The long term objective of this project is to characterize the multiplicity of Ca2+ -signaling in normal and abnormal cardiac function. This research is focused on a novel mitochondrial Ca2+-signaling pathway that is triggered by exposing cardiac cells to biologically relevant jets of 'pressurized fluid'(PF). Considering that the strength of the normal heartbeat is mainly determined by Ca2+-induced Ca2+ release (CICR) from the SR, the PF-triggered response may add fine control and plasticity to interaction of CICR with mitochondrial energy production which may become critical in various pressure-flow related myopathies. Our recent results show that PF pulses activate relatively slow (~1.5 s) Ca2+ transients that originate from a small intracellular Ca2+ pool that appear to be independent of transmembrane entry or release of SR Ca2+, nitric oxide (NO) or IP3-signaling pathways, but depend on the integrity of the mitochondrial function as metabolic uncoupler FCCP or mitochondrial Ca2+ uniporter (mCU) blocker, Ru360 suppress them. Reducing agent DTT and reactive oxygen species (ROS) scavenger Tempol, as well as the mitochondrial NCX (mNCX) blocker CGP-37157, also specifically inhibit these Ca2+ transients. In Rhod-2 AM-loaded and permeabilized cells, confocal imaging show mitochondrial gain of Ca2+ on release of SR Ca2+ by caffeine and a loss of mitochondrial Ca2+ on application of PF pulses. These signals are suppressed by Na+-free or CGP-37157- containing solutions, implicating mNCX in mediating the Ca2+ release process. We hypothesize that mitochondrial Ca2+ stores contribute significantly to normal and pathological cardiac Ca2+-signaling in response to mechanical stimuli, release of ROS and Ca2+- overload. Using freshly dissociated cardiomyocytes, we shall test this hypothesis using electrophysiological, confocal and TIRF fluorescence imaging and molecular techniques:
Aim 1. To characterize the involvement of reactive oxygen species (ROS) in activating PF-triggered Ca2+ transients and explore if they are enhanced by conditions of Ca2+ loading.
Aim 2. To characterize the PF-induced Ca2+ release with respect to the mitochondrial membrane potential (m), location and ionic mechanism.
Aim 3. To characterize the molecular nature of the mechano-sensor and the signaling pathway that leads to mitochondrial Ca2+ releases. The proposed research investigates a novel Ca2+-signaling pathway that may be activated by the stresses and strains that occur in normally working atrial and ventricular myocardia and gains prominence when mitochondrial function is compromised. Since mitochondria play an important role in apoptosis, cardiomyopathy, ischemia-reperfusion injury, Leigh syndrome, and fatal infantile acidosis, it is likely that such a specific mitochondrial Ca2+ -signaling pathway plays a critical role in these pathologies.
Human heart has great energetic needs because of its large workload (equivalent to pushing 1 ton the length of a football field per day);where the required energy supply is provided by mitochondria occupying ~30% of the cellular volume. Since cardiac contractility in normal and abnormal heart is mediated by cycling of Ca2+ in the cell, it is likely that Ca2+ signaling in the heart may also be the mechanism by which energy supply from the mitochondria is regulated. The nature of the signals that transmit information between the generation of energy supply per beat from the mitochondria and the Ca2+ released from the SR to meet the contractile needs of the myocyte is the subject of the proposed research in both healthy and diseased hearts.
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|N'gouemo, Prosper; Faingold, Carl L; Morad, Martin (2009) Calcium channel dysfunction in inferior colliculus neurons of the genetically epilepsy-prone rat. Neuropharmacology 56:665-75|
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