Axograph software

Isolated cardiomyocytes were impaled with double-barreled Ca²⁺-selective microelectrodes for the determination [Ca²⁺]_d, and the potentials were recorded via a high-impedance amplifier (WPI 773 electrometer, FL, United States) as described previously (Lopez et al., 2000 (link); Lopez et al., 2017 (link)). The potential from the 3M KCl microelectrode barrel (V_m) was subtracted electronically from the potential recorded by the Ca²⁺ ion-selective barrel (V_CaE) to produce a differential Ca²⁺-specific potential (V_DiffCa), which represent the cardiomyocyte [Ca²⁺]_d. V_m and V_DiffCa potentials were acquired at a frequency of 1,000 Hz with AxoGraph software (version 4.6; Axon Instruments, CA, United States) and stored in a computer for further analysis. The following criteria were used to accept individual cardiomyocytes [Ca²⁺]_d measurements from control and db/db mice: 1) a quick drop to a steady level of Vm; 2) a stable recording potential (V_m and V_DiffCa) for no less than 60 s; 3) a quick return to baseline of Vm and V_DiffCa potential on the exit of the microelectrodes from the cell. These criteria were not met in 15% of the total impalements carried out in control and 25% in db/db cardiomyocytes, and these data were rejected from our analysis.

Double-barreled Ca²⁺-selective microelectrodes were prepared from thin-walled borosilicate with 1.2 and 1.5 mm outside diameter (OD) (PB150F-4, World Precision Instruments, Sarasota, FL, USA). The ion-selective barrel (1.5-mm OD) was silanized with dimethyldichlorosilane vapor and then backfilled with the Ca²⁺ ionophore II (ETH 129) Sigma-Aldrich in Saint Louis, MO, USA. The remaining portion of the ion-selective barrel was backfilled with pCa7, as previously described in [10 (link)]. The resting membrane potential (RMP) barrel (1.2-mm OD) was backfilled with 3 M KCl before the measurements. The RPM- and Ca²⁺-specific potentials were acquired at a frequency of 1000 Hz using AxoGraph software (version 4.6; Axon Instruments, San Jose, CA, USA), then and stored on a computer for further analysis. Before and after each measurement, individual calibration of the Ca²⁺-selective microelectrodes was performed by following the previously described protocol [10 (link)]. If the calibration curves obtained before and after the measurement differed by more than 3 mV, the data from that microelectrode were excluded from further analysis [10 (link)].

Double-barreled, Ca²⁺ selective microelectrodes were prepared from thin-walled 1.2 and 1.5 mm outside diameter (OD) borosilicate HCl-washed glass capillaries (PB150F-4, World Precision Instruments, FL, United States). The tip of the ion-selective barrel (1.5 mm OD) was silanized with dimethyldichlorosilane vapor and then filled with Ca²⁺ ionophore II (ETH 129, Sigma-Aldrich, MO, United States); the remainder of the barrel was filled with pCa7 (Eltit et al., 2013 (link)). The membrane potential barrel (1.2 mm OD) was filled with 3 M KCl. The double-barreled Ca²⁺ microelectrode was mounted on a modified plastic holder containing Ag/AgCl wires, which was attached to a head stage (input impedance >1011Ω), connected to a Duo 773 electrometer (World Precision Instruments, FL, United States). The Vm and Ca²⁺ specific potentials were acquired at a frequency of 1,000 Hz with the AxoGraph software (version 4.6; Axon Instruments, CA, United States) and stored on a computer for further analysis. Each Ca²⁺ selective microelectrode was individually calibrated before and after measurements, as previously described, and if the two calibration curves did not agree within 3 mV, the data from that microelectrode were discarded (Lopez et al., 1983 (link); Eltit et al., 2013 (link)).