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Role of the sarcoplasmic reticulum in calcium dynamics of the ventricular myocardium of Lepidosiren paradoxa (Dipnoi) at different temperatures
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Journal of Thermal Biology 29 (2004) 81–89
Role of the sarcoplasmic reticulum in calcium dynamics of the ventricular myocardium of Lepidosiren paradoxa (Dipnoi) at different temperatures M.J. Costa, C.D. Olle, A.L. Kalinin, F.T. Rantin* Department of Physiological Sciences, Federal University of Sao * Carlos, Via Washington Luiz, km 235, Sao * Carlos, SP 13565-905, Brazil Received 10 August 2003; accepted 12 November 2003
Abstract This study analyzed the impact of changes in stimulation frequency on the inotropic responses of the South American lungfish Lepidosiren paradoxa ventricle strips. The effects of prolonged diastolic pauses and stepwise increases in stimulation frequency upon force development were determined at different temperatures (15 C, 25 C, and 35 C) and in response to 10 mM of ryanodine. A post-rest potentiation of force was observed only at 15 C, but ryanodine was able to depress twitch force at low stimulation frequencies, irrespective of temperature. Moreover, when in vivo stimulation frequencies were reached, the sarcoplasmic reticulum was relevant to the Ca2+ management only at 15 C and 25 C. In contrast, the lack of effect of ryanodine at the in vivo frequencies at 35 C indicates that the excitation–contraction coupling depends exclusively on the transarcolemmal Ca2+ influx. Therefore, this organelle seems to have a slower Ca2+-cycling capacity, in spite of being anatomically well developed and potentially functional in all the temperatures tested. r 2003 Elsevier Ltd. All rights reserved. Keywords: Excitation–contraction coupling; Ventricle strips; Sarcoplasmic reticulum; Temperature; Ryanodine; Post-rest force; Force–frequency relationship; Lungfish; Lepidosiren paradoxa; Fish; Calcium
1. Introduction The prime determinants of force development by heart muscle are the intracellular Ca2+ activity and the time available for Ca2+ to interact with the contractile apparatus (Yue, 1987). 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 ultraAbbreviations: E–C coupling—excitation–contraction coupling, Fc —contractile force, NCX—Na+/Ca2+ exchanger, SL—Sarcolemma, SR—Sarcoplasmic reticulum *Corresponding author. Tel.: +55-16-260-8328; fax: +5516-260-8314. E-mail address: [email protected] (F.T. Rantin).
structural distinctions between hearts of different species underlie important physiological differences, particularly in the dynamics of Ca2+ delivery to the contractile apparatus (Tibbits et al., 1992a). On a beat-to-beat basis, Ca2+ may originate from the extracellular space or from intracellular stores (Driedzic and Gesser, 1988), specifically the sarcoplasmic reticulum (SR). The putative contribution of SR Ca2+ stores to excitation–contraction (E–C) coupling has a profound impact on Ca2+ management, since this organelle is able to significantly reduce diffusional distances and accelerate both contraction and relaxation rates. The relative importance of Ca2+ release from the SR in activation of cardiac muscle contraction varies considerably among different species, stages of development,
0306-4565/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2003.11.002
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stimulation frequency and temperature (Fabiato, 1982; Bers, 1991). Fish, like most of lower vertebrates, but unlike mammals, appear to be less able to generate free Ca2+ from the SR. Several studies have demonstrated that this organelle is usually poorly developed and does not seem to directly contribute to the activation of the contractile apparatus at physiological stimulation frequencies and temperatures in ventricle strips of most species (Gesser and Poupa, 1978; Vornanen, 1989; Tibbits et al., 1991). In animals with a poorly developed SR, heart contractility is triggered by increases in the intracellular Ca2+ concentration primarily due to its influx across the sarcolemma (SL). However, the appropriate diffusional distances for extracellular Ca2+ to the myofilaments can be assured due to their smaller myocyte diameter (Tibbits et al., 1992a) in spite of the absence of a functional SR. Since a change in environmental temperature results in an immediate change in body temperature (Bowler and Tirri, 1990), the effect of this parameter in cardiac E–C coupling should also be considered beside these interspecific differences in the main source of Ca2+ for myofilament activation. It has been widely demonstrated that the importance of the SR to force generation decreases at low temperatures in most fish (HoveMadsen, 1992; M^ller-Nielsen and Gesser, 1992; Gesser, 1996), because ryanodine-sensitive Ca2+-release channels of SR spend a greater proportion of time in the open-state as temperature decreases (Bers, 2001). On the other hand, the Na+/Ca2+ exchanger (NCX) is relatively temperature insensitive in many fish, which could be an adaptative response to cold (McKnight et al., 1989; Tibbits et al., 1992b; Xue et al, 1999). Considering that most of the studies on cardiac E–C coupling were carried out on temperate fishes, the goal of this work was to extend the knowledge to the neotropical lungfish Lepidosiren paradoxa. Moreover, in vitro experiments were performed at physiologically relevant contraction frequencies and temperatures, allowing a comparison to the in vivo situation. The South American lungfish inhabits rivers and lakes in both tropical and subtropical regions of South America, where it is frequently subjected to large daily temperature changes. 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 lungfish (Burggren and Johansen, 1986), this group is supposed to provide a unique opportunity to study the physiological adaptations correlated with the emergence of air-breathing.
2. Materials and methods 2.1. Animals Active, non-aestivating specimens of the South American lungfish, Lepidosiren paradoxa, of either sex weighing between 480 and 1030 g and body length from 50 to 86 cm were obtained from water filled clay pits near Cuiab!a River, in the Brazilian Pantanal area, Mato Grosso State. For at least 4 weeks prior to experimentation, the fish were maintained in 1000 l holding tanks equipped with continuous supply of dechlorinated water (1.2 l min1) at constant temperature (2571 C). The holding tanks were covered with floating macrophytes in order to reduce light intensity and to minimize stress. The animals were fed weekly on chopped chicken liver. 2.2. Ventricle strip preparation Fish were sacrificed by decapitation, and the heart were excised and placed in ice-cooled saline. Pairs of strips with a thickness of maximally 1 mm and weight of 11.8770.98 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 25 C and bubbled throughout the experiment with a gas mixture of 98% O2 and 2% CO2 (pH 7.5). Strips kept in the saline at +4 C could be used for up to 3 days without detectable changes in responses. The temperature of the muscle bath was kept at 25 C, unless otherwise stated. Preparations were suspended using surgical silk to connect to a NARCO F-60 isometric force transducer placed around a platinum electrode. This electrode and another 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 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 at 0.2 Hz before each experimental protocol. In the first experimental protocol, the force developed by ventricle strips upon the first stimulation following increasing rest periods was determined at 15 C, 25 C and 35 C. This approach details the capacity for the storage of intracellular Ca2+ during a non-physiological diastolic pause. After a steady-state condition at 12 contractions min1 was achieved, stimulation ceased for successive increased periods of 5, 10, 30, 60, 300 and 600 s. Force development of the first contraction following the resting period was compared to the last contraction in a steady-state train. These experiments
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were conducted with and without 10 mM ryanodine (Sigmas). Several studies on mammalian and fish myocardium (Nayler et al., 1970; Rousseau et al., 1987; Driedzic and Gesser, 1988; Tibbits et al., 1991; Hove-Madsen, 1992; Keen et al., 1994; Gesser, 1996; Vornanen, 1996; Bers, 2001) demonstrated that this ryanodine concentration reduces the functional importance of SR for the E–C coupling. When applied, ryanodine was added to the medium at least 40 min before the diastolic pauses. In further experimental trials, ventricle strip preparations were subjected to increases in imposed contraction frequency from 0.1 Hz until the frequency in which at least 80% of the strips were still able to contract regularly. To analyze the potential contribution of the SR at more physiological frequencies, 10 mM of ryanodine was added to the bath 40 min before alterations in frequency. These protocols were performed at 15 C, 25 C and 35 C. The in vivo physiological heart frequencies presented as the dashed areas in Fig. 3 correspond to those registered by Costa et al. (2002) for L. paradoxa.
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Fig. 1. Effect of ryanodine on the ventricular post-rest force development (Fc —%; mean values7SE) of the South American lungfish, Lepidosiren paradoxa, at different resting intervals and experimental temperatures. (A) 15 C ðn ¼ 10Þ; (B) 25 C (acclimation temperature; n ¼ 10); (C) 35 C ðn ¼ 10Þ: Tension is normalized to the last steady-state twitch prior to rest (0.2 Hz). Asterisks on top of bars indicate a significant ðpo0:05Þ difference in relation to the last contraction force before rest, while asterisks above horizontal lines represent a significant effect ðpo0:05Þ of ryanodine on post-rest force.
Fig. 1 shows the effect of prolonged diastolic pauses on post-rest contraction force at different experimental temperatures with and without addition of ryanodine to the medium. At 15 C, control preparations showed a post-rest potentiation of twitch force (% steady-state force) following rest periods above 60 min, which was completely abolished by ryanodine (Fig. 1A). Conversely, at 25 C and 35 C, the post-rest tension of control
preparations decreased significantly after diastolic pauses of 300 and 60 s, respectively. This post-rest decay of twitch force was further pronounced by the addition of ryanodine after 600 s of rest at acclimation temperature (Fig. 1B) and by diastolic pauses above 60 s at 35 C (Fig. 1C). The relative contribution of the Ca2+ from the SR to force generation as a function of experimental
3. Results
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Fig. 2. Effect of temperature on force development (Fc —%; mean values7SE) of ventricle strips of the South American lungfish, Lepidosiren paradoxa, after 10 min of resting period in the absence (top; n ¼ 10) and presence (bottom; n ¼ 10) of 10 mM ryanodine. Shading indicates ryanodine-sensitive force (SR contribution to force generation).
temperature after a diastolic pause of 600 s is presented in Fig. 2 (dashed area). In spite of the postrest potentiation of twitch force observed at 15 C, pre-treatment with ryanodine resulted in a post-rest force about 21%, 17% and 38% lower than that observed for control preparations at 15 C, 25 C and 35 C, respectively. 3.2. Force–frequency relationship Fig. 3A shows that the positive force–frequency relationship observed in all stimulation frequencies for control preparations at 15 C was shifted to the left (from 0.4 to 0.2 Hz) in response to ryanodine. The dashed area in Fig. 2A indicates that twitch force of control preparations increased significantly at physiological stimulation frequencies (0.2970.01 Hz) at 15 C. In contrast, ventricle strips treated with ryanodine started to contract irregularly above 0.2 Hz, a lower frequency compared to the physiological range. At the acclimation temperature, in spite of the tendency for a positive relationship, especially between 0.4 and 1.0 Hz, control preparations responded to increases in stimulation frequency with changes in force development only at the highest frequency (2.0 Hz), when a significant ðpo0:05Þ decrease in twitch force was observed (Fig. 3B). However, this relationship was shifted downwards by the addition of ryanodine. Additionally, when force–frequency responses were analyzed at frequencies within the in vivo range at 25 C (0.5470.03 Hz; dashed area in Fig. 3B), ryanodine treatment resulted in a decrease in twitch force of about 10%.
The tendency for a positive force–frequency relationship observed at lower temperatures for control preparations disappeared completely at 35 C, becoming negative above 1.8 Hz (Fig. 3C). This negative relationship contrasts with the response to ryanodine, which resulted in a significant decrease in twitch force only at the highest frequency observed for this treatment. Moreover, the response curve was shifted to the left (from 4.0 to 2.0 Hz) after addition of ryanodine. Finally, when the responses were analyzed at in vivo frequencies (1.1770.03 Hz; dashed area in Fig. 2C), ryanodine had no effect on force development at 35 C.
4. Discussion The twitch tension developed by cardiac muscle is affected in large measure by variations in frequency and regularity of stimulation (Hajdu, 1969). While the force– frequency relationship probably involves several mechanisms associated with Ca2+ uptake and storage (Wohlfart and Noble, 1982), the relative importance of individual components of the process may vary among species (Driedzic and Gesser, 1988). A pause in regular stimulation of cardiac muscle produces characteristic changes in contraction during the post-rest recovery. The characterization of the postrest response can provide information on the basic cellular mechanisms involved in the control of cardiac contraction (Mill et al., 1992). These mechanisms include the relative participation of intracellular stores that liberate Ca2+ during contraction and also the activity of NCX at different experimental conditions (Sutko et al., 1986).
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Fig. 3. Effect of increases in stimulation frequency on twitch force (Fc —% of initial values7SE) of ventricle strips of the South American lungfish, Lepidosiren paradoxa, at control preparations (squares—2.5 mM of Ca2+ and without ryanodine) and after the pretreatment with 10 mM of ryanodine (triangles) at (A) 15 C (n ¼ 10), (B) 25 C (n ¼ 10) and (C) 35 C (n ¼ 10). For each treatment, open symbols indicate a significant difference ðpo0:05Þ in relation to the Fc observed initially (at 0.1 Hz) and the asterisks denote a difference between the two treatments (control and ryanodine) in the same frequency ðpo0; 05Þ: The dashed area indicates the heart frequency measured in vivo at each temperature by Costa et al. (2002).
During the pause, Ca2+ can be pumped out of the cell by the SL Ca2+-pump and/or by the NCX and can also be stored at intracellular sites such as the SR (Mill et al., 1992). The net result is a variable amount of Ca2+ activator stored within the cell that can be liberated upon post-rest contraction (Bers et al., 1993; HoveMadsen et al., 2000). If the amount of intracellular Ca2+ increases, a post-rest potentiation of twitch force is observed, while a post-rest decay results from a more
pronounced Ca2+ efflux from the cell (Bers, 2001). Since SL Ca2+-pump does not seem to play a central role in Ca2+ management in most fish due to its very low Ca2+-transporting capacity (Tibbits et al., 1991), the two main mechanisms that influence this process are the SR and NCX activities. Several mammalian hearts present a post-rest potentiation of twitch force in response to short pauses, which decreases as the rest period is increased (Vassalo and
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Mill, 1988), while in rat myocardium this post-rest potentiation is maintained even after long pauses (Vassalo et al., 1990). This potentiation of twitch force has been related to a greater accumulation of Ca2+ at the higher priming frequencies, which, in turn, is released during the test beat (Edman and Johannsson, 1976; Rumberger and Reichel, 1972). Conversely, most fish exhibit a minimal post-rest potentiation of force (Driedzic and Gesser, 1988), suggesting a less developed SR. Moreover, ryanodine has no effect on the development of post-rest potentiation in most teleost species (Driedzic and Gesser, 1988). An exception of the teleost pattern is the rainbow trout heart, in which the SR is anatomically much more developed (Santer, 1985) and the ventricle strips show a post-rest potentiation of force that is strongly reduced by ryanodine, especially at high temperatures (HoveMadsen and Gesser, 1989; Hove-Madsen, 1992; M^llerNielsen and Gesser, 1992). Despite this, ryanodine has no effect on force development when physiological temperatures are considered (Keen et al., 1994; Gesser, 1996; Shiels et al., 1998). Our results differ considerably from those previously reported, since ryanodine reduced significantly the postrest force of lungfish ventricle strips not only at 25 C, a common temperature faced by the species in its habitat, but also in a wider temperature range (from 15 C to 35 C, Figs. 1 and 2). Another interesting finding was the post-rest decay of twitch force at 25 C and 35 C as the resting period was increased to 300 and 600 s (Figs. 1B and C). A post-rest Ca2+ depletion has also been consistently observed in rabbit ventricle (Ferraz et al., 2001), suggesting an increased Ca2+ efflux from the cell as resting period is prolonged. In lungfish, a temperature-dependent increase of NCX activity may account for this response. This result contrasts with those described for most fish species, since in these animals the NCX presents relative temperature insensitivity (Xue et al., 1999). This adaptative response to cold allows the animal to maintain the cardiac performance at low temperatures when the SR does not seem to be functional. The NCX temperature-insensitivity was only described for temperate fish (Bersohn et al., 1991; Tibbits et al., 1992b; Marengo et al., 1997), but one could expect a pronounced temperature-sensitivity of this Ca2+transporting mechanism in tropical species as a strategy to face higher temperatures. When physiological stimulation frequencies are considered, the temperature sensitivity of the NCX would allow the species to increase its cardiac performance as temperature is augmented even without a significant increase in SR activity (Fig. 2). This fact could partially explain the positive inotropic response observed by Costa et al. (2002) for L. paradoxa ventricle strips when temperature was acutely increased from 25 C to 35 C. However,
more specific inotropic interventions are needed to fully explain the effect of temperature on the lungfish NCX activity. An increase in stimulation frequency, within the physiological range, increases the myocardium force of contraction in most mammalian species (Koch-Weser and Blinks, 1963), turtles and frogs (Driedzic and Gesser, 1985) and also in elasmobranchs (Maylie et al., 1979; Driedzic and Gesser, 1988). In contrast, such increases in stimulation frequency result in a stepwise decrease in developed tension in rats and mice myocardium at physiological frequencies (Benfonaro, 1958; Stemmer and Akera, 1986) in spite of their highly developed SR network (Bers, 1991). Similarly, a negative force–frequency relationship was reported for most temperate (Driedzic and Gesser, 1985, 1988; Hove-Madsen and Gesser, 1989; Vornanen, 1989; Bailey and Driedzic, 1990; Hove-Madsen, 1992; Shiels and Farrell, 1997) and tropical (Rantin et al., 1998; Costa et al., 2000) teleost fish. However, contrasting to rat and mouse, the SR of fish generally does not contribute significantly to tension development at in vivo stimulation frequencies and temperatures. Even though, species such as tunas (Keen et al., 1992; Shiels et al., 1999), the Atlantic cod (Driedzic and Gesser, 1985), and mackerels (Driedzic and Gesser, 1988; Shiels and Farrell, 2000) did not show this negative force– frequency response. In tunas, the positive force– frequency relationship, associated with a pronounced effect of ryanodine in force development by the atrial (Shiels et al., 1999) and ventricle strips (Tibbits, 1996), suggests a higher reliance on SR Ca2+ for contraction over its physiological temperatures and heart rates. A negative force–frequency relationship was also observed in the present study for L. paradoxa ventricle strips at 25 C and 35 C when high stimulation frequencies were achieved. However, this negative staircase was not observed when the interval comprehended between 0.1 Hz and that recorded in vivo was considered at each experimental temperature (dashed area in Fig. 3), suggesting an absence of a frequency-dependent decrease in intracellular Ca2+ concentration at these conditions. This finding suggests that L. paradoxa possesses an efficient ventricular Ca2+-transporting mechanism, which would allow an adequate delivery of Ca2+ to the myofilaments even after the temperature and/or frequency-induced curtailment of the time to peak force at physiological conditions. The main Ca2+-transporting mechanisms responsible for Ca2+ management at each temperature were assessed by the use of ryanodine (to block the SR Ca2+-release channels), as discussed below. At the highest temperature (35 C), and in vivo stimulation range (dashed area in Fig. 3C), the ryanodine application had no effect on tension development. This result, associated with the absence of a
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decrease in twitch force when this stimulation frequencies were achieved for control preparations, suggests that the apparent enhance in NCX activity during increases in temperature (as indicated by the post-rest decay of twitch force at higher temperatures in Figs. 1B and C) can assure an appropriate inotropism at physiological frequencies, even without the SR participation at 35 C. The positive force–frequency relationship observed for control preparations at the lowest temperature corroborates the temperature-dependency of the NCX, indicating a net increase of intracellular Ca2+ accumulation as temperature is decreased. Moreover, it can be proposed that at 15 C the SR plays a functional role in the Ca2+ management at the frequencies observed in vivo, since ryanodine shifted the force–frequency relationship to the left (Fig. 3A). Moreover, this organelle also seems to play some role on Ca2+ management at in vivo frequencies and acclimation temperature (Fig. 3B), as ryanodine reduced tension development in almost 10%. On the other hand, at 35 C ryanodine did not reduce either the contractile force or the maximal frequency to values bellow the in vivo range. This implies that the SR, in spite of being potentially functional at 35 C (Fig. 1C), probably does not play a role in Ca2+ management at in vivo condition. Taken together, the present results suggest a low Ca2+-cycling capacity of the ventricular SR of L. paradoxa, implying that this organelle does not play a significant role at high heart rates, in spite of its high anatomical development (Hochachka and Hulbert, 1978) and apparent functionality at lower temperatures and/or lower stimulation frequencies. Therefore, as temperature decreases, with a concomitant temperature-dependent decrease in chronotropism, the SR plays a central role in Ca2+ regulation, compensating the apparent temperature-dependent decrease in NCX activity. These results contrast with those described for the ventricle strips of most fish already studied (Keen et al., 1994; Thomas et al., 1996; Shiels and Farrell, 1997; Shiels et al., 1998) in which ryanodine does not change significantly the force development when physiological frequencies and/or temperatures are considered.
Acknowledgements The authors would like to thank the field technician Mr. Nelson Santos Alves Matos for the collection and maintenance of the lungfish. We are also thankful to Dr. Jay A. Nelson (Towson University, MD, USA) for revising the text. This study was supported by FAPESP (Ph.D. fellowship and grant to Monica J. Costa, No. 98/ 11846-6). All the experiments were performed complying with the Brazilian laws.
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Hajdu, S., 1969. Mechanism of the woodworth staircase phenomenon in heart and skeletal muscle. Am. J. Physiol. 216, 206–214. Hochachka, P., Hulbert, W., 1978. Glycogen ‘seas’, glycogen bodies, and glycogen granules in heart and skeletal muscle of two air-breathing, burrowing fishes. Can. J. Zool. 56, 774–786. Hove-Madsen, L., 1992. The influence of temperature on ryanodine sensitivity and the force–frequency relationship in the myocardium of rainbow trout. J. Exp. Biol. 167, 47–60. Hove-Madsen, L., Gesser, H., 1989. Force–frequency relation in the myocardium of rainbow trout: effects of K+ and adrenaline. J. Comp. Physiol. B 159, 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. Am. J. Physiol. 279, R1856–R1864. Johansen, K., Hanson, D., 1968. Functional anatomy of the hearts of lungfishes and amphibians. Am. Zool. 8, 191–210. Johansen, K., Lenfant, C., Hanson, D., 1968. Cardiovascular dynamics in the lungfishes. Z. Vegl. Physiol. 59, 205–218. 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. Can. J. Zool. 70, 1211–1217. Keen, J.E., Vianzon, D.M., Farrell, A.P., Tibbits, G.F., 1994. Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium. J. Comp. Physiol. B 164, 438–443. Koch-Weser, J., Blinks, J.R., 1963. The influence of the exterval between beats on myocardial contractility. Pharmacol. Rev. 15, 601–652. Marengo, F.D., Wang, S-Y., Langer, G.A., 1997. The effects of temperature upon calcium exchange in intact cultured cardiac myocytes. Cell Calcium 21, 263–273. Maylie, J.M., Nunzi, M.G., Morad, M., 1979. Excitation– contraction coupling in ventricular muscle of dogfish (Squalus acanthias). Mt. Desert Island Biol. Lab. Bull. 19, 84–87. McKnight, K.J., Kashihara, H., Tibbits, G.F., 1989. Na+/Ca2+ exchange in sarcolemma from a salmonid heart: seasonal variations. Can. Fed. Biol. Soc. Proc. 32, 150. Mill, J.G., Vassallo, D.V., Leite, C.M., 1992. Mechanisms underlying the genesis of post-rest contractions in cardiac muscle. Braz. J. Med. Biol. Res. 25, 399–150408. M^ller-Nielsen, T., Gesser, H., 1992. Sarcoplasmic reticulum and excitation–contraction coupling at 20 and 10 C in rainbow trout myocardium. J. Comp. Physiol. B 162, 526–534. Nayler, W.G., Daile, P., Chipperfield, D., Gan, K., 1970. Effect of ryanodine on calcium in cardiac muscle. Am. J. Physiol. 219, 1620–1626. 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.
Rousseau, E., Smith, J.S., Meissner, G., 1987. Ryanodine modifies conductance and gating behavior of single Ca2+ release channel. Am. J. Physiol. 253, C364–C368. Rumberger, E., Reichel, H., 1972. The force–frequency relationship: a comparative study between warm- and cold-blooded animals. Pflugers . Arch. Gesamte Physiol. 332, 206–217. Santer, R.M., 1985. Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell. Biol. 89, 1–102. Shiels, H.A., Farrell, A.P., 1997. The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing ca2+ to force development in isolated ventricular trabeculae from rainbow trout. J. Exp. Biol. 200, 1607–1621. Shiels, H.A., Farrell, A.P., 2000. The effect of ryanodine on isometric tension development in isolated ventricular trabeculae from Pacific mackerel (Scomber japonicus). Comp. Biochem. Physiol. 125A, 331–341. Shiels, H.A., Stevens, E.D., Farrell, A.P., 1998. Effect of temperature, adrenaline and ryanodine on power production in trout (Oncorhynchus mykiss) ventricular trabeculae. J. Exp. Biol. 201, 2701–2710. Shiels, H.A., Freund, E.V., Farrell, A.P., Block, B.A., 1999. The sarcoplasmic reticulum plays a major role in isometric contraction in atrial muscle of yellowfin tuna. J. Exp. Biol. 202, 881–890. Stemmer, P., Akera, T., 1986. Concealed positive force– frequency relationships in rat and mouse cardiac muscle revealed by ryanodine. Am. J. Physiol. 251, H1106–H1110. Sutko, J.L., Bers, D.M., Reeves, J.P., 1986. Post-rest inotropy in rabbit ventricle: Na+–Ca2+ exchange determines sarcoplasmic reticulum Ca2+ content. Am. J. Physiol. 250, H654–H661. Thomas, M.J., Hamman, B.N., Tibbits, G.F., 1996. Dihydropyridine and ryanodine binding in ventricles from rat, trout, dogfish and hagfish. J. Exp. Biol. 199, 1999–2009. Tibbits, G.F., 1996. Towards a molecular explanation of the high performance of the tuna heart. Comp. Biochem. Physiol. 113A, 77–82. 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. Can. J. Zool. 69, 2014–2019. Tibbits, G.F., Moyes, C.D., Hove-Madsen, L., 1992a. Excitation–contraction coupling in the teleost heart. In: Hoar, W.S., Randall, D.J., Farrell, A.P. (Eds.), Fish Physiology: The Cardiovascular System, Vol. 12A. Academic Press Inc., New York, pp. 267–303. Tibbits, G.F., Philipson, K.D., Kashihara, H., 1992b. Characterization of myocardial Na+–Ca2+ exchange in rainbow trout. Am. J. Physiol. 262, C411–C417. Vassalo, D.V., Mill, J.G., 1988. Mechanical behavior of rest contractions in cardiac muscle. Acta Physiol. Pharm. Lat. Am. 38, 99–114. Vassalo, D.V., Mill, J.G., Abreu, G.R., 1990. Control of rest potentiation in the ventricular myocardium by the Na–Ca exchange mechanism. Acta. Physiol. Pharm. Lat. Am. 40, 129–136. Vornanen, M., 1989. Regulation of contractility of the fish (Carassius carassius L.) heart ventricle. Comp. Biochem. Physiol. 94C, 477–483.
ARTICLE IN PRESS M.J. Costa et al. / Journal of Thermal Biology 29 (2004) 81–89 Vornanen, M., 1996. Effect of extracellular calcium on the contractility of warm- and cold-acclimated crucian carp heart. J. Comp. Physiol. B 165, 507–517. Wohlfart, B., Noble, M.I.M., 1982. The cardiac excitation– contraction cycle. Pharmalog. Ther. 16, 1–43. Xue, X.H., Hryshko, L.V., Nicoll, D.A., Philipson, K.D., Tibbits, G.F., 1999. Cloning, expression, and characteriza-
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tion of the trout cardiac Na+/Ca2+ exchanger. Am. J. Physiol. 277, C693–C700. Yue, D.T., 1987. Intracellular [Ca2+] related to rate of force development in twitch contraction of heart. Am. J. Physiol. 252, H760–H770.
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Role of the sarcoplasmic reticulum in calcium dynamics of the ventricular myocardium of Lepidosiren paradoxa (Dipnoi) at different temperatures M.J. Costa, C.D. Olle, A.L. Kalinin, F.T. Rantin* Department of Physiological Sciences, Federal University of Sao * Carlos, Via Washington Luiz, km 235, Sao * Carlos, SP 13565-905, Brazil Received 10 August 2003; accepted 12 November 2003
Abstract This study analyzed the impact of changes in stimulation frequency on the inotropic responses of the South American lungfish Lepidosiren paradoxa ventricle strips. The effects of prolonged diastolic pauses and stepwise increases in stimulation frequency upon force development were determined at different temperatures (15 C, 25 C, and 35 C) and in response to 10 mM of ryanodine. A post-rest potentiation of force was observed only at 15 C, but ryanodine was able to depress twitch force at low stimulation frequencies, irrespective of temperature. Moreover, when in vivo stimulation frequencies were reached, the sarcoplasmic reticulum was relevant to the Ca2+ management only at 15 C and 25 C. In contrast, the lack of effect of ryanodine at the in vivo frequencies at 35 C indicates that the excitation–contraction coupling depends exclusively on the transarcolemmal Ca2+ influx. Therefore, this organelle seems to have a slower Ca2+-cycling capacity, in spite of being anatomically well developed and potentially functional in all the temperatures tested. r 2003 Elsevier Ltd. All rights reserved. Keywords: Excitation–contraction coupling; Ventricle strips; Sarcoplasmic reticulum; Temperature; Ryanodine; Post-rest force; Force–frequency relationship; Lungfish; Lepidosiren paradoxa; Fish; Calcium
1. Introduction The prime determinants of force development by heart muscle are the intracellular Ca2+ activity and the time available for Ca2+ to interact with the contractile apparatus (Yue, 1987). 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 ultraAbbreviations: E–C coupling—excitation–contraction coupling, Fc —contractile force, NCX—Na+/Ca2+ exchanger, SL—Sarcolemma, SR—Sarcoplasmic reticulum *Corresponding author. Tel.: +55-16-260-8328; fax: +5516-260-8314. E-mail address: [email protected] (F.T. Rantin).
structural distinctions between hearts of different species underlie important physiological differences, particularly in the dynamics of Ca2+ delivery to the contractile apparatus (Tibbits et al., 1992a). On a beat-to-beat basis, Ca2+ may originate from the extracellular space or from intracellular stores (Driedzic and Gesser, 1988), specifically the sarcoplasmic reticulum (SR). The putative contribution of SR Ca2+ stores to excitation–contraction (E–C) coupling has a profound impact on Ca2+ management, since this organelle is able to significantly reduce diffusional distances and accelerate both contraction and relaxation rates. The relative importance of Ca2+ release from the SR in activation of cardiac muscle contraction varies considerably among different species, stages of development,
0306-4565/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2003.11.002
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stimulation frequency and temperature (Fabiato, 1982; Bers, 1991). Fish, like most of lower vertebrates, but unlike mammals, appear to be less able to generate free Ca2+ from the SR. Several studies have demonstrated that this organelle is usually poorly developed and does not seem to directly contribute to the activation of the contractile apparatus at physiological stimulation frequencies and temperatures in ventricle strips of most species (Gesser and Poupa, 1978; Vornanen, 1989; Tibbits et al., 1991). In animals with a poorly developed SR, heart contractility is triggered by increases in the intracellular Ca2+ concentration primarily due to its influx across the sarcolemma (SL). However, the appropriate diffusional distances for extracellular Ca2+ to the myofilaments can be assured due to their smaller myocyte diameter (Tibbits et al., 1992a) in spite of the absence of a functional SR. Since a change in environmental temperature results in an immediate change in body temperature (Bowler and Tirri, 1990), the effect of this parameter in cardiac E–C coupling should also be considered beside these interspecific differences in the main source of Ca2+ for myofilament activation. It has been widely demonstrated that the importance of the SR to force generation decreases at low temperatures in most fish (HoveMadsen, 1992; M^ller-Nielsen and Gesser, 1992; Gesser, 1996), because ryanodine-sensitive Ca2+-release channels of SR spend a greater proportion of time in the open-state as temperature decreases (Bers, 2001). On the other hand, the Na+/Ca2+ exchanger (NCX) is relatively temperature insensitive in many fish, which could be an adaptative response to cold (McKnight et al., 1989; Tibbits et al., 1992b; Xue et al, 1999). Considering that most of the studies on cardiac E–C coupling were carried out on temperate fishes, the goal of this work was to extend the knowledge to the neotropical lungfish Lepidosiren paradoxa. Moreover, in vitro experiments were performed at physiologically relevant contraction frequencies and temperatures, allowing a comparison to the in vivo situation. The South American lungfish inhabits rivers and lakes in both tropical and subtropical regions of South America, where it is frequently subjected to large daily temperature changes. 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 lungfish (Burggren and Johansen, 1986), this group is supposed to provide a unique opportunity to study the physiological adaptations correlated with the emergence of air-breathing.
2. Materials and methods 2.1. Animals Active, non-aestivating specimens of the South American lungfish, Lepidosiren paradoxa, of either sex weighing between 480 and 1030 g and body length from 50 to 86 cm were obtained from water filled clay pits near Cuiab!a River, in the Brazilian Pantanal area, Mato Grosso State. For at least 4 weeks prior to experimentation, the fish were maintained in 1000 l holding tanks equipped with continuous supply of dechlorinated water (1.2 l min1) at constant temperature (2571 C). The holding tanks were covered with floating macrophytes in order to reduce light intensity and to minimize stress. The animals were fed weekly on chopped chicken liver. 2.2. Ventricle strip preparation Fish were sacrificed by decapitation, and the heart were excised and placed in ice-cooled saline. Pairs of strips with a thickness of maximally 1 mm and weight of 11.8770.98 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 25 C and bubbled throughout the experiment with a gas mixture of 98% O2 and 2% CO2 (pH 7.5). Strips kept in the saline at +4 C could be used for up to 3 days without detectable changes in responses. The temperature of the muscle bath was kept at 25 C, unless otherwise stated. Preparations were suspended using surgical silk to connect to a NARCO F-60 isometric force transducer placed around a platinum electrode. This electrode and another 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 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 at 0.2 Hz before each experimental protocol. In the first experimental protocol, the force developed by ventricle strips upon the first stimulation following increasing rest periods was determined at 15 C, 25 C and 35 C. This approach details the capacity for the storage of intracellular Ca2+ during a non-physiological diastolic pause. After a steady-state condition at 12 contractions min1 was achieved, stimulation ceased for successive increased periods of 5, 10, 30, 60, 300 and 600 s. Force development of the first contraction following the resting period was compared to the last contraction in a steady-state train. These experiments
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were conducted with and without 10 mM ryanodine (Sigmas). Several studies on mammalian and fish myocardium (Nayler et al., 1970; Rousseau et al., 1987; Driedzic and Gesser, 1988; Tibbits et al., 1991; Hove-Madsen, 1992; Keen et al., 1994; Gesser, 1996; Vornanen, 1996; Bers, 2001) demonstrated that this ryanodine concentration reduces the functional importance of SR for the E–C coupling. When applied, ryanodine was added to the medium at least 40 min before the diastolic pauses. In further experimental trials, ventricle strip preparations were subjected to increases in imposed contraction frequency from 0.1 Hz until the frequency in which at least 80% of the strips were still able to contract regularly. To analyze the potential contribution of the SR at more physiological frequencies, 10 mM of ryanodine was added to the bath 40 min before alterations in frequency. These protocols were performed at 15 C, 25 C and 35 C. The in vivo physiological heart frequencies presented as the dashed areas in Fig. 3 correspond to those registered by Costa et al. (2002) for L. paradoxa.
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Fig. 1. Effect of ryanodine on the ventricular post-rest force development (Fc —%; mean values7SE) of the South American lungfish, Lepidosiren paradoxa, at different resting intervals and experimental temperatures. (A) 15 C ðn ¼ 10Þ; (B) 25 C (acclimation temperature; n ¼ 10); (C) 35 C ðn ¼ 10Þ: Tension is normalized to the last steady-state twitch prior to rest (0.2 Hz). Asterisks on top of bars indicate a significant ðpo0:05Þ difference in relation to the last contraction force before rest, while asterisks above horizontal lines represent a significant effect ðpo0:05Þ of ryanodine on post-rest force.
Fig. 1 shows the effect of prolonged diastolic pauses on post-rest contraction force at different experimental temperatures with and without addition of ryanodine to the medium. At 15 C, control preparations showed a post-rest potentiation of twitch force (% steady-state force) following rest periods above 60 min, which was completely abolished by ryanodine (Fig. 1A). Conversely, at 25 C and 35 C, the post-rest tension of control
preparations decreased significantly after diastolic pauses of 300 and 60 s, respectively. This post-rest decay of twitch force was further pronounced by the addition of ryanodine after 600 s of rest at acclimation temperature (Fig. 1B) and by diastolic pauses above 60 s at 35 C (Fig. 1C). The relative contribution of the Ca2+ from the SR to force generation as a function of experimental
3. Results
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Fig. 2. Effect of temperature on force development (Fc —%; mean values7SE) of ventricle strips of the South American lungfish, Lepidosiren paradoxa, after 10 min of resting period in the absence (top; n ¼ 10) and presence (bottom; n ¼ 10) of 10 mM ryanodine. Shading indicates ryanodine-sensitive force (SR contribution to force generation).
temperature after a diastolic pause of 600 s is presented in Fig. 2 (dashed area). In spite of the postrest potentiation of twitch force observed at 15 C, pre-treatment with ryanodine resulted in a post-rest force about 21%, 17% and 38% lower than that observed for control preparations at 15 C, 25 C and 35 C, respectively. 3.2. Force–frequency relationship Fig. 3A shows that the positive force–frequency relationship observed in all stimulation frequencies for control preparations at 15 C was shifted to the left (from 0.4 to 0.2 Hz) in response to ryanodine. The dashed area in Fig. 2A indicates that twitch force of control preparations increased significantly at physiological stimulation frequencies (0.2970.01 Hz) at 15 C. In contrast, ventricle strips treated with ryanodine started to contract irregularly above 0.2 Hz, a lower frequency compared to the physiological range. At the acclimation temperature, in spite of the tendency for a positive relationship, especially between 0.4 and 1.0 Hz, control preparations responded to increases in stimulation frequency with changes in force development only at the highest frequency (2.0 Hz), when a significant ðpo0:05Þ decrease in twitch force was observed (Fig. 3B). However, this relationship was shifted downwards by the addition of ryanodine. Additionally, when force–frequency responses were analyzed at frequencies within the in vivo range at 25 C (0.5470.03 Hz; dashed area in Fig. 3B), ryanodine treatment resulted in a decrease in twitch force of about 10%.
The tendency for a positive force–frequency relationship observed at lower temperatures for control preparations disappeared completely at 35 C, becoming negative above 1.8 Hz (Fig. 3C). This negative relationship contrasts with the response to ryanodine, which resulted in a significant decrease in twitch force only at the highest frequency observed for this treatment. Moreover, the response curve was shifted to the left (from 4.0 to 2.0 Hz) after addition of ryanodine. Finally, when the responses were analyzed at in vivo frequencies (1.1770.03 Hz; dashed area in Fig. 2C), ryanodine had no effect on force development at 35 C.
4. Discussion The twitch tension developed by cardiac muscle is affected in large measure by variations in frequency and regularity of stimulation (Hajdu, 1969). While the force– frequency relationship probably involves several mechanisms associated with Ca2+ uptake and storage (Wohlfart and Noble, 1982), the relative importance of individual components of the process may vary among species (Driedzic and Gesser, 1988). A pause in regular stimulation of cardiac muscle produces characteristic changes in contraction during the post-rest recovery. The characterization of the postrest response can provide information on the basic cellular mechanisms involved in the control of cardiac contraction (Mill et al., 1992). These mechanisms include the relative participation of intracellular stores that liberate Ca2+ during contraction and also the activity of NCX at different experimental conditions (Sutko et al., 1986).
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Fig. 3. Effect of increases in stimulation frequency on twitch force (Fc —% of initial values7SE) of ventricle strips of the South American lungfish, Lepidosiren paradoxa, at control preparations (squares—2.5 mM of Ca2+ and without ryanodine) and after the pretreatment with 10 mM of ryanodine (triangles) at (A) 15 C (n ¼ 10), (B) 25 C (n ¼ 10) and (C) 35 C (n ¼ 10). For each treatment, open symbols indicate a significant difference ðpo0:05Þ in relation to the Fc observed initially (at 0.1 Hz) and the asterisks denote a difference between the two treatments (control and ryanodine) in the same frequency ðpo0; 05Þ: The dashed area indicates the heart frequency measured in vivo at each temperature by Costa et al. (2002).
During the pause, Ca2+ can be pumped out of the cell by the SL Ca2+-pump and/or by the NCX and can also be stored at intracellular sites such as the SR (Mill et al., 1992). The net result is a variable amount of Ca2+ activator stored within the cell that can be liberated upon post-rest contraction (Bers et al., 1993; HoveMadsen et al., 2000). If the amount of intracellular Ca2+ increases, a post-rest potentiation of twitch force is observed, while a post-rest decay results from a more
pronounced Ca2+ efflux from the cell (Bers, 2001). Since SL Ca2+-pump does not seem to play a central role in Ca2+ management in most fish due to its very low Ca2+-transporting capacity (Tibbits et al., 1991), the two main mechanisms that influence this process are the SR and NCX activities. Several mammalian hearts present a post-rest potentiation of twitch force in response to short pauses, which decreases as the rest period is increased (Vassalo and
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Mill, 1988), while in rat myocardium this post-rest potentiation is maintained even after long pauses (Vassalo et al., 1990). This potentiation of twitch force has been related to a greater accumulation of Ca2+ at the higher priming frequencies, which, in turn, is released during the test beat (Edman and Johannsson, 1976; Rumberger and Reichel, 1972). Conversely, most fish exhibit a minimal post-rest potentiation of force (Driedzic and Gesser, 1988), suggesting a less developed SR. Moreover, ryanodine has no effect on the development of post-rest potentiation in most teleost species (Driedzic and Gesser, 1988). An exception of the teleost pattern is the rainbow trout heart, in which the SR is anatomically much more developed (Santer, 1985) and the ventricle strips show a post-rest potentiation of force that is strongly reduced by ryanodine, especially at high temperatures (HoveMadsen and Gesser, 1989; Hove-Madsen, 1992; M^llerNielsen and Gesser, 1992). Despite this, ryanodine has no effect on force development when physiological temperatures are considered (Keen et al., 1994; Gesser, 1996; Shiels et al., 1998). Our results differ considerably from those previously reported, since ryanodine reduced significantly the postrest force of lungfish ventricle strips not only at 25 C, a common temperature faced by the species in its habitat, but also in a wider temperature range (from 15 C to 35 C, Figs. 1 and 2). Another interesting finding was the post-rest decay of twitch force at 25 C and 35 C as the resting period was increased to 300 and 600 s (Figs. 1B and C). A post-rest Ca2+ depletion has also been consistently observed in rabbit ventricle (Ferraz et al., 2001), suggesting an increased Ca2+ efflux from the cell as resting period is prolonged. In lungfish, a temperature-dependent increase of NCX activity may account for this response. This result contrasts with those described for most fish species, since in these animals the NCX presents relative temperature insensitivity (Xue et al., 1999). This adaptative response to cold allows the animal to maintain the cardiac performance at low temperatures when the SR does not seem to be functional. The NCX temperature-insensitivity was only described for temperate fish (Bersohn et al., 1991; Tibbits et al., 1992b; Marengo et al., 1997), but one could expect a pronounced temperature-sensitivity of this Ca2+transporting mechanism in tropical species as a strategy to face higher temperatures. When physiological stimulation frequencies are considered, the temperature sensitivity of the NCX would allow the species to increase its cardiac performance as temperature is augmented even without a significant increase in SR activity (Fig. 2). This fact could partially explain the positive inotropic response observed by Costa et al. (2002) for L. paradoxa ventricle strips when temperature was acutely increased from 25 C to 35 C. However,
more specific inotropic interventions are needed to fully explain the effect of temperature on the lungfish NCX activity. An increase in stimulation frequency, within the physiological range, increases the myocardium force of contraction in most mammalian species (Koch-Weser and Blinks, 1963), turtles and frogs (Driedzic and Gesser, 1985) and also in elasmobranchs (Maylie et al., 1979; Driedzic and Gesser, 1988). In contrast, such increases in stimulation frequency result in a stepwise decrease in developed tension in rats and mice myocardium at physiological frequencies (Benfonaro, 1958; Stemmer and Akera, 1986) in spite of their highly developed SR network (Bers, 1991). Similarly, a negative force–frequency relationship was reported for most temperate (Driedzic and Gesser, 1985, 1988; Hove-Madsen and Gesser, 1989; Vornanen, 1989; Bailey and Driedzic, 1990; Hove-Madsen, 1992; Shiels and Farrell, 1997) and tropical (Rantin et al., 1998; Costa et al., 2000) teleost fish. However, contrasting to rat and mouse, the SR of fish generally does not contribute significantly to tension development at in vivo stimulation frequencies and temperatures. Even though, species such as tunas (Keen et al., 1992; Shiels et al., 1999), the Atlantic cod (Driedzic and Gesser, 1985), and mackerels (Driedzic and Gesser, 1988; Shiels and Farrell, 2000) did not show this negative force– frequency response. In tunas, the positive force– frequency relationship, associated with a pronounced effect of ryanodine in force development by the atrial (Shiels et al., 1999) and ventricle strips (Tibbits, 1996), suggests a higher reliance on SR Ca2+ for contraction over its physiological temperatures and heart rates. A negative force–frequency relationship was also observed in the present study for L. paradoxa ventricle strips at 25 C and 35 C when high stimulation frequencies were achieved. However, this negative staircase was not observed when the interval comprehended between 0.1 Hz and that recorded in vivo was considered at each experimental temperature (dashed area in Fig. 3), suggesting an absence of a frequency-dependent decrease in intracellular Ca2+ concentration at these conditions. This finding suggests that L. paradoxa possesses an efficient ventricular Ca2+-transporting mechanism, which would allow an adequate delivery of Ca2+ to the myofilaments even after the temperature and/or frequency-induced curtailment of the time to peak force at physiological conditions. The main Ca2+-transporting mechanisms responsible for Ca2+ management at each temperature were assessed by the use of ryanodine (to block the SR Ca2+-release channels), as discussed below. At the highest temperature (35 C), and in vivo stimulation range (dashed area in Fig. 3C), the ryanodine application had no effect on tension development. This result, associated with the absence of a
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decrease in twitch force when this stimulation frequencies were achieved for control preparations, suggests that the apparent enhance in NCX activity during increases in temperature (as indicated by the post-rest decay of twitch force at higher temperatures in Figs. 1B and C) can assure an appropriate inotropism at physiological frequencies, even without the SR participation at 35 C. The positive force–frequency relationship observed for control preparations at the lowest temperature corroborates the temperature-dependency of the NCX, indicating a net increase of intracellular Ca2+ accumulation as temperature is decreased. Moreover, it can be proposed that at 15 C the SR plays a functional role in the Ca2+ management at the frequencies observed in vivo, since ryanodine shifted the force–frequency relationship to the left (Fig. 3A). Moreover, this organelle also seems to play some role on Ca2+ management at in vivo frequencies and acclimation temperature (Fig. 3B), as ryanodine reduced tension development in almost 10%. On the other hand, at 35 C ryanodine did not reduce either the contractile force or the maximal frequency to values bellow the in vivo range. This implies that the SR, in spite of being potentially functional at 35 C (Fig. 1C), probably does not play a role in Ca2+ management at in vivo condition. Taken together, the present results suggest a low Ca2+-cycling capacity of the ventricular SR of L. paradoxa, implying that this organelle does not play a significant role at high heart rates, in spite of its high anatomical development (Hochachka and Hulbert, 1978) and apparent functionality at lower temperatures and/or lower stimulation frequencies. Therefore, as temperature decreases, with a concomitant temperature-dependent decrease in chronotropism, the SR plays a central role in Ca2+ regulation, compensating the apparent temperature-dependent decrease in NCX activity. These results contrast with those described for the ventricle strips of most fish already studied (Keen et al., 1994; Thomas et al., 1996; Shiels and Farrell, 1997; Shiels et al., 1998) in which ryanodine does not change significantly the force development when physiological frequencies and/or temperatures are considered.
Acknowledgements The authors would like to thank the field technician Mr. Nelson Santos Alves Matos for the collection and maintenance of the lungfish. We are also thankful to Dr. Jay A. Nelson (Towson University, MD, USA) for revising the text. This study was supported by FAPESP (Ph.D. fellowship and grant to Monica J. Costa, No. 98/ 11846-6). All the experiments were performed complying with the Brazilian laws.
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