Resistance to atrial fibrillation domestication and mitochondrial dysfunction in sheep: a potential key role of the TCA cycle and mitochondrial redox state

Figures

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  Fig. 1. AF properties, electrophysiological and structural remodeling. A: Tachypacing bursts per 24-h as a function of follow-up time. Results shown for AF-Sensitive (AF-S, N = 12) and AF-Resistant (AF-R, N = 8) sheep. B-C: P wave duration (B) and PR interval (C) recorded before starting the RAP protocol. D: Pseudo left anterior oblique (LAO) view of representative electro-anatomical maps of the bi-atrial local activation time, for the three groups (RA, right atrium, LA, left atrium). Scalebar range is 11-80 ms for all maps and groups. E: Atrial conduction velocity calculated from the 3D maps obtained during contact mapping of the right (RA) and left (LA) atrium. F: Representative images of left atria (LA) posterior wall histological preparations, for the three groups, after Masson trichrome stain and scanning. Scale bar = 2 mm. G: Endomysial fibrosis measured from histological sections stained with Masson Trichrome. H: CM cross-sectional area. I-M: LAA tissue fold change of mRNA transcripts expression level of Sodium channel protein type 5 subunit alpha (Scn5a, I), Voltage-dependent L-type calcium channel subunit alpha-1C (Cacna1c, J), Connexin 43 (Cx43, K), Connexin 40 (Cx40, L), and…

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  Fig. 2. Proteomic, enzymatic and metabolomic analysis of LA-appendage. LAA samples freeze-clamped in vivo were analyzed by mass spectrometry for proteins identification. The computational analysis suggested dysregulation of multiple proteins involved in mitochondrial function, metabolism and bioenergetics. A: Volcano-plot obtained by comparing AF-R to AF-S sheep. B: Pathway enrichment analysis results for the comparisons between AF-R and AF-S – red dots represent the pathways with false discovery rate <0.05. C to F: Hexokinase activity (HK, panel C), Glucose-6-phosphate (G6P, panel D), pyruvate to l-Lactate ratio (panel E), and Glycogen content (panel F) assessed in frozen LAA. Panel G: scheme summarizing the glycolytic remodeling assessed by proteomic, enzymatic and metabolomic studies. Complementary results are shown in supplemental figures 2, 3 and 4. C to F show individual points and mean ± SD. Statistical analysis was performed with Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. Numbers in brackets correspond to individual experiments for each group.

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  Fig. 3. Metabolite content and enzyme activity in LA Appendage (LAA). Samples of LAA freeze-clamped in vivo were analyzed by high-pressure ion chromatography (HPIC) or enzymatic assay for metabolites belonging to the TCA cycle (see methods). Metabolite content is expressed in pmol/mg wet weight (ww). Enzymes' activity was assessed on isolated mitochondria except for Citrate Synthase (CS) which was analyzed in LAA homogenate to evaluate mitochondrial content. In accordance to the graph plots red and orange arrows represent the changes in metabolites or enzyme activity in the AF-S and AF-R, respectively. For TCA cycle intermediates, citrate (a), isocitrate (b), α-Ketoglutarate (c), succinate (f), fumarate (g), and malae (h) content were assessed. For mitochondrial enzymes, Pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (α-KGDH, 4), fumarase (FH, 7), succinyl-CoA synthetase (SCS, 5), l-malate dehydrogenase isoform 2 (MDH2, 8), and D/L-2-hydroxyglutarate dehydrogenase (2HGDH). Acetyl-CoA, Oxaloacetate and succinyl-CoA could not be detected in our samples. Red arrows and orange arrows show the changes in AF-S and AF-R, respectively. Panels show individual data points and mean…

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  Fig. 4. O2-consumption, respiratory coupling, rate of ATP synthesis, and H2O2 emission from mitochondria. Mitochondria were isolated and purified from LAA of Sham, AF-Sensitive (AF-S), and AF-Resistant (AF-R) sheep to evaluate (i) electron transport chain function, (panels A: State 2 non-phosphorylating respiration, B: respiration coupled to maximal ATP-synthesis (State 3, phosphorylating respiration) C: State 3 to State 2 ratio, D: rate of ATP synthesis, and E: P to O ratio reflecting OXPHOS efficiency. F-I: rate of hydrogen peroxide emission (H2O2 emission) assessed with pyruvate + malate or palmitoyl-carnitine + malate (PM or PCM, Panels F (raw traces) and G), or glutamate + malate + succinate in absence or presence of S1QEL (GMS or GMS + S1QEL, panels H (typical traces) and I). J: autofluorescence of the mitochondrial NAD(P)H pool obtained during state 3 respiration measurement (B). Panels show individual data points and mean ± SD. Statistical analysis was performed with t-test using the Holm-Sidak method to correct for multiple comparisons. The numbers in brackets represent individual experiments in each group. #p < 0.05 GMS + S1QEL vs. GMS, by Wilcoxon test for paired samples.…

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  Fig. 5. Complexes I and II specific activity, and LAA energetic and redox status. A - C: Mitochondrial complex I (A) and complex II (B) specific activity, and complex I/II activity ratio (C) studied in isolated mitochondrial from Sham, AF-S, and AF-R. D - I. ATP to ADP ratio (D), PCr content (E), Total carbonyl content (F), Total NAD+ (G), Total NADH (H), and NAD to NADH ratio (I) were studied in the LAA of the three groups by HPIC (see methods). Panels show individual data-points and mean ± SD. Statistical analysis in panels A to D, was performed with t-tests using the Holm-Sidak method for multiple comparisons. Statistical analysis in panels E to I, was performed by Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. The numbers in brackets represent individual experiments.

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  Fig. 6. Differential mitochondrial redox regulation within AF-R and AF-S LAA. A and B: cytosolic (A) and mitochondrial (B) NAD+ to NADH ratio calculated using the l-lactate to pyruvate and β-hydroxybutyrate to acetoacetate ratio under thermodynamic equilibrium postulate for the reaction catalyzed by lactate dehydrogenase and β-hydroxybutyrate dehydrogenase. C. Graph representing the mean measured LAA tissue NAD + to NADH ratio as a function of the mean calculated NAD + to NADH ratio. D: calculated mitochondrial proton motive force (Δp) within the LAA tissue while considering the system at thermodynamic equilibrium. E and F: calculated mitochondrial oxaloacetate (E, [OAA]) and α-ketoglutarate (F, [α-KG]) concentrations. Panels show individual data-points and mean ± SD. Statistical analysis was performed by Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons. The numbers in brackets represent individual experiments.

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  Fig. 7. Effect of exogenous succinate application on atrial cardiomyocytes. A-D: Isolated rat atrial cardiomyocytes (RACMs) were incubated with 5 mmol/L of succinate and intracellular metabolites content was assessed by mass spectrometry. Signals correction-quantification and normalization were performed using both deuterium-labelled succinic acid and oxalic acid (A), respectively (see methods). Intracellular content of succinate (B), fumarate (C) and l-malate (D) is represented. E: Representative images illustrating ROS emission measurement by CellROX Green in RACM treated with either succinate at 5 mmol/L or vehicle (BF: bright field, FITC: Fluorescein). F: Quantification of intracellular ROS emission in presence of succinate measured with CellROX, with respect to the background. G-J: Evolution of LAA NAD(P)H (G (time course), H (quantification)) and MitoPY1 (H2O2 sensitive fluorescent probe, I (time course), J (quantification)) fluorescence measured in the ex vivo perfused rat heart, using an optical fiber positioned close to the LAA, upon perfusion with 5 mmol/L succinate. A-D: Statistical analysis via Mann-Whitney, N = 4 in each group Test. F, H and I: Statistical analysis via…

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  Fig. 8. Effect of succinate on arrhythmia susceptibility and carbonylation in vivo and effect of RAP on blood metabolites. Atrial arrhythmia susceptibility was assessed in vivo by transesophageal burst-pacing following intravenous injection of vehicle, succinate or succinate + S1QEL. A: Number of animals with inducible AF/AT versus no arrhythmia. B: Cumulative probability of AF/AT as function of number of induction-bursts. C: AF/AT duration in response to a burst-pacing challenge. D: Carbonyl content of in vivo hearts measured by Oxyblot. E-I: Blood was harvested before (baseline) or after in vivo atrial tachystimulation by transesophageal burst-pacing (RAP), and citrate (E), adenosine monophosphate (AMP, F), inorganic phosphate (G) were assessed by mass spectrometry (N = 6). H: Succinate to l-malate ratio. I: Succinate to fumarate ratio. Statistical analysis by Fischer exact test (A), Log-rank Mantel-Cox test (B), Kruskal-Wallis test and Dunn's post-hoc test for multiple comparisons (C, D), and Wilcoxon test (E-I). Complementary analysis is presented in Supplementary Fig. 11.

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  Fig. 9. Scheme summarizing the study findings. Electrically-maintained AF led to domestication in AF-sensitive (AF-S) animals within 3 weeks, but some animals were AF resistant (AF-R) and failed to develop self-sustained AF despite 4 months of electrically maintained AF. Specific patterns of mitochondrial and metabolic remodeling characterized AF-S. In the presence of succinate, AF-S mitochondria showed increased ROS emission, decreased P to O ratio, increased complex II to complex I activity ratio. AF-R and AF-S mitochondria showed differential regulation of the TCA cycle. At the whole-tissue level, AF-S atria showed more oxidized proteins compared to Sham, along with an increased NAD+ to NADH ratio and impaired energy availability reflected by a decreased ATP to ADP ratio relative to both Sham and AF-R values. AF-R LAA showed a higher PCr paralleled by increased CKMT2 expression and a more reduced mitochondrial matrix secondary to increased ketone body metabolism compared to AF-S. AF-S also showed differential TCA cycle enzymes regulation compared to AF-R, likely contributing to succinate accumulation in the AF-S group… (Created with BioRender.com.)