Pclamp8

Ca²⁺ transients and sarcoplasmic reticulum Ca²⁺ content were assessed in cardiac ventricular myocytes isolated from 12‐week‐old male MK2^+/+ and MK2^−/− mice as described previously.⁴³ Briefly, myocytes were incubated with 10 μmol/L Fluo‐4 AM (Molecular Probes, Carlsbad, CA), then transferred to a perfusion chamber on the stage of a Zeiss LSM 510 microscope (Carl Zeiss AG, Jena, Germany), and perfused with a Tyrode solution. The perfusion chamber was fitted with bipolar platinum electrodes attached to a Grass SD9 stimulator and maintained at 37°C. To assess Ca²⁺ transients, myocytes were continuously field stimulated at a rate of 2 Hz. To assess the sarcoplasmic reticulum Ca²⁺ content, myocytes first received 10 conditioning pulses at 2 Hz to ensure that each cell had a similar activation history. Upon completion of the conditioning pulses, 10 μmol/L caffeine was applied to the myocyte for 10 seconds via a rapid solution switcher. Changes in free Ca²⁺ were measured in line scan mode with excitation at 488 nm and emission measured at 505 to 530 nm. Myocytes were scanned repeatedly along the length of the cell at 1.5‐ms intervals for 7 seconds. Sequential scans were stacked to create a two‐dimensional image. Image J (National Institutes of Health, Bethesda, MD) was used to visualize the Ca²⁺ transients, and the data were analyzed with pCLAMP 8.2 (Molecular Devices).

Calcium channel currents were recorded with either an EPC7 (HEKA Electronik) or an Axopatch 200 (Molecular Devices) amplifier. Data were sampled at 20 kHz and filtered at 10 kHz (−3 dB) using either PatchMaster (HEKA Electronik) or pCLAMP8.2 (Molecular Devices) software. Access resistance and input resistance were monitored by a step of −10 mV. Recordings in which the access resistance increased by >20% were discarded. Leak current was subtracted online using a P/−4 protocol, and recordings with leak currents over 150 pA at holding potential were discarded.