Isolation of single PV cardiomyocytes
All experiments were performed according to institutional guidelines. Male cardiomyopathic hamsters (Bio14.6) and normal F1B hamsters purchased from Bio Breeders Inc. (Fitchburg, MA, USA) and aged 36 to 57 weeks, were used for the experiments. Hamsters were anesthetized with intraperitoneal injection of sodium pentobarbital (50 mg/kg). A mid-line thoracotomy was quickly performed along with removal of heart and lungs. The PVs were perfused in a retrograde manner via polyethylene tube passed through the aorta and left ventricle into the left atrium. The proximal end of the polyethylene tubing was connected to a Langendorff apparatus for perfusion with oxygenated Tyrode's solution at 37°C. The Tyrode's solution contained (in mmol/L) NaCl 137, KCl 5.4, CaCl2 1.8, MgCl2 0.5, HEPES 10 and glucose 11 (pH was adjusted to 7.4 with NaOH) for about 15 minutes until efferent fluid was without blood.
The perfusate was then replaced with oxygenated Ca2+-free Tyrode's solution containing 1 mg/ml collagenase (Sigma, Type I) and 0.01 mg/ml protease (Sigma, Type XIV) for 30-40 minutes. Afterwards, the heart was washed with oxygenated Ca2+-free Tyrode's solution for 10 minutes. After that, the LA-PV area was removed from the heart, cut into fine pieces and gently shaken in 5-10 ml high-K+ storage solution until single cardiomyocytes were obtained.
Cellular electrophysiology
Only LA-PV cardiomyocytes with clear cross striations obtained from 19 myopathic hamsters and 22 healthy hamsters were used for electrophysiological studies. Action potentials and ionic currents were recorded by means of whole-cell patch-clamp techniques with an Axopatch 1D amplifier (Axon Instruments, CA, U.S.A) as described in detail recently [11, 13, 15]. The standard pipette solution contained (in mmol/L) KCl 20, K aspartate 110, MgCl2 1, EGTA 0.5, Mg2ATP 5, Na2phosphocreatine 5, LiGTP 0.1, and HEPES 10, adjusted to pH 7.2 with KOH. The standard extracellular solution was the normal Tyrode's solution also used in cell isolation. Ionic currents were recorded in voltage-clamp mode. For the recording of the calcium currents ICa,L and ICa,T, tetraethylammonium (TEA) chloride and CsCl replaced NaCl and KCl, respectively. For ICa,T recording, tetrodotoxin (5 μmol/L) was added to block the fast Na+ current (INa). For K+ current measurement, CdCl2 (200 μmol/L) was added to block ICa,L. A 30-ms prepulse from -80 to -40 mV was used to inactivate the sodium channel, followed by a 300-ms test pulses in 10-mV increments to +60 mV. IK1 was quantified as 1 mmol/L Ba2+-sensitive current.
Action potentials (APs) were recorded in current-clamp mode. The tip potentials were zeroed before formation of the membrane-pipette seal in Tyrode's solution. After rupture, junction potential (8 mV) was corrected for AP recording. A small hyperpolarizing step from a holding potential of -50 mV to a testing potential of -55 mV was used to obtaining the total cell capacitance at the beginning of each experiment. The area under the capacitative current was divided by the applied voltage step to obtain the total cell capacitance. Normally 60-80% of series resistance (RS) was electronically compensated. After compensation, the average time constant was 71 ± 6 μs, (cell capacitance 33 ± 2 pF, for 83 cells in healthy group; and 47 ± 3 pF for 67 cells in myopathic group). The average RS was 1.8 ± 0.1 MΩ for all cells examined. Currents rarely exceeded 1.2 nA, and the maximal voltage error did not exceed 3 mV.
Voltage command pulses were generated by a 12-bit digital-to-analog converter controlled by pCLAMP software (Axon Instruments). APs were elicited by pulses of 2 ms and 90 mV at a rate of 1 Hz. AP measurements were begun 3 minutes after cell membrane rupture, and the steady-state AP duration was measured at 20% (APD20), 50% (APD50) and 90% (APD90) of full repolarization. Depolarization-induced currents included the transient outward K+ current (ITO) and the delayed rectifier outward K+ current (IK), and were elicited by applying steps from a holding potential (Vh) of -40 mV in 10 mV increment up to +60 mV at a frequency of 0.1 Hz.
In recording calcium currents, to minimize the effect of "rundown" [13], the depolarizing steps were applied from a holding potential which was alternated between -90 mV and -50 mV. Also the steps were applied between 5 and 15 minutes after rupturing the membrane patch. ICa,L was measured as inward current during depolarizing 300 ms steps applied from the holding potential of -50 mV in 10 mV increments up to +60 mV. ICa,T was separated from ICa,L by subtraction of currents elicited by 300 ms depolarizing steps from holding potentials of -50 and of -90 mV in increments of 10-mV up to +60 mV. The calcium currents were measured as the difference between the inward peak and the current remaining at the end of the voltage step.
The background current IK1 was measured during steps from holding potential of -40 mV in 10 mV increments to test potentials ranging from -20 to -120 mV at a frequency of 0.1 Hz. The holding potential of -40 mV was used to inactivate the sodium channel. For measurement of Na+-Ca2+ exchange current (NCX), the external solution (in mM) consisted of NaCl 140, CaCl2 2, MgCl2 1, HEPES 5, and glucose 10 with a pH of 7.4, and contained strophanthidin (4 μM) nitredipine (10 μM) and niflumic acid (15μM). Test steps were applied from a holding potential of -40 mV to test potentials of -100, -80, -60, -40, -20, 0, +20, +40, +60, +80 and +100 mV.
Statistics
All quantitative data are expressed as mean ± S.E.M. The differences between healthy and myopathic cardiomyocytes were analyzed by one-way ANOVA. The χ2 test with Yates' correction of Fisher's exact test was used for the categorical data. A value of p < 0.05 was considered to be statistically significant.