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. 2010 Apr 13;121(14):1606-13.
doi: 10.1161/CIRCULATIONAHA.109.914911. Epub 2010 Mar 29.

Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species in failing cardiac myocytes

Michael Kohlhaas  1 , Ting Liu, Andreas Knopp, Tanja Zeller, Mei Fang Ong, Michael Böhm, Brian O'Rourke, Christoph Maack
Affiliations

Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species in failing cardiac myocytes

Michael Kohlhaas et al. Circulation. .

Abstract

Background: Oxidative stress is causally linked to the progression of heart failure, and mitochondria are critical sources of reactive oxygen species in failing myocardium. We previously observed that in heart failure, elevated cytosolic Na(+) ([Na(+)](i)) reduces mitochondrial Ca(2+) ([Ca(2+)](m)) by accelerating Ca(2+) efflux via the mitochondrial Na(+)/Ca(2+) exchanger. Because the regeneration of antioxidative enzymes requires NADPH, which is indirectly regenerated by the Krebs cycle, and Krebs cycle dehydrogenases are activated by [Ca(2+)](m), we speculated that in failing myocytes, elevated [Na(+)](i) promotes oxidative stress.

Methods and results: We used a patch-clamp-based approach to simultaneously monitor cytosolic and mitochondrial Ca(2+) and, alternatively, mitochondrial H(2)O(2) together with NAD(P)H in guinea pig cardiac myocytes. Cells were depolarized in a voltage-clamp mode (3 Hz), and a transition of workload was induced by beta-adrenergic stimulation. During this transition, NAD(P)H initially oxidized but recovered when [Ca(2+)](m) increased. The transient oxidation of NAD(P)H was closely associated with an increase in mitochondrial H(2)O(2) formation. This reactive oxygen species formation was potentiated when mitochondrial Ca(2+) uptake was blocked (by Ru360) or Ca(2+) efflux was accelerated (by elevation of [Na(+)](i)). In failing myocytes, H(2)O(2) formation was increased, which was prevented by reducing mitochondrial Ca(2+) efflux via the mitochondrial Na(+)/Ca(2+) exchanger.

Conclusions: Besides matching energy supply and demand, mitochondrial Ca(2+) uptake critically regulates mitochondrial reactive oxygen species production. In heart failure, elevated [Na(+)](i) promotes reactive oxygen species formation by reducing mitochondrial Ca(2+) uptake. This novel mechanism, by which defects in ion homeostasis induce oxidative stress, represents a potential drug target to reduce reactive oxygen species production in the failing heart.

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Figures

Figure 1
Figure 1
Regulation of oxidative phosphorylation and ROS formation by NAD(P)H and Ca2+. The Krebs cycle is fueled by metabolic substrates (glucose, fatty acids) via acetyl-coenzyme A and mediates the recovery of oxidized NAD+ to NADH, which donates electrons to the ETC for oxidative phosphorylation of ATP. Regeneration of antioxidative capacity is also coupled to the Krebs cycle, because regeneration of NADPH requires products of the Krebs cycle (isocitrate, malate, NADH). Three rate-controlling enzymes of the Krebs cycle (pyruvate, isocitrate, and α-ketoglutarate dehydrogenase) are activated by Ca2+. ΔΨm indicates mitochondrial membrane potential; Nnt, nicotinamide nucleotide transhydrogenase; Mn-SOD, Mn2+-dependent superoxide dismutase; PRX, peroxiredoxin; GPX, glutathione peroxidase; TRXr/o, reduced/oxidized thioredoxin; GSH/GSSG, reduced/oxidized glutathione; TR, thioredoxin reductase; GR, glutathione reductase; IDPm, mitochondrial NADP+-dependent isocitrate dehydrogenase; MEP, mitochondrial malic enzyme; α-KG, α-ketoglutarate; and mNHE, mitochondrial Na+/H+ exchanger.
Figure 2
Figure 2
Mitochondrial Ca2+ uptake during transitions of workload. Myocytes were depolarized from −80 to 10 mV for 80 ms at 3 Hz and superfused with isoproterenol. Pipette solution contained 5 mmol/L [Na+]i, in the absence (control, n=13) and presence of the MCU-blocker Ru360 (1 μmol/L; n=11). A–C, left panels: Time courses of L-type Ca2+ currents (ICa,L; A), [Ca2+]c (B), and [Ca2+]m (C) during the whole experiment; right panels, averaged currents, [Ca2+]c, and [Ca2+]m after 10 minutes of the protocol, respectively (see arrow in left panels). Con indicates control; sys, systole; and dias, diastole. D, Averaged amplitudes of [Ca2+]m (Δ[Ca2+]m) plotted against the respective Δ[Ca2+]c, in the absence (Con) and presence of Ru360 (Ru). E, [Ca2+]m plotted against [Ca2+]c during a single Ca2+ transient (averaged data at 10 minutes of the protocol). F, Time to peak (TTP) and G, time constant of decay (τ) of [Ca2+]c (Cyto) and [Ca2+]m (Mito) in the absence (C) and presence (Ru) of Ru360, respectively. *P<0.05 from minute 9 to 12 and P<0.01 from minute 12 to 30; **P<0.01 from minute 6 to 30 in C, and as indicated in F and G (2-way ANOVA, respectively).
Figure 3
Figure 3
Dynamic regulation of mitochondrial ROS by [Ca2+]m and NAD(P)H. Myocytes (n=16) were loaded with CM-H2DCF to monitor H2O2 together with the autofluorescence of NAD(P)H. A similar protocol as in Figure 2 was performed (voltage-clamp pulses from −80 to 10 mV at 3 Hz, [Na+]i=5 mmol/L). Changes in ICa,L (A), NAD(P)H (D), and H2O2 (E) are displayed together with changes in Δ[Ca2+]c (B) and diastolic [Ca2+]m (C) from the experiments in Figure 2 (control group). The gray trace in E indicates H2O2 in unpatched cells that were not paced. F, NAD(P)H correlated to diastolic [Ca2+]m after β-adrenergic stimulation and initial NAD(P)H oxidation (starting at minute 6). G, Rates of CM-DCF oxidation, indicating the net H2O2 formation (ΔF/F0×min−1), averaged over 1 minute, respectively, and correlated to the respective (averaged) NAD(P)H levels after β-adrenergic stimulation with isoproterenol at the indicated time points. For minutes 12 to 17, 1 average value was calculated. Stim indicates stimulation.
Figure 4
Figure 4
Inhibition of mitochondrial Ca2+ uptake increases mitochondrial ROS production. The same protocol was used as in Figures 2 and 3, respectively. Amplitudes of [Ca2+]c (Δ[Ca2+]c; A) and diastolic [Ca2+]m (B), in the absence (Con, n=13) and presence (Ru, n=11; 1 μmol/L in the pipette solution) of Ru360 are displayed. Ca2+ data are taken from the series of experiments in Figure 2. NAD(P)H (C) and H2O2 (D) in the absence (n=16) and presence of Ru360 (n=15; 1 μmol/L in the pipette solution). E, NAD(P)H plotted against diastolic [Ca2+]m. F, Net mitochondrial formation of H2O2 plotted versus NAD(P)H. *P<0.05 at minute 8 to 10 and P<0.01 from minute 12 to 17; **P<0.05 from minute 10 to 15; †P<0.05 at minute 14 to 17 and P<0.07 at minute 7 to 14 (1-way ANOVA, respectively).
Figure 5
Figure 5
Elevated [Na+]i increases ROS production in normal myocytes. A similar protocol as in Figure 2 was performed, except that isoproterenol was washed in starting 40 seconds after the onset of pacing. Pipette solution was 5 or 15 mmol/L [Na+]i as indicated. A and B, [Ca2+]c and [Ca2+]m were measured in normal myocytes (5 mmol/L [Na+]i, n=15; 15 mmol/L [Na+]i, n=20). A, Systolic [Ca2+]m plotted against systolic [Ca2+]c; B, diastolic [Ca2+]m plotted against diastolic [Ca2+]c; the numbers indicate the time (in minutes) after the start of the experiment. C and D, Net H2O2 formation was determined by CM-DCF in a separate set of cells (n=15/12). CM-DCF oxidation, indicating levels of H2O2, is given as F/F0 (C) or ΔF/F0 per minute (D). *P<0.05 for 5 vs 15 mmol/L [Na+]i at minute 6 to 18 (1-way ANOVA).
Figure 6
Figure 6
Increased ROS production in failing myocytes is related to deficient mitochondrial Ca2+ uptake. A, Heart weight/body weight (HW/BW) ratio in guinea pigs 4 weeks after ascending aortic constriction (failing, F; n=3) compared with age- and sex-matched control animals (nonfailing, NF; n=4). B, In vivo left ventricular ejection fraction (LVEF) before (BLN) and 4 weeks after (4w) aortic banding (n=3). C, H2O2 levels in intact, field-stimulated myocytes (4 Hz) from normal (NF, n=8) or failing (F) myocytes, in the absence (Con; n=8) or presence (CGP) of CGP-37157 (1 μmol/L; n=6), an inhibitor of the mNCE. D, Cumulative H2O2 formation in failing myocytes that were voltage clamped (4 Hz) and equilibrated with a pipette solution that contained either 5 or 15 mmol/L [Na+] (n=7/3). *P<0.05 F vs NF; **P<0.01 4w vs BLN; †P<0.05 F/Con vs NF/Con and F/CGP vs F/Con at minutes 4 to 7, respectively; ‡P<0.001 for 5 vs 15 mmol/L [Na+]i at minutes 3 to 7 by unpaired t test (A), paired t test (B), and 1-way ANOVA (C and D), respectively.
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