Isolation of ventricular myocytes. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals [20]. Male and female C57Bl6/J mice, (11 of male and 8 of female animals, Jackson Laboratories) were housed according to approved IACUC guidelines. Mice aged between 2–3 months were anesthetized using Isoflurane (1%). Following thoracotomy, hearts were quickly excised, immersed in Ca2+ free buffer, mounted on a Langendorff apparatus, and retrogradely perfused with the zero Ca2+ Tyrode buffer (in mM: NaCl 140; KCl 4; MgCl2 1; glucose 10; Hepes 10; pH 7.4). (37°C) containing Liberase H (Roche), according to a procedure described previously [9, 14]. The left ventricle was excised from the digested heart, placed in stop buffer containing BSA 1 mg/mL, cut into several pieces (average size 1 mm) and gently triturated into single cells. Myocytes were pelleted by gravity and resuspended in the low-Ca2+ Tyrode buffer (in mM: NaCl 140; KCl 4; CaCl2 0.1; MgCl2 1; glucose 10; Hepes 10; pH 7.4). [Ca2+] was gradually adjusted to 1 mM. Isolated cardiomyocytes were stored at room temperature (20°C). All chemicals and reagents were purchased from Sigma-Aldrich (St Louis, USA).
Measurement of [Ca 2+ ] i . Changes in the cytosolic [Ca2+] ([Ca2+]i) were measured with laser scanning confocal microscopy (Radiance 2000 MP, Bio-Rad, UK) equipped with a ×40 oil-immersion objective lens (N.A.=1.3) as described previously [38, 39]. To record [Ca2+]i, we used the high-affinity Ca2+ indicator Fluo-4 (Molecular Probes/Invitrogen, Carlsbad, CA, USA). To load the cytosol with Fluo-4, ventricular myocytes were incubated at room temperature with 10 µM Fluo-4 AM for 15 min in Tyrode solution (in mM: NaCl 140; KCl 4; CaCl2 1; MgCl2 1; glucose 10; Hepes 10; pH 7.4), followed by a 20 min wash. Fluo-4 was excited with the 488 nm line of an argon laser and the emission signal collected at wavelengths above 515 nm. Changes in [Ca2+]i were expressed as changes in F/F0, where F0 is the Fluo-4 signal at the resting condition.
Ca2+ spark measurements were conducted in permeabilized ventricular myocytes as described previously [38–40]. After the surface membrane permeabilization with saponin, Ca2+ sparks were studied in an internal solution composed of (in mM): K-aspartate 100; KCl 15; KH2PO4 5; MgATP 5; EGTA 0.35; CaCl2 0.1; MgCl2 0.75; phosphocreatine 10; HEPES 10; Fluo-4 pentapotassium salt 0.04 mM; dextran (MW: 40,000) 8%, and pH 7.2 (KOH). Free [Ca2+] and [Mg2+] in this solution were 100 nM and 1 mM, respectively. Sparks were detected and analyzed using the SparkMaster algorithm [27].
Measurement of [Na + ] i . Changes in [Na+]i were monitored using inverted fluorescence microscope (Nikon Ti2 Eclipse) equipped with air immersion 40x objective and Lumencore Spectra X excitation system. The fluorescent sodium indicator SBFI/AM (Molecular Probes/Invitrogen, Carlsbad, CA, USA) was used to trace the Na+-dependent fluorescent signal. To load the cytosol with SBFI, ventricular myocytes were incubated at room temperature with 20 µM SBFI/AM for 30 min in Tyrode solution, followed by a 20 min wash. The SBFI signal was detected using the stream acquisition tool in Metamorph software (Molecular Devices) with 100 ms excitation (excitation 325/25 nm, emission 530 nm). Emitted light was passed through a dichroic emission filter cube (DAPI/GFP/TagRFP Spectra X emission filter set) and detected with Photometrics Prime 95B 25 mm camera. Two-dimensional images were obtained at 15 s intervals. Changes of [Na+]i are presented as background-subtracted normalized fluorescence (1-F/F0). The experiments were conducted in Tyrode solution. All 2-D images and line scan measurements for [Ca2+]SR were analyzed with ImageJ software (NIH, USA).
Preparation of purified NKA. A purified sodium pump was prepared from the pig's outer medulla as described previously [23]. The SDS-solubilized microsomal membranes were separated using differential ultracentrifugation and the final protein was aliquoted at a concentration of 1 mg/ml and stored at -80°C.
Measurements of NKA-specific ATPase activity. NKA ATPase activity was determined by the Baginsky method as described previously [3]. The method detects the presence of inorganic phosphate in the sample by measuring absorbance at 710 nm. Specifically, 2 µl of purified NKA was mixed with 48 µl ATPase buffer (in mM: Na-ATP 3.3, NaCl 192, MgCl2 4, KCl 20; pH 7.2) and incubated for 6 minutes at room temperature. The inorganic phosphate was stained with 75 µl of staining solution (160 mM ascorbic acid, 3.7% (v/v) HCl, 3% (w/v) SDS, and 0.5% ammonium molybdate) and the reaction was stopped after 8 minutes by addition of 125 µl of stopping solution (0.9% (w/v) bismuth citrate, 0.9% (w/v) sodium citrate and 3.7% HCl). Immediately after stopping the reaction, the plate was read using the microplate reader Clario Star (BMG Labtech, Germany). The calibration line was determined using the set of KH2PO4 solutions with concentrations ranging between 0-37.5 nM. To estimate the NKA-specific ATPase activity, the signal in the presence of the NKA inhibitor ouabain (50 µM) was subtracted from the total ATPase activity.
Statistics. Data are presented as mean ± standard error of the mean (SEM) of n measurements. Statistical comparisons between groups were performed using Student's t test for unpaired data sets. Differences were considered statistically significant at P < 0.05. Statistical analysis and graphical representation of averaged data was carried out using the OriginPro7.5 software (OriginLab, USA).