Mg(2+) differentially regulates two modes of mitochondrial Ca(2+) uptake in isolated cardiac mitochondria: implications for mitochondrial Ca(2+) sequestration

Abstract

The manner in which mitochondria take up and store Ca(2+) remains highly debated. Recent experimental and computational evidence has suggested the presence of at least two modes of Ca(2+) uptake and a complex Ca(2+) sequestration mechanism in mitochondria. But how Mg(2+) regulates these different modes of Ca(2+) uptake as well as mitochondrial Ca(2+) sequestration is not known. In this study, we investigated two different ways by which mitochondria take up and sequester Ca(2+) by using two different protocols. Isolated guinea pig cardiac mitochondria were exposed to varying concentrations of CaCl2 in the presence or absence of MgCl2. In the first protocol, A, CaCl2 was added to the respiration buffer containing isolated mitochondria, whereas in the second protocol, B, mitochondria were added to the respiration buffer with CaCl2 already present. Protocol A resulted first in a fast transitory uptake followed by a slow gradual uptake. In contrast, protocol B only revealed a slow and gradual Ca(2+) uptake, which was approximately 40 % of the slow uptake rate observed in protocol A. These two types of Ca(2+) uptake modes were differentially modulated by extra-matrix Mg(2+). That is, Mg(2+) markedly inhibited the slow mode of Ca(2+) uptake in both protocols in a concentration-dependent manner, but not the fast mode of uptake exhibited in protocol A. Mg(2+) also inhibited Na(+)-dependent Ca(2+) extrusion. The general Ca(2+) binding properties of the mitochondrial Ca(2+) sequestration system were reaffirmed and shown to be independent of the mode of Ca(2+) uptake, i.e. through the fast or slow mode of uptake. In addition, extra-matrix Mg(2+) hindered Ca(2+) sequestration. Our results indicate that mitochondria exhibit different modes of Ca(2+) uptake depending on the nature of exposure to extra-matrix Ca(2+), which are differentially sensitive to Mg(2+). The implications of these findings in cardiomyocytes are discussed.

Keywords: Calcium efflux; Calcium sequestration; Calcium uptake; Cardiac; Mitochondria.

Fig. 1

Timelines show the two experimental protocols used to characterize and quantify mitochondrial Ca²⁺ handling (influx, efflux, sequestration) in isolated guinea pig cardiac mitochondria. In protocol A, mitochondria were added to the experimental buffer before CaCl₂ was added. In protocol B, mitochondria were added to respiration buffer with CaCl₂ already present. All other additions were identical between protocols. 40 μM EGTA was present in all the experimental buffers; 0.5 μM CsA was added to all mitochondrial suspensions. Inset axes are the same as the main figure panels

Fig. 2

Extra-matrix free Ca²⁺ ([Ca²⁺]_e) dynamics. Ca²⁺ uptake and Ca²⁺ release for each combination of CaCl₂ and MgCl₂ concentrations are shown using the protocol depicted in Fig. 1. CaCl₂ was added to respiring mitochondrial suspension (left column; protocol A) or mitochondria were added to the buffer containing a given CaCl₂ concentration (right column; protocol B) at 60 s followed by ruthenium red (RR) at 300 s and NaCl at 360 s. The results for protocol A are shown in the left column and the results for protocol B are shown in the right column. Each row corresponds to the buffer MgCl₂ indicated on the left of each row. Insets show [Ca²⁺]_e dynamics for 0, 10, and 20 μM CaCl₂ in more detail. The axes are the same as the axes in the main figure panels. Error bars signify standard error of the mean

Fig. 3

Two modes of Ca²⁺ uptake. For CaCl₂ concentrations of 20 μM and greater, a double exponential function was fit between 65 s and 150 s to the [Ca²⁺]_e dynamics observed in protocol A (Fig. 2). The fit time constants show that there was a fast and slow component of Ca²⁺ uptake associated with each bolus of CaCl₂ administered. The inset axes labels are the same as the main figure panel. Error bars signify propagated standard deviations

Fig. 4

Matrix free Ca²⁺ ([Ca²⁺]_m) dynamics. Ca²⁺ uptake and Ca²⁺ release for each combination of CaCl₂ and MgCl₂ concentrations are shown using the protocol depicted in Fig. 1. CaCl₂ was added to the respiring mitochondrial suspension (left column; protocol A) or mitochondria were added to the buffer containing a given CaCl₂ concentration (right column; protocol B) at 60 s followed by ruthenium red (RR) at 300 s and NaCl at 360 s. The results for protocol A are shown in the left column and the results for protocol B in the right column. In protocol B when CaCl₂ was 40 μM, the fluorescent signal was close to Rmax, so the calculated [Ca²⁺]_m is likely an overestimation of the true value of [Ca²⁺]_m. Each row corresponds to the buffer MgCl₂ indicated on the left of each row. Insets show [Ca²⁺]_m dynamics for 0, 10, and 20 μM CaCl₂ in more detail. The axes are the same as the axes in the main figure panels. Error bars signify standard error of the mean

Fig. 5

Ca²⁺ uptake by mitochondria. The amount of Ca²⁺ uptake by mitochondria depends on method of CaCl₂ delivery. The bar plots show [Ca²⁺]_m just after addition of CaCl₂ to mitochondria (protocol A, left bar) or after addition of mitochondria to buffer containing CaCl₂ (protocol B, right bar). These data correspond to a time of approximately 65 s. For all 20 and 30 μM CaCl₂ conditions, the rate of Ca²⁺ uptake in protocol A was significantly different from that of protocol B (p ≤ 0.05) for most of the [CaCl₂] and even in the presence of Mg²⁺. Error bars signify standard error of the mean

Fig. 6

ΔΨ_m and NADH dynamics. The bioenergetics responses during protocols A and B were monitored in parallel using the ΔΨ_m sensitive dye TMRM and NADH autofluorescence. Traces are individual recordings. In averaged data there were no significant differences between the two MgCl₂ and CaCl₂ groups. The increases in signals mark the addition of mitochondria at 30 s (protocol A) and at 60 s (protocol B). The number of experimental groups was reduced to consist of only 0 and 1 mM MgCl₂ and 0 and 40 μM CaCl₂

Fig. 7

Mitochondrial Ca²⁺ buffering power. The Ca²⁺ sequestration system consists of at least two classes of buffers that bind Ca²⁺ with a differential affinity and capacity. Class 1 buffers are of the prototypical type whereby a single Ca²⁺ ion binds to a single site in an uncooperative manner. Class 2 buffers are atypical and bind multiple Ca²⁺ ions in a cooperative fashion. Class 1 buffers are not affected by Mg²⁺; however, in the presence of Mg²⁺, both the binding capacity and affinity of the Class 2 buffers are compromised. The blue, yellow and red colors correspond to the added 0 mM MgCl₂, 0.5 mM MgCl₂ and 1 mM MgCl₂ conditions, respectively. The circles (O) and x’s (x) represent rates obtained using the data from Protocols A and B, respectively. The lines correspond to model simulations of the two classes of Ca²⁺ buffers using Eq. 2 with the parameters listed in Table 1. Error bars signify propagated standard deviations. Dashed lines represent contributions to mitochondrial buffering power from the class 2 buffers only

Fig. 8

Mg²⁺ inhibition of Ca²⁺ uptake and extrusion. Panel 8a shows slow mode of Ca²⁺ uptake (via MCU) was attenuated by extra-matrix Mg²⁺ in the physiological concentration range. The model parameters for the simplified MCU model are: V_max, 900 nmol/mg/min; K_Ca, 6 μM; and K_Mg, 0.3 mM. Solid lines correspond to slow mode of MCU rates observed during protocol A, and dotted lines correspond to the slow mode of MCU rates observed during protocol B. The rates of Ca²⁺ uptake in protocol B were approximately 40 % of the rates observed in protocol A. Panel 8b shows buffer Mg²⁺ in the physiological range used in this study also affected the rate of Ca²⁺ efflux (via mNCE). The model parameters for the simplified NCE are: V_max, 40 nmol/mg/min; K_Ca, 5 μM; and K_Mg, 1 mM. The blue, yellow and red colors correspond to the 0 mM MgCl₂, 0.5 mM MgCl₂ and 1 mM MgCl₂ conditions, respectively. Extra-matrix [Na⁺] was assumed constant (10 mM) and thus not included in the equation. The circles (O) and x’s (x) represent rates obtained using the data from Protocols A and B, respectively. The lines correspond to model simulations with the equations given above their respective panels. Error bars signify propagated standard deviations