Tracking Membrane Potential, Calcium and REDOX Activity in Single Mitochondria During Normal Cardiac Cell Stimulation

Abstract

Mitochondria are well recognized as “the powerhouse” of the cell. In cardiac cells they may also play a role in Ca2+ signaling, redox signaling and cell death. They are therefore important in maintaining cardiac cellular homeostasis. By coupling fuel oxidation to ion transport across their inner membrane they create an electrochemical gradient. This electrochemical gradient is used to drive energy production to meet the cellular needs; it also drives the transport of ions, such as Ca2+, across the inner mitochondrial membrane, in turn, Ca2+ regulates mitochondrial activity. The mitochondrial membrane potential is therefore an accurate indicator of the energetic state of the cell. The cardiac action potential continuously changes the potential of the cell membrane resulting in Ca2+ entry and further Ca2+ release from the sarcoplasmic reticulum (SR) triggering cell contraction. In freshly dissociated rat ventricular myocytes, we use confocal imaging of the fluorescent membrane potential indicator TMRE and the fluorescent Ca2+ indicator Rhod-2 trapped in the mitochondrial matrix to tracked changes in mitochondrial membrane potential and mitochondrial Ca2+ ([Ca2+]m) in individual mitochondria in response to electrical stimulation and caffeine induced SR Ca2+ release. We also tracked changes in mitochondrial NADH by measuring NADH auto fluorescence in single mitochondria under the same experimental conditions. We conclude that the intrinsic functional link between the electrical activity of the cardiac cell (due to SR Ca2+ release) and mitochondrial dynamics can be faithfully studied in real time by tracking mitochondrial membrane potential, Ca2+ or NADH in single mitochondria.