George S.B. Williams

Mitochondrial calcium uptake

Calcium (Ca2+) uptake into the mitochondrial matrix is critically important to cellular function. As a regulator of matrix Ca2+ levels, this flux influences energy production and can initiate cell death. If large, this flux could potentially alter intracellular Ca2+ ([Ca2+]i) signals. Despite years of study, fundamental disagreements on the extent and speed of mitochondrial Ca2+ uptake still exist. Here, we review and quantitatively analyze mitochondrial Ca2+ uptake fluxes from different tissues and interpret the results with respect to the recently proposed mitochondrial Ca2+ uniporter (MCU) candidate. This quantitative analysis yields four clear results: (i) under physiological conditions, Ca2+ influx into the mitochondria via the MCU is small relative to other cytosolic Ca2+ extrusion pathways; (ii) single MCU conductance is ∼6–7 pS (105 mM [Ca2+]), and MCU flux appears to be modulated by [Ca2+]i, suggesting Ca2+ regulation of MCU open probability (PO); (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 Ca2+ critically regulates mitochondrial function, the mitochondria do not act as a significant dynamic buffer of cytosolic Ca2+ under physiological conditions. Nevertheless, with prolonged (superphysiological) elevations of [Ca2+]i, mitochondrial Ca2+ uptake can increase 10- to 1,000-fold and begin to shape [Ca2+]i dynamics.

Stimulated Emission Depletion Live-Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

STED Live Cell Super-Resolution Imaging Shows Proliferative Remodeling of T-Tubule Membrane Structures After Myocardial Infarction

Rationale: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca2+ release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). Objectives: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. Methods and Results: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca2+ release and action potential prolongation. Conclusions: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca2+ release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Tachycardia-induced silencing of subcellular Ca2+ signaling in atrial myocytes

Atrial fibrillation (AF) is characterized by sustained high atrial activation rates and arrhythmogenic cellular Ca2+ signaling instability; however, it is not clear how a high atrial rate and Ca2+ instability may be related. Here, we characterized subcellular Ca2+ signaling after 5 days of high atrial rates in a rabbit model. While some changes were similar to those in persistent AF, we identified a distinct pattern of stabilized subcellular Ca2+ signaling. Ca2+ sparks, arrhythmogenic Ca2+ waves, sarcoplasmic reticulum (SR) Ca2+ leak, and SR Ca2+ content were largely unaltered. Based on computational analysis, these findings were consistent with a higher Ca2+ leak due to PKA-dependent phosphorylation of SR Ca2+ channels (RyR2s), fewer RyR2s, and smaller RyR2 clusters in the SR. We determined that less Ca2+ release per [Ca2+]i transient, increased Ca2+ buffering strength, shortened action potentials, and reduced L-type Ca2+ current contribute to a stunning reduction of intracellular Na+ concentration following rapid atrial pacing. In both patients with AF and in our rabbit model, this silencing led to failed propagation of the [Ca2+]i signal to the myocyte center. We conclude that sustained high atrial rates alone silence Ca2+ signaling and do not produce Ca2+ signaling instability, consistent with an adaptive molecular and cellular response to atrial tachycardia.

NCLX: the mitochondrial sodium calcium exchanger.

The free Ca(2+) concentration within the mitochondrial matrix ([Ca(2+)]m) regulates the rate of ATP production and other [Ca(2+)]m sensitive processes. It is set by the balance between total Ca(2+) influx (through the mitochondrial Ca(2+) uniporter (MCU) and any other influx pathways) and the total Ca(2+) efflux (by the mitochondrial Na(+)/Ca(2+) exchanger and any other efflux pathways). Here we review and analyze the experimental evidence reported over the past 40years which suggest that in the heart and many other mammalian tissues a putative Na(+)/Ca(2+) exchanger is the major pathway for Ca(2+) efflux from the mitochondrial matrix. We discuss those reports with respect to a recent discovery that the protein product of the human FLJ22233 gene mediates such Na(+)/Ca(2+) exchange across the mitochondrial inner membrane. Among its many functional similarities to other Na(+)/Ca(2+) exchanger proteins is a unique feature: it efficiently mediates Li(+)/Ca(2+) exchange (as well as Na(+)/Ca(2+) exchange) and was therefore named NCLX. The discovery of NCLX provides both the identity of a novel protein and new molecular means of studying various unresolved quantitative aspects of mitochondrial Ca(2+) movement out of the matrix. Quantitative and qualitative features of NCLX are discussed as is the controversy regarding the stoichiometry of the NCLX Na(+)/Ca(2+) exchange, the electrogenicity of NCLX, the [Na(+)]i dependency of NCLX and the magnitude of NCLX Ca(2+) efflux. Metabolic features attributable to NCLX and the physiological implication of the Ca(2+) efflux rate via NCLX during systole and diastole are also briefly discussed.

Models of cardiac excitation–contraction coupling in ventricular myocytes

Mathematical and computational modeling of cardiac excitation-contraction coupling has produced considerable insights into how the heart muscle contracts. With the increase in biophysical and physiological data available, the modeling has become more sophisticated with investigations spanning in scale from molecular components to whole cells. These modeling efforts have provided insight into cardiac excitation-contraction coupling that advanced and complemented experimental studies. One goal is to extend these detailed cellular models to model the whole heart. While this has been done with mechanical and electrophysiological models, the complexity and fast time course of calcium dynamics have made inclusion of detailed calcium dynamics in whole heart models impractical. Novel methods such as the probability density approach and moment closure technique which increase computational efficiency might make this tractable.

Superresolution Modeling of Calcium Release in the Heart

Stable calcium-induced calcium release (CICR) is critical for maintaining normal cellular contraction during cardiac excitation-contraction coupling. The fundamental element of CICR in the heart is the calcium (Ca2+) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca2+ from the sarcoplasmic reticulum (SR). The Ca2+ leak out of the SR is an important process for cellular Ca2+ management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca2+ spark. Here, we present a detailed, three-dimensional model of a cardiac Ca2+ release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca2+ sparks and robust Ca2+ spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca2+ spark and nonspark-based SR Ca2+ leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca2+-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca2+ leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca2+ spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca2+ release properties due to variations in inter-RyR coupling via local subspace Ca2+ concentration ([Ca2+]ss). These results are illustrated for RyR clusters based on super-resolution stimulated emission depletion microscopy. Finally, we present a believed-novel approach by which the spark fidelity of a RyR cluster can be predicted from structural information of the cluster using the maximum eigenvalue of its adjacency matrix. These results provide critical insights into CICR dynamics in heart, under normal and pathological conditions.

Moment Closure for Local Control Models of Calcium-Induced Calcium Release in Cardiac Myocytes

In prior work, we introduced a probability density approach to modeling local control of Ca2+-induced Ca2+ release in cardiac myocytes, where we derived coupled advection-reaction equations for the time-dependent bivariate probability density of subsarcolemmal subspace and junctional sarcoplasmic reticulum (SR) [Ca2+] conditioned on Ca2+ release unit (CaRU) state. When coupled to ordinary differential equations (ODEs) for the bulk myoplasmic and network SR [Ca2+], a realistic but minimal model of cardiac excitation-contraction coupling was produced that avoids the computationally demanding task of resolving spatial aspects of global Ca2+ signaling, while accurately representing heterogeneous local Ca2+ signals in a population of diadic subspaces and junctional SR depletion domains. Here we introduce a computationally efficient method for simulating such whole cell models when the dynamics of subspace [Ca2+] are much faster than those of junctional SR [Ca2+]. The method begins with the derivation of a system of ODEs describing the time-evolution of the moments of the univariate probability density functions for junctional SR [Ca2+] jointly distributed with CaRU state. This open system of ODEs is then closed using an algebraic relationship that expresses the third moment of junctional SR [Ca2+] in terms of the first and second moments. In simulated voltage-clamp protocols using 12-state CaRUs that respond to the dynamics of both subspace and junctional SR [Ca2+], this moment-closure approach to simulating local control of excitation-contraction coupling produces high-gain Ca2+ release that is graded with changes in membrane potential, a phenomenon not exhibited by common pool models. Benchmark simulations indicate that the moment-closure approach is nearly 10,000-times more computationally efficient than corresponding Monte Carlo simulations while leading to nearly identical results. We conclude by applying the moment-closure approach to study the restitution of Ca2+-induced Ca2+ release during simulated two-pulse voltage-clamp protocols.

Ca2+ dynamics in the mitochondria - state of the art.

The importance of [Ca2+] in the mitochondrial matrix, [Ca2+]mito, had been proposed by early work of Carafoli and others [1], [2] and [3]. The key suggestion in the 1970s [4] was that regulatory [Ca2+]mito played a role in controlling the rate of activation of tricarboxylic acid cycle dehydrogenases, important in the regulation of ATP production by the electron transport chain (ETC) during oxidative phosphorylation. This view is now established [5] and [6] and the key questions currently debated are to what extent do the mitochondria acquire and release Ca2+, and what impact do mitochondria have on the dynamic Ca2+ signal in the cardiac ventricular myocyte [7]. Although investigations of Ca2+ dynamics in mitochondria have been problematic, disparate and inconclusive, they have also been both provocative and exciting. A recent special issue of this journal presented contrasting perspectives on the speed, extent and mechanisms of changes in [Ca2+]mito, and how these changes may influence cellular spatio-temporal [Ca2+]i dynamics [8]. An audio discussion is also available online [9]. The uncertain nature of the signaling pathways is noted in Table 1 (see below) which shows mitochondrial proteins and processes that are of current focus and which remain contentious. Each of the “items” listed is largely unsettled, or is a “work in progress”. There may be advocates for opposing positions noted or recent discoveries that must still be tested at multiple levels by diverse laboratories. Currently, the first item, the mitochondrial sodium/calcium exchanger (NCLX) [10], appears the most solid with respect to the molecular identification and physiological function, whereas, the recently described candidates of the mitochondrial Ca2+ uniporter (MCU) [11] and [12] still need to be verified and broadly examined by the scientific community.

Ambiguous interactions between diastolic and SR Ca2+ in the regulation of cardiac Ca2+ release

Sobie et al. highlight unresolved issues concerning the regulation of sarcoplasmic reticulum calcium release in cardiac myocytes.