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Temperature dependence of mitoflash biogenesis in cardiac mitochondria
Archives of Biochemistry and Biophysics 666 (2019) 8–15
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Temperature dependence of mitoflash biogenesis in cardiac mitochondria Peng Yu, Wenfeng Qi, Bahetiyaer Huwatibieke, Jinghang Li, Xianhua Wang, Heping Cheng
∗
T
State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
A B S T R A C T
Mitochondrial flashes (mitoflashes) represent fundamental biochemical and biophysical dynamics of the organelle, involving sudden depolarization of mitochondrial membrane potential (ΔΨm), bursting production of reactive oxygen species (ROS), and accelerated extrusion of matrix protons. Here we investigated temperature dependence of mitoflash biogenesis as well as ΔΨm oscillations, a subset of which overlapping with mitoflashes, in both cardiac myocytes and isolated respiring cardiac mitochondria. Unexpectedly, we found that mitoflash biogenesis was essentially temperature-independent in intact cardiac myocytes, evidenced by the constancy of frequency as well as amplitude and rise speed over 5 °C–40 °C. Moderate temperature dependence was found in single mitochondria charged by respiratory substrates, where mitoflash frequency was decreased over 5 °C–20 °C with Q10 of 0.74 for Complex I substrates and 0.83 for Complex II substrate. In contrast, ΔΨm oscillation frequency displayed a negative temperature dependence at 5 °C–20 °C with Q10 of 0.82 in intact cells, but a positive temperature dependence at 25 °C - 40 °C with Q10 of 1.62 in isolated mitochondria charged with either Complex I or Complex II substrates. Moreover, the recovery speed of individual mitoflashes exhibited mild temperature dependence (Q10 = 1.14–1.22). These results suggest a temperature compensation of mitoflash frequency at both the mitochondrial and extra-organelle levels, and underscore that mitoflashes and ΔΨm oscillations are related but distinctly different mitochondrial functional dynamics.
1. Introduction It has been widely appreciated that mitochondria as the cellular power houses synthesize ATP through oxidative phosphorylation mediated by the inner membrane-bound electron transfer chain (ETC) and ATP synthase (Complex V) [1]. Vigorous research in the past two and a half decades, however, has unveiled a distinctive role of the organelle as a signaling hub in vital processes ranging from programmed cell death to innate immunity, from Ca2+ regulation to redox and ROS signaling [2–6]. In particular, we and others have shown that respiring mitochondria generate 10-s electrochemical pulses in the form of “mitochondrial flashes (mitoflashes)”, which comprise manifold signals including dissipation of ΔΨm, bursting ROS production, and transient matrix alkalization [7–10]. The mitoflash activity is universally present and highly conserved across all the eukaryotic cells and organisms examined [7,11–13]. As a signaling entity, mitoflashes are digital, local (single-mitochondrion), and stochastic, and operate in primary frequency-modulatory manner in response to regulatory factors (local protons [9], Ca2+ [14–16] and basal ROS [14,17,18]), altered metabolic rate and energy demand [19,20], and physiological activities such as synaptic transmission in hippocampal neurons [21]. Temperature is a fundamental biological variable that regulates diverse biophysical and biochemical processes ranging from diffusion and transport of metabolites to enzymatic activity and to compound
∗
physiological phenomena such as action potentials. As a rule of thumb, a diffusion-limited process displays a temperature coefficient (Q10) between 1.1 and 1.5 and enzymatic activity typically displays a Q10 of 2 and higher [22]. For compound parameters of a complex process, however, Q10 value varies over different temperature ranges because of differential temperature effects on the underlying sub-processes. In squid giant axons, the action potential duration is markedly shortened and its amplitude holds nearly constant below 30 °C and decreases rapidly with increasing temperature until the action potential is completely abolished at 40 °C [23]. Over different temperature ranges, the Q10 values of the rise speed vary from 1.5 to 2.7 and the Q10 values of the recovery speed from 2.1 to 5.3 [23]. In isolated mitochondria from rat ventricular myocytes with pyruvate and malate as substrates, the oxygen consumption rate increases from 5 °C to 35 °C, with Q10 values varying from 4.9 to 1.7, and decreases from 35 °C to 40 °C, with Q10 value of 0.8 [24]. For individual enzymes of pyruvate dehydrogenase, Complex I, and Complex IV, their activities increase monotonically with increasing temperature, while the activities of Complex II and Complex III are optimal around 30 °C [24]. The activity of ATP synthase increases by 7-fold from 10 °C to 25 °C and less than 2-fold from 25 °C to 37 °C [25]. These findings illustrate that characterizing the temperature sensitivity of mitoflash biogenesis should be revealing as to its underlying mechanisms and physiological regulation. The biogenesis of mitoflashes is an excitable phenomenon
Corresponding author. E-mail address: [email protected] (H. Cheng).
https://doi.org/10.1016/j.abb.2019.03.002 Received 7 December 2018; Received in revised form 1 March 2019; Accepted 3 March 2019 Available online 18 March 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.
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2.4. Isolation of cardiac mitochondria
comprising ignition, autonomous evolvement, and termination subprocesses. In a working model recently refined [13], stochastic and flickering openings of the mitochondrial permeability transition pore (mPTP) is thought to cause plummeting depolarization and ignite mitoflashes, through a Poisson process [26]. Once activated, depolarization accelerates electron transfer from the donor pool to ETC acceptors (e.g., from NADH to Complex I and from FADH2 to Complex II) and shifts the redox potential toward oxidation. Water and ionic influxes also mechanically stretch the inner mitochondrial membrane (IMM) and lead to subtle dislocation of the exquisite ETC molecular assembly. The resultant dis-insulation of the normal electron path at the intermolecular junctures promotes a burst of superoxide formation [13,27]. The depolarization accelerates ETC activity and its coupled proton pumping across the IMM, raising matrix pH. Termination of a mitoflash ensues upon the closure of the mPTP, restoration of ionic homeostasis, re-insulation of the ETC path, and restoration of normal ETC activity as ΔΨm repolarizes [13,27]. In this way, mitoflash biogenesis reflects multifaceted interlinked biophysical and biochemical processes, each likely displaying distinctive temperature sensitivity. In the present study, we characterized temperature dependence of mitoflash biogenesis in two systems, intact cardiac myocytes and isolated cardiac mitochondria supported with different substrates. We also examined ΔΨm oscillations as related mitochondrial dynamics for comparison. We revealed a remarkable invariance of mitoflash frequency in intact cells over a broad temperature range, but a moderate temperature-dependent change in isolated cardiac mitochondria. In addition, we showed that ΔΨm oscillation exhibited distinctively different patterns of temperature dependence. Overall, our results reveal a homeostatic regulation of mitoflash biogenesis in terms of temperature compensation and underscore an important role of the mitoflash as a signaling entity operating in the frequency modulatory mode.
Cardiac mitochondria were isolated from mt-cpYFP transgenic mice as previously reported with some modifications [7,10]. Briefly, the mouse heart was washed with ice-cold isolation buffer (300 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.5 mg/ml BSA, pH 7.2), minced and homogenized. The homogenate was centrifuged at 4 °C for 10 min at 600 g. The supernatant was collected and further centrifuged at 4 °C for 10 min at 6000 g. The pellet was re-suspended in isolation buffer and centrifuged at 4 °C for 10 min at 4000 g to make them adhere to coverslips for confocal microscopy imaging. 2.5. Temperature control A CherryTemp™ heater/cooler stage (Cherry BioTech) was used to control the temperature from 5 °C to 40 °C. The isolated cardiomyocytes or cardiac mitochondria were adhered to a coverslip and a silicone spacer of about 1 mm in thickness with a round hole of 1 cm in diameter was placed on the coverslip to form about 80 μl volume for bath solution. The heater/cooler block was placed above the spacer to form a sandwich structure and connected to the temperature controller by a closed fluidic loop. The temperature was then controlled by the flowing liquid in the closed fluidic loop. The temperature was altered alternatively from 5 °C to 40 °C and from 40 °C to 5 °C to avoid time-dependent effects. At each temperature, the cells or mitochondria were allowed at least 5 min for equilibrium. 2.6. Confocal imaging An inverted confocal microscope (Zeiss LSM 710) with a 40 × , 1.3 NA oil-immersion objective was used for imaging. The isolated cardiomyocytes and mitochondria were adhered to the coverslip which was temperately controlled by the temperature controller. The cardiomyocytes were incubated in Tyrode's solution (137 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose and 20 mM HEPES, pH 7.35) and mitochondria were incubated in an experimental buffer (125 mM KCl, 2 mM K2HPO4, 5 mM MgCl2, 10 mM HEPES, 1 mg/ml BSA, pH 7.4) in the presence of 2.5 mM succinate or 5 mM glutamate/malate for imaging. For TMRM measurement, cells or mitochondria were loaded with 100 nM TMRM at 37 °C for 15 min and then in 50 nM TMRM during imaging. To obtain mt-cpYFP and TMRM signals simultaneously, images were taken by tandem scanline excitation at 488, 405 and 543 nm, and emission collection at 505–530, 505–530, and > 560 nm, respectively. In a typical time-series recordings, 100 frames of 900 × 256 (for cardiomyocytes) or 512 × 512 pixels (for isolated mitochondria) were collected at 80–120 pixels/ 10 μm in bidirectional scanning mode. The frame rate was 1 frame/ second.
2. Material and methods 2.1. Animal care All animal experiments were carried out according to the rules of the American Association for the Accreditation of Laboratory Animal Care International (AAALAC) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All procedures were approved by the Animal Care Committee of Peking University accredited by AAALAC International (IMM-ChengHP-14). 2.2. Reagents Laminin were from Sigma-Aldrich Corporation. Tetramethylrhodamine methyl ester (TMRM) was from Molecular Probes (ThermoFisher). Other reagents were obtained commercially at the highest grade available.
2.7. Image processing and mitoflash analysis
2.3. Cardiomyocytes isolation
The time lapse image series were analyzed by a custom-developed program similar to FlashSniper written in Matlab [26]. Mitoflashes were identified manually by inspecting the peaks in the traces. Once the peak was identified the nearest minimum point prior to the peak point was marked as the start point and the first point reaching the 0.9-fold of peak was marked as end point automatically. The rise time (denoted as tonset) was defined as the time interval between the start point and peak point and the recovery time (denoted as t50) was defined as the interval between the peak point and the point whose amplitude is the half of peak in decaying phase. The same algorithm was used for parametric measurement of ΔΨm oscillations.
Single ventricular myocytes were enzymatically isolated from the hearts of mt-cpYFP transgenic mice as described previously [28]. Briefly, the mouse hearts were rapidly excised from the animals under anesthesia and perfused with a calcium-free buffer (120 mM NaCl, 5.4 mM KCl, 1.2 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES, 1.6 mM MgCl2, 5 mM taurine and 10 mM BDM, aerated with 95% O2 and 5% CO2, pH 7.35) for 2–3 min at 37 °C. The heart was then digested in a buffer containing 1 mg/ml collagenase (Worthington type II) and 1 mg/ ml bovine serum albumin and 20 μM Ca2+ for 25 min. The ventricle was then cut into small pieces and incubated in digesting solution. Finally the myocytes were harvested and placed on coverslip coated with laminin and incubated in Dulbecco Minimal Essential Medium (DMEM) for 1 h at 37 °C and then used for confocal microscopy imaging.
2.8. Q10 calculation Q10 was the fold change of a parameter for a 10 °C change in 9
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dissect possible regulation at the extra-organelle level, we also detected mitoflashes in isolated mitochondria from the transgenic mouse hearts charged with different respiratory substrates (glutamate/malate for Complex I or succinate for Complex II) in the absence of ADP (i.e., at state II/IV respiration) (Fig. 1C and D). Each and every mitoflash was accompanied by a transient downward deflection of TMRM signal representing depolarization of ΔΨm, namely ΔΨm oscillation (Fig. 1). However, a significant subpopulation of ΔΨm oscillations went without discernible companion mitoflashes in both experimental systems (Fig. 1), in agreement with previous reports [7,26]. To investigate the temperature dependence of these two types of functional dynamics of the mitochondria, we used a temperature control system (CherryTemp™ heater/cooler stage) which enables precise control of the bath chamber temperature over the range of 5 °C–40 °C at 5 °C interval (Fig. 1A). We anticipated that mitoflash biogenesis might be highly temperature-sensitive because of the high temperature sensitivity of enzymatic activities of pertinent ETC complexes [24]. To our surprise, by quantifying frequencies of mitoflashes at varying temperatures, we found that the mitoflash frequency in cardiomyocytes was nearly constant over the large temperature range examined (Fig. 2A, Table 1). Quantitatively, the temperature coefficient (Q10) was 0.98 at low temperature (5 °C - 20 °C) and 1.18 at higher temperature (25 °C - 40 °C), indicating little or no temperature-dependence of mitoflash biogenesis in cardiomyocytes. Moreover, we examined the temperature effect on properties of individual mitoflash by averaging mitoflash time courses (Fig. 3A), and revealed a slight temperature-
temperature. It was calculated by
X2 ⎞ Q10 = ⎛ ⎝ X1 ⎠
10 (T2− T1)
Where X2 is the parameter value at the higher temperature T2, X1 is the parameter value at the lower temperature T1. Operationally, we usually extracted Q10 by plotting the data on semi-log axes, drawing a straight line through the points by linear regression, and determining its slope. 2.9. Statistics Data are expressed as mean ± s.e.m. Student's t-test was applied to determine the statistical significance. P < 0.05 was considered statistically significant. 3. Results 3.1. Temperature effects on mitoflashes and ΔΨm oscillations in cardiac myocytes In single cardiac myocytes isolated from mice with transgenic expression of the mitochondrial targeted cpYFP, mitoflashes indexed with cpYFP and ΔΨm oscillations measured with TMRM were detected simultaneously. As reported previously [7,8,10], discrete mitoflashes, which were reflected by sudden, ∼10 s long cpYFP fluorescent transients, occurred spontaneously in intact cardiac myocytes (Fig. 1B). To
Fig. 1. Mitoflashes and ΔΨm oscillations in cardiomyocytes and isolated cardiac mitochondria. (A) Diagram of the temperature control device. (B) Upper, representative confocal microscopy images of cardiomyocytes expressing mt-cpYFP (green) and stained with TMRM (red). Scale bar, 10 μm. Lower, representative traces of mitoflashes indexed with cpYFP and ΔΨm oscillations indexed with TMRM. Arrows indicate ΔΨm oscillations with companion mitoflashes. Arrowhead indicates ΔΨm oscillation without companion mitoflashes. (C-D) Upper, representative images of isolated cardiac mitochondria supported by Complex I substrates glutamate/malate (5 mM) (C), or Complex II substrate succinate (2.5 mM) (D). Scale bars, 5 μm. Lower: representative traces of mitoflashes and ΔΨm oscillations. 10
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Fig. 2. Effect of temperature on the frequency of mitoflashes and ΔΨm oscillations. Upper: Frequencies of mitoflashes and ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 14–43 cells in (A), n = 8–22 frames in (B), n = 8–35 frames in (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The mitoflash or ΔΨm oscillation frequency was plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated.
was specific for the subpopulation of ΔΨm oscillations with no discernible companion mitoflashes. Indeed, the frequency of uncoupled ΔΨm oscillations showed a significant temperature-dependence with Q10 of 0.76 at 5 °C - 20 °C while Q10 for the coupled ones was 0.98 at the lower temperature (Fig. 5A). At 25 °C–40 °C, both coupled and uncoupled ΔΨm oscillations showed little temperature dependence (Fig. 5A). Moreover, the percentage of coupled ΔΨm oscillation was increased with Q10 of 1.36 at 5 °C - 20 °C and Q10 of 1.11 at 25 °C–40 °C (Fig. 6A). The percentage of uncoupled ΔΨm oscillation was decreased with Q10 of 0.89 at 5 °C - 20 °C and Q10 of 0.93 at 25 °C–40 °C (Fig. 6A).
dependent increase of mitoflash recovery speed with little change in mitoflash magnitude with increasing temperature (Figs. 3A and 4). Parametric measurements showed near-unity Q10 for amplitude and rise speed (indexed by 1/tonset) and a mild temperature dependence for recovery speed (indexed by 1/t50) with Q10 of 1.21 (Table 2). Thus, mitoflash biogenesis in cardiomyocytes is temperature-invariant in terms of frequency, amplitude, and onset kinetics, except for a mild temperature-dependence for mitoflash relaxation kinetics. A different pattern of temperature dependence was found for ΔΨm oscillations. Along with a trend of increase with lowering temperature, the ΔΨm oscillation frequency displayed a significant increase at 5 °C compared to 25 °C (Fig. 2A, Table 1). The Q10 for ΔΨm oscillation frequency was 0.82 at lower temperature (5 °C - 20 °C) (Fig. 2A), indicating a negative temperature-dependence over this temperature range. Except for a trend of temperature-dependent increase of ΔΨm oscillation amplitude (Q10 = 1.09) (Table 2), the kinetic parameters including rise speed and recovery speed exhibited little temperaturedependent changes (Table 2) such that the time courses of ΔΨm oscillations were nearly identical at different temperatures (Figs. 3A and 4). Because mitoflash frequency was largely temperature-independent, this increase of ΔΨm oscillation frequency with lowering temperature
3.2. Temperature effects on mitoflashes and ΔΨm oscillations in isolated cardiac mitochondria Respiring mitochondria in cell-free systems retain the ability to generate mitoflashes and ΔΨm oscillations in the absence of any extraorganelle regulatory mechanisms [10]. We determined the temperature effects on isolated cardiac mitochondria at state II/IV respiration. With either Complex I or Complex II substrates added, lowering temperature from 25 °C significantly increased mitoflash frequency (Fig. 2B and C, Table 1), indicating an intrinsically negative temperature dependence
Table 1 Frequencies of mitoflashes and ΔΨm oscillations as a function of temperature. Cardiomyocytes mitoflashes 5 °C 10 °C 15 °C 20 °C 25 °C 30 °C 35 °C 40 °C
1.14 1.33 1.13 1.17 1.00 1.02 1.04 1.31
± ± ± ± ± ± ± ±
0.16 0.30 0.34 0.20 0.26 0.26 0.19 0.35
Mitochondria, glutamate/malate ΔΨm oscillations 1.62 1.40 1.25 1.20 1.00 1.00 0.94 1.11
± ± ± ± ± ± ± ±
∗∗
0.11 0.14 0.15 0.12 0.18 0.16 0.13 0.19
ΔΨm oscillations
mitoflashes 1.76 1.41 1.35 1.07 1.00 0.94 0.98 1.23
± ± ± ± ± ± ± ±
Mitochondria, succinate
∗
0.24 0.19 0.20 0.25 0.11 0.14 0.12 0.30
0.88 0.83 0.95 1.00 1.00 1.14 1.72 1.95
± ± ± ± ± ± ± ±
0.08 0.07 0.14 0.11 0.07 0.10 0.11∗∗ 0.18∗∗
ΔΨm oscillations
mitoflashes 1.83 1.68 1.59 1.36 1.00 1.01 1.00 1.54
± ± ± ± ± ± ± ±
∗∗
0.14 0.15∗∗ 0.14∗∗ 0.20 0.13 0.23 0.16 0.34
1.14 1.04 1.11 1.05 1.00 1.19 1.55 2.05
± ± ± ± ± ± ± ±
0.06 0.05 0.08 0.12 0.07 0.13 0.13∗∗ 0.19∗∗
The frequencies at different temperatures are normalized to that of 25 °C. Data are graphically shown in Fig. 2 and expressed as mean ± s.e.m. ∗p < 0.05, ∗∗ p < 0.01 versus 25 °C group. 11
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Fig. 3. Averaged traces of mitoflashes and ΔΨm oscillations at different temperatures. Averaged traces of mitoflashes and ΔΨm oscillations at 5 °C–40 °C in cardiomyocytes (A) and isolated mitochondria supported with glutamate/malate (B) or succinate (C). n = 43–68 events for mitoflashes and 118–200 events for ΔΨm oscillations in (A), n = 38–82 events for mitoflashes and 110–319 events for ΔΨm oscillations in (B), n = 69–315 events for mitoflashes and 310–589 events for ΔΨm oscillations in (C). Fig. 4. Semi-log plots for the parameters of mitoflashes and ΔΨm oscillations. Semi-log plots of the amplitude (A), rising speed indexed with 1/tonset (B), and recovery speed indexed with 1/t50 (C) of mitoflashes (left) and ΔΨm oscillations (right) in cardiomyocytes or isolated mitochondria against temperature. n = 43–68 events for mitoflashes and n = 118–200 events for ΔΨm oscillations in cardiomyocytes, n = 38–82 events for mitoflashes and n = 110–319 events for ΔΨm oscillations in isolated mitochondria supported with glutamate/malate, and n = 69–315 events for mitoflashes and n = 310–589 events for ΔΨm oscillations in mitochondria supported with succinate.
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Table 2 Q10 values for the parameters of mitoflashes and ΔΨm oscillations under different experimental conditions. Δψm oscillations
mitoflashes
Cardiomyocytes Mitochondria, glutamate/malate Mitochondria, succinate
Amplitude
Rise speed
Recovery speed
Amplitude
Rise speed
Recovery speed
1.01 0.98 1.02
1.06 1.04 1.08
1.21 1.14 1.22
1.09 1.04 1.03
1.05 1.05 1.06
1.06 1.18 1.08
The Q10s are derived from Fig. 4.
and Table 2). Taken together, we conclude that the temperature effects on the two types of mitochondrial functional dynamics are temperature range- and experimental system-dependent, and that mitoflash biogenesis and ΔΨm oscillation display distinctly different behaviors in their response to varying temperatures.
of mitoflash biogenesis at the organelle level. The Q10 values were 0.74 with Complex I substrates glutamate/malate and 0.83 with Complex II substrate succinate (Fig. 2B and C). At the higher temperature range (25 °C - 40 °C), we observed a modest positive temperature-dependence (Q10 = 1.14 for Complex I substrates and Q10 = 1.29 for Complex II substrate). In contrast, lowering temperature from 25 °C showed no effect on ΔΨm oscillation frequency, but raising temperature from 25 °C significantly enhanced it with Q10 of 1.62 in either condition (Fig. 2B and C, Table 1). Moreover, the mitoflash uncoupled ΔΨm oscillation frequency showed a higher temperature dependence at either 5 °C 20 °C or 25 °C - 40 °C (Fig. 5B and C). The percentage of coupled ΔΨm oscillation was decreased with increasing temperature with Q10s of 0.65 (in the presence of glutamate/malate) and 0.86 (in the presence of succinate) at 5 °C - 20 °C and 0.68 (in the presence of glutamate/malate) and 0.75 (in the presence of succinate) at 25 °C–40 °C (Fig. 6B and C). The percentage of uncoupled ΔΨm oscillation was increased with increasing temperature with Q10s of 1.32 (in the presence of glutamate/ malate) and 1.23 (in the presence of succinate) at 5 °C - 20 °C (Fig. 6B and C). Regarding the amplitude and onset properties, however, little or very mild changes were observed over the temperature range examined, for both mitoflashes and ΔΨm oscillations, as was the case in intact cells (Table 2 and Fig. 4). The recovery of both mitoflashes and ΔΨm oscillations showed mild temperature-dependence (Fig. 4B and C
4. Discussion Mitoflash biogenesis involves multifaceted processes such as sudden ΔΨm depolarization, accelerated proton pumping catalyzed by ETC complexes as well as bursting ROS production. In essence, mitoflash reflects electrical and chemical excitation at the single-organelle level, and a pre-requisite is that the mitochondria should be charged bioenergetically, i.e., the establishment of proper proton electrochemical potential [9]. Moreover, ignition of mitoflashes is thought to be determined by the flickering opening of mPTP which by itself is the point of convergence of many intra-organelle and extra-organelle signals such as Ca2+, basal ROS, and microdomain protons. Termination of a mitoflash reflects the closure of the mPTP, restoration of ionic homeostasis, re-insulation of the ETC path, and restoration of normal ETC activity, and repolarization of ΔΨm [13,27]. Since many of these processes are known to be temperature-sensitive [22,29,30], the present study showed that mitoflash biogenesis displayed complex,
Fig. 5. Effect of temperature on the frequency of mitoflash coupled and uncoupled ΔΨm oscillations. Upper: Frequencies of mitoflash coupled and uncoupled ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 14–43 cells in (A), n = 8–22 frames in (B), n = 8–35 frames in (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The frequency of mitoflash coupled or uncoupled ΔΨm oscillations was plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated. 13
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Fig. 6. Effect of temperature on the percentages of mitoflash coupled and uncoupled ΔΨm oscillations. Upper: The percentages of mitoflash coupled and uncoupled ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 118–200 events in (A), n = 110–319 events in (B), n = 310–589 events (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The percentages of mitoflash coupled and uncoupled ΔΨm oscillations were plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated.
transporters. In this scenario, we found that mitoflash biogenesis and ΔΨm oscillation exhibited strikingly different temperature dependence. This result reflects that temperature may differentially regulate the gating of mPTP and the gating of other ionic channels underlying ΔΨm oscillations. Moreover, we demonstrate opposing pattern of temperature dependence of ΔΨm oscillation frequency in cardiomyocytes versus single mitochondrion. For instance, over 25 °C - 40 °C, there was substantial positive temperature dependence with Q10 1.62 in isolated mitochondria but a Q10 near unity in intact cardiomyocytes. Again, this experimental system-specific temperature dependence is suggestive of regulation of ΔΨm oscillations by some extra-organelle mechanisms. In summary, we have demonstrated the surprising temperature invariance of mitoflash frequency in intact cardiomyocytes. Together with the stereotyped mitoflash magnitude and onset kinetics, this result suggests not only a homeostatic regulation of mitoflash biogenesis but also a near perfect temperature compensation among the constituent processes underlying mitoflash biogenesis. Both intra- and extra-mitochondrial mechanisms likely contribute to this temperature-compensatory behavior, because, a mild to moderate, negative or positive, temperature-dependence of mitoflash frequency was unmasked over certain temperature range in isolated mitochondria. Further, the temperature dependence as well as the frequency of ΔΨm oscillations, a subset of which is coupled with mitoflash biogenesis, was strikingly different. This result indicates that mitoflash biogenesis and ΔΨm oscillation are mechanistically different, albeit related, mitochondrial functional dynamics. Taken together, characterization of mitolfash temperature dependence sheds new light on the mitoflash biogenesis and its physiological regulation.
experimental system- and temperature range-dependent features in response to temperature changes. Paradoxically, we found that mitoflash frequency in intact cardiomyocytes displayed no or very mild temperature dependence, depending on temperature range examined, in contrast to mitochondrial metabolic and respiratory activities which are highly sensitive to temperature [24,29]. The relative constancy of mitoflash frequency over a broad temperature range in intact cells suggests a temperature compensation of mitoflash biogenesis, resulting in a functional homeostatic regulation of this digital phenomenon. However, the temperature effect on isolated mitochondria was quite different: mitoflash frequency appeared to be mildly temperature-dependent at higher temperature, but was negatively and moderately temperature-dependent at lower temperature regardless of the respiratory substrates used. The disparity of temperature dependent mitoflash biogenesis in cells versus isolated mitochondria suggests the presence of extra-mitochondrial mechanisms that regulate the mitoflash production under physiological conditions. In both experimental systems across the temperature range examined, mitoflash amplitude was highly invariant, so was mitoflash rise speed, while the relaxation kinetics of mitoflash displayed modest positive temperature dependence. Averaged time courses of mitoflashes were stereotypical at different temperatures, except for temporal kinetics. Since the mitoflash amplitude is collectively determined by the duration and conductance of mPTP opening, the rate of proton pumping mediated by ETC, and the rate of electron leakage for ROS production, this result suggests that a near-perfect compensation among the processes to increase and those to decrease the mitoflash magnitude. It has been shown that ΔΨm oscillations could arise from different types of ionic channels as well as electrogenic transporters in the inner mitochondrial membrane [31–33]. Of them, mitoflash-linked ΔΨm oscillations only represent a subset arising from transient mPTP openings, while the uncoupled counterpart might be induced by the opening of other types of ionic channels or the activation of electrogenic
Author contributions Heping Cheng and Xianhua Wang conceived and supervised the study. Peng Yu performed experiments and analyzed data. Peng Yu, Wenfeng Qi, and Bahetiyaer Huwatibieke developed the temperature 14
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controlling system. Jinghang Li developed imaging processing algorithms and software. Xianhua Wang and Heping Cheng wrote the paper with input from all authors. All participated in data interpretation.
[16] [17]
Acknowledgments
[18]
This work was supported by the National Key Basic Research Program of China (2016YFA0500403, 2017YFA0504002, and 2013CB531200) and the National Science Foundation of China (31470811, 31521062, and 31821091). The authors declare no competing financial interests.
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Temperature dependence of mitoflash biogenesis in cardiac mitochondria Peng Yu, Wenfeng Qi, Bahetiyaer Huwatibieke, Jinghang Li, Xianhua Wang, Heping Cheng
∗
T
State Key Laboratory of Membrane Biology, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing, 100871, China
A B S T R A C T
Mitochondrial flashes (mitoflashes) represent fundamental biochemical and biophysical dynamics of the organelle, involving sudden depolarization of mitochondrial membrane potential (ΔΨm), bursting production of reactive oxygen species (ROS), and accelerated extrusion of matrix protons. Here we investigated temperature dependence of mitoflash biogenesis as well as ΔΨm oscillations, a subset of which overlapping with mitoflashes, in both cardiac myocytes and isolated respiring cardiac mitochondria. Unexpectedly, we found that mitoflash biogenesis was essentially temperature-independent in intact cardiac myocytes, evidenced by the constancy of frequency as well as amplitude and rise speed over 5 °C–40 °C. Moderate temperature dependence was found in single mitochondria charged by respiratory substrates, where mitoflash frequency was decreased over 5 °C–20 °C with Q10 of 0.74 for Complex I substrates and 0.83 for Complex II substrate. In contrast, ΔΨm oscillation frequency displayed a negative temperature dependence at 5 °C–20 °C with Q10 of 0.82 in intact cells, but a positive temperature dependence at 25 °C - 40 °C with Q10 of 1.62 in isolated mitochondria charged with either Complex I or Complex II substrates. Moreover, the recovery speed of individual mitoflashes exhibited mild temperature dependence (Q10 = 1.14–1.22). These results suggest a temperature compensation of mitoflash frequency at both the mitochondrial and extra-organelle levels, and underscore that mitoflashes and ΔΨm oscillations are related but distinctly different mitochondrial functional dynamics.
1. Introduction It has been widely appreciated that mitochondria as the cellular power houses synthesize ATP through oxidative phosphorylation mediated by the inner membrane-bound electron transfer chain (ETC) and ATP synthase (Complex V) [1]. Vigorous research in the past two and a half decades, however, has unveiled a distinctive role of the organelle as a signaling hub in vital processes ranging from programmed cell death to innate immunity, from Ca2+ regulation to redox and ROS signaling [2–6]. In particular, we and others have shown that respiring mitochondria generate 10-s electrochemical pulses in the form of “mitochondrial flashes (mitoflashes)”, which comprise manifold signals including dissipation of ΔΨm, bursting ROS production, and transient matrix alkalization [7–10]. The mitoflash activity is universally present and highly conserved across all the eukaryotic cells and organisms examined [7,11–13]. As a signaling entity, mitoflashes are digital, local (single-mitochondrion), and stochastic, and operate in primary frequency-modulatory manner in response to regulatory factors (local protons [9], Ca2+ [14–16] and basal ROS [14,17,18]), altered metabolic rate and energy demand [19,20], and physiological activities such as synaptic transmission in hippocampal neurons [21]. Temperature is a fundamental biological variable that regulates diverse biophysical and biochemical processes ranging from diffusion and transport of metabolites to enzymatic activity and to compound
∗
physiological phenomena such as action potentials. As a rule of thumb, a diffusion-limited process displays a temperature coefficient (Q10) between 1.1 and 1.5 and enzymatic activity typically displays a Q10 of 2 and higher [22]. For compound parameters of a complex process, however, Q10 value varies over different temperature ranges because of differential temperature effects on the underlying sub-processes. In squid giant axons, the action potential duration is markedly shortened and its amplitude holds nearly constant below 30 °C and decreases rapidly with increasing temperature until the action potential is completely abolished at 40 °C [23]. Over different temperature ranges, the Q10 values of the rise speed vary from 1.5 to 2.7 and the Q10 values of the recovery speed from 2.1 to 5.3 [23]. In isolated mitochondria from rat ventricular myocytes with pyruvate and malate as substrates, the oxygen consumption rate increases from 5 °C to 35 °C, with Q10 values varying from 4.9 to 1.7, and decreases from 35 °C to 40 °C, with Q10 value of 0.8 [24]. For individual enzymes of pyruvate dehydrogenase, Complex I, and Complex IV, their activities increase monotonically with increasing temperature, while the activities of Complex II and Complex III are optimal around 30 °C [24]. The activity of ATP synthase increases by 7-fold from 10 °C to 25 °C and less than 2-fold from 25 °C to 37 °C [25]. These findings illustrate that characterizing the temperature sensitivity of mitoflash biogenesis should be revealing as to its underlying mechanisms and physiological regulation. The biogenesis of mitoflashes is an excitable phenomenon
Corresponding author. E-mail address: [email protected] (H. Cheng).
https://doi.org/10.1016/j.abb.2019.03.002 Received 7 December 2018; Received in revised form 1 March 2019; Accepted 3 March 2019 Available online 18 March 2019 0003-9861/ © 2019 Elsevier Inc. All rights reserved.
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2.4. Isolation of cardiac mitochondria
comprising ignition, autonomous evolvement, and termination subprocesses. In a working model recently refined [13], stochastic and flickering openings of the mitochondrial permeability transition pore (mPTP) is thought to cause plummeting depolarization and ignite mitoflashes, through a Poisson process [26]. Once activated, depolarization accelerates electron transfer from the donor pool to ETC acceptors (e.g., from NADH to Complex I and from FADH2 to Complex II) and shifts the redox potential toward oxidation. Water and ionic influxes also mechanically stretch the inner mitochondrial membrane (IMM) and lead to subtle dislocation of the exquisite ETC molecular assembly. The resultant dis-insulation of the normal electron path at the intermolecular junctures promotes a burst of superoxide formation [13,27]. The depolarization accelerates ETC activity and its coupled proton pumping across the IMM, raising matrix pH. Termination of a mitoflash ensues upon the closure of the mPTP, restoration of ionic homeostasis, re-insulation of the ETC path, and restoration of normal ETC activity as ΔΨm repolarizes [13,27]. In this way, mitoflash biogenesis reflects multifaceted interlinked biophysical and biochemical processes, each likely displaying distinctive temperature sensitivity. In the present study, we characterized temperature dependence of mitoflash biogenesis in two systems, intact cardiac myocytes and isolated cardiac mitochondria supported with different substrates. We also examined ΔΨm oscillations as related mitochondrial dynamics for comparison. We revealed a remarkable invariance of mitoflash frequency in intact cells over a broad temperature range, but a moderate temperature-dependent change in isolated cardiac mitochondria. In addition, we showed that ΔΨm oscillation exhibited distinctively different patterns of temperature dependence. Overall, our results reveal a homeostatic regulation of mitoflash biogenesis in terms of temperature compensation and underscore an important role of the mitoflash as a signaling entity operating in the frequency modulatory mode.
Cardiac mitochondria were isolated from mt-cpYFP transgenic mice as previously reported with some modifications [7,10]. Briefly, the mouse heart was washed with ice-cold isolation buffer (300 mM sucrose, 5 mM HEPES, 1 mM EGTA, 0.5 mg/ml BSA, pH 7.2), minced and homogenized. The homogenate was centrifuged at 4 °C for 10 min at 600 g. The supernatant was collected and further centrifuged at 4 °C for 10 min at 6000 g. The pellet was re-suspended in isolation buffer and centrifuged at 4 °C for 10 min at 4000 g to make them adhere to coverslips for confocal microscopy imaging. 2.5. Temperature control A CherryTemp™ heater/cooler stage (Cherry BioTech) was used to control the temperature from 5 °C to 40 °C. The isolated cardiomyocytes or cardiac mitochondria were adhered to a coverslip and a silicone spacer of about 1 mm in thickness with a round hole of 1 cm in diameter was placed on the coverslip to form about 80 μl volume for bath solution. The heater/cooler block was placed above the spacer to form a sandwich structure and connected to the temperature controller by a closed fluidic loop. The temperature was then controlled by the flowing liquid in the closed fluidic loop. The temperature was altered alternatively from 5 °C to 40 °C and from 40 °C to 5 °C to avoid time-dependent effects. At each temperature, the cells or mitochondria were allowed at least 5 min for equilibrium. 2.6. Confocal imaging An inverted confocal microscope (Zeiss LSM 710) with a 40 × , 1.3 NA oil-immersion objective was used for imaging. The isolated cardiomyocytes and mitochondria were adhered to the coverslip which was temperately controlled by the temperature controller. The cardiomyocytes were incubated in Tyrode's solution (137 mM NaCl, 5.4 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 1.8 mM CaCl2, 10 mM glucose and 20 mM HEPES, pH 7.35) and mitochondria were incubated in an experimental buffer (125 mM KCl, 2 mM K2HPO4, 5 mM MgCl2, 10 mM HEPES, 1 mg/ml BSA, pH 7.4) in the presence of 2.5 mM succinate or 5 mM glutamate/malate for imaging. For TMRM measurement, cells or mitochondria were loaded with 100 nM TMRM at 37 °C for 15 min and then in 50 nM TMRM during imaging. To obtain mt-cpYFP and TMRM signals simultaneously, images were taken by tandem scanline excitation at 488, 405 and 543 nm, and emission collection at 505–530, 505–530, and > 560 nm, respectively. In a typical time-series recordings, 100 frames of 900 × 256 (for cardiomyocytes) or 512 × 512 pixels (for isolated mitochondria) were collected at 80–120 pixels/ 10 μm in bidirectional scanning mode. The frame rate was 1 frame/ second.
2. Material and methods 2.1. Animal care All animal experiments were carried out according to the rules of the American Association for the Accreditation of Laboratory Animal Care International (AAALAC) and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). All procedures were approved by the Animal Care Committee of Peking University accredited by AAALAC International (IMM-ChengHP-14). 2.2. Reagents Laminin were from Sigma-Aldrich Corporation. Tetramethylrhodamine methyl ester (TMRM) was from Molecular Probes (ThermoFisher). Other reagents were obtained commercially at the highest grade available.
2.7. Image processing and mitoflash analysis
2.3. Cardiomyocytes isolation
The time lapse image series were analyzed by a custom-developed program similar to FlashSniper written in Matlab [26]. Mitoflashes were identified manually by inspecting the peaks in the traces. Once the peak was identified the nearest minimum point prior to the peak point was marked as the start point and the first point reaching the 0.9-fold of peak was marked as end point automatically. The rise time (denoted as tonset) was defined as the time interval between the start point and peak point and the recovery time (denoted as t50) was defined as the interval between the peak point and the point whose amplitude is the half of peak in decaying phase. The same algorithm was used for parametric measurement of ΔΨm oscillations.
Single ventricular myocytes were enzymatically isolated from the hearts of mt-cpYFP transgenic mice as described previously [28]. Briefly, the mouse hearts were rapidly excised from the animals under anesthesia and perfused with a calcium-free buffer (120 mM NaCl, 5.4 mM KCl, 1.2 mM NaH2PO4, 5.6 mM glucose, 10 mM HEPES, 1.6 mM MgCl2, 5 mM taurine and 10 mM BDM, aerated with 95% O2 and 5% CO2, pH 7.35) for 2–3 min at 37 °C. The heart was then digested in a buffer containing 1 mg/ml collagenase (Worthington type II) and 1 mg/ ml bovine serum albumin and 20 μM Ca2+ for 25 min. The ventricle was then cut into small pieces and incubated in digesting solution. Finally the myocytes were harvested and placed on coverslip coated with laminin and incubated in Dulbecco Minimal Essential Medium (DMEM) for 1 h at 37 °C and then used for confocal microscopy imaging.
2.8. Q10 calculation Q10 was the fold change of a parameter for a 10 °C change in 9
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dissect possible regulation at the extra-organelle level, we also detected mitoflashes in isolated mitochondria from the transgenic mouse hearts charged with different respiratory substrates (glutamate/malate for Complex I or succinate for Complex II) in the absence of ADP (i.e., at state II/IV respiration) (Fig. 1C and D). Each and every mitoflash was accompanied by a transient downward deflection of TMRM signal representing depolarization of ΔΨm, namely ΔΨm oscillation (Fig. 1). However, a significant subpopulation of ΔΨm oscillations went without discernible companion mitoflashes in both experimental systems (Fig. 1), in agreement with previous reports [7,26]. To investigate the temperature dependence of these two types of functional dynamics of the mitochondria, we used a temperature control system (CherryTemp™ heater/cooler stage) which enables precise control of the bath chamber temperature over the range of 5 °C–40 °C at 5 °C interval (Fig. 1A). We anticipated that mitoflash biogenesis might be highly temperature-sensitive because of the high temperature sensitivity of enzymatic activities of pertinent ETC complexes [24]. To our surprise, by quantifying frequencies of mitoflashes at varying temperatures, we found that the mitoflash frequency in cardiomyocytes was nearly constant over the large temperature range examined (Fig. 2A, Table 1). Quantitatively, the temperature coefficient (Q10) was 0.98 at low temperature (5 °C - 20 °C) and 1.18 at higher temperature (25 °C - 40 °C), indicating little or no temperature-dependence of mitoflash biogenesis in cardiomyocytes. Moreover, we examined the temperature effect on properties of individual mitoflash by averaging mitoflash time courses (Fig. 3A), and revealed a slight temperature-
temperature. It was calculated by
X2 ⎞ Q10 = ⎛ ⎝ X1 ⎠
10 (T2− T1)
Where X2 is the parameter value at the higher temperature T2, X1 is the parameter value at the lower temperature T1. Operationally, we usually extracted Q10 by plotting the data on semi-log axes, drawing a straight line through the points by linear regression, and determining its slope. 2.9. Statistics Data are expressed as mean ± s.e.m. Student's t-test was applied to determine the statistical significance. P < 0.05 was considered statistically significant. 3. Results 3.1. Temperature effects on mitoflashes and ΔΨm oscillations in cardiac myocytes In single cardiac myocytes isolated from mice with transgenic expression of the mitochondrial targeted cpYFP, mitoflashes indexed with cpYFP and ΔΨm oscillations measured with TMRM were detected simultaneously. As reported previously [7,8,10], discrete mitoflashes, which were reflected by sudden, ∼10 s long cpYFP fluorescent transients, occurred spontaneously in intact cardiac myocytes (Fig. 1B). To
Fig. 1. Mitoflashes and ΔΨm oscillations in cardiomyocytes and isolated cardiac mitochondria. (A) Diagram of the temperature control device. (B) Upper, representative confocal microscopy images of cardiomyocytes expressing mt-cpYFP (green) and stained with TMRM (red). Scale bar, 10 μm. Lower, representative traces of mitoflashes indexed with cpYFP and ΔΨm oscillations indexed with TMRM. Arrows indicate ΔΨm oscillations with companion mitoflashes. Arrowhead indicates ΔΨm oscillation without companion mitoflashes. (C-D) Upper, representative images of isolated cardiac mitochondria supported by Complex I substrates glutamate/malate (5 mM) (C), or Complex II substrate succinate (2.5 mM) (D). Scale bars, 5 μm. Lower: representative traces of mitoflashes and ΔΨm oscillations. 10
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Fig. 2. Effect of temperature on the frequency of mitoflashes and ΔΨm oscillations. Upper: Frequencies of mitoflashes and ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 14–43 cells in (A), n = 8–22 frames in (B), n = 8–35 frames in (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The mitoflash or ΔΨm oscillation frequency was plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated.
was specific for the subpopulation of ΔΨm oscillations with no discernible companion mitoflashes. Indeed, the frequency of uncoupled ΔΨm oscillations showed a significant temperature-dependence with Q10 of 0.76 at 5 °C - 20 °C while Q10 for the coupled ones was 0.98 at the lower temperature (Fig. 5A). At 25 °C–40 °C, both coupled and uncoupled ΔΨm oscillations showed little temperature dependence (Fig. 5A). Moreover, the percentage of coupled ΔΨm oscillation was increased with Q10 of 1.36 at 5 °C - 20 °C and Q10 of 1.11 at 25 °C–40 °C (Fig. 6A). The percentage of uncoupled ΔΨm oscillation was decreased with Q10 of 0.89 at 5 °C - 20 °C and Q10 of 0.93 at 25 °C–40 °C (Fig. 6A).
dependent increase of mitoflash recovery speed with little change in mitoflash magnitude with increasing temperature (Figs. 3A and 4). Parametric measurements showed near-unity Q10 for amplitude and rise speed (indexed by 1/tonset) and a mild temperature dependence for recovery speed (indexed by 1/t50) with Q10 of 1.21 (Table 2). Thus, mitoflash biogenesis in cardiomyocytes is temperature-invariant in terms of frequency, amplitude, and onset kinetics, except for a mild temperature-dependence for mitoflash relaxation kinetics. A different pattern of temperature dependence was found for ΔΨm oscillations. Along with a trend of increase with lowering temperature, the ΔΨm oscillation frequency displayed a significant increase at 5 °C compared to 25 °C (Fig. 2A, Table 1). The Q10 for ΔΨm oscillation frequency was 0.82 at lower temperature (5 °C - 20 °C) (Fig. 2A), indicating a negative temperature-dependence over this temperature range. Except for a trend of temperature-dependent increase of ΔΨm oscillation amplitude (Q10 = 1.09) (Table 2), the kinetic parameters including rise speed and recovery speed exhibited little temperaturedependent changes (Table 2) such that the time courses of ΔΨm oscillations were nearly identical at different temperatures (Figs. 3A and 4). Because mitoflash frequency was largely temperature-independent, this increase of ΔΨm oscillation frequency with lowering temperature
3.2. Temperature effects on mitoflashes and ΔΨm oscillations in isolated cardiac mitochondria Respiring mitochondria in cell-free systems retain the ability to generate mitoflashes and ΔΨm oscillations in the absence of any extraorganelle regulatory mechanisms [10]. We determined the temperature effects on isolated cardiac mitochondria at state II/IV respiration. With either Complex I or Complex II substrates added, lowering temperature from 25 °C significantly increased mitoflash frequency (Fig. 2B and C, Table 1), indicating an intrinsically negative temperature dependence
Table 1 Frequencies of mitoflashes and ΔΨm oscillations as a function of temperature. Cardiomyocytes mitoflashes 5 °C 10 °C 15 °C 20 °C 25 °C 30 °C 35 °C 40 °C
1.14 1.33 1.13 1.17 1.00 1.02 1.04 1.31
± ± ± ± ± ± ± ±
0.16 0.30 0.34 0.20 0.26 0.26 0.19 0.35
Mitochondria, glutamate/malate ΔΨm oscillations 1.62 1.40 1.25 1.20 1.00 1.00 0.94 1.11
± ± ± ± ± ± ± ±
∗∗
0.11 0.14 0.15 0.12 0.18 0.16 0.13 0.19
ΔΨm oscillations
mitoflashes 1.76 1.41 1.35 1.07 1.00 0.94 0.98 1.23
± ± ± ± ± ± ± ±
Mitochondria, succinate
∗
0.24 0.19 0.20 0.25 0.11 0.14 0.12 0.30
0.88 0.83 0.95 1.00 1.00 1.14 1.72 1.95
± ± ± ± ± ± ± ±
0.08 0.07 0.14 0.11 0.07 0.10 0.11∗∗ 0.18∗∗
ΔΨm oscillations
mitoflashes 1.83 1.68 1.59 1.36 1.00 1.01 1.00 1.54
± ± ± ± ± ± ± ±
∗∗
0.14 0.15∗∗ 0.14∗∗ 0.20 0.13 0.23 0.16 0.34
1.14 1.04 1.11 1.05 1.00 1.19 1.55 2.05
± ± ± ± ± ± ± ±
0.06 0.05 0.08 0.12 0.07 0.13 0.13∗∗ 0.19∗∗
The frequencies at different temperatures are normalized to that of 25 °C. Data are graphically shown in Fig. 2 and expressed as mean ± s.e.m. ∗p < 0.05, ∗∗ p < 0.01 versus 25 °C group. 11
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Fig. 3. Averaged traces of mitoflashes and ΔΨm oscillations at different temperatures. Averaged traces of mitoflashes and ΔΨm oscillations at 5 °C–40 °C in cardiomyocytes (A) and isolated mitochondria supported with glutamate/malate (B) or succinate (C). n = 43–68 events for mitoflashes and 118–200 events for ΔΨm oscillations in (A), n = 38–82 events for mitoflashes and 110–319 events for ΔΨm oscillations in (B), n = 69–315 events for mitoflashes and 310–589 events for ΔΨm oscillations in (C). Fig. 4. Semi-log plots for the parameters of mitoflashes and ΔΨm oscillations. Semi-log plots of the amplitude (A), rising speed indexed with 1/tonset (B), and recovery speed indexed with 1/t50 (C) of mitoflashes (left) and ΔΨm oscillations (right) in cardiomyocytes or isolated mitochondria against temperature. n = 43–68 events for mitoflashes and n = 118–200 events for ΔΨm oscillations in cardiomyocytes, n = 38–82 events for mitoflashes and n = 110–319 events for ΔΨm oscillations in isolated mitochondria supported with glutamate/malate, and n = 69–315 events for mitoflashes and n = 310–589 events for ΔΨm oscillations in mitochondria supported with succinate.
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Table 2 Q10 values for the parameters of mitoflashes and ΔΨm oscillations under different experimental conditions. Δψm oscillations
mitoflashes
Cardiomyocytes Mitochondria, glutamate/malate Mitochondria, succinate
Amplitude
Rise speed
Recovery speed
Amplitude
Rise speed
Recovery speed
1.01 0.98 1.02
1.06 1.04 1.08
1.21 1.14 1.22
1.09 1.04 1.03
1.05 1.05 1.06
1.06 1.18 1.08
The Q10s are derived from Fig. 4.
and Table 2). Taken together, we conclude that the temperature effects on the two types of mitochondrial functional dynamics are temperature range- and experimental system-dependent, and that mitoflash biogenesis and ΔΨm oscillation display distinctly different behaviors in their response to varying temperatures.
of mitoflash biogenesis at the organelle level. The Q10 values were 0.74 with Complex I substrates glutamate/malate and 0.83 with Complex II substrate succinate (Fig. 2B and C). At the higher temperature range (25 °C - 40 °C), we observed a modest positive temperature-dependence (Q10 = 1.14 for Complex I substrates and Q10 = 1.29 for Complex II substrate). In contrast, lowering temperature from 25 °C showed no effect on ΔΨm oscillation frequency, but raising temperature from 25 °C significantly enhanced it with Q10 of 1.62 in either condition (Fig. 2B and C, Table 1). Moreover, the mitoflash uncoupled ΔΨm oscillation frequency showed a higher temperature dependence at either 5 °C 20 °C or 25 °C - 40 °C (Fig. 5B and C). The percentage of coupled ΔΨm oscillation was decreased with increasing temperature with Q10s of 0.65 (in the presence of glutamate/malate) and 0.86 (in the presence of succinate) at 5 °C - 20 °C and 0.68 (in the presence of glutamate/malate) and 0.75 (in the presence of succinate) at 25 °C–40 °C (Fig. 6B and C). The percentage of uncoupled ΔΨm oscillation was increased with increasing temperature with Q10s of 1.32 (in the presence of glutamate/ malate) and 1.23 (in the presence of succinate) at 5 °C - 20 °C (Fig. 6B and C). Regarding the amplitude and onset properties, however, little or very mild changes were observed over the temperature range examined, for both mitoflashes and ΔΨm oscillations, as was the case in intact cells (Table 2 and Fig. 4). The recovery of both mitoflashes and ΔΨm oscillations showed mild temperature-dependence (Fig. 4B and C
4. Discussion Mitoflash biogenesis involves multifaceted processes such as sudden ΔΨm depolarization, accelerated proton pumping catalyzed by ETC complexes as well as bursting ROS production. In essence, mitoflash reflects electrical and chemical excitation at the single-organelle level, and a pre-requisite is that the mitochondria should be charged bioenergetically, i.e., the establishment of proper proton electrochemical potential [9]. Moreover, ignition of mitoflashes is thought to be determined by the flickering opening of mPTP which by itself is the point of convergence of many intra-organelle and extra-organelle signals such as Ca2+, basal ROS, and microdomain protons. Termination of a mitoflash reflects the closure of the mPTP, restoration of ionic homeostasis, re-insulation of the ETC path, and restoration of normal ETC activity, and repolarization of ΔΨm [13,27]. Since many of these processes are known to be temperature-sensitive [22,29,30], the present study showed that mitoflash biogenesis displayed complex,
Fig. 5. Effect of temperature on the frequency of mitoflash coupled and uncoupled ΔΨm oscillations. Upper: Frequencies of mitoflash coupled and uncoupled ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 14–43 cells in (A), n = 8–22 frames in (B), n = 8–35 frames in (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The frequency of mitoflash coupled or uncoupled ΔΨm oscillations was plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated. 13
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Fig. 6. Effect of temperature on the percentages of mitoflash coupled and uncoupled ΔΨm oscillations. Upper: The percentages of mitoflash coupled and uncoupled ΔΨm oscillations at different temperatures in cardiomyocytes (A), isolated mitochondria supported with glutamate/malate (B) or succinate (C). Data were mean ± s.e.m. n = 118–200 events in (A), n = 110–319 events in (B), n = 310–589 events (C). *p < 0.05, **p < 0.01 versus 25 °C group. Lower: Semi-log plot of data in A. The percentages of mitoflash coupled and uncoupled ΔΨm oscillations were plotted against temperature. The slopes were determined by linear regression at lower temperature range (5 °C - 20 °C) and higher temperature range (25 °C–40 °C) and the corresponding Q10s were indicated.
transporters. In this scenario, we found that mitoflash biogenesis and ΔΨm oscillation exhibited strikingly different temperature dependence. This result reflects that temperature may differentially regulate the gating of mPTP and the gating of other ionic channels underlying ΔΨm oscillations. Moreover, we demonstrate opposing pattern of temperature dependence of ΔΨm oscillation frequency in cardiomyocytes versus single mitochondrion. For instance, over 25 °C - 40 °C, there was substantial positive temperature dependence with Q10 1.62 in isolated mitochondria but a Q10 near unity in intact cardiomyocytes. Again, this experimental system-specific temperature dependence is suggestive of regulation of ΔΨm oscillations by some extra-organelle mechanisms. In summary, we have demonstrated the surprising temperature invariance of mitoflash frequency in intact cardiomyocytes. Together with the stereotyped mitoflash magnitude and onset kinetics, this result suggests not only a homeostatic regulation of mitoflash biogenesis but also a near perfect temperature compensation among the constituent processes underlying mitoflash biogenesis. Both intra- and extra-mitochondrial mechanisms likely contribute to this temperature-compensatory behavior, because, a mild to moderate, negative or positive, temperature-dependence of mitoflash frequency was unmasked over certain temperature range in isolated mitochondria. Further, the temperature dependence as well as the frequency of ΔΨm oscillations, a subset of which is coupled with mitoflash biogenesis, was strikingly different. This result indicates that mitoflash biogenesis and ΔΨm oscillation are mechanistically different, albeit related, mitochondrial functional dynamics. Taken together, characterization of mitolfash temperature dependence sheds new light on the mitoflash biogenesis and its physiological regulation.
experimental system- and temperature range-dependent features in response to temperature changes. Paradoxically, we found that mitoflash frequency in intact cardiomyocytes displayed no or very mild temperature dependence, depending on temperature range examined, in contrast to mitochondrial metabolic and respiratory activities which are highly sensitive to temperature [24,29]. The relative constancy of mitoflash frequency over a broad temperature range in intact cells suggests a temperature compensation of mitoflash biogenesis, resulting in a functional homeostatic regulation of this digital phenomenon. However, the temperature effect on isolated mitochondria was quite different: mitoflash frequency appeared to be mildly temperature-dependent at higher temperature, but was negatively and moderately temperature-dependent at lower temperature regardless of the respiratory substrates used. The disparity of temperature dependent mitoflash biogenesis in cells versus isolated mitochondria suggests the presence of extra-mitochondrial mechanisms that regulate the mitoflash production under physiological conditions. In both experimental systems across the temperature range examined, mitoflash amplitude was highly invariant, so was mitoflash rise speed, while the relaxation kinetics of mitoflash displayed modest positive temperature dependence. Averaged time courses of mitoflashes were stereotypical at different temperatures, except for temporal kinetics. Since the mitoflash amplitude is collectively determined by the duration and conductance of mPTP opening, the rate of proton pumping mediated by ETC, and the rate of electron leakage for ROS production, this result suggests that a near-perfect compensation among the processes to increase and those to decrease the mitoflash magnitude. It has been shown that ΔΨm oscillations could arise from different types of ionic channels as well as electrogenic transporters in the inner mitochondrial membrane [31–33]. Of them, mitoflash-linked ΔΨm oscillations only represent a subset arising from transient mPTP openings, while the uncoupled counterpart might be induced by the opening of other types of ionic channels or the activation of electrogenic
Author contributions Heping Cheng and Xianhua Wang conceived and supervised the study. Peng Yu performed experiments and analyzed data. Peng Yu, Wenfeng Qi, and Bahetiyaer Huwatibieke developed the temperature 14
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controlling system. Jinghang Li developed imaging processing algorithms and software. Xianhua Wang and Heping Cheng wrote the paper with input from all authors. All participated in data interpretation.
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Acknowledgments
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This work was supported by the National Key Basic Research Program of China (2016YFA0500403, 2017YFA0504002, and 2013CB531200) and the National Science Foundation of China (31470811, 31521062, and 31821091). The authors declare no competing financial interests.
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