Changes of Cytosolic Ca<SUP>2&#8290;</SUP> under Metabolic Inhibition in Isolated Rat Ventricular Myocytes

Changes of Cytosolic Ca<SUP>2&#8290;</SUP> under Metabolic Inhibition in Isolated Rat Ventricular Myocytes

To characterize cytosolic Ca<SUP>2&#8290;</SUP> fluctuations under metabolic inhibition, rat ventricular myocytes were exposed to 200μM 2,4-dinitrophenol (DNP), and mitochondrial Ca<SUP>2&#8290;</SUP>, mitochondrial membrane potential (ΔΨ<SUB>m</SUB>), and cytosolic Ca<SUP>2&#8290;</SUP> 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<SUP>&#8290;</SUP>/Ca<SUP>2&#8290;</SUP> exchange (NCX) in cytosolic Ca<SUP>2&#8290;</SUP> efflux was studied in KB-R7943 and Na<SUP>&#8290;</SUP>-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&#8273;10% within 70&#8273;10 s, and later by 400&#8273;200% at 850&#8273;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) Ca<SUP>2&#8290;</SUP> was depleted by 1μM thapsigargin plus 10μM 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 Ca<SUP>2&#8290;</SUP> overload, while Na<SUP>&#8290;</SUP>-free solution accelerated the second. The above results suggest that: 1) the initial rise in cytosolic Ca<SUP>2&#8290;</SUP> under DNP results from mitochondrial depolarization; 2) the secondary increase is caused by progressive Ca<SUP>2&#8290;</SUP> release from SR; 3) NCX plays an important role in transient cytosolic Ca<SUP>2&#8290;</SUP> shifts under metabolic inhibition with DNP.