Changes of cytosolic Ca2+ under metabolic inhibition in isolated rat ventricular myocytes

To characterize cytosolic Ca2+ fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200 μM 2,4-dinitrophenol (DNP), and mitochondrial CaT+, mitochondrial membrane potential (Δψm), and cytosolic Ca2+ were measured, using Rhod-2 AM, TMRE, and Fluo-4 AM fluorescent dyes, respectively, by Laser Scanning Confocal Microscopy (LSCM). Furthermore, the role of sarcolemmal Na+/Ca2+ exchange (NCX) in cytosolic Ca2+ efflux was studied in KB-R7943 and Na+-free normal Tyrode's solution (143 mM LiCl). When DNP was applied to cells loaded with Fluo-4 AM, Fluo-4 AM fluorescence intensity initially increased by 70 ± 10% within 70 ± 10 s, and later by 400 ± 200% at 850 ± 46 s. Fluorescence intensity of both Rhod-2 AM and TMRE were initially decreased by DNP, coincident with the initial increase of Fluo-4 AM fluorescence intensity. When sarcoplasmic reticulum (SR) Ca2+ was depleted by 1 μM thapsigargin plus 10 μM ryanodine, the initial increase of Fluo-4 AM fluorescence intensity was unaffected, however, the subsequent progressive increase was abolished. KB-R7943 delayed both the first and the second phases of cytosolic Ca2+ overload, while Na+-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca2+ under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca2+ release from SR; 3) NCX plays an important role in transient cytosolic Ca2+ shifts under metabolic inhibition with DNP.