches of the vgus nerve. This procedure left the pericrdium intct, while isolting the hert in terms of sline input nd output. Once the surgery hd een completed (15 20 min), the fish ws immersed in temperture-controlled sline th t 15, 18 or 22 C. The input cnnul ws ttched to constnt-pressure reservoir nd the output cnnul ws connected to constnt pressure hed. Output pressure ws initilly set t 5 kp to simulte resting in vivo ventrl ortic lood pressure (Stevens nd Rndll, 1967), nd filling (input) pressure ws djusted to give crdic output of 20 ml min 1 kg 1 ody mss for the 15 nd 18 C groups. Crdic output ws set t 25 ml min 1 kg 1 ody mss for the 22 C group to ccount for temperture effects on in vivo resting crdic output (Frrell nd Jones, 1992). At ll tempertures, the hert mintined this initil control level of performnce for period of 20 min to llow for recovery from surgery nd for equilirtion to the orgn th. The sline in the orgn th nd the perfusion reservoirs ws mintined t the desired cclimtion temperture y Lud cooling unit (Brinkmnn Instruments, Rexdle, Ontrio, Cnd). The sline (ph 7.8 t 15 C) contined (in mmol l 1 ): NCl, 124; KCl, 3.1; MgSO 4. 7H 2 O, 0.93; CCl 2. 2H 2 O, 2.52; glucose, 5.6; Tes slt, 6.4; nd Tes cid, 3.6 (Keen et l. 1994). The Tes uffer system ws selected to simulte the uffering cpcity of trout plsm nd the norml chnge in lood ph with temperture ( pk/dt=0.016 ph units C 1 ). The sline ws equilirted with % O 2 for t lest 30 min prior to experimenttion. The coronry rtery, which supplies the outer compct myocrdium of the ventricle, ws not perfused nd so oxygented sline ws used to ensure tht sufficient mount of oxygen diffused from the ventriculr lumen to the compct myocrdium. The oxygen grdient from the lumen to the mycrdium of our perfused hert ws t lest 20 times greter thn tht in vivo. The control sline contined 30 nmol l 1 drenline itrtrte ecuse Grhm nd Frrell (1989) hve estlished tht tonic drenergic stimultion with 10 nmol l 1 drenline is essentil for long-term viility of perfused herts t 5 C. In ddition, Keen et l. (1994) showed tht trout cclimted to high tempertures (18 C) hve decresed crdic sensitivity to drenline. Experimentl protocols The mximum pumping ility of the hert ws ssessed y mesuring the following: (1) the ility of the hert to mintin stroke volume when exposed to increses in output pressure (i.e. homeometric regultion); (2) mximum crdic output; (3) mximum power output; nd (4) output pressure t mximum power output. Homeometric regultion ws investigted y incresing distolic output pressure from 4 to 8 kp in increments of 1 kp, or until crdic output fell y 40 %. During homeometric regultion, the input pressure ws mintined t control levels. Output pressure ws not incresed further to ensure tht the hert ws not dmged prior to the mesurement of mximum crdic output nd mximum power output. In fish swimming mximlly, or exposed to high drenline doses, distolic ventrl ortic pressure is unlikely to exceed 8 kp (Kiceniuk nd Jones, 1977; Gmperl et l. 1994). Mximum crdic output ws determined under control conditions y incresing filling pressure in 8 12 steps (in increments of kp) until crdic output reched mximum vlue. Once mximum crdic output hd een ttined, output distolic pressure ws rised in steps of kp until the mximum power output ws reched. The output pressure t this point ws noted. Ech step in filling nd output pressure ws mintined for pproximtely 1 2 min. The hert ws returned to the control work lod for recovery period of 15 min fter the determintion of homeometric regultion nd following the determintion of mximum power output. This llowed the hert to recover fully etween tests nd/or to equilirte to new drenline concentrtions. This series of experimentl procedures required pproximtely 1 h to complete. All crdiovsculr mesurements were repeted t two dditionl drenline concentrtions ( nd 200 nmol l 1 ) to

3 Trout crdic performnce 665 cover the rnge for circulting ctecholmine levels oserved in stressed rinow trout (Millign et l. 1989; Gmperl et l. 1994; Rndll nd Perry, 1992). In ddition, preliminry experiments t 15 C (Fig. 1) showed tht 200 nmol l 1 drenline chieved mximum drenergic stimultion of the in situ preprtion nd tht nmol l 1 drenline ws ner the EC 50 for mximum stimultion. Instrumenttion nd nlysis An in-line electromgnetic flow proe (Zeped instruments, Hert rte (ets min 1 ) Mximum power output (mw g 1 ) Mximum crdic output (ml min 1 kg 1 ) [Adrenline] (nmol l 1 ) Fig. 1. Reltionship etween drenline concentrtion nd crdiovsculr vriles for in situ perfused trout herts t 15 C. Open circles represent individul herts nd filled circles represent group mens (N=3). Verticl rs represent ± 1 S.E.M. Settle, Wshington, USA) ws used to record men crdic output, nd pressure trnsducers (Nrco Life Sciences, Houston, TX, USA) were used to mesure input nd output pressures through sline-filled side-rms. Prior to experimenttion, pressure chnges due to cnnul resistnce were clculted t known flow rtes. These vlues were then used to djust input nd output pressure to the levels experienced y the sinus venosus nd ulus rteriosus, respectively. Clirtion of the pressure trnsducers ws performed dily ginst sttic wter column. Pressure nd flow signls were mplified nd displyed on four-chnnel chrt recorder (Gould, Clevelnd, OH, USA) in conjunction with microcomputer running Ltech Noteook (Lortory Technologies Corportion, Wilmington, MA). Dt were collected t 5 Hz, nd lock verges were clculted every 15 s. Hert rte ws mesured y counting the numer of systolic peks recorded during 10 s period. Stroke volume nd power output were clculted s follows: VS =(Q. /fh)/m, (1) p=[q. (P o P i ) ]/M v, (2) where Q. (ml min 1 ) is crdic output, P o is men output pressure (kp), P i is men filling pressure (kp), VS is stroke volume (ml kg 1 ody mss), fh is hert rte (ets min 1 ), M is ody mss (kg), p is power output (mw g 1 ventricle mss), M v is ventriculr mss (g) nd is mw min ml 1 kp 1. Within ech temperture group, pired t-tests were used to identify sttisticl differences etween crdiovsculr vriles recorded t 30, nd 200 nmol l 1. The effect of temperture within prticulr drenline concentrtion ws ssessed using one-wy nlysis of vrince (ANOVA). A covrint nlysis of vrince (ANCOVA) ws pplied to the stroke volume hert rte reltionship to isolte the interctive effects of temperture nd drenline. A generl liner model (Zr, 1974) ws used to exmine whether temperture nd drenline ffected the reltionship etween filling pressure nd stroke volume (i.e. the Strling curve) (Proc GLM, SAS Institute). For ll sttisticl nlyses, the fiducil limit of significnce ws chosen s 5 %. Vlues throughout the text re expressed s mens ± S.E.M. Results Homeometric regultion In our experience, crdic filure is not normlly prolem once the surgery hs een completed. This fct is illustrted y the success of ll seven of the preprtions ttempted t 15 C. However, t tempertures ove 15 C, increses in output pressure either during the initil elevtion to control conditions (5 kp finl pressure) or during the first homeometric regultion test cused crdic filure (indicted y sustined crdic rrhythmi) in some herts. At 18 C, two of the 13 ttempts filed, nd t 22 C the filure rte reched 40 % (six out of 15 preprtions). In these filing preprtions, incresing the drenline concentrtion to nmol l 1 occsionlly

4 666 A. P. FARRELL AND OTHERS restored the norml et frequency, ut only temporrily. These oservtions suggest tht ove 15 C there ws prticulrly strong negtive effect of temperture on the pressure-generting ility of certin herts. As result, it should e rememered tht the men vlues we report for crdic vriles do not tke into ccount the fct tht proportion of hert preprtions filed t 18 C nd 22 C. At ll tempertures, n increse in distolic output pressure significntly decresed resting stroke volume (Fig. 2). There ws lso significnt effect of temperture on the reltionship etween resting stroke volume nd temperture. At 15 C, stroke volume ws mintined ove % of the resting vlue even t n output pressure of 8 kp. In contrst, t 18 C stroke volume ws reduced to less thn % of the resting vlue t n output pressure of only 7 kp (Fig. 2). There ws no significnt difference etween the curves for 15 C nd 22 C. Adrenergic stimultion hd no mrked effect on the generl shpe of the homeometric reltionships (Fig. 2). However, drenline consistently displced the reltionship downwrds ecuse drenergiclly medited increses in hert rte ment tht the set point for resting stroke volume ws lower (see elow). Hert rte Hert rte incresed significntly with oth temperture nd drenline concentrtion (Fig. 3A). Incresing the temperture from 15 to 22 C incresed hert rte y 13.9 ets min 1 (from 69.9 to 83.8 ets min 1 ) with 30 nmol l 1 drenline, nd y 20.8 ets min 1 (from 81.5 to ets min 1 ) with 200 nmol l 1 drenline. The Q 10 vlues for 30 nmol l 1 nd 200 nmol l 1 drenline were clculted s 1.28 nd 1.36, respectively. Incresing the drenline concentrtion from 30 to 200 nmol l 1 significntly incresed hert rte t ll tempertures. This increse ws 18.5 ets min 1 (22 %) t 22 C nd pproximtely 10 ets min 1 t 15 C (17 %) nd 18 C (13 %). Stroke volume In lmost ll cses, the filling pressures t the routine work lods were sumient nd there were no significnt effects of temperture on the filling pressure required to generte routine crdic output (Tle 1). Incresing the filling pressure generted typicl Strling curve for stroke volume t ll tempertures. However, cclimtion temperture significntly ltered the shpe of the Strling curve (Fig. 4). Acclimtion to higher tempertures (18 nd 22 C) cused significnt downwrd shift in the upper rm of the Strling curve (Fig. 4) nd significntly decresed mximum stroke volume (Figs 3B, 4). Mximum stroke volumes were pproximtely 1 ml kg 1 t 15 C nd 0.8 ml kg 1 t 22 C. Adrenline hd no significnt effect on mximum stroke volume regrdless of the cclimtion temperture (Fig. 3B). Mximum crdic output Although there ws some vriility in crdic output Stroke volume (% of resting vlue) Stroke volume (% of resting vlue) Stroke volume (% of resting vlue) nmol l 1 nmol l nmol l Output pressure (kp) etween temperture groups, there were no significnt differences etween vlues. Furthermore, it is cler tht mximum crdic output with nd 200 nmol l 1 drenline ws unchnged y cclimtion temperture (Fig. 3C). This indictes tht the temperture-induced increses in hert rte 15 C 18 C 22 C Fig. 2. Reltionship etween stroke volume nd output pressure for in situ herts exposed to vrious tempertures (15 C, N=7; 18 C, N=11; 18 C, N=8) nd drenline concentrtions. Mesurements for herts t 18 C nd 8 kp of output pressure re not shown ecuse further increses in output pressure gretly reduced crdic output (see Mterils nd methods). A dgger denotes significnt (P<0.05) decrese in the stroke volume t the highest output pressure tested when compred with the resting vlue. An sterisk indictes significnt difference from the vlue for stroke volume of the 15 C fish tested t the highest output pressure. Verticl rs represent ± 1 S.E.M.

5 Trout crdic performnce 667 Resting hert rte (ets min 1 ) 110 A 30 nmol l 1 nmol l nmol l Mximum stroke volume (ml kg 1 ) B Mximum crdic output (ml min 1 kg 1 ) C Mximum power output (mw g 1 hert) D Acclimtion temperture ( C) Acclimtion temperture ( C) Fig. 3. Reltionship etween cclimtion temperture nd (A) resting hert rte, (B) mximum stroke volume, (C) mximum crdic output nd (D) mximum power output for in situ perfused trout herts t 15 (N=7), 18 (N=11) nd 22 C (N=8). Verticl rs represent 1 S.E.M. Dissimilr letters indicte vlues tht re significntly different (P<0.05) etween cclimtion tempertures within ech drenline concentrtion. Within ech temperture, mens with n unequl numer of sterisks indicte significnt differences etween drenline concentrtions (P<0.05). were offset y equl nd opposite chnges in mximum stroke volume. This point is illustrted in Fig. 5, where the 29 % increse in hert rte etween 15 nd 22 C ws ssocited with 23 % reduction in stroke volume. Within the 15 nd 22 C temperture groups, there were sttisticlly significnt effects of drenline (Fig. 3C). Incresing the drenline concentrtion from 30 to 200 nmol l 1 incresed mximum crdic output y 10 ml min 1 kg 1 (15 %) t 15 C nd y 7.5 ml min 1 kg 1 (10 %) t 22 C. There ws no significnt effect of drenline on crdic output t 18 C. Mximum power output While the homeometric regultion test nd the filure of high proportion of herts hinted t poorer inotropic performnce under resting conditions t 22 C, reduced mximum power output ws cler indictor of inotropic filure t this cclimtion temperture. Mximum power Tle 1. Morphometric nd crdiovsculr vriles for rinow trout (Oncorhynchus mykiss) cclimted to 15, 18 nd 22 C Test Resting P i (kp) P o t mximum power (kp) temperture Body mss Hert mss RVM ( C) (g) (g) (%) 30 nmol l 1 nmol l nmol l 1 30 nmol l 1 nmol l nmol l 1 15 (N=7) 493.1± ± ± ± ± ± ± ± ± (N=11) 515.1±28.9, 0.40± ± ± ± ± ±0.15, 6.±0.16, 6.14± (N=9) 6.5± ± ± ± ± ± ± ± ±0.24 Resting input pressure, nd output pressure t mximum power output, were recorded t three different drenline concentrtions (30, nd 200 nmol l 1 ) using n in situ hert preprtion. Vlues re expressed s mens ± 1 S.E.M. RVM, reltive ventriculr mss. Dissimilr letters indicte significntly different vlues (P<0.05) within column.

6 668 A. P. FARRELL AND OTHERS Stroke volume (ml kg 1 ) C 1 18 C C Increse in input pressure (kp) Mximum stroke volume (ml kg 1 ) C c 18 C c 30 nmol l 1 nmol l nmol l 1 22 C Fig. 4. Strling curves for in situ perfused trout herts t cclimtion tempertures of 15 (N=7, ), 18 (N=11, ) nd 22 C (N=9, ). Within ech temperture, ech point represents dt for n individul hert t drenline concentrtions of 30, nd 200 nmol l 1. Best-fitting equtions for ech cclimtion temperture were: 15 C, y= 2.439x x (r 2 =0.942); 18 C, y= 2.454x x (r 2 =0.829); 22 C, y= 1.887x x (r 2 =0.824). Anlysis of covrince showed tht the stroke volume input pressure reltionships t ll tempertures were significntly different from ech other (P<0.05). output ws significntly lower t 22 C tht t 15 C, irrespective of the drenline concentrtion (Fig. 3D). This reduction in mximum power output occurred primrily ecuse the mximum pressure-generting ility of the hert ws significntly lower (Tle 1), since mximum crdic output ws unffected (Fig. 3C). Discussion The present study, which is the first to mesure the mximum performnce of perfused slmonid hert t tempertures ner the upper lethl temperture, clerly shows 110 Hert rte (ets min 1 ) Fig. 5. The reltionship etween mximum stroke volume nd hert rte for in situ perfused trout herts t vrious tempertures nd drenline concentrtions. Vlues re expressed s mens ± 1 S.E.M. Dissimilr letters within temperture groups indicte significnt differences (P<0.05) etween drenline concentrtions. Mens for ech drenline concentrtion were significntly different etween tempertures (P<0.05). tht mximum crdic output reches plteu t pproximtely 15 C nd tht tempertures ove 18 C re ssocited with reduced pressure-generting ility. This conclusion is consistent with in vivo mesurements mde on nother slmonid, the sockeye slmon (Oncorhynchus nerk). Dvis (1968) nd Brett (1971) found tht crdic output in swimming sockeye slmon ws essentilly unchnged etween 15 nd 22 C. In ddition, they estimted tht crdic work during ctivity hd pek t 15 C ecuse ventrl ortic lood pressure ws lower t 22 C thn t 15 C. If it is ssumed tht mximl prolonged swimming ctivity elicits mximl Tle 2. A comprison of mximum crdic performnce vriles t vrious tempertures using in situ hert preprtions with intct pericrdi nd tonic (5 30 nmol l 1 drenline) or mximl levels of drenergic stimultion (vlues in prentheses) Temperture Hert rte Stroke volume Crdic output Power output ( C) (ets min 1 ) (ml kg 1 ) (ml min 1 kg 1 ) (mw g 1 ) Reference Keen nd Frrell (1994) Frrell et l. (1988) Millign nd Frrell (1991) (73) 0.89 (0.86) 56 (63) 5.2 (6.9) Frrell et l. (1991) 10 (TR) 66 (75) 1.05 (1.05) 67 (75) 6.7 (8.7) Frrell et l. (1991) 15 (82) 0.99 (1.02) 66 (76) 8.00 (9.3) Present study Keen nd Frrell (1994) (88) 0.97 (0.91) 78 (76) 9.3 (8.6) Present study (102) 0.81 (0.77) 71 (78) 7.2 (8.0) Present study TR indictes tht this group of fish ws exercise-trined for 1 month.

7 Trout crdic performnce 669 crdic response in sockeye slmon, then the sockeye slmon s mximl in vivo crdic performnce peks t its preferred temperture (12 14 C; Brett, 1971) nd well elow its upper lethl limit (24 C). We elieve tht similr conclusion cn e drwn for the rinow trout, whose preferred temperture nd upper lethl temperture re lmost identicl to those of the sockeye slmon (Blck, 1953; Grside nd Tit, 1958). When using perfused trout hert preprtion without coronry circultion, the exchnge of gses nd solutes etween the myocrdium nd the perfuste will e ffected. This is of prticulr concern in the present study for two resons. First, the experimentl conditions promoted crdic filure nd, second, ny prolems with diffusion my hve een excerted in the herts of the 22 C fish ecuse their ventricles were 30 % lrger thn those of the 15 C fish. Nonetheless, there re severl importnt resons why the sence of coronry circultion ws unlikely to is the experiments towrds the poorer crdic performnce oserved t 22 C. Foremost, there is good greement etween the crdic performnce mesures for our perfused in situ herts nd reported in vivo vlues for swimming sockeye slmon (Dvis, 1968; Brett, 1971). In oth studies, hert rte incresed t 22 C while stroke volume, crdic output, pressure genertion nd crdic power output decresed. Second, y limiting fish size to less thn 750 g nd oxygenting the perfuste, we elieve tht ny prolem with oxygen diffusion into the myocrdium ws lrgely eliminted. The oxygen prtil pressure grdient ws t lest 20 times tht normlly found in venous lood, nd the thickness of the compct myocrdium in our fish (<1 mm) ws no more thn tht used in exmining crdic performnce with electriclly pced, isolted strips. Third, Dvie nd Frrell (1991) were unle to improve the performnce of normoxic dogfish herts y perfusing the coronry circultion with ir-sturted sline. Although we my hve eliminted the possiility of n oxygen limittion, there is n dditionl concern regrding solute trnsfer, prticulrly the removl of H + nd K +, which in themselves could reduce hert contrctility. We know from previous studies tht lctte nd H + re relesed into the lumen nd cn e mesured in the perfuste leving the trout hert (Frrell nd Millign, 1986). Therefore, trnsfers of solute from the trout myocrdium re fr from completely inhiited. If the lrger herts of the 22 C group did led to poorer crdic performnce, we would predict negtive correltion etween hert size nd crdic power output. However, no significnt reltionship exists etween these two vriles (r 2 =0.30). In view of the ove discussion, we feel confident in extrpolting our oservtions to the in vivo sitution nd in providing mechnistic explntions. To illustrte the point tht pek crdic performnce occurs t pproximtely 15 C in rinow trout, Tle 2 summrizes the ville dt on mximum crdic performnce of perfused rinow trout herts t vrious tempertures. The dt in Tle 2 re comprle ecuse they were collected in the sme lortory using the sme type of hert preprtion (i.e. n in situ hert with n intct pericrdium) nd n initil tonic drenergic stimultion (5 30 nmol l 1 drenline). Tle 2 clerly shows tht the mximum stroke volume under conditions of tonic drenergic stimultion occurs etween 10 nd 15 C, wheres mximum crdic output nd mximum power output occur t 18 C. Although these dt suggest tht mximum crdic performnce in rinow trout occurs t 18 C, it is unlikely tht in vivo mximl crdic performnce is chieved without significnt drenergic stimultion (humorl nd/or sympthetic). Under conditions of mximl drenergic stimultion, crdic output remins constnt etween 10 nd 22 C, n effect which shifts the optimum temperture for mximum power output to 15 C (Tle 2; Fig. 3D). Becuse power output is the most pproprite index of integrted crdic performnce, it ppers tht mximum performnce of rinow trout herts is chieved t 15 C. Temperture is generlly regrded s hving positive chronotropic nd negtive inotropic effects on the teleost myocrdium (Lennrd nd Huddrt, 1992; Mtikinen nd Vornnen, 1992). The present study supports this generliztion with regrd to oth the chronotropic nd inotropic effects of temperture. Mtikinen nd Vornnen (1992) nicely illustrted the simultneous nd opposing effects of temperture-relted negtive inotropy nd positive chronotropy using isolted crp crdic muscle. By deriving mximum tissue pumping cpcity term (the product of the spontneous hert rte nd the mximum isometric force; gmg 1 tissue min 1 ), they demonstrted pek tissue pumping cpcity t pproximtely 20 C, temperture well elow the upper lethl temperture of crp (pproximtely 35 C). The performnce curve for isolted crp crdic muscle strips hd n inverted U shpe s function of temperture. Consequently, the decline in tissue pumping cpcity of the crp hert t wrm tempertures ers striking resemlnce to the decline in mximum power output in in situ rinow trout herts (see Tle 2; see Fig. 6) nd in vivo in sockeye slmon (Brett, 1971). In ll three instnces, there ws decrese in inotropic performnce nd/or decresed mximum stroke volume t higher tempertures. Inotropic filure in our rinow trout herts t tempertures greter thn 15 C ws demonstrted y lower vlues for mximum power output nd mximum output pressure t 18 nd 22 C. In ddition, the significnce of this result is mgnified when one considers tht the filure of numer of preprtions t these tempertures resulted in only the stronger herts eing tested (this is my in fct explin why the homeometric regultion curves were similr for 15 nd 22 C herts). The finding tht rinow trout herts hd poorer pressure-generting ility t tempertures ove 15 C hs indirect support from in vivo studies. For exmple, Dvis (1968) reported reduced ventrl nd dorsl ortic lood pressures in swimming sockeye slmon t 22 C compred with vlues t 15 C, even though crdic output ws the sme. Also, Wood et l. (1979) found significnt ttenution of the increse in dorsl ortic lood pressure in rinow trout in response to drenline injections t 22 C compred with 12 C. Thus, in oth of these studies, the hert performed less

8 6 A. P. FARRELL AND OTHERS pressure work t 22 C. These in vivo oservtions could e relted to incresed temperture directly ffecting the norml neurl, hormonl nd locl control of vsomotor sttus in the systemic circultion (i.e. either greter vsodiltory cpcity or weker vsoconstrictory cpcity). However, on the sis of the present oservtions, we cn include nother possiility. In response to poorer crdic pressure-generting ility t 22 C, the vsomotor system my produce vsodilttion to llow for the mintennce of crdic output. Interestingly, Gmperl et l. (1994) reported tht drenline injection into rinow trout resulted in significntly lower in vivo dorsl ortic pressures if the pericrdium ws opened. Opening the pericrdium is known to cuse poorer pressure genertion in oth the rinow trout (Frrell et l. 1988) nd the eel (Anguill dieffencchi) (Frnklin nd Dvie, 1991), nd reduces mximum power output in the rinow trout hert y pproximtely 45 %. Work on isolted crdic muscle strips from teleost fish clerly shows tht mximum tension decreses with incresing pcing frequencies, negtive stircse effect (Driedzic nd Gesser, 1985; Vornnen, 1989; Biley nd Driedzic, 19). It seems likely tht this negtive stircse effect would explin the negtive inotropic effect of wrm temperture in our herts. Indeed, there is decrese in force when the spontneous et frequency increses with temperture (Ask, 1983; Mtikinen nd Vornnen, 1992), nd indictions re tht fctors ssocited with either shortening of the durtion of the ctive stte or reduction in the intensity of the ctive stte my ecome limiting t high et frequencies (Vornnen, 1989; Driedzic nd Gesser, 1994). The fctors could include the inility of the contrctile proteins to generte mximl force t shortened ctive sttes or impired clcium delivery to nd removl from the contrctile proteins (Vornnen, 1989; Mtikinen nd Vornnen, 1992). Nevertheless, lterntive explntions for the reduction in mximum power output nd mximum pressure-generting ility with incresing temperture should not e excluded t this time. For exmple, decreses in -receptor numer nd/or ffinity could hve diminished the positive inotropic influence of drenline. In ventriculr strips, Keen et l. (1993) showed tht it tkes pproximtely 10 times the drenline concentrtion t 18 C to chieve the sme level of tension genertion mesured t 8 C, nd tht this effect ws ssocited with fewer srcolemml -drenoreceptors. In ddition, Ask et l. (1981), using tril tissue, showed tht the contrctile force elicited y mximlly effective dose of drenline (1.4 mol l 1 ) t 14 C ws only 30 % of tht produced t 2 C. Although the oservtion tht hert rte incresed with incresing drenline concentrtion t ll tempertures (Fig. 3A) is pprently inconsistent with diminished drenergic influence t high tempertures, it must e rememered tht positive chronotropy is medited primrily through effects on the hert s pcemker cells (Hung, 1973), wheres inotropic effects occur primrily ecuse of drenergic stimultion of the ventricle. Thus, there could e differentil temperture effects on drenergic sensitivity for these two regions of the hert. It is cler from the present study tht the ppliction of Q 10 vlues to mximum crdic output hs limited vlue. At tempertures ove the preferred temperture, Q 10 vlues could e misleding ecuse of the plteu in mximum crdic output. Furthermore, ecuse tempertures ove 15 C re ssocited with incresed hert rtes ut constnt crdic output nd flling power outputs, hert rte must e considered to e very poor predictor of crdic performnce t these tempertures. Mximum stroke volume decresed with incresing temperture (Fig. 3B). Previous studies with in situ trout herts hve lso reported tht mximum stroke volume decresed with incresing temperture (Grhm nd Frrell, 19; Keen nd Frrell, 1994). Likewise, Ymmitsu nd Itzw (19) showed tht stroke volume decresed with incresing temperture in the isolted crp hert, lthough it is unlikely tht they mesured mximum performnce. The dt presented in Tle 2 suggest tht mximum stroke volume of the in situ rinow trout hert (pproximtely 1 ml kg 1 ) occurs t tempertures of 15 C nd elow. In hert preprtion with punctured pericrdium, Grhm nd Frrell (19) showed tht stroke volume decresed from 1 ml kg 1 t 5 C to 0.7 ml kg 1 t 15 C. Becuse stroke volume in our in situ hert with n intct pericrdium ws still 1 ml kg 1 t 15 C, it is possile tht the pericrdium plys n importnt role in mintining mximum stroke volume t wrm tempertures. Hert rte clerly hd n importnt influence on mximum stroke volume (Fig. 5). This grees with numerous previous studies where temperture-induced decreses in stroke volume were ssocited with concomitnt increses in hert rte (Grhm nd Frrell, 1989; Lennrd nd Huddrt, 1992; Keen nd Frrell, 1994). There re two possile explntions for this inverse reltionship etween hert rte nd mximum stroke volume: either limittion on crdic filling or the negtive stircse effect on crdic contrctility referred to ove. To wht degree these two fctors influence stroke volume t higher hert rtes cn e resolved only y direct mesurements of hert chmer volumes during the crdic cycle. Using echocrdiogrphy, Frnklin nd Dvie (1992) showed tht ventriculr end-systolic volume in rinow trout is normlly ner zero. Therefore, if negtive stircse effect is involved in the reduced stroke volume t high hert rtes, end-systolic volume would e found to increse. In contrst, lower enddistolic volume would ccount for the decrese in stroke volume if filling time ws prolem, s suggested y Frrell et l. (1989) to explin decrese in mximum stroke volume of 0.2 ml kg 1 when isolted trout herts were pced t ets min 1. One piece of evidence which suggests tht limittions on crdic filling my contriute to the decrese in stroke volume t high hert rtes comes from studies on in situ herts with intct (present study) versus punctured pericrdi (Grhm nd Frrell, 19). Stroke volume decresed y 0.3 ml kg 1 (from 1 ml kg 1 ) when intrinsic hert rte reched ets min 1 in herts with punctured pericrdium. In contrst, herts with n intct pericrdium were le to mintin mximum stroke volume until hert rte exceeded

9 Trout crdic performnce 671 Percentge of mximl vlue VS Power output Q. fh Acclimtion temperture ( C) Fig. 6. Proposed reltionship etween cclimtion temperture nd the mximl level of crdiovsculr vriles for the rinow trout. The % vlues for stroke volume (VS), hert rte (fh), power output nd crdic output (Q. ) re 1 ml kg 1, 120 ets min 1, 9.5 mw g 1 nd ml min 1 kg 1, respectively. ets min 1. A mechnistic explntion for the enhnced mintennce of mximum stroke volume in herts with n intct pericrdium is tht the pericrdium in rinow trout permits vis--fronte filling of the hert (Frrell et l. 1988) nd this type of crdic filling is likely to e fster thn vis--tergo filling (Frrell nd Jones, 1992). Fig. 6, while somewht specultive t this time, summrizes our ides on crdic performnce in rinow trout s function of cclimtion temperture. We hope tht it will provide useful frmework for further reserch in this re. Hert rte follows Q 10 reltionship up to the upper lethl temperture, where it reches its mximum level of pproximtely 120 ets min 1. Mximum stroke volume (pproximtely 1mlkg 1 ) is mintined up to the preferred temperture, ove which it decreses. For severl degrees ove the preferred temperture, the decrese in stroke volume is mtched y the increse in hert rte. Consequently, mximum crdic output (pproximtely ml min 1 kg 1 ) hs rod plteu extending for severl degrees higher thn the preferred temperture. In contrst, the pressure-generting ility of the hert decreses t tempertures higher thn the preferred temperture such tht pek mximum power output (round 9.5 mw g 1 ) occurs round the preferred temperture. Mechnistic explntions for the decline in mximum performnce ove the preferred temperture require further work t oth the orgn nd tissue level. However, the oservtion tht mximum stroke volume ws not mintined t high tempertures suggests tht myocrdil dpttions re quite limited ove the preferred temperture. This is not the cse t colder tempertures. For exmple, cold cclimtion results in lrger crdic mss (Grhm nd Frrell, 1989) nd greter numer of srcolemml drenoceptors tht increse the sensitivity of the trout hert to drenline (Keen et l. 1993). As result of cold-cclimtion, mximum stroke volume nd power output tend to e higher thn otherwise possile with the ccompnying temperture-dependent decrese in hert rte. Whether ny of the ove generliztions pply to other teleost species, such s sockeye slmon nd crp, remins to e determined. However, it is cler for the rinow trout (1) tht mximum crdic performnce declines t tempertures ove the preferred temperture; (2) tht the usefulness of Q 10 reltionships for crdic functions other thn hert rte is highly dependent upon the section of the therml regime of the fish under considertion; nd (3) tht hert rte is poor indictor of integrted crdic function t tempertures ove the preferred temperture. This work ws supported y Nturl Sciences nd Engineering Reserch Council of Cnd operting grnt to A.P.F. nd post-doctorl fellowship to A.K.G. References ASK, J. A. (1983). Comprtive spects of drenergic receptors in the herts of lower vertertes. Comp. Biochem. Physiol. 76A, ASK, J. A., STENE-LARSEN, G. AND HELLE, K. B. (1981). Temperture effects on the B 2-drenoceptors of the trout trium. J. comp. Physiol. 143B, BAILEY, J. R. AND DRIEDZIC, W. R. (19). Enhnced mximum frequency nd force development of fish herts following temperture cclimtion. J. exp. Biol. 149, BLACK, E. C. (1953). Upper lethl tempertures of some British Columin freshwter fishes. J. Fish. Res. Bd Cn. 10, BRETT, J. R. (1971). Energetic responses of slmon to temperture. A study of some therml reltions in the physiology nd freshwter ecology of sockeye slmon. Am. Zool. 11, DAVIE, P. S. AND FARRELL, A. P. (1991). Crdic performnce of n isolted hert preprtion from the dogfish (Squlus cnthis): the effects of hypoxi nd coronry rtery perfusion. Cn. J. Zool. 69, DAVIS, J. C. (1968). The influence of temperture nd ctivity on certin crdiovsculr nd respirtory prmeters in dult sockeye slmon. MSc thesis, University of British Columi. 114pp. DRIEDZIC, W. R. AND GESSER, H. (1985). C 2+ protection from the negtive inotropic effect of contrction frequency on teleost herts. J. comp. Physiol. 156B, DRIEDZIC, W. R. AND GESSER, H. (1988). Differences in force frequency reltionships nd clcium dependency etween elsmornch nd teleost herts. J. exp. Biol. 140, DRIEDZIC, W. R. AND GESSER, H. (1994). Energy metolism nd contrctility in ecotothermic verterte herts: Hypoxi, cidosis nd low temperture. Physiol. Rev. 74, FARRELL, A. P. (1991). From hgfish to tun perspective on crdic function. Physiol. Zool. 64, FARRELL, A. P., HAMMONS, A. M., GRAHAM, M. S. AND TIBBITS, G. F. (1988). Crdic growth in rinow trout, Slmo girdneri. Cn. J. Zool. 66, FARRELL, A. P., JOHANSEN, J. A. AND SUAREZ, R. K. (1991). Effects of exercise-trining on crdic performnce nd muscle enzymes

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