Heterogeneity of mitochondrial matrix free Ca2+:: resolution of Ca2+ dynamics in individual mitochondria in situ

The role of mitochondria in Ca2+ homeostasis is controversial. We employed the Ca2+-sensitive dye rhod 2 with novel, high temporal and spatial resolution imaging to evaluate changes in the matrix free Ca2+ concentration of individual mitochondria ([Ca2+](m)) in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 mu M serotonin (5-HT) evoked modest cytosolic Ca2+ transients [cytosolic free Ca2+ concentration ([Ca2+](cyt)) <500 nM; measured with fura 2] and triggered contractions in short-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca2+](m)) in only 4% of cells. This revealed heterogeneity in the responses of individual mitochondria, all of which stained with MitoTracker Green FM. In contrast, stimulation with 100 mu M ATP evoked large cytosolic Ca2+ transients (>1,000 nM) and induced pronounced, reversible elevation of [Ca2+](m) (measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca2+ uptake usually lagged behind the cytosolic Ca2+ transient peak by 3-5 s, and [Ca2+], declined more slowly than did bulk [Ca2+](cyt). The uptake delay may prevent mitochondria from interfering with rapid signaling events while enhancing the mitochondrial response to large, long-duration elevations of [Ca2+](cyt). The responses of arterial myocytes to modest physiological stimulation do not, however, depend on such marked changes in [Ca2+](m).