Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury

Abstract

Aims: No effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R.

Methods and results: Panx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1-/- mice, but leukocyte infiltration in the infarct area was similar between Panx1-/- and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1-/- hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1-/- hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1-/- hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1-/- hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1-/- hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1-/- SSM. Finally, Panx1-/- cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content.

Conclusion: Panx1-/- mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage.

Keywords: Cardioprotection; Heart; Ischemia/reperfusion; Mitochondria; Pannexin1.

Graphical Abstract

Figure 1

Panx1 deletion protects against I/R injury. Immunofluorescent staining on cardiac cryosections against Panx1 (green) confirmed endothelial expression of Panx1 in the cardiac microvasculature and SMCs of arterioles (A). The signal was absent upon omission of primary antibody (B). After I/R, the Panx1 signal remained present in the endothelium of coronary vessels and the cardiac microvasculature (C, D), as well as in the neutrophils in capillaries and infiltrating the myocardium (D–E). DAPI (blue), Evans blue (red). (A–E) are representative photographs of three independent experiments. Ligation of LAD resulted in a comparable AAR in WT and Panx1^−/− mice (F), but Panx1-deficient animals displayed smaller IA (G) and decreased serum cardiac Troponin-I (cTnI; H). N = 10, unpaired t-test. Representative photographs (I) and quantification (J) of immunofluorescent staining against the neutrophil marker Ly6G (green). No differences were observed in neutrophil (J) and macrophage (K) recruitment between WT and Panx1^−/− mice (N = 4–5, unpaired t-test). In vivo I/R in mice with neutrophil-specific Panx1 deletion (Panx1^Ndel; N = 10) and controls (Panx1^fl/fl; N = 9, unpaired t-test) resulted in similar AAR (L), IA (M), and cardiac cTnI (N) between both groups. Scale bars = 50 μm.

Figure 2

Panx1 deletion improves the recovery of LV function after ex vivo I/R. Immunolabelling of cardiomyocytes isolated from WT adult mice demonstrated Panx1 (green) staining in plasma membranes and subsarcolemmal regions (A, B). The signal was absent in Panx1^−/− cardiomyocytes (C). DAPI (blue), Evans blue (red). Scale bar = 50 μm. (A–C) are representative photographs of three independent experiments. qPCR confirmed Panx1 mRNA expression in WT cardiomyocytes while absent in Panx1^−/− cardiomyocytes. N = 3. B16-BL6 cells were used as a positive control (D). Representative myocardial contractile activity trace during Langendorff perfusion: WT and Panx1^−/− hearts were stabilized for 20 min, and then subjected to global no-flow ischaemia (30 min) and reperfusion (60 min) (E). The physiological myocardial function measured at the end of the stabilization, i.e. left ventricular developed pressure (LVDP; F), contractility (+dP/dt; G), relaxation (–dP/dt; H), HR (I), and RPP (J) were not different between both genotypes. N = 20, unpaired t-test. The sensitivity to ischaemia measured as time to onset of contracture (TTOC; K) and to maximal contracture (TTMC; L) was increased in Panx1^−/− mice while the ischaemic rigour (M) remained unchanged. N = 20, unpaired t-test. After I/R, Panx1^−/− hearts displayed improved recovery of LVDP (N), +dP/dt (O), –dP/dt (P), and RPP (R), whereas HR (Q) remained similar between both genotypes. N = 20, unpaired t-test.

Figure 3

Panx1 deletion abolishes the cardioprotective effects of IPC. Mouse hearts were subjected to three cycles of brief ischaemia (5 min) followed by reperfusion (5 min) before the period of prolonged ischaemia (30 min) and reperfusion (60 min). The recovery of LVDP (A), +dP/dt (B), –dP/dt (C), HR (D), and RPP (E) was not changed between WT (N = 9) and Panx1^−/− (N = 10, unpaired t-test) mice following IPC and I/R. Moreover, no differences were observed in TTOC (F), TTMC (G), and ischaemic rigour (H) under these conditions. N = 10, unpaired t-test.

Figure 4

Panx1 is present in cardiac SSM. qPCR shows Panx1 mRNA in mitochondria derived from WT ventricular tissue while absent in mitochondria of Panx1^−/− ventricles. N = 4. mRNA extracted from WT and Panx1^−/− hearts were used as positive and negative control, respectively (A). Panx1 protein is present in mitochondria isolated from WT ventricular tissue while absent in cardiomyocyte mitochondria of Panx1^−/− ventricles (B). RNAscope in situ hybridization on murine heart cryosections demonstrated colocalization of Panx1 mRNA (red) with mitochondrial COX-I mRNA (green) (C–D). DAPI (blue). Scale bar = 5 μm. Co-localization is revealed by the yellow signal. (C and D) are representative photographs of three independent experiments. Western blots showing Panx1 protein in SSM fractions and not in IFM fractions isolated from ventricles of WT hearts (E). The absence of Na⁺/K ⁺ -ATPase confirmed the purity of mitochondrial extraction, the presence of Cx43 confirmed the correct subfractioning and MnSOD served as loading control. LV represents a total protein sample of LV tissue of the WT heart. Representative EM images of WT (F) and Panx1^−/− (G) hearts. Scale bars = 2 µm. Morphological parameters, i.e. average area per SSM (H) or IFM (I) and circularity of SSM (J) or IFM (K), were not different between genotypes (WT: 44 images analysed from three hearts; Panx1^−/−: 43 images analysed from three hearts). The total number of mitochondria analysed was 542 SSM and 668 IFM for WT hearts, and 356 SSM and 635 IFM for Panx1^−/− hearts. The total SSM area relative to the length of the sarcolemma (L) was also not different between WT and Panx1^−/− hearts. Statistical analysis of EM data was performed using a linear mixed model with either repeated measures and compound symmetry (mitochondrial area per sarcolemmal length) or a nested design (area per mitochondrion and shape circularity). The ATP content in Panx1^−/− mitochondria was higher than in WT mitochondria. N = 4 (M). A hypo-osmotic shock induced more ATP release from Panx1^−/− mitochondria than from WT mitochondria. N = 4, unpaired t-test (N).

Figure 5

Panx1 modulates mitochondrial respiration. Western blots revealed lower Panx1 expression in B16-F10 (F10) cells as compared to isogenic B16-BL6 (BL6) cells (A). Representative traces of OCR measured by Seahorse in untreated B16-BL6 (black) and B16-F10 (grey) cells or after VDAC1 inhibition with 10 μM NSC15364 (B16-BL6: red; B16-F10: green) (B). Basal respiration was reduced in B16-F10 compared to B16-BL6 cells. N = 15, unpaired t-test. (C). Likewise, the maximal respiration rate was reduced in B16-F10 compared to B16-BL6 cells after VDAC1 inhibition with 10 μM of TRO19622 (N = 5, unpaired t-test) (D) or NSC15364 (N = 5, unpaired t-test) (E). Representative traces of calcium-induced mPTP opening in cardiac SSM from WT (black) and Panx1^−/− (grey) hearts (F). The mPTP inhibitor—cyclosporin A (CsA) served as internal quality control (WT—violet; Panx1^−/−—light green) (F). The quantification of a number of calcium waves before complete mPTP opening (arrows in F) showed no differences between WT and Panx1^−/− mitochondria at both basal conditions and after CsA-treatment. N = 6, 2-way ANOVA (G). Representative traces of ROS production from WT and Panx1^−/− SSM (H). An increase in slope was observed upon rotenone (rot-) treatment (blue and yellow) compared to the basal level in WT (black) and Panx1-deficient (grey) cardiac mitochondria (H). ROS production, measured as the slope, was comparable between the two genotypes at both conditions. N = 9, two-way ANOVA (I). Basal respiration was similar between WT and Panx1^−/− SSM measured at complex 1 (J) and complex 2 (K). The ADP-stimulated respiration showed an increased OCR in WT samples in complex 1 and complex 2, while no differences upon ADP stimulation were observed in Panx1^−/− mitochondria (J and K, respectively). N = 5, two-way ANOVA. Panx1^−/− cardiomyocytes (N = 61 cardiomyocytes from three heart cell isolations) had a lower mitochondrial membrane potential (ΔΨm) than WT cardiomyocytes (N = 30 cardiomyocytes from three heart cell isolations, unpaired t-test) (L).