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Effect of acute temperature transitions on chronotropic and inotropic responses of the South American lungfish Lepidosiren paradoxa
Journal of Thermal Biology 27 (2002) 39–45
Effect of acute temperature transitions on chronotropic and inotropic responses of the South American lungfish Lepidosiren paradoxa Monica Jones Costa, Claudio Dalle Olle, Jacqueline Aparecida Ratto, Lu!ıs Carlos Anelli Jr., Ana Lu! cia Kalinin, Francisco Tadeu Rantin* Department of Physiological Sciences, Federal University of Sa*o Carlos, Via Washington Luiz, km 235, 13565-905 Sa*o Carlos (SP), Brazil Received 16 January 2001; accepted 6 February 2001
Abstract The in vivo and in vitro cardiac responses of Lepidosiren paradoxa were analyzed during temperature variations from 258C (acclimation temperature) to 158C and 358C, and subsequent return to 258C. Chronotropic (heart frequency) and inotropic (twitch force) responses varied directly with temperature, decreasing from 258C to 158C and increasing from 258C to 358C. However, time to peak tension (TPT) and time to half relaxation (THR) showed an inverse tendency. The results indicate that the myocardium of L. paradoxa responds more appropriately to acute elevations in temperature, which results in an increased cardiac performance due to both positive chronotropism and inotropism, in spite of the temperature-induced curtailment of TPT and THR. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Temperature; Heart function; Ventricle strips; Excitation–contraction coupling; Calcium management; Lungfish; Lepidosiren paradoxa
1. Introduction The scope of fishes cardiovascular function and the wide-ranging environmental temperatures impose unique demands on the regulation of cardiac contractility and hence to the excitation–contraction (E–C) coupling. Contractile mechanisms (i.e., the actin-myosin interaction and its regulation by Ca2+) appear to be similar in all vertebrate hearts (Driedzic and Gesser, 1994). However, anatomical and ultra-structural distinctions between hearts of different species underlie important physiological differences, particularly to the regulation of Ca2+ delivery to contractile apparatus (Tibbits et al., 1992).
*Corresponding author. Tel.: +55-16-2608314; fax: +55-162608328. E-mail address: [email protected] (F.T. Rantin).
The lungfish, Lepidosiren paradoxa, inhabits rivers and lakes in both tropical (Amazon) and subtropical (Pantanal) regions of South America. This species has evolved farthest towards complete air-breathing and distinct pulmonary and systemic circuits (Johansen et al., 1968). Among the three recent genera of Dipnoi, the South American lungfish is structurally the most advanced, showing the greatest degree of atrial subdivision and ventricular septation (Johansen and Hanson, 1968). Presuming that the tetrapods ancestors were physiologically very similar to the modern lungfishes (Burggren and Johansen, 1986), this group is thought to provide a unique opportunity to study the physiological adaptations correlated with the emergence of air-breathing. This study was the first to analyze the impact of an acute temperature transition on the chronotropic and inotropic responses of the ventricular myocardium of a lungfish.
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 3 - 4
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M.J. Costa et al. / Journal of Thermal Biology 27 (2002) 39–45
2. Materials and methods Active, non-aestivating specimens of the South American lungfish, L. paradoxa, weighing between 480 and 1190 g and body lengths from 51 to 97 cm were obtained from water filled clay pits near Rio Cuiab!a in Pantanal area, Cuiab!a, MT, Brazil. For at least 4 weeks prior to experimentation, the fish were maintained in 1000 l holding tanks equipped with a continuous supply of dechlorinated water (1.2 l/min) at a constant temperature (25 18C). The holding tanks were covered with macrophytes in order to reduce light intensity and to minimize stress. The animals were fed weekly on chopped chicken liver. 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 recorded by electrocardiography. Two ECG electrodes were placed in a ventral position between the gills perpendicularly to each other. A reference electrode was located in the water of the experimental chamber (for details see Rantin et al., 1993; Harder et al., 1999). The electrode set was connected to the universal coupler of a Narco Narcotrace 40 recorder (Narco Bio-Systems, Houston, TX, USA) in order to obtain ECG recordings similar to those observed to the lead DI of the standard electrocardiography. For implantation of the electrodes, the fish were immersed in a benzocaine solution (0.5%). This allowed a sufficient level of anesthesia while spontaneous breathing was maintained. After surgery, the fish was placed into a restraining U-shaped PVC chamber allowing the lungfish to breath air. After a recovery period of at least 12 h, fH was measured at 258C and the temperature was increased (108C/h) until 358C or decreased in the same ratio to 158C and then returned to the acclimation temperature. Heart frequency was recorded at intervals of 2.58C. 2.2. Ventricle strip preparation Fish were killed by a sharp blow to the head, and the heart was quickly removed and placed in ice-cooled saline. Pairs of strips with a thickness of maximally 1 mm and weighting 11.87 0.98 mg were excised from the ventricle and placed into a bathing medium containing 100 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.5 mM NaH2PO4, 27 mM NaHCO3, 2.5 mM CaCl2 and 10 mM glucose. The saline was kept at 258C and bubbled throughout the experiment with 98% O2/2% CO2 to result in a pH of 7.6. Strips kept in the saline at +48C could be used for up to 3 days without detectable changes in responses. 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. 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 before temperature changes. In the first experimental protocol, ventricle strips preparations were subjected to a temperature transition from 258C to 158C over a 25–30 min time period. Thereafter, the temperature was increased in the same ratio back to 258C. In the second protocol, the temperature of the water bath was increased until 358C over a 25–30 min time period and subsequently decreased to the initial temperature. The inotropic responsiveness of the ventricle strips to such changes in temperature was measured by means of the twitch force (Fc}% initial values). Moreover, to evaluate the effect of the temperature on time to peak tension (TPT}ms) and time to half relaxation (THR}ms), recordings at high speed (25 mm/s) were performed (for details see Maitikanen and Vornanen, 1992). In order to compare the effects of temperature changes on TPT and THR the curves were also normalized (% of the initial values). 2.3. Data presentation and analysis Results are presented as mean values SE. In all experiments, significance levels with respect to parameters of the same experimental protocol were assessed with One-way Analysis of Variance (ANOVA) followed by Bartlett’s test for homogeneity of variances and Tukey–Kramer multiple comparisons test. Additionally, Mann–Whitney test was employed to compare parameters of different values of TPT and THR at the same temperature. Significance levels are indicated in the figures as follows: * p50:05, ** p50:01, *** p50:001 (GraphPad Instat version 3.00, GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Heart rate Fig. 1 shows the effects of the decreases in temperature from 258C to 158C and subsequent return to 258C
M.J. Costa et al. / Journal of Thermal Biology 27 (2002) 39–45
on fH (bpm). The fH decreased significantly ðp50:05Þ only from 17.58C to 158C, at which it reached the minimum value of 17.1 0.8 bpm ðQ10 ¼ 2:02 0:15Þ. After the return to 258C, the fH increased to a value similar to that initially recorded in this temperature. Significant differences in fH between similar temperatures were observed only at 208C. The effects of the acute increase in temperature from 258C to 358C and the subsequent return to 258C on fH are shown in Fig. 2. The fH increased gradually and significantly since 25 until 358C, at which it reached the maximum value of 70.3 1.7 bpm ðQ10 ¼ 2:31 0:14Þ. Significant differences in fH values of similar temperatures were not observed. These results indicate that warmer temperatures have a more pronounced effect on the chronotropic responses of the species heart, since Q10 values for fH were
41
significantly higher ð p50:01Þ after the increase than when temperature was decreased. 3.2. ‘‘In vitro’’ measurements
Fig. 1. The effect of temperature transition from 258C to 158C and subsequent return to 258C on in vivo heart rate (fH }bpm) of L. paradoxa. Mean values SE ðn ¼ 20Þ.
After the initial stabilization at acclimation temperature (258C), the twitch force developed by ventricle strips of L. paradoxa was 6.29 0.88 mN/mg wet weight ðn ¼ 20Þ. The temperature transition from 258C to 158C resulted in a significant decrease ðp50:05Þ in twitch force at the lowest temperature (Fig. 3). During the subsequent return to 258C, twitch force increased again and achieved the initial values at 208C. All temperatures observed during the subsequent return to acclimation temperature were similar to those initially observed, including at 258C. The effect of the acute transition to a high temperature on twitch force can be observed in Fig. 4. The increase in temperature resulted in a progressive and significant ð p50:01Þ positive inotropic response at 32.58C and reached its maximum values at 358C ð p50:001Þ. An acute temperature transition of 108C does not seem to have any deleterious effect on heart tissue, since after the subsequent return to acclimation temperature, the Fc initially observed in this temperature was restored for both experimental protocols. Similarly to what was observed for heart frequency, the inotropic responsiveness of the species heart is under a more pronounced influence of increases in temperature, since Q10 values for Fc were significantly higher ð p50:001Þ when the temperature was increased ðQ10 ¼ 1:52 0:09Þ than when the temperature was decreased ðQ10 ¼ 1:14 0:04Þ. However, the opposite response was observed (Fig. 5) when the time parameters measured in vitro are
Fig. 2. The effect of temperature transition from 258C to 358C and subsequent return to 258C on in vivo heart rate (fH }bpm) of L. paradoxa. Mean values SE ðn ¼ 20Þ.
Fig. 3. The effect of temperature transition from 258C to 158C and subsequent return to 258C on twitch force (Fc}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
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considered (i.e., TPT and THR varied indirectly with temperature). The decreases in temperature from 258C to 158C (Fig. 5A) resulted in a progressive and significant ð p50:001Þ prolongation in both TPT (from 0.86 0.03 to 1.70 0.04 ms) and THR (from 0.35 0.02 to 0.79 0.02 ms). Moreover, the normalization of the curves (Fig. 6) indicated that the decrease in temperature had a more pronounced effect on THR than TPT ð p50:05Þ, suggesting that the negative inotropism observed after the decreases in temperature is mainly due to a direct effect of low temperature on the Ca2+ transporting mechanism responsible for cytosolic Ca2+ lowering during diastole. Conversely, increases in temperature from 258C until 358C (Fig. 5B) resulted in a progressive and significant ð p50:001Þ curtailment in both TPT (from 0.78 0.03 to 0.41 0.02 ms) and THR (from 0.37 0.02 to 0.20 0.01 ms). However, the normalization of these curves (Fig. 7) indicates that the elevation in temperature results in a similar degree of curtailment in both TPT and THR.
The results described above, associated with the lack of significance between the Q10 values obtained to TPT after decreases (2.00 0.02) and increases (1.92 0.06) in temperature, in contrast to the significant difference
Fig. 4. The effect of temperature transition from 258C to 358C and subsequent return to 258C on twitch force (Fc}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 7. The effect of temperature transition from 258C to 358C and subsequent return to 258C on TPT (&}% of the initial values) and THR (4}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 6. The effect of temperature transition from 258C to 158C and subsequent return to 258C on TPT (&}% of the initial values) and THR (4}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 5. The effect of temperature transition from 258C to 158C (A) or to 358C (B) and subsequent returns to 258C on TPT (&}MS) and THR (4}MS) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
M.J. Costa et al. / Journal of Thermal Biology 27 (2002) 39–45
ð p50:01Þ between the Q10 values observed to THR after decreases (2.34 0.13) and increases (1.80 0.08) in temperature, indicate that L. paradoxa heart relaxation is a limiting step to the maintenance of the cardiac performance at cold temperatures.
4. Discussion Cardiac output must be well regulated to maintain sufficient blood flow in order to assure the distribution of essential nutrients, metabolites, gases and other substances to or from the various body tissues in different conditions (Lillywhite et al., 1999). Consequently, efficient adjustments between stroke volume and heart rate after an acute change in temperature are necessary in order to allow a poikilothermic animal to face thermal transitions. The heart capacity to respond appropriately to acute temperature change is a crucial factor and determines if the animal can survive in a new thermal condition. In most of the lower vertebrates studied so far, resting heart rate does not exceed 120 bpm (Farrell, 1991), ranging between 10 and 60 bpm in fish (Driedzic and Gesser, 1994). Exceptions to this pattern are the tuna, Katsuwonus pelamis (Keen et al., 1992) and Bathygobius soporator (Rantin et al., 1998). These species present heart rates more like those of small mammals than fish. According to Burggren et al. (1997), temperature is the single most important environmental determinant of heart rate, directly affecting the intrinsic pacemaker rate. Farrell and Jones (1991) pointed out that, in most of the fish, the cardiac output regulation is mediated by alterations in the stroke volume, while in mammals it is regulated mainly through the control of heart rate. On the other hand, several studies indicate that an increase in cardiac output in response to temperature elevations may occur exclusively by increases in heart rate (Randall, 1968), while stroke volume is increased by low temperatures (Vornanen, 1994). In the present study, the Q10 values for fH were about 2, similar to those described for temperate fish in response to acute temperature changes (Rantin et al., 1998). Therefore, L. paradoxa probably relies on other mechanisms than only chronotropic adjustments to maintain its cardiac performance in response to acute temperature transitions. Tibbits et al. (1991) reported that the activation of the cardiac E–C coupling occurs in response to an increase in cytosolic Ca2+ concentration. This increase can depend on both internal Ca2+ mobilization from the sarcoplasmic reticulum (SR) and external Ca2+ influx across the sarcolemma (SL) through voltage-dependent calcium channels (L-type) and Na+/Ca2+ exchange (Hove-Madsen et al., 2000). Conversely, cardiac muscle relaxes by lowering cytosolic Ca2+ concentrations back
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to the diastolic levels mainly by its transport to extracellular space through SL (Na+/Ca2+ exchange) or into SR by Ca2+-ATPase activity. The magnitude of the increase and decrease in cytosolic [Ca2+] depends on the degree of activity of Ca2+}transporting mechanisms from each compartment, which varies among species and with temperature (Gwathmey and Morgan, 1991). The most striking finding was the positive inotropism associated with the positive chonotropic response presented by L. paradoxa ventricle after temperature increases (Fig. 4). This positive inotropism differs from the responses reported for most of the ectothermic vertebrates and mammals, since these animals show a decrease in myocardial force (e.g. flounder, frogs, turtle, rats, rabbits) or a lack of change in twitch force (e.g. trout) at higher temperatures (Shi and Jackson, 1997; Costa et al., 2000). These variations of the cardiac muscle force responses to temperature changes may depend on both maximal Ca2+}activated force and Ca2+}sensitivity differences among species (Kim et al., 2000). The heart capacity of developing a positive inotropism, even after the temperature-induced increase in chronotropism, can also be suggested by the curtailment of TPT and THR observed in vitro after temperature elevation (Figs. 5 and 7). This maintenance of a positive inotropism at higher temperatures, even after the temperature-induced curtailment of TPT and THR, indicates that the species possesses very efficient mechanisms of Ca2+ transportation from and into the cytosol in the cardiac E–C coupling. This allows faster rates of contraction and relaxation to be achieved at higher temperatures. One possibility is that the species has a functional SR, which would allow a very fast Ca2+ sequestration and liberation from and to the myoplasm. Hochachka and Hulbert (1978) found a very well developed SR in the ventricular myocardium of L. paradoxa. However, the physiological role of this organella remains to be elucidated. Another potential mechanisms would be the presence of a very active Na+/Ca2+ exchanger, resulting in a fast Ca2+ efflux from the cell during cardiac relaxation. This hypothesis is of special relevance since a post-rest decay of twitch force was observed for ventricle strips of L. paradoxa after non-physiological prolonged diastolic pauses ranging from 7 to 350 s (unpublished results). Accordingly to Bers (1991), a decreased amplitude of the first postrest contraction with increased resting periods occurs in ventricular myocardium of several mammalian species, and is linked to transsarcolemmal Ca2+ movements via Na+/Ca2+ exchange that reduces cytosolic Ca2+ during relaxation. It can also be proposed that the mechanisms responsible by the Ca2+ influx and efflux to and from the cell, and possibly to SR (if functional for Ca2+
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management), are greatly affected by temperature in the ventricular myocytes of L. paradoxa. This high sensibility to temperature is similar to that reported for the heart of mammals and birds, but contrasts with most of the fish species already studied. In fish, particularly the temperate species, these mechanisms generally show a minimum degree of temperature sensitivity, indicating an adaptive response to cold (Kim et al., 2000). Conversely, the unexpected negative inotropism observed after the decrease in temperature (Fig. 3) indicates that an acute drop in temperature does not allow the expression of Ca2+ transporting proteins and/ or myosin isoforms more adapted to low temperatures. This fact may be responsible by the decrease in twitch force observed when temperature was reduced, even after the prolongation of TPT and THR (Fig. 5). This suggests that in a situation of acute decrease in temperature, as observed in the present study, L. paradoxa will not be able to maintain its cardiac performance, mostly due to a temperature-induced impairment of its cardiac relaxation (Fig. 6). The strong positive inotropic response observed during the acute temperature elevation corroborates this hypothesis. It indicates that the contractile proteins and/or Ca2+ transporting proteins isoforms, expressed in the acclimation temperature (258C), probably reflect those expressed in the species natural habitat. However, the negative inotropism observed in vitro during cold exposure could be improved in vivo by a thermal stress-induced catecholamines release. There are several mechanisms of adrenergic modulation for the heart performance and some of them are relevant to the inotropism regulation. The stimulation of b-adrenergic receptors causes the phosphorylation of the SL L-type Ca2+ channels (Shields et al., 1998) and also stimulates Na+/K+ exchanger (Hove-Madsen and Gesser, 1989), increasing the transsarcolemmal Ca2+ influx. Additionally, Boller and Pott (1989) demonstrated that Ca2+ transportation through SR is also increased by catecholamines in mammalian heart. The lower Q10 values for both Fc and fH when temperature was decreased indicate that lower temperatures have a less pronounced effect than the higher ones on both ino- and chronotropic responses of the species heart. This may prevent the occurrence of an acute drop in cardiac output at low temperatures allowing, within physiological limits, this sedentary and aestivating fish to face acute reductions in environmental temperatures without great impairments in the cardiac performance. These results, associated with the typical chronotropic response of fish, indicate that the species is able to respond more appropriately to an acute elevation in temperature, resulting in a significant increase in cardiac performance due to both positive chrono and inotropic responses, even after the temperature-induced curtailment of TPT and THR. There is coherence between the
data of this work and the thermal conditions faced by the species in its natural habitat. Temperatures of 308C or higher are frequently recorded in the ponds where specimens of L. paradoxa are collected during sunny days of summer. However, under natural conditions L. paradoxa rarely faces acute transitions to low temperatures like the ones experienced in the present study.
Acknowledgements This study was supported by FAPESP (Proc. 1998/ 11846-6).
References Bers, D.M., 1991. Excitation–contraction coupling and cardiac contractile force. Kluwer Academic Publishers, Dordrecht, 258p. Boller, M., Pott, L., 1989. b-Adrenergic modulation of transient inward current in guinea-pig cardiac myocytes. Pflu. gers Archiv 415, 276–288. Burggren, W., Farrell, A., Lillywhite, H., 1997. Vertebrate cardiovascular systems. In: Dantzler, W.H. (Ed.), Handbook of Physiology, Vol. I: Comparative Physiology. American Physiological Society, Bethesda, pp. 215–308. Burggren, W., Johansen, K., 1986. Circulation and respiration in lungfishes (Dipnoi). Journal of Morphology, Supplement 1, 217–236. Costa, M.J., Rivaroli, L., Rantin, F.T., Kalinin, A.L., 2000. Cardiac tissue function of the teleost fish Oreochromis niloticus under different thermal conditions. Journal of Thermal Biology 25, 373–379. Driedzic, W.R., Gesser, H., 1994. Energy metabolism and contractility in ectothermic vertebrate hearts: Hypoxia, acidosis, and low temperature. Physiological Reviews 74 (1), 221–258. Farrell, A.P., 1991. From hagfish to tuna: a perspective on cardiac function in fish. Physiological Zoology 64, 1137–1164. 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. Gwathmey, J.K., Morgan, J.P., 1991. Calcium handling in myocardium from amphibian, avian, and mammalian species; the search for two components. Journal of Comparative Physiology 161 (B), 19–25. Harder, V., Souza, R.H.S., Severi, W., Rantin, F.T., Bridges, C.R., 1999. The South American lungfish}adaptations to an extreme habitat. In: Val, A.L., Almeida-Val, V.M.F. (Eds.), Biology of Tropical Fishes. INPA, Manaus, pp. 87–98. Hochachka, P., Hulbert, W., 1978. Glycogen ‘seas’, glycogen bodies, and glycogen granules in heart and skeletal muscle of two air-breathing, burrowing fishes. Canadian Journal of Zoology 56, 774–786.
M.J. Costa et al. / Journal of Thermal Biology 27 (2002) 39–45 Hove-Madsen, L., Gesser, H., 1989. Force-frequency relation in the myocardium of rainbow trout: effects of K+ and adrenaline. Journal of Comparative Physiology 159 (B), 61–69. Hove-Madsen, L., Llach, A., Tort, L., 2000. Na+/Ca2+} exchange activity regulates contraction and SR Ca2+ content in rainbow trout atrial myocytes. American Journal of Physiology 279, R1856–R1864. Johansen, K., Hanson, D., 1968. Functional anatomy of the hearts of lungfishes and amphibians. American Zoologist 8, 191–210. Johansen, K., Lenfant, C., Hanson, D., 1968. Cardiovascular dynamics in lungfishes. Zeitschrift fuer Vergleichende Physiologie 59, 157–186. Keen, J.E., Farrell, A.P., Tibbits, G.F., Brill, R.W., 1992. Cardiac physiology in tunas. II. Effect of ryanodine, calcium, and adrenaline on force-frequency relationship in atrial strips from skipjack tuna, Katsuwonus pelamis. Canadian Journal of Zoology 70, 1211–1217. Kim, C., Coyne, M., Gwathmey, J., 2000. Voltage-dependent calcium channels in ventricular cells of rainbow trout: effect of temperature changes in vitro. American Journal of Physiology 278, R1524–R1534. Lillywhite, H.B., Zippel, K.C., Farrell, A.P., 1999. Resting and maximal heart rates in ectothermic vertebrates. Comparative Biochemistry and Physiology 124 (A), 369–382. Maitikanen, N., Vornanen, M., 1992. Effect of season and temperature acclimation on the function of crucian carp (Carassius carassius) heart. Journal of Experimental Biology 167, 203–220.
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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. Journal of Thermal Biology 23, 31–39. 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. Environmental Biology of Fishes 36, 93–97. Shi, H., Jackson, D.C., 1997. Effects of anoxia, acidosis and temperature on the contractile properties of turtle cardiac muscle strips. Journal of Experimental Biology 200, 1965–1973. Shields, H.A., Stevens, E.D., Farrell, A.P., 1998. Effects of temperature, adrenaline and ryanodine on power production in rainbow trout Oncohynchus mykiss ventricular trabeculae. Journal of Experimetal Biology 201, 2701–2710. Tibbits, G.F., Hove-Madsen, L., Bers, D.M., 1991. Calcium transport and the regulation of cardiac contractility in teleosts: a comparison with higher vertebrates. Canadian Journal of Zoology 69, 2014–2019. 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. American Journal of Physiology 267 (36), 1567–1573.
Effect of acute temperature transitions on chronotropic and inotropic responses of the South American lungfish Lepidosiren paradoxa Monica Jones Costa, Claudio Dalle Olle, Jacqueline Aparecida Ratto, Lu!ıs Carlos Anelli Jr., Ana Lu! cia Kalinin, Francisco Tadeu Rantin* Department of Physiological Sciences, Federal University of Sa*o Carlos, Via Washington Luiz, km 235, 13565-905 Sa*o Carlos (SP), Brazil Received 16 January 2001; accepted 6 February 2001
Abstract The in vivo and in vitro cardiac responses of Lepidosiren paradoxa were analyzed during temperature variations from 258C (acclimation temperature) to 158C and 358C, and subsequent return to 258C. Chronotropic (heart frequency) and inotropic (twitch force) responses varied directly with temperature, decreasing from 258C to 158C and increasing from 258C to 358C. However, time to peak tension (TPT) and time to half relaxation (THR) showed an inverse tendency. The results indicate that the myocardium of L. paradoxa responds more appropriately to acute elevations in temperature, which results in an increased cardiac performance due to both positive chronotropism and inotropism, in spite of the temperature-induced curtailment of TPT and THR. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Temperature; Heart function; Ventricle strips; Excitation–contraction coupling; Calcium management; Lungfish; Lepidosiren paradoxa
1. Introduction The scope of fishes cardiovascular function and the wide-ranging environmental temperatures impose unique demands on the regulation of cardiac contractility and hence to the excitation–contraction (E–C) coupling. Contractile mechanisms (i.e., the actin-myosin interaction and its regulation by Ca2+) appear to be similar in all vertebrate hearts (Driedzic and Gesser, 1994). However, anatomical and ultra-structural distinctions between hearts of different species underlie important physiological differences, particularly to the regulation of Ca2+ delivery to contractile apparatus (Tibbits et al., 1992).
*Corresponding author. Tel.: +55-16-2608314; fax: +55-162608328. E-mail address: [email protected] (F.T. Rantin).
The lungfish, Lepidosiren paradoxa, inhabits rivers and lakes in both tropical (Amazon) and subtropical (Pantanal) regions of South America. This species has evolved farthest towards complete air-breathing and distinct pulmonary and systemic circuits (Johansen et al., 1968). Among the three recent genera of Dipnoi, the South American lungfish is structurally the most advanced, showing the greatest degree of atrial subdivision and ventricular septation (Johansen and Hanson, 1968). Presuming that the tetrapods ancestors were physiologically very similar to the modern lungfishes (Burggren and Johansen, 1986), this group is thought to provide a unique opportunity to study the physiological adaptations correlated with the emergence of air-breathing. This study was the first to analyze the impact of an acute temperature transition on the chronotropic and inotropic responses of the ventricular myocardium of a lungfish.
0306-4565/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 6 - 4 5 6 5 ( 0 1 ) 0 0 0 1 3 - 4
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2. Materials and methods Active, non-aestivating specimens of the South American lungfish, L. paradoxa, weighing between 480 and 1190 g and body lengths from 51 to 97 cm were obtained from water filled clay pits near Rio Cuiab!a in Pantanal area, Cuiab!a, MT, Brazil. For at least 4 weeks prior to experimentation, the fish were maintained in 1000 l holding tanks equipped with a continuous supply of dechlorinated water (1.2 l/min) at a constant temperature (25 18C). The holding tanks were covered with macrophytes in order to reduce light intensity and to minimize stress. The animals were fed weekly on chopped chicken liver. 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 recorded by electrocardiography. Two ECG electrodes were placed in a ventral position between the gills perpendicularly to each other. A reference electrode was located in the water of the experimental chamber (for details see Rantin et al., 1993; Harder et al., 1999). The electrode set was connected to the universal coupler of a Narco Narcotrace 40 recorder (Narco Bio-Systems, Houston, TX, USA) in order to obtain ECG recordings similar to those observed to the lead DI of the standard electrocardiography. For implantation of the electrodes, the fish were immersed in a benzocaine solution (0.5%). This allowed a sufficient level of anesthesia while spontaneous breathing was maintained. After surgery, the fish was placed into a restraining U-shaped PVC chamber allowing the lungfish to breath air. After a recovery period of at least 12 h, fH was measured at 258C and the temperature was increased (108C/h) until 358C or decreased in the same ratio to 158C and then returned to the acclimation temperature. Heart frequency was recorded at intervals of 2.58C. 2.2. Ventricle strip preparation Fish were killed by a sharp blow to the head, and the heart was quickly removed and placed in ice-cooled saline. Pairs of strips with a thickness of maximally 1 mm and weighting 11.87 0.98 mg were excised from the ventricle and placed into a bathing medium containing 100 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.5 mM NaH2PO4, 27 mM NaHCO3, 2.5 mM CaCl2 and 10 mM glucose. The saline was kept at 258C and bubbled throughout the experiment with 98% O2/2% CO2 to result in a pH of 7.6. Strips kept in the saline at +48C could be used for up to 3 days without detectable changes in responses. 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. 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 before temperature changes. In the first experimental protocol, ventricle strips preparations were subjected to a temperature transition from 258C to 158C over a 25–30 min time period. Thereafter, the temperature was increased in the same ratio back to 258C. In the second protocol, the temperature of the water bath was increased until 358C over a 25–30 min time period and subsequently decreased to the initial temperature. The inotropic responsiveness of the ventricle strips to such changes in temperature was measured by means of the twitch force (Fc}% initial values). Moreover, to evaluate the effect of the temperature on time to peak tension (TPT}ms) and time to half relaxation (THR}ms), recordings at high speed (25 mm/s) were performed (for details see Maitikanen and Vornanen, 1992). In order to compare the effects of temperature changes on TPT and THR the curves were also normalized (% of the initial values). 2.3. Data presentation and analysis Results are presented as mean values SE. In all experiments, significance levels with respect to parameters of the same experimental protocol were assessed with One-way Analysis of Variance (ANOVA) followed by Bartlett’s test for homogeneity of variances and Tukey–Kramer multiple comparisons test. Additionally, Mann–Whitney test was employed to compare parameters of different values of TPT and THR at the same temperature. Significance levels are indicated in the figures as follows: * p50:05, ** p50:01, *** p50:001 (GraphPad Instat version 3.00, GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Heart rate Fig. 1 shows the effects of the decreases in temperature from 258C to 158C and subsequent return to 258C
M.J. Costa et al. / Journal of Thermal Biology 27 (2002) 39–45
on fH (bpm). The fH decreased significantly ðp50:05Þ only from 17.58C to 158C, at which it reached the minimum value of 17.1 0.8 bpm ðQ10 ¼ 2:02 0:15Þ. After the return to 258C, the fH increased to a value similar to that initially recorded in this temperature. Significant differences in fH between similar temperatures were observed only at 208C. The effects of the acute increase in temperature from 258C to 358C and the subsequent return to 258C on fH are shown in Fig. 2. The fH increased gradually and significantly since 25 until 358C, at which it reached the maximum value of 70.3 1.7 bpm ðQ10 ¼ 2:31 0:14Þ. Significant differences in fH values of similar temperatures were not observed. These results indicate that warmer temperatures have a more pronounced effect on the chronotropic responses of the species heart, since Q10 values for fH were
41
significantly higher ð p50:01Þ after the increase than when temperature was decreased. 3.2. ‘‘In vitro’’ measurements
Fig. 1. The effect of temperature transition from 258C to 158C and subsequent return to 258C on in vivo heart rate (fH }bpm) of L. paradoxa. Mean values SE ðn ¼ 20Þ.
After the initial stabilization at acclimation temperature (258C), the twitch force developed by ventricle strips of L. paradoxa was 6.29 0.88 mN/mg wet weight ðn ¼ 20Þ. The temperature transition from 258C to 158C resulted in a significant decrease ðp50:05Þ in twitch force at the lowest temperature (Fig. 3). During the subsequent return to 258C, twitch force increased again and achieved the initial values at 208C. All temperatures observed during the subsequent return to acclimation temperature were similar to those initially observed, including at 258C. The effect of the acute transition to a high temperature on twitch force can be observed in Fig. 4. The increase in temperature resulted in a progressive and significant ð p50:01Þ positive inotropic response at 32.58C and reached its maximum values at 358C ð p50:001Þ. An acute temperature transition of 108C does not seem to have any deleterious effect on heart tissue, since after the subsequent return to acclimation temperature, the Fc initially observed in this temperature was restored for both experimental protocols. Similarly to what was observed for heart frequency, the inotropic responsiveness of the species heart is under a more pronounced influence of increases in temperature, since Q10 values for Fc were significantly higher ð p50:001Þ when the temperature was increased ðQ10 ¼ 1:52 0:09Þ than when the temperature was decreased ðQ10 ¼ 1:14 0:04Þ. However, the opposite response was observed (Fig. 5) when the time parameters measured in vitro are
Fig. 2. The effect of temperature transition from 258C to 358C and subsequent return to 258C on in vivo heart rate (fH }bpm) of L. paradoxa. Mean values SE ðn ¼ 20Þ.
Fig. 3. The effect of temperature transition from 258C to 158C and subsequent return to 258C on twitch force (Fc}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
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considered (i.e., TPT and THR varied indirectly with temperature). The decreases in temperature from 258C to 158C (Fig. 5A) resulted in a progressive and significant ð p50:001Þ prolongation in both TPT (from 0.86 0.03 to 1.70 0.04 ms) and THR (from 0.35 0.02 to 0.79 0.02 ms). Moreover, the normalization of the curves (Fig. 6) indicated that the decrease in temperature had a more pronounced effect on THR than TPT ð p50:05Þ, suggesting that the negative inotropism observed after the decreases in temperature is mainly due to a direct effect of low temperature on the Ca2+ transporting mechanism responsible for cytosolic Ca2+ lowering during diastole. Conversely, increases in temperature from 258C until 358C (Fig. 5B) resulted in a progressive and significant ð p50:001Þ curtailment in both TPT (from 0.78 0.03 to 0.41 0.02 ms) and THR (from 0.37 0.02 to 0.20 0.01 ms). However, the normalization of these curves (Fig. 7) indicates that the elevation in temperature results in a similar degree of curtailment in both TPT and THR.
The results described above, associated with the lack of significance between the Q10 values obtained to TPT after decreases (2.00 0.02) and increases (1.92 0.06) in temperature, in contrast to the significant difference
Fig. 4. The effect of temperature transition from 258C to 358C and subsequent return to 258C on twitch force (Fc}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 7. The effect of temperature transition from 258C to 358C and subsequent return to 258C on TPT (&}% of the initial values) and THR (4}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 6. The effect of temperature transition from 258C to 158C and subsequent return to 258C on TPT (&}% of the initial values) and THR (4}% of the initial values) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
Fig. 5. The effect of temperature transition from 258C to 158C (A) or to 358C (B) and subsequent returns to 258C on TPT (&}MS) and THR (4}MS) of ventricle strips of L. paradoxa. Mean values SE ðn ¼ 10Þ.
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ð p50:01Þ between the Q10 values observed to THR after decreases (2.34 0.13) and increases (1.80 0.08) in temperature, indicate that L. paradoxa heart relaxation is a limiting step to the maintenance of the cardiac performance at cold temperatures.
4. Discussion Cardiac output must be well regulated to maintain sufficient blood flow in order to assure the distribution of essential nutrients, metabolites, gases and other substances to or from the various body tissues in different conditions (Lillywhite et al., 1999). Consequently, efficient adjustments between stroke volume and heart rate after an acute change in temperature are necessary in order to allow a poikilothermic animal to face thermal transitions. The heart capacity to respond appropriately to acute temperature change is a crucial factor and determines if the animal can survive in a new thermal condition. In most of the lower vertebrates studied so far, resting heart rate does not exceed 120 bpm (Farrell, 1991), ranging between 10 and 60 bpm in fish (Driedzic and Gesser, 1994). Exceptions to this pattern are the tuna, Katsuwonus pelamis (Keen et al., 1992) and Bathygobius soporator (Rantin et al., 1998). These species present heart rates more like those of small mammals than fish. According to Burggren et al. (1997), temperature is the single most important environmental determinant of heart rate, directly affecting the intrinsic pacemaker rate. Farrell and Jones (1991) pointed out that, in most of the fish, the cardiac output regulation is mediated by alterations in the stroke volume, while in mammals it is regulated mainly through the control of heart rate. On the other hand, several studies indicate that an increase in cardiac output in response to temperature elevations may occur exclusively by increases in heart rate (Randall, 1968), while stroke volume is increased by low temperatures (Vornanen, 1994). In the present study, the Q10 values for fH were about 2, similar to those described for temperate fish in response to acute temperature changes (Rantin et al., 1998). Therefore, L. paradoxa probably relies on other mechanisms than only chronotropic adjustments to maintain its cardiac performance in response to acute temperature transitions. Tibbits et al. (1991) reported that the activation of the cardiac E–C coupling occurs in response to an increase in cytosolic Ca2+ concentration. This increase can depend on both internal Ca2+ mobilization from the sarcoplasmic reticulum (SR) and external Ca2+ influx across the sarcolemma (SL) through voltage-dependent calcium channels (L-type) and Na+/Ca2+ exchange (Hove-Madsen et al., 2000). Conversely, cardiac muscle relaxes by lowering cytosolic Ca2+ concentrations back
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to the diastolic levels mainly by its transport to extracellular space through SL (Na+/Ca2+ exchange) or into SR by Ca2+-ATPase activity. The magnitude of the increase and decrease in cytosolic [Ca2+] depends on the degree of activity of Ca2+}transporting mechanisms from each compartment, which varies among species and with temperature (Gwathmey and Morgan, 1991). The most striking finding was the positive inotropism associated with the positive chonotropic response presented by L. paradoxa ventricle after temperature increases (Fig. 4). This positive inotropism differs from the responses reported for most of the ectothermic vertebrates and mammals, since these animals show a decrease in myocardial force (e.g. flounder, frogs, turtle, rats, rabbits) or a lack of change in twitch force (e.g. trout) at higher temperatures (Shi and Jackson, 1997; Costa et al., 2000). These variations of the cardiac muscle force responses to temperature changes may depend on both maximal Ca2+}activated force and Ca2+}sensitivity differences among species (Kim et al., 2000). The heart capacity of developing a positive inotropism, even after the temperature-induced increase in chronotropism, can also be suggested by the curtailment of TPT and THR observed in vitro after temperature elevation (Figs. 5 and 7). This maintenance of a positive inotropism at higher temperatures, even after the temperature-induced curtailment of TPT and THR, indicates that the species possesses very efficient mechanisms of Ca2+ transportation from and into the cytosol in the cardiac E–C coupling. This allows faster rates of contraction and relaxation to be achieved at higher temperatures. One possibility is that the species has a functional SR, which would allow a very fast Ca2+ sequestration and liberation from and to the myoplasm. Hochachka and Hulbert (1978) found a very well developed SR in the ventricular myocardium of L. paradoxa. However, the physiological role of this organella remains to be elucidated. Another potential mechanisms would be the presence of a very active Na+/Ca2+ exchanger, resulting in a fast Ca2+ efflux from the cell during cardiac relaxation. This hypothesis is of special relevance since a post-rest decay of twitch force was observed for ventricle strips of L. paradoxa after non-physiological prolonged diastolic pauses ranging from 7 to 350 s (unpublished results). Accordingly to Bers (1991), a decreased amplitude of the first postrest contraction with increased resting periods occurs in ventricular myocardium of several mammalian species, and is linked to transsarcolemmal Ca2+ movements via Na+/Ca2+ exchange that reduces cytosolic Ca2+ during relaxation. It can also be proposed that the mechanisms responsible by the Ca2+ influx and efflux to and from the cell, and possibly to SR (if functional for Ca2+
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management), are greatly affected by temperature in the ventricular myocytes of L. paradoxa. This high sensibility to temperature is similar to that reported for the heart of mammals and birds, but contrasts with most of the fish species already studied. In fish, particularly the temperate species, these mechanisms generally show a minimum degree of temperature sensitivity, indicating an adaptive response to cold (Kim et al., 2000). Conversely, the unexpected negative inotropism observed after the decrease in temperature (Fig. 3) indicates that an acute drop in temperature does not allow the expression of Ca2+ transporting proteins and/ or myosin isoforms more adapted to low temperatures. This fact may be responsible by the decrease in twitch force observed when temperature was reduced, even after the prolongation of TPT and THR (Fig. 5). This suggests that in a situation of acute decrease in temperature, as observed in the present study, L. paradoxa will not be able to maintain its cardiac performance, mostly due to a temperature-induced impairment of its cardiac relaxation (Fig. 6). The strong positive inotropic response observed during the acute temperature elevation corroborates this hypothesis. It indicates that the contractile proteins and/or Ca2+ transporting proteins isoforms, expressed in the acclimation temperature (258C), probably reflect those expressed in the species natural habitat. However, the negative inotropism observed in vitro during cold exposure could be improved in vivo by a thermal stress-induced catecholamines release. There are several mechanisms of adrenergic modulation for the heart performance and some of them are relevant to the inotropism regulation. The stimulation of b-adrenergic receptors causes the phosphorylation of the SL L-type Ca2+ channels (Shields et al., 1998) and also stimulates Na+/K+ exchanger (Hove-Madsen and Gesser, 1989), increasing the transsarcolemmal Ca2+ influx. Additionally, Boller and Pott (1989) demonstrated that Ca2+ transportation through SR is also increased by catecholamines in mammalian heart. The lower Q10 values for both Fc and fH when temperature was decreased indicate that lower temperatures have a less pronounced effect than the higher ones on both ino- and chronotropic responses of the species heart. This may prevent the occurrence of an acute drop in cardiac output at low temperatures allowing, within physiological limits, this sedentary and aestivating fish to face acute reductions in environmental temperatures without great impairments in the cardiac performance. These results, associated with the typical chronotropic response of fish, indicate that the species is able to respond more appropriately to an acute elevation in temperature, resulting in a significant increase in cardiac performance due to both positive chrono and inotropic responses, even after the temperature-induced curtailment of TPT and THR. There is coherence between the
data of this work and the thermal conditions faced by the species in its natural habitat. Temperatures of 308C or higher are frequently recorded in the ponds where specimens of L. paradoxa are collected during sunny days of summer. However, under natural conditions L. paradoxa rarely faces acute transitions to low temperatures like the ones experienced in the present study.
Acknowledgements This study was supported by FAPESP (Proc. 1998/ 11846-6).
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