Prolonged anoxia tolerance of facultative anaerobes is based on metabolic depression and thus on controlled reduction of energy-utilizing processes. One proposed survival mechanism is the closing of ion channels to decrease energetic cost of ion pumping (Hochachka PW. Science 231: 234–241, 1986). To test this hypothesis, the involvement of L-type Ca2+ channels in seasonal anoxia tolerance of the vertebrate heart was examined by determining the number of [methyl-3H]PN200–110 (a ligand of L-type Ca2+ channel α-subunit) binding sites of the cardiac tissue and the density of Ca2+ current in ventricular myocytes of an anoxia-resistant fish species, the crucian carp. In their natural environment, the fish were exposed for >3 mo of hypoxia (O2 <2.5 mg/l) followed by almost 8 wk of anoxia that resulted in abrupt depletion of cardiac glycogen stores in late spring. Unexpectedly, however, the number of [methyl-3H]PN200–110 binding sites did not decline in hypoxia/anoxia as predicted by the channel arrest hypothesis but remained constant for most of the year. However, in early summer, the number of [methyl-3H]PN200–110 binding sites doubled for a period of ∼2 mo, which functionally appeared as a 74% larger Ca2+ current density. Thus the anoxia tolerance of the carp heart cannot be based on downregulation of Ca2+ channel units in myocytes but is likely to depend on suppressed heart rate, i.e., regulation of the heart at the systemic level, and direct depressive effects of low temperature on Ca2+ current to achieve savings in cardiac work load and ion pumping. The summer peak in the number of functional Ca2+ channels indicates a short period of high cardiac activity possibly associated with reproduction and active perfusion of tissues after the winter stresses.
- L-type calcium channels
- channel arrest
- calcium current
because of the urgent dependence of heart and brain on a continuous supply of oxygen, exposure to anoxia is a disastrous event for most vertebrates, generally leading to death within a few minutes. A few ectothermic species are, however, remarkably anoxia resistant and able to withstand complete oxygen shortage for several months without any functional damage. These animals are useful targets for physiological research as they can reveal molecular, cellular, and systemic-level mechanisms of physiological plasticity required for such prolonged anoxia tolerance. Crucian carp (Carassius carassius L.), a teleost fish species, is one of the most anoxia-tolerant vertebrates with abilities to survive several months of complete anoxia at low temperatures (3, 10) and is therefore a good model species for anoxia research.
Although large glycogen stores and alternative metabolic pathways are often necessary for successful anaerobiosis, the exceptional anoxia tolerance of crucian carp and other facultative anaerobes is generally based on metabolic suppression, i.e., reduction of energy-utilizing processes (9, 20). To achieve a balance between energy demand and reduced energy supply, downregulation of physical activity and energy-consuming processes of tissue cells is inevitable under prolonged anoxia. The two major energy sinks in animal cells are protein synthesis and ion pumping (6, 26), the latter being particularly important in excitable tissues such as heart and brain (26). Almost 20 years ago the late Peter Hochachka presented a testable hypothesis called “channel arrest” that might account for the energy balance of excitable cells and still enable the maintenance of ion homeostasis and prevent anoxic death of cells (8). According to the channel arrest hypothesis, in hypoxic conditions facultative anaerobes compensate for the reduced ATP-dependent ion pumping capacities by reducing the densities of functional channels per unit membrane surface area in proportion to declining metabolic rate (8). Indeed, disturbed ion homeostasis with cytosolic overload of Na+, K+, Ca2+, and H+, which is typical for hypoxic heart cells of hypoxia-sensitive vertebrates, would be detrimental for heart and brain and thus for survival of facultative anaerobes. Although it is expected that the ion gradients are maintained in anoxic cardiac myocytes of anoxia-tolerant vertebrates, it is not clear how this is achieved. For example, if channel arrest is involved, it is not known which ion channels might be reduced and how the number of functional channels may be regulated.
In our previous study (21), we indicated that the inward rectifier K+ channels are not arrested in the heart of crucian carp under prolonged anoxia. In the present paper, we extend those studies to the L-type Ca2+ channels, which could be targets for channel arrest for a number of reasons. Ca2+ channels are intimately involved in excitation-contraction coupling, which regulates the force of cardiac contraction at myocyte level by changes in the amount of free intracellular Ca2+. Ca2+ channels allow sarcolemmal Ca2+ entry, which may directly contribute to the cytosolic free Ca2+ concentration or function as a trigger for Ca2+ efflux from the sarcoplasmic reticulum (2, 5, 14, 19, 32). Furthermore, Ca2+ channels maintain the long plateau duration of the cardiac action potential, thereby supporting Ca2+ influx by a third mechanism, the Na+/Ca2+ exchange (34). Indeed, pharmacological blockade of Ca2+ channels strongly reduces the force of cardiac contraction in crucian carp (28). We hypothesized that the number of dihydropyridine receptors (DHPRs), i.e., the pore-forming α-subunit of the L-type cardiac channel, and the density of L-type Ca2+ current (ICa,L) would be downregulated so that Ca2+ entry would be less in fish exposed to prolonged anoxia than in normoxic animals. To maximize the physiological relevance of the data, the experiments were conducted with animals that were directly caught from the wild in different seasons. This is important because several environmental cues like temperature, oxygen availability, and photoperiod may be needed to trigger physiological responses (4, 24), and in particular because anoxia tolerance of the crucian carp and the function of the crucian carp heart are strongly correlated with the seasons of the year (17, 23, 28, 31)
MATERIALS AND METHODS
Crucian carp (C. carassius L.), 10–220 g in body mass (n = 766), were captured regularly from a local pond around the year beginning in May 2002 and ending in June 2003. The fish traps were kept in the same locations throughout the year and were checked once or twice each month. Temperature and oxygen content of the pond water were measured each time at the site of traps with a battery-operated analyzer (Cellox 325 with WTW Multiline P4). The fish were brought immediately into the lab and held in 500-liter metal tanks with circulating groundwater at the temperature of the pond water until used for sample preparation or for the experiments. Glycogen content of the hearts was determined (15), and cardiac homogenates for DHPR determinations were prepared (18) within 24 h from the capture of the fish. All experiments were made with the permission of the local committee for animal experimentation.
Preparation of cardiac samples.
Crude ventricular membranes were prepared for ligand-binding experiments according to the procedure of Milnes and MacLeod (18). Fish were stunned by a blow to the head and killed by cutting the spine. Hearts were carefully trimmed away, and ventricular muscle was separated, blotted dry, and weighted to the nearest 0.1 mg. Ventricles from 3–30 fish were pooled for each cardiac homogenate. Tissue was diced with scissors and then homogenized using a Teflon-glass homogenizor (Heidolph) for four 20-s bursts at maximum speed (2,200 rpm) in 15 vol of ice-cold buffer (in mmol/l: 500 KCl, 25 HEPES, 0.005 PMSF, pH 7.2 at 20°C). Homogenate was centrifuged at 1,000 g for 15 min at 4°C; the pellet was discarded and the supernatant was stored frozen at −40°C until used in ligand binding experiments. Protein concentration of the samples was determined by the method of Lowry et al. (16).
Determination of DHPRs.
The number (Bmax) and affinity (Kd) of DHPR binding sites were determined with [methyl-3H]PN200–110 (Amersham Pharmacia Biotech) by saturation binding experiments. About 20 μg of homogenate protein was incubated with different concentrations (10–600 pmol/l) of [methyl-3H]PN200–110 (Amersham, Little Chalfont, UK) in 0.5 ml of incubation buffer (composition in mmol/l: 25 Tris, 10 Na-HEPES, 1 Na2EDTA, 1.1 MgCl2, 0.005 PMSF, pH 7.4 at 20°C) to obtain total binding. Nonspecific binding was detected by measuring [methyl-3H]PN200–110 binding in the presence of 10 μmol/l of nonlabeled nifedipine (Sigma) and was subtracted from the total binding to calculate specific binding, which accounted for >70% of the total binding at the highest concentration of [methyl-3H]PN200–110. Binding experiments were done at dim red light to prevent photobleaching of [methyl-3H]PN200–110 and nifedipine. Preliminary experiments indicated that equilibrium binding of [methyl-3H]PN200–110 was attained within <4 h. Accordingly, reaction was terminated after 4-h incubation at room temperature (20°C) with 6 ml of the ice-cold wash buffer (10 mmol/l Na-HEPES, pH 7.4 at 20°C), and samples were immediately filtered under vacuum (Millipore 1225 Sampling Manifold, UK) through Whatman GF/B filters (Merck, Poole, UK) with three 6-ml washes of ice-cold buffer. Filters were soaked in 10 ml of scintillant (Ready Protein+, Beckman), and radioactivity of the samples was measured using a Wallac 1414 WinSpectral liquid scintillation counter for 10 min. Sample counts were corrected for background and quench using the external standard ratio method. Specific binding for each cardiac sample was plotted as a function of the ligand concentration, and a rectangular hyperbola for one binding site was fitted to the plot to obtain the maximum number of binding sites and the dissociation constant where Bs = substrate binding (pmol/mg), Bmax = number of DHPR sites (pmol/mg), Kd = affinity of DHPR sites (nmol/l), and Cs = substrate ([methyl-3H]PN200–110) concentration (nmol/l). Furthermore, Scatchard plots were constructed to see if more than one ligand binding site was involved.
Isolation of cardiac myocytes.
Ventricular myocytes were isolated using the established protocols (21, 32). Briefly, the fish was stunned by a blow to the head and pithed. The whole heart was carefully excised and cannulated through the bulbus arteriosus. The heart was retrogradely perfused, using a hydrostatic pressure head of 50 cmH2O, first with Ca2+-free saline for 8 min and then with proteolytic enzymes for 18 min. Both solutions were oxygenated with 100% O2. The Ca2+-free saline contained (in mM) 100 NaCl, 10 KCl, 1.2 KH2PO4, 3 MgSO4, 50 taurine, 20 glucose, and 10 HEPES adjusted to pH 6.9 with KOH. For enzymatic digestion 0.75 mg/ml collagenase (type 1A), 0.50 mg/ml trypsin (type IX), and 0.75 mg/ml fatty acid-free albumin (all from Sigma, St. Louis, MO) were added to Ca2+-free saline that was recirculated using a peristaltic pump. After enzymatic digestion, the ventricle was separated, and single cells were liberated by agitation through the opening of a glass pipette. Myocytes were stored at 6°C and used within 5 h after isolation.
Whole cell patch clamp of Ca2+ current.
For whole cell patch clamp, a small aliquot of cells was taken into the recording chamber (RC-26, Warner Instrument; 150 μl). After the myocytes had settled, they were superfused at a constant 1.5 ml/min flow of physiological saline that contained (in mM) 150 NaCl, 5.4 CsCl, 1.2 MgSO4, 0.4 NaH2PO4, 1.8 CaCl2, 10 glucose, and 10 HEPES adjusted to pH 7.6 with CsOH. Tetrodotoxin citrate (0.5 μM; Tocris Cookson) was added in the external solution to prevent Na+ currents. Patch pipettes were pulled from borosilicate glass (Garner, Claremont, CA) using a two-stage vertical puller (L/M-3P-A, List-Medical, Darmstadt, Germany) and filled with Cs+-based electrode solution that contained (in mM) 130 CsCl, 15 tetraethylammonium chloride, 1 MgCl2, 5 oxaloacetate, 5 EGTA, 5 MgATP, 10 HEPES, and 0.03 disodium GTP, pH adjusted to 7.2 with CsOH. Temperature of the physiological saline was regulated to desired values by two water baths that chilled or heated the inflow tube carrying the perfusion solution to the recording chamber. Ca2+ currents (ICa) were elicited from a holding potential of −80 mV by 500-ms square wave pulses to voltages between −70 and +70 mV with 10-mV steps, and the currents were leakage corrected on line using the P/N procedure of the acquisition software (pCLAMP 8.2, Axon Instruments).
The annual changes in glycogen content and DHPR binding were tested by ANOVA and Kruskal-Wallis test, respectively. The densities of ICa between winter and summer fish were compared by unpaired t-test.
Seasonality in environment and animals.
Figure 1 depicts annual changes in temperature and oxygen content in the pond recorded at the dates of fish catching. The oxygen limit (2.5 mg/l O2; Ref. 3) for the initiation of anaerobic energy production in crucian carp at 5°C is indicated by the dotted line in Fig. 1A. According to this threshold value, the hypoxic period began at the end of November and lasted for ∼5.5 mo until the end of April. The last 8 wk of this time period were completely anoxic. Thus the fish were clearly experiencing strong seasonal changes in both temperature and oxygen content during the study period.
There was also a clear seasonality in the glycogen content of the heart (P < 0.001) (Fig. 1B). Glycogen stores were smallest in May and increased first slowly in July–September and then more rapidly in late autumn so that the peak value was attained in the beginning of January. During the hypoxic midwinter the glycogen content remained relatively constant and then abruptly dropped during the anoxic period in March. These findings indicate that the crucian carp heart reacted to prolonged anoxia by mobilizing carbohydrate stores of the heart.
Seasonality of DHPR binding.
[methyl-3H]PN200–110 binding in crude cardiac membranes was determined by saturation binding experiments in each month if sufficient numbers of fish were caught. Figure 2 shows the association binding of [methyl-3H]PN200–110 and their Scatchard plots for four different months. The binding curves in different times of the year follow typical saturation binding kinetics, and the linearity of Scatchard plots suggests that only a single type of DHPR binding site is present in the crucian carp heart. The number of DHPR binding sites remained constant for most of the year with the exception of early summer, May–July, when the number of receptors approximately doubled (P < 0.001) (Fig. 3A). The increase in DHPR binding sites between mid-May and mid-July occurred both in 2002 and 2003. In contrast, there were no differences in Kd values or protein content of the preparations in any month (P > 0.05) (Fig. 3, B and C, respectively). Thus the binding experiments indicate that the number of DHPRs is approximately doubled for a short period of time in midsummer and remains at a lower and constant level for the rest of the year.
The Bmax of [methyl-3H]PN200–110 binding represents the total cellular pool of Ca2+ channel α-subunits and does not necessarily equal the number of functional Ca2+ channels in the sarcolemma. To see if the differences in Bmax also appear at functional level, we compared the densities of cardiac ICa in winter- and summer-acclimatized carp. Although there were no differences in myocyte size between summer and winter fish, the ICa was bigger in summer. At 11°C, the peak ICa density was 74% larger in May–June than in January–February (P < 0.001), thus confirming that the increase in DHPR binding sites represents an increase in the number of functional L-type Ca2+ channels in the summer-acclimatized carp (Fig. 4). When measured at physiological body temperature of the fish, the peak density of ICa was 6.1 times larger in May–June than in January–February (Table 1).
It is shown that the number of cardiac DHPRs and ICa of the anoxia-tolerant ectothermic vertebrate, the crucian carp, do not follow seasonal changes in water oxygen content but instead show a short duration peak in early summer. These findings do not support the hypothesis that the number or activity of ion channels is downregulated in the hypoxic/anoxic tissues of the facultative anaerobe, and therefore alternative mechanisms must be sought for seasonal metabolic downregulation and anoxia resistance of the crucian carp heart.
Environmental conditions in the study pond followed a typical pattern for the small water bodies of north-temperate climates, including seasonal changes in water temperature, a long hypoxic period in winter, and finally a completely anoxic period in spring. Seasonal changes affected the glycogen content of the carp heart such that large glycogen stores were accumulated during late summer and autumn to act as a substrate for the anaerobic energy production under the oxygen-deficient time period (13, 30). It is notable, however, that glycogen reserves did not diminish during the hypoxic winter but abruptly reduced after several weeks of anoxia. This suggests that the cardiac glycogen is mainly used as an emergency store of energy under complete anoxia, and therefore the hypoxic energy production was possibly based on aerobic metabolism or on the fermentation of the large glycogen stores of the liver (12). The fish heart comprises only ∼0.1% of the body mass (1), and it is not, therefore, a large burden to the whole body energy stores. The depletion of cardiac glycogen stores at the end of March does, however, indicate that the crucian carp heart was under the threat of energy insufficiency and thus in a situation that might have required channel arrest to stabilize the energy supply-demand ratio to a lower level (8).
Unlike glycogen stores, the Bmax of [methyl-3H]PN200–110 binding did not react to anoxia in March–April but remained at the same level where it was in the previous July. This indicates that cardiac L-type Ca2+ channels are not downregulated by seasonal anoxia in the natural environment of the crucian carp. Accordingly, differential expression of the Ca2+ channel protein is not the means by which energy savings are obtained in ion pumping or in reduced cardiac contractility. These results accord with our previous findings from another cation conductance, the inward rectifier K+ current (IK1), of the crucian carp ventricular myocytes, which was also unaffected by prolonged anoxia (21). Furthermore, recent in vivo recordings of cardiac function indicate that crucian carp retain normal cardiac performance for at least 5 days of anoxia at 8°C (J. Stecyk, personal communication). Together these findings suggest that anoxic channel arrest is not an active mechanism in the crucian carp heart, at least in the form that involves changes in the number of ion channels or alteration in their catalytic activity.
Although both the number of cardiac Ca2+ channels and their activity in isolated myocytes were measured, neither of these methods is able to reveal covalent modification of ICa, which might be activated or inactivated during anoxia (7). Thus the possibility remains that the arrest of Ca2+ channels could have gone unnoticed. However, we regard this possibility very unlikely, because maximal activation of the β-adrenergic receptor pathway, the strongest modifier of the L-type Ca2+ channels, increases sarcolemmal Ca2+ influx in crucian carp myocytes by 22% and the force of ventricular contraction by 12%, respectively (29, 33). Thus even complete abolition of the adrenergic tone in anoxic carp could cause only marginal changes in ICa.
Because cardiac glycogen reserves were partially depleted in anoxia, the heart was under the threat of energy insufficiency. Accordingly, alternative solutions for channel arrest must exist to avoid anoxic death of the cardiac myocytes. In winter, low temperature (<4°C) will suppress heart rate and thus cardiac energy consumption to a small fraction of its value in normoxic and warm summer waters (17). Furthermore, the depressive effects of low temperature on heart rate and cardiac contractility are maximally exploited through the inverse thermal compensation that is typical for crucian carp heart (17, 28). Because each heartbeat is elicited by an action potential, depressed heart rate means fewer action potentials per unit time, i.e., an action potential arrest (21). Due to action potential arrest, Ca2+ channels will be recruited less frequently and the contractility of the heart will be diminished, rationing glycogen stores of liver and heart. Action potential arrest is conceptually different from channel arrest because heart rate is regulated at systemic level by the autonomic nervous system (25) and, unlike channel arrest, does not require any changes in the function of ion channels at myocyte level.
In principle, temperature-dependent reduction of ICa density could also reduce ion leakage and cardiac contractility, but the effect is likely to be smaller than the direct impact of temperature on ICa because low temperature will prolong action potential duration and thus keep channels open for a longer time (27). In crucian carp ventricle, the duration of action potential is prolonged more than twofold, from 1.3 to 2.8 s, by a temperature drop from 18° to 4°C (22), which is much less than 6.1-fold change in the peak density of ICa, suggesting that a direct temperature effect on ICa might reduce ion leakage and alleviate the anoxic energy insufficiency. Thus action potential arrest combined with the reduced ICa in the cold will probably provide sufficient reduction in cation leakage through the sarcolemma so that energy cost in ATP-dependent ion pumping is diminished (1) and a balance between energy supply and demand is attained at lower level. Inverse thermal acclimatization obviously preconditions the heart for winter such that low temperatures effectively reduce cardiac contractility and provides protection against anoxia (28).
Although the Bmax of [3H]PN200–110 binding remained fairly constant for a major part of the year irrespective of changes in oxygen content, a prominent upregulation of DHPRs and ICa occurred in May–July. This is reminiscent of the seasonal changes in the expression of myosin heavy chains of the crucian carp ventricle, which is characterized by transient expression of the fast myosin isoform in early summer (31). Therefore, it seems that there is a remodeling of cardiac protein expression in early summer at the level of both sarcolemma and myofibrils that tends to support high cardiac activity. This cardiac remodeling may be associated with energy allocation to growth and reproduction that occur in carp from late May to mid-July in Central Finland (62°) (11). Indeed, when waters warm, a switch from anaerobic to aerobic metabolism is necessary to fuel the increased cardiac activity required for replenishment of starved tissues (12), for growth, and for reproduction.
In conclusion, [methyl-3H]PN200–110 binding indicates that the number of cardiac L-type Ca2+ channels does not diminish under seasonal anoxia as predicted by the channel arrest hypothesis. However, the action potential arrest due to low temperature can be regarded as functional channel arrest because ion channels are less frequently recruited and therefore impose smaller load on ATP-driven ion pumping. Direct impact of low temperature on ICa will provide further savings in energy consumption. Thus the seasonal depression of the heart in facultative anaerobes seems to involve physiological amendments mainly at the systemic level, e.g., in the form of depressed heart rate or as a result of inverse thermal compensation, which will secondarily bring savings in ion pumping without the need to downregulate ion channel densities. Although anoxic channel arrest has not been found in the crucian carp heart, it needs to be emphasized that cardiac function is, however, strongly seasonal. In fact, it appears that function of the heart is upregulated for a few months in summer rather than being downregulated under anoxia.
The study was financially supported by the Academy of Finland (Grant No. 78045).
We are grateful to A. Kervinen for technical assistance.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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