Mitochondrial connexin 43 impacts on respiratory complex I activity and mitochondrial oxygen consumption

Abstract

Connexin 43 (Cx43) is present at the sarcolemma and the inner membrane of cardiomyocyte subsarcolemmal mitochondria (SSM). Lack or inhibition of mitochondrial Cx43 is associated with reduced mitochondrial potassium influx, which might affect mitochondrial respiration. Therefore, we analysed the importance of mitochondrial Cx43 for oxygen consumption. Acute inhibition of Cx43 in rat left ventricular (LV) SSM by 18α glycyrrhetinic acid (GA) or Cx43 mimetic peptides (Cx43-MP) reduced ADP-stimulated complex I respiration and ATP generation. Chronic reduction of Cx43 in conditional knockout mice (Cx43(Cre-ER(T)/fl) + 4-OHT, 5-10% of Cx43 protein compared with control Cx43(fl/fl) mitochondria) reduced ADP-stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O(2)/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O(2)/min.*mg protein in Cx43(fl/fl) mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43(Cre-ER(T)/fl) +4-OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43(fl/fl) mice (19.8 ± 1.3%, n = 8, P < 0.05); complex II activities were similar between genotypes. Supporting the importance of Cx43 for respiration, in Cx43-overexpressing HL-1 cardiomyocytes complex I respiration was increased, whereas complex II respiration remained unaffected. Taken together, mitochondrial Cx43 is required for optimal complex I activity and respiration and thus mitochondrial ATP-production.

Fig 1

Effect of acute Cx43-inhibition using 18αGA on mitochondrial function. (A) Mitochondrial membrane potential was measured using rhodamine 123, and the difference in mitochondrial rhodamine 123 fluorescence 1 min. before and 1 min. after addition of 18αGA (1 μmol/l: n = 7, 10 μmol/l: n = 6, 100 μmol/l: n = 7) or DMSO (n = 5), respectively, was calculated. An enhanced difference in fluorescence values indicates loss of membrane potential. *P < 0.05 versus DMSO. (B) Quantification of the difference between mitochondrial NAD(P)H autofluorescence 1 min. before and 1 min. after addition of 18αGA (1 μmol/l: n = 7, 10 μmol/l: n = 6, 100 μmol/l: n = 7) or DMSO (n = 5), respectively. An enhanced difference in fluorescence values indicates loss of NAD(P)H autofluorescence. *P < 0.05 versus DMSO. Basal (C, n = 6) and ADP-stimulated respiration (D, n = 13) were measured in rat LV SSM using substrates for complexes 1 or 2, respectively, before and after addition of 1 μmol/l 18αGA or DMSO as vehicle, respectively. *P < 0.05 versus before 18αGA.

Fig 2

Effect of connexin mimetic peptides on mitochondrial oxygen consumption and ATP production. (A) Basal and ADP-stimulated respiration using substrates for complex I or complex II, respectively, were measured in untreated rat LV SSM (n = 12), or SSM incubated with 250 μmol/l Cx40- (n = 12) or Cx43-MP (n = 12), respectively. *P < 0.05 versus untreated, ^#P < 0.05 versus Cx40-MP. (B) Original traces demonstrating luciferase activities of isolated rat SSM before and after addition of 500 μmol/l ADP under control conditions (untreated) and with Cx43 inhibition by 250 μmol/l Cx43-MP. Oligomycin, which inhibits the ATP synthase, was used as negative control. (C) ATP generation was quantified as the area under the curve for the first minute after addition of ADP in untreated or Cx43-MP-treated rat SSM (n = 15), *P < 0.05.

Fig 3

Effect of chronic Cx43-inhibition on oxygen consumption. (A) Western blot analysis was performed for Cx43 and GAPDH on right ventricular proteins of control mice (Cx43^fl/fl), control mice treated with 4-hydroxytamoxifen (Cx43^{fl/fl + 4-OHT}), and conditional Cx43-knockout mice (Cx43^{Cre-ER(T)/fl + 4-OHT}), respectively. (B) Quantification of the Cx43 expression normalized to that of GAPDH. *P < 0.05 versus Cx43^fl/fl. Basal and ADP-stimulated respiration were measured using substrates for complex I (C) or complex II (D), respectively, on LV SSM of Cx43^fl/fl, Cx43^{fl/fl + 4-OHT} and Cx43^{Cre-ER(T)/fl + 4-OHT} mice, respectively. *P < 0.05 versus Cx43^fl/fl.

Fig 4

Cx43-overexpression in mitochondria. (A) Western blot analysis was performed for Cx43 and the 70 kD subunit of complex II (CII) as mitochondrial marker protein on mitochondria isolated from HL-1 cells, which were transfected with the empty vector pBabe-puro or the Cx43-overexpressing vector pBabe-Cx43, respectively (n = 5). (B) Oxygen consumption was measured using substrates for complex I (cells in suspension, left panel) or substrates for complex II (isolated mitochondria, right panel) from HL-1 cells transfected with the empty vector pBabe-puro or the Cx43 overexpressing vector pBabe-Cx43, respectively. *P < 0.05.