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Temperature effect on contractile activity of the Ambystoma dumerilii heart previously treated with isoproterenol
Comparative Biochemistry and Physiology, Part A 147 (2007) 743 – 749 www.elsevier.com/locate/cbpa
Temperature effect on contractile activity of the Ambystoma dumerilii heart previously treated with isoproterenol ☆ A. Cano-Martínez ⁎, A. Vargas-González, V. Guarner-Lans Departamento de Fisiología, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano # 1, Colonia Sección XVI, Tlalpan, México D.F. 14080, Mexico Received 31 March 2006; received in revised form 20 October 2006; accepted 22 October 2006 Available online 27 October 2006
Abstract The spontaneous heart rate (HR) and ventricular (V) and atrium (A) tensions (T) were evaluated through isolated organ assays at different temperatures in hearts from Ambystoma dumerilii control and treated with isoproterenol (ISO) [(150 mg/kg i.p. each 24 h, for 3 days)] on days 1, 5, 30 and 90 after ISO. In control hearts, the HR increased and the T decreased when temperature was augmented. One day after ISO the HR (43– 24%) and T (50–25%) decreased with respect to control, between 8 and 24 °C. Five, 30 and 90 days after ISO, HR showed a gradual recovery with similar effect when the temperature was changed; but the AT increased and VT decreased at temperatures between 8 and 12 °C and were only recovered at temperatures above 12 °C. Our results indicate that the HR recovers after ISO in A. dumerilii independently of temperature. The recovery of AT and VT is similar to HR at temperatures higher than 12 °C and the increases in VT could be compensating the decrease in VT caused by ISO, at temperatures lower than 12 °C. The changes in heart contractile activity of A. dumerilii after insult show the thermic plasticity that is observed in ectothermic vertebrates. © 2006 Elsevier Inc. All rights reserved. Keywords: Amphibian; Heart activity; Isoproterenol; Temperature
1. Introduction In ectothermic animals, such as amphibians, body temperature varies with environmental temperature and this results in a change in metabolic and cardiac activity (Burggren et al., 1997). An increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults, in order to meet the oxygen demand of body tissues (Pelster, 1999). There is evidence demonstrating that a linkage exists ☆
This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. Tel.: +52 55 73 29 11; fax: +52 55730926. E-mail addresses: [email protected], [email protected] (A. Cano-Martínez). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.10.030
between metabolism and cardiac activity in adult vertebrates, which represents the main drive for adaptations and adjustment of cardiac activity to changing environmental conditions, but it is not established in embryonic and early larval stages of fishes and amphibians (Schonweger et al., 2000; Jacob et al., 2002; Pelster, 2003). Therefore, when a depression of cardiac activity occurs, in the embryonic or larval heart, it is most likely a direct effect of oxygen deficiency on cardiac myocytes; and the regulated cardiovascular response to hypoxia appears only in late stages of development becoming similar to that found in adult organisms (Pelster, 1999). Variation of HR is influenced by numerous factors, including temperature which affects the intrinsic pacemaker rate, as well as the adrenergic and cholinergic modulations (Lillywhite et al., 1999). Cardiac ventricular pressure overload related to adrenergic stimulation in mammals produces several instances of myocardial hypertrophy in which an increase in collagen content, and interstitial fibrosis have been observed (Weber et al., 1990). Isoproterenol (ISO), is a β-adrenergic agonist acting as a powerful cardio-accelerator that increases heart rate and blood
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pressure in different species when administered systemically. The heart overstimulation by ISO administration in endotherms (Rona, 1985; Benjamin et al., 1989) and ectotherms (Poupa and Carlsten, 1970; Carlsten et al., 1982; Chiu and Chu, 1989; Ostadal et al., 1995) produces cardiac hypertrophy, myocyte necrosis, and interstitial fibrosis like that observed in infarcted human heart. Different evidence from mammals has suggested that apoptosis could be mediating the injury to cardiac muscle by β-adrenergic receptor stimulation (Shizukuda and Buttrick, 2001; Leone et al., 2002; Li et al., 2006) in correlation with oxidative stress (Balta et al., 1999; Narang et al., 2005; Ishizawa et al., 2006; Yogeeta et al., 2006) and Ca+2i increases (DiazMunoz et al., 2006). Considering that the heart contractile activity can be altered after damage with ISO and that, in isolated heart assays, the pacemaker or the contractility of the cardiac myocytes may be subjected to direct influence of temperature, with a poor influence of the nervous and hormone systems, in the present work we evaluated the effect of temperature on contractile activity of isolated hearts from an ectothermic vertebrate, Ambystoma dumerilii control and at different times after ISO administration, including the injury and recovery periods. 2. Materials and methods 2.1. Animals The present study was carried out with adult A. dumerilii (12 months old) that had been bred in captivity by the “Estación Biológica para el Estudio Integral y Aprovechamiento Sustentable del Achoque (Ambystoma dumerilii) Jimbani Erandi” at the Monastery of Ntra. Sra. María Inmaculada de la Salud of Dominic Monks in Pátzcuaro, Michoacán, México, with the SEMARNAT environment permission MDOSY1606611. The animals were transported to our laboratory in Mexico City, by the beginning of spring, where they were kept in fresh water at room temperature with a natural light/dark cycle. The experiments were started 2 weeks after the arrival of the animals to the laboratory, and when the reproductive period for A. dumerilii is off. Food before and during experiments consisted of live fish ad libitum. Care and handling of animals were carried out according to the principles of laboratory animal care (NIH publication No. 86-23, revised 1985). 2.2. Induction of myocardial injury The animals were randomly assigned to 4 experimental or 4 control groups (A, B, C, D) (n = 8 per group), Table 1. Each experimental organism received 150 mg/kg b.w., i.p. of ISO (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 100 μL of amphibian Ringer (NaCl 119.7, KCl 5.6 and CaCl2 1.63 mM, pH 7.6), each, 24 h for three days. The animals of the control group received the equivalent volume of Ringer solution; the administration of ISO to groups A, B, C and D were begun on consecutive days to assure the sacrifice of no more that 4 animals for recording on experiment days (Table 1). The general activity and swimming were recorded during 1 h after administration.
2.3. Sacrifice and in vitro recording of cardiac activity The animals were sacrificed by decapitation 1, 5, 30 and 90 days after the last ISO or Ringer administration according to Table 1. The procedure considered the injection and recording in 4 consecutive days, starting the injection for each recording time in the first week of experiment. In this way 2 ISO and 2 Ringer randomly selected animals were recorded each day as indicated in Table 1 between days 4 and 97 of the experiment (3 months and 1 week). The animals on each control and experimental group (n = 8) (1, 5, 30, 90 days after last ISO) were recorded during 4 consecutive days (Table 1). The heart was isolated by thoracotomy and immediately placed in a chamber with amphibian Ringer (concentrations in mM) (NaCl 111, KCl 1.9, NaHCO3 2.4, NaH2PO4 0.07 and CaCl2 0.8, pH 7.8). The atrium was attached to the bottom of the chamber with surgical thread # 6 and to a tension transducer (model FTO3, Grass), connected to polygraph (model 79D, Grass) by the ventricular apex. A passive tension of 50 to 80 mg was applied with an initial 5 min for recording stabilization. We
Table 1 Classification of groups of Ambystoma dumerilii for days of ISO administration and days of sacrifice and recording after ISO Days of experiment Ringer/ISO administration
1 2 3 Day of sacrifice and recording for group 1 day after last injection 4 5 6 7 Day of sacrifice and recording for group 5 days after last injection 8 9 10 11 Day of sacrifice and recording for group 30 days after last injection 34 35 36 37 Day of sacrifice and recording for group 90 days after last injection 94 95 96 97
Group A (8C, 8E)
B (8C, 8E)
C (8C, 8E)
D (8C, 8E)
Day Day Day C (n)
1 2 3 E (n)
Day 2 Day 3 Day 4 C E (n) (n)
Day 3 Day 4 Day 5 C E (n) (n)
Day Day Day C (n)
4 5 6 E (n)
(2)
(2)
(2) C (n)
(2) E (n)
(2) C (n)
(2) E (n)
(2) C (n)
(2) E (n)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C = control, E = experimental (n = number of animals for each day of recording) from each condition.
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3. Results 3.1. Effect of isoproterenol on A. dumerilii After the administration of ISO, the animals adopted a floating position and inhaled “mouthfuls” of air from the surface for a period of 30–45 min. After this time, no other relevant changes were observed, in comparison with control animals. After day 1 of the first injection, a red color in the water of animals treated with ISO appeared, which increased in intensity with the next injections. An extensive skin vasodilation was also observed. Similar observations for skin vasodilation in toads (Burggren and Vitalis, 2005) and for water color had been previously reported for anuran amphibians and interpreted as the presence of ISO oxidized metabolites (Carlsten and Poupa, 1977). 3.2. Effect of temperature on contractile activity of isolated adult hearts
Fig. 1. In vitro effect of temperature on heart rate (HR) (a) and tension (b) of heart from Ambystoma dumerilii. Temperature was increased degree by degree from 6 to 24 °C in the incubation chamber, recording 10 min in each case. Notice that with the temperature increase, HR increased while atrium tension (AT) and ventricular tension (VT) decreased. The insert in the right side of (a) shows a representative trace of the spontaneous contractile activity of the heart, and corresponds to a record at 16 ± 2 °C in the incubation chamber. Values are means ± S.E.M; n = 8.
3.2.1. Control hearts In the heart of the control animals no evidence of injury was found. The insert of Fig. 1a shows a representative trace of the spontaneous contractile activity of the heart of A. dumerilii in the control group. In control hearts, an increase in HR (Fig. 1a) and a decrease in AT and VT (Fig. 1b) were observed as a response to increases in the temperature. The changes in the atrium were more evident. 3.2.2. Experimental hearts In hearts of animals treated with ISO, atrium congestion, ruggedness and a red color in the surface of left ventricle were observed, mainly 1 to 5 days after ISO. These alterations diminished 30 and 90 days after treatment, when the superficial characteristics of the heart were similar to the control.
recorded the cardiac activity during 10 min at each temperature value from 6 to 24 °C, with a minimum of 5 min for stabilization between each recording. The changes of temperature were applied always from the lowest to the highest temperature, and were controlled by a regulator in the bath. The activity was recorded during all times of the experiment. The recordings in the polygraph paper showed the spontaneous activity of the AT and VT as two different and integrated spikes, a long spike for AT and a short spike for VT (insert in Fig. 1a). It was possible to measure the HR by counting the number of spikes and AT and VT by measuring the spike amplitude. All spikes for each temperature values were counted and measured, except those of stabilization period. 2.4. Statistical analysis Values (mean ± S.E.M) are presented for cardiac activity variables (HR, AT and VT) at each temperature (Fig. 1). After ISO, comparisons between the five temperature groups were performed using a one-way ANOVA, and multiple comparisons were performed using Student–Newman–Keuls (SNK) test and in all instances P ≤ 0.05 was used as the level of significance.
Fig. 2. Effect of temperature on heart rate (HR) of isolated heart from Ambystoma dumerilii control (C) and 1, 5, 30, and 90 days (d) after myocardial damage induced by ISO. The increases in the temperature between 8 and 24 °C increased the HR in C and in injured hearts (1 d after ISO) and during the restoration of contractile activity (5, 30 and 90 d after ISO). Values are means ± S.E.M. Significant differences (P ≤ 0.05) between times after ISO administration for HR at each recorded temperature are indicated by dissimilar letters. After one way ANOVA and post-hoc SNK test.
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3.2.3. Heart rate The effect of temperature on HR after the lesion with ISO and its comparison with the control are shown in Fig. 2. One day after the last ISO administration, the HR was 57–76% of control between 8 and 24 °C. Five days after ISO, HR corresponded to 70 to 94% of the control values in the temperature range indicated previously. Thirty days after ISO, the HR was from 64 to 101% of the control. At 90 days after ISO, the values of HR were similar to those in the control, when the recording was performed at 12, 16, 20 and 24 °C.
Table 2 Comparison of heart rate between anuran amphibian and Ambystoma dumerilii
3.2.4. Atrium and ventricular tension One day after ISO, the values for AT were 45 to 78% (Fig. 3a) and for VT were 49 to 78% (Fig. 3b) of the control values in a temperature range from 8 to 24 °C. When the recording was performed at temperatures higher than 12 °C, the AT and VT values were recovered gradually at 5, 30 and 90 days after ISO
until they reached the control values on day 90. But when the AT was recorded at 12 °C or lower temperature the values were 22 and 32% higher than in the control; while the VT values remained in 50% (8 °C) and 80% (12 °C) of control values at days 5, 30 and 90 after last ISO administration.
Rana catesbeiana (Herman et al., 1986) Rana tigrina (Chiu and Chu, 1989) A. dumerilii (Present study)
6 °C
8 °C
12 °C
14 °C
22 °C
24 °C
–
–
11 ± 1
–
34 ± 2
–
–
–
22 ± 1
–
39 ± 2
6±1
7±1
9±1
28 ± 2
31 ± 2
4.5 ± 0.3 5±1
4. Discussion 4.1. Effect of temperature on the heart contractile activity in A. dumerilii
Fig. 3. Effect of temperature on atrium tension (AT) (a) and ventricular tension (VT) (b) in isolated heart from Ambystoma dumerilii control (C) and 1, 5, 30, and 90 days (d) after ISO administration. In all temperatures recorded, 1 d after ISO the AT and VT were decreased and a gradual recovery was observed between 5 and 90 after ISO. Except at temperatures below 12 °C where the values of AT were higher and those of VT lower in comparison with C. Values are means ± S.E.M. Significant differences (P ≤ 0.05) between times after ISO administration for atrium tension (AT) and ventricular tension (VT) at each recorded temperature are indicated by dissimilar letters. After one way ANOVA and post-hoc SNK test.
In contrast to previous studies carried out in anuran amphibians, where only the temperature effect on the atrium contractile activity was reported (Ask, 1983; Chiu and Chu, 1989), in the present study we simultaneously evaluated the contractile activity developed by the atrium and ventricle (insert in Fig. 1a). In our traces the evaluation of HR and both AT and VT was possible. The values of HR found in the present study in control hearts are similar to those reported for the HR of other ectothermic vertebrates (Farrell, 1991; Fritsche, 1997; Lillywhite et al., 1999). However, our values tend to be lower than those reported for Rana tigrina (Chiu and Chu, 1989) and R. catesbeiana (Herman et al., 1986) in vitro (Herman et al., 1986; Chiu and Chu, 1989) and in vivo preparations (Herman et al., 1986) recorded at temperatures from 6 to 24 °C (Table 2). At temperatures between 6 and 14 °C, the values in A. dumerilii are 57% of the values reported for R. catesbeiana and R. tigrina (Chiu and Chu, 1989), between 22 and 24 °C, the values of A. dumerilii are 81% of the values recorded in frogs. The differences on HR values could be related to the mean body mass difference between R. catesbeiana (127 ± 5.4 g) (Broch et al., 2002) and R. tigrina (250–650 g) (Hoque and Saidapur, 1994) in comparison with A. dumerilii (51 ± 3 g) in the present study. Even more, we could speculate that the differences are related with the fact that anura and caudate are different phylogenetic groups or with the fact that A. dumerilii is neotenic, aspects interesting to tackle in future studies. Independent of the cause of the differences in the HR between these species, the data suggest that the variations in the HR between anuran and neotenic urodele amphibians at lower temperatures are more pronounced (Table 2). The changes of HR in opposite sense to AT and VT, when temperature was increased (Fig. 1) are consistent with the data reported in the literature for segments of cardiac muscle of ectothermic vertebrates (Ask, 1983; Burggren et al., 1997). These
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series of results reflect the metabolic capacity of ectotherms to adjust to environmental changes. When temperature is low, metabolic demands decrease and the heart can pump higher blood volume, at lower frequency. In contrast, when the temperature increases and oxygen availability decreases (Dejours, 1989; Pelster, 1999), the heart meets the metabolic demand by increasing the HR and decreasing the pumped blood volume per beat through less tension development in order to maintain cardiac output (Pelster, 1999). The rate of increase in the HR with temperature elevations limits the myocardial ability to develop tension through a decrease in oxygen availability, which is crucial to muscular work (Farrell, 1991). This response can be interpreted as an evolutionary constraint in ectothermic organisms to develop fast heart contraction rates (Burggren et al., 1997), which is reflected in the fact that the heart of birds and mammals can beat at considerably higher frequencies than those observed in most ectothermic animals (no more that 120 beats/min) at the same temperatures (Lillywhite et al., 1999). 4.2. Heart injury with isoproterenol in A. dumerilii The administration of ISO produces cardiomyocyte necrosis, interstitial fibrosis and cardiac hypertrophy in the heart of mammals (Rona, 1985; Benjamin et al., 1989) reptiles (Ostadal et al., 1995) and anuran amphibians (Poupa and Carlsten, 1970; Carlsten et al., 1982). Moreover, we have obtained evidence showing the appearance of interstitial fibrosis produced by ISO in the urodele amphibian A. mexicanum (data no shown). In the present study we found that the proportion of heart activity of A. dumerilii was dependent of temperature, even when the contractile activity of isolated heart is recorded after injury with ISO, resulting in a reduction in HR (Fig. 2), AT and VT values (Fig. 3). This tendency was observed even after 1 day of damage when the HR values increased, AT and VT values decreased, with the increase in temperature. The damage effect was detectable in the isolated heart at temperatures from 8 to 24 °C. The damage in the heart of A. dumerilii was reflected in HR, AT, VT for only a short period of time after ISO administration, and from 5 to 90 days a gradual recovery was observed, as has also been observed in anuran amphibians (Carlsten and Poupa, 1977). This suggests that the mechanism of injury by ISO in anuran and urodele amphibians could be similar. The mechanism by which ISO damages the amphibian heart is unknown; however, considering the actual evidence we suggest that the cardiac sensitivity to ISO in ectotherms is lower than the one observed in endothermic vertebrates (Ostadal et al., 1995). To produce an injury equivalent to the one observed in endotherms, a higher dose of ISO is necessary (Carlsten and Poupa, 1977; Ostadal et al., 1995). Furthermore to β-adrenergic receptor activation in cardiomyocytes, the binding of ISO to β-adrenergic receptors in the coronary arteries has been proposed as the principal way by which heart damage is produced in mammals (Trivella et al., 1990). However, the hearts of A. dumerilii and other amphibians lack coronary arteries (Ghiara et al., 1984; Farrell, 1992; Adler et al., 2004). The trabeculed structure of the ventricle of A. dumerilii could allow an efficient nutrient diffusion
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which makes coronary circulation unnecessary (Ghiara et al., 1984; Farrell, 1992). This may be correlated with a lower oxygen demand in ectothermic than in endothermic vertebrates, being the latter characterized by higher cardiac output (Lillywhite et al., 1999). An alternative explanation for ISO-damage in amphibians is based on reports supporting the presence of β-adrenergic receptors in the ventricular myocardium of anuran amphibians (Chiu and Chu, 1989; Herman et al., 1996; Jurevicius and Fischmeister, 1997) and in the urodele amphibian, A. mexicanum (Cano-Martínez et al., 2004). We suggest that the cardiac damage by ISO in A. dumerilii, could be due to its binding on β-adrenergic receptors located directly in the myocardium. The linking between the myocardial damage and the adrenergic stimulation is unclear. For mammals, there is evidence of apoptosis mediated injury to cardiac muscle by β-adrenergic receptor stimulation (Shizukuda and Buttrick, 2001; Leone et al., 2002; Li et al., 2006) in correlation with oxidative stress (Balta et al., 1999; Narang et al., 2005; Ishizawa et al., 2006; Yogeeta et al., 2006) and Ca+2i increases (Diaz-Munoz et al., 2006). It is probable that a similar mechanism is responsible for the ISO damage in the A. dumerilii heart; this may be demonstrated in future studies. 4.3. Effect of temperature in the contractile activity of the isolated heart after damage with ISO The increase of HR parallel to a decrease in AT and VT values, when the temperature bath increased was consistent for hearts from 5, 30 and 90 days after the last ISO administration when the records were performed from 12 °C to 24 °C (Figs. 2 and 3). In contrast, when the temperature bath was 12 °C or lower this relation was lost. This suggests that the environmental changes in temperature had no effect in the heart contractile activity recovery when the animals are in environmental temperatures above 12 °C, since this response was similar to that in the control hearts. But at temperatures lower than 12 °C, the heart response could be altered and we cannot discard the integrated effect of ISO and low temperatures. These results are important to be considered when the in vitro studies are performed. The biological significance of changes in heart activity at lower temperatures during recovery period in our study is unknown, but we can suggest that the adjustment between HR, AT and VT, could be relevant for A. dumerilii when in its natural habitat the mean temperatures are less than 12 °C in the presence of some cardiotoxic agents. This possibility cannot be discarded because recent surveys of aquatic habitats suggest that organic wastewater contaminants (OWCs) may be common in aquatic ecosystems (Smith and Burgett, 2005) as could be the case in Pátzcuaro Lake. Therefore, it is interesting to consider the ecological evaluation of the habitat of A. dumerilii and other Ambystoma of North America. This possibility has been considered as a probable explanation to frog malformations related with water pollution, when studying the lethal and sublethal effects of three OWCs (acetaminophen, caffeine, and triclosan) on American toad (Bufo americanus) tadpoles (Smith and Burgett, 2005).
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If the possible adjustment in AT and VT after an hypothetic heart damage in the natural habitat of A. dumerilii occurs, we can suggest that this adjustment could form part of mechanisms that preserve the life of animals by blocking the consecutive damage in other tissues as the brain after heart injury, as has been found in comatose humans (Felberg et al., 2001; Holzer et al., 2006) and dogs (Wu et al., 2006), surviving a cardiac arrest. Therapeutic hypothermia improved short-term neurological recovery and survival when compared with standard treatment (Felberg et al., 2001; Holzer et al., 2006; Wu et al., 2006). The possible protection from damage may be related with cell intracellular Ca2+ release in the heart and/or other tissues, and supported by the phenomena of Ca2+-paradox occurring in isolated frog hearts, and in frog and mouse ventricle strips, during Ca2+-free perfusion (phase I), when PCa of the sarcolemma is increased. Low temperature (4 °C) provides complete protection against these molecular changes in frog and a partial protection in mouse tissue. The re-introduction of extracellular Ca2+ (phase II) causes typical Ca2+-triggered damage which is markedly reduced at 4 °C in both species (Rudge and Duncan, 1984). Other studies considering loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release to define cell damage, found that after 30 min of Ca2+-free perfusion at 37 °C, ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase and the total amount of protein lost were increased, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase (Touraki and Beis, 1990). In summary, the present work shows the first evidence of the temperature effect on cardiac contractile activity in A. dumerilii in controls and after ISO. Our results indicate that the heart of A. dumerilii has a gradual functional restoration after ISO damage. HR recovered at temperatures between 12 and 24 °C and AT and VT at temperatures between 16 and 24 °C. However, at temperatures lower than 12 °C the temperature effect observed in the control hearts was different from that of animals previously treated with ISO. This indicated that the injured and recovering heart responds depending on the thermic plasticity observed in the ectotherms. Acknowledgements We thank Esteban Prado-Zayago for his help in the obtention of the animals. This work was supported by the grant 00-303 to AC-M from Instituto Nacional de Cardiología “Ignacio Chávez". References Adler, A., Huang, H., Wang, H., Wang, Z., Conetta, J., Levee, E., Zhang, X., Hitze, H.T., 2004. Endocardial endothelium in the avascular frog heart: role for diffusion of ON in control of cardiac O2 consumption. Am. J. Physiol, Heart Circ. Physiol. 287, 14–21. Ask, J.A., 1983. Comparative aspects of adrenergic receptors in the hearts of lower vertebrates. Comp. Biochem. Physiol. A 76, 543–552. Balta, N., Stoian, I., Petec, C., Petec, G., 1999. Decreased SOD activity and increased nitrates level in rat heart with left ventricular hypertrophy induced by isoproterenol. Rom. J. Physiol. 36, 175–182.
Benjamin, I.J., Jalil, J.E., Tan, L.B., Cho, K., Weber, K.T., Clark, W.A., 1989. Isoproterenol-induced myocardial fibrosis in relation to myocyte necrosis. Circ. Res. 65, 657–670. Broch, L., Morales, R.D., Sandoval, A.V., Hedrick, M.S., 2002. Regulation of the respiratory central pattern generator by chloride-dependent inhibition during development in the bullfrog (Rana catesbeiana). J. Exp. Biol. 205 (Pt8), 1161–1169. Burggren, W.W., Vitalis, T.Z., 2005. The interplay of cutaneous water loss gas exchange and blood flow in the toad, Bufo woodhousei: adaptations in a terrestrially adapted amphibians. J. Exp. Biol. 208 (Pt1), 105–112. Burggren, W.W., Farrell, A.P., Lillywhite, H.B., 1997. Vertebrate cardiovascular systems. In: Dantzler, W.H. (Ed.), Handbook of Physiology. Sec 13. Comparative Physiology, vol. I. 199. American Physiology Society, Bethesda, MD, pp. 215–308. Cano-Martínez, A., Vargas-González, A., Guarner-Lans, V., Prado-Zayago, E., 2004. Isoproterenol-produced damage in amphibian heart could be mediated by adrenergic receptors located in the heart muscle. Proc. West. Pharmacol. Soc. 47, 63–66. Carlsten, A., Poupa, P., 1977. Effect of time on the response to cyanide anoxia of frog heart damaged by isoproterenol. Acta Pharmacol. Toxicol. (Copenh) 41, 18–24. Carlsten, A., Ericson, L.E., Poupa, O., 1982. The healing of frog heart lesions induced by isoproterenol injections. Acta Pathol. Microbiol. Immunol. Scand. A 90, 57–65. Chiu, K.W., Chu, J.Y., 1989. Temperature and adrenoceptors in the frog heart. Comp. Biochem. Physiol. C 94, 149–157. Dejours, P., 1989. From comparative physiology of respiration to several problems of environmental adaptations and to evolution. J. Physiol. 410, 1–19. Diaz-Munoz, M., Alvarez-Perez, M.A., Yanez, L., Vidrio, S., Martinez, L., Rosas, G., Yanez, M., Ramirez, S., de Sanchez, V.C., 2006. Correlation between oxidative stress and alteration of intracellular calcium handling in isoproterenol-induced myocardial infarction. Mol. Cell. Biochem. 289, 125–136. Farrell, A.P., 1991. Cardiac scope in lower vertebrates. Can. J. Zool. 69, 1981–1984. Farrell, A.P., 1992. Cardiac output: regulation and limitations. In: Bicudo, E. (Ed.), The Vertebrate Gas Transport Cascade: Adaptations to Environment and Mode of Life. CRC Press, Boca Raton, pp. 208–214. Felberg, R.A., Krieger, D.W., Chuang, R., Persse, D.E., Burgin, W.S., Hickenbottom, S.L., Morgenstern, L.B., Rosales, O., Grotta, J.C., 2001. Hypothermia after cardiac arrest: feasibility and safety of an external cooling protocol. Circulation 104, 1799–1804. Fritsche, R., 1997. Ontogeny of cardiovascular control in amphibians. Am. Zool. 37, 23–30. Ghiara, P., Parente, L., Piomelli, D., 1984. The cyclo-oxygenase pathway in the avascular heart of the frog, Rana esculenta L. Gen. Pharmacol. 15, 309–313. Herman, C.A., Robleto, D.O., Mata, P.L., Heller, R.S., 1986. Cardiovascular responses to catecholamines at 12 Celsius in American bullfrog (Rana catesbeiana). J. Exp. Zool. 240, 17–23. Herman, C.A., Luczy, G., Wikberg, J.E., Uhlén, S., 1996. Characterization of adrenoceptor types and subtypes in American bullfrogs acclimated to warm or cold temperature. Gen. Comp. Endocrinol. 104, 168–178. Holzer, M., Mullner, M., Sterz, F., Robak, O., Kliegel, A., Losert, H., Sodeck, G., Uray, T., Zeiner, A., Laggner, A.N., 2006. Efficacy and safety of endovascular cooling after cardiac arrest: cohort study and Bayesian approach. Stroke 37, 1792–1797. Hoque, B., Saidapur, S.K., 1994. Dynamics of oogenesis in the tropical anuran Rana tigrina (Amphibia: Ranidae) with special reference to vitellogenic cycles in wild-caught and captive frogs. J. Biosci. 19, 339–352. Ishizawa, M., Mizushige, K., Noma, T., Namba, T., Guo, P., Murakami, K., Tsuji, T., Miyatake, A., Ohmori, K., Kohno, M., 2006. An antioxidant treatment potentially protects myocardial energy metabolism by regulating uncoupling protein 2 expression in a chronic beta-adrenergic stimulation rat model. Life Sci. 15, 2974–2982. Jacob, E., Drexel, D., Schwerte, T., Pelster, B., 2002. Influence of hypoxia and hypoxemia on the development of cardiac activity in zebrafish larvae. Am. J. Physiol., Regul. Integr. Comp. Physiol. 283, 911–917.
A. Cano-Martínez et al. / Comparative Biochemistry and Physiology, Part A 147 (2007) 743–749 Jurevicius, J., Fischmeister, R., 1997. Longitudinal distribution of Na+ and Ca2+ channels and β-adrenoceptors on the sarcolemmal membrane of frog cardiomyocytes. J. Physiol. 503, 471–477. Leone, M., Albanese, J., Martin, C., 2002. Positive inotropic stimulation. Curr. Opin. Crit. Care 8, 395–403. Li, D., Qu, Y., Tao, L., Liu, H., Hu, A., Gao, F., Sharifi-Azad, S., Grunwald, Z., Ma, X.L., Sun, J.Z., 2006. Inhibition of iNOS protects the aging heart against beta-adrenergic receptor. J. Surg. Res. 131, 64–72. Lillywhite, H.B., Zippel, K.C., Farrell, A.P., 1999. Resting and maximal heart rates in ectothermic vertebrates. Comp. Biochem. Physiol. A 124, 369–382. Narang, D., Sood, S., Thomas, M., Dinda, A.K., Maulik, S.K., 2005. Dietary palm olein oil augments cardiac antioxidant enzymes and protects against isoproterenol-induced myocardial necrosis in rats. J. Pharm. Pharmacol. 57, 1445–1451. Ostadal, B., Pelouch, V., Ostadalova, I., Novakova, O., 1995. Structural and biochemical remodeling in catecholamine-induced cardiomyopathy: comparative and ontogenetic aspects. Mol. Cell. Biochem. 147, 83–88. Pelster, B., 1999. Environmental influences on the development of the cardiac system in fish and amphibians. Comp. Biochem. Physiol. A 124, 407–412. Pelster, B., 2003. Developmental plasticity in the cardiovascular system of fish, with special reference to the zebrafish. Comp. Biochem. Physiol. A 133, 547–553. Poupa, O., Carlsten, A., 1970. Isoproterenol-induced cardiac lesions in frog observed in vivo. Can. J. Physiol. Pharmacol. 48, 306–311. Rona, G., 1985. Catecholamine cardiotoxicity. J. Mol. Cell. Cardiol. 17, 291–306. Rudge, M.F., Duncan, C.J., 1984. Comparative studies on the calcium paradox in cardiac muscle: the effect of temperature on the different phases. Comp. Biochem. Physiol. A 79, 393–398.
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Schonweger, G., Schewerte, T., Pelster, B., 2000. Temperature-dependent development cardiac activity in unrestrained larvae of the minnow Phoxinus phoxinus. Am. J. Physiol., Regul. Integr. Comp. Physiol. 279, R1634–R1640. Shizukuda, Y., Buttrick, P.M., 2001. Protein kinase C(epsilon) modulates apoptosis induced by beta-adrenergic stimulation in adult rat ventricular myocytes via extracellular signal-regulated kinase (ERK) activity. J. Mol. Cell. Cardiol. 33, 1791–1803. Smith, G.R., Burgett, A.A., 2005. Effects of three organic wastewater contaminants on American toad, Bufo americanus, tadpoles. Ecotoxicology 14, 477–784. Touraki, M., Beis, I., 1990. Characterization of the calcium paradox in the isolated perfused frog heart: enzymatic, ionic, contractile and electrophysiological studies. J. Comp. Physiol. B 160, 113–118. Trivella, M.G., Broten, T.P., Feigl, E.O., 1990. Beta-receptor subtypes in the canine coronary circulation. Am. J. Physiol. 259, H1575–H1585. Weber, K.T., Janicki, J.S., Pick, R., Capasso, J., Anversa, P., 1990. Myocardial fibrosis and pathologic hypertrophy in the rat with renovascular hypertension. Am. J. Cardiol. 65, 1G–7G. Wu, X., Drabek, T., Kochanek, P.M., Henchir, J., Stezoski, S.W., Stezoski, J., Cochran, K., Garman, R., Tisherman, S.A., 2006. Induction of profound hypothermia for emergency preservation and resuscitation allows intact survival cardiac arrest resulting from prolonged lethal hemorrhage and trauma in dog. Circulation 113, 1974–1982. Yogeeta, S.K., Gnanapragasam, A., Kumar, S.S., Subhashini, R., Sathivel, A., Devaki, T., 2006. Synergistic interactions of ferulic acid with ascorbic acid: its cardioprotective role during isoproterenol induced myocardial infarction in rats. Mol. Cell. Biochem. 283, 139–146.
Temperature effect on contractile activity of the Ambystoma dumerilii heart previously treated with isoproterenol ☆ A. Cano-Martínez ⁎, A. Vargas-González, V. Guarner-Lans Departamento de Fisiología, Instituto Nacional de Cardiología “Ignacio Chávez”, Juan Badiano # 1, Colonia Sección XVI, Tlalpan, México D.F. 14080, Mexico Received 31 March 2006; received in revised form 20 October 2006; accepted 22 October 2006 Available online 27 October 2006
Abstract The spontaneous heart rate (HR) and ventricular (V) and atrium (A) tensions (T) were evaluated through isolated organ assays at different temperatures in hearts from Ambystoma dumerilii control and treated with isoproterenol (ISO) [(150 mg/kg i.p. each 24 h, for 3 days)] on days 1, 5, 30 and 90 after ISO. In control hearts, the HR increased and the T decreased when temperature was augmented. One day after ISO the HR (43– 24%) and T (50–25%) decreased with respect to control, between 8 and 24 °C. Five, 30 and 90 days after ISO, HR showed a gradual recovery with similar effect when the temperature was changed; but the AT increased and VT decreased at temperatures between 8 and 12 °C and were only recovered at temperatures above 12 °C. Our results indicate that the HR recovers after ISO in A. dumerilii independently of temperature. The recovery of AT and VT is similar to HR at temperatures higher than 12 °C and the increases in VT could be compensating the decrease in VT caused by ISO, at temperatures lower than 12 °C. The changes in heart contractile activity of A. dumerilii after insult show the thermic plasticity that is observed in ectothermic vertebrates. © 2006 Elsevier Inc. All rights reserved. Keywords: Amphibian; Heart activity; Isoproterenol; Temperature
1. Introduction In ectothermic animals, such as amphibians, body temperature varies with environmental temperature and this results in a change in metabolic and cardiac activity (Burggren et al., 1997). An increase in temperature typically requires adjustments in cardiac activity because ventilatory and convectional transport of respiratory gases usually are tightly coupled in adults, in order to meet the oxygen demand of body tissues (Pelster, 1999). There is evidence demonstrating that a linkage exists ☆
This paper is part of the 3rd special issue of CBP dedicated to The Face of Latin American Comparative Biochemistry and Physiology organized by Marcelo Hermes-Lima (Brazil) and co-edited by Carlos Navas (Brazil), Rene Beleboni (Brazil), Rodrigo Stabeli (Brazil), Tania Zenteno-Savín (Mexico) and the editors of CBP. This issue is dedicated to the memory of two exceptional men, Peter L. Lutz, one of the pioneers of comparative and integrative physiology, and Cicero Lima, journalist, science lover and Hermes-Lima's dad. ⁎ Corresponding author. Tel.: +52 55 73 29 11; fax: +52 55730926. E-mail addresses: [email protected], [email protected] (A. Cano-Martínez). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.10.030
between metabolism and cardiac activity in adult vertebrates, which represents the main drive for adaptations and adjustment of cardiac activity to changing environmental conditions, but it is not established in embryonic and early larval stages of fishes and amphibians (Schonweger et al., 2000; Jacob et al., 2002; Pelster, 2003). Therefore, when a depression of cardiac activity occurs, in the embryonic or larval heart, it is most likely a direct effect of oxygen deficiency on cardiac myocytes; and the regulated cardiovascular response to hypoxia appears only in late stages of development becoming similar to that found in adult organisms (Pelster, 1999). Variation of HR is influenced by numerous factors, including temperature which affects the intrinsic pacemaker rate, as well as the adrenergic and cholinergic modulations (Lillywhite et al., 1999). Cardiac ventricular pressure overload related to adrenergic stimulation in mammals produces several instances of myocardial hypertrophy in which an increase in collagen content, and interstitial fibrosis have been observed (Weber et al., 1990). Isoproterenol (ISO), is a β-adrenergic agonist acting as a powerful cardio-accelerator that increases heart rate and blood
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pressure in different species when administered systemically. The heart overstimulation by ISO administration in endotherms (Rona, 1985; Benjamin et al., 1989) and ectotherms (Poupa and Carlsten, 1970; Carlsten et al., 1982; Chiu and Chu, 1989; Ostadal et al., 1995) produces cardiac hypertrophy, myocyte necrosis, and interstitial fibrosis like that observed in infarcted human heart. Different evidence from mammals has suggested that apoptosis could be mediating the injury to cardiac muscle by β-adrenergic receptor stimulation (Shizukuda and Buttrick, 2001; Leone et al., 2002; Li et al., 2006) in correlation with oxidative stress (Balta et al., 1999; Narang et al., 2005; Ishizawa et al., 2006; Yogeeta et al., 2006) and Ca+2i increases (DiazMunoz et al., 2006). Considering that the heart contractile activity can be altered after damage with ISO and that, in isolated heart assays, the pacemaker or the contractility of the cardiac myocytes may be subjected to direct influence of temperature, with a poor influence of the nervous and hormone systems, in the present work we evaluated the effect of temperature on contractile activity of isolated hearts from an ectothermic vertebrate, Ambystoma dumerilii control and at different times after ISO administration, including the injury and recovery periods. 2. Materials and methods 2.1. Animals The present study was carried out with adult A. dumerilii (12 months old) that had been bred in captivity by the “Estación Biológica para el Estudio Integral y Aprovechamiento Sustentable del Achoque (Ambystoma dumerilii) Jimbani Erandi” at the Monastery of Ntra. Sra. María Inmaculada de la Salud of Dominic Monks in Pátzcuaro, Michoacán, México, with the SEMARNAT environment permission MDOSY1606611. The animals were transported to our laboratory in Mexico City, by the beginning of spring, where they were kept in fresh water at room temperature with a natural light/dark cycle. The experiments were started 2 weeks after the arrival of the animals to the laboratory, and when the reproductive period for A. dumerilii is off. Food before and during experiments consisted of live fish ad libitum. Care and handling of animals were carried out according to the principles of laboratory animal care (NIH publication No. 86-23, revised 1985). 2.2. Induction of myocardial injury The animals were randomly assigned to 4 experimental or 4 control groups (A, B, C, D) (n = 8 per group), Table 1. Each experimental organism received 150 mg/kg b.w., i.p. of ISO (Sigma-Aldrich, St. Louis, MO, USA), dissolved in 100 μL of amphibian Ringer (NaCl 119.7, KCl 5.6 and CaCl2 1.63 mM, pH 7.6), each, 24 h for three days. The animals of the control group received the equivalent volume of Ringer solution; the administration of ISO to groups A, B, C and D were begun on consecutive days to assure the sacrifice of no more that 4 animals for recording on experiment days (Table 1). The general activity and swimming were recorded during 1 h after administration.
2.3. Sacrifice and in vitro recording of cardiac activity The animals were sacrificed by decapitation 1, 5, 30 and 90 days after the last ISO or Ringer administration according to Table 1. The procedure considered the injection and recording in 4 consecutive days, starting the injection for each recording time in the first week of experiment. In this way 2 ISO and 2 Ringer randomly selected animals were recorded each day as indicated in Table 1 between days 4 and 97 of the experiment (3 months and 1 week). The animals on each control and experimental group (n = 8) (1, 5, 30, 90 days after last ISO) were recorded during 4 consecutive days (Table 1). The heart was isolated by thoracotomy and immediately placed in a chamber with amphibian Ringer (concentrations in mM) (NaCl 111, KCl 1.9, NaHCO3 2.4, NaH2PO4 0.07 and CaCl2 0.8, pH 7.8). The atrium was attached to the bottom of the chamber with surgical thread # 6 and to a tension transducer (model FTO3, Grass), connected to polygraph (model 79D, Grass) by the ventricular apex. A passive tension of 50 to 80 mg was applied with an initial 5 min for recording stabilization. We
Table 1 Classification of groups of Ambystoma dumerilii for days of ISO administration and days of sacrifice and recording after ISO Days of experiment Ringer/ISO administration
1 2 3 Day of sacrifice and recording for group 1 day after last injection 4 5 6 7 Day of sacrifice and recording for group 5 days after last injection 8 9 10 11 Day of sacrifice and recording for group 30 days after last injection 34 35 36 37 Day of sacrifice and recording for group 90 days after last injection 94 95 96 97
Group A (8C, 8E)
B (8C, 8E)
C (8C, 8E)
D (8C, 8E)
Day Day Day C (n)
1 2 3 E (n)
Day 2 Day 3 Day 4 C E (n) (n)
Day 3 Day 4 Day 5 C E (n) (n)
Day Day Day C (n)
4 5 6 E (n)
(2)
(2)
(2) C (n)
(2) E (n)
(2) C (n)
(2) E (n)
(2) C (n)
(2) E (n)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C (n)
E (n)
(2)
(2)
C = control, E = experimental (n = number of animals for each day of recording) from each condition.
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3. Results 3.1. Effect of isoproterenol on A. dumerilii After the administration of ISO, the animals adopted a floating position and inhaled “mouthfuls” of air from the surface for a period of 30–45 min. After this time, no other relevant changes were observed, in comparison with control animals. After day 1 of the first injection, a red color in the water of animals treated with ISO appeared, which increased in intensity with the next injections. An extensive skin vasodilation was also observed. Similar observations for skin vasodilation in toads (Burggren and Vitalis, 2005) and for water color had been previously reported for anuran amphibians and interpreted as the presence of ISO oxidized metabolites (Carlsten and Poupa, 1977). 3.2. Effect of temperature on contractile activity of isolated adult hearts
Fig. 1. In vitro effect of temperature on heart rate (HR) (a) and tension (b) of heart from Ambystoma dumerilii. Temperature was increased degree by degree from 6 to 24 °C in the incubation chamber, recording 10 min in each case. Notice that with the temperature increase, HR increased while atrium tension (AT) and ventricular tension (VT) decreased. The insert in the right side of (a) shows a representative trace of the spontaneous contractile activity of the heart, and corresponds to a record at 16 ± 2 °C in the incubation chamber. Values are means ± S.E.M; n = 8.
3.2.1. Control hearts In the heart of the control animals no evidence of injury was found. The insert of Fig. 1a shows a representative trace of the spontaneous contractile activity of the heart of A. dumerilii in the control group. In control hearts, an increase in HR (Fig. 1a) and a decrease in AT and VT (Fig. 1b) were observed as a response to increases in the temperature. The changes in the atrium were more evident. 3.2.2. Experimental hearts In hearts of animals treated with ISO, atrium congestion, ruggedness and a red color in the surface of left ventricle were observed, mainly 1 to 5 days after ISO. These alterations diminished 30 and 90 days after treatment, when the superficial characteristics of the heart were similar to the control.
recorded the cardiac activity during 10 min at each temperature value from 6 to 24 °C, with a minimum of 5 min for stabilization between each recording. The changes of temperature were applied always from the lowest to the highest temperature, and were controlled by a regulator in the bath. The activity was recorded during all times of the experiment. The recordings in the polygraph paper showed the spontaneous activity of the AT and VT as two different and integrated spikes, a long spike for AT and a short spike for VT (insert in Fig. 1a). It was possible to measure the HR by counting the number of spikes and AT and VT by measuring the spike amplitude. All spikes for each temperature values were counted and measured, except those of stabilization period. 2.4. Statistical analysis Values (mean ± S.E.M) are presented for cardiac activity variables (HR, AT and VT) at each temperature (Fig. 1). After ISO, comparisons between the five temperature groups were performed using a one-way ANOVA, and multiple comparisons were performed using Student–Newman–Keuls (SNK) test and in all instances P ≤ 0.05 was used as the level of significance.
Fig. 2. Effect of temperature on heart rate (HR) of isolated heart from Ambystoma dumerilii control (C) and 1, 5, 30, and 90 days (d) after myocardial damage induced by ISO. The increases in the temperature between 8 and 24 °C increased the HR in C and in injured hearts (1 d after ISO) and during the restoration of contractile activity (5, 30 and 90 d after ISO). Values are means ± S.E.M. Significant differences (P ≤ 0.05) between times after ISO administration for HR at each recorded temperature are indicated by dissimilar letters. After one way ANOVA and post-hoc SNK test.
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3.2.3. Heart rate The effect of temperature on HR after the lesion with ISO and its comparison with the control are shown in Fig. 2. One day after the last ISO administration, the HR was 57–76% of control between 8 and 24 °C. Five days after ISO, HR corresponded to 70 to 94% of the control values in the temperature range indicated previously. Thirty days after ISO, the HR was from 64 to 101% of the control. At 90 days after ISO, the values of HR were similar to those in the control, when the recording was performed at 12, 16, 20 and 24 °C.
Table 2 Comparison of heart rate between anuran amphibian and Ambystoma dumerilii
3.2.4. Atrium and ventricular tension One day after ISO, the values for AT were 45 to 78% (Fig. 3a) and for VT were 49 to 78% (Fig. 3b) of the control values in a temperature range from 8 to 24 °C. When the recording was performed at temperatures higher than 12 °C, the AT and VT values were recovered gradually at 5, 30 and 90 days after ISO
until they reached the control values on day 90. But when the AT was recorded at 12 °C or lower temperature the values were 22 and 32% higher than in the control; while the VT values remained in 50% (8 °C) and 80% (12 °C) of control values at days 5, 30 and 90 after last ISO administration.
Rana catesbeiana (Herman et al., 1986) Rana tigrina (Chiu and Chu, 1989) A. dumerilii (Present study)
6 °C
8 °C
12 °C
14 °C
22 °C
24 °C
–
–
11 ± 1
–
34 ± 2
–
–
–
22 ± 1
–
39 ± 2
6±1
7±1
9±1
28 ± 2
31 ± 2
4.5 ± 0.3 5±1
4. Discussion 4.1. Effect of temperature on the heart contractile activity in A. dumerilii
Fig. 3. Effect of temperature on atrium tension (AT) (a) and ventricular tension (VT) (b) in isolated heart from Ambystoma dumerilii control (C) and 1, 5, 30, and 90 days (d) after ISO administration. In all temperatures recorded, 1 d after ISO the AT and VT were decreased and a gradual recovery was observed between 5 and 90 after ISO. Except at temperatures below 12 °C where the values of AT were higher and those of VT lower in comparison with C. Values are means ± S.E.M. Significant differences (P ≤ 0.05) between times after ISO administration for atrium tension (AT) and ventricular tension (VT) at each recorded temperature are indicated by dissimilar letters. After one way ANOVA and post-hoc SNK test.
In contrast to previous studies carried out in anuran amphibians, where only the temperature effect on the atrium contractile activity was reported (Ask, 1983; Chiu and Chu, 1989), in the present study we simultaneously evaluated the contractile activity developed by the atrium and ventricle (insert in Fig. 1a). In our traces the evaluation of HR and both AT and VT was possible. The values of HR found in the present study in control hearts are similar to those reported for the HR of other ectothermic vertebrates (Farrell, 1991; Fritsche, 1997; Lillywhite et al., 1999). However, our values tend to be lower than those reported for Rana tigrina (Chiu and Chu, 1989) and R. catesbeiana (Herman et al., 1986) in vitro (Herman et al., 1986; Chiu and Chu, 1989) and in vivo preparations (Herman et al., 1986) recorded at temperatures from 6 to 24 °C (Table 2). At temperatures between 6 and 14 °C, the values in A. dumerilii are 57% of the values reported for R. catesbeiana and R. tigrina (Chiu and Chu, 1989), between 22 and 24 °C, the values of A. dumerilii are 81% of the values recorded in frogs. The differences on HR values could be related to the mean body mass difference between R. catesbeiana (127 ± 5.4 g) (Broch et al., 2002) and R. tigrina (250–650 g) (Hoque and Saidapur, 1994) in comparison with A. dumerilii (51 ± 3 g) in the present study. Even more, we could speculate that the differences are related with the fact that anura and caudate are different phylogenetic groups or with the fact that A. dumerilii is neotenic, aspects interesting to tackle in future studies. Independent of the cause of the differences in the HR between these species, the data suggest that the variations in the HR between anuran and neotenic urodele amphibians at lower temperatures are more pronounced (Table 2). The changes of HR in opposite sense to AT and VT, when temperature was increased (Fig. 1) are consistent with the data reported in the literature for segments of cardiac muscle of ectothermic vertebrates (Ask, 1983; Burggren et al., 1997). These
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series of results reflect the metabolic capacity of ectotherms to adjust to environmental changes. When temperature is low, metabolic demands decrease and the heart can pump higher blood volume, at lower frequency. In contrast, when the temperature increases and oxygen availability decreases (Dejours, 1989; Pelster, 1999), the heart meets the metabolic demand by increasing the HR and decreasing the pumped blood volume per beat through less tension development in order to maintain cardiac output (Pelster, 1999). The rate of increase in the HR with temperature elevations limits the myocardial ability to develop tension through a decrease in oxygen availability, which is crucial to muscular work (Farrell, 1991). This response can be interpreted as an evolutionary constraint in ectothermic organisms to develop fast heart contraction rates (Burggren et al., 1997), which is reflected in the fact that the heart of birds and mammals can beat at considerably higher frequencies than those observed in most ectothermic animals (no more that 120 beats/min) at the same temperatures (Lillywhite et al., 1999). 4.2. Heart injury with isoproterenol in A. dumerilii The administration of ISO produces cardiomyocyte necrosis, interstitial fibrosis and cardiac hypertrophy in the heart of mammals (Rona, 1985; Benjamin et al., 1989) reptiles (Ostadal et al., 1995) and anuran amphibians (Poupa and Carlsten, 1970; Carlsten et al., 1982). Moreover, we have obtained evidence showing the appearance of interstitial fibrosis produced by ISO in the urodele amphibian A. mexicanum (data no shown). In the present study we found that the proportion of heart activity of A. dumerilii was dependent of temperature, even when the contractile activity of isolated heart is recorded after injury with ISO, resulting in a reduction in HR (Fig. 2), AT and VT values (Fig. 3). This tendency was observed even after 1 day of damage when the HR values increased, AT and VT values decreased, with the increase in temperature. The damage effect was detectable in the isolated heart at temperatures from 8 to 24 °C. The damage in the heart of A. dumerilii was reflected in HR, AT, VT for only a short period of time after ISO administration, and from 5 to 90 days a gradual recovery was observed, as has also been observed in anuran amphibians (Carlsten and Poupa, 1977). This suggests that the mechanism of injury by ISO in anuran and urodele amphibians could be similar. The mechanism by which ISO damages the amphibian heart is unknown; however, considering the actual evidence we suggest that the cardiac sensitivity to ISO in ectotherms is lower than the one observed in endothermic vertebrates (Ostadal et al., 1995). To produce an injury equivalent to the one observed in endotherms, a higher dose of ISO is necessary (Carlsten and Poupa, 1977; Ostadal et al., 1995). Furthermore to β-adrenergic receptor activation in cardiomyocytes, the binding of ISO to β-adrenergic receptors in the coronary arteries has been proposed as the principal way by which heart damage is produced in mammals (Trivella et al., 1990). However, the hearts of A. dumerilii and other amphibians lack coronary arteries (Ghiara et al., 1984; Farrell, 1992; Adler et al., 2004). The trabeculed structure of the ventricle of A. dumerilii could allow an efficient nutrient diffusion
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which makes coronary circulation unnecessary (Ghiara et al., 1984; Farrell, 1992). This may be correlated with a lower oxygen demand in ectothermic than in endothermic vertebrates, being the latter characterized by higher cardiac output (Lillywhite et al., 1999). An alternative explanation for ISO-damage in amphibians is based on reports supporting the presence of β-adrenergic receptors in the ventricular myocardium of anuran amphibians (Chiu and Chu, 1989; Herman et al., 1996; Jurevicius and Fischmeister, 1997) and in the urodele amphibian, A. mexicanum (Cano-Martínez et al., 2004). We suggest that the cardiac damage by ISO in A. dumerilii, could be due to its binding on β-adrenergic receptors located directly in the myocardium. The linking between the myocardial damage and the adrenergic stimulation is unclear. For mammals, there is evidence of apoptosis mediated injury to cardiac muscle by β-adrenergic receptor stimulation (Shizukuda and Buttrick, 2001; Leone et al., 2002; Li et al., 2006) in correlation with oxidative stress (Balta et al., 1999; Narang et al., 2005; Ishizawa et al., 2006; Yogeeta et al., 2006) and Ca+2i increases (Diaz-Munoz et al., 2006). It is probable that a similar mechanism is responsible for the ISO damage in the A. dumerilii heart; this may be demonstrated in future studies. 4.3. Effect of temperature in the contractile activity of the isolated heart after damage with ISO The increase of HR parallel to a decrease in AT and VT values, when the temperature bath increased was consistent for hearts from 5, 30 and 90 days after the last ISO administration when the records were performed from 12 °C to 24 °C (Figs. 2 and 3). In contrast, when the temperature bath was 12 °C or lower this relation was lost. This suggests that the environmental changes in temperature had no effect in the heart contractile activity recovery when the animals are in environmental temperatures above 12 °C, since this response was similar to that in the control hearts. But at temperatures lower than 12 °C, the heart response could be altered and we cannot discard the integrated effect of ISO and low temperatures. These results are important to be considered when the in vitro studies are performed. The biological significance of changes in heart activity at lower temperatures during recovery period in our study is unknown, but we can suggest that the adjustment between HR, AT and VT, could be relevant for A. dumerilii when in its natural habitat the mean temperatures are less than 12 °C in the presence of some cardiotoxic agents. This possibility cannot be discarded because recent surveys of aquatic habitats suggest that organic wastewater contaminants (OWCs) may be common in aquatic ecosystems (Smith and Burgett, 2005) as could be the case in Pátzcuaro Lake. Therefore, it is interesting to consider the ecological evaluation of the habitat of A. dumerilii and other Ambystoma of North America. This possibility has been considered as a probable explanation to frog malformations related with water pollution, when studying the lethal and sublethal effects of three OWCs (acetaminophen, caffeine, and triclosan) on American toad (Bufo americanus) tadpoles (Smith and Burgett, 2005).
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If the possible adjustment in AT and VT after an hypothetic heart damage in the natural habitat of A. dumerilii occurs, we can suggest that this adjustment could form part of mechanisms that preserve the life of animals by blocking the consecutive damage in other tissues as the brain after heart injury, as has been found in comatose humans (Felberg et al., 2001; Holzer et al., 2006) and dogs (Wu et al., 2006), surviving a cardiac arrest. Therapeutic hypothermia improved short-term neurological recovery and survival when compared with standard treatment (Felberg et al., 2001; Holzer et al., 2006; Wu et al., 2006). The possible protection from damage may be related with cell intracellular Ca2+ release in the heart and/or other tissues, and supported by the phenomena of Ca2+-paradox occurring in isolated frog hearts, and in frog and mouse ventricle strips, during Ca2+-free perfusion (phase I), when PCa of the sarcolemma is increased. Low temperature (4 °C) provides complete protection against these molecular changes in frog and a partial protection in mouse tissue. The re-introduction of extracellular Ca2+ (phase II) causes typical Ca2+-triggered damage which is markedly reduced at 4 °C in both species (Rudge and Duncan, 1984). Other studies considering loss of electrical and mechanical activity, ion fluxes, creatine kinase and protein release to define cell damage, found that after 30 min of Ca2+-free perfusion at 37 °C, ventricular activity ceased and a major contraction occurred followed by an increase in resting tension. During the 15-min re-perfusion period the release of creatine kinase and the total amount of protein lost were increased, while lower perfusion temperatures resulted in a decreased loss of protein and creatine kinase (Touraki and Beis, 1990). In summary, the present work shows the first evidence of the temperature effect on cardiac contractile activity in A. dumerilii in controls and after ISO. Our results indicate that the heart of A. dumerilii has a gradual functional restoration after ISO damage. HR recovered at temperatures between 12 and 24 °C and AT and VT at temperatures between 16 and 24 °C. However, at temperatures lower than 12 °C the temperature effect observed in the control hearts was different from that of animals previously treated with ISO. This indicated that the injured and recovering heart responds depending on the thermic plasticity observed in the ectotherms. Acknowledgements We thank Esteban Prado-Zayago for his help in the obtention of the animals. This work was supported by the grant 00-303 to AC-M from Instituto Nacional de Cardiología “Ignacio Chávez". References Adler, A., Huang, H., Wang, H., Wang, Z., Conetta, J., Levee, E., Zhang, X., Hitze, H.T., 2004. Endocardial endothelium in the avascular frog heart: role for diffusion of ON in control of cardiac O2 consumption. Am. J. Physiol, Heart Circ. Physiol. 287, 14–21. Ask, J.A., 1983. Comparative aspects of adrenergic receptors in the hearts of lower vertebrates. Comp. Biochem. Physiol. A 76, 543–552. Balta, N., Stoian, I., Petec, C., Petec, G., 1999. Decreased SOD activity and increased nitrates level in rat heart with left ventricular hypertrophy induced by isoproterenol. Rom. J. Physiol. 36, 175–182.
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