Mitochondrial calcium uptake.

Calcium (Ca(2+)) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca(2+) levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca(2+) ([Ca(2+)]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca(2+) uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca(2+) uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca(2+) uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca(2+) influx into the mitochondria via the MCU is small relative to other cytosolic Ca(2+) extrusion pathways; (ii) single MCU conductance is ∼6-7 pS (105 mM [Ca(2+)]), and MCU flux appears to be modulated by [Ca(2+)]i, suggesting Ca(2+) regulation of MCU open probability (P(O)); (iii) in the heart, two features are clear: the number of MCU channels per mitochondrion can be calculated, and MCU probability is low under normal conditions; and (iv) in skeletal muscle and liver cells, uptake per mitochondrion varies in magnitude but total uptake per cell still appears to be modest. Based on our analysis of available quantitative data, we conclude that although Ca(2+) critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca(2+) under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca(2+)]i, mitochondrial Ca(2+) uptake can increase 10- to 1,000-fold and begin to shape [Ca(2+)]i dynamics.

Calcium movement in cardiac mitochondria.

Existing theory suggests that mitochondria act as significant, dynamic buffers of cytosolic calcium ([Ca(2+)]i) in heart. These buffers can remove up to one-third of the Ca(2+) that enters the cytosol during the [Ca(2+)]i transients that underlie contractions. However, few quantitative experiments have been presented to test this hypothesis. Here, we investigate the influence of Ca(2+) movement across the inner mitochondrial membrane during both subcellular and global cellular cytosolic Ca(2+) signals (i.e., Ca(2+) sparks and [Ca(2+)]i transients, respectively) in isolated rat cardiomyocytes. By rapidly turning off the mitochondria using depolarization of the inner mitochondrial membrane potential (ΔΨm), the role of the mitochondria in buffering cytosolic Ca(2+) signals was investigated. We show here that rapid loss of ΔΨm leads to no significant changes in cytosolic Ca(2+) signals. Second, we make direct measurements of mitochondrial [Ca(2+)] ([Ca(2+)]m) using a mitochondrially targeted Ca(2+) probe (MityCam) and these data suggest that [Ca(2+)]m is near the [Ca(2+)]i level (∼100 nM) under quiescent conditions. These two findings indicate that although the mitochondrial matrix is fully buffer-capable under quiescent conditions, it does not function as a significant dynamic buffer during physiological Ca(2+) signaling. Finally, quantitative analysis using a computational model of mitochondrial Ca(2+) cycling suggests that mitochondrial Ca(2+) uptake would need to be at least ∼100-fold greater than the current estimates of Ca(2+) influx for mitochondria to influence measurably cytosolic [Ca(2+)] signals under physiological conditions. Combined, these experiments and computational investigations show that mitochondrial Ca(2+) uptake does not significantly alter cytosolic Ca(2+) signals under normal conditions and indicates that mitochondria do not act as important dynamic buffers of [Ca(2+)]i under physiological conditions in heart.

Dynamics of calcium sparks and calcium leak in the heart.

We present what we believe to be a new mathematical model of Ca(2+) leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca(2+)-release units, each containing a cluster of stochastically gating Ca(2+) channels (RyRs), whose biophysical properties (e.g., Ca(2+) sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca(2+)-release properties, and shows how this balances reuptake by the SR Ca(2+) pump. Simulations reveal that SR Ca(2+) leak consists of brief but frequent single RyR openings (~3000 cell(-1) s(-1)) that are likely to be experimentally undetectable, and are, therefore, "invisible". We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca(2+)), and occasionally lead to the generation of Ca(2+) sparks (~130 cell(-1) s(-1)). Furthermore, this physiological formulation of "invisible" leak allows for the removal of the ad hoc, non-RyR mediated Ca(2+) leak terms present in prior models. Finally, our model shows how Ca(2+) sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.

Stochastic simulation of cardiac ventricular myocyte calcium dynamics and waves.

A three dimensional model of calcium dynamics in the rat ventricular myocyte was developed to study the mechanism of calcium homeostasis and pathological calcium dynamics during calcium overload. The model contains 20,000 calcium release units (CRUs) each containing 49 ryanodine receptors. The model simulates calcium sparks with a realistic spontaneous calcium spark rate. It suggests that in addition to the calcium spark-based leak, there is an invisible calcium leak caused by the stochastic opening of a small number of ryanodine receptors in each CRU without triggering a calcium spark. The model also explores the mechanism of calcium wave propagation between release sites under the conditions of calcium overload.