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Cardiac tissue function of the teleost fish Oreochromis niloticus under different thermal conditions
Journal of Thermal Biology 25 (2000) 373±379
www.elsevier.com/locate/jtherbio
Cardiac tissue function of the teleost ®sh Oreochromis niloticus under dierent thermal conditions Monica Jones Costa, Luciano Rivaroli, Francisco Tadeu Rantin, Ana LuÂcia Kalinin* Department of Physiological Sciences, Federal University of SaÄo Carlos, Via Washington Luiz, km 235-13565-905, SaÄo Carlos, SP, Brazil Received 24 September 1999; accepted 4 November 1999
Abstract The cardiac responses of Oreochromis niloticus acclimated to 258C were assessed using ventricle strips mounted for isometric force recording (Fc) and in vivo heart rate ( fH). fH increased progressively from 25 to 408C. At extracellular Ca2+ concentrations of 1.25 and 9.25 mM, a transition from 25 to 408C resulted in a decreased Fc. At both 25 and 408C, Fc rose when [Ca2+] was increased from 1.25 to 9.25 mM. Fc remained constant at 72 and 120 contractionsminÿ1 at 25 and 408C, respectively, and declined thereafter. The post-rest potentiation was not in¯uenced by ryanodine, indicating that the sarcoplasmic reticulum is not important to the excitation±contraction coupling. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Acclimation; Temperature; Heart function; Ventricle strips; Excitation-contraction coupling; Calcium management; Sarcoplasmic reticulum; Nile tilapia; Oreochromis niloticus
1. Introduction The ability of the cardiac muscle to maintain pump performance under dierent physiological conditions is one of the most important characteristics that allow vertebrates to survive under extreme conditions (Driedzic and Gesser, 1994). It is well established that changes in environmental temperature have an immediate and substantial eect upon cardiac performance in north-temperate teleost ®sh (Bailey and Driedzic, 1990). In these species, rest-
* Corresponding author. Tel.: +55-16-2608314; fax: +5516-2608328. E-mail address: [email protected] (A.L. Kalinin).
ing heart rates are typically under 100 beatsminÿ1 and follow a Q10 of about 2 during acute temperature transitions. Heart contractility is usually triggered by increases in the intracellular Ca2+ concentration due to an in¯ux across the sarcolemma on a beat to beat basis, although the functional importance of the cardiac sarcoplasmic reticulum in supplying Ca2+ for contractility increases with temperature in some species (Rantin et al., 1998). However, there is little information about the impact of temperature on tropical species. The present study extends ®ndings to the freshwater cichlid Oreochromis niloticus, a tropical species tolerant to a wide range of temperatures (from 010± 408C; Fernandes and Rantin, 1986) in dierent Brazilian regions.
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M.J. Costa et al. / Journal of Thermal Biology 25 (2000) 373±379
2. Materials and methods Specimens of Oreochromis niloticus weighing between 254 2 17.6 g were collected at the Tropical Fish Research Center (CEPTA/IBAMA), Pirassununga, SP, Brazil. For at least 4 weeks prior to experimentation, the ®sh were maintained in holding tanks equipped with a continuous supply of well-aerated and dechlorinated water at a constant temperature (258C). They were fed with commercial ®sh food pellets ad libitum. For the specimens used in the in vivo experiments, food was withheld 24 h before experimentation. 2.1. In vivo heart rate Heart rate ( fH-bpm) was obtained by electrocardiography. An electrode was placed in a ventral position between the gills and the heart and a second electrode in a ventral position close to the pelvic ®ns. A reference electrode was located in the water of the experimental chamber (for details see Glass et al., 1991; Rantin et al., 1993). The electrode set was connected to a universal coupler of a Narco Narcotrace 40 recorder (Narco Bio Systems, Houston, TX, USA). For implantation of the electrodes, the ®sh were immersed in a benzocaine solution (1 g of benzocaine dissolved in 0.1 l of 99% ethanol and diluted in 10 l of water). This level of anesthesia allowed spontaneous breathing. After surgery, the ®sh was placed into a holding PVC tube. The oxygen tension of the ingoing (PinO2-mmHg) water was measured continuously. Water was siphoned via polyethylene catheters to O2 electrodes (FAC 001-O2, FAC-SaÄo Carlos, SP, Brazil) housed within temperature controlled cuvettes and connected to a FAC-204A O2 Analyzer. The PinO2 was maintained at normoxic levels (140 mmHg). After a recovery period of 12 h, the fH was measured at 258C. The temperature was then increased (108Chÿ1) to 30, 35 and 408C and subsequently reduced to 258C. Each experimental temperature was maintained for 30 min and measurements were obtained during the last 5 min. 2.2. Ventricle strip preparation Animals were killed by a blow on the head. Pairs of strips with a thickness of maximally 1 mm were excised from the ventricle and placed into an oxygenated bathing medium containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 0.5 mM NaH2PO4, 1.25 mM CaCl2, 10 mM glucose and 10 mM imidazole±HEPES buer to give pH 7.6. The temperature of the muscle bath was kept at 258C in a temperature-controlled water bath, unless otherwise stated. The preparations were suspended using surgical silk to connect to a NARCO
F-60 isometric force transducer placed around one platinum electrode. This electrode and one placed in the bath were connected to a stimulator (Grass SD9, Quincy, MA, USA) delivering electrical square pulses having a duration of 8 ms and a voltage 50% above that of the threshold value in order to provide a security margin resulting in maximal stimulation throughout the experiment. The stimulation rate was 0.2 Hz unless otherwise stated. The preparations were stretched to provide a twitch tension at the maximum of the length±twitch tension relation. Twitch tension was allowed to stabilize for about 30 min. In the ®rst experimental protocol, muscle preparations were subjected to a temperature transition from 25 to 408C over a 25±30 min time period. Preparations were maintained at 408C for 30 min. Thereafter, the temperature was decreased over a period of 15±20 min back to 258C and held at that temperature for a further 30 min. To evaluate the dependence of the cardiac muscle responses resulting from the eects of temperature changes on the excitation±contraction coupling, an experimental series was performed at a physiological level of 1.25 mM extracellular Ca2+ and also at an elevated level of 9.25 mM Ca2+. In further experimental protocols, preparations were allowed to initially stabilize at 25 or 408C. In a third series of experiments, the maximal capacity of hearts to develop force was assessed trough the addition of either Ca2+ or adrenaline to bathing medium. To evaluate the eect of the time exposure to 25 and 408C, an additional series was performed without adrenaline, using 1.25 or 9.25 mM Ca2+. In a fourth series, preparations were subjected to an increase in imposed contraction frequency coupling. Finally, the force development upon the ®rst stimulation following a rest period was determined at 25, 35 and 408C. This approach can inform on the capacity for the storage of intracellular Ca2+ during resting period. After a steady-state condition at 12 contractionsminÿ1 was achieved, stimulation ceased for a period of 5 min. Force development of the ®rst contraction following the resting period was compared to the last contraction in a steady-state train. These experiments were conducted with and without 10 mM ryanodine in the medium. According to Coronado et al. (1994), in mammalian muscle, ryanodine stimulates Ca2+ release from sarcoplasmic reticulum (SR) at low concentrations (10±300 mM). When applied, ryanodine was added to the medium 30±45 min before the 5 min interval. 2.3. Data presentation and analysis Results are presented as mean values2SE. In all experiments, signi®cance levels with respect to parameters of the same experimental protocol were
M.J. Costa et al. / Journal of Thermal Biology 25 (2000) 373±379
375
assessed with One-way Analysis of Variance (ANOVA) followed by Bartlett's test for homogeneity of variances and Tukey±Kramer multiple comparisons test. Additionally, the Mann±Whitney test was employed to compare parameters of dierent experimental protocols at the same temperature. Signi®cance levels are indicated in the ®gures as follows: p < 0.05, p < 0.01, p < 0.001 (GraphPad Instat version 2.01, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. In vivo heart rates Fig. 1 shows the eects of the increases in temperature from 25 to 408C and subsequent recovery to 258C on the fH (bpm). The fH increased progressively and signi®cantly from 30 to 408C, at which fH reached its maximum value of 97.226.5 bpm. This value was 2.9fold higher than that at 258C. After the return to 258C, the fH decreased to a value similar to that recorded previously at the same temperature. 3.2. Ventricle strip preparation At extracellular Ca2+ concentrations of 1.25 and 9.25 mM, a transition from 25 to 408C resulted in a decrease in twitch force (Fig. 2). During the subsequent restoration to 258C, twitch force increased in both Ca2+ concentrations but did not achieve the initial values. However, at the Ca2+ concentration of 9.25 mM the Fc was signi®cantly higher than at 1.25 mM, in the experimental temperatures of 30, 35 and 408C. The scope of activity at 25 and 408C was examined by increasing levels of extracellular Ca2+ and adrenaline. The eects of extracellular Ca2+ on the twitch
Fig. 1. The eect of a change in temperature from 25 to 408C on heart rate ( fH Ð bpm) of Oreochromis niloticus. Mean values2SE (n = 10).
Fig. 2. The eect of changes in temperature from 25 to 408C and back to 258C on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at an extracellular Ca2+ concentration of 1.25 mM (q) and 9.25 mM (Q). Mean values2SE (n = 10).
force of the cardiac muscle of O. niloticus is presented in Fig. 3. At 258C, increments in extracellular Ca2+ from 1.25 to 9.25 mM resulted in increases in twitch force development. However, at 408C, the twitch force increased progressively reaching maximum values at 9.25 mM, indicating a large dependence on extracellular Ca2+. Increased concentrations of adrenaline caused signi®cant increases in twitch force only at 408C (Fig. 4). This experiment involves 10 min of exposure to adrenaline at each condition. Control experiments at 1.25 mM or 9.25 Ca2+ and zero adrenaline were performed to determine if there was deterioration of force contraction over this time period (Fig. 5). Changes in twitch force with time were observed only at 408C and 9.25 mM Ca2+. In this condition, the Fc decreased
Fig. 3. The eect of extracellular Ca2+ on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at 258C (q) and 408C (Q). Mean values2SE (n = 10).
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Fig. 4. The eect of adrenaline on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at 258C (q) and 408C (Q). Mean values2SE (n = 10).
progressive and signi®cantly along the time, reaching minimum values after 40 min of exposure (2.4-fold lower than those initially recorded). The impact of increases in the imposed contraction frequency is presented in Fig. 6. At 258C, twitch force immediately declined as frequency was increased above 30 contractionsminÿ1. Preparations became irregular above 72 contractionsminÿ1. At 408C twitch force development remained constant at frequencies up to 120 contractionsminÿ1 and declined with further increase of frequency. Preparations were able to maintain a rhythmic response up to about 180 contractionsminÿ1. At 408C, resting tension tended to increase, i.e., relaxation became incomplete with rates above 60 contractionsminÿ1. Fig. 7 shows the force development of the ®rst contraction following a rest period at 25, 35 or 408C, in
Fig. 5. Eect of time on twitch force of cardiac muscle of O. niloticus at 258C (q) and 408C (Q). Mean values 2 SE (n = 10).
Fig. 6. Eect of stimulation frequency at 25 and 408C on twitch force (upper panel) and resting tension (lower panel). Twitch force was allowed to stabilize before the next increment in frequency was applied. Mean values2SE (n = 10).
the presence or absence of ryanodine. Post-rest potentiation occurred only at 258C and was not in¯uenced by ryanodine. At 358C, force contraction remained unchanged either with or without ryanodine. A postrest decay was recorded at 408C in both treatments. In all cases, no signi®cant dierences were observed
Fig. 7. Twitch force of the ®rst contraction following a rest period. A stimulation rate of 0.2 Hz was followed by a 5 min rest period. q Ð control values; Q Ð 10 mM ryanodine. Asterisks above bars indicate signi®cance level between the bar and the last contraction prior to the rest period. Mean values2SE (n = 10).
M.J. Costa et al. / Journal of Thermal Biology 25 (2000) 373±379
between the results obtained with and without ryanodine, indicating that, in this species, the sarcoplasmic reticulum does not seem to be of importance to the excitation±contraction coupling of heart muscle. 4. Discussion The cardiac output must be regulated to maintain an adequate blood ¯ow for the muscle in exercise, brain and other tissues under a variety of conditions. According to Farrell and Jones (1991), in most of the ®sh the regulation of the cardiac output is mediated by alterations in the cardiac stroke volume instead of through the control of the heart rate, as in mammals. On the other hand, according to Randall (1968), following increases in temperature, an increase in the cardiac output in ®sh heart may occur exclusively by increased heart rate, which results in direct eect of temperature on the pacemaker cells. This alters the membrane permeability and, consequently, increases the intrinsic rate of depolarization of these cells. Additionally, Vornanen (1994), working on crucian carp, described that high temperatures increase its heart rate while low temperatures increase stroke volume. According to Driedzic and Gesser (1994), the resting heart rates in ®sh are typically under 100 beatsminÿ1 (10±60 bpm, not exceeding 120 bpm). Exceptions of this pattern are the tuna (Katsuwonus pelamis ), that presents resting fH around 120 bpm, and surpasses 200 bpm during exercise (Keen et al., 1992), and Bathygobius soporator, in which the resting fH is about 140 bpm at 258C (Rantin et al., 1998), which is comparable to mammalian fH. Tropical teleost ®sh have Q10 values for the fH around 2 during acute changes in temperature (Rantin et al., 1998), similar to those obtained in the present study. Therefore, in spite of the high capacity to survive at elevated temperatures, the temperature-dependent variations of the fH of O. niloticus did not dier signi®cantly from those presented by temperate species. This result suggests that O. niloticus triggers other mechanisms than increases in fH to maintain the heart performance during increases in temperature. The negative inotropic eect presented by O. niloticus after temperature increases is consistent with that previously recorded by Bailey and Driedzic (1990) and Rantin et al. (1998) for Perca ¯uviatilis and Bathygobius soporator, respectively. These authors described progressive reductions of the contractile activity of the ventricular strips during increments of 158C in the experimental temperature. The reduction in twitch force during the elevation in temperature from 35 to 408C in the present study could indicate a possible irreversible damage in myocardial function after reaching the highest temperature,
377
since the initial force was not recovered after the return to 258C in both physiological and high Ca2+ concentrations. These results dier from those recorded by Rantin et al. (1998), where the twitch force of B. soporator at 1.25 mM Ca2+ was completely recovered upon the return to 258C. This suggests that B. soporator has special adaptations to deal with rapid and substantial temperature transitions. According to these authors, a possible explanation for the negative inotropic temperature eect could be pH related, since increases in temperature should lower pH and, consequently, the peak force. Several studies report on the dependence of heart contractility on extracellular calcium concentration and emphasize the importance of the calcium ¯ux across the sarcolemma for the excitation±contraction coupling (Driedzic and Gesser, 1994; Tibbits et al., 1992; Vornanen, 1996). Therefore, the elevation in Fc by the increments in the levels of extracellular Ca2+ indicates a marked dependence of heart performance on extracellular Ca2+, in spite of the intracellular stores. According to Bailey and Driedzic (1990), the increased contractility due to calcium increments may be a short duration response to facilitate heart performance when an animal previously acclimated to low temperature is subject to an acute transition to a higher temperature. This is consistent with the results obtained in the present study, in which the ventricle strips at an extracellular Ca2+ of 9.25 mM, presented a reduction of force contraction after an elevation of temperature from 25 to 408C (3-fold lower than those at extracellular Ca2+ concentrations of 1.25 mM), i.e., there was an attenuation in the negative inotropic eect. However, even when using a high concentration of calcium, the return to the initial temperature did not facilitate a recovery of the initial force contraction indicating an impairment of the ventricle muscle function after exposure to high temperature. The importance of the extracellular Ca2+ for the heart contraction of O. niloticus was evident from the addition of crescent concentrations of calcium to the medium (from 1.25 to 11.25 mM). Such increments resulted in increases of Fc from 100% (control values) to about 150% (258C) and 200% (408C), at Ca2+ concentrations of 9.25 mM. The lack of signi®cant dierences between the Fc values recorded at 25 and 408C could be related to an ineciency of mechanisms other than those involved in the calcium supply, possibly an incomplete relaxation. On the other hand, a signi®cant loss of force was observed after 40 min of exposure to 9.25 mM Ca2+ at 408C, relative to values obtained at 258C and the same Ca2+ concentration and also relative to the performance of the strips exposed to low Ca2+ (1.25 mM) at 408C. Such results are in agreement with those reported by Rantin et al. (1998) for Bathygobius
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soporator, suggesting that a combination between high temperatures and high extracellular calcium levels should be deleterious to the heart. The positive inotropic eect of adrenaline, that occurred only at 408C, suggests that the catecholamines enhance the cardiac performance during the thermal stress, improving the Ca2+ eux and, consequently, the relaxation. The relationship between maximum isometric force and frequency of contraction was documented for several species. There is a direct correlation between the development of the sarcoplasmic reticulum (SR) and the maximum frequency of contraction, seeming that the largest contraction rates are found in the tissues that present a highly developed SR and/or ecient mechanisms to transport calcium across the sarcolemma. In ventricular preparations of mammals, reptiles, amphibians, several elasmobranch species (Driedzic and Gesser, 1985, 1988) and also in the tuna atrial preparations (Keen et al., 1992), there is a positive relationship between the contraction frequency and the maximum Fc at low frequencies, while at high frequencies this relationship becomes negative. In the present study, the frequencies of contraction recorded in vitro superposes the maximum frequencies recorded in vivo both at 25 and 408C, consistent with Driedzic and Gesser (1985). This indicates that the myocites are not functioning, in vivo, at the limit of their capacities of E±C coupling, suggesting the presence of extrinsic mechanisms modulating fH and/or the occurrence of physiological limitations not related to the Ca2+ regulation in the myocites. The maximum pacing rate was higher at 408C, consistent with the results obtained by Rantin et al. (1998) for B. soporator. However, relaxation appears to be a rate limiting step as judged from the increases in resting tension (i.e., incomplete relaxation). This suggests an inability to decrease intracellular Ca2+. The present study indicates that O. niloticus presents a slow calcium turnover, possibly related to the absence of a functional SR, similar to most of the teleosts already studied, and to an ineciency in reducing the intracellular Ca2+ levels, mainly at high temperatures. Studies involving heart tissues of mammal demonstrated that the ryanodine channels of the SR are highly temperature-dependent (Bers, 1987, 1989). Therefore, possible increases in the importance of the SR to the generation of force in heart tissue preparations could became evident during acute elevation of the temperature. In trout cardiac muscle, the contribution of the SR to excitation±contraction coupling increases with temperature in the range of 10±208C (Hove-Madsen, 1992; Mùller-Nielsen and Gesser, 1992; Gesser, 1996). The heart of O. niloticus, in contrast to
that of rainbow trout, did not show strong post-rest potentiation that could be inhibited by ryanodine, a well established inhibitor of the SR. Hence, the SR does not seem to be of importance to the excitation± contraction coupling of the heart muscle of O. niloticus, irrespective of temperature. This implies that Ca2+ availability for contraction is directly dependent upon in¯ux and eux across the sarcolemma. This feature may limit maximal heart rates. In summary, heart performance is compromised at high temperature and under conditions leading to high intracellular Ca2+ (high extracellular Ca2+, high stimulation frequency) and does not appear to involve a functional SR to sequester Ca2+ during relaxation. A controlled increase in heart rate at high temperature may have a protective eect in maintaining low levels of intracellular Ca2+.
Acknowledgements This work was supported by CNPq (Proc. 301298/ 96-3) CAPES and FAPESP (Proc. 1997/1674-0). Specimens of Nile tilapia were kindly provided by CEPTA/ IBAMA, Pirassununga, SP, Brazil.
References Bailey, J.R., Driedzic, W.R., 1990. Enhanced maximum frequency and force development of ®sh hearts following temperature acclimation. J. Exp. Biol. 149, 239±254. Bers, D.M., 1987. Ca in¯ux and sarcoplasmic reticulum Ca release in cardiac muscle activation during post-rest recovery. Am. J. Physiol. 248 (17), 366±381. Bers, D.M., 1989. SR Ca loading in cardiac muscle preparations based on rapid cooling contractures. Am. J. Physiol. 256 (C), 109±120. Coronado, R., Morrissette, J., Sukhareva, M., Vaughan, D.M., 1994. Structure and function of ryanodine receptors. Am. J. Physiol. 246 (35), C1485±C1504. Driedzic, W.R., Gesser, H., 1985. Ca2+ protection from negative inotropic eect of contraction frequency on teleost hearts. J. Comp. Physiol. 156, 135±142. Driedzic, W.R., Gesser, H., 1988. Dierences in force-frequency relationships and calcium dependency between elasmobranch and teleost hearts. J. Exp. Biol. 140, 227± 241. Driedzic, W.R., Gesser, H., 1994. Energy metabolism and contractility in ectothermic vertebrate hearts: Hypoxia, acidosis, and low temperature. Physiol. Rev. 74 (1), 221± 258. Farrell, A.P., Jones, D.R., 1991. The heart. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish Physiology, vol. XII(A): The Cardiovascular System. Academic Press, New York, pp. 1±88. Fernandes, M.N., Rantin, F.T., 1986. Lethal temperatures of
M.J. Costa et al. / Journal of Thermal Biology 25 (2000) 373±379 Oreochromis niloticus (Pisces, Cichlidae). Rev. Brasil. Biol. 46 (3), 589±595. Gesser, H., 1996. Cardiac force-interval relationship, adrenaline and sarcoplasmic reticulum in rainbow trout. J. Comp. Physiol. 166 (B), 278±285. Glass, M.L., Rantin, F.T., Verzola, R.M.M., Fernandes, M.N., Kalinin, A.L., 1991. Cardio-respiratory synchronization and myocardial function in hypoxic carp (Cyprinus carpio ) L. J. Fish Biol. 39, 143±149. Hove-Madsen, L., 1992. The in¯uence of temperature on ryanodine sensitivity and the force-frequency relationship in the myocardium of rainbow trout. J. Exp. Biol. 167, 47± 60. Keen, J.E., Farrell, A.P., Tibbits, G.F., Brill, R.W., 1992. Cardiac physiology in tunas. II. Eect of ryanodine, calcium, and adrenaline on force-frequency relationship in atrial strips from skipjack tuna, Katsuwonus pelamis. Can. J. Zool. 70, 1211±1217. Mùller-Nielsen, T., Gesser, H., 1992. Sarcoplasmic reticulum and excitation±contraction coupling at 20 and 108C in rainbow trout myocardium. J. Comp. Physiol. 162 (B), 526±534.
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Randall, D.J., 1968. Functional morphology of the heart in ®shes. Am. J. Zool. 8, 179±189. Rantin, F.T., Glass, M.L., Kalinin, A.L., Verzola, R.M.M., Fernandes, M.N., 1993. Cardiorespiratory responses in two ecologically distinct erythrinids (Hoplias malabaricus and Hoplias lacerdae ) exposed to graded environmental hypoxia. Environ. Biol. Fish. 36, 93±97. Rantin, F.T., Gesser, H., Kalinin, A.L., Guerra, C.D.R., Freitas, J.C., Driedzic, W.R., 1998. Heart performance, Ca2+ regulation and energy metabolism at high temperatures in Bathygobius soporator, a tropical marine teleost. J. Therm. Biol. 23, 31±39. Tibbits, G.F., Moyes, C.D., Hove-Madsen, L., 1992. Excitation±contraction coupling in the teleost heart. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish Physiology, vol. XII(A): The Cardiovascular System. Academic Press, New York, pp. 267±303. Vornanen, M., 1994. Seasonal and temperature-induced changes in myosin heavy chain composition of crucian carp hearts. Am. J. Physiol. 267 (36), 1567±1573. Vornanen, M., 1996. Eect of extracellular calcium on the contractility of warm- and cold-acclimated crucian carp heart. J. Comp. Physiol. 165, 507±517.
www.elsevier.com/locate/jtherbio
Cardiac tissue function of the teleost ®sh Oreochromis niloticus under dierent thermal conditions Monica Jones Costa, Luciano Rivaroli, Francisco Tadeu Rantin, Ana LuÂcia Kalinin* Department of Physiological Sciences, Federal University of SaÄo Carlos, Via Washington Luiz, km 235-13565-905, SaÄo Carlos, SP, Brazil Received 24 September 1999; accepted 4 November 1999
Abstract The cardiac responses of Oreochromis niloticus acclimated to 258C were assessed using ventricle strips mounted for isometric force recording (Fc) and in vivo heart rate ( fH). fH increased progressively from 25 to 408C. At extracellular Ca2+ concentrations of 1.25 and 9.25 mM, a transition from 25 to 408C resulted in a decreased Fc. At both 25 and 408C, Fc rose when [Ca2+] was increased from 1.25 to 9.25 mM. Fc remained constant at 72 and 120 contractionsminÿ1 at 25 and 408C, respectively, and declined thereafter. The post-rest potentiation was not in¯uenced by ryanodine, indicating that the sarcoplasmic reticulum is not important to the excitation±contraction coupling. 7 2000 Elsevier Science Ltd. All rights reserved. Keywords: Acclimation; Temperature; Heart function; Ventricle strips; Excitation-contraction coupling; Calcium management; Sarcoplasmic reticulum; Nile tilapia; Oreochromis niloticus
1. Introduction The ability of the cardiac muscle to maintain pump performance under dierent physiological conditions is one of the most important characteristics that allow vertebrates to survive under extreme conditions (Driedzic and Gesser, 1994). It is well established that changes in environmental temperature have an immediate and substantial eect upon cardiac performance in north-temperate teleost ®sh (Bailey and Driedzic, 1990). In these species, rest-
* Corresponding author. Tel.: +55-16-2608314; fax: +5516-2608328. E-mail address: [email protected] (A.L. Kalinin).
ing heart rates are typically under 100 beatsminÿ1 and follow a Q10 of about 2 during acute temperature transitions. Heart contractility is usually triggered by increases in the intracellular Ca2+ concentration due to an in¯ux across the sarcolemma on a beat to beat basis, although the functional importance of the cardiac sarcoplasmic reticulum in supplying Ca2+ for contractility increases with temperature in some species (Rantin et al., 1998). However, there is little information about the impact of temperature on tropical species. The present study extends ®ndings to the freshwater cichlid Oreochromis niloticus, a tropical species tolerant to a wide range of temperatures (from 010± 408C; Fernandes and Rantin, 1986) in dierent Brazilian regions.
0306-4565/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 9 9 ) 0 0 1 0 9 - 6
374
M.J. Costa et al. / Journal of Thermal Biology 25 (2000) 373±379
2. Materials and methods Specimens of Oreochromis niloticus weighing between 254 2 17.6 g were collected at the Tropical Fish Research Center (CEPTA/IBAMA), Pirassununga, SP, Brazil. For at least 4 weeks prior to experimentation, the ®sh were maintained in holding tanks equipped with a continuous supply of well-aerated and dechlorinated water at a constant temperature (258C). They were fed with commercial ®sh food pellets ad libitum. For the specimens used in the in vivo experiments, food was withheld 24 h before experimentation. 2.1. In vivo heart rate Heart rate ( fH-bpm) was obtained by electrocardiography. An electrode was placed in a ventral position between the gills and the heart and a second electrode in a ventral position close to the pelvic ®ns. A reference electrode was located in the water of the experimental chamber (for details see Glass et al., 1991; Rantin et al., 1993). The electrode set was connected to a universal coupler of a Narco Narcotrace 40 recorder (Narco Bio Systems, Houston, TX, USA). For implantation of the electrodes, the ®sh were immersed in a benzocaine solution (1 g of benzocaine dissolved in 0.1 l of 99% ethanol and diluted in 10 l of water). This level of anesthesia allowed spontaneous breathing. After surgery, the ®sh was placed into a holding PVC tube. The oxygen tension of the ingoing (PinO2-mmHg) water was measured continuously. Water was siphoned via polyethylene catheters to O2 electrodes (FAC 001-O2, FAC-SaÄo Carlos, SP, Brazil) housed within temperature controlled cuvettes and connected to a FAC-204A O2 Analyzer. The PinO2 was maintained at normoxic levels (140 mmHg). After a recovery period of 12 h, the fH was measured at 258C. The temperature was then increased (108Chÿ1) to 30, 35 and 408C and subsequently reduced to 258C. Each experimental temperature was maintained for 30 min and measurements were obtained during the last 5 min. 2.2. Ventricle strip preparation Animals were killed by a blow on the head. Pairs of strips with a thickness of maximally 1 mm were excised from the ventricle and placed into an oxygenated bathing medium containing 125 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 0.5 mM NaH2PO4, 1.25 mM CaCl2, 10 mM glucose and 10 mM imidazole±HEPES buer to give pH 7.6. The temperature of the muscle bath was kept at 258C in a temperature-controlled water bath, unless otherwise stated. The preparations were suspended using surgical silk to connect to a NARCO
F-60 isometric force transducer placed around one platinum electrode. This electrode and one placed in the bath were connected to a stimulator (Grass SD9, Quincy, MA, USA) delivering electrical square pulses having a duration of 8 ms and a voltage 50% above that of the threshold value in order to provide a security margin resulting in maximal stimulation throughout the experiment. The stimulation rate was 0.2 Hz unless otherwise stated. The preparations were stretched to provide a twitch tension at the maximum of the length±twitch tension relation. Twitch tension was allowed to stabilize for about 30 min. In the ®rst experimental protocol, muscle preparations were subjected to a temperature transition from 25 to 408C over a 25±30 min time period. Preparations were maintained at 408C for 30 min. Thereafter, the temperature was decreased over a period of 15±20 min back to 258C and held at that temperature for a further 30 min. To evaluate the dependence of the cardiac muscle responses resulting from the eects of temperature changes on the excitation±contraction coupling, an experimental series was performed at a physiological level of 1.25 mM extracellular Ca2+ and also at an elevated level of 9.25 mM Ca2+. In further experimental protocols, preparations were allowed to initially stabilize at 25 or 408C. In a third series of experiments, the maximal capacity of hearts to develop force was assessed trough the addition of either Ca2+ or adrenaline to bathing medium. To evaluate the eect of the time exposure to 25 and 408C, an additional series was performed without adrenaline, using 1.25 or 9.25 mM Ca2+. In a fourth series, preparations were subjected to an increase in imposed contraction frequency coupling. Finally, the force development upon the ®rst stimulation following a rest period was determined at 25, 35 and 408C. This approach can inform on the capacity for the storage of intracellular Ca2+ during resting period. After a steady-state condition at 12 contractionsminÿ1 was achieved, stimulation ceased for a period of 5 min. Force development of the ®rst contraction following the resting period was compared to the last contraction in a steady-state train. These experiments were conducted with and without 10 mM ryanodine in the medium. According to Coronado et al. (1994), in mammalian muscle, ryanodine stimulates Ca2+ release from sarcoplasmic reticulum (SR) at low concentrations (10±300 mM). When applied, ryanodine was added to the medium 30±45 min before the 5 min interval. 2.3. Data presentation and analysis Results are presented as mean values2SE. In all experiments, signi®cance levels with respect to parameters of the same experimental protocol were
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assessed with One-way Analysis of Variance (ANOVA) followed by Bartlett's test for homogeneity of variances and Tukey±Kramer multiple comparisons test. Additionally, the Mann±Whitney test was employed to compare parameters of dierent experimental protocols at the same temperature. Signi®cance levels are indicated in the ®gures as follows: p < 0.05, p < 0.01, p < 0.001 (GraphPad Instat version 2.01, GraphPad Software, San Diego, CA, USA). 3. Results 3.1. In vivo heart rates Fig. 1 shows the eects of the increases in temperature from 25 to 408C and subsequent recovery to 258C on the fH (bpm). The fH increased progressively and signi®cantly from 30 to 408C, at which fH reached its maximum value of 97.226.5 bpm. This value was 2.9fold higher than that at 258C. After the return to 258C, the fH decreased to a value similar to that recorded previously at the same temperature. 3.2. Ventricle strip preparation At extracellular Ca2+ concentrations of 1.25 and 9.25 mM, a transition from 25 to 408C resulted in a decrease in twitch force (Fig. 2). During the subsequent restoration to 258C, twitch force increased in both Ca2+ concentrations but did not achieve the initial values. However, at the Ca2+ concentration of 9.25 mM the Fc was signi®cantly higher than at 1.25 mM, in the experimental temperatures of 30, 35 and 408C. The scope of activity at 25 and 408C was examined by increasing levels of extracellular Ca2+ and adrenaline. The eects of extracellular Ca2+ on the twitch
Fig. 1. The eect of a change in temperature from 25 to 408C on heart rate ( fH Ð bpm) of Oreochromis niloticus. Mean values2SE (n = 10).
Fig. 2. The eect of changes in temperature from 25 to 408C and back to 258C on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at an extracellular Ca2+ concentration of 1.25 mM (q) and 9.25 mM (Q). Mean values2SE (n = 10).
force of the cardiac muscle of O. niloticus is presented in Fig. 3. At 258C, increments in extracellular Ca2+ from 1.25 to 9.25 mM resulted in increases in twitch force development. However, at 408C, the twitch force increased progressively reaching maximum values at 9.25 mM, indicating a large dependence on extracellular Ca2+. Increased concentrations of adrenaline caused signi®cant increases in twitch force only at 408C (Fig. 4). This experiment involves 10 min of exposure to adrenaline at each condition. Control experiments at 1.25 mM or 9.25 Ca2+ and zero adrenaline were performed to determine if there was deterioration of force contraction over this time period (Fig. 5). Changes in twitch force with time were observed only at 408C and 9.25 mM Ca2+. In this condition, the Fc decreased
Fig. 3. The eect of extracellular Ca2+ on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at 258C (q) and 408C (Q). Mean values2SE (n = 10).
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Fig. 4. The eect of adrenaline on twitch force (Fc Ð % of the initial values) of cardiac muscle of Oreochromis niloticus at 258C (q) and 408C (Q). Mean values2SE (n = 10).
progressive and signi®cantly along the time, reaching minimum values after 40 min of exposure (2.4-fold lower than those initially recorded). The impact of increases in the imposed contraction frequency is presented in Fig. 6. At 258C, twitch force immediately declined as frequency was increased above 30 contractionsminÿ1. Preparations became irregular above 72 contractionsminÿ1. At 408C twitch force development remained constant at frequencies up to 120 contractionsminÿ1 and declined with further increase of frequency. Preparations were able to maintain a rhythmic response up to about 180 contractionsminÿ1. At 408C, resting tension tended to increase, i.e., relaxation became incomplete with rates above 60 contractionsminÿ1. Fig. 7 shows the force development of the ®rst contraction following a rest period at 25, 35 or 408C, in
Fig. 5. Eect of time on twitch force of cardiac muscle of O. niloticus at 258C (q) and 408C (Q). Mean values 2 SE (n = 10).
Fig. 6. Eect of stimulation frequency at 25 and 408C on twitch force (upper panel) and resting tension (lower panel). Twitch force was allowed to stabilize before the next increment in frequency was applied. Mean values2SE (n = 10).
the presence or absence of ryanodine. Post-rest potentiation occurred only at 258C and was not in¯uenced by ryanodine. At 358C, force contraction remained unchanged either with or without ryanodine. A postrest decay was recorded at 408C in both treatments. In all cases, no signi®cant dierences were observed
Fig. 7. Twitch force of the ®rst contraction following a rest period. A stimulation rate of 0.2 Hz was followed by a 5 min rest period. q Ð control values; Q Ð 10 mM ryanodine. Asterisks above bars indicate signi®cance level between the bar and the last contraction prior to the rest period. Mean values2SE (n = 10).
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between the results obtained with and without ryanodine, indicating that, in this species, the sarcoplasmic reticulum does not seem to be of importance to the excitation±contraction coupling of heart muscle. 4. Discussion The cardiac output must be regulated to maintain an adequate blood ¯ow for the muscle in exercise, brain and other tissues under a variety of conditions. According to Farrell and Jones (1991), in most of the ®sh the regulation of the cardiac output is mediated by alterations in the cardiac stroke volume instead of through the control of the heart rate, as in mammals. On the other hand, according to Randall (1968), following increases in temperature, an increase in the cardiac output in ®sh heart may occur exclusively by increased heart rate, which results in direct eect of temperature on the pacemaker cells. This alters the membrane permeability and, consequently, increases the intrinsic rate of depolarization of these cells. Additionally, Vornanen (1994), working on crucian carp, described that high temperatures increase its heart rate while low temperatures increase stroke volume. According to Driedzic and Gesser (1994), the resting heart rates in ®sh are typically under 100 beatsminÿ1 (10±60 bpm, not exceeding 120 bpm). Exceptions of this pattern are the tuna (Katsuwonus pelamis ), that presents resting fH around 120 bpm, and surpasses 200 bpm during exercise (Keen et al., 1992), and Bathygobius soporator, in which the resting fH is about 140 bpm at 258C (Rantin et al., 1998), which is comparable to mammalian fH. Tropical teleost ®sh have Q10 values for the fH around 2 during acute changes in temperature (Rantin et al., 1998), similar to those obtained in the present study. Therefore, in spite of the high capacity to survive at elevated temperatures, the temperature-dependent variations of the fH of O. niloticus did not dier signi®cantly from those presented by temperate species. This result suggests that O. niloticus triggers other mechanisms than increases in fH to maintain the heart performance during increases in temperature. The negative inotropic eect presented by O. niloticus after temperature increases is consistent with that previously recorded by Bailey and Driedzic (1990) and Rantin et al. (1998) for Perca ¯uviatilis and Bathygobius soporator, respectively. These authors described progressive reductions of the contractile activity of the ventricular strips during increments of 158C in the experimental temperature. The reduction in twitch force during the elevation in temperature from 35 to 408C in the present study could indicate a possible irreversible damage in myocardial function after reaching the highest temperature,
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since the initial force was not recovered after the return to 258C in both physiological and high Ca2+ concentrations. These results dier from those recorded by Rantin et al. (1998), where the twitch force of B. soporator at 1.25 mM Ca2+ was completely recovered upon the return to 258C. This suggests that B. soporator has special adaptations to deal with rapid and substantial temperature transitions. According to these authors, a possible explanation for the negative inotropic temperature eect could be pH related, since increases in temperature should lower pH and, consequently, the peak force. Several studies report on the dependence of heart contractility on extracellular calcium concentration and emphasize the importance of the calcium ¯ux across the sarcolemma for the excitation±contraction coupling (Driedzic and Gesser, 1994; Tibbits et al., 1992; Vornanen, 1996). Therefore, the elevation in Fc by the increments in the levels of extracellular Ca2+ indicates a marked dependence of heart performance on extracellular Ca2+, in spite of the intracellular stores. According to Bailey and Driedzic (1990), the increased contractility due to calcium increments may be a short duration response to facilitate heart performance when an animal previously acclimated to low temperature is subject to an acute transition to a higher temperature. This is consistent with the results obtained in the present study, in which the ventricle strips at an extracellular Ca2+ of 9.25 mM, presented a reduction of force contraction after an elevation of temperature from 25 to 408C (3-fold lower than those at extracellular Ca2+ concentrations of 1.25 mM), i.e., there was an attenuation in the negative inotropic eect. However, even when using a high concentration of calcium, the return to the initial temperature did not facilitate a recovery of the initial force contraction indicating an impairment of the ventricle muscle function after exposure to high temperature. The importance of the extracellular Ca2+ for the heart contraction of O. niloticus was evident from the addition of crescent concentrations of calcium to the medium (from 1.25 to 11.25 mM). Such increments resulted in increases of Fc from 100% (control values) to about 150% (258C) and 200% (408C), at Ca2+ concentrations of 9.25 mM. The lack of signi®cant dierences between the Fc values recorded at 25 and 408C could be related to an ineciency of mechanisms other than those involved in the calcium supply, possibly an incomplete relaxation. On the other hand, a signi®cant loss of force was observed after 40 min of exposure to 9.25 mM Ca2+ at 408C, relative to values obtained at 258C and the same Ca2+ concentration and also relative to the performance of the strips exposed to low Ca2+ (1.25 mM) at 408C. Such results are in agreement with those reported by Rantin et al. (1998) for Bathygobius
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soporator, suggesting that a combination between high temperatures and high extracellular calcium levels should be deleterious to the heart. The positive inotropic eect of adrenaline, that occurred only at 408C, suggests that the catecholamines enhance the cardiac performance during the thermal stress, improving the Ca2+ eux and, consequently, the relaxation. The relationship between maximum isometric force and frequency of contraction was documented for several species. There is a direct correlation between the development of the sarcoplasmic reticulum (SR) and the maximum frequency of contraction, seeming that the largest contraction rates are found in the tissues that present a highly developed SR and/or ecient mechanisms to transport calcium across the sarcolemma. In ventricular preparations of mammals, reptiles, amphibians, several elasmobranch species (Driedzic and Gesser, 1985, 1988) and also in the tuna atrial preparations (Keen et al., 1992), there is a positive relationship between the contraction frequency and the maximum Fc at low frequencies, while at high frequencies this relationship becomes negative. In the present study, the frequencies of contraction recorded in vitro superposes the maximum frequencies recorded in vivo both at 25 and 408C, consistent with Driedzic and Gesser (1985). This indicates that the myocites are not functioning, in vivo, at the limit of their capacities of E±C coupling, suggesting the presence of extrinsic mechanisms modulating fH and/or the occurrence of physiological limitations not related to the Ca2+ regulation in the myocites. The maximum pacing rate was higher at 408C, consistent with the results obtained by Rantin et al. (1998) for B. soporator. However, relaxation appears to be a rate limiting step as judged from the increases in resting tension (i.e., incomplete relaxation). This suggests an inability to decrease intracellular Ca2+. The present study indicates that O. niloticus presents a slow calcium turnover, possibly related to the absence of a functional SR, similar to most of the teleosts already studied, and to an ineciency in reducing the intracellular Ca2+ levels, mainly at high temperatures. Studies involving heart tissues of mammal demonstrated that the ryanodine channels of the SR are highly temperature-dependent (Bers, 1987, 1989). Therefore, possible increases in the importance of the SR to the generation of force in heart tissue preparations could became evident during acute elevation of the temperature. In trout cardiac muscle, the contribution of the SR to excitation±contraction coupling increases with temperature in the range of 10±208C (Hove-Madsen, 1992; Mùller-Nielsen and Gesser, 1992; Gesser, 1996). The heart of O. niloticus, in contrast to
that of rainbow trout, did not show strong post-rest potentiation that could be inhibited by ryanodine, a well established inhibitor of the SR. Hence, the SR does not seem to be of importance to the excitation± contraction coupling of the heart muscle of O. niloticus, irrespective of temperature. This implies that Ca2+ availability for contraction is directly dependent upon in¯ux and eux across the sarcolemma. This feature may limit maximal heart rates. In summary, heart performance is compromised at high temperature and under conditions leading to high intracellular Ca2+ (high extracellular Ca2+, high stimulation frequency) and does not appear to involve a functional SR to sequester Ca2+ during relaxation. A controlled increase in heart rate at high temperature may have a protective eect in maintaining low levels of intracellular Ca2+.
Acknowledgements This work was supported by CNPq (Proc. 301298/ 96-3) CAPES and FAPESP (Proc. 1997/1674-0). Specimens of Nile tilapia were kindly provided by CEPTA/ IBAMA, Pirassununga, SP, Brazil.
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