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.
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.
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 (Ca(2+)) spark, which arises from a cluster of ryanodine receptors (RyR). Opening of these RyR clusters is triggered to produce a local, regenerative release of Ca(2+) from the sarcoplasmic reticulum (SR). The Ca(2+) leak out of the SR is an important process for cellular Ca(2+) management, and it is critically influenced by spark fidelity, i.e., the probability that a spontaneous RyR opening triggers a Ca(2+) spark. Here, we present a detailed, three-dimensional model of a cardiac Ca(2+) release unit that incorporates diffusion, intracellular buffering systems, and stochastically gated ion channels. The model exhibits realistic Ca(2+) sparks and robust Ca(2+) spark termination across a wide range of geometries and conditions. Furthermore, the model captures the details of Ca(2+) spark and nonspark-based SR Ca(2+) leak, and it produces normal excitation-contraction coupling gain. We show that SR luminal Ca(2+)-dependent regulation of the RyR is not critical for spark termination, but it can explain the exponential rise in the SR Ca(2+) leak-load relationship demonstrated in previous experimental work. Perturbations to subspace dimensions, which have been observed in experimental models of disease, strongly alter Ca(2+) spark dynamics. In addition, we find that the structure of RyR clusters also influences Ca(2+) release properties due to variations in inter-RyR coupling via local subspace Ca(2+) concentration ([Ca(2+)]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.
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.
Consumption of adenosine triphosphate (ATP) by the heart can change dramatically as the energetic demands increase from a period of rest to strenuous activity. Mitochondrial ATP production is central to this metabolic response since the heart relies largely on oxidative phosphorylation as its source of intracellular ATP. Significant evidence has been acquired indicating that Ca(2+) plays a critical role in regulating ATP production by the mitochondria. Here the evidence that the Ca(2+) concentration in the mitochondrial matrix ([Ca(2+)]m) plays a pivotal role in regulating ATP production by the mitochondria is critically reviewed and aspects of this process that are under current active investigation are highlighted. Importantly, current quantitative information on the bidirectional Ca(2+) movement across the inner mitochondrial membrane (IMM) is examined in two parts. First, we review how Ca(2+) influx into the mitochondrial matrix depends on the mitochondrial Ca(2+) channel (i.e., the mitochondrial calcium uniporter or MCU). This discussion includes how the MCU open probability (PO) depends on the cytosolic Ca(2+) concentration ([Ca(2+)]i) and on the mitochondrial membrane potential (ΔΨm). Second, we discuss how steady-state [Ca(2+)]m is determined by the dynamic balance between this MCU-based Ca(2+) influx and mitochondrial Na(+)/Ca(2+) exchanger (NCLX) based Ca(2+) efflux. These steady-state [Ca(2+)]m levels are suggested to regulate the metabolic energy supply due to Ca(2+)-dependent regulation of mitochondrial enzymes of the tricarboxylic acid cycle (TCA), the proteins of the electron transport chain (ETC), and the F1F0 ATP synthase itself. We conclude by discussing the roles played by [Ca(2+)]m in influencing mitochondrial responses under pathological conditions. This article is part of a Special Issue entitled "Mitochondria: From BasicMitochondrial Biology to Cardiovascular Disease."
The free Ca2+ concentration within the mitochondrial matrix ([Ca2+]m) regulates the rate of ATP production and other [Ca2+]m sensitive processes. It is set by the balance between total Ca2+ influx (through the mitochondrial Ca2+ uniporter (MCU) and any other influx pathways) and the total Ca2+ efflux (by the mitochondrial Na+/Ca2+ exchanger and any other efflux pathways). Here we review and analyze the experimental evidence reported over the past 40 years which suggest that in the heart and many other mammalian tissues a putative Na+/Ca2+ exchanger is the major pathway for Ca2+ 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+/Ca2+ exchange across the mitochondrial inner membrane. Among its many functional similarities to other Na+/Ca2+ exchanger proteins is a unique feature: it efficiently mediates Li+/Ca2+ exchange (as well as Na+/Ca2+ 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 Ca2+ movement out of the matrix. Quantitative and qualitative features of NCLX are discussed as is the controversy regarding the stoichiometry of the NCLX Na+/Ca2+ exchange, the electrogenicity of NCLX, the [Na+]i dependency of NCLX and the magnitude of NCLX Ca2+ efflux. Metabolic features attributable to NCLX and the physiological implication of the Ca2+ efflux rate via NCLX during systole and diastole are also briefly discussed.
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