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 (Cx43Cre-ER(T)/fl + 4-OHT, 5–10% of Cx43 protein compared with control Cx43fl/fl mitochondria) reduced ADP-stimulated complex I respiration of LV SSM to 47.8 ± 2.4 nmol O2/min.*mg protein (n = 8) from 61.9 ± 7.4 nmol O2/min.*mg protein in Cx43fl/fl mitochondria (n = 10, P < 0.05), while complex II respiration remained unchanged. The LV complex I activities (% of citrate synthase activity) of Cx43Cre-ER(T)/fl+4-OHT mice (16.1 ± 0.9%, n = 9) were lower than in Cx43fl/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.


Introduction
The transmembrane protein Cx43 is constitutive for the formation of gap junctions between cardiomyocytes and thus essential for cell-cell communication. Connexin 43 is also present at the inner membrane of cardiomyocyte SSM, but not in cardiomyocyte interfibrillar mitochondria (IFM) [1][2][3]. The cardioprotection by ischaemic pre-condi-tioning or pharmacological pre-conditioning with diazoxide, an opener of mitochondrial ATP-dependent potassium channels (mito K ATPchannels) [4], depends on the presence of Cx43 [5][6][7][8]. In murine mitochondria in which Cx43 was replaced by connexin 32 (which forms hemichannels with lower potassium conductance than Cx43 [9]), mitochondrial potassium influx is reduced [10]. Potassium uptake of mitochondria from wild-type mice is also decreased by the gap junction blocker 18a-glycyrrhetinic acid (18aGA) [10]. As mitochondrial potassium flux is involved in respiratory control of mitochondria [11,12], we now investigated the role of Cx43 for mitochondrial oxygen consumption and respiratory chain complex activities by its inhibition, reduction, or overexpression, respectively.

Animal model
The present study was approved by the Bioethical Committee of the state Nordrhein-Westfalen, Germany. It conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996).
A conditional knockout of Cx43 was achieved by intraperitoneal injection of 3 mg 4-hydroxytamoxifen (4-OHT) once per day on five consecutive days in Cx43 Cre-ER(T)/fl mice. Untreated Cx43 fl/fl and 4-OHT-treated (Cx43 fl/fl + 4-OHT) mice served as controls. The mice were 10-to 16-weeks old and sacrificed on day 11 after the first injection. The right ventricles were used to control the reduction of Cx43 by Western blot analysis. The decreased amount of Cx43 in total right ventricular protein extracts is paralleled by a reduction in mitochondrial Cx43 [1]. Left ventricles (LV) were used for the isolation of mitochondria. Lewis rats (250-350 g) were anaesthetized with enflurane, the hearts were removed, and the LV were used for isolation of mitochondria.
Overexpression of Cx43 in HL-1 cardiomyocytes HL-1 cells, a cell line derived from mouse atrial cardiomyocytes, were cultured under 5% CO 2 atmosphere in Claycomb Medium supplemented with 10% foetal bovine serum (JRH Biosciences, Lenexa, KS, USA), 4 mmol/l L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomycin and 100 lmol/l norepinephrine, and were plated in culture flasks pre-coated with 25 lg/ml fibronectin/0.02% gelatin solution. To obtain HL-1 cells overexpressing Cx43, the coding sequence of rat Cx43 was inserted into a pBABEpuro retroviral plasmid under the control of the retroviral promoter (pBABEpuro-Cx43). Empty vector was used as control. To produce retroviral particles with the VSV-G virus envelope, which do not require any specific receptor in the target cells, the 293GPG packing cell line was transfected. After 48 hrs, supernatant rich in viral particles was collected and added to HL-1  Fig. 1 Effect of acute Cx43-inhibition using 18aGA 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 18aGA (1 lmol/l: n = 7, 10 lmol/l: n = 6, 100 lmol/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 18aGA (1 lmol/l: n = 7, 10 lmol/l: n = 6, 100 lmol/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 lmol/l 18aGA or DMSO as vehicle, respectively. *P < 0.05 versus before 18aGA. cells. HL-1 cardiomyocytes were allowed to grow for additional 48 hrs before starting the selection with puromycin at 2 lg/ml (P8833; Sigma-Aldrich, St. Loius, MO, USA). Overexpression of Cx43 was confirmed by Western blot analysis on isolated mitochondria.

Isolation of mitochondria
Subsarcolemmal mitochondria were isolated from tissue samples of mouse and rat LV by differential centrifugation as described previously [1].
For isolation of both SSM and IFM, rat LV tissue was processed as already described [3].

Mitochondrial oxygen consumption
Mitochondrial respiration of mouse or rat LV SSM was measured with a Clark-type oxygen electrode (Oxygen meter 782, Strathkelvin, Glasgow, UK) at 25°C in incubation buffer (containing in mmol/l: 125 KCl, 10 Tris-MOPS, 1.2 Pi-Tris, 1.2 MgCl 2 , 0.02 EGTA-Tris, pH 7.4) as described previously [13]. Five mmol/l glutamate and 2.5 mmol/l malate were used as substrates for complex I, whereas complex II-mediated respiration was measured in the presence of 2 lmol/l rotenone (inhibits complex I) and 5 mmol/l succinate.
Oxygen consumption was determined for 100 lg LV mouse SSM or rat SSM incubated for 30 min. at 4°C in isolation buffer, without or with 250 lmol/l Cx43-mimetic peptides (SRPTEKTIFII) or Cx40-mimetic peptides (SRPTEKNVFIV). This concentration of mimetic peptides (MP) has been demonstrated to block Cx43-formed hemichannels [14]. Cx40-MP was chosen as control, since the amino acids are similar to that of the Cx43-MP and Cx40 is present in atrial but not in ventricular mitochondria [15]. After recording of basal oxygen consumption, respiration was stimulated by the addition of 40 lmol/l ADP.
One-hundred micrograms rat LV SSM proteins were added to incubation buffer supplemented with 200 lmol/l ADP. One lmol/l, then either 10 lmol/l 18aGA, which inhibits gap junctional cell communication, or the respective volume of DMSO as vehicle was added after 5 min., and the ADP-stimulated respiration was recorded for further 5 min. Oxygen consumption was quantified 1 min. before and 1 min. after addition of 18aGA or DMSO, respectively. Mitochondrial respiration was calculated as the oxygen consumption in nmol O 2 /min.*mg protein. In HL-1 cardiomyocytes, complex I-mediated respiration was measured at 25°C using intact cells (200,000-250,000 cells/ml) resuspended in cell-assay buffer (in mmol/l: NaCl 140, KCl 3.6, MgSO 4 1.2, CaCl 2 2, HEPES 20 pH 7.4, glucose 5 mmol/l). Oxygen consumption was expressed as nmol O 2 /min.*10 6 cells. Complex II-mediated respiration was measured in isolated mitochondria at 25°C in incubation buffer, using rotenone and succinate as substrate, as described for mouse and rat mitochondria.

Mitochondrial ATP generation
Fifty lg rat LV SSM were diluted in 100 ll incubation buffer supplemented with 5 mmol/l glutamate and 2.5 mmol/l malate as substrates for complex I and 0.1 mmol/l di-adenosine-pentaphosphate. Considering the lack of effect of Cx43-MP on complex II respiration, ATP generation was measured using substrates for complex I only. One-hundred microlitres of the ATP bioluminescent assay kit (1:5 diluted with incubation buffer; Sigma-Aldrich) was added and the bioluminescence was recorded for 1 min. with a Cary Eclipse spectrophotometer (Varian) at room temperature. ATP generation was initiated by the addition of 500 mmol/l ADP, and the bioluminescence was recorded for another minute. The bioluminescence of a sample without mitochondria was subtracted. Rat LV SSM were studied under control conditions (untreated) or after incubation for 30 min. at 4°C with 250 lmol/l Cx43-MP (n = 15). In addition, ATP generation was measured in the presence of 1 lmol/l 18aGA or DMSO as vehicle (n = 4). Twenty-five lg/ml oligomycin was used to inhibit the ATP synthase and to prevent ATP generation.

Activity of respiratory chain complexes
The LV of Cx43 fl/fl (n = 8) and Cx43 Cre-ER(T)/fl + 4-OHT mice (n = 9) were homogenized in a solution containing 50 mmol/l Tris (pH 7.5), 100 mmol/l potassium chloride, 5 mmol/l MgCl 2 , and 1 mmol/l ethylenediaminetetraacetic acid. Enzyme activities were spectrophotometrically monitored at 30°C (DU 640 photometer; Beckmann Instruments, Palo Alto, CA, USA) and normalized to citrate synthase activity. The analysis of the activity of mitochondrial respiratory chain complex I, complex II and citrate synthase as mitochondrial marker enzyme was performed as described previously [16].

Statistics
Data are reported as mean ± S.E.M. Oxygen consumption in HL-1 cells, basal respiration with 10 lM 18aGA, and activities of respiratory complexes were compared using unpaired Student's t-test. Western blot data for Cx43, mitochondrial membrane potential and mitochondrial autofluorescence were compared by one-way ANOVA and Fisher's least-significant difference tests were used for post-hoc comparisons. Oxygen consumption without or with ADP-stimulation and without or with 1 lmol/l 18aGA or mimetic peptides, respectively, was compared by two-way ANOVA with repeated measures followed by Bonferroni tests. A P < 0.05 was considered to indicate a significant difference.
ATP generation of rat LV SSM treated with 250 lmol/l Cx43-MP was reduced to 92.7 ± 1.9% of untreated mitochondria (n = 15, Fig. 2B and C). In the presence of oligomycin, which inhibits the mitochondrial ATP synthase, almost no ATP was produced (Fig. 2B). 4-OHT reduced the Cx43 content to about 10% in Cx43 Cre-ER(T)/fl mice, but did not influence Cx43 expression in Cx43 fl/fl mice ( Fig. 3A and B). ADP-stimulated oxygen consumption with substrates for complexes I or II ( Fig. 3C and D) was unaffected by 4-OHT in Cx43 fl/fl mice, but ADP-stimulated complex I respiration was lower in Cx43 Cre-ER(T)/fl + 4-OHT than in Cx43 fl/fl SSM; ADP-stimulated complex II respiration was comparable between groups. Basal respiration (complexes I and II) was not modified by Cx43-deficiency ( Fig. 3C  and D).

Discussion
The present study demonstrates that mitochondrial Cx43 impacts on ADP-stimulated complex I respiration and ATP production.
The effects of Cx43 overexpression as well as acute and chronic Cx43 inhibition/reduction on mitochondrial oxygen consumption were investigated. In isolated mitochondria from HL-1 cells, complex I respiration was below the detection level and was therefore measured in intact cells only. Here, overexpression of Cx43 in HL-1 cardiomyocytes increased complex I respiration compared with control-transfected cells. Cx43 overexpression had no effect on complex II respiration. As glucose, which was used as substrate in the experiments with intact cells, does not exclusively activate complex I, it is not possible to attribute the effects of Cx43-overexpression to enhanced activity of this complex only. However, the finding that Cx43-overexpression had no impact on complex II respiration in isolated mitochondria supports the role of mitochondrial Cx43 for complex I-driven oxygen consumption.
In isolated rat SSM, 1 lmol/l 18aGA, a concentration without toxic effects, reduced ADP-stimulated complex I, but not complex II respiration; higher concentrations of 18aGA (10 and 100 lmol/l) damaged mitochondria by inducing loss of membrane potential and NAD(P)H autofluorescence and by uncoupling basal respiration. Ten lmol/l of the 18aGA isomer 18bGA has previously been reported to induce mitochondrial transition pore opening in rat heart mitochondria [17].
The effects of 18aGA were more pronounced than those measured with Cx43-MP. Apart from blocking Cx43-mediated effects, 18aGA may react directly with a subunit of complex I [17], explaining its greater potency.
Cx43-MP block Cx43-formed hemichannels and gap junctions [14]. While Cx40 and Cx43 are both expressed in endothelial cells, only Cx43 is present in ventricular cardiomyocytes and in isolated ventricular mitochondria [15]. Cx40 is present in atrial cardiomyocytes and was detected in atrial mitochondria as well [15]. Thus, any effect observed with Cx40-MP served as control for non-cardiomyo-cyte mitochondria isolated from LV myocardium, and indeed, the Cx40-MP decreased mitochondrial respiration slightly. While the localization of Cx40 in mitochondria from endothelial cells has not been demonstrated yet, the putative presence of Cx40 in these organelles may be responsible for the reduced oxygen consumption induced by Cx40-MP. Importantly, the reduction of complex I respiration, but not complex II respiration, by Cx43-MP was significantly greater than by Cx40-MP in SSM (containing Cx43) but not IFM (lacking Cx43), highlighting the specificity of the observed effect.
To investigate the effect of a more chronic Cx43 reduction on respiration, we studied the oxygen consumption of mitochondria from conditional Cx43-knockout mice and found reduced ADP-stimulated complex I respiration. It is possible that Cx43-deficiency affects the composition and hence activity of complex I, as the Cx43-carboxyterminus has also been detected in cardiomyocyte nuclei [18] and Cx43deficient hearts have decreased transcript levels of 11 nuclear genes encoding subunits of complex I, whereas mRNAs encoding complex II subunits are not differentially expressed [19]. Of note, specifically complex I activity was reduced in Cx43-knockout mice. However, as not only chronic reduction but also acute inhibition of Cx43 reduced complex I respiration, Cx43 may directly affect complex I by interfering with complex I subunits. However, co-immunoprecipitation studies did not detect an interaction of subunits of complex I with Cx43 (data not shown).
In conclusion, the present study demonstrates the importance of mitochondrial Cx43 for oxygen consumption and ATP production. Inhibition or reduction of mitochondrial Cx43 specifically decreases complex I respiration.