Total Matrix Ca2+ Modulates Ca2+ Efflux via the Ca2+/H+ Exchanger in Cardiac Mitochondria.

Gayathri K Natarajan,Jyotsna Mishra,Amadou K S Camara,Wai-Meng Kwok,David F Stowe,Lyall Glait

doi:10.3389/fphys.2020.510600

Gayathri K Natarajan, Jyotsna Mishra + Show 4 more

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https://doi.org/10.3389/fphys.2020.510600

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Abstract

Mitochondrial Ca2+ handling is accomplished by balancing Ca2+ uptake, primarily via the Ru360-sensitive mitochondrial calcium uniporter (MCU), Ca2+ buffering in the matrix and Ca2+ efflux mainly via Ca2+ ion exchangers, such as the Na+/Ca2+ exchanger (NCLX) and the Ca2+/H+ exchanger (CHE). The mechanism of CHE in cardiac mitochondria is not well-understood and its contribution to matrix Ca2+ regulation is thought to be negligible, despite higher expression of the putative CHE protein, LETM1, compared to hepatic mitochondria. In this study, Ca2+ efflux via the CHE was investigated in isolated rat cardiac mitochondria and permeabilized H9c2 cells. Mitochondria were exposed to (a) increasing matrix Ca2+ load via repetitive application of a finite CaCl2 bolus to the external medium and (b) change in the pH gradient across the inner mitochondrial membrane (IMM). Ca2+ efflux at different matrix Ca2+ loads was revealed by inhibiting Ca2+ uptake or reuptake with Ru360 after increasing number of CaCl2 boluses. In Na+-free experimental buffer and with Ca2+ uptake inhibited, the rate of Ca2+ efflux and steady-state free matrix Ca2+ [mCa2+]ss increased as the number of administered CaCl2 boluses increased. ADP and cyclosporine A (CsA), which are known to increase Ca2+ buffering while maintaining a constant [mCa2+]ss, decreased the rate of Ca2+ efflux via the CHE, with a significantly greater decrease in the presence of ADP. ADP also increased Ca2+ buffering rate and decreased [mCa2+]ss. A change in the pH of the external medium to a more acidic value from 7.15 to 6.8∼6.9 caused a twofold increase in the Ca2+ efflux rate, while an alkaline change in pH from 7.15 to 7.4∼7.5 did not change the Ca2+ efflux rate. In addition, CHE activation was associated with membrane depolarization. Targeted transient knockdown of LETM1 in permeabilized H9c2 cells modulated Ca2+ efflux. The results indicate that Ca2+ efflux via the CHE in cardiac mitochondria is modulated by acidic buffer pH and by total matrix Ca2+. A mechanism is proposed whereby activation of CHE is sensitive to changes in both the matrix Ca2+ buffering system and the matrix free Ca2+ concentration.

Highlights

Ca2+ handling in mitochondria is critical for cell survival.Recent research has focused on the role of mitochondria in dynamically regulating intracellular and steady-state free matrix Ca2+ ([mCa2+]ss) in response to increases in cytosolic Ca2+ (Duchen, 2000; Rizzuto et al, 2000; Csordás et al, 2012; Takeuchi et al, 2015; De Stefani et al, 2016)
Na+-Independent Ca2+ Efflux in Cardiac Mitochondria Increases With Total Matrix Ca2+ Content
The Ca2+ efflux rate measured after Ru360 addition at 1400 s, approached the Ca2+ efflux rate observed upon opening of the mitochondrial permeability transition pore (mPTP) at the same time in control (Figure 1A, black trace and Figure 1B, black circle)

Summary

Introduction

Ca2+ handling in mitochondria is critical for cell survival.Recent research has focused on the role of mitochondria in dynamically regulating intracellular and steady-state free matrix Ca2+ ([mCa2+]ss) in response to increases in cytosolic Ca2+ (Duchen, 2000; Rizzuto et al, 2000; Csordás et al, 2012; Takeuchi et al, 2015; De Stefani et al, 2016). To achieve this, [mCa2+]ss is tightly maintained through mechanisms that balance Ca2+ uptake from the cytosol, Ca2+ buffering in the matrix and Ca2+ efflux from the mitochondria (Finkel et al., 2015; Giorgi et al, 2018). Ca2+ transport from the cytosol across the outer mitochondrial membrane (OMM) occurs mainly through the voltage-dependent anion channel (VDAC), which allows movement of a variety of ions, including both influx and efflux of Ca2+ (Tan and Colombini, 2007; Camara et al, 2017). MCU allows a selective influx of Ca2+ down its electrochemical gradient and is blocked by ruthenium red (RuR) and Ru360 (Kirichok et al, 2004; Gunter and Sheu, 2009; Baughman et al, 2011; De Stefani et al, 2011). Ca2+ is buffered primarily by precipitating with inorganic phosphate compounds, allowing for the uptake of large amounts of Ca2+ without disrupting the mitochondrial membrane potential, m (Starkov, 2010; Wei et al, 2012)

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