IK1 measurements

Applying the whole cell patch clamp technique and established pulse protocols, I_K1 was recorded at room temperature (22 ± 1.5°C) from ventricular cardiomyocytes up to 8 hours after preparation using an Axoclamp 200B patch clamp amplifier (Axon Instruments, Union City, CA). Pipettes were formed from aluminosilicate glass (AF150-100-10; Science Products, Hofheim, Germany) with a P-97 horizontal puller (Sutter Instruments, Novato, CA). Their resistances were between 0.8 and 2 MΩ when filled with pipette solution (see below). Data acquisition was performed with pClamp 6.0 software (Axon Instruments) through a 12-bit A-D/D-A interface (Digidata 1200; Axon Instruments). Data were low-pass filtered with 1–10 kHz (−3 dB) and digitized at 10–100 kHz. Data analysis was performed using Clampfit 10.2 (Axon Instruments) and GraphPad Prism 5.01 (San Diego, USA) software. The cells were bathed in 140 mM NaCl, 4 mM KCl, 2 mM CaCl₂, 2 mM MgCl₂, 5 mM HEPES, 5 mM Glucose, pH = 7.4 (adjusted with NaOH). The pipette solution contained 10 mM NaCl, 140 mM KCl, 2 mM EGTA, 1 mM MgCl₂, 0.1 mM Na-GTP, 5 mM Mg-ATP, 10 mM HEPES, pH = 7.2 (adjusted with KOH). Potassium currents were elicited by 500-ms hyper- and depolarizing voltage steps between −140 and −30 mV from a holding potential of −100 mV (see inset in Fig. 1B). Around the resting membrane potential and at more hyperpolarized voltages, the ventricular I_K1 conductance is much larger than that of any other potassium current.²¹ In murine ventricular cardiomyocytes, currents through Kir2.1 channels represent the major component of I_K1 consistent with the finding that myocytes isolated from Kir2.1−/− mice completely lacked detectable whole cell I_K1 in 4 mM external potassium.¹⁹ A potential “contamination” of I_K1 by other inward rectifying potassium currents (I_K,ATP,³⁴ I_K,ACh,³⁵ and I_K,Ca³⁵) in our experiments was excluded by the composition of our experimental solutions. Thus, I_K,ATP was inhibited by the presence of mM concentrations of ATP in the pipette solution,^36,37 and activation of I_K,ACh was prevented by the lack of acetylcholine. I_K,Ca activation could be excluded because calcium channels do not activate at potentials more negative than −60 mV at which significant I_K1 was detectable. Further, calcium was absent from the pipette solution which additionally contained 2 mM of the calcium buffer EGTA (see above). For the determination of I_K1 density-voltage relations, the current amplitudes at the end of the test pulses to various potentials were measured. These were then divided by the cell capacitance to obtain current densities. I_K1 recordings from both wt and dystrophic cardiomyocytes were always started 5 min after whole cell access was attained to avoid potential artifacts due to time-dependent shifts in current properties.