INTRODUCTION

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THE TIME OF EMBRYONIC and larval development, characterized by rapid cell proliferation combined with migration and differentiation of cells and organs, certainly represents one of the most exciting periods in an animal's life cycle. This is not only true with respect to developmental phenomena, it also applies to the physiology of the organisms. In vertebrate embryos, the heart is the first functional organ, and, with the onset of cardiac activity, blood flow is observed very early in development. The autonomic nervous system, however, gains control over cardiac performance later in larval development, although adrenergic and cholinergic receptors are found in cardiac muscle cells much earlier (17, 18, 24). This observation certainly prompts the question, whether early larvae are able to modify heart performance in response to environmental challenges like hypoxia or temperature changes.

Whereas for adult vertebrates, the tight linkage between metabolic demand of tissues and cardiac activity is well established, this linkage apparently does not yet exist in early larval stages. Several studies demonstrated that the rate of oxygen uptake and also cardiac performance of fish or amphibian larvae are hardly affected by chemical destruction of hemoglobin with phenylhydrazine or by raising the animals in an atmosphere containing CO to block the hemoglobin oxygen transport (12, 21, 27, 28). Furthermore, mutants of the zebrafish have been described that develop for up to 5 days after fertilization without a beating heart (1). Whereas the lack of a coupling between cardiac performance and convective oxygen transport might be due to the fact that in these small larvae, diffusive oxygen transport through the skin and tissues is sufficient to assure a proper oxygen supply to tissues; the fact that zebrafish mutants survive without a beating heart even challenges the idea that nutrient transport between cells and organs is dependent on convective transport. Nutrient transport from yolk to tissues and also between cells, however, appears to be very important to meet the rapid growth rates typically observed during larval development (23, 25). Thus the role of the cardiac system in this situation is not easily defined.

The present study, therefore, was set out to analyze the changes in cardiac performance with development under a wide range of temperatures and thus, under a wide range of metabolic activities, in completely unrestrained minnow (Phoxinus phoxinus). With a Q₁₀ for enzymatic reactions in the range of 2, temperature has a profound influence on metabolic activity. Accordingly, metabolic activity of animals increases with temperature, and this appears to be especially pronounced in larval stages, where the Q₁₀ for oxygen uptake typically is in the range of 3 (18, 25). If, in larvae, the connection between metabolic demand and cardiac performance is not yet established, this temperature-induced increase in oxygen uptake may be met without any increase in cardiac activity. On the other hand, the pacemaker or the contractility of the cardiomyocytes may be subject to a direct influence of temperature, which is independent of a nerve- or hormone-dependent regulated response. The results show, that during the initiation of cardiac activity, cardiac output is almost independent of incubation temperature and thus of metabolic activity, whereas at the time of swim bladder inflation, cardiac output significantly increases with incubation temperature.

	MATERIALS AND METHODS

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Animals. Eggs of the minnow (Phoxinus phoxinus) were obtained from our own breeding colony. Parent animals were caught from various Alpine lakes near Innsbruck and kept in a freshwater aquarium. To prolong the breeding season, animals were kept at a temperature of 7°C to mimic the cold season. The animals were fed twice per day.

The imaging system. The imaging system consisted of an inverted microscope (Leica Leitz DMIL) equipped with a [2/3]-in. machine vision charge-coupled device camera (Hamamatsu C 2400 without infrared cut-off filter). The camera was connected to the luminance input of a SVHS video recorder (Sony S-9500). The VCR was remote controlled via the RS232 serial communication port. The setting of the video recorder as well as the recorded images were digitized by a monochrome frame-grabber card (Imagenation PX-610) with a personal computer (Pentium III 450 MHz). The illumination could be reduced to infrared light with a wavelength of 780 or 913 nm to prevent light-induced stress reactions of the animals. The microscope desk was temperature controlled.

Recording of cardiac activity. Every 24 h after spawning, the eggs or larvae were transferred into the temperature-controlled incubation chamber of the microscope stage. The temperature was set to the incubation temperature of the eggs. First, the anatomical development of the animals was recorded by careful inspection of the eggs and larvae. Animals showing any developmental irregularities were removed. Eggs and larvae typically rested on the bottom of the incubation chamber so that video recordings of cardiac activity could be taken without any anesthesia and without any physical restriction of swimming activity. Long-term recordings, in which cardiac activity was recorded for up to 1 h after transfer into the observation chamber of the microscope desk demonstrated that the transfer of the eggs or larvae into this chamber did not influence cardiac activity. After initial inflation of the swim bladder, however, this method of observation could not be employed any longer, because swimming activity of the larvae increased significantly at this point, and the animals only rarely settled in a position that allowed for a stable recording of cardiac activity.

Determination of heart rate, stroke volume, and cardiac output. Heart rate was determined by measuring the time interval for 30 heart beats. Determination of stroke volume, using digital image analysis, followed the method described by Hou and Burggren (11). Video sequences of the ventricle were saved in computer memory. The perimeter of the ventricle image was outlined manually during end diastole and during end systole using a mouse or a graphic tablet. The perimeter was analyzed with a "fit-to-ellipse" algorithm that first calculated the center of mass of the perimeter and subsequently the best-fitting ellipse. The major and minor axes of the ellipse were extracted and directly transferred into a Microsoft Excel worksheet for calculation of the stroke volume using the formula for a prolate spheroid ([4/3] ×  × a×b²) (11). For analysis, five diastoles and systoles were analyzed, and mean stroke volume was calculated as the difference between diastolic and systolic ventricular volume. Cardiac output was calculated as the product of stroke volume times heart rate.

Measurement of oxygen consumption. Oxygen consumption of a group of 10 larvae was measured until day 6 after spawning at 25°C and until day 14 after spawning at 15°C, respectively, in a Cyclobios Twinflow (Cyclobios, Innsbruck, Austria) (7). Larvae were transferred into the respirometer chamber ~7 h after spawning, and oxygen consumption was continuously measured for 20-22 h every day. Average values obtained for each 60-min period were used to calculate the daily mean oxygen consumption. To correct for possible microbial respiration, background respiration of the respirometer system (including oxygen uptake of the Clark oxygen electrode) was measured once every day after removal of the animals (3). Data were analyzed using a custom-made software package Datgraph (Cyclobios).

Statistical analysis. The acquired data and also the extracted data were automatically exported into an ASCII file for statistical analysis. Statistical analysis was performed using the software package Statistica. Data are presented as means ± SE with n = 13, except when stated otherwise.

	RESULTS

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For minnows (Phoxinus phoxinus) raised at temperatures between 10°C and 25°C, the development time significantly decreased at higher temperatures. Figure 1 shows the temperature-dependent appearance of some anatomical and physiological landmarks, such as gill development and hatching and of the initiation of cardiac activity and of blood flow. Whereas initiation of cardiac activity was observed at day 2 at 25°C, at 12.5°C it was only observed at day 4. At 25°C, circulation of erythrocytes was established within a few hours after the first heart beat, but at 12.5°C, circulating red cells were observed 1 day after the first heart beat. Similarly, initial inflation of the swim bladder occurred at day 6 in animals raised at 25°C, but at day 21 in animals raised at 12.5°C.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   The appearance of circulation-related anatomical and physiological landmarks in minnow (Phoxinus phoxinus) raised at 5 different temperatures (n = 13).

Animals raised at 10°C showed an extremely delayed development. Even at day 14 no hatching was observed, and many animals showed developmental abnormalities such as a malformation of the eye. Therefore, this temperature series was terminated, and no physiological measurements were performed.

Figure 2 summarizes the changes in body weight measured in animals raised at 15°C and at 25°C. The weight of the eggs was the same at 3.34 ± 0.10 and at 3.39 ± 0.10 mg for animals raised at 15°C and at 25°C, respectively. At the time of hatching, the egg mass had increased by ~0.4 mg at 15°C, whereas no change was observed at 25°C. After hatching, the weight of the larvae was ~50% of the egg mass (1.81 ± 0.09 and 1.76 ± 0.14 mg, respectively). During subsequent development until inflation of the swim bladder, body mass increased by ~0.8 mg in both groups so that the weight of the hatchlings as well as the weight of the larvae at the time of swim bladder inflation were not significantly different between animals raised at 15°C or at 25°C.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Changes in body mass of eggs and larvae of the minnow raised at 15°C and at 25°C (n = 10).

Oxygen consumption in animals raised at 15°C increased with development (Fig. 3A). Overall, the rate of oxygen consumption increased approximately sixfold within the observation period. Similar results were obtained in animals raised at 25°C. Oxygen consumption in animals raised at 25°C increased from 5.6 to 47.6 nmol/h per larva (Fig. 3B). This resulted in a Q₁₀ of 1.7 for the egg and of 2.4 for the larvae at the time of swim bladder inflation.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Changes in oxygen consumption of a minnow embryo developing at 15°C (A) and at 25°C (B). Oxygen uptake was measured in a twin-flow respirometer for a group of 10 embryos/larvae for 20-22 h every day between spawning and opening of the swim bladder. Daily means for a single animal were calculated by averaging the oxygen consumption values obtained for each 1-h interval of a day and dividing by the number of animals within the respirometer chamber. Values are means ± SD. , the time of hatching.

Heart rate significantly increased with development and with increasing incubation temperatures (Fig. 4). At 12.5°C, mean heart rate was 92.8 ± 1.38 beats/min at the time of swim bladder filling and 233 ± 1.73 beats/min at 25°C.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Changes in heart rate with development in the minnow (Phoxinus phoxinus) raised at temperatures between 12.5°C and 25°C (n = 13).

Q₁₀ for heart rate was in the range of 1.7 to 1.9 for incubation temperatures of 15°C and above (Table 1). This was not only true when comparing values of neighboring incubation temperatures (e.g., 15°C and 17.5°C; 17.5°C and 20°C; 20°C and 25°C), but also when comparing the 15°C and the 25°C group. Between 12.5°C and 15°C, however, the temperature sensitivity of heart rate was significantly higher with a Q₁₀ of 2.8 (Table 1).

The comparison of diastolic ventricular volumes at the onset of cardiac activity demonstrated that diastolic volumes were higher in fish raised at low temperatures (Fig. 5). With proceeding development, diastolic volume significantly increased in fish raised at 15°C and above, but this increase was less pronounced at 20°C and at 25°C. This was also true for systolic ventricular volume (data not shown), but the increase was much smaller than in diastolic volume. In animals raised at 12.5°C, however, diastolic ventricular volume decreased with development, and systolic volume remained almost unchanged.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Diastolic ventricular volume at the onset of cardiac activity (initial) and at the time of initial swim bladder inflation (final) in minnow larvae raised at 5 different temperatures (n = 13). *Significantly different from initial volume (P < 0.05).

At incubation temperatures of 15°C and above, stroke volume increased with development, but at 12.5°C, stroke volume decreased with development (Fig. 6, A and B). Initial stroke volume was significantly higher at low temperatures. Between 42% and 58% of the diastolic volume was ejected (Fig. 6C). Whereas in animals raised at temperatures of 17.5°C and above, the ratio of stroke volume to diastolic volume remained constant during development, it decreased in animals raised at 12.5°C and at 15°C.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   A: the development of ventricular stroke volume in the minnow (Phoxinus phoxinus) raised at temperatures between 12.5°C and 25°C. B: change in stroke volume between the first day of cardiac activity and the final measurement at the time of initial swim bladder inflation. *The increase in stroke volume at 17.5°C was significantly larger than at other temperatures; #the decrease in stroke volume observed at 12.5°C was significantly different from the changes observed at all other temperatures (P < 0.05). C: stroke volume calculated as fraction of diastolic ventricular volume at the onset of cardiac activity (initial) and at the time of initial swim bladder inflation (final) in minnow larvae (n = 13). *Significantly lower than the initial value (P < 0.05).

Cardiac output increased with development at incubation temperatures of 15°C and above, whereas at 12.5°C, cardiac output remained almost constant throughout development until initial inflation of the swim bladder (Fig. 7A). A comparison of cardiac output values at the time of the first circulation of blood revealed that at this stage, cardiac output varied between 130 and 210 nl/min, and there was no correlation with incubation temperature (Fig. 7B). At the time of swim bladder inflation, cardiac output was up to twice as high as at the initial stage in animals raised at 15°C and above, whereas in animals raised at 12.5°C, cardiac output remained constant at ~150 nl/min (Fig. 7B). With the use of the average body mass (see Fig. 2) weight-specific cardiac output can be calculated as 72.1 and 95.5 nl · mg¹ · min¹ for 15°C hatchlings and larvae at the time of initial swim bladder inflation, respectively. For animals raised at 25°C, weight-specific cardiac output amounted to 104.7 and 149.6 nl · mg¹ · min¹ for hatchlings and larvae at the time of initial swim bladder inflation, respectively. This resulted in a Q₁₀ value of 1.45 for hatchlings and of 1.56 for larvae. Calculating Q₁₀ values on the basis of cardiac output determined for individuals, similar values were obtained with 1.41 and 1.48 for hatchlings and larvae, respectively.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   A: the development of cardiac output in the minnow (Phoxinus phoxinus) raised at temperatures between 12.5°C and 25°C. B: change in cardiac output between the first day of cardiac activity and the final measurement at the time of initial swim bladder inflation (n = 13). *Significantly different from initial value (P < 0.05).

	DISCUSSION

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Critique of methods. In previous studies, cardiac activity of fish or amphibian larvae has been analyzed using MS-222 (Tricaine) as a water-soluble anesthetic (6, 15, 19, 20). Although there is a bulk of evidence suggesting that in very early stages, MS-222 has no significant effect on cardiac activity, it may still exert some yet unknown effects. Cellulose wadding or nylon meshes have also been used to physically restrict the animals and reduce body movements (2, 16) that may interfere with the gas exchange because of an increase in the thickness of unstirred layers surrounding the body. Our method, using an inverted microscope and infrared illumination, circumvents all the problems associated with these methods, and cardiac performance can be recorded in free-swimming larvae. Because the early stages of the minnow, and also of the zebrafish (unpublished results), typically settle at the bottom of the tank and pigmentation does not yet obscure ventral or lateral view of the ventricle, stable recordings can be obtained in a reasonable time. Only after inflation of the swim bladder does this method become very tedious and inefficient.

Changes in heart rate. A Q₁₀ of ~2 indicates complete temperature conformity, and a value ~1 indicates complete compensation (4). In adult fish, chronic temperature changes typically are accompanied by a partial compensation of resting heart rate, and Q₁₀ values, therefore, often are below 2. However, in isolated adult hearts and also after inhibition of adrenergic and cholinergic modulation of cardiac activity, the Q₁₀ value is close to a value of 2. This suggests that the temperature compensation is mainly achieved by extrinsic modulation of cardiac activity and not by the intrinsic properties of the pacemaker itself (4, 8). In minnow larvae, the Q₁₀ was ~1.8 in the temperature range between 15°C and 25°C, which is close to temperature conformity according to Farrell and Jones (4). This suggests that extrinsic control mechanisms, which are responsible for a temperature compensation in adult fish, are not yet functional. Indeed, a number of studies indicate that nervous control of cardiac activity is achieved late in development (14, 17, 24), even though adrenergic and cholinergic receptors are present and the ventricle can respond to external application of these hormones (5, 13, 22). The results of this study thus suggest that minnow larvae at this stage of development do not use the hormonal pathway for temperature compensation.

Changes in stroke volume and cardiac output. Whereas heart rate of minnow larvae increased with temperature and development, initial stroke volume was higher at lower temperatures. In isolated adult trout hearts, stroke volume was about the same at 5°C and at 15°C, whereas ventricle mass at 5°C was higher than at 15°C (8). A comparison of the morphological development at different temperatures indeed revealed that higher temperatures accelerate differentiation, but animals raised at higher temperatures typically are smaller than animals raised at lower temperatures (9, 10, 18, 26). Although no difference in body mass was observed between animals raised at 15°C and at 25°C in our study, diastolic ventricular volume at the onset of cardiac activity was significantly higher in 12.5°C animals compared with animals raised at 20°C or 25°C. If resting diastolic ventricular volume can be accepted as an index of heart size, this suggests that at low temperatures, an increase in heart size compensates for a decrease in heart rate, or vice versa, so that cardiac output remains constant. Initial cardiac output showed no significant correlation to incubation temperature, but between 15°C and 25°C the Q₁₀ was ~1.4.