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Biophysical Journal
Volume 110
February 2016
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Article Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2D Kalyan C. Vinnakota,1,* Abhishek Singhal,1 Franc¸oise Van den Bergh,1 Masoumeh Bagher-Oskouei,1 Robert W. Wiseman,2 and Daniel A. Beard1 1 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; and 2Department of Physiology, Michigan State University, East Lansing, Michigan
ABSTRACT In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.
INTRODUCTION Mitochondria transduce the chemical potential from oxidation of carbon substrates into the chemical potential of the ATP hydrolysis reaction though oxidative phosphorylation. Most of the chemical energy from substrate oxidation in this process is obtained in the terminal oxidative pathway of the Krebs cycle in the generation of reducing equivalents NADH and FADH2 in reactions catalyzed by dehydrogenases, and from the transfer of electrons from those reducing equivalents to oxygen. Therefore, the relationship between the degree of reduction of the total NADH pool and rate of respiration (VO2) in a steady state constitutes an important quantitative descriptor that captures the balance between the processes generating and consuming NADH. Modulation of this relationship by feedback and open-loop controllers of oxidative phosphorylation can provide insight into regulation of the overall process of terminal oxidation of substrates and oxidative phosphorylation in response to fluxes of ATP hydrolysis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the system, whereas feedback control is mediated by reactants and products of the biochemical reactions in the system. Calcium (Ca2þ) is known to stimulate the activities of several mitochondrial dehydrogenases including pyruvate
Submitted November 26, 2014, and accepted for publication December 14, 2015. *Correspondence: [email protected]
dehydrogenase complex, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase (1,2). Partly on this basis, Ca2þ is speculated to stimulate the flux of mitochondrial ATP synthesis in an open-loop control to meet cellular ATP demand in the heart over a range of cellular contractile work states that is also regulated by Ca2þ (3). However, recent work showed that feedback from products of ATP hydrolysis was sufficient in explaining the relationship between steady-state metabolite concentrations and the flux of oxygen consumption in vivo in the heart (4,5). Furthermore, observations in intact cardiac mitochondria demonstrate that the effects of Ca2þ on the flux of NAD(P)H synthesis and respiration are minor and even potentially insignificant at physiologically relevant conditions in Ca2þ, Mg2þ, temperature, pyruvate, and phosphate (6,7). Skeletal muscle has a larger dynamic range in ATP demand from rest to maximal exercise, and its mitochondrial oxidative phosphorylation capacity is fully utilized within this range of ATP demand (8). A recent report on mitochondrial Ca2þ uniporter knockout mice showed a reduced maximal work on an inclined treadmill when compared with wild-type mice (9). A more recent follow-up study from the same group showed no reduction in cardiac function in the mitochondrial Ca2þ uniporter knockout mice, which implies that the reduced exercise performance of the knockout mice may be because of the loss of Ca2þ modulation of skeletal muscle mitochondrial function (7). Additionally, theoretical and empirical
Editor: Godfrey Smith Ó 2016 by the Biophysical Society 0006-3495/16/02/0954/8
http://dx.doi.org/10.1016/j.bpj.2015.12.018
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studies in skeletal muscle in vivo showed that feedback control alone was insufficient in explaining steady-state inorganic phosphate measurements from rest to high workloads (10). Our study reports a test of the hypothesis that Ca2þ exerts open-loop control on oxidative phosphorylation in skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate in the presence of malate. The experiments conducted were aimed at determining whether the relationship between NADH and flux of respiration and its modulation by Ca2þ was different between mitochondria from cardiac and skeletal muscle. The experimental approach for this study was derived from Vinnakota et al. (6), where respiration and NADH fluorescence were measured when extramitochondrial Ca2þ was varied. The assay in our study was improved over that of Vinnakota et al. (6) by using apyrase (ATPase) to achieve distinct fluxes of ATP hydrolysis and of mitochondrial respiratory rates as a consequence. The fluorescence of rhodamine was also measured to assess mitochondrial uptake and release of the dye because of changes in inner membrane potential. Analysis of NADH and respiration data showed that 1) Ca2þ exerts open-loop control on oxidative phosphorylation in skeletal muscle mitochondria but not in cardiac mitochondria, and 2) kinetics of NADH synthesis and respiration in cardiac mitochondria are similar to skeletal muscle mitochondria at high Ca2þ.
MATERIALS AND METHODS Isolation of mitochondria and determination of citrate synthase activity Procedures for extracting cardiac tissue and isolating mitochondria were adapted from Vinnakota et al. (6). Fifteen- to seventeen-week-old Wistar rats were anesthetized with an intraperitoneal injection of 0.3–0.5 mL ketamine (90 mg/kg) and 0.3–0.5 mL dexmedetomidine (0.5 mg/kg) followed by 0.5 mL heparin (1000 USP units/mL). The heart was perfused with ice-cold cardioplegia solution (25 mM KCl, 100 mM NaCl, 10 mM Dextrose, 25 mM MOPS, 1 mM EGTA, pH 7.2) for 5 min by cannulating the aorta. The ventricles were dissected and placed in isolation buffer on ice (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 1 mM EGTA, 0.1% BSA, pH 7.2). Hind-limb muscle soleus and oxidative portions of the gastrocnemius were dissected and trimmed of fat and connective tissue in cold isolation buffer. Subsequently the ventricles and the skeletal muscles were separately minced, added to separate beakers each with 2.5 mL isolation buffer with 5 units of protease/mL (Bacillus licheniformis), and homogenized for 40–60 s on ice. Isolation buffer was added to the homogenates to a final volume of 25 mL each in separate tubes and centrifuged (Eppendorf F-34-6-38 rotor and 5810R centrifuge) at 8000 g for 10 min twice discarding the supernatant each time to remove the protease and resuspending the pellets in isolation buffer to a 25 mL volume. The resuspended pellets from the second spin were centrifuged at 700 g, and the supernatant was centrifuged at 8000 g to yield a mitochondrial rich fraction in the final pellet. The final pellet was resuspended in 0.3 mL isolation buffer, and the activity of citrate synthase per microliter of the suspension was determined using the protocol of Eigentler et al. (11) based on the assay of Srere (12). Briefly, the initial velocity of citrate syn-
955 thase reaction was determined at pH 8.1, 0.5 mM oxaloacetate, and 0.3 mM Acetyl CoA at 30 C by measuring the rate of formation of 5-thio-2-nitrobenzoic acid (absorbance at 412 nm). 5-thio-2-nitrobenzoic acid is formed by the chemical reaction of CoA produced by the citrate synthase reaction with 5,50 -dithiobis (2-nitrobenzoic acid), which was added to the reaction medium at an initial concentration of 0.1 mM.
Respirometry, states of respiration, and the control of extramitochondrial Ca2D concentration Respiration measurements were made using a high-resolution respirometer (Oxygraph 2K, Oroboros Instruments, Innsbruck, Austria) with mitochondria suspended at a concentration of 0.337 U CS/mL (which corresponds to ~0.1 mg protein/mL in a Bradford assay calibrated against bovine serum albumin standards), in a respiration buffer at pH 7.2 and 37 C. The respiration buffer was composed of 50 mM MOPS, 107.5 mM KCl, 5 mM K2HPO4, 1mM EGTA, and 0.1% w/w BSA, with additional components added according to the desired state of respiration in the assay. The quality of the mitochondrial preparation was assessed by the respiratory control index defined as the ratio of respiratory rate obtained upon adding 375 mM of ADP (state 3) in the presence of substrates (10 mM pyruvate and 10 mM malate) to that obtained upon adding substrates alone (LEAK) at 37 C. Mitochondrial preparations with respiratory control indices >10 were deemed sufficient for our experiments. Actual values ranged from 10–12 for cardiac mitochondria and 15–19 for skeletal muscle mitochondria. Four states of respiration were obtained for this study listed in the order of increasing respiration rates: 1) LEAK (generates protonmotive force to compensate for loss because of proton leak, mitochondrial Ca2þ influx, and other ion circuits); 2) state 3.5 (because of ATPase activity in the mitochondrial preparation); 3) submaximal respiratory state with the addition of 0.2 U/mL apyrase; and 4) maximal respiratory state with the addition of 0.8 U/mL apyrase (from potatoes, Grade VI). For experiments in the LEAK 2, the buffer included 1 mM MgCl2, 0.25 mM pyruvate, and 0.25 mM malate. For experiments in state 3.5, submaximal, and maximal respiratory states, the buffer contained 5mM ATP and 6 mM MgCl2 in addition to all of the preceding components. In all cases the buffer pH was adjusted to 7.2 at 37 C using KOH, the final concentration of Naþ was 10 mM and that of free Mg2þ was 1 mM. Free Ca2þ concentration in the respiration buffer was buffered with 1 mM EGTA as described in Vinnakota et al. (6). Additional buffer components in our study did not affect the total Ca2þ added to the buffer for achieving the desired free extramitochondrial Ca2þ concentration [Ca2þ]e. The corresponding total Ca2þ and [Ca2þ]e values employed in this study are 0 mM to 0 nM, 300 mM to 50 nM, 630 mM to 200 nM, 750 mM to 350 nM, and 870 mM to 750 nM. The buffering capacity of EGTA for Ca2þ beyond 750 nM [Ca2þ]e is greatly reduced, compromising the ability to maintain [Ca2þ]e in the presence of mitochondrial Ca2þ uptake.
Measurement of mitochondrial NAD(P)H fluorescence NAD(P)H fluorescence was recorded at 470 nm with excitation at 350 nm and a 12 nm bandwidth in a multimode plate reader (Varioskan Flash, Thermo Scientific) with the samples placed in 24-well microplate wells with thermal incubation to maintain the sample temperature at 37 C. Mitochondria were suspended in 1 mL preheated respiration buffer to obtain a final concentration of 0.337 U CS/mL in each of six wells amounting to six technical replicates per biological sample for each combination of ATPase load and [Ca2þ]e. NAD(P)H fluorescence (FNADH) was recorded every 12 s in each well and the samples were kept well mixed by 600 shakes/min (orbital shaking) between successive readings. A time course recording was obtained in the following manner: 1) 0 s, introduction Biophysical Journal 110(4) 954–961
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of the samples with enzymes and Ca2þ into the plate reader and start of recording for 17 successive readings; 2) 240 or 300 s, addition of rotenone (10 mM) or carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP: 4 mM); 3) 17 successive readings; and 4) 496 s, end of recording. The recorded time courses of FNADH were expressed as a fraction (fNADH) of the range of FNADH attainable between maximally reduced (obtained after rotenone addition, FRot NADH ) and oxidized states (obtained after FCCP FCCP Rot FCCP addition, FFCCP NADH ), i.e., fNADH ¼ ðFNADH hFNADH iÞ=ðhFNADH i hFNADH iÞ, where hi denotes averaging over time.
Qualitative assessment of changes in inner membrane potential (DJm) using rhodamine 123 Changes in rhodamine 123 fluorescence (excitation wavelength lex ¼ 503 nm, fluorescence emission wavelength lem ¼ 527 nm, monochromator-based wavelength selection) with reference to a maximally depolarized state were used to qualitatively assess changes in DJm (13). This was performed simultaneously with the NAD(P)H measurement by adding rhodamine123 to the respiration buffer at a final concentration of 50 nM. The rhodamine123 fluorescence (FRh) was measured every 12 s as described for the NAD(P)H fluorescence. For normalization, the rhodamine123 fluorescence was also recorded in maximally depolarized states obtained by adding FCCP to a final concentration of 4 mM (FFCCP Rh ). We define fRh ¼ 1 FRh =hFFCCP Rh i, where hi denotes averaging over time. fRh increases monotonically with mitochondrial inner membrane potential.
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Data analysis Fig. 1 shows representative time courses of fluxes of respiration in mitochondria from cardiac (Fig. 1 A) and skeletal muscles (Fig. 1 B) for different states of respiration. The average respiration rate for a given state (VO2 ) was calculated by averaging the oxygen consumption rates between 160 and 180 s for skeletal muscle mitochondria, and 200 and 220 s for cardiac mitochondria, where they were approaching steady states before all of the oxygen in the respirometer chamber was consumed. The interval for averaging was constrained by the time taken for the respirometer chamber to become anoxic, which was sooner for skeletal muscle mitochondria especially in the Ca2þ stimulated states. fNADH and fRh were also averaged during the same time interval for each respiratory state to plot them against VO2 . fNADH time course data were tested for approach to a steady state by fitting the data to a line and performing a t-test for the null hypothesis that the slope of the line was not different from zero. The estimated fNADH was used for further analysis. The symbols fNADH and fRh refer to the averaged values through the remainder of this article and in Figs. 2, 3, and 4. Direct comparison of fNADH and VO2 with the aim of testing for the exertion of open-loop control by [Ca2þ]e is not possible from the experimental data because it requires the clamping of fNADH to compare VO2 between different [Ca2þ]e and vice versa. Because the clamping of either variable is experimentally infeasible, we use a model-based bapproach. fNADH versus VO2 plots (see Fig. 3) were fitted to a function eaVO2 to test whether there are qualitative differences in the fNADH versus VO2 relationships because of
FIGURE 1 Representative time courses of respiration rates, fNADH and fRh. (A), (C), and (E) show VO2 , fNADH, and fRh time course data, respectively, from cardiac mitochondria. (B), (D), and (F) show data on the same variables for skeletal muscle mitochondria. Time zero in the recordings corresponds to the addition of mitochondria with Ca2þ and appropriate quantity of apyrase to the respiration buffer. The end points for fNADH (0 with FCCP treatment and 1 with rotenone treatment) and fRh (0 with FCCP treatment) are obtained at 240 or 300 s, which are not shown in the plots. The shaded area in each plot represents the interval of averaging for each variable. All data are shown for one replicate.
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D FIGURE 2 Effect of [Ca2þ]e on VO2 , fNADH, and fRh. (A), (C), and (E) show VO2 , fNADH, and fRh data from cardiac mitochondria plotted against [Ca2þ]e. (B), (D), and (F) show data on the same variables plotted against [Ca2þ]e for skeletal muscle mitochondria. In each plot, data acquired during respiratory states: LEAK, state 3.5, submaximal state, and maximal state. The data are presented as the mean 5 SE estimated from three to four biological replicates for each data point.
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[Ca2þ]e or source tissue of mitochondria. The function eaVO2 , which was determined to be a parsimonious representation of the data, has the necessary theoretical attribute of yielding a value of 1 or no oxidation of NADH when there is no respiration (VO2 ¼ 0). Initial model fits by ordinary least squares using a common b for all data sets showed a high correlation coefficient (>0.8) between a and b, which means that a and b are not independent. Therefore, we fixed the value of b nominally at the estimated value 0.3745 for all data sets under consideration and treated only a as the single adjustable parameter for each fNADH versus VO2 data set. Biochemically the parameter a determines the relative extent of oxidation of NADH as the rate of respiration increases. Changes in parameter a because of an open-loop controller indicates modulation of NADH synthesis fluxes relative to the flux of its consumption, i.e., oxidative phosphorylation. Mean data were
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fitted taking into account uncertainties in both VO2 and fNADH by orthogonal distance regression (ODR) using ODRPACK 2.01 (14,15) provided in SciPy (16), a Python-based open source software library for scientific computing. The computations were performed using the IPython environment (17). The estimated 95% confidence intervals of parameter a were examined for statistically significant differences between ODR fits to each of the fNADH versus VO2 data sets (see Table 1).
RESULTS AND DISCUSSION The primary data consisting of VO2 , fNADH, and fRh were obtained at rest and three fluxes of ATP hydrolysis at 5 mM initial
B FIGURE 3 Modulation of fNADH versus VO2 and fRh versus VO2 plots by [Ca2þ]e. (A) shows fNADH and fRh plotted against VO2 for cardiac mitochondria. (B) shows the same variables plotted against VO2 for skeletal muscle mitochondria, where the error bars represent standard errors estimated from three to four biological replicates. The solid and dashed lines, respectively, show fNADH versus 0:3745 VO2 data fit to expðaVO Þ at 350 nM and 2 50 nM [Ca2þ]e. To see this figure in color, go online.
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B FIGURE 4 Modulation of fNADH VO2 versus VO2 and fRh VO2 versus VO2 plots by [Ca2þ]e. (A) shows fNADH VO2 and fRh VO2 plotted against VO2 for cardiac mitochondria. (B) shows the same variables plotted against VO2 for skeletal muscle mitochondria. In both panels, the solid lines in red and black, respectively, show model fits for fNADH VO2 at 50 nM and 350 nM [Ca2þ]e. To see this figure in color, go online.
ATP and 5 mM initial Pi concentrations while varying [Ca2þ]e between 0–750 nM in the presence of 1 mM free Mg2þ and 10 mM Naþ (see Fig. 2). The initial Pi concentration of 5 mM is supraphysiological for the heart resulting in respiration rates of higher magnitude than those reported in the Vinnakota study where the maximum initial Pi concentration was 2.5 mM (6). Additionally, the 5 mM initial Pi concentration was chosen to facilitate comparison with skeletal muscle respiration at the same initial Pi, which represents the resting physiological level for oxidative skeletal muscle (18,19). Finally, our study utilizes 0.25 mM pyruvate to avoid pyruvate substrate limitation (6) while being close to reported in situ pyruvate concentrations (20–22) and to avoid pyruvate dependent activation of pyruvate dehydrogenase complex (20,23). [Ca2D]e does not exert open-loop control on isolated cardiac mitochondrial oxidative phosphorylation VO2 data as a function of [Ca2þ]e are shown in Fig. 2, A and B, for cardiac and skeletal muscle mitochondria, respectively. The LEAK state respiration did not change significantly with [Ca2þ]e indicating insignificant contribution of membrane potential dissipation, because of [Ca2þ]e current, to the measured effect of [Ca2þ]e on respiration in mitochondria from both tissues. TABLE 1 Parameter Estimates, Their Standard Deviations, and 95% Confidence Intervals from Orthogonal Distance Regression of fNADH versus VO2 Data to the Function expð aV0:3745 Þ O2 Tissue
[Ca2þ]e (nM)
a 5 SD, (95% Confidence Interval)
Cardiac
0 50 200 350 0 50 200 350
2.1169 5 0.0603, (1.9251, 2.3087) 2.0801 5 0.0517, (1.9157, 2.2446)# 2.1597 5 0.0960, (1.8543, 2.4650) 2.0257 5 0.1391, (1.5829, 2.4684) 2.3558 5 0.0994, (2.0395, 2.6721)* 2.2684 5 0.0525, (2.1012, 2.4355)*,# 2.1562 5 0.1124, (1.7985, 2.5140) 2.0344 5 0.0547, (1.8604, 2.2084)*
Skeletal
*p < 0.05, two-tailed t-test between high and low [Ca2þ]e in skeletal muscle mitochondria, power of one for three biological replicates. # p < 0.05, two-tailed t-test between cardiac and skeletal muscle mitochondria at 50 nM [Ca2þ]e. Biophysical Journal 110(4) 954–961
In cardiac mitochondria, a modest 15.6% but statistically significant increase of VO2 was observed at the highest flux of ATP hydrolysis with [Ca2þ]e changing from 0 to 750 nM (two-tailed t-test, p ¼ 0.025), with no statistically significant effects in the remaining respiratory states. The fNADH and fRh data for cardiac mitochondria shown in Fig. 2, C and E, show no significant change with [Ca2þ]e for all of the respiratory states examined in this study. To characterize the relationships between respiration and NAD(P)H and inner membrane potential and their modulation by [Ca2þ]e we plotted fNADH and fRh against VO2 in Fig. 3 A at low (50 nM) and high (350 nM) [Ca2þ]e. The value for low [Ca2þ]e is a representative resting value in cardiac (24) and oxidative skeletal muscle (25) and that for high value was chosen in accordance with Vinnakota et al. (6). [Ca2þ] values in the myoplasm have been observed to approach values between 1 and 2 mM in isolated oxidative skeletal muscle fibers (26). The high [Ca2þ]e value in our study, which is below the maximum values observed in situ, was chosen to facilitate comparison with measurements on cardiac mitochondria as well as because of a practical constraint on buffering [Ca2þ]e reliably above 1 mM in our experiments. Data from cardiac mitochondria in Fig. 3 A show that NAD(P)H is oxidized and the inner mitochondrial membrane is depolarized with increasing respiration rate and that this relationship does not differ between low and high [Ca2þ]e concentrations. The 0:3745 model fits to fNADH versus aV VO2 data, using the function e O2 , and yielded no statistically significant differences between parameter estimates for low and high [Ca2þ]e (see Table 1). These results reaffirm previously published findings that Ca2þ modulation of mitochondrial NAD(P)H generation relative to consumption over a range of respiratory fluxes in cardiac mitochondria utilizing pyruvate in the presence of malate is insignificant to be physiologically relevant (6,27–29). Previous studies demonstrated significant [Ca2þ]e stimulation of the fNADH versus VO2 relation under experimental conditions designed to maximize this effect. Those experimental conditions include depletion of Ca2þ by chelators before the application of a Ca2þ stimulus, and the utilization of substrates such as glutamate/malate and submillimolar alphaketoglutarate (6,27,28,30,31). The observed stimulatory effects of [Ca2þ]e become insignificant under more
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physiologically relevant conditions and substrates because of saturation of the overall system response and not the lack of sensitivity of individual dehydrogenases to matrix Ca2þ (29). [Ca2D]e exerts open-loop control on isolated skeletal muscle mitochondrial oxidative phosphorylation Skeletal muscle mitochondria attain a larger range of VO2 than cardiac mitochondria per unit citrate synthase activity (two-tailed t-test, p < 0.05, maximal respiratory state, [Ca2þ]e, 0–350 nM). Additionally, they also show an increase in VO2 in the maximal and submaximal respiratory states till 350 nM [Ca2þ]e (Fig. 2 B, two-tailed t-test, p < 0.05). In the maximal respiratory state, a 10% decrease (two-tailed t-test, p ¼ 0.22) in VO2 was observed when [Ca2þ]e changed from 350 to 750 nM. This decline in respiratory rate at higher Ca2þ concentrations has also been observed by Glancy et al. (32) in skeletal muscle mitochondria and by Mildaziene et al. (27) in cardiac mitochondria. The reasons for this phenomenon are yet unclear. We may at least exclude the possibility of decrease in respiration because of damage of a subpopulation of mitochondria because of Ca2þ overload because changes of similar magnitude were not observed in fNADH. The effects of [Ca2þ]e on fNADH and fRh alone are not apparent from the plots in Fig. 2, D and F, but are examined in fNADH versus VO2 plots. The relationship between terminal substrate oxidation and mitochondrial respiration in response to ATP demand is reflected in fNADH versus VO2 plot in Fig. 3 B, which aV0:3745 was fitted to the same function e O2 used for data from cardiac mitochondria. The estimates for parameter a were found to decrease as [Ca2þ]e increased from 50– 350 nM, which implies an increase in the flux of NADH synthesis relative to oxidation because of an increase in [Ca2þ]e. This conclusion was found to be valid between 0 and 350 nM [Ca2þ]e. Additionally, the value of a for cardiac mitochondria is smaller than that of skeletal muscle mitochondria at low [Ca2þ]e but not different from the estimates in both tissues at high [Ca2þ]e (see Table 1). An increase in respiration because of an increase [Ca2þ]e may be because of [Ca2þ]e modulation of extra mitochondrial ATPases and the resulting mitochondrial response, or because of an increase in dissipation of proton motive force from mitochondrial Ca2þ uptake. From the LEAK state data in Fig. 2, A and B, we may exclude the dissipation of mitochondrial protonmotive force because of mitochondrial Ca2þ uptake alone. If the turnover rate of the mitochondrial dehydrogenases is not modulated by matrix calcium, the values of fNADH will fall on the same fNADH versus VO2 curve. A displacement of [Ca2þ]e modulated (fNADH, VO2 ) from the original curve in the direction of increasing fNADH and VO2 must mean that net mitochondrial dehydrogenase flux is
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indeed modulated by Ca2þ, which may include a stoichiometrically linked flux of generation of FADH2 along with NADH. fNADH VO2 and fRh VO2 , which are analogous to power dissipation and are representative of free energy input into generating phosphorylation potential per unit time, are plotted against VO2 in Fig. 4 for cardiac (Fig. 4 A) and skeletal muscle mitochondria (Fig. 4 B). The plotted variables increase with VO2 as expected in mitochondria from both tissues but fNADH VO2 is modulated in a stimulatory manner by [Ca2þ]e in skeletal muscle mitochondria, further reaffirming the difference between Ca2þ modulation of NADH generation in cardiac and skeletal muscle mitochondria. A potential reason for this difference in Ca2þ modulation of NADH generation in cardiac and skeletal muscle mitochondria might be the markedly lower mitochondrial Ca2þ uniporter current density in cardiac mitochondria when compared with skeletal muscle mitochondria (33). Physiological implications of [Ca2D]e control on mitochondrial oxidative phosphorylation Cardiac mitochondria therefore operate at high relative NADH generation fluxes, which are not affected by [Ca2þ]e dependent open-loop control. In vivo cardiac mitochondria operate at a higher resting ATPase rate with a fourfold increase at maximal workload (4,5). Skeletal muscle mitochondria on the other hand are subject to a much lower ATPase flux at rest and may reach their maximal capacity for oxidative phosphorylation at high workloads. Cytosolic free Ca2þ in muscle, which regulates the contractile ATPase flux, could potentially extend the range of oxidative ATP synthesis. An in vivo metabolic control analysis by Jeneson et al. (34) showed that the control of oxidative ATP synthesis in muscle shifts increasingly from extramitochondrial ATPase to mitochondrial ATP synthesis flux as the ATP demand increases, which points to a potential role for open-loop control by [Ca2þ]e at high workloads. The role of Ca2þ in this context would be to decrease the response time of the fluxes generating NADH such that net NADH oxidation is reduced when VO2 increases to a new steady state. Skeletal muscle utilizes all of its mitochondrial capacity, so even a modest impact of Ca2þ on substrate dehydrogenation could contribute to the range of oxygen consumption although by a small fraction. Indeed, mice lacking the mitochondrial Ca2þ uniporter, which facilitates the entry of Ca2þ into the mitochondrial matrix, were reported to achieve 20% smaller maximal work on an inclined treadmill when compared with wild-type mice (9). Our study was performed using mitochondria from oxidative muscles consisting of mainly type I fibers. Further characterization of control of mitochondrial oxidative phosphorylation in various fiber types may be necessary to achieve a more complete understanding of in vivo muscle function and to test the generality of the results reported in our study. Biophysical Journal 110(4) 954–961
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Although out results show [Ca2þ]e modulation of NADH synthesis fluxes in response to demand, the distribution of open-loop control among potential sites such as Ca2þ sensitive dehydrogenases in the mitochondrial matrix is unclear. Studies on the distribution of flux control of mitochondrial ATP synthesis and respiration in skeletal muscle (35) showed that pyruvate uptake and oxidation was found to have a control coefficient of 0.2 at maximal ADP stimulated respiration in vitro, which means that [Ca2þ]e modulation of pyruvate oxidation via the pyruvate dehydrogenase complex could potentially contribute to the observed increase in NADH synthesis flux in skeletal muscle mitochondria. Simulation studies adapting a cardiac model of mitochondrial oxidative phosphorylation to describe skeletal muscle energetics could not describe the steady-state relationship between steady-state Pi and ATPase flux over the entire range of the ATPase flux (10). Particularly the model under predicts the Pi data at low ATPase flux values implying high mitochondrial ATP synthesis flux at low ATP demand fluxes as a consequence of assuming constant enzyme activities and feedback control as the sole mechanism. Our results demonstrating [Ca2þ]e modulation of skeletal muscle mitochondrial ATP synthesis fluxes could be a potential mechanism that could reconcile the mitochondrial model with the data. AUTHOR CONTRIBUTIONS D.A.B., K.C.V., and R.W.W. conceived and designed the research. A.S., F.V.dB., K.C.V., and M.B.O. performed the research. A.S., D.A.B., F.V.dB., and K.C.V. analyzed the data. D.A.B., F.V.dB., K.C.V., and R.W.W. wrote the article.
ACKNOWLEDGMENTS The authors thank Dr. Ronald A. Meyer (Michigan State University) for suggesting parametric analysis of fNADH versus VO2 plots. Research was funded by NIH grant R01 DK095210 (D.A.B. and R.W.W.).
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4. Wu, F., E. Y. Zhang, ., D. A. Beard. 2008. Phosphate metabolite concentrations and ATP hydrolysis potential in normal and ischaemic hearts. J. Physiol. 586:4193–4208.
24. Dibb, K. M., D. A. Eisner, and A. W. Trafford. 2007. Regulation of systolic [Ca2þ]i and cellular Ca2þ flux balance in rat ventricular myocytes by SR Ca2þ, L-type Ca2þ current and diastolic [Ca2þ]i. J. Physiol. 585:579–592.
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Biophysical Journal 110(4) 954–961
Biophysical Journal
Volume 110
February 2016
954–961
Article Open-Loop Control of Oxidative Phosphorylation in Skeletal and Cardiac Muscle Mitochondria by Ca2D Kalyan C. Vinnakota,1,* Abhishek Singhal,1 Franc¸oise Van den Bergh,1 Masoumeh Bagher-Oskouei,1 Robert W. Wiseman,2 and Daniel A. Beard1 1 Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, Michigan; and 2Department of Physiology, Michigan State University, East Lansing, Michigan
ABSTRACT In cardiac muscle, mitochondrial ATP synthesis is driven by demand for ATP through feedback from the products of ATP hydrolysis. However, in skeletal muscle at higher workloads there is an apparent contribution of open-loop stimulation of ATP synthesis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the pathway. The role of calcium, which is known to stimulate the activity of mitochondrial dehydrogenases, as an open-loop controller, was investigated in isolated cardiac and skeletal muscle mitochondria. The kinetics of NADH synthesis and respiration, feedback from ATP hydrolysis products, and stimulation by calcium were characterized in isolated mitochondria to test the hypothesis that calcium has a stimulatory role in skeletal muscle mitochondria not apparent in cardiac mitochondria. A range of respiratory states were obtained in cardiac and skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate and malate, and flux of respiration, NAD(P)H fluorescence, and rhodamine 123 fluorescence were measured over a range of extra mitochondrial calcium concentrations. We found that under these conditions calcium stimulates NADH synthesis in skeletal muscle mitochondria but not in cardiac mitochondria.
INTRODUCTION Mitochondria transduce the chemical potential from oxidation of carbon substrates into the chemical potential of the ATP hydrolysis reaction though oxidative phosphorylation. Most of the chemical energy from substrate oxidation in this process is obtained in the terminal oxidative pathway of the Krebs cycle in the generation of reducing equivalents NADH and FADH2 in reactions catalyzed by dehydrogenases, and from the transfer of electrons from those reducing equivalents to oxygen. Therefore, the relationship between the degree of reduction of the total NADH pool and rate of respiration (VO2) in a steady state constitutes an important quantitative descriptor that captures the balance between the processes generating and consuming NADH. Modulation of this relationship by feedback and open-loop controllers of oxidative phosphorylation can provide insight into regulation of the overall process of terminal oxidation of substrates and oxidative phosphorylation in response to fluxes of ATP hydrolysis. Open-loop control is defined as modulation of flux through a biochemical pathway by a moiety, which is not a reactant or a product of the biochemical reactions in the system, whereas feedback control is mediated by reactants and products of the biochemical reactions in the system. Calcium (Ca2þ) is known to stimulate the activities of several mitochondrial dehydrogenases including pyruvate
Submitted November 26, 2014, and accepted for publication December 14, 2015. *Correspondence: [email protected]
dehydrogenase complex, isocitrate dehydrogenase, and 2-oxoglutarate dehydrogenase (1,2). Partly on this basis, Ca2þ is speculated to stimulate the flux of mitochondrial ATP synthesis in an open-loop control to meet cellular ATP demand in the heart over a range of cellular contractile work states that is also regulated by Ca2þ (3). However, recent work showed that feedback from products of ATP hydrolysis was sufficient in explaining the relationship between steady-state metabolite concentrations and the flux of oxygen consumption in vivo in the heart (4,5). Furthermore, observations in intact cardiac mitochondria demonstrate that the effects of Ca2þ on the flux of NAD(P)H synthesis and respiration are minor and even potentially insignificant at physiologically relevant conditions in Ca2þ, Mg2þ, temperature, pyruvate, and phosphate (6,7). Skeletal muscle has a larger dynamic range in ATP demand from rest to maximal exercise, and its mitochondrial oxidative phosphorylation capacity is fully utilized within this range of ATP demand (8). A recent report on mitochondrial Ca2þ uniporter knockout mice showed a reduced maximal work on an inclined treadmill when compared with wild-type mice (9). A more recent follow-up study from the same group showed no reduction in cardiac function in the mitochondrial Ca2þ uniporter knockout mice, which implies that the reduced exercise performance of the knockout mice may be because of the loss of Ca2þ modulation of skeletal muscle mitochondrial function (7). Additionally, theoretical and empirical
Editor: Godfrey Smith Ó 2016 by the Biophysical Society 0006-3495/16/02/0954/8
http://dx.doi.org/10.1016/j.bpj.2015.12.018
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studies in skeletal muscle in vivo showed that feedback control alone was insufficient in explaining steady-state inorganic phosphate measurements from rest to high workloads (10). Our study reports a test of the hypothesis that Ca2þ exerts open-loop control on oxidative phosphorylation in skeletal muscle mitochondria utilizing physiologically relevant concentrations of pyruvate in the presence of malate. The experiments conducted were aimed at determining whether the relationship between NADH and flux of respiration and its modulation by Ca2þ was different between mitochondria from cardiac and skeletal muscle. The experimental approach for this study was derived from Vinnakota et al. (6), where respiration and NADH fluorescence were measured when extramitochondrial Ca2þ was varied. The assay in our study was improved over that of Vinnakota et al. (6) by using apyrase (ATPase) to achieve distinct fluxes of ATP hydrolysis and of mitochondrial respiratory rates as a consequence. The fluorescence of rhodamine was also measured to assess mitochondrial uptake and release of the dye because of changes in inner membrane potential. Analysis of NADH and respiration data showed that 1) Ca2þ exerts open-loop control on oxidative phosphorylation in skeletal muscle mitochondria but not in cardiac mitochondria, and 2) kinetics of NADH synthesis and respiration in cardiac mitochondria are similar to skeletal muscle mitochondria at high Ca2þ.
MATERIALS AND METHODS Isolation of mitochondria and determination of citrate synthase activity Procedures for extracting cardiac tissue and isolating mitochondria were adapted from Vinnakota et al. (6). Fifteen- to seventeen-week-old Wistar rats were anesthetized with an intraperitoneal injection of 0.3–0.5 mL ketamine (90 mg/kg) and 0.3–0.5 mL dexmedetomidine (0.5 mg/kg) followed by 0.5 mL heparin (1000 USP units/mL). The heart was perfused with ice-cold cardioplegia solution (25 mM KCl, 100 mM NaCl, 10 mM Dextrose, 25 mM MOPS, 1 mM EGTA, pH 7.2) for 5 min by cannulating the aorta. The ventricles were dissected and placed in isolation buffer on ice (200 mM mannitol, 50 mM sucrose, 5 mM KH2PO4, 5 mM MOPS, 1 mM EGTA, 0.1% BSA, pH 7.2). Hind-limb muscle soleus and oxidative portions of the gastrocnemius were dissected and trimmed of fat and connective tissue in cold isolation buffer. Subsequently the ventricles and the skeletal muscles were separately minced, added to separate beakers each with 2.5 mL isolation buffer with 5 units of protease/mL (Bacillus licheniformis), and homogenized for 40–60 s on ice. Isolation buffer was added to the homogenates to a final volume of 25 mL each in separate tubes and centrifuged (Eppendorf F-34-6-38 rotor and 5810R centrifuge) at 8000 g for 10 min twice discarding the supernatant each time to remove the protease and resuspending the pellets in isolation buffer to a 25 mL volume. The resuspended pellets from the second spin were centrifuged at 700 g, and the supernatant was centrifuged at 8000 g to yield a mitochondrial rich fraction in the final pellet. The final pellet was resuspended in 0.3 mL isolation buffer, and the activity of citrate synthase per microliter of the suspension was determined using the protocol of Eigentler et al. (11) based on the assay of Srere (12). Briefly, the initial velocity of citrate syn-
955 thase reaction was determined at pH 8.1, 0.5 mM oxaloacetate, and 0.3 mM Acetyl CoA at 30 C by measuring the rate of formation of 5-thio-2-nitrobenzoic acid (absorbance at 412 nm). 5-thio-2-nitrobenzoic acid is formed by the chemical reaction of CoA produced by the citrate synthase reaction with 5,50 -dithiobis (2-nitrobenzoic acid), which was added to the reaction medium at an initial concentration of 0.1 mM.
Respirometry, states of respiration, and the control of extramitochondrial Ca2D concentration Respiration measurements were made using a high-resolution respirometer (Oxygraph 2K, Oroboros Instruments, Innsbruck, Austria) with mitochondria suspended at a concentration of 0.337 U CS/mL (which corresponds to ~0.1 mg protein/mL in a Bradford assay calibrated against bovine serum albumin standards), in a respiration buffer at pH 7.2 and 37 C. The respiration buffer was composed of 50 mM MOPS, 107.5 mM KCl, 5 mM K2HPO4, 1mM EGTA, and 0.1% w/w BSA, with additional components added according to the desired state of respiration in the assay. The quality of the mitochondrial preparation was assessed by the respiratory control index defined as the ratio of respiratory rate obtained upon adding 375 mM of ADP (state 3) in the presence of substrates (10 mM pyruvate and 10 mM malate) to that obtained upon adding substrates alone (LEAK) at 37 C. Mitochondrial preparations with respiratory control indices >10 were deemed sufficient for our experiments. Actual values ranged from 10–12 for cardiac mitochondria and 15–19 for skeletal muscle mitochondria. Four states of respiration were obtained for this study listed in the order of increasing respiration rates: 1) LEAK (generates protonmotive force to compensate for loss because of proton leak, mitochondrial Ca2þ influx, and other ion circuits); 2) state 3.5 (because of ATPase activity in the mitochondrial preparation); 3) submaximal respiratory state with the addition of 0.2 U/mL apyrase; and 4) maximal respiratory state with the addition of 0.8 U/mL apyrase (from potatoes, Grade VI). For experiments in the LEAK 2, the buffer included 1 mM MgCl2, 0.25 mM pyruvate, and 0.25 mM malate. For experiments in state 3.5, submaximal, and maximal respiratory states, the buffer contained 5mM ATP and 6 mM MgCl2 in addition to all of the preceding components. In all cases the buffer pH was adjusted to 7.2 at 37 C using KOH, the final concentration of Naþ was 10 mM and that of free Mg2þ was 1 mM. Free Ca2þ concentration in the respiration buffer was buffered with 1 mM EGTA as described in Vinnakota et al. (6). Additional buffer components in our study did not affect the total Ca2þ added to the buffer for achieving the desired free extramitochondrial Ca2þ concentration [Ca2þ]e. The corresponding total Ca2þ and [Ca2þ]e values employed in this study are 0 mM to 0 nM, 300 mM to 50 nM, 630 mM to 200 nM, 750 mM to 350 nM, and 870 mM to 750 nM. The buffering capacity of EGTA for Ca2þ beyond 750 nM [Ca2þ]e is greatly reduced, compromising the ability to maintain [Ca2þ]e in the presence of mitochondrial Ca2þ uptake.
Measurement of mitochondrial NAD(P)H fluorescence NAD(P)H fluorescence was recorded at 470 nm with excitation at 350 nm and a 12 nm bandwidth in a multimode plate reader (Varioskan Flash, Thermo Scientific) with the samples placed in 24-well microplate wells with thermal incubation to maintain the sample temperature at 37 C. Mitochondria were suspended in 1 mL preheated respiration buffer to obtain a final concentration of 0.337 U CS/mL in each of six wells amounting to six technical replicates per biological sample for each combination of ATPase load and [Ca2þ]e. NAD(P)H fluorescence (FNADH) was recorded every 12 s in each well and the samples were kept well mixed by 600 shakes/min (orbital shaking) between successive readings. A time course recording was obtained in the following manner: 1) 0 s, introduction Biophysical Journal 110(4) 954–961
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of the samples with enzymes and Ca2þ into the plate reader and start of recording for 17 successive readings; 2) 240 or 300 s, addition of rotenone (10 mM) or carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP: 4 mM); 3) 17 successive readings; and 4) 496 s, end of recording. The recorded time courses of FNADH were expressed as a fraction (fNADH) of the range of FNADH attainable between maximally reduced (obtained after rotenone addition, FRot NADH ) and oxidized states (obtained after FCCP FCCP Rot FCCP addition, FFCCP NADH ), i.e., fNADH ¼ ðFNADH hFNADH iÞ=ðhFNADH i hFNADH iÞ, where hi denotes averaging over time.
Qualitative assessment of changes in inner membrane potential (DJm) using rhodamine 123 Changes in rhodamine 123 fluorescence (excitation wavelength lex ¼ 503 nm, fluorescence emission wavelength lem ¼ 527 nm, monochromator-based wavelength selection) with reference to a maximally depolarized state were used to qualitatively assess changes in DJm (13). This was performed simultaneously with the NAD(P)H measurement by adding rhodamine123 to the respiration buffer at a final concentration of 50 nM. The rhodamine123 fluorescence (FRh) was measured every 12 s as described for the NAD(P)H fluorescence. For normalization, the rhodamine123 fluorescence was also recorded in maximally depolarized states obtained by adding FCCP to a final concentration of 4 mM (FFCCP Rh ). We define fRh ¼ 1 FRh =hFFCCP Rh i, where hi denotes averaging over time. fRh increases monotonically with mitochondrial inner membrane potential.
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Data analysis Fig. 1 shows representative time courses of fluxes of respiration in mitochondria from cardiac (Fig. 1 A) and skeletal muscles (Fig. 1 B) for different states of respiration. The average respiration rate for a given state (VO2 ) was calculated by averaging the oxygen consumption rates between 160 and 180 s for skeletal muscle mitochondria, and 200 and 220 s for cardiac mitochondria, where they were approaching steady states before all of the oxygen in the respirometer chamber was consumed. The interval for averaging was constrained by the time taken for the respirometer chamber to become anoxic, which was sooner for skeletal muscle mitochondria especially in the Ca2þ stimulated states. fNADH and fRh were also averaged during the same time interval for each respiratory state to plot them against VO2 . fNADH time course data were tested for approach to a steady state by fitting the data to a line and performing a t-test for the null hypothesis that the slope of the line was not different from zero. The estimated fNADH was used for further analysis. The symbols fNADH and fRh refer to the averaged values through the remainder of this article and in Figs. 2, 3, and 4. Direct comparison of fNADH and VO2 with the aim of testing for the exertion of open-loop control by [Ca2þ]e is not possible from the experimental data because it requires the clamping of fNADH to compare VO2 between different [Ca2þ]e and vice versa. Because the clamping of either variable is experimentally infeasible, we use a model-based bapproach. fNADH versus VO2 plots (see Fig. 3) were fitted to a function eaVO2 to test whether there are qualitative differences in the fNADH versus VO2 relationships because of
FIGURE 1 Representative time courses of respiration rates, fNADH and fRh. (A), (C), and (E) show VO2 , fNADH, and fRh time course data, respectively, from cardiac mitochondria. (B), (D), and (F) show data on the same variables for skeletal muscle mitochondria. Time zero in the recordings corresponds to the addition of mitochondria with Ca2þ and appropriate quantity of apyrase to the respiration buffer. The end points for fNADH (0 with FCCP treatment and 1 with rotenone treatment) and fRh (0 with FCCP treatment) are obtained at 240 or 300 s, which are not shown in the plots. The shaded area in each plot represents the interval of averaging for each variable. All data are shown for one replicate.
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D FIGURE 2 Effect of [Ca2þ]e on VO2 , fNADH, and fRh. (A), (C), and (E) show VO2 , fNADH, and fRh data from cardiac mitochondria plotted against [Ca2þ]e. (B), (D), and (F) show data on the same variables plotted against [Ca2þ]e for skeletal muscle mitochondria. In each plot, data acquired during respiratory states: LEAK, state 3.5, submaximal state, and maximal state. The data are presented as the mean 5 SE estimated from three to four biological replicates for each data point.
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[Ca2þ]e or source tissue of mitochondria. The function eaVO2 , which was determined to be a parsimonious representation of the data, has the necessary theoretical attribute of yielding a value of 1 or no oxidation of NADH when there is no respiration (VO2 ¼ 0). Initial model fits by ordinary least squares using a common b for all data sets showed a high correlation coefficient (>0.8) between a and b, which means that a and b are not independent. Therefore, we fixed the value of b nominally at the estimated value 0.3745 for all data sets under consideration and treated only a as the single adjustable parameter for each fNADH versus VO2 data set. Biochemically the parameter a determines the relative extent of oxidation of NADH as the rate of respiration increases. Changes in parameter a because of an open-loop controller indicates modulation of NADH synthesis fluxes relative to the flux of its consumption, i.e., oxidative phosphorylation. Mean data were
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fitted taking into account uncertainties in both VO2 and fNADH by orthogonal distance regression (ODR) using ODRPACK 2.01 (14,15) provided in SciPy (16), a Python-based open source software library for scientific computing. The computations were performed using the IPython environment (17). The estimated 95% confidence intervals of parameter a were examined for statistically significant differences between ODR fits to each of the fNADH versus VO2 data sets (see Table 1).
RESULTS AND DISCUSSION The primary data consisting of VO2 , fNADH, and fRh were obtained at rest and three fluxes of ATP hydrolysis at 5 mM initial
B FIGURE 3 Modulation of fNADH versus VO2 and fRh versus VO2 plots by [Ca2þ]e. (A) shows fNADH and fRh plotted against VO2 for cardiac mitochondria. (B) shows the same variables plotted against VO2 for skeletal muscle mitochondria, where the error bars represent standard errors estimated from three to four biological replicates. The solid and dashed lines, respectively, show fNADH versus 0:3745 VO2 data fit to expðaVO Þ at 350 nM and 2 50 nM [Ca2þ]e. To see this figure in color, go online.
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B FIGURE 4 Modulation of fNADH VO2 versus VO2 and fRh VO2 versus VO2 plots by [Ca2þ]e. (A) shows fNADH VO2 and fRh VO2 plotted against VO2 for cardiac mitochondria. (B) shows the same variables plotted against VO2 for skeletal muscle mitochondria. In both panels, the solid lines in red and black, respectively, show model fits for fNADH VO2 at 50 nM and 350 nM [Ca2þ]e. To see this figure in color, go online.
ATP and 5 mM initial Pi concentrations while varying [Ca2þ]e between 0–750 nM in the presence of 1 mM free Mg2þ and 10 mM Naþ (see Fig. 2). The initial Pi concentration of 5 mM is supraphysiological for the heart resulting in respiration rates of higher magnitude than those reported in the Vinnakota study where the maximum initial Pi concentration was 2.5 mM (6). Additionally, the 5 mM initial Pi concentration was chosen to facilitate comparison with skeletal muscle respiration at the same initial Pi, which represents the resting physiological level for oxidative skeletal muscle (18,19). Finally, our study utilizes 0.25 mM pyruvate to avoid pyruvate substrate limitation (6) while being close to reported in situ pyruvate concentrations (20–22) and to avoid pyruvate dependent activation of pyruvate dehydrogenase complex (20,23). [Ca2D]e does not exert open-loop control on isolated cardiac mitochondrial oxidative phosphorylation VO2 data as a function of [Ca2þ]e are shown in Fig. 2, A and B, for cardiac and skeletal muscle mitochondria, respectively. The LEAK state respiration did not change significantly with [Ca2þ]e indicating insignificant contribution of membrane potential dissipation, because of [Ca2þ]e current, to the measured effect of [Ca2þ]e on respiration in mitochondria from both tissues. TABLE 1 Parameter Estimates, Their Standard Deviations, and 95% Confidence Intervals from Orthogonal Distance Regression of fNADH versus VO2 Data to the Function expð aV0:3745 Þ O2 Tissue
[Ca2þ]e (nM)
a 5 SD, (95% Confidence Interval)
Cardiac
0 50 200 350 0 50 200 350
2.1169 5 0.0603, (1.9251, 2.3087) 2.0801 5 0.0517, (1.9157, 2.2446)# 2.1597 5 0.0960, (1.8543, 2.4650) 2.0257 5 0.1391, (1.5829, 2.4684) 2.3558 5 0.0994, (2.0395, 2.6721)* 2.2684 5 0.0525, (2.1012, 2.4355)*,# 2.1562 5 0.1124, (1.7985, 2.5140) 2.0344 5 0.0547, (1.8604, 2.2084)*
Skeletal
*p < 0.05, two-tailed t-test between high and low [Ca2þ]e in skeletal muscle mitochondria, power of one for three biological replicates. # p < 0.05, two-tailed t-test between cardiac and skeletal muscle mitochondria at 50 nM [Ca2þ]e. Biophysical Journal 110(4) 954–961
In cardiac mitochondria, a modest 15.6% but statistically significant increase of VO2 was observed at the highest flux of ATP hydrolysis with [Ca2þ]e changing from 0 to 750 nM (two-tailed t-test, p ¼ 0.025), with no statistically significant effects in the remaining respiratory states. The fNADH and fRh data for cardiac mitochondria shown in Fig. 2, C and E, show no significant change with [Ca2þ]e for all of the respiratory states examined in this study. To characterize the relationships between respiration and NAD(P)H and inner membrane potential and their modulation by [Ca2þ]e we plotted fNADH and fRh against VO2 in Fig. 3 A at low (50 nM) and high (350 nM) [Ca2þ]e. The value for low [Ca2þ]e is a representative resting value in cardiac (24) and oxidative skeletal muscle (25) and that for high value was chosen in accordance with Vinnakota et al. (6). [Ca2þ] values in the myoplasm have been observed to approach values between 1 and 2 mM in isolated oxidative skeletal muscle fibers (26). The high [Ca2þ]e value in our study, which is below the maximum values observed in situ, was chosen to facilitate comparison with measurements on cardiac mitochondria as well as because of a practical constraint on buffering [Ca2þ]e reliably above 1 mM in our experiments. Data from cardiac mitochondria in Fig. 3 A show that NAD(P)H is oxidized and the inner mitochondrial membrane is depolarized with increasing respiration rate and that this relationship does not differ between low and high [Ca2þ]e concentrations. The 0:3745 model fits to fNADH versus aV VO2 data, using the function e O2 , and yielded no statistically significant differences between parameter estimates for low and high [Ca2þ]e (see Table 1). These results reaffirm previously published findings that Ca2þ modulation of mitochondrial NAD(P)H generation relative to consumption over a range of respiratory fluxes in cardiac mitochondria utilizing pyruvate in the presence of malate is insignificant to be physiologically relevant (6,27–29). Previous studies demonstrated significant [Ca2þ]e stimulation of the fNADH versus VO2 relation under experimental conditions designed to maximize this effect. Those experimental conditions include depletion of Ca2þ by chelators before the application of a Ca2þ stimulus, and the utilization of substrates such as glutamate/malate and submillimolar alphaketoglutarate (6,27,28,30,31). The observed stimulatory effects of [Ca2þ]e become insignificant under more
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physiologically relevant conditions and substrates because of saturation of the overall system response and not the lack of sensitivity of individual dehydrogenases to matrix Ca2þ (29). [Ca2D]e exerts open-loop control on isolated skeletal muscle mitochondrial oxidative phosphorylation Skeletal muscle mitochondria attain a larger range of VO2 than cardiac mitochondria per unit citrate synthase activity (two-tailed t-test, p < 0.05, maximal respiratory state, [Ca2þ]e, 0–350 nM). Additionally, they also show an increase in VO2 in the maximal and submaximal respiratory states till 350 nM [Ca2þ]e (Fig. 2 B, two-tailed t-test, p < 0.05). In the maximal respiratory state, a 10% decrease (two-tailed t-test, p ¼ 0.22) in VO2 was observed when [Ca2þ]e changed from 350 to 750 nM. This decline in respiratory rate at higher Ca2þ concentrations has also been observed by Glancy et al. (32) in skeletal muscle mitochondria and by Mildaziene et al. (27) in cardiac mitochondria. The reasons for this phenomenon are yet unclear. We may at least exclude the possibility of decrease in respiration because of damage of a subpopulation of mitochondria because of Ca2þ overload because changes of similar magnitude were not observed in fNADH. The effects of [Ca2þ]e on fNADH and fRh alone are not apparent from the plots in Fig. 2, D and F, but are examined in fNADH versus VO2 plots. The relationship between terminal substrate oxidation and mitochondrial respiration in response to ATP demand is reflected in fNADH versus VO2 plot in Fig. 3 B, which aV0:3745 was fitted to the same function e O2 used for data from cardiac mitochondria. The estimates for parameter a were found to decrease as [Ca2þ]e increased from 50– 350 nM, which implies an increase in the flux of NADH synthesis relative to oxidation because of an increase in [Ca2þ]e. This conclusion was found to be valid between 0 and 350 nM [Ca2þ]e. Additionally, the value of a for cardiac mitochondria is smaller than that of skeletal muscle mitochondria at low [Ca2þ]e but not different from the estimates in both tissues at high [Ca2þ]e (see Table 1). An increase in respiration because of an increase [Ca2þ]e may be because of [Ca2þ]e modulation of extra mitochondrial ATPases and the resulting mitochondrial response, or because of an increase in dissipation of proton motive force from mitochondrial Ca2þ uptake. From the LEAK state data in Fig. 2, A and B, we may exclude the dissipation of mitochondrial protonmotive force because of mitochondrial Ca2þ uptake alone. If the turnover rate of the mitochondrial dehydrogenases is not modulated by matrix calcium, the values of fNADH will fall on the same fNADH versus VO2 curve. A displacement of [Ca2þ]e modulated (fNADH, VO2 ) from the original curve in the direction of increasing fNADH and VO2 must mean that net mitochondrial dehydrogenase flux is
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indeed modulated by Ca2þ, which may include a stoichiometrically linked flux of generation of FADH2 along with NADH. fNADH VO2 and fRh VO2 , which are analogous to power dissipation and are representative of free energy input into generating phosphorylation potential per unit time, are plotted against VO2 in Fig. 4 for cardiac (Fig. 4 A) and skeletal muscle mitochondria (Fig. 4 B). The plotted variables increase with VO2 as expected in mitochondria from both tissues but fNADH VO2 is modulated in a stimulatory manner by [Ca2þ]e in skeletal muscle mitochondria, further reaffirming the difference between Ca2þ modulation of NADH generation in cardiac and skeletal muscle mitochondria. A potential reason for this difference in Ca2þ modulation of NADH generation in cardiac and skeletal muscle mitochondria might be the markedly lower mitochondrial Ca2þ uniporter current density in cardiac mitochondria when compared with skeletal muscle mitochondria (33). Physiological implications of [Ca2D]e control on mitochondrial oxidative phosphorylation Cardiac mitochondria therefore operate at high relative NADH generation fluxes, which are not affected by [Ca2þ]e dependent open-loop control. In vivo cardiac mitochondria operate at a higher resting ATPase rate with a fourfold increase at maximal workload (4,5). Skeletal muscle mitochondria on the other hand are subject to a much lower ATPase flux at rest and may reach their maximal capacity for oxidative phosphorylation at high workloads. Cytosolic free Ca2þ in muscle, which regulates the contractile ATPase flux, could potentially extend the range of oxidative ATP synthesis. An in vivo metabolic control analysis by Jeneson et al. (34) showed that the control of oxidative ATP synthesis in muscle shifts increasingly from extramitochondrial ATPase to mitochondrial ATP synthesis flux as the ATP demand increases, which points to a potential role for open-loop control by [Ca2þ]e at high workloads. The role of Ca2þ in this context would be to decrease the response time of the fluxes generating NADH such that net NADH oxidation is reduced when VO2 increases to a new steady state. Skeletal muscle utilizes all of its mitochondrial capacity, so even a modest impact of Ca2þ on substrate dehydrogenation could contribute to the range of oxygen consumption although by a small fraction. Indeed, mice lacking the mitochondrial Ca2þ uniporter, which facilitates the entry of Ca2þ into the mitochondrial matrix, were reported to achieve 20% smaller maximal work on an inclined treadmill when compared with wild-type mice (9). Our study was performed using mitochondria from oxidative muscles consisting of mainly type I fibers. Further characterization of control of mitochondrial oxidative phosphorylation in various fiber types may be necessary to achieve a more complete understanding of in vivo muscle function and to test the generality of the results reported in our study. Biophysical Journal 110(4) 954–961
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Although out results show [Ca2þ]e modulation of NADH synthesis fluxes in response to demand, the distribution of open-loop control among potential sites such as Ca2þ sensitive dehydrogenases in the mitochondrial matrix is unclear. Studies on the distribution of flux control of mitochondrial ATP synthesis and respiration in skeletal muscle (35) showed that pyruvate uptake and oxidation was found to have a control coefficient of 0.2 at maximal ADP stimulated respiration in vitro, which means that [Ca2þ]e modulation of pyruvate oxidation via the pyruvate dehydrogenase complex could potentially contribute to the observed increase in NADH synthesis flux in skeletal muscle mitochondria. Simulation studies adapting a cardiac model of mitochondrial oxidative phosphorylation to describe skeletal muscle energetics could not describe the steady-state relationship between steady-state Pi and ATPase flux over the entire range of the ATPase flux (10). Particularly the model under predicts the Pi data at low ATPase flux values implying high mitochondrial ATP synthesis flux at low ATP demand fluxes as a consequence of assuming constant enzyme activities and feedback control as the sole mechanism. Our results demonstrating [Ca2þ]e modulation of skeletal muscle mitochondrial ATP synthesis fluxes could be a potential mechanism that could reconcile the mitochondrial model with the data. AUTHOR CONTRIBUTIONS D.A.B., K.C.V., and R.W.W. conceived and designed the research. A.S., F.V.dB., K.C.V., and M.B.O. performed the research. A.S., D.A.B., F.V.dB., and K.C.V. analyzed the data. D.A.B., F.V.dB., K.C.V., and R.W.W. wrote the article.
ACKNOWLEDGMENTS The authors thank Dr. Ronald A. Meyer (Michigan State University) for suggesting parametric analysis of fNADH versus VO2 plots. Research was funded by NIH grant R01 DK095210 (D.A.B. and R.W.W.).
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