SUMMARY

Specialisations in excitation–contraction coupling may have played an important role in the evolution of endothermy and high cardiac performance in scombrid fishes. We examined aspects of Ca2+ handling in cardiomyocytes from Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). The whole-cell voltage-clamp technique was used to measure the temperature sensitivity of the L-type Ca2+ channel current (ICa), density, and steady-state and maximal sarcoplasmic reticulum (SR) Ca2+ content (ssSRload and maxSRload). Current–voltage relations, peak ICa density and charge density of ICa were greatest in mackerel and yellowfin at all temperatures tested. ICa density and kinetics were temperature sensitive in all species studied, and the magnitude of this response was not related to the thermal preference of the species. SRload was greater in atrial than in ventricular myocytes in the Pacific bluefin tuna, and in species that are more cold tolerant (bluefin tuna and mackerel). ICa and SRload were particularly small in bonito, suggesting the Na+/Ca2+ exchanger plays a more pivotal role in Ca2+ entry into cardiomyocytes of this species. Our comparative approach reveals that the SR of cold-tolerant scombrid fishes has a greater capacity for Ca2+ storage. This specialisation may contribute to the temperature tolerance and thermal niche expansion of the bluefin tuna and mackerel.

INTRODUCTION

One of the most important events in vertebrate evolution is the transition from ectothermy to endothermy. This transition is difficult to study because endothermy is a derived characteristic in birds and mammals. The family Scombridae includes highly active regionally endothermic (tunas) and ectothermic (mackerel and bonito) fishes that occupy diverse habitats, including cold, temperate and tropical waters (Gunn and Block, 2001). In addition, within the family Scombridae, the tribe Thunnini contains guilds of closely related tunas with recent common ancestry such as the yellowfin (tropical) and bluefin (cold temperate) tunas (Chow and Kishino, 2010). Thus, scombrid fishes provide a model lineage to investigate specialisations that evolved in response to endothermy, and those that developed in response to thermal niche expansion.

Among tunas, the bluefin tunas have evolved the highest degree of endothermy, possessing internalised slow twitch oxidative (red) muscle and countercurrent heat exchangers that conserve metabolic heat produced in the locomotor musculature, viscera, brain and eyes (Carey and Lawson, 1973; Carey et al., 1984; Linthecum and Carey, 1971). Electronic tagging studies indicate that bluefin tunas occupy a wide thermal niche. Atlantic bluefin tuna range from the frigid waters off Canada in swimming sea surface temperatures (SST) as low as 0.02°C, to the warmer spawning grounds in the Gulf of Mexico (SST as high as 30°C) (Teo et al., 2007; Walli et al., 2009; Lawson et al., 2010). Owing to a high degree of endothermy, the bluefin tuna maintains an elevated body temperature with a mean visceral temperature of 21–23°C and a maximum of over 33°C (Block et al., 2001; Block et al., 2005; Walli et al., 2009). Electronic tagging of juvenile Pacific bluefin in the eastern Pacific demonstrates occupation of waters that average 17.8°C (Kitagawa et al., 2004; Boustany et al., 2010). Maintenance of a thermal excess of 1–10°C above ambient is routine in these juvenile bluefin despite the cool California Current waters (Marcinek et al., 2001a; Kitagawa et al., 2007).

Yellowfin tunas also have internalised slow-twitch muscles but fewer countercurrent heat exchangers (Graham and Dickson, 2004). These fish have a more tropical distribution (Weng et al., 2009; Shaefer et al., 2007). Juvenile yellowfin tunas tracked simultaneously with similar sized Pacific bluefin tunas in the California Current tend to occupy warmer waters to the south (mean SST of 22°C) and are limited in their northern geographic range (Shaefer et al., 2007). The capacity to maintain a thermal excess is not present in the ectothermic sister taxa of the Thunnus, such as Scomber (mackerel) and Sarda (bonito), because they lack countercurrent heat exchangers. Thus, comparative analyses of these fishes with tuna provide an excellent opportunity to examine the evolutionary sequence of events leading to endothermy and concomitant changes in performance and niche utilisation in extant teleosts.

Despite the ability to conserve metabolic heat, tunas face unique physiological challenges; their heart is close to the gills and separate from the supply of the vascular countercurrent heat exchangers (Carey et al., 1984; Brill et al., 1994). The heart also receives cold coronary blood directly from the gills (Brill, 1987; Brill et al., 2001). Thus, as with mackerel and bonito, the hearts of even the largest tunas operate at ambient water temperatures. Therefore, the tuna heart has an extra physiological burden compared with the mackerel and the bonito, as it must be able to function over a broad thermal range while delivering blood to muscle, viscera and cranial tissues that are often warmer than ambient water temperature and have high metabolic demands.

Tunas that have been studied (bluefin, yellowfin and skipjack) have evolved a robust cardiovascular system. Relative to body size, yellowfin and bluefin tuna hearts are significantly larger than other teleosts, have a thicker ventricular wall and an increased percentage of compact myocardium (Farrell et al., 1992). Furthermore, the tuna heart has large myoglobin reserves (Giovane et al., 1980), a higher oxygen affinity (bluefin tunas) (Marcinek et al., 2001b), high anaerobic enzyme activities (Dickson, 1995; Driedzic and Gesser, 1994) and a highly developed coronary circulation (Farrell et al., 1992), which appears to be reduced in the bonito and almost absent in the mackerel (Davie, 1991). Previous studies have also characterised yellowfin and skipjack tunas as possessing significantly higher intrinsic maximal heart rates (up to 200 beats min–1), which are almost double those found in most teleosts (Brill et al., 2005; Farrell et al., 1992; Keen et al., 1995; Roberts and Graham, 1979). However, recent in situ heart measurements in bluefin, yellowfin and albacore tunas (Blank et al., 2004), and in vivo measurements in bluefin tunas (Clark et al., 2008), indicate heart rates are primarily 40–130 beats min–1, which is not exceptional among teleosts (Farrell, 1991).

Variation in scombrid cardiovascular performance may be explained in part by specialisations in excitation–contraction (E–C) coupling. It was previously thought that most teleosts cycle Ca2+ across the sarcolemma primarily through the L-type Ca2+ channel or the Na+/Ca2+ exchanger (NCX) (Keen et al., 1992; Shiels and Farrell, 1997; Shiels et al., 2006; Vornanen et al., 2002) with the sarcoplasmic reticulum (SR) contributing negligible amounts of Ca2+ for contraction. However, more recent evidence suggests that the role of the L-type Ca2+ channel and the SR in teleost E–C coupling depends on a number of factors, including temperature, frequency of contraction, phylogeny and cardiovascular capacity (Hove-Madsen, 1992; Moller-Nielsen and Gesser, 1992; Keen et al., 1994; Shiels and Farrell, 1997; Shiels et al., 1998; Aho and Vornanen, 1999; Rivaroli et al., 2006; Galli et al., 2008). With regards to scombrids, recent physiological and electron microscopy work has demonstrated that the bluefin ventricle and atrium, the bigeye tuna ventricle and the yellowfin tuna atrium have evolved a more advanced ‘mammalian-like’ form of E–C coupling, with a significant proportion (40–50%) of activator Ca2+ used for contraction originating in the SR (DiMaio and Block, 2008; Galli et al., 2008; Shiels et al., 1999; Shiels et al., 2011). Furthermore, biochemical studies indicate bluefin tuna have increased SR Ca2+ ATPase (SERCA-2) activity and expression over a range of temperatures when compared with other scombrids (Landeira-Fernandez et al., 2004; Castilho et al., 2007). Lastly, Ca2+ entry through the L-type Ca2+ channel is greater and inactivation kinetics are faster in atrial myocytes of bluefin tuna than of Pacific mackerel (Shiels et al., 2004). Collectively, these results suggest enhanced Ca2+ cycling through E–C coupling pathways may have evolved within the scombrid lineage and may be crucial in the evolution of increased cardiac performance, endothermy and niche expansion. Importantly, these Ca2+ pumps and ion channels are all known to be sensitive to acute temperature change (Shiels et al., 2000; Shiels et al., 2002a; Shiels et al., 2002b; Landeira-Fernandez et al., 2004; Castilho et al., 2007; Shiels et al., 2011).

To gain a greater understanding of the evolution of endothermy and cardiovascular performance within scombrids, it is crucial to obtain information on myocyte E–C coupling in a phylogenetic selection of scombrid species. The present study examined the effects of physiologically relevant acute temperature change on aspects of two major E–C coupling pathways [L-type Ca2+ channel current (ICa) density and SR Ca2+ content] in ventricular myocytes from four members of the family Scombridae: Pacific bonito (Sarda chiliensis), Pacific mackerel (Scomber japonicus), yellowfin tuna (Thunnus albacares) and Pacific bluefin tuna (Thunnus orientalis). Atrial myocytes were also examined in Pacific bluefin tuna. These species encompass both ectothermic (mackerel and bonito) and endothermic (yellowfin and bluefin tuna) species, as well as cold-tolerant (mackerel and bluefin tuna), temperate (bonito) and tropical (yellowfin) species. By using a comparative approach, we aimed to identify specialisations in E–C coupling pathways that could have permitted increased cardiac performance, endothermy and/or increased thermal tolerance.

MATERIALS AND METHODS

Animals

Bluefin tuna (Thunnus orientalis, Temminck and Schlegel 1844, n=6, fish mass=11.1±0.5 kg), yellowfin tuna (Thunnus albacares, Bonnaterre 1788, n=4, fish mass=4.33±0.3 kg) and Pacific bonito (Sarda chiliensis, Girard 1858, n=4, fish mass=1.84±0.4 kg) were caught at sea, by purse or seine or by hook and line, off the coast of San Diego, CA, USA. Fish were held aboard the Shogun fishing vessel in large wells flooded by seawater, or in captive farm pens (bluefin tuna) at Mariculture Del Norte, Ensenada, Mexico. All fish were transported by truck during a 9–11 h transport to the Tuna Research and Conservation Centre (TRCC) in pacific Grove, CA, USA. Pacific mackerel (Scomber japonicus, Houttuyn 1782, n=10, fish mass=0.52±0.1 kg) were caught in Monterey Bay, CA, USA and held in captivity at Monterey Bay Aquarium and the TRCC. Fish were kept for no less than 3 months prior to experimentation in 109 m3 tanks at 20±1°C and fed a diet of krill, squid, sardines and enriched gelatine, as previously described (Farwell, 2001). All experimental procedures were conducted in accordance with Stanford University institutional animal use protocols.

Cardiomyocyte preparation

Ventricular myocytes from all species were isolated by adaptation of protocols previously developed for bluefin tuna (Shiels et al., 2004; Galli et al., 2009). Because atrial tissue is known to be more SR dependent than ventricular tissue in rainbow trout (Aho and Vornanen, 1999), we also isolated atrial myocytes from bluefin tuna to test whether the same holds true for this species and to serve as a measure of the sensitivity of our analyses of SR Ca2+ content. Fish were killed by pithing and decapitation (for mackerel) and severing of the spinal cord (for tuna), and the heart was removed intact [mean mass (in g): bluefin tuna, 37.6±1.4; mackerel, 1.14±0.2; bonito=3.99±0.5; yellowfin tuna, 17.1±4.9]. The bulbus arteriosus was cannulated and the heart was retrogradely perfused with an oxygenated, Ca2+-free isolation solution to promote cardiomyocyte relaxation and clear the heart of blood. For the tuna and bonito, the coronary artery was also cannulated and perfused, whereas that of the mackerel was not, because of size constraints. Isolation solution was perfused from a height of 60 cm for ∼10 min for mackerel, ∼15 min for bonito, and ∼20 min for tuna. Proteolytic enzymes were then added to the isolation solution and run through the cannulated bulbus arteriosus for ∼20 min in mackerel and ∼30 min in tuna and bonito. After enzymatic digestion, the ventricle and atrium were dissected and placed into separate Petri dishes containing fresh isolation solution. Tissues were gently triturated with a Pasteur pipette to free cardiomyocytes, and cells were stored at 20°C for the duration of the experiment (up to 8 h).

Solutions

All solutions used were adapted from those previously developed for Pacific bluefin tuna (Shiels et al., 2004). The isolation solution consisted of (in mmol l–1) 100 NaCl, 10 KCl, 1.2 KH2PO4, 4 MgSO4, 50 taurine, 20 glucose and 10 Hepes, with pH adjusted to 6.9 with KOH at 20°C. The enzymatic digestion solution comprised collagenase (type IA), trypsin (type IX) and fatty-acid-free BSA added to the isolation solution. The extracellular solution used for current recordings contained (in mmol l–1) 150 NaCl, 5.4 CsCl, 1.2 MgSO4, 0.4 NaH2PO4, 3.2 CaCl2, 10 glucose and 10 Hepes, adjusted to pH 7.7 with CsOH. Tetrodotoxin (TTX; 1 μmol l–1) was added to the extracellular solution just before perfusion of cells in the bath in order to block fast Na+ channels. Caffeine (10 mmol l–1) was similarly added just before use to promote release of SR Ca2+ stores. The pipette solution consisted of (in mmol l–1) 130 CsCl, 5 MgATP, 15 tetraethylammonium chloride, 1 MgCl2, 5 sodium phosphocreatine, 10 Hepes and 0.025 EGTA with pH adjusted to 7.2 with CsOH. CsCl and tetraethylammonium chloride were incorporated into the solutions in order to block K+ currents. Thapsigargin (2 μmol l–1) and ryanodine (10 μmol l–1) were added to the extracellular solution to block caffeine transient currents for control purposes. Nifedipine (2.5 μmol l–1) was similarly added to block ICa. Except for TTX (purchased from Tocris Bioscience, Ellisville, MO, USA), all chemicals were purchased from Sigma-Aldrich (Dorset, UK).

Experimental procedure

Procedures for obtaining and analysing ICa and SR Ca2+ characteristics were adapted from established methods for fish preparations (Shiels et al., 2004; Shiels et al., 2006; Vornanen, 1997; Vornanen, 1998). A sample of cardiomyocytes, suspended in isolation solution, were added to the recording chamber and allowed to settle and attach to the bottom for ∼5 min. The cell chamber was superperfused at 23°C with extracellular solution containing TTX at a rate of ∼2 ml min–1. Voltage clamp experiments were conducted in the whole-cell configuration using a PC 505B amplifier (Warner Instruments, Hamden, CT, USA) with a 202B headstage (Warner Instruments). Micropipettes were made from borosilicate glass (Garner Glass, Claremont, CA, USA) using a P-87 pipette puller (Sutter Instrument Company, Novato, CA, USA) and had resistances of 2.35±0.03 MΩ when filled with pipette solution. Junction potentials were zeroed prior to seal formation, and pipette capacitance was compensated for once a gigaohm seal was achieved. Access to the cell was obtained by rupturing the membrane patch with a brief voltage pulse. Capacitive transients were compensated for by adjusting the series resistance and cell capacitance compensation circuits. Mean series resistance was 9.7±0.5 MΩ. Currents were low-pass filtered at a frequency of 2 kHz and analysed offline using Clampfit 9.2 (Axon Instruments, Sunnyvale, CA, USA).

Measurement of ICa

ICa was activated using the protocol illustrated in Fig. 1. Current–voltage relationships were elicited by depolarizing the cell from –80 mV to +60 mV using 500 ms square pulses in 10 mV increments. The amplitude of the L-type Ca2+ channel was calculated as the difference between the maximal inward current deflection and the current at the end of the depolarizing pulse. ICa was normalised for cell size by dividing ICa by cell capacitance to give ICa density in pA pF–1. Current–voltage curves were obtained at 15, 19 and 23°C by adjusting superfusate temperature through a temperature-controlled rapid solution changer (SC-20 in-line heater/cooler, CL-100 bipolar temperature controller; Warner Instruments). When possible, the effects of all three temperatures were measured in each cell. However, because of problems in maintaining a high quality seal (in the gigaohm range), some cardiomyocytes were only tested at one or two of the experimental temperatures. The order of temperature exposure was reversed (perfused first at 15°C and then warmed to 19 and 23°C) in some experiments, and this did not affect the results. Inactivation kinetics of ICa and the time constants of fast (τf) and slow inactivation (τs) were obtained by fitting a second-order (mackerel and tuna) or first-order (bonito, τ) exponential curve to the decaying portion of the ICa using the Chebyshev procedure (Clampfit software, Axon Instruments). Charge density of ICa (QCa) was calculated as the integral of the peak ICa for each current–voltage curve and is given in pC pF–1. This value was used to calculate the change in total cellular Ca2+ resulting from Ca2+ influx through L-type Ca2+ channels (Δ[Ca2+]) using cell volume (Vornanen, 1997). Cell volume was calculated from the measured cell capacitance and the surface:volume ratio of the cells. Cardiomyocytes were considered to be flat elliptical cylinders with an axis ratio of 1:2 for the elliptical cross section (Vornanen, 1997; Vornanen, 1998). Δ[Ca2+] was expressed as a function of non-mitochondrial volume (Vornanen, 1997; Vornanen, 1998).

Measurement of SR Ca2+ content

By rapidly changing between ports on the rapid solution changer, caffeine (10 mmol l–1) was briefly (∼1 s) administered to induce SR Ca2+ release. Ca2+ released by the SR is extruded from the cell via the NCX, resulting in an inward Na+ current (INCX). This Na+ current is proportional (3:1) to the Ca2+ released by the SR, and thus the time integral of the INCX can be used to calculate the Ca2+ released (in pC) by the SR (Varro et al., 1993). This value was adjusted for cell size by dividing it by the cell capacitance (pC pF–1). Integrating the INCX and cell volume gave total Ca2+ released by the SR (in μmol l–1). Cell volumes were calculated by extrapolating surface area measurements from cell capacitance (in pF) and using previously developed calculations for surface:volume ratios and specific membrane capacitance (Vornanen, 1997). Preliminary studies in each species and at each temperature showed that at least 25 pulses from –80 mV to 0 mV (500 ms) were necessary to achieve a steady-state SR Ca2+ load (ssSRload). Therefore, 25 pulses were used in all subsequent experiments when measuring ssSRload. Maximal SR Ca2+ load (maxSRload) was calculated as the caffeine-induced INCX after a square wave pulse from –80 to +50 mV for 6–9 s. ssSRload and maxSRload Ca2+ loads were measured in ventricular myocytes from all species at each test temperature (15, 19 and 23°C, as described in the previous section). Additionally, as SR dependence is known to be higher in atrial than in ventricular muscle, we measured ssSRload and maxSRload in atrial myocytes from one of the scombrid species, the bluefin tuna. This allowed assessment of the sensitivity of our SRload measurements.

Statistics

With the exception of original traces and voltage protocols, data are given as means ± s.e.m. N-values are the number of cells and are given in each figure legend. A one-way ANOVA with the Student–Newman–Keuls test was used to evaluate the effects of temperature on maximal and steady-state SR Ca2+ content, ICa density and ICa inactivation kinetics. Data were considered significantly different if P<0.05.

Fig. 1.

Effect of acute temperature changes on L-type Ca2+ channel current–voltage relationships in ventricular myocytes from the Pacific mackerel, bonito, yellowfin tuna and Pacific bluefin tuna. (A) Representative trace and voltage protocol for examining the temperature sensitivity of the L-type Ca2+ channel current (ICa) in a ventricular myocyte from the mackerel (cell capacitance was 34 pF). ICa was completely abolished with 2.5 μmol l–1 nifedipine (dotted line). (B–E) The current–voltage relationship for ICa for each scombrid. The voltage protocol is given in the inset between D and E. Red circles, 23°C; dark blue circles, 19°C, light blue circles, 15°C. Statistical analysis is discussed in the text. Values are means ± s.e.m. The number of cells examined at 23, 19 and 15°C, respectively were: bluefin tuna 13, 10, 9; mackerel 15, 16, 10; bonito 16, 17, 15; yellowfin tuna 20, 18, 17. Vm, membrane potential.

Fig. 1.

Effect of acute temperature changes on L-type Ca2+ channel current–voltage relationships in ventricular myocytes from the Pacific mackerel, bonito, yellowfin tuna and Pacific bluefin tuna. (A) Representative trace and voltage protocol for examining the temperature sensitivity of the L-type Ca2+ channel current (ICa) in a ventricular myocyte from the mackerel (cell capacitance was 34 pF). ICa was completely abolished with 2.5 μmol l–1 nifedipine (dotted line). (B–E) The current–voltage relationship for ICa for each scombrid. The voltage protocol is given in the inset between D and E. Red circles, 23°C; dark blue circles, 19°C, light blue circles, 15°C. Statistical analysis is discussed in the text. Values are means ± s.e.m. The number of cells examined at 23, 19 and 15°C, respectively were: bluefin tuna 13, 10, 9; mackerel 15, 16, 10; bonito 16, 17, 15; yellowfin tuna 20, 18, 17. Vm, membrane potential.

RESULTS

Cardiomyocyte size

The size of scombrid cardiomyocytes was compared by measuring the cell capacitance. Bluefin and yellowfin ventricular cardiomyocytes had a significantly smaller cell capacitance than the bonito and the mackerel (Table 1).

Table 1.

L-type Ca2+ channel charge density and inactivation kinetics from scombrid ventricular myocytes

L-type Ca2+ channel charge density and inactivation kinetics from scombrid ventricular myocytes
L-type Ca2+ channel charge density and inactivation kinetics from scombrid ventricular myocytes

Characterisation of ICa in scombrid cardiomyocytes

In all species a voltage step from –80 mV to 0 mV in the presence of TTX gave rise to a slow activating and inactivating ICa that varied in amplitude and kinetics according to temperature (Fig. 1A). To confirm this current originated from the L-type Ca2+ channel we applied nifedipine (2.5 μmol l–1), a specific L-type Ca2+ channel inhibitor, that completely abolished ICa (Fig. 1A dotted line).

ICa–voltage relationships and kinetics

The temperature dependence of the ICa–voltage relationships for each scombrid species tested are shown in Fig. 1B–E. Activation of ICa began at –40 mV, peaked at ∼0 mV and reversed at approximately –50 mV. Peak ICa amplitude was similar between mackerel and yellowfin tuna at each temperature tested (Fig. 1B,D). ICa inactivation kinetics for these species were also similar (Table 1), and as a result, QCa (the charge carried by ICa) was not significantly different between the two species over the range of temperatures tested (Table 1). Peak ICa density and QCa in bonito myocytes was significantly smaller than in bluefin tuna, yellowfin tuna and mackerel myocytes at all temperatures tested (Fig. 1B–D and Table 1). The decay of bonito ICa was better fit by a single exponential equation. Bluefin tuna peak ICa was larger than that of bonito, but significantly smaller than the mackerel and yellowfin tuna peaks (Fig. 1E). Furthermore, bluefin tuna ICa inactivation kinetics were generally slower than those of mackerel and yellowfin tuna, leading to a smaller QCa at 19 and 15°C (Fig. 1B–D, Table 1).

Effect of temperature on ICa–voltage relationships and kinetics

In this and subsequent sections, Q10 values have been calculated across the range of 23 to 15°C. In all species tested, acute reductions in temperature from 23 to 19 to 15°C led to a significant decrease in peak ICa density (Fig. 1). Although the temperature sensitivity of ICa in mackerel and yellowfin tuna was relatively high (Q10=3.1 and 2.7, respectively), a cold-induced slowing of ICa kinetics (τs and τf) allowed QCa to remain unaffected by acute temperature reductions (Q10=1.1 and 1.2, respectively; Table 1). In bluefin tuna, the cold-induced slowing of ICa kinetics was not significant, and as a result, reductions in temperature led to both a significant decrease in peak ICa (Q10=2.6) and QCa (Q10=1.8; Table 1). In bonito, although τ was significantly slower at cold temperatures, the dramatic effect of cold temperature on peak ICa (Q10=11.0) caused QCa to decline significantly (Q10=4.23; Table 1).

Steady state and maximal SRload

Atrial cardiomyocytes are known to more SR dependent than ventricular cardiomyocytes, relying more strongly on SR Ca2+ contributions. We assessed ssSRload and maxSRload in both ventricular and atrial cardiomyocytes from the bluefin tuna. Application of caffeine caused the cells to contract strongly and induced a large inward current (INCX), as shown in Fig. 2A (ventricular myocyte) and B (atrial myocyte). This current was completely abolished by pre-incubation with SR inhibitors (10 μmol l–1 ryanodine and 2 μmol l–1 thapsigargin) for 5 min (not shown). The time integral of this current, as shown in Fig. 2C (ventricular myocyte) and D (atrial myocyte), was used to assess SR Ca2+ content at the time of caffeine application at each temperature.

Under stead-state conditions, bluefin tuna atrial myocytes had significantly larger SRload values compared with ventricular myocytes (Fig. 2E,G), consistent with increased SR content in atrial cells. This result was also apparent under maximal conditions, but only significant at 23°C (Fig. 2F,H).

Having verified our technique, we went on to measure SRload in the other scombrid cardiomyocytes. ssSRload and maxSRload was greater in mackerel and bluefin ventricular myocytes compared with yellowfin tuna and bonito (Figs 2 and 3). This difference was found to be statistically significant at all temperatures tested, except at 19°C, where bluefin ssSRload was not significantly different from that of bonito (P>0.05). No significant differences in ssSRload or maxSRload were observed between mackerel and bluefin tuna, or between yellowfin tuna and bonito, at any temperature tested (Figs 2 and 3).

Effects of temperature on SRload

The ssSRload in ventricular and atrial cardiomyocytes from the bluefin tuna decreased significantly when the temperature was reduced from 21 to 15°C (Q10=2.1 and 2.2, respectively), and was more temperature sensitive within the upper temperature range (19–21°C; Fig. 2E,G). Ventricular maxSRload was significantly decreased between 21 and 15°C (Q10=1.8), whereas atrial maxSRload was only temperature sensitive at the lower temperature range (15–19°C; Fig. 2F,H). For the other scombrids, ssSRload in ventricular myocytes was only sensitive to temperature in bonito (Q10=1.3), whereas maxSRload decreased significantly with a temperature reduction from 21 to 15°C in the bonito and yellowfin (Q10=1.7 and 1.8, respectively) and was more temperature sensitive in the upper thermal range (Fig. 3).

DISCUSSION

The major findings from our study are, (1) the magnitude of ventricular ICa density does not correlate with endothermy, cardiovascular capacity or thermal niche expansion, (2) SR Ca2+ content is greater in the more cold-tolerant species (bluefin tuna and mackerel), and (3) bonito utilises a different E–C coupling strategy from mackerel and tunas.

Scombrid ICa–voltage relationships, kinetics

Species-specific variability in the relative contribution of ICa to force development has previously been demonstrated in scombrids and supports the hypothesis that differences in cardiac performance may be related to differences in ICa density (Shiels et al., 2004). However, in the present study ICa and QCa density were greatest in mackerel and yellowfin at all temperatures tested. This result suggests the magnitude of ICa density does not correlate with the evolution of cardiovascular capacity, endothermy or thermal niche expansion.

Fig. 2.

Effects of acute temperature change on steady-state and maximal sarcoplasmic reticulum Ca2+ content (SRload) in atrial and ventricular myocytes from the Pacific bluefin tuna. (A–D) Representative steady-state Na+/Ca2+ exchange current (INCX) recordings in pA (A,B), and corresponding charge transfer (integral of INCX) in pC (C,D). (E–H) Mean data (± s.e.m.) for ventricular and atrial steady-state and maximal SRload and corresponding charge transfer. *Difference between the ventricular and atrial myocytes. Significant effects of temperature on SRload within a species are shown by different letters, a23 vs 15, b23 vs 19, c19 vs 15°C. P<0.05, one-way analysis of variance, Student–Newman–Keuls post hoc test. The number of cells (N) at 21, 19 and 15°C, respectively, for steady-state SRload were: ventricle N=12, 10 and 8, atrium N=11, 7 and 6; for maximal SRload, ventricle N=10, 11, 9 and atrium N=6, 7, 6.

Fig. 2.

Effects of acute temperature change on steady-state and maximal sarcoplasmic reticulum Ca2+ content (SRload) in atrial and ventricular myocytes from the Pacific bluefin tuna. (A–D) Representative steady-state Na+/Ca2+ exchange current (INCX) recordings in pA (A,B), and corresponding charge transfer (integral of INCX) in pC (C,D). (E–H) Mean data (± s.e.m.) for ventricular and atrial steady-state and maximal SRload and corresponding charge transfer. *Difference between the ventricular and atrial myocytes. Significant effects of temperature on SRload within a species are shown by different letters, a23 vs 15, b23 vs 19, c19 vs 15°C. P<0.05, one-way analysis of variance, Student–Newman–Keuls post hoc test. The number of cells (N) at 21, 19 and 15°C, respectively, for steady-state SRload were: ventricle N=12, 10 and 8, atrium N=11, 7 and 6; for maximal SRload, ventricle N=10, 11, 9 and atrium N=6, 7, 6.

Interestingly, bonito peak ICa and QCa was dramatically lower than the other scombrids at all temperatures tested. In fact, peak ICa density in bonito ventricular myocytes tested at 23°C was three times and six times lower than that recorded in ventricular myocytes of trout (tested at 21°C and acclimated to 17°C) and carp (tested at 21°C and acclimated to 23°C) (Vornanen, 1997; Vornanen, 1998). Similar results on L-type Ca2+ channel characteristics found in bonito have been reported in burbot ventricular myocytes (Shiels et al., 2006). Although direct comparisons are difficult because of different experimental conditions, low ICa and QCa density in burbot was related to a smaller density of sarcolemmal L-type Ca2+ channels (Tiitu and Vornanen, 2003), whereas INCX density was robust and thought to be the primary pathway for sarcolemmal Ca2+ influx (Shiels et al., 2006). We propose that the NCX might be the primary pathway for sarcolemmal Ca2+ influx in the bonito, and might provide the majority of activator Ca2+ to the myofilaments (see SR discussion below).

Scombrid ICa temperature sensitivity

All scombrid fishes showed significant reductions in ICa with decreasing temperature. In the mackerel and yellowfin, the slowing of inactivation kinetics of ICa offset the negative effects of temperature, resulting in a temperature-insensitive Ca2+ flux (Q10=1.2 for mackerel, Q10=1.1 for yellowfin). This effect has been observed in most other teleosts examined to date (Shiels et al., 2000), and may be a common protective mechanism against the depressive effects of the cold. In contrast, the Ca2+ carried by ICa in the bluefin tuna and mackerel appears to be temperature sensitive with distinctly different affects (Q10=1.7 and 4.2, respectively). It is possible that temperature-induced alterations in heart rate and action potential shape and/or duration in these species may offset the negative effects of cold temperature on ICa (see Limitations of the study and future directions).

Fig. 3.

Effects of acute temperature change on steady-state and maximal sarcoplasmic reticulum Ca2+ content (SRload) in mackerel, yellowfin tuna, bonito and Pacific bluefin tuna ventricular myocytes. Here we have converted charge transfer (in pC) to the total change in intracellular Ca2+ (in μmol l–1) as explained in the Materials and methods. Data are means ± s.e.m. * A significant difference between mackerel and bluefin vs yellowfin and bonito. No significant differences were observed between yellowfin and bonito or bluefin and mackerel. Significant effects of temperature on SRload within a species are shown by different letters; a23 vs 15, b23 vs 19, c19 vs 15°C. P<0.05, one-way analysis of variance, Student–Newman–Keuls post hoc test. The number of cells (N) at 21, 19 and 15°C, respectively, steady-state SRload were: mackerel N=11, 13, 8; yellowfin tuna N=10, 13 and 10; bonito N=10, 8 and 9; bluefin tuna N=12, 10 and 8; for maximal SRload; mackerel N=12, 12 and 9; yellowfin tuna N=10, 12 and 11; bonito N=8, 9, 10, bluefin tuna N=10, 11 and 9.

Fig. 3.

Effects of acute temperature change on steady-state and maximal sarcoplasmic reticulum Ca2+ content (SRload) in mackerel, yellowfin tuna, bonito and Pacific bluefin tuna ventricular myocytes. Here we have converted charge transfer (in pC) to the total change in intracellular Ca2+ (in μmol l–1) as explained in the Materials and methods. Data are means ± s.e.m. * A significant difference between mackerel and bluefin vs yellowfin and bonito. No significant differences were observed between yellowfin and bonito or bluefin and mackerel. Significant effects of temperature on SRload within a species are shown by different letters; a23 vs 15, b23 vs 19, c19 vs 15°C. P<0.05, one-way analysis of variance, Student–Newman–Keuls post hoc test. The number of cells (N) at 21, 19 and 15°C, respectively, steady-state SRload were: mackerel N=11, 13, 8; yellowfin tuna N=10, 13 and 10; bonito N=10, 8 and 9; bluefin tuna N=12, 10 and 8; for maximal SRload; mackerel N=12, 12 and 9; yellowfin tuna N=10, 12 and 11; bonito N=8, 9, 10, bluefin tuna N=10, 11 and 9.

SR Ca2+ content across species and temperatures

The ssSRload in scombrid ventricular myocytes at 23°C ranged between 229 and 578 μmol l–1 Ca2+. These values are similar to recently published data from warm-acclimated (18°C) freshwater teleosts: ssSRload in ventricular myocytes from the rainbow trout, burbot and crucian carp ranged between 332 and 540 μmol l–1 at 18°C (Shiels et al., 2002b; Haverinen and Vornanen, 2009). Although these results suggest scombrid ssSRload is not exceptional among teleosts, the values presented here are three- to ninefold larger than those reported for rabbit (87 μmol l–1 at 22°C) and rat (64 μmol l–1 at 23°C) (Walden et al., 2009) ventricular myocytes. Furthermore, our values for maxSRload suggest scombrid ventricular myocytes can store up to 628 μmol l–1 of Ca2+ in the SR, which is tenfold larger than mammalian SRload under steady-state conditions. Thus, bluefin tunas and cold-tolerant active teleosts in general, appear to have an exceptional capacity to store Ca2+ in the SR.

The large SR Ca2+ storage capacity of fish compared with mammalian cardiomyocytes does not appear to relate to differences in total SR volume (DiMaio and Block, 2008; Shiels et al., 2011; Bowler and Tirri, 1990). Rather, Haverinen and Vornanen suggest functional differences in Ca2+ management within the SR results in a higher total Ca2+ capacity (Haverinen and Vornanen, 2009). For example, the fish ryanodine receptor appears to have a lower affinity for Ca2+ binding than mammals, which would stabilise SR Ca2+ content (Vornanen, 2006). Potentially the cooler ambient temperatures experienced by fish cardiomyocytes (compared with mammals) requires higher Ca2+ content to initiate and maintain Ca2+-release events (Haverinen and Vornanen, 2009). Support for this contention comes from recent imaging of intracellular Ca2+ in bluefin tuna myocytes, which shows both steady-state SR Ca2+ utilisation during E–C coupling and increased SR Ca2+ utilisation in the cold (Shiels et al., 2011).

Species-specific differences in SR Ca2+ content do show significant variations within the scombrid lineage, which provides some evidence for linkage between thermal tolerance and niche expansion. Ventricular steady-state and maximal SR Ca2+ loads decrease in the order of bluefin tuna>mackerel>yellowfin tuna>bonito, with no statistical difference between bluefin tuna and mackerel. As far as the bluefin tuna is concerned, our finding of a comparatively large SR Ca2+ storage capacity is consistent with previous studies indicating an enhanced role for the SR in E–C coupling. Ultrastructural studies have revealed a significant SR density in bluefin tuna cardiomyocytes (DiMaio and Block, 2008), and protein work has demonstrated enhanced expression of SERCA-2 compared with other scombrids (Landeira-Fernandez et al., 2004; Landeira-Fernandez et al., 2007). Most recently, fluorescent microscopy has confirmed that the bluefin tuna SR releases Ca2+ on a beat-to-beat basis, contributing activator Ca2+ for contraction (Shiels et al., 2011). Thus, the bluefin tuna SR has an enhanced capacity to store, release and reuptake Ca2+ from the cytosol, which contributes to the generation of increased cardiac force and frequencies of contraction in this species.

Also consistent with previous studies on the significance of the SR in scombrids, the yellowfin tuna had significantly smaller SRload values than the bluefin tuna. Isolated ventricular muscle from the yellowfin tuna has been shown to be unresponsive to SR inhibition with ryanodine and thapsigargin (Galli et al., 2008). Furthermore, SERCA-2 protein expression in the yellowfin tuna ventricle is significantly less than in the bluefin tuna (Landeira-Fernandez et al., 2004). Thus, the geographical distribution in warm warmer waters of the yellowfin tuna may not require the specialisations in E–C coupling that are seen in the bluefin tuna heart.

The equally large SR Ca2+ storage capacity of the mackerel and bluefin tuna was a somewhat surprising result in our study. Prior work suggested the Pacific mackerel has a lower expression of SERCA-2 than yellowfin, albacore and bluefin tuna (Landeira-Fernandez et al., 2004). However, ryanodine receptors have been identified in the mackerel, using western blot analysis (Castilho et al., 2007), and ryanodine has been shown to inhibit ventricular force of contraction by 20% (Shiels and Farrell, 2000). Although our results suggest SR Ca2+ loading was more temperature sensitive in the bluefin tuna and mackerel (higher Q10 values), ssSRload and maxSRload were larger at any given temperature than in the more tropical species, the bonito and yellowfin. A large and maintained capacity to store Ca2+ in the SR at cold temperatures may allow these species to maintain Ca2+ cycling and cardiac function during exploitation of cold waters.

Limitations of the study and future directions

Our study aimed at identifying differences between scombrids in the capacity of their cardiomyocytes to move Ca2+ through the L-type Ca2+ channel and store Ca2+ in the SR. By using square-wave pulses at a single frequency to measure ICa, we were able to isolate these differences without interspecific variation in action potential shape or frequency. However, temperature independently affects the frequency of contraction and action potential shape and/or duration, which in turn can have profound affects on ICa, (Shiels et al., 2000) and SR loading (Shiels et al., 2002b). Thus, future experiments should be aimed at assessing ICa properties with physiologically relevant frequencies of contraction and action potentials as the command waveform.

This study was supported by a grant from the National Oceanic and Atmospheric Administration to B.A.B. and by the generous support of the Monterey Bay Aquarium Foundation. G.L.J.G. was funded in part through a University of Manchester Wellcome Trust VIP award. We thank Charles Farwell, Jake Noguiera, Luis Rodriguez, Ty Brandt and the staff of the Tuna Research and Conservation Center and the Monterey Bay Aquarium for helping to maintain the captive scombrid collection. We thank Captains Ted Dunn, Norm Kagawa and Bruce Smith, and the crew of the F/V Shogun for their help collecting bluefin at sea. We are indebted to the owners of Mariculture Del Norte, Ensenada, Mexico for their cooperation and for the gift of Pacific bluefin tuna for this study. We thank the Mexican Government for allowing bluefin collection for research purposes. Deposited in PMC for release after 6 months.

REFERENCES

Aho
E.
,
Vornanen
M.
(
1999
).
Contractile properties of atrial and ventricular myocardium of the heart of rainbow trout (Oncorhynchus mykiss): effects of thermal acclimation
.
J. Exp. Biol.
202
,
2663
-
2677
.
Blank
J. M.
,
Morrissette
J. M.
,
Landeira-Fernandez
A. M.
,
Blackwell
S. B.
,
Williams
T. D.
,
Block
B. A.
(
2004
).
In situ cardiac performance of Pacific bluefin tuna hearts in response to acute temperature change
.
J. Exp. Biol.
207
,
881
-
890
.
Block
B. A.
,
Dewar
H.
,
Blackwell
S. B.
,
Williams
T. D.
,
Prince
E. D.
,
Farwell
C. J.
,
Boustany
A.
,
Teo
S. L. H.
,
Seitz
A.
,
Walli
A.
, et al. 
. (
2001
).
Migratory movements, depth preferences, and thermal biology of Atlantic bluefin tuna
.
Science
293
,
1310
-
1314
.
Block
B. A.
,
Teo
S. L. H.
,
Walli
A.
,
Boustany
A.
,
Stokesbury
M. J. W.
,
Farwell
C. J.
,
Weng
K. C.
,
Dewar
H.
,
Williams
T. D.
(
2005
).
Electronic tagging and population structure of Atlantic bluefin tuna
.
Nature
434
,
1121
-
1127
.
Boustany
A. M.
,
Matteson
R.
,
Castleton
M.
,
Farwell
C.
,
Block
B. A.
(
2010
).
Movements of pacific bluefin tuna (Thunnus orientalis) in the Eastern North Pacific revealed with archival tags
.
Prog. Oceanog.
86
,
94
-
104
.
Bowler
K.
,
Tirri
R.
(
1990
).
Temperature dependence of the heart isolated from the cold or warm acclimated perch (Perca fluviatilis)
.
Comp. Biochem. Physiol.
96
,
177
-
180
.
Brill
R.
(
1987
).
On the standard metabolic rates of tropical tunas, including the effect of body size and acute temperature change
.
Fish. Bull.
85
,
4254
-
4261
.
Brill
R. W.
,
Bushnell
P. G.
(
2001
).
Tunas: Physiology, Ecology and Evolution
(ed.
Block
B. A.
,
Stevens
E. D.
), pp.
79
-
112
.
San Diego, CA
:
Academic Press
.
Brill
R. W.
,
Dewar
H.
,
Graham
J. B.
(
1994
).
Basic concepts relevant to heat-transfer in fishes, and their use in measuring the physiological thermoregulatory abilities of tunas
.
Environ. Biol. Fishes
40
,
1573
-
5133
.
Brill
R. W.
,
Bigelow
K. A.
,
Musyl
M. K.
,
Fritsches
K. A.
,
Warrant
E. J.
(
2005
).
Bigeye tuna behavior and physiology and their relevance to stock assessments and fishery biology
.
Col. Vol. Sci. Pap. ICCAT
57
,
142
-
161
.
Carey
F. G.
,
Lawson
K. D.
(
1973
).
Temperature regulation in free-swimming bluefin tuna
.
Comp. Biochem. Physiol.
44
,
375
-
392
.
Carey
F. G.
,
Kanwisher
J. W.
,
Stevens
E. D.
(
1984
).
Bluefin tuna warm their viscera during digestion
.
J. Exp. Biol.
109
,
1
-
20
.
Castilho
P. C.
,
Landeira-Fernandez
A. M.
,
Morrisette
J. M.
,
Block
B. A.
(
2007
).
Elevated Ca2+ ATPase (SERCA2) activity in tuna hearts: comparative aspects of temperature dependence
.
Comp. Biochem. Physiol.
148A
,
124
-
132
.
Chow
S.
,
Kishino
H.
(
2010
).
Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): inconsistent implications from morphology, nuclear and mitochondrial genomes
.
J. Mol. Evol.
41
,
714
-
718
.
Clark
T. D.
,
Taylor
B. D.
,
Seymour
R. S.
,
Ellis
D.
,
Buchanan
J.
,
Fitzgibbon
Q. P.
,
Frappell
P. B.
(
2008
).
Moving with the beat: heart rate and visceral temperature of free-swimming and feeding bluefin tuna
.
Proc. Biol. Sci.
22
,
2841
-
2850
.
Davie
P.
,
Farrell
A. P.
(
1991
).
The coronary and luminal circulations of the myocardium of fishes
.
Can. J. Zool.
69
,
1993
-
2001
.
Dickson
K.
(
1995
).
Unique adaptations of the metabolic biochemistry of tunas and billfishes for life in the pelagic environment
.
Environ. Biol. Fishes
42
,
65
-
97
.
DiMaio
A.
,
Block
B. A.
(
2008
).
Ultrastructure of the sarcoplasmic reticulum in cardiac myocytes from Pacific bluefin tuna
.
Cell Tissue Res.
334
,
121
-
134
.
Driedzic
W. R.
,
Gesser
H.
(
1994
).
Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis, and low temperature
.
Physiol. Rev.
74
,
221
-
258
.
Farrell
A. P.
(
1991
).
From hagfish to tuna: a perspective on cardiac function in fish
.
Physiol. Zool.
64
,
1137
-
1164
.
Farrell
A. P.
,
Davie
P. S.
,
Franklin
C. E.
,
Johansen
J. A.
,
Brill
R. W.
(
1992
).
Cardiac physiology in tunas. 1. In vitro perfused heart preparations from yellowfin and skipjack tunas
.
Can. J. Zool.
70
,
1200
-
1210
.
Farwell
C. J.
(
2001
).
Tunas: Physiology, Ecology and Evolution
(ed.
Block
B. A.
,
Stevens
E. D.
), pp.
391
-
410
.
San Diego, CA
:
Academic Press
.
Galli
G.
,
Shiels
H.
,
Brill
R.
(
2008
).
Temperature sensitivity of cardiac function in pelagic fishes with different vertical mobilities: yellowfin tuna (Thunnus albacares), bigeye tuna (T. obesus), mahimahi (Coryphaena hippurus) and swordfish (Xiphias gladius)
.
Phys. Biochem. Zool.
82
,
280
-
290
.
Galli
G. L. J.
,
Lipnick
M. S.
,
Block
B. A.
(
2009
).
Effect of thermal acclimation on action potentials and sarcolemmal K+ channels from Pacific bluefin tuna cardiomyocytes
.
Am. J. Physiol.
297
,
502
-
509
.
Giovane
A.
,
Greco
G.
,
Maresca
A.
,
Tota
B.
(
1980
).
Myoglobin in the heart ventricle of tuna and other fishes
.
Experientia
36
,
219
-
220
.
Graham
J. B.
,
Dickson
K. A.
(
2004
).
Tuna comparative physiology
.
J. Exp. Biol.
207
,
4015
-
4024
.
Gunn
J.
,
Block
B. A.
(
2001
).
Tunas: Physiology, Ecology and Evolution
(ed.
Block
B. A.
,
Stevens
E. D.
), pp.
167
-
219
.
San Diego, CA
:
Academic Press
.
Haverinen
J.
,
Vornanen
M.
(
2009
).
Comparison of sarcoplasmic reticulum calcium content in atrial and ventricular myocytes of three fish species
.
Am. J. Physiol.
297
,
R1180
-
R1190
.
Hove-Madsen
L.
(
1992
).
The influence of temperature on ryanodine sensitivity and the force–frequency relationship in the myocardium of rainbow trout
.
J. Exp. Biol.
167
,
47
-
60
.
Keen
J. E.
,
Farrell
A. P.
,
Tibbits
G. F.
,
Brill
R. W.
(
1992
).
Cardiac physiology in tunas. 2. Effect of ryanodine, calcium, and adrenaline on force frequency relationships in atrial strips from skipjack tuna, Katsuwonus Pelamis
.
Can. J. Zool.
70
,
1211
-
1217
.
Keen
J. E.
,
Vianzon
D. M.
,
Farrell
A. P.
,
Tibbits
G. F.
(
1994
).
Effect of temperature and temperature acclimation on the ryanodine sensitivity of the trout myocardium
.
J. Comp. Physiol.
164B
,
438
-
443
.
Keen
J. E.
,
Brill
R. W.
,
Aota
S.
,
Farrell
A. P.
,
Randall
D. J.
(
1995
).
Cholinergic and adrenergic regulation of heart rate and ventral aortic pressure in 2 species of tropical tunas; Katsuwonus pelamis and Thunnus albacares
.
Can. J. Zool.
73
,
1681
-
1688
.
Kitagawa
T.
,
Kimura
S.
,
Nakata
H.
,
Yamada
H.
(
2004
).
Diving behaviour of immature, feeding Pacific bluefin tuna (Thunnus thynnus orientalis) in relation to season and area: the East China Sea and Kuroshio-Oyashio transition region
.
Fish. Oceanogr.
13
,
161
-
180
.
Kitagawa
T.
,
Boustany
A.
,
Farwell
C.
,
Williams
T. D.
,
Castleton
M. C.
,
Block
B. A.
(
2007
).
Horizontal and vertical movements of juvenile bluefin tuna (Thunnus orientalis) in relation to seasons and oceanographic conditions in the eastern Pacific Ocean
.
Fish. Oceanogr.
16
,
409
-
421
.
Landeira-Fernandez
A. M.
,
Morrisette
J. M.
,
Blank
J. M.
,
Block
B. A.
(
2004
).
Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel
.
Am. J. Physiol.
286
,
R398
-
R404
.
Lawson
G.
,
Castleton
M. C.
,
Block
B. A.
(
2010
).
Movements and diving behavior of Atlantic bluefin tuna (Thunnus thynnus) in relation to water column structure in the northwestern Atlantic
.
Mar. Ecol. Prog. Ser.
400
,
245
-
265
.
Linthicum
S. C.
,
Carey
F. G.
(
1972
).
Regulation of brain and eye temperatures by the bluefin tuna
.
Comp. Biochem. Physiol.
43A
,
425
-
433
.
Marcinek
D. J.
,
Bonaventura
J.
,
Wittenberg
B.
,
Block
B. A.
(
2001b
).
Oxygen affinity and amino acid sequence of myoglobins from endothermic and ectothermic fish
.
Am. J. Physiol.
280
,
1123
-
1133
.
Møller-Nielsen
T.
,
Gesser
H.
(
1992
).
Sarcoplasmic reticulum and excitation coupling at 10 and 20°C in rainbow trout myocardium
.
J. Comp. Physiol.
162
,
526
-
534
.
Rivaroli
L.
,
Rantin
F. T.
,
Kalinin
A. L.
(
2006
).
Cardiac function of two ecologically distinct Neotropical freshwater fish: curimbata, Prochilodus lineatus (Teleostei, Prochilodontidae), and trahira, Hoplias malabaricus (Teleostei, Erythrinidae)
.
Comp. Biochem. Physiol.
145A
,
322
-
327
.
Roberts
J.
,
Graham
J.
(
1979
).
Effects of swimming speed on the excess temperatures and activities of heart and red and white muscle in the mackerel, Scomber japonicus
.
Fish. Bull. NOAA
76
,
861
-
867
.
Shaefer
K.
,
Fuller
D.
,
Block
B. A.
(
2007
).
Movements, behavior, and habitat utilization of yellowfin tuna (Thunnus albacares) in the northeastern Pacific Ocean, ascertained through archival tag data
.
Mar. Biol.
152
,
503
-
525
.
Shiels
H. A.
,
Farrell
A. P.
(
1997
).
The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing Ca2+ to force development in isolated ventricular trabeculae from rainbow trout
.
J. Exp. Biol.
200
,
1607
-
1621
.
Shiels
H. A.
,
Farrell
A. P.
(
2000
).
The effect of ryanodine on isometric tension development in isolated ventricular trabeculae from Pacific mackerel (Scomber japonicus)
.
Comp. Biochem. Physiol.
125
,
331
-
341
.
Shiels
H. A.
,
Stevens
E. D.
,
Farrell
A. P.
(
1998
).
Effects of temperature, adrenaline and ryanodine on power production in rainbow trout Oncorhynchus mykiss ventricular trabeculae
.
J. Exp. Biol.
201
,
2701
-
2710
.
Shiels
H. A.
,
Freund
E. V.
,
Farrell
A. P.
,
Block
B. A.
(
1999
).
The sarcoplasmic reticulum plays a major role in isometric contraction in atrial muscle of yellowfin tuna
.
J. Exp. Biol.
202
,
881
-
890
.
Shiels
H. A.
,
Vornanen
M.
,
Farrell
A. P.
(
2000
).
Temperature dependence of L-type Ca2+ channel current in atrial myocytes from rainbow trout
.
J. Exp. Biol.
203
,
2771
-
2780
.
Shiels
H. A.
,
Vornanen
M.
,
Farrell
A. P.
(
2002a
).
Effects of temperature on intracellular Ca2+ in trout atrial myocytes
.
J. Exp. Biol.
205
,
3641
-
3650
.
Shiels
H. A.
,
Vornanen
M.
,
Farrell
A. P.
(
2002b
).
Temperature dependence of cardiac sarcoplasmic reticulum function in rainbow trout myocytes
.
J. Exp. Biol.
205
,
3631
-
3639
.
Shiels
H. A.
,
Blank
J. M.
,
Farrell
A. P.
,
Block
B. A.
(
2004
).
Electrophysiological properties of the L-type Ca2+ current in cardiomyocytes from bluefin tuna and Pacific mackerel
.
Am. J. Physiol.
286
,
659
-
668
.
Shiels
H. A.
,
Paajanen
V.
,
Vornanen
M.
(
2006
).
Sarcolemmal ion currents and sarcoplasmic reticulum Ca2+ content in ventricular myocytes from the cold stenothermic fish, the burbot (Lota lota)
.
J. Exp. Biol.
209
,
3091
-
3100
.
Shiels
H. A.
,
Thompson
S.
,
Block
B. A.
(
2011
).
Warm fish with cold hearts: thermal plasticity and excitation-contraction coupling in bluefin tuna
.
Proc. R. Soc. Lond. B Biol. Sci.
278
,
18
-
27
.
Teo
S. L.
,
Boustany
A.
,
Dewar
H.
,
Stokesbury
M. J.
,
Weng
K. C.
,
Beemer
S.
,
Seitz
A.
,
Farwell
C.
,
Prince
E. D.
,
Block
B. A.
(
2007
).
Annual migrations, diving behavior, and thermal biology of Atlantic bluefin tuna, Thunnus thynnus, on their Gulf of Mexico breeding grounds
.
Mar. Biol.
151
,
1
-
18
.
Tiitu
V.
,
Vornanen
M.
(
2003
).
Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences
.
J. Comp. Physiol. B
173
,
285
-
291
.
Varro
A.
,
Negretti
N.
,
Hester
S. B.
,
Eisner
D. A.
(
1993
).
An estimate of the calcium content of the sarcoplasmic reticulum in rat ventricular myocytes
.
Pflug. Arch.
423
,
158
-
160
.
Vornanen
M.
(
1997
).
Sarcolemmal Ca influx through L-type Ca channels in ventricular myocytes of a teleost fish
.
Am. J. Physiol.
41
,
1432
-
1140
.
Vornanen
M.
(
1998
).
L-type Ca2+ current in fish cardiac myocytes: effects of thermal acclimation and beta-adrenergic stimulation
.
J. Exp. Biol.
201
,
543
-
547
.
Vornanen
M.
(
2006
).
Temperature and Ca2+ dependence of [3H]ryanodine binding in the burbot (Lota lota L.) heart
.
Am. J. Physiol.
290
,
R345
-
R351
.
Vornanen
M.
,
Shiels
H. A.
,
Farrell
A. P.
(
2002
).
Plasticity of excitation-contraction coupling in fish cardiac myocytes
.
Comp. Biochem. Physiol.
132A
,
827
-
846
.
Walden
A. P.
,
Dibb
K. M.
,
Trafford
A. W.
(
2009
).
Differences in intracellular calcium homeostasis between atrial and ventricular myocytes
.
J. Mol. Cell. Cardiol.
46
,
463
-
473
.
Walli
A.
,
Teo
S. L.
,
Boustany
A.
,
Farwell
C.
,
Williams
T.
,
Dewar
H.
,
Prince
E. D.
,
Block
B. A.
(
2009
).
Seasonal movements, aggregations and diving behavior of Atlantic bluefin tuna (Thunnus thynnus) revealed with archival tags
.
PloS One
4
,
e651
.
Weng
K. C.
,
Stokesbury
M. J.
,
Boustany
A.
,
Seitz Teo
S. L.
,
Miller
S. K.
,
Block
B. A.
(
2009
).
Habitat and behaviour of yellowfin tuna Thunnus albacares in the Gulf of Mexico determined using pop-up satellite archival tags
.
J. Fish Biol.
74
,
1434
-
1449
.