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SUPPLEMENTAL METHODS
(a) Myocyte isolation
Full methods for myocyte isolation have been previously described [1]. Briefly, fish were
sacrificed by destruction of the brain via pithing. The heart (mean mass, 30.7 ± 4.9 g, n = 5) was quickly excised, and retrograde perfused through the bulbus arteriosus with isolating solution from a height of 50 cm for 20 min. The coronary artery was also perfused. Proteolytic enzymes were then added to the isolating solution and retrograde luminal perfusion was continued for
35-40 min (depending on heart size). After enzymatic treatment, the ventricle was dissected from the rest of the heart, placed in a separate dish containing fresh isolating solution, cut into small pieces and then triturated through the opening of a Pasteur pipette to free individual myocytes. Myocytes were stored in fresh isolating solution for up to 8 hr at the acclimation temperature of 14 o C. The isolation solution contained (in mmol): 100 NaCl, 10 KCl, 1.2 KH
2
PO
4
,
4 MgSO
4
, 50 taurine, 20 glucose, and 10 Hepes, adjusted to pH 6.9 with KOH. For enzymatic digestion, 0.75 mg ml -1 collagenase (Type 1A), 0.25 mg ml -1 trypsin (Type IX) and 0.75 mg ml -1 fatty-acid bovine serum albumin (BSA) were added to this solution.
(b) Confocal microscopy
An Olympus 100x water-immersion objective was used in all measurements. The pin-hole
aperture was set to the size of the Airy disk to optimize z-axis resolution. In accordance with [2,
3] fluorescence (F)/ background fluorescence (Fo) was converted to Δ[Ca
2+ ] i
by [Ca 2+ ] i
=
K d
(F/Fo)/(K d
/[Ca 2+ ] i-rest
+1-F/Fo), with [Ca 2+ ] i-rest
at 100 nM which is close to the resting [Ca 2+ ] i as
calibrated by in vivo ratiometric (Fura-2) recordings for rainbow trout cardiomyocytes [4]. The
K d
of Fluo-4 is temperature sensitive [5] and thus the K
d
measured in vivo at room temperature
in rat myocytes [3] was adjusted for the in vivo temperature dependence reported by [5] to
provide K d
’s of 829 nM at 28 °C, 1594 at 14 °C and 1922 nM at 8 °C for calibration of the Ca 2+ signals in the current study. In some myocytes, there were intense, localized and time invariant fluorescent signals detected. As these probably represent dye accumulating in intracellular compartments these areas where avoided when focusing the line scans. The position of the transverse line scan was moved slightly over the course of the experiment to avoid bleaching.
Ca 2+ transient amplitude, transient rise time, and rate of rise were measured between diastolic and peak [Ca 2+ ] i
. Decay time was calculated as the time taken for the transient to decay by 63%

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(i.e. tau) from the peak. Rate of decay is calculated as the straight line regression over this time.
This method was preferred over calculating single exponential fits to the decay as the fit was not always faithful at the colder temperatures.
(b) Electrophysiology
Ventricular myocytes were placed in a recording chamber (RC-24, Warner Instruments) and superfused with same extracellular solution as those used for the confocal imaging.
Experiments were performed using a Warner Instruments patch-clamp amplifier, PC-505B, and a
202B headstage. Pipettes had a resistance of 2.7 ± 0.2 when filled with pipette solution.
Junction potentials were zeroed prior to seal formation. Pipette capacitance was compensated after formation of a GΩ seal. Mean series resistance was 10.1 ± 0.5 MΩ. Membrane capacitance was measured using the calibrated capacity compensation circuit of the amplifier.
Signals were low-pass filtered (2 kHz), and analyzed off-line using pClamp 9.0 software (Axon
Instruments). APs from isolated myocytes were recorded in current-clamp mode of the amplifier. APs were elicited by 2 ms depolarizing voltage pulses (0.1-0.7 mV).
L-type Ca 2+ channel currents (I
Ca
) were recorded in voltage-clamp configuration. Tetrodotoxin
(TTX) (0.5 μM), CsCl and tetraethylammonium (TEA) chloride were incorporated into the extracellular solution to block fast Na + channels and K + channels, respectively. 1 μM isoprenaline was used to assess the effect of adrenergic stimulation on I
Ca.
Isoprenaline is a synthetic analogue of adrenaline. The doses of isoprenaline (1 μM) and adrenaline (500 nM) used
throughout this study should both provide near maximal stimulation (see [6]; [7]).
Current-voltage relationships were elicited by depolarizing the cell from -80 mV to +60 mV using
500 ms square pulses in 10 mV incremental increases. The amplitude of the L-type Ca 2+ channel was calculated as the difference between the maximal inward current deflection and the current at the end of the depolarizing pulse. I
Ca
was normalised for cell size by dividing I
Ca
by cell capacitance to give I
Ca
density in pA pF -1 . Inactivation kinetics of I
Ca
, tau fast (τ f
) and tau slow (τ s
), were obtained by fitting a second-order exponential curve to the decaying portion of the I
Ca using the Chebyshev procedure (Clampfit software, Axon Instruments). Charge density of I
Ca
(Q
Ca
) was calculated as the integral of the peak I
Ca
for each current-voltage curve and given in pC pF -1 . This value was used to calculate the change in total cellular Ca 2+ due to Ca 2+ influx through

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L-type Ca 2+
channels using cell volume [8]. Cell volume was calculated from the measured cell
capacitance and the surface-to-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. Ca 2+ entry on I
Ca
was expressed as a function of myofibrillar volume [8]. AP upstroke velocity (mV
.
ms -1 ) was calculated as ΔVm (mV)/Δt (ms) where t is time. Steady-state kinetic parameters were obtained by fitting steady-state activation and inactivation data to Boltzmann equations to determine the half-activating or half-inactivating potential (Vh) and the slope of activation and inactivation (k), as previously described [6] and shown in supplemental figure 1 and Table 3. The overlap of the activation and inactivation curves was used to calculate the non-inactivating component of I
Ca
, known as the window current [6] shown in figure 5.
1. Shiels H.A., Blank J.M., Farrell A.P., Block B.A. 2004 Electrophysiological properties of the Ltype Ca 2+ current in cardiomyocytes from bluefin tuna and Pacific mackerel. Am J Physiol
RegulIntegrComp Physiol 286(4), R659-R668.
2. Cheng H., Lederer W.J., Cannell M.B. 1993 Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science 262(5134), 740-744.
3. Loughrey C.M., MacEachern K.E., Cooper J., Smith G.L. 2003 Measurement of the dissociation constant of Fluo-3 for Ca2+ in isolated rabbit cardiomyocytes using Ca 2+ wave characteristics.
Cell Calcium 34(1), 1-9.
4. Shiels H.A., Vornanen M., Farrell A.P. 2002 Effects of temperature on intracellular [Ca 2+ ] in trout atrial myocytes. J Exp Biol 205(Pt 23), 3641-3650.
5. Woodruff M.L., Sampath A.P., Matthews H.R., Krasnoperova N.V., Lem J., Fain G.L. 2002
Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice. J Physiol 542, 843-854.
6. Vornanen M. 1998 L-type Ca 2+ current in fish cardiac myocytes: Effects of thermal acclimation and beta-adrenergic stimulation. J Exp Biol 201, 533-547.
7. Shiels H.A., Vornanen M., Farrell A.P. 2003 Acute temperature change modulates the response of I Ca to adrenergic stimulation in fish cardiomyocytes. Physiol Biochem Zool 76,
816-24.
8. Vornanen M. 1997 Sarcolemmal Ca influx through L-type Ca channels in ventricular myocytes of a teleost fish. Am J Physiol 41, R1432-R1440.

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