Mitochondrial Calcium Regulation In Cardiac Myocytes: Local Gradients Of Intra-Mitochondrial Calcium, Mitochondrial Calcium Uniporter And Permeability Transition Pore-Mediated Calcium Homeostasis

摘要

[Ca2+]mito regulates mitochondrial energy production, provides transient Ca2+ buffering under stress, and can be involved in cell death. Mitochondria are near the sarcoplasmic reticulum (SR) in cardiac myocytes, and evidence for crosstalk exists. However, quantitative measurements of Ca2+]mito are limited, and spatial Ca2+]mito gradients have not been directly measured. To directly measure local Ca2+]mito during normal SR Ca release in intact myocytes, and evaluate potential subsarcomeric spatial Ca2+]mito gradients.;Methods and Results: Using the mitochondrially targeted inverse pericam indicator Mitycam, calibrated in situ, we directly measured Ca2+] mito during SR Ca release in intact rabbit ventricular myocytes by confocal microscopy. During steady state pacing, Ca2+]mito amplitude was 29+/-3 nM, rising rapidly (similar to cytosolic free [Ca 2+]) but declining much more slowly. Taking advantage of the structural periodicity of cardiac sarcomeres, we found that Ca2+] mito near SR Ca2+ release sites (Z-line) versus mid-sarcomere (M-line) reached a high peak amplitude (37+/-4 versus 26+/-4 nM, respectively P<0.05) which occurred earlier in time. This difference was attributed to ends of mitochondria being physically closer to SR Ca release sites, because the mitochondrial Ca2+ uniporter was homogeneously distributed, and elevated [Ca2+] applied laterally did not produce longitudinal Ca2+]mito gradients. We developed methods to measure spatiotemporal Ca2+]mito gradients quantitatively during excitation--contraction coupling. The amplitude and kinetics of Ca2+]mito transients differ significantly from those in the cytosol and are within the mitochondria respectively higher and faster near the Z-line versus M-line. This approach will help clarify SR-mitochondrial Ca2+ signaling.;Mitochondria are strategically positioned in cell to coordinate ATP production with metabolic demand. In the beat-to-beat heart, mitochondrial Ca 2+ load is the critical factor tuning the bioenergetics. However, the pathological condition of mitochondrial Ca2+ overload, which can occur after stressful situations such as after ischemic injury, can trigger mitochondrial permeability transition pore (mPTP) opening and cardiomyocyte death. Mitochondrial calcium uniporter (MCU) has been proposed as a predominant pathway of mitochondrial Ca2+ uptake. However, in cardiac myocytes, the actual pathway of mitochondrial Ca2+ uptake and mitochondrial Ca2+ buffering capacity are still under debate in both physiological and pathological conditions. To test the functional role of MCU in the heart, our collaborators (Dr. Molkentin's lab) generated a genetic mouse model with inducible and cardiomyocyte-specific deletion of this gene. As expected, mitochondria from cardiac-specific Mcu-deleted mice were refractory to acute Ca2+ uptake without affecting the cytosolic Ca2+ homeostasis during the normal SR Ca 2+ cycle. These mice in the adult heart were also protected from acute ischemia-reperfusion injury. Resting mitochondrial Ca2+ levels were normal in hearts of Mcu-deleted mice, and Mcu-deleted mice did not show a cardiac phenotype with up to 1 year of aging. These results suggest that the MCU is not required for long-term mitochondrial Ca2+ homeostasis but instead serves as a "fight-or-flight" mediator.;Mitochondria produce most cellular ATP, and are especially critical for survival of highly aerobic cells such as cardiac myocytes and neurons. Opening of high-conductance and long-lasting mitochondrial permeability transition pores (mPTP) causes uncoupling of respiration, mitochondrial injury and cell death. Conversely, low-conductance and transient mPTP openings (tPTP) have been proposed to limit mitochondrial Ca2+ load and be cardioprotective, but direct evidence for tPTP in cells is limited. Here, we measured tPTP directly as transient drops in mitochondrial [Ca2+] (Ca2+] mito) and membrane potential (DeltaPsim) in adult cardiac myocytes during cyclical sarcoplasmic reticulum Ca2+ release, by simultaneous live imaging of 500-1,000 individual mitochondria. The frequency of tPTPs rose at higher Ca2+]mito, [Ca2+]i, with 1 microM peroxide exposure and in myocyte from failing hearts. The tPTPs were suppressed by preventing mitochondrial Ca2+ influx, by mPTP inhibitor cyclosporine A (CsA) and in cyclophilin D knockout mice.;These tPTP events average 57 +/- 5 s in duration, but were rare (occurring in <0.1% of the cell's mitochondria at any moment) such that the overall energetic cost to the cell is minimal. The tPTP pore size is much smaller than for permanent mPTP, as neither Rhod-2 nor calcien (600 Da) were lost. Thus, proteins and even molecules the size of NADH (663 Da) will be retained during these tPTP. We conclude that tPTP openings (MitoWinks) may be molecularly related to pathological mPTP, but are likely to be physiologically beneficial to mitochondrial (and cell) survival by allowing individual mitochondria to reset themselves with little overall energetic cost.