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The Journal of Experimental Biology 203, 493–504 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JEB2320
RATE-DEPENDENT CHANGES IN CELL SHORTENING, INTRACELLULAR Ca2+ LEVELS AND MEMBRANE POTENTIAL IN SINGLE, ISOLATED RAINBOW TROUT (ONCORHYNCHUS MYKISS) VENTRICULAR MYOCYTES CLAIRE L. HARWOOD2,*, F. CHRIS HOWARTH1,‡, JOHN D. ALTRINGHAM2 AND ED WHITE1 1School of Biomedical Sciences and 2School of Biology, University of Leeds, Leeds LS2 9JT, UK *Present address: Department of Biology, Leidy Laboratories, University of Pennsylvania, Philadelphia, PA 19104, USA (e-mail:
[email protected]) ‡Present address: Department of Physiology, United Arab Emirates University, PO Box 17666, Al Ain, United Arab Emirates
Accepted 3 November 1999; published on WWW 17 January 2000 Summary The effects of increasing stimulation frequency (from stimulation frequency. At 0.6 Hz, electrically evoked 0.2 to 1.4 Hz) on the contractility, intracellular Ca2+ [Ca2+]i transients in the presence of 10 mmol l−1 caffeine 2+ concentration ([Ca ]i) and membrane potential of single or 10 µmol l−1 ryanodine and 2 µmol l−1 thapsigargin were ventricular myocytes isolated from the heart of rainbow reduced by approximately 15 %. trout (Oncorhynchus mykiss) were measured. Cell We have described the changes in contractility, [Ca2+]i shortening, expressed as a percentage of resting cell length, and action potential configuration in a fish cardiac muscle was our index of contractility. The fluorescent Ca2+ system. Under the conditions tested (0.6 Hz, 15 °C), we indicator Fura-2 was used to monitor changes in [Ca2+]i. conclude that the sarcoplasmic reticulum contributes at Action potentials and L-type Ca2+ currents (ICa) were least 15 % of the Ca2+ associated with the [Ca2+]i transient. recorded using the whole-cell patch-clamp technique. The rate-dependent decrease in contraction amplitude Experiments were performed at 15 °C. appears to be associated with the fall in the amplitude of Increasing the stimulation frequency caused a the [Ca2+]i transient. This, in turn, may be influenced 2+ significant increase in diastolic [Ca ]i and a significant by changes in the action potential configuration via decrease in diastolic cell length and membrane potential. mechanisms such as altered Ca2+ efflux and Ca2+ influx. In During systole, there was a significant fall in the support of our conclusions, we present evidence that there amplitude of the [Ca2+]i transient, cell shortening and is a rate-dependent decrease in Ca2+ influx via ICa but that action potential with a decrease in the duration of the the Ca2+ load of the sarcoplasmic reticulum is not reduced action potential at both 20 % and 90 % repolarisation. at increased contraction frequencies. Caffeine was used to assess the Ca2+ content of the sarcoplasmic reticulum. We observed that sarcoplasmic reticulum Ca2+ load was greater at 1.0 Hz than at 0.6 Hz, Key words: heart, rainbow trout, Oncorhynchus mykiss, myocyte, stimulation frequency, Ca2+ transient, Fura-2, action potential, despite a smaller electrically evoked [Ca2+]i transient. The sarcoplasmic reticulum, excitation–contraction coupling. amplitude of ICa was found to decrease with increased
Introduction Cardiac output is the product of heart rate and stroke volume. In fish, cardiac muscle contractility is known to influence stroke volume and is, in turn, heavily influenced by heart rate (Farrell and Jones, 1992). In ventricular strips from rainbow trout and a variety of other teleost species, an increase in stimulation frequency has been reported to decrease (Driezdic and Gesser, 1985, 1988; Bailey and Driezdic, 1990; Hove-Madsen, 1992; Shiels and Farrell, 1997) or increase (Hove-Madsen and Gesser, 1989; Hove-Madsen 1992; Matikainen and Vornanen, 1992) the isometric force of contraction. Mechanisms that underlie the contraction/frequency
relationship in fish cardiac muscle may be related to the process of excitation–contraction coupling. Our understanding of excitation–contraction coupling in teleost hearts is incomplete (see Tibbits et al., 1992). Cardiac muscle contractility is largely determined by the amplitude of the systolic intracellular Ca2+ ([Ca2+]i) transient (Lewatowski and Pytkowski, 1987; Bers, 1991). In mammals, most of this activating Ca2+ originates from the sarcoplasmic reticulum and is released via Ca2+-induced Ca2+ release (CICR; e.g. Fabiato, 1985). However, Ca2+ can also enter the sarcoplasm through the sarcolemma, via the L-type Ca2+ current (ICa) and possibly the Na+/Ca2+ exchanger (Bers, 1991). The relative contribution
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to the systolic [Ca2+]i transient varies with species (Negretti et al., 1993; Cleeman and Morad, 1991; Tibbits et al., 1991; Varro et al., 1993). Previous studies in teleost fish suggest that release of Ca2+ from the sarcoplasmic reticulum is not necessary to activate contraction, which is instead initiated by a large transsarcolemmal Ca2+ influx (e.g. Tibbits et al., 1990). This is further supported by ultrastructural studies showing that fish cardiac muscle has a sparse sarcoplasmic reticulum and lacks t-tubules (Santer, 1985). In addition, studies on multicellular preparations have failed to show any effect of ryanodine (which impairs the function of the sarcoplasmic reticulum Ca2+-release channels) on contraction under physiological conditions (Dreidzic and Gesser, 1988; Hove-Madsen and Gesser, 1989). Furthermore, patch-clamp analysis of ICa currents indicates that the trans-sarcolemmal Ca2+ influx may contribute significantly to the activation of contraction in carp heart muscle (Vornanen, 1997, 1998). However, other recent studies on trout atrial and ventricular myocytes concluded that ICa was not sufficient to activate contraction fully (HoveMadsen and Tort, 1998). This finding, together with the measurement of sarcoplasmic reticulum Ca2+ uptake rates, suggested that, in trout, the sarcoplasmic reticulum may be capable of participating in Ca2+ regulation during excitation–contraction coupling (Aho and Vornanen, 1998; Hove-Madsen et al., 1998). It is possible that an increase in stimulation frequency may alter the shape and duration of the action potential. Such changes have been shown to be a major determinant of the force/frequency relationship in rabbit, guinea pig and rat cardiac muscle (Szigligeti et al., 1996). An increase in stimulation frequency may also influence the levels of [Ca2+]i via effects on ICa (Li et al., 1999) and/or the sarcoplasmic reticulum (Smith et al., 1988). Hove-Madsen et al. (1998) demonstrated a similar effect of stimulation frequency on ICa in fish. Shiels and Farrell (1997) concluded that the contribution of the sarcoplasmic reticulum to activating [Ca2+] was inversely proportional to pacing frequency at 22 °C, but that ryanodine was without effect at stimulation frequencies above 0.2 Hz at 12 °C. Changes in membrane ionic currents and [Ca2+]i are likely to affect and to be affected by changes in the time course of the action potential (see Boyett et al., 1993). The aim of the present study was to characterise the effects of changes in stimulation frequency upon contractility, [Ca2+]i transients and action potential configuration in ventricular myocytes isolated from rainbow trout (Oncorhynchus mykiss) heart. To our knowledge, the effects of stimulation frequency upon action potentials and [Ca2+]i transients in fish cardiac muscle have not previously been reported. By measuring these variables within a single system we were able to compare their magnitude and temporal changes and thus gain greater insights into the interactions between the mechanisms involved. A preliminary account of this work has been presented in abstract form (Harwood et al., 1998a).
Materials and methods Fish origin and maintenance Female rainbow trout [Oncorhynchus mykiss (Walbaum)] (140±5 g, mean ± S.E.M., N=20) were purchased from Washburn Valley Trout Farm, North Yorkshire, UK. They were held for up to 12 weeks in indoor, 2 m diameter, fibreglass tanks containing filtered, recirculated aerated fresh water. The water temperature was maintained at 12–15 °C. The trout were exposed to a 16 h:8 h L:D photoperiod and were fed commercial trout pellets ad libitum. Preparation of Ca2+-tolerant rainbow trout ventricular myocytes Ventricular myocytes were obtained by enzymatic digestion using a modified version of the method described by Vornanen (1998) and techniques for isolating mammalian cardiac myocytes (e.g. White et al., 1995). Trout were killed by a sharp blow to the head, and the spinal cord and brain were destroyed. The heart was carefully removed and placed in a Petri dish containing a nominally Ca2+-free Tyrode solution (for composition, see below). A glass cannula was inserted through the bulbus arteriosus into the ventricle (Moon et al., 1996) and held in place by suture thread. It was found to be important to penetrate the ventricle to obtain a good yield of cells. The atrium was cut to prevent fluid build-up in the ventricle (Milligan, 1994). The heart was mounted on a Langendorff perfusion apparatus and perfused from a height of 10 cm with a nominally Ca2+-free, low-Na+ Tyrode solution to lower intraand extracellular [Ca2+] and to close intercalated disc gap junctions. This initial perfusion lasted for 7 min and was at a flow rate of 11 ml min−1. The composition of the Ca2+-free Tyrode solution was (in mmol l−1): NaCl, 100; KCl, 10; KH2PO4, 1.2; MgSO4, 4; taurine, 50; pyruvate, 20; Hepes, 10; pH was adjusted to 6.9 with KOH. Milli-Q ultrapure water was used because water quality greatly affects the yield of myocytes. The heart was then perfused for 40 min with a recirculating, Ca2+-free Tyrode solution additionally containing proteolytic enzymes to dissolve intracellular connective tissue (0.75 mg ml−1 collagenase, Worthington type II, activity 200–230 units mg−1; 0.1 mg ml−1 protease, Sigma type XXIV), 1.0 mg ml−1 bovine serum albumin (BSA) and 32 µmol l−1 Ca2+. Following superfusion, the bulbus arteriosus and the atrium were removed. The ventricle was placed in Ca2+-free Tyrode solution and cut into small pieces, and the tissue was gently dispersed using forceps and repeated aspiration with a plastic Pasteur pipette. The suspension of cells was filtered through nylon gauze (pore size 250 µm) into a roundbottomed test-tube. The remaining tissue was resuspended, and the dispersion, aspiration and filtering processes were repeated. After filtration, the cells were allowed to settle to the bottom of the test-tube. The supernatant was poured off, and the cells were resuspended in the Ca2+-free Tyrode solution containing progressively higher concentrations of Ca2+ up to a final concentration of 750 µmol l−1. The cells
Effects of stimulation frequency on trout ventricular myocytes were stored in a Petri dish and refrigerated at 4–6 °C, the storage procedure used for mammalian myocytes in our laboratory. Cells were used within 8 h of isolation. All isolation procedures and subsequent experiments were carried out at 15±1.0 °C, using a Grant LTD water bath (Grant, Cambridge, UK) as a cooler. The experimental chamber and measurement of cell shortening The myocytes were allowed to settle on the glass coverslip forming the bottom of a chamber (volume 0.7 ml) mounted on the stage of a Nikon Diaphot inverted microscope (White et al., 1995). The cells were superfused with a control solution containing (in mmol l−1): NaCl, 124.1; KCl, 3.1; CaCl2, 2.5; MgSO4, 0.9; pyruvate, 5.0; Tes sodium salt, 11.8; Tes free acid, 8.2; pH 7.8 at 15 °C), which is routinely used in experiments on in situ (e.g. Olson et al., 1994) and in multicellular (e.g. Harwood et al., 1998b) fish cardiac muscle preparations, thus giving consistency with previous experiments. Solution flowed through the chamber by means of gravity feed, and the level was controlled by suction. Cells were stimulated using 10 ms current pulses delivered by external platinum electrodes connected to a Grass (SD9) stimulator. Stimulation frequency was progressively increased in increments of 0.2 Hz from 0.2 to 1.4 Hz and then decreased back to 0.2 Hz in similar steps. This range of frequencies covers the physiological heart rates found in trout (Priede, 1974). Each rate was maintained until steady-state shortening was reached. Cell shortening was measured on-line from a video image of the cell using an edge detection device (Crystal Biotech, Northborough, MA, USA) which sampled cell length at 50 Hz. The cell image could be optimally aligned for measurement by rotating a CCD camera attached to the side-arm of the microscope. Fish cardiac myocytes are long and narrow, making it difficult to record consistently from both edges of the cell. When this was possible, cell shortening was expressed as a percentage of resting cell length. When it was only possible to record from one edge of the cell, contractility was expressed in terms of the relative amplitude of contraction. The mean length of cells measured in this study was 143±7 µm (mean ± S.E.M., N=20 cells). Measurement of [Ca2+]i using Fura-2 Changes in [Ca2+]i were recorded in Fura-2-loaded myocytes using a spectrophotometer (Cairn Research, Faversham, UK) as previously described in detail by Frampton et al. (1991). Briefly, ventricular myocytes were loaded with the acetoxymethyl ester (AM) of the Ca2+-selective fluorescent dye Fura-2 (Molecular Probes, Eugene, OR, USA). To load the cells, 50 µg of Fura-2 AM was dissolved in 50 µl of dimethyl sulphoxide (DMSO) to give a 1.0 mmol l−1 stock solution. A sample (6.25 µl) of this stock solution was then added to 2.5 ml of cells, giving a final Fura-2 concentration of 3.0 µmol l−1. Cells were gently shaken for 4 min and then left to settle to the bottom of the test-tube for a further 6 min. The supernatant was
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removed, and the cells were resuspended and stored in a Tyrode solution containing 750 µmol l−1 Ca2+. Fura-2-loaded myocytes were alternately illuminated (every 2 ms) by 340 and 380 nm light using a rotating filter wheel. This excitation light was passed to the cell under study via a 430 nm dichroic mirror beneath the microscope nosepiece and a ×40 oil-immersion objective lens. The resultant fluorescence emission was collected by the objective lens and transmitted to the microscope side-port via a 580 nm dichroic mirror and a 510 nm emission filter for detection by a photomultiplier tube. A variable diaphragm was used to ensure that only fluorescence from the cell under study was collected. The output of the photomultiplier tube was passed to the spectrophotometer. The ratio of the emitted fluorescence at the two excitation wavelengths (340 nm:380 nm ratio) was calculated to give an index of [Ca2+]i. Cells were also illuminated with red light, which passed via the diochroic mirror to the camera in the side-arm to give a video image of the cell. Electrophysiological measurements To monitor membrane potential and membrane currents in fish myocytes (see Vornanen, 1997, 1998; Hove-Madsen and Tort, 1998), experiments were performed in patch-clamped myocytes using the whole-cell configuration. Experiments were performed using an Axoclamp-2B amplifier (Axon Instruments Inc.) controlled by a 1401-plus CED interface and software (Cambridge Electronic Design Ltd, Cambridge, UK). Glass pipettes with a resistance of 2.5–5.0 MΩ were pulled from non-heparinized haematocrit tubes using a vertical pipette puller (List Medical; type LM3PA). The pipette solution contained (in mmol−1): potassium aspartate, 110; KCl, 10; NaCl, 10; MgCl2, 8; K2ATP, 8; Hepes, 10; EGTA, 0.05, pH 7.1. The level of intracellular Ca2+ buffering by this solution was sufficiently low that, under whole-cell conditions, cells contracted vigorously in response to triggered membrane depolarisations. Gigaohm seals sometimes formed spontaneously on contact with the myocytes but more usually following application of suction to the pipette via a syringe. Once the whole-cell configuration had been achieved, action potentials were recorded in bridge mode in the absence of added hyperpolarising current. ICa was measured by voltageclamping cells at −70 mV. The cells were then step depolarised to −40 mV for 150 ms to inactivate Na+ current, then depolarised to 0 mV for 500 ms to invoke inward ICa (e.g. White et al., 1995) (see Fig. 5). The amplitude of ICa, after steady state had been reached, was measured as the difference between the peak inward current and the current at the end of the depolarisation to 0 mV. Experiments investigating the role of the sarcoplasmic reticulum 2+ After steady-state [Ca ]i transients had been obtained, stimulation was stopped for 10 s and the cells were exposed to 10 mmol l−1 caffeine using a rapid solution changing device
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(Levi et al., 1996). Briefly, cells were locally superfused with normal Tyrode solution via a small delivery tube positioned within 100 µm of the cell. This solution could be rapidly exchanged for a caffeine-containing solution. Caffeine is thought to release Ca2+ from the sarcoplasmic reticulum, and the amplitude of the induced [Ca2+]i transient can be used as an index of sarcoplasmic reticulum Ca2+ content (Bassani et al., 1995). Electrical stimulation was resumed in the presence of caffeine until steady-state transients were achieved, before caffeine was washed out with control solution. To complement the studies with caffeine, experiments were also performed with ryanodine and thapsigargin (Calbiochem). Ryanodine, at concentrations up to 10 µmol l−1, irreversibly locks the Ca2+-release channels in a partially open (but subconductive) state (Rousseau et al., 1987). This facilitates a continuous leak of Ca2+ from the sarcoplasmic reticulum and prevents the sarcoplasmic reticulum from acting as a functional Ca2+ store. Thapsigargin is an irreversible inhibitor of the sarcoplasmic reticulum Ca2+-ATPase pump and will prevent sarcoplasmic reticulum Ca2+ uptake. After steady-state [Ca2+]i transients had been obtained, ryanodine (10 µmol l−1) and thapsigargin (2 µmol l−1) were applied to the cell using the rapid switching method described above. Measurements were taken after the decline of the [Ca2+]i transient had reached a stable level (usually within 5 min). Data recording and statistical analyses Analogue signals were passed to a Neuro-corder DR-890 (Cygnus Technology, Inc, PA, USA) A/D converter for storage on digital tape. All data are presented as means ± S.E.M. Statistical significance of the results was tested using Spearman rank order correlation, Mann–Whitney rank sum tests and
Student’s t-tests (Sigmastat Statistical Software, SPSS) as appropriate. Significance levels were set to P
