Dynamic modulation of Ca2+ sparks by mitochondrial oscillations in isolated guinea pig cardiomyocytes under oxidative stress.

All protocols involving animals conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996) and were approved by the Johns Hopkins Animal Care and Use Committee.

Cardiomyocyte isolation and loading of fluorescent probes

All experiments were carried out at 37 °C on freshly isolated adult guinea pig ventricular myocytes prepared by enzymatic dispersion as previously described.[ 18 ] Briefly, animals of either sex were anaesthetized with sodium pentobarbital (30 mg/kg I.P.). Following thoracotomy, hearts were quickly excised, mounted on a Langendorff apparatus, and perfused with collagenase-containing solution at 37°C. After isolation, cells were stored in a high K + solution (in mmol/L: 120 Glutamate, 25 KCl, 1 MgCl 2 , 10 HEPES, 1 EGTA, and pH 7.5 with KOH) temporarily. The cationic potentiometric fluorescent dye tetramethyl rhodamine methyl ester (TMRM) was used to monitor changes in ΔΨ m . ROS production was monitored with MitoSOX (Invitrogen), a superoxide-sensitive fluorescent indicator. The localizations of TMRM and MitoSOX within the mitochondria were shown in the supplemental materials ( figure S1 ). To image the distribution of ΔΨ m (or ROS) and Ca 2+ simultaneously, 100 nM TMRM (or 2 µmol/L MitoSOX) and 4 µmol/L fluo-4 AM were added to the external solution and allowed to equilibrate for at least 25 min at 37°C. After loading, the cells were resuspended in the experimental solution for 20 min to permit de-esterification of the dye before recording images. To monitor the intracellular reduced glutathione (GSH) and Δψ m simultaneously, cells were loaded with 50 µmol/L monochlorobimane (MCB) and TMRM as described previously [ 14 ].

Image Acquisition and Analysis

The dish containing the cardiomyocytes was equilibrated at 37°C with unrestricted access to atmospheric oxygen on the stage of a Nikon E600FN upright microscope. Images were recorded using a two-photon laser scanning microscope (Bio-Rad MRC-1024MP) with excitation at 760 nm (Tsunami Ti:Sa laser, Spectra Physics) as described previously [ 15 ]. Because of the overlap in the cross-sections for two-photon excitation of the three fluorophores of interest (NADH or GSB, Fluo-4, and TMRM or MitoSOX), this wavelength permitted recording of redox, Ca 2+ , and ΔΨ m or ROS simultaneously. A three channel photomultiplier detector assembly with appropriate dichroic mirrors and bandpass filters was used to separate the fluorescence emissions of the blue (