1. Shivendra G. Tewari 1, Ranjan K. Pradhan 1,2, Jason N. Bazil 1,2, Amadou K.S. Camara 3, David F. Stowe 2,3, Daniel A. Beard 1,2, and Ranjan K. Dash 1,2 1 Biotechnology and Bioengineering Center, 2 Department of Physiology, and 3 Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI Characterization of Different Modes of Ca 2+ Uptake under Different Physiological Conditions in the Heart Mitochondria Model of Mitochondrial Bioenergetics and Ca 2+ Handling The model is developed from the Beard’s model of mitochondrial respiratory system and oxidative phosphorylation (1) to account for the dynamics of mitochondrial Na + -Ca 2+ cycle and interaction between extra-matrix Mg 2+ and Ca 2 (2, 3), and additional pathway (fCU) for mitochondrial Ca 2+ uptake. The Ca 2+ sequestration model consists of four types of buffering proteins : BP1, BP2, BP3 and BP4 in the matrix. The integrated model includes the reactions at complex I, III, IV, and F0F1 ATPase of the electron transport system; the substrate transporters (ANT and PHT), cation transporters (sCU, fCU, NCE, NHE, CHE, and KHE), and passive K + and H + permeations across the mitochondrial inner membrane; and the passive substrate transport fluxes of adenines and phosphates across the mitochondrial outer membrane. Both uniporters sCU and fCU are inhibited by extra-matrix Mg 2+. The flux through the TCA cycle producing NADH is expressed in terms of a phenomenological dehydrogenase flux. ANT: adenine nucleotide translocase, PHT: phosphate-hydrogen cotransporter, sCU: slow Ca 2+ uniporter, fCU: fast Ca 2+ uniporter, NCE: 3Na + /1Ca 2+ exchanger, NHE: Na + /H + exchanger, CHE: Ca 2+ /H + exchanger, and KHE: K + /H + exchanger. (1): Beard, PLoS Comput Biol 1(4): e36, 2005; (2): Dash and Beard, J Physiol 586(13): 3267-3285, 2008; (3): Pradhan et al., Biophysical J 101(9):2071-2081, 2011. Kinetic Mechanism of Ca 2+ Influx via Ca 2+ Uniporter with Mg 2+ Inhibition The uniporter T is assumed to have two binding sites for both Ca 2+ on either side of the inner mitochondrial membrane (IMM) and two distinct binding sites for Mg 2+ on the cytoplasmic side of the IMM. (A) Two Ca 2+ ions from the cytoplasmic side cooperatively bind to the uniporter in two steps to form the complex T2Ca 2+ e which then undergoes conformal changes to form the complex 2Ca 2+ x T. This complex goes through the reverse process where it dissociates in two steps to form the unbound uniporter T and two Ca 2+ ions in the matrix side. (B) A linear mixed-type scheme for Mg 2+ inhibition of Ca 2+ influx via the uniporter. The Mg 2+ interacts with the Ca 2+ only in the cytoplasmic side of the IMM. There is no Mg 2+ binding to uniporter T from the matrix side of the IMM. But in the matrix side, Ca 2+ binding is symmetrical to that of the cytoplasmic side as shown in this figure. The transport of Ca 2+ via the uniporter is limited by the rate constants k i and k o which are dependent on  m, and modulated by extra-matrix free [Ca 2+ ], matrix free [Ca 2+ ] and extra-matrix free [Mg 2+ ].  The figure is reproduced from Pradhan et al., Biophysical J 101(9):2071-2081, 2011. Abstract / Summary Cardiac mitochondria can act as a significant Ca 2+ sink and shape the cytosolic Ca 2+ signals affecting various cellular processes such as energy metabolism and excitation-contraction coupling. However, mitochondrial Ca 2+ uptake mechanisms under different (patho)physio- logical conditions are not well understood. Characterization of these mechanisms is crucial in developing a quantitative understanding of Ca 2+ signals in the heart. For this purpose, we performed Ca 2+ uptake experiments in isolated guinea pig heart mitochondria under different experimental concentrations of extra-matrix Mg 2+ and Ca 2+. The Na + free respiration buffer contained 1 mM EGTA with variable levels of Mg 2+ (0 mM–2 mM) and CaCl 2 (0.0 mM–0.6 mM) was added as a pulse after mitochondrial energization with substrate was pyruvic acid. Experimental data were analyzed using an integrated model of mitochondrial bioenergetics and cation handling. Our model analyses of the data reveal the existence of two Ca 2+ uniporters or Ca 2+ uptake pathways, namely a fast CU (fCU) and a slow CU (sCU), which exhibit contrasting differences in [Ca 2+ ] e and [Mg 2+ ] e sensitivities. fCU is a time-dependent high affinity Ca 2+ uniporter, while sCU is a time-independent low affinity Ca 2+ uniporter. Both uniporters are inhibited by extra-matrix Mg 2+. The binding affinity of fCU for Mg 2+ is higher as compared to that of sCU. This work was supported by NIH/R01-HL095122. Matrix and Extra-matrix Free Ca 2+ Dynamics with Mg 2+ Inhibition Model Simulations of Fluxes through Two Types of Uniporter: sCU and fCU (A) (B) Comparison of model simulations to data on the dynamics of buffer (A) and matrix (B, C, D) free [Ca 2+ ] in the absence ([Mg 2+ ] e = 0) and presence of 0.5 and 1 mM Mg 2+ in Na + free buffer mediums. Adding CaCl 2 (0, 0.25, 0.5, 0.6 mM) caused a dose-dependent increase in [Ca 2+ ] m, consistent with the increase in [Ca 2+ ] e. Two different phase of uptake (slow and fast) were observed during and after adding CaCl 2. The slow phase of Ca 2+ uptake was through the low affinity Ca 2+ uniporter and the fast phase was through the high affinity Ca 2+ uniporter. Model accurately describes the data for all Mg 2+ levels and Mg 2+ reduces [Ca 2+ ] m. CaCl 2 was added at t = 120 s. TypeDefinitionValueUnits BP1 [B Ca,1 ]Total protein concentration30.0mM K Ca,1 Ca 2+ binding constant6.00µM n1n1 Number of binding sites1- BP2 [B Ca,2 ]Total protein concentration35.0mM K Ca,2 Ca 2+ binding constant5.9µM n2n2 Number of binding sites2- BP3 [B Ca,3 ]Total protein concentration40.0mM K Ca,3 Ca 2+ binding constant5.8µM n3n3 Number of binding sites3- BP4 [B Ca,4 ]Total protein concentration45.0mM K Ca,4 Ca 2+ binding constant5.7µM n4n4 Number of binding sites4- Ca 2+ Buffering Power (β Ca ) is a manifestation of the Ca 2+ sequestration system. For every Ca 2+ ion entering (or leaving) the mitochondria, only a fraction remain free. This can be written as: For highly buffered conditions, β Ca is large. Mathematically: Table 1. Buffering Model Parameters Ca 2+ Sequestration System in Mitochondrial Matrix Simulations of slow (time- independent) and fast (time- dependent) Ca 2+ uniporter fluxes for three levels of extra-matrix MgCl 2 and four levels of extra-matrix CaCl 2. NCE was inactivated. (A,B,C): Dynamics of slow, low affinity uniporter fluxes (sCU) in response to added CaCl 2 (0, 0.25, 0.5, 0.6 mM), in the absence (A) and presence of 0.5 mM MgCl 2 (B) and 1 mM MgCl 2 (C) added to the buffer medium. (D,E,F): Dynamics of fast, high affinity uniporter fluxes (sCU) in response to added CaCl 2 (0, 0.25, 0.5, 0.6 mM), in the absence (A) and presence of 0.5 mM MgCl 2 (B) and 1 mM MgCl 2 (C) added to the buffer medium. In all the experiments and simulations, MgCl 2 was added at t = 0 s and CaCl 2 was added at t = 120 s. Ca 2+ fluxes by both the slow and fast uniporters were obser- ved to be inhibited by extra- matrix Mg 2+. In addition, the fast uniporter is slowly inactivating depending on the matrix free [Ca 2+ ]. Blue: 0 mM CaCl 2 Green: 0.25 mM CaCl 2 Red : 0.5 mM CaCl 2 Pink: 0.6 mM CaCl 2 (C) (A) (D) (B) (A)(D) (B) (E) (C) (F)