Fig.5.

Simultaneously recorded TIRF images of mitochondrial and cytosolic Ca²⁺ signals in a single patch-clamped hiPSC-CM, in response to Na⁺ withdrawal (Na⁺ replaced with TEA) and 5mM caffeine applications. hiPSC-CMs were infected with GCaMP3-mito probe for 72 hours and dialyzed with 0.1mM Fura-2 salt through patch pipet during the experiment. Withdrawal of Na⁺ activates an outward I_NCX and its replacement generates a large slowly decaying inward current, that accompanies the rise and fall of cytosolic calcium. The mitochondrial Ca²⁺ signals recorded from a single mitochondrion (white circle ROI.01 show significant delays when compared to the activation of I_NCX or rise of cytosolic Ca²⁺. The blue traces represent cytosolic Fura-2 Ca²⁺ signal from the same cellular locations. Note that the rise of mitochondrial Ca²⁺ lags significantly behind the rise in mitochondrial Ca²⁺. The delays in mitochondrial Ca²⁺ uptake is shorter on release of SR Ca²⁺ compared to withdrawal of Na⁺. The black traces represent the I_NCX currents activated by Na⁺ withdraw and caffeine application. GCaMP3-mito probe was excited at 488nm, where increasing fluorescence signal indicates increases in mitochondrial Ca²⁺. Fura2 was emission signals measured at 405 nm (decreasing fluorescence indicates increase in cytosolic Ca²⁺). Temperature 35 degrees C.