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COMPARATIVE AND EVOLUTIONARY PHYSIOLOGY

Calcium flux in turtle ventricular myocytes

¹Faculty of Life Sciences, The University of Manchester, Core Technology Facility, Manchester, United Kingdom; and ²School of Biosciences, The University of Birmingham, Birmingham, United Kingdom

Submitted 15 June 2006 ; accepted in final form 2 August 2006

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The relative contribution of the sarcoplasmic reticulum (SR), the L-type Ca²⁺ channel and the Na⁺/Ca²⁺ exchanger (NCX) were assessed in turtle ventricular myocytes using epifluorescent microscopy and electrophysiology. Confocal microscopy images of turtle myocytes revealed spindle-shaped cells, which lacked T-tubules and had a large surface area-to-volume ratio. Myocytes loaded with the fluorescent Ca²⁺-sensitive dye Fura-2 elicited Ca²⁺ transients, which were insensitive to ryanodine and thapsigargin, indicating the SR plays a small role in the regulation of contraction and relaxation in the turtle ventricle. Sarcolemmal Ca²⁺ currents were measured using the perforated-patch voltage-clamp technique. Depolarizing voltage steps to 0 mV elicited an inward current that could be blocked by nifedipine, indicating the presence of Ca²⁺ currents originating from L-type Ca²⁺ channels (I_Ca). The density of I_Ca was 3.2 ± 0.5 pA/pF, which led to an overall total Ca²⁺ influx of 64.1 ± 9.3 µM/l. NCX activity was measured as the Ni⁺-sensitive current at two concentrations of intracellular Na⁺ (7 and 14 mM). Total Ca²⁺ influx through the NCX during depolarizing voltage steps to 0 mV was 58.5 ± 7.7 µmol/l and 26.7 ± 3.2 µmol/l at 14 and 7 mM intracellular Na⁺, respectively. In the absence of the SR and L-type Ca²⁺ channels, the NCX is able to support myocyte contraction independently. Our results indicate turtle ventricular myocytes are primed for sarcolemmal Ca²⁺ transport, and most of the Ca²⁺ used for contraction originates from the L-type Ca²⁺ channel.

reptile; excitation-contraction coupling; sarcoplasmic reticulum; Na⁺/Ca²⁺ exchanger; L-type Ca²⁺ channel

SR and sarcolemmal Ca²⁺ cycling can vary between species (3, 6, 16, 21, 32, 33, 36), different stages of development (14, 27, 28), and regionally within the heart (6, 25). In most fish and amphibians, transsarcolemmal Ca²⁺ influx is the primary source of activator Ca²⁺ responsible for initiating contraction (1, 26, 31, 39, 44). The majority of this extracellular Ca²⁺ enters the cell through L-type Ca²⁺ channels, although in some species, the NCX may also contribute significant amounts of activator Ca²⁺ (19, 34, 42). The contribution of intracellular Ca²⁺ cycling through the sarcoplasmic reticulum (SR) is minimal in most ectothermic vertebrates and varies subject to experimental conditions (6, 12, 13, 21, 31). In contrast, activation of the myofilaments in most adult mammalian hearts occurs mainly through the mobilization of the intracellular Ca²⁺ stores of the SR, with sarcolemmal Ca²⁺ influx primarily acting as a trigger for SR Ca²⁺ release (6).

The regulation of contraction with respect to Ca²⁺ cycling has never been studied on a cellular level in any reptilian species. Recently, we have shown isolated turtle ventricular muscle to be relatively insensitive to ryanodine, a compound that inhibits the SR Ca²⁺ release channel. These results suggest that, similar to fish, the SR contributes little Ca²⁺ to turtle heart contraction and relaxation (12). In the absence of a functional SR, we would hypothesize that sarcolemmal Ca²⁺ transport is the primary source of Ca²⁺ and that the NCX is the primary Ca²⁺ efflux pathway. The aim of the present study was to confirm this hypothesis experimentally. Here, we have examined the contribution of the cardiac L-type Ca²⁺ channel, the NCX, and the SR to contraction and relaxation in single isolated ventricular myocytes from the turtle heart. Our results confirm our hypothesis that transsarcolemmal flux is the primary route for Ca²⁺ transport in turtle ventricular myocytes. Moreover, our data provide the groundwork for future studies on ion regulation of contractility in reptilian cardiomyocytes.

	MATERIALS AND METHODS

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Animal Origin and Care

Yellow-bellied turtles, Trachemys scripta scripta (body mass = 218.5 ± 23.9 g, heart mass = 0.61 ± 0.1 g, n = 12) were obtained from Monkfield Nutrition Ltd (Hertfordshire, UK). Turtles were held in 1.5 x 0.5 x 0.5 m plastic tanks containing water maintained at 20–21°C (40 cm depth) and dry basking platforms, allowing access to heating lamps for behavioral thermoregulation.

Solutions

The isolation solution contained (in mM) 100 NaCl, 10 KCl, 1.2 KH₂PO₄, 4 MgSO₄, 50 taurine, 20 glucose, and 10 HEPES, with pH adjusted to 6.9 via KOH. For enzymatic digestion, 1.5 mg/ml collagenase (type 1A), 1 mg/ml trypsin (type IX), and 1.5 mg/ml fatty acid-free BSA were added to the solution. The extracellular solution perfusing the myocytes contained (in mM) 150 NaCl, 5.4 CsCl, 1.5 MgSO₄, 0.4 NaH₂PO₄, 2 CaCl₂, 10 glucose, 10 HEPES, with pH adjusted to 7.7 via CsOH. For whole cell voltage-clamp electrophysiological measurements, the pipette solution contained (in mM) 130 CsCl, 1 MgCl₂, 5 MgATP, 5 Na₂-phosphocreatine (unless stated otherwise), 10 HEPES, 15 tetraethylammonium chloride (TEA), and 0.03 Na₂GTP. The EGTA concentration was 0.025 mM unless stated otherwise. The pH was adjusted to 7.2 with CsOH. Including TEA and CsOH abolished all K⁺ currents. In perforated-patch, voltage-clamp experiments, 240 µg/ml amphoteracin was added to the pipette solution. To be certain that cells were indeed perforated and not in the whole cell configuration, a high concentration of CaCl₂ (10 mM) was also included in the pipette solution. All drugs were purchased from Sigma Aldrich.

Isolated Myocyte Preparation

Myocytes were obtained by adaptation of isolation protocols previously described for fish (32, 34, 42). All procedures were made in accordance with UK Home Office regulations. Briefly, turtles were euthanized by decapitation, and the head was immediately submerged into liquid nitrogen to fully destroy all neural connections. A 2 cm x 2 cm piece of the ventral plastron was removed above the heart using a bone saw. The heart was excised and a cannula was inserted into the right aortic arch and advanced into the ventricle for perfusion. The heart was retrogradely perfused for 10 min at room temperature first with isolation solution to clear the heart of blood and to stop the heart contracting and then with proteolytic enzymes (BSA, collagenase, and trypsin) for a further 20 min. The enzyme solution was retained for use later. Ventricular tissue was separated from the atria, cut into small pieces with scissors, and placed in the collected enzyme solution and shaken at 34°C (Grant OLS200 water bath shaker). To check for viable myocytes, aliquots of tissue suspension were taken every 5 min and placed under a microscope. Once viable myocytes were observed, the suspension was removed from the shaker, left to settle, and resuspended in fresh isolation solution. Healthy viable cells were usually obtained after 15–20 min of heating and shaking. Myocytes were agitated gently, filtered through a nylon mesh, and stored in fresh isolation solution at room temperature for up to 8 h.

Myocyte Morphometrics

To obtain measurements of myocyte dimensions, cells were imaged using confocal microscopy. The sarcolemmal membrane of the myocytes were visualized by loading cells for 10 min with the lipophilic fluorescent indicator di-8-ANNEPS (5 µM, Molecular Probes). Cells were then resuspended in fresh isolation solution and imaged using a laser scanning confocal microscope (Leica, Germany) with 488-nm excitation light and detection at >505 nm. Consecutive plane scans (x-y) were made through the cell to make a three-dimensional model (z stack), from which cell length, width, depth, and volume were calculated using the Zeiss LSM image browser 5.0 software program (see Table 1).

To record changes in [Ca²⁺]_i, ventricular myocytes were loaded with the AM form of the Ca²⁺-sensitive fluorescent dye Fura-2 (Molecular Probes) to a final concentration of 4 µmol/l. Myocytes were shaken gently for 10 min to allow loading; then a sample of the cells were placed on the stage and left to settle for a further 5 min. Cells were then perfused for 15–20 min with extracellular solution for deesterification. All fluorescent measurements were conducted at room temperature. To examine the contribution of the SR to Ca²⁺ flux in turtle myocytes, cells were field stimulated to contract at 0.2 and 0.5 Hz in the presence and absence of SR blockade [10 µmol/l ryanodine and 2 µmol/l thapsigargin (Sigma Aldrich)]. To ensure complete inhibition of the SR, cells were perfused with ryanodine and thapsigargin for a period of 5 min before measurements were commenced. The ratio of the fluorescence emitted at 510 nm in response to alternate illumination with light at 340 and 380 nm (Cairn Research) was used as our index of [Ca²⁺]_i.

Electrophysiological Measurements of Sarcolemmal Currents

Samples of myocytes were added to the recording chamber and left to settle and attach to the bottom. Cells were perfused with extracellular solution at a rate of 1–2 ml/min at room temperature (21°C). Both whole cell and perforated-patch, voltage-clamp experiments were performed using an Axopatch 200B amplifier (Axon Instruments) with a CV 203BU headstage (Axon Instruments). Patch pipettes were pulled with borosilicate glass (Harvard Apparatus) and had a resistance of 2.4 ± 0.02 M when filled with pipette solution. In perforated-patch, voltage-clamp experiments, once a gigaohm seal had formed, patch pipette resistance (5–6 M) was compensated for, and Ra was monitored using the membrane test function to assess the extent of perforation. Once electrical access to the cell was gained, the cell capacitive currents were compensated for by manually adjusting series resistance (Ra) and the cell capacitance compensation circuits. Series resistance [whole cell Ra = 8.2 ± 0.9 M (n = 38); perforated Ra = 17.2 ± 1.5 M (n = 40)] and capacitance [Cm = 42.4 ± 1.9 pF (n = 78)] were measured using the membrane test function of pClamp 9.0 software (Axon Instruments). In the perforated-patch configuration, Ra was monitored throughout the experiments. Signals were analyzed offline using Clampfit 9.0 software (Axon Instruments).

The voltage-clamp waveform protocols for each experiment are provided in the figures. The amplitude of I_Ca was calculated as the difference between peak inward current and the current at the end of the depolarizing pulse. I_Ca was normalized to the cell area by dividing the amplitude of I_Ca by the cell capacitance to give pA/pF. To assess the rate of inactivation of I_Ca, tau fast (f) and tau slow (s) inactivation components were derived by fitting a second-order exponential function to the decaying portion of I_Ca using the Chebyshev procedure (Clampfit software, Axon Instruments). Steady-state kinetic parameters were determined by fitting steady-state activation and inactivation data to Boltzmann equations to calculate the half-activating and half-inactivating potential (V_h) and the slope of activation and inactivation (k), as previously described (44). Recovery from inactivation of I_Ca was assessed by normalizing current amplitude at a constant test pulse (500 ms, –70 to 0 mV) to the constant prepulse value (500 ms, –70 to 0 mV) after various interpulse durations (50–350 ms, –70 mV, see Fig. 6). The contribution of I_Ca to total cellular [Ca²⁺] was calculated from the transferred charges and cell volume. Charge transfer was determined by integrating the inactivating portion of the Ca²⁺ current for 500-ms square-wave voltage pulses from –70 mV to 0 mV. Cell volume was calculated from the measured cell capacitance (42.4 ± 1.9) and the surface-to-volume ratio of the cells. The myocytes were considered to be flat elliptical cylinders with an axis ratio of 1.2 for the elliptical cross section (34, 43, 44). The change in total cellular Ca²⁺ due to Ca²⁺ influx through L-type Ca²⁺ channels was expressed as a function of nonmitochondrial volume (34, 44).

View larger version (18K): [in this window] [in a new window]   Fig. 1. Morphology of live ventricular myocytes from the turtle heart. A: light microscopy image. B: confocal microscopy image. Scale bar applies to both images. Mean morphometric data are provided in Table 1.

View larger version (7K): [in this window] [in a new window]   Fig. 2. The effect of inhibiting the sarcoplasmic reticulum (SR) on the turtle intracellular calcium transient [Ca2+]i. A: relative reduction in [Ca2+]i due to SR inhibition with ryanodine (10 µM) and thapsigargin (2 µM). Values are means ± SE (n = 6). B: original traces of turtle [Ca2+]i in the presence and absence of SR blockade. Statistical analysis (rank sum test) revealed no significant differences between control or SR-inhibited transients (P > 0.05).

View larger version (5K): [in this window] [in a new window]   Fig. 3. Isolation of L-type Ca2+ current (ICa). A: effect of 25 µM TTX, a specific Na+ channel blocker, on INa in a turtle ventricular myocyte (capacitance = 19.65 pF). B: effect of 50 µM nifedipine, a specific blocker of the L-type Ca2+ channel, on ICa in a turtle ventricular myocyte (capacitance = 44.9 pF) pretreated with 25 µM TTX. The waveform protocol used to activate ICa and INa is depicted above each current recording.

View larger version (10K): [in this window] [in a new window]   Fig. 4. Time-dependent change in amplitude of L-type Ca2+ current (ICa). Rundown of ICa was assessed using the whole cell voltage-clamp technique (W/C) at two different pipette concentrations of EGTA (0.025 mM, n = 4; 5 mM, n = 4), with BAPTA (5 mM, n = 5), and with the perforated-patch voltage-clamp technique (P/P) (n = 6). Because rundown of ICa was minimal in perforated-patch, this configuration of the voltage-clamp technique was used in all further experiments for assessment of ICa. Values are expressed as means ± SE.

View larger version (6K): [in this window] [in a new window]   Fig. 5. L-type Ca2+ channel current-voltage relationships in turtle ventricular myocytes. ICa was normalized to myocyte capacitance (pF) and expressed as current density (pA pF–1). Values are expressed as means ± SE (n = 15).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Steady-state activation and inactivation of ICa in turtle ventricular cardiac myocytes. A: voltage protocol used to measure steady-state activation and inactivation. B: representative current recording from a turtle ventricular myocyte (capacitance = 78 pF) subjected to the voltage protocol given in A; C: mean steady-state inactivation and activation profiles. Values are means ± SE (n = 10). Inactivation () is measured by depolarizing from –70 mV to a test potential for 1 s and then testing the remaining available ICa at 0 mV. Activation () is calculated by dividing peak current by apparent driving force [applied membrane potential (Em) – reversal potential (Erev)] according to Ohm's law. Inset: L-type Ca2+ channel window current (product of activation and inactivation at each voltage).

With the exception of original traces and voltage protocols, data are given as mean values ± SE. N values are for number of cells in which the minimum number of animals is n = 4. Statistical tests are supplied in the appropriate figure legends.

	RESULTS

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Cell Morphology

Dissociation of the turtle heart required a higher concentration of digestive enzymes, longer perfusion times, and higher temperatures than those used previously for various fish species (32, 34, 43, 44). These differences may be due to variation in the structure of the extracellular matrix of the turtle heart compared with the fish heart or may relate to the complication of perfusing a three-chambered ventricle. Light and confocal microscopy images of isolated turtle ventricular myocytes are displayed in Fig. 1. Ventricular myocytes were typically spindle-shaped, being 190 µm in length and 5–7 µm in width and depth (Table 1). When comparing the light and confocal images, it is apparent that the sarcomeres of the myocytes are not associated with T-tubules. Myocytes had a small cell volume (2 pl), leading to a large surface area-to-volume ratio, typical of ectothermic vertebrates.

Turtle Ca²⁺ Transients and the Functional Significance of the SR

The functional significance of the SR was assessed via pharmacological blockade with ryanodine and thapsigargin (10 µM and 2 µM, respectively). Turtle myocytes were loaded with Fura 2 and field stimulated at 0.2 Hz in the absence and presence of SR blockade (Fig. 2). The amplitude of [Ca²⁺]_i was not significantly altered following inhibition of the SR (Fig. 2B), indicating the SR plays a small role in Ca²⁺ cycling on a beat-to-beat basis.

L-Type Ca²⁺ Channel Properties

Characterization of inward currents. A depolarizing voltage step from –80 mV to –40 mV elicited a fast-inactivating Na⁺ current (I_Na) (Fig. 3A). As both I_Na and the L-type Ca²⁺ current (I_Ca) are activated within the same voltage range, we used TTX, a specific Na⁺ channel blocker, to inhibit I_Na (Fig. 3A). Thus a voltage step from –80 mV to 0 mV in the presence of TTX gave rise to a slower activating and inactivating I_Ca (Fig. 3B). In previous studies on ectotherms, less than 1 µM TTX is sufficient to totally abolish I_Na (20, 32, 43). However, in turtle ventricular myocytes, we found much higher doses (25–40 µM) were necessary to abolish I_Na completely. Similar doses are used in mammalian preparations (6). Therefore, rather than blocking I_Na pharmacologically with high [TTX], prepulses from –70 mV to –40 mV were applied before each test pulse in all subsequent protocols to fully inactivate I_Na and experimentally isolate I_Ca. To be certain that the remaining current originated from L-type Ca²⁺ channels, we used 50 µM nifedipine, a specific L-type Ca²⁺ channel blocker, to inhibit I_Ca (Fig. 3B). A similar inhibition of I_Ca could be achieved with 100 µM CdCl₂ or 1 mM NiCl₂. The required dose of nifedipine is unusual for cardiac L-type Ca²⁺ channels (20, 32, 43) and may relate to the subunit composition of the L-type Ca²⁺ channel (see discussion).

In many species, I_Ca is known to deteriorate or "rundown" over time, particularly when measured in the whole-cell configuration. We assessed I_Ca rundown in turtle myocytes using the whole cell voltage-clamp technique (with various intracellular Ca²⁺ buffering) compared with the perforated-patch, voltage-clamp method (Fig. 4). Cells were depolarized from –70 mV to 0 mV, and I_Ca density was measured over a period of 12 min. In the whole cell configuration, I_Ca deteriorated to 50% of its original value within 2 min, and to 20% after 12 min, regardless of the level of intracellular Ca²⁺ buffering (Fig. 4). In the perforated-patch configuration, I_Ca remained relatively stable and was only reduced by 10% over the entire 12-min period. Therefore, the perforated-patch, voltage-clamp technique was used in all subsequent experiments when measuring I_Ca. Interestingly, when using 5 mM EGTA or 5 mM BAPTA, the I_Ca density (3.8 ± 1.0 pA/pF and 4.2 ± 0.3 pA/pF, respectively) was similar to perforated-patch I_Ca density (3.2 ± 0.5 pA/pF). However, when using 25 µM EGTA, I_Ca density was considerably lower (1.54 ± 0.36 pA/pF). This suggests the buffering capacity of turtle myocytes is greater than 25 µM EGTA.

I_Ca density, kinetics and voltage relations. The current-voltage relationship for turtle ventricular myocytes is shown in Fig. 5. I_Ca activated at approximately –40 mV, peaked at 0 mV, and reversed at 60 mV. At peak I_Ca density (–3.2 ± 0.5 pA/pF), the time constant for fast inactivation (f) and slow inactivation (s) was 28.7 ± 1.5 ms and 169.2 ± 8.6 ms, respectively (n = 15). Because of this relatively slow inactivation time, charge transfer, and therefore total Ca²⁺ influx through L-type Ca²⁺ channels, was particularly high in turtle myocytes (Table 2).

I_Ca recovery from inactivation. The recovery of I_Ca from inactivation at –70 mV following 1-s prepulses to 0 mV is shown in Fig. 7. The number of recovered channels increased as the duration between the prepulse and the test pulse was lengthened. The time constant of recovery from inactivation () was 157.3 ± 18.6 ms, thus at physiologically relevant frequencies of contraction (0.2–1 Hz), incomplete restitution of turtle L-type Ca²⁺ channels is unlikely to occur.

View larger version (20K): [in this window] [in a new window]   Fig. 7. Recovery of ICa from inactivation in turtle ventricular myocytes. A: voltage protocol used to measure recovery from inactivation. B: representative trace from a turtle ventricular myocyte (capacitance = 78.0) subjected to the displayed voltage protocol. C: relationship between current recovery and interpulse duration for ventricular myocytes. Values are expressed as means ± SE, n = 10.

Characterization of I_NCX. The capacity of the NCX to transfer charge is highly dependent on the intracellular [Na⁺]. In mammalian cardiac myocytes, resting intracellular Na⁺ concentration ([Na⁺]_i) varies between species with a range of 4–15 mM (7) Because in vivo [Na⁺]_i is not known for turtle myocytes, we investigated the capacity of the NCX at a low (7 mM) and a high (14 mM) amount of Na⁺ in the patch pipette. Membrane current was measured in the presence of 50 µM nifedipine to block I_Ca, and a combination of 10 µM ryanodine and 2 µM thapsigargin was used to inhibit possible SR Ca²⁺ release and reuptake. Under these conditions, a square-wave voltage pulse from –70 mV to 0 mV for 500 ms gave rise to a maintained outward current, which could be blocked with 10 mM NiCl₂, confirming the presence of a Ni⁺-sensitive Na⁺/Ca²⁺ exchange current (I_NCX). The Ni²⁺-sensitive currents elicited with 14 mM and 7 mM [Na⁺]_i are shown in Fig. 8. I_NCX was integrated to give a measure of charge transfer so that total Ca²⁺ influx through the NCX could be calculated. At 14 and 7 mM, intracellular Na⁺, NCX charge transfer was 0.24 ± 0.1 and 0.12 ± 0.1, respectively, giving a total Ca²⁺ influx through the NCX at 0 mV of 58.5 ± 7.7 µmol/l and 26.7 ± 3.2 µmol/l, respectively (n = 9 and 7, Fig. 8). Total Ca²⁺ influx through the NCX was significantly greater with 14 mM than with 7 mM [Na⁺]_i (P = 0.004).

View larger version (6K): [in this window] [in a new window]   Fig. 8. Measurement of the Na+/Ca2+ exchange current (INCX) using square wave voltage steps in turtle ventricular myocytes. A: voltage waveform protocol was applied in the absence and then the presence of NiCl2 (10 mM), a blocker of the NCX. B: INCX was identified as the Ni2+-sensitive current at each intracellular concentrations of Na+: 14 and 7 mM.

View larger version (10K): [in this window] [in a new window]   Fig. 9. Measurement of the INCX using ramp protocols (inset) in turtle ventricular myocytes. INCX was identified as the Ni2+-sensitive current during the hyperpolarizing phase of the ramp pulse and was measured at two different intracellular concentrations of Na+: 7 mM Na+ (n = 5) and 14 mM Na+ (n = 5). Inset shows voltage ramp protocol. Values are means ± SE. *Significant difference between data at 7 mM Na+ and 14 mM Na+ (one-way repeated-measures ANOVA; P < 0.05).

	DISCUSSION

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In the hearts of most ectothermic vertebrates, the SR seems to contribute little to contraction and relaxation (see Ref. 45). The cardiac muscle of many ectothermic species, including the turtle, are ryanodine insensitive and exhibit a postrest decay of force, suggesting SR independence, at least under normal physiological conditions (10, 12, 17, 41). In the present study, we tested SR involvement in cellular Ca²⁺ flux directly, and we show that inhibition of SR function with a combination of ryanodine and thapsigargin had little effect on [Ca²⁺]_i in turtle ventricular myocytes, supporting earlier findings on isolated muscle preparations (12). Thus, in the absence of a functional SR, the turtle heart will depend strongly on transsarcolemmal Ca²⁺ cycling for both the contraction and relaxation of the myocyte.

The importance of extracellular Ca²⁺ cycling in turtles, and the apparent lack of SR involvement has a clear ultrastructural basis. The morphological design of turtle myocytes reveals a system primed for transsarcolemmal Ca²⁺ flux. Turtle myocytes are spindle-shaped (long and thin) and lack T-tubules, which is typical of ectothermic vertebrates (45). The surface area-to-volume ratio (18.3) is high compared with mammalian ventricular myocytes (rabbit, 4.6; rat, 6–8) (2, 30), but similar to other ectotherms (trout, 18.2; crucian carp, 19.2; bluefin tuna, 14–17; mackerel, 18–22) (32, 42). This large surface area-to-volume ratio will enhance the efficacy of sarcolemmal Ca²⁺ transport by reducing the diffusional distance that Ca²⁺ has to travel to the myofilaments. Moreover, the myofilaments of ectothermic myocytes are subsarcolemmal (42), further promoting the close association of the sarcolemmal membrane and the contractile elements of the cell.

In most ectotherms, the majority of extracellular Ca²⁺ enters the cell through L-type Ca²⁺ channels (18, 34, 42). The central role of these channels can be demonstrated by an almost complete inhibition of force production by L-type Ca²⁺ channel blockers in fish (1). Our study demonstrates the turtle is no exception to this trend and relies strongly on this form of Ca²⁺ influx for contraction. The density and kinetics of turtle I_Ca are typical of other ectothermic species (20, 32, 42, 43). Steady-state activation and inactivation curves and the time taken for recovery from inactivation also coincide with those found in fish (32, 34, 42, 43). A sizeable window current exists at room temperature, which probably contributes to the prolongation of the action potential, and may be important in Ca²⁺ cycling at lower body temperatures (see Ref. 34).

Interestingly, although the density and kinetics of turtle I_Ca, with the exception of I_Ca time course of inactivation (see below), may be similar to other ectothermic species, the pharmacology suggests the turtle heart may predominantly contain L-type Ca²⁺ channels composed of a different subunit than most vertebrate cardiac L-type channels. The L-type Ca²⁺ channel is made up of a number of subunits, but the ₁-subunit is the main functional unit of the channel, responsible for pore formation and voltage sensitivity (35). In cardiac muscle, it exists as two main isoforms: _1C and _1D (8). The _1C subunit is most commonly found in the mammalian myocardium and is characteristically sensitive to dihydropyridines (24). Conversely, the _1D is found abundantly in neurons or pacemaker regions of the heart and requires a higher dose of dihydropyridines (10–20-fold higher) to completely inhibit I_Ca (4, 22, 38, 46). Thus the pharmacology of the turtle L-type Ca²⁺ channel current may suggest the heart contains a large complement of _1D subunits in their L-type Ca²⁺ channels. It is not clear what the functional significance of these channels may be in turtle ventricular myocytes; however, L-type Ca²⁺ channels containing the _1D subunit have been linked with conduction of the action potential in the mammalian heart (23, 37, 47).

Our results indicate the turtle L-type Ca²⁺ channel is the predominant source of extracellular Ca²⁺, with a total Ca²⁺ influx of 64.1 ± 9.3 of nonmitochondrial (n = 15) space. This value is 5 times that found in adult mammalian ventricular myocytes (5, 29) and approximately double that of certain fish ventricular cells (34, 42, 43). This difference is probably due to the relatively slow time course of inactivation of turtle I_Ca and may be a direct result of less Ca²⁺-induced inactivation of the L-type Ca²⁺ channel and the lack of a functional SR. However, it must be noted that in similar experiments with fish, measurements were made in the whole cell configuration with intracellular buffering, which can increase the amplitude of I_Ca and therefore the rate of Ca²⁺-dependent inactivation of the channel. Nevertheless, when compared with mammalian studies using a perforated-patch configuration, our results indicate the turtle L-type Ca²⁺ channel can contribute an enormous amount of Ca²⁺ for contraction, probably enough to support myocyte contraction independently of other cellular cycling mechanisms.

The large amount of Ca²⁺ that enters the cell during contraction via the L-type channels has to be removed to allow relaxation, and if the SR is not involved in the relaxation process, then another mechanism must be in place. In mammals, the SR and the NCX compete for the removal of Ca²⁺ during relaxation (6). Thus, in the absence of a functional SR, it is expected that the NCX will now act as the primary Ca²⁺ efflux pathway in turtle ventricular myocytes. In some species the NCX also contributes to contraction by entering "reverse mode" (Ca²⁺ in, Na⁺ out) during the upsweep of the action potential. In the hearts of neonatal mammals, which are similar to turtle cardiac myocytes in both structure and function, the NCX accounts for 65–75% of total Ca²⁺ influx (27, 28), and among ectotherms, the NCX alone can activate contraction in the crucian carp or rainbow trout, with up to 50% of Ca²⁺ entry mediated through this reverse NCX activity (19, 44). Thus, in ectothermic vertebrates, and also mammalian neonates, the NCX may provide a substantial proportion of the activator Ca²⁺ necessary for contraction.

Measurement of NCX activity is complicated by the lack of information regarding the intracellular concentration of Na⁺ in turtle ventricular myocytes. In fish, varying [Na⁺]_i greatly influences the activity of the NCX, with a reduction from 16 to 10 mM [Na⁺]_i leading to a 58% reduction in Ca²⁺ entry through the NCX (19). Thus we have measured the capacity of the NCX at two different concentrations of [Na⁺]_i; 14 and 7 mM. Depolarizing voltage steps to 0 mV in the presence of L-type channel and SR blockade led to a nickel-sensitive outward current corresponding to a total Ca²⁺ influx of 58.5 ± 7.7 µmol/l (n = 9) and 26.7 ± 3.2 µmol/l (n = 7) at 14 and 7 mM [Na⁺]_i, respectively. Importantly, at either concentration of [Na⁺]_i, NCX Ca²⁺ entry was sufficient to support myocyte contraction independently. If we speculate that [Na⁺]_i in turtle ventricular myocytes is somewhere between 7 and 14 mM and we combine these figures with Ca²⁺ entry via L-type channels, we could expect a total Ca²⁺ influx at 0 mV via both mechanisms to amount to 100 µmol/l. Of this, 35% of activator Ca²⁺ originates from the NCX. This percentage is slightly higher than that seen in the crucian carp (26%) when NCX activity was measured using similar voltage protocols and intracellular constituents. It is important to note that in the present study, L-type Ca²⁺ channels were inhibited with nifedipine, while measuring NCX activity. Previous studies measuring NCX activity in fish have suggested the presence of an intact Ca²⁺ current via L-type channels will increase forward mode NCX activity, due to the concentration-dependent nature of the exchanger and the reduced driving force for Ca²⁺ entry (19, 44). Thus our study may have overestimated the amount of Ca²⁺ entry via the NCX, although Hove-Madsen et al. (19) found that preserving I_Ca during depolarizing voltage steps had little effect on NCX Ca²⁺ entry at 0 mV. In any case, it seems clear that the NCX is capable of contributing activator Ca²⁺ for contraction in turtle myocytes, and importantly, it is able to support myocyte contraction independent of the SR and L-type Ca²⁺ channels.

Perspectives

This is the first study to address cellular Ca²⁺ cycling in any reptilian species. Our results show that turtles, similar to most fish, rely predominantly on the L-type Ca²⁺ channel for delivering extracellular Ca²⁺ for contraction under routine environmental conditions. We also show that the NCX is a powerful route for both Ca²⁺ entry and Ca²⁺ removal. Freshwater turtles are renowned for their ability to tolerate a wide range of environmental challenges, and some species hibernate in aquatic habitats for up to 6 mo, subjecting themselves to large temperature fluctuations, long periods of hypoxia and anoxia, and consequential acidosis (15, 40). The mammalian myocardium is particularly sensitive to environmental change (6, 9, 11), which suggests the turtle heart may have physiological specializations in E-C coupling, which allow them to cope with their changing environment. The present study provides the groundwork for cellular studies on ion regulation of contractility in reptilian cardiomyocytes. Thus future studies should be aimed at investigating how these Ca²⁺ flux pathways are affected by temperature, hypoxia, and acidosis to provide mechanistic insight into the stress tolerance of the turtle heart.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This study was funded by The BBSRC, The Welcome Trust, The Company of Biologists, and The Anglo-Danish Society.

	ACKNOWLEDGMENTS

 
We especially thank Prof. David Eisner and Dr Andrew Trafford for their helpful advice and technical expertise.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. L. J. Galli, Faculty of Life Sciemces, The Univ. of Manchester, Core Technology Facility, 46 Grafton St., Manchester, M13 9NT UK (e-mail: ginaljgalli{at}hotmail.com)

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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