PITX2C deficiency promotes arrhythmogenic remodelling via oxidative stress in atrial myocytes

Figures

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  Fig. 1. PITX2C transcriptionally regulates oxidative phosphorylation pathway genes. Bulk RNA-seq was performed on neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2 (KD) siRNA. [A] Principal component analysis of samples used in RNA-seq with ellipses depicting 95% confidence intervals. [B] Volcano plot showing significantly (p < 0.05) enriched genes with at least a |1.5| fold change. [C] Gene set enrichment analysis (GSEA) of significantly (FDR >0.25) enriched pathways. [D] Top 25 genes enriched in oxidative phosphorylation pathway from GSEA.

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  Fig. 2. Pitx2c deficiency impairs mitochondrial oxidative phosphorylation and elevates mitochondrial derived reactive oxygen species. Mitochondrial respiration was assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA using the Seahorse XFp MitoStress test. [A] Representative oxygen consumption rate (OCR) trace is shown with injections of oligomycin, carbonyl cyanite-4 (trifluoromethoxy) phenylhydrazone (FCCP), and rotenone/antimycin A. [B] Quantification of mitochondrial function relative to Ctl cells including non-mitochondrial respiration (Non-Mito Resp.), basal respiration (Basal Resp.), maximal respiration (Max Resp.), Proton Leak, ATP production (ATP Prod.), spare respiratory capacity (Spare Resp. Cap.), and coupling efficiency (Coupling Eff.). N = 4, n = 12 wells. [C] mtDNA content relative to Ctl cells was quantified by qPCR and are expressed as mt-co1/polg (mtDNA/nDNA). N = 4, n = 11 wells. [D] Representative western blot for electron transport chain subunits NDUFS1 (Complex I), SDHA (Complex II), UQCRFS1 (Complex III), MT-CO1 (Complex IV), and ATP5A1 (Complex V). [E] Quantification of electron transport chain subunits. N = 4.…

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  Fig. 3. Pitx2c deficiency promotes ectopic beats through dysregulated intracellular calcium cycling. Spontaneous intracellular calcium dynamics were assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA using a fluorescent calcium indicator, Fluo-8. [A] Representative time course of Fluo-8/baseline fluorescence (Fluo-8/F0) reporting rises in cytosolic calcium. Rate kinetics were quantified including [B] beat rate, [C] time to peak, [D] time to 90% decay, and [E] time to 50% decay. Arrhythmogenic calcium traces were observed and classified as early [F] or delayed [G] afterdepolarization-like calcium events and quantified [H,I]. Statistical analyses were performed using nested t-test [B-E] or Fisher's exact test [H, I] to study differences between groups. N = 3, Ctl: n = 121 cells, KD: n = 123. Data are expressed as means ± SEM.

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  Fig. 4. Sarcomere structure is perturbed with the loss of PITX2C in neonatal rat atrial cardiomyocytes. Sarcomere structure was assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA. [A] Confocal microscopy of immunofluorescently labelled Myomesin (M-band), F-actin (Z-disk), and DAPI (nuclei). Scale bars depict 50 μm. Quantification of [B] sarcomere length [C] sarcomere organization and [D] M-band density (as a % of total cell area) [A,B] N = 3, Ctl: n = 105 cells, KD: n = 106 cells. [D] Ctl: n = 124 cells, KD: n = 126 cells [E] Confocal microscopy of immunofluorescently labelled Titin, F-actin, and DAPI. Scale bars depict 50 μm and [E'] representation of co-localization of Titin and F-actin signal along one myofibril. [F] Quantification of Titin and F-actin co-localization. N = 3, Ctl: n = 154 cells, KD: 140 cells. Statistical analyses were performed using nested t-test [B—D, F] to study differences between groups. Data are expressed as mean ± SEM.

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  Fig. 5. Antioxidant treatment partially attenuates deleterious phenotypes in Pitx2c deficient atrial cardiomyocytes. Neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA were treated with 1 mM N-acetylcysteine (NAC) for 24 h. [A] Quantification of mitochondrial specific reactive oxygen species using MitoSOX relative to Ctl cells in Ctl or KD NRAMs. N = 4, Ctl: 76 cells, KD: 79 cells, KD + NAC: 96 cells. Intracellular calcium rate kinetics were quantified including [B] beat rate, [C] time to peak, [D] time to 90% decay, and [E] time to 50% decay. Early [F] and delayed [G] afterdepolarization-like calcium events were quantified. N = 4, Ctl: n = 68 cells, KD: n = 47, KD + NAC: 72 cells. [H] Quantification of Titin and F-actin co-localization. N = 3, Ctl: 60 cells, KD: 91 cells, KD + NAC: 76 cells. Statistical analyses were performed using nested one-way ANOVA followed by Tukey's multiple comparison test [A-E, H] or Fisher's exact test [F,G] to study differences between groups. Data are expressed as mean ± SEM.

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  Fig. S1. Neonatal rat atrial myocytes transfected with non-targeting (Ctl) siRNA or one of two Pitx2c (548, 549) siRNAs at various doses. RT-qPCR was performed at 48- and 72-h post-transfection and Pitx2c mRNA levels were normalized to B-actin. Statistical analyses were performed using 2-way ANOVA followed by Tukey's multiple comparison test. Data are expressed as mean ± SEM. N = 3, n = 2–8 wells.

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  Fig. S2. Full uncropped western blot images for Fig. 2D.

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  Fig. S3. Pitx2c deficiency does not impair glycolytic function. Glycolysis was assessed in neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA using the Seahorse XFp Glycolytic Rate assay. [A] Representative proton efflux rate (PER) trace is shown with injections of rotenone & antimycin A and 2-deoxyglucose (2-DG). [B] Quantification of glycolytic function relative to Ctl cells including basal glycolysis, basal PER, % PER from glycolysis, compensatory glycolysis, mitochondrial oxygen consumption rate (mitoOCR)/glycolysis PER (glycoPER), and post 2-DG acidification (post 2-DG acid.). N = 2, n = 6 wells. Statistical analyses were performed using Student's t-test [B, C, E] to study differences between groups. Data are expressed as mean ± SEM.

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  Fig. S4. Pitx2c deficiency results in no changes in cell surface area or overall morphology. Cell morphology was assessed in fluorescently labelled neonatal rat atrial myocytes (NRAMs) transfected with non-targeting (Ctl) or Pitx2c (KD) siRNA stained for F-actin. [A] Cell surface area, [B] cell elongation, and [C] cell roundness were quantified. N = 3, n = 310–340 cells. Statistical analyses performed using Student's t-test to study differences between groups. RT-qPCR of quantify [D] total Titin (exon 49–50) mRNA levels. Total Titin levels were normalized to B-actin. N = 3, n = 6 wells. Data are expressed as means ± SEM. Statistical analyses performed using nested t-test (Fig. S4A-C) or Student's t-test (Fig. S4D) to study differences between groups.