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. 2024 Jan 24;14(2):144.
doi: 10.3390/biom14020144.

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes

Yuriana Oropeza-Almazán  1 , Lothar A Blatter  1
Affiliations

Role of Mitochondrial ROS for Calcium Alternans in Atrial Myocytes

Yuriana Oropeza-Almazán et al. Biomolecules. .

Abstract

Atrial calcium transient (CaT) alternans is defined as beat-to-beat alternations in CaT amplitude and is causally linked to atrial fibrillation (AF). Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling through redox environment regulation. In isolated rabbit atrial myocytes, ROS production is enhanced during CaT alternans, measured by fluorescence microscopy. Exogenous ROS (tert-butyl hydroperoxide) enhanced CaT alternans, whereas ROS scavengers (dithiothreitol, MnTBAP, quercetin, tempol) alleviated CaT alternans. While the inhibition of cellular NADPH oxidases had no effect on CaT alternans, interference with mitochondrial ROS (ROSm) production had profound effects: (1) the superoxide dismutase mimetic MitoTempo diminished CaT alternans and shifted the pacing threshold to higher frequencies; (2) the inhibition of cyt c peroxidase by SS-31, and inhibitors of ROSm production by complexes of the electron transport chain S1QEL1.1 and S3QEL2, decreased the severity of CaT alternans; however (3) the impairment of mitochondrial antioxidant defense by the inhibition of nicotinamide nucleotide transhydrogenase with NBD-Cl and thioredoxin reductase-2 with auranofin enhanced CaT alternans. Our results suggest that intact mitochondrial antioxidant defense provides crucial protection against pro-arrhythmic CaT alternans. Thus, modulating the mitochondrial redox state represents a potential therapeutic approach for alternans-associated arrhythmias, including AF.

Keywords: Ca alternans; atrial fibrillation; cellular redox regulation; electron transport chain; excitation–contraction coupling; mitochondrial ROS; mitochondrial dysfunction.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cellular ROS production during CaT alternans. (A) Simultaneous [Ca]i and cellular ROS generation rate measurements in an atrial myocyte during increasing pacing frequencies (N/n = 23/45). (B) CaT AR and fraction of alternating cells as a function of stimulation frequency (0.5–2.5 Hz). (C) Average ROS production rate in non-alternating (AR = 0–0.1; 103 measurements) and alternating cells (AR = 0.1–1; 58 measurements). Statistical analysis was performed using the Mann–Whitney U test. (D) ROS production rate in non-alternating and alternating cells as a function of stimulation frequency.
Figure 2
Figure 2
CaT alternans modulation by redox environment. (A) CaT alternans recordings (left) from atrial cells before (Ctrl) and during exposure to tert-butyl hydroperoxide (tBHP, 1 mM), paced at 1.7 Hz. Right: mean ± SEM and individual AR measurements in Ctrl and H2O2 (N/n = 7/19). (B) CaT alternans in Ctrl and during dithiothreitol (DTT, 1 mM) exposure (left), paced at 1 Hz. Right: mean ± SEM and individual AR measurements in Ctrl and DTT (N/n = 2/6). (C) CaT alternans in Ctrl, in the presence of tBHP and tBHP + DTT recorded from the same atrial myocyte, paced at 1 Hz. Statistical analysis (A,B) performed using Student’s t-test for paired data.
Figure 3
Figure 3
Effect of antioxidants on CaT alternans. CaT alternans recordings from atrial myocytes (left panels) before (Ctrl) and during the application of (A) superoxide scavenger quercetin (10 µM), paced at 2.1 Hz; (B) SOD mimetic MnTBAP (50 µM), paced at 1.1 Hz; and (C) SOD mimetic Tempol (1.6 mM), paced at 1.1 Hz. Right panels: mean ± SEM and individual AR measurements in Ctrl and quercetin (N/n = 2/6), MnTBAP (N/n = 2/6), and Tempol (N/n = 4/8), respectively. Statistical analysis performed using Student’s t-test for paired data.
Figure 4
Figure 4
Effects of inhibition of NADPH oxidases on CaT alternans. (A) CaT recordings from atrial cells (left panels) before (Ctrl) and during the application of (A) Apocynin (1 µM), paced at 1.1 Hz; (B) NOX2 inhibitor GSK2795039 (5 µM), paced at 1.25 Hz; and (C) NOX4 inhibitor GLX351322 (5 µM), paced at 1.3 Hz. Right panels: mean ± SEM and individual AR measurements in Ctrl and apocynin (N/n = 3/6), GSK2795039 (N/n = 6/13) and GLX351322 (N/n = 5/8). Statistical analysis performed using Student’s t-test for paired data.
Figure 5
Figure 5
Effect of inhibition of ROSm production on CaT alternans. (A) CaT recordings from atrial myocytes during electrical pacing at 1, 1.5 and 2 Hz in Ctrl and in the presence of the mitochondrial SOD mimetic MitoTempo (20 µM). Cells were incubated with MitoTempo for 5 min. (B) Average ARs in Ctrl (N/n = 5/10) and MitoTempo (N/n = 5/9) as a function of stimulation frequency (0.5–2 Hz). The horizontal dashed line marks the alternans threshold (AR > 0.1). (C–E). CaT alternans recordings (left panels) before (Ctrl) and during the application of (C) cyt c peroxidase inhibitor SS-31 (20 µM), paced at 2 Hz; (D) S1QEL1.1 (suppressor of site IQ electron leak from mitochondrial Complex I; 5 µM), paced at 1.1 Hz; and (E) S3QEL2 (suppressor of site IIIQO electron leak of mitochondrial Complex III; 15 μM), paced at 1.3 Hz. Right panels (C–E): mean ± SEM and individual AR measurements in Ctrl and SS-31 (N/n = 2/6), S1QEL1.1 (N/n = 3/8) and S3QEL2 (N/n = 8/11). Statistical analysis performed using Student’s t-test for paired data.
Figure 6
Figure 6
Effect of impairment of mitochondrial antioxidant defense on CaT alternans. (A) CaT recordings from atrial cells during electrical pacing at 1, 1.5 and 2 Hz in Ctrl (N/n = 5/12) and in the presence of the mitochondrial transhydrogenase (THD) inhibitor NBD-Cl (4-chloro-7-nitrobenzo-2-oxa-1,3 diazole; 2.5 µM; N/n = 5/9). (B) Average ARs in Ctrl and NBD-Cl as a function of stimulation frequency (0.5–2 Hz). The horizontal dashed line marks the alternans threshold (AR > 0.1). Statistical analysis performed using ANOVA. (C) Left: CaT alternans in Ctrl and during application of the mitochondrial TR2 inhibitor auranofin (10 μM), paced at 1.5 Hz. Right: mean ± SEM and individual AR measurements in Ctrl and auranofin (N/n = 3/7). Statistical analysis performed using Student’s t-test for paired data.
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