All rodents were treated according 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) and approved by the Institutional Laboratory Animal Care and Use Committee of the NIH, Bethesda, MD, USA or according to the European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Council of Europe Treaty Series No. 123). For Langendorff heart perfusion experiments male Sprague–Dawley rats (190–210 g and 7–8 weeks old) were anesthetized with pentobarbital and anti-coagulated with heparin. Male Wistar–Janvier rats (190–210 g and 8–10 weeks old) were used for mitochondrial permeability experiments and were anesthetized with 2.5 % v/v isoflurane. Male C57BL/6J mice, 11–14 weeks of age with a body weight ranging from 25 to 35 g, were anesthetized by intraperitoneal (i.p.) injection of ketamine (45 mg/kg) and xylazine (Rompun, 10 mg/kg).

Excised rat hearts were placed in ice-cold Krebs-Henseleit buffer (120 mmol NaCl, 11 mmol d-glucose, 25 mmol NaHCO₃, 1.75 mmol CaCl₂, 4.7 mmol KCl, 1.2 mmol MgSO₄, and 1.2 mmol KH₂PO₄) and the hearts were Langendorff perfused in retrograde fashion with oxygenated Krebs–Henseleit buffer (95 % O₂/5 % CO₂, pH 7.4) at a constant pressure of 100 cm water at 37 °C. Hearts were randomly assigned to either a control group perfused for 40 min under normoxic conditions or an ischemic preconditioned (IPC) group where the hearts were perfused for 20 min under normoxic conditions followed by four cycles of 5 min ischemia and 5 min reperfusion [35, 69]. Perfusion was performed in the dark to prevent breakdown of SNO modifications. Control experiments were also performed by daylight confirming light sensitivity of SNO modifications.

C57BL/6 mice were anesthetized by intraperitoneal injection of ketamine (45 mg/kg) and xylazine (Rompun 10 mg/kg). A tracheal tube was inserted for mechanical ventilation, which was performed according to the individual body weight at a tidal volume of 2.1–2.5 ml and a respiratory rate of 140 breaths per minute. The mice were supplemented with 100 % oxygen via a rodent ventilator (Minivent) side port. Pharmacological preconditioning was induced by injection of 48 nmol sodium nitrite into the cavity of the left ventricle, whereas the control group received an equal volume of 50 µl physiological saline. The mouse chests were opened through a midline sternotomy. Subsequent mouse hearts were excised, mitochondria were isolated, and SNO of mtCx43 was quantified by using the modified biotin switch method. For details of the preparation as well as the extent of infarct size reduction achieved by this protocol see Rassaf et al. [46].

All procedures were performed at 4 °C to maintain mitochondrial integrity. To prevent light-induced SNO breakdown, samples were kept in the dark during the isolation process. SSM were isolated from left ventricles of male rats as described previously [1]. For functional analysis, left rat ventricles were minced in isolation buffer containing 250 mmol sucrose, 10 mmol HEPES, 1 mmol EDTA, and 0.5 % BSA (pH 7.4 adjusted with Tris base), homogenized with an Ultra Turrax and centrifuged at 800g for 10 min. The resulting supernatant was centrifuged for 10 min at 10,780g and the pellet was resuspended in BSA-free isolation buffer and centrifuged twice at 7,650g for 5 min.

Interfibrillar mitochondria (IFM), which lack Cx43, were used as negative controls and isolated as previously described [5]. Minced rat left ventricles were weighed, homogenized and centrifuged at 800g for 10 min. The resulting pellet was resuspended in 5 ml isolation buffer additional containing 0.5 % BSA and Nargase (8 U/g) and incubated on ice for 1 min. The tissue was homogenized and centrifuged at 800g for 10 min and the supernatant was centrifuged for 10 min at 10,780g. The resulting pellet was resuspended in BSA free isolation buffer and centrifuged twice at 7,650g for 5 min.

For quantitative SNO analysis, a BSA-free isolation buffer with SNO modifications protecting agents was used containing 250 mmol sucrose, 10 mmol HEPES, 1 mmol EDTA, TRIS-base (pH 7.4), 0.1 mmol neocuproine, EDTA free complete protease inhibitor (Roche Diagnostics, Grenzach, Germany), and phosphatase inhibitor (Roche Diagnostics, Grenzach, Germany). The mitochondria were isolated as described above and then layered on a 30 % Percoll gradient and centrifuged for 30 min at 35,000g. This resulted in a lower fraction containing pure SSM and an upper fraction containing cell debris. The lower fraction was collected and washed three times with isolation buffer by centrifuged at 10,200g for 5 min.

Protein concentrations were determined by the Lowry assay using BSA as a standard. For quantitative analysis, the purity of isolated mitochondria was validated by Western blot analysis confirming the absence of non-mitochondrial cellular proteins and the enrichment of mitochondrial proteins. Mitochondria were stored at −80 °C.

Dye permeation experiments were performed according to Miro-Casas et al. [36]. Freshly isolated mitochondria were pelleted by centrifugation at 10,200g for 3 min at 4 °C and resuspended at a concentration of 400 µg/ml in isosmotic succinate buffer (150 mmol KCl, 7 mmol NaCl, 2 mmol KH₂PO₄, 1 mmol MgCl₂, 6 mmol MOPS, pH 7.2, 6 mmol succinate, 0.25 ADP, and 0.5 µmol rotenone). Mitochondria were assigned to six groups and were either supplemented with 1 or 25 µmol of the hemichannel blocker carbenoxolone (CBX; Sigma-Aldrich, Heidenheim, Germany), 0.5 mmol NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP; Life Technologies, Darmstadt, Germany), 1 mmol of NO donor S-nitrosoglutathione (GSNO; Santa Cruz, Heidelberg, Germany), a combination of NO donor and CBX, or 5 µl dimethyl sulfoxide (DMSO; used as solvent, Sigma-Aldrich, Heidenheim, Germany). Additional mitochondria from Langendorff perfused hearts receiving either IPC or control perfusion were assigned to dye permeation experiments. As negative control, mitochondria were exposed to UV light for 5 min to remove light sensitive SNO modifications.

After a 20 min incubation period at 25 °C and 650 rpm, 50 µmol/l of the Cx43 hemichannel-permeable dye Lucifer Yellow CH dilithium salt (LY; Sigma-Aldrich, Heidenheim, Germany) and 25 µg/ml of the hemichannel-impermeable dye Rhodamine B isothicyanate-dextran 10S (RITC-dextran; Sigma-Aldrich, Heidenheim, Germany) were added and samples were incubated for 25 min at 25 °C and 650 rpm. Subsequently, mitochondria were washed and resuspended in 200 µl succinate buffer. Fluorescence of LY (λ _ex 430 nm, λ _em 535 nm) and RITC-dextran (λ _ex 545 nm, λ _em 600 nm) was measured by a 96-microplate fluorometer (Cary Eclipse spectrophotometer, Varian, Mulgrave, Australia) at high sensitivity [36]. As a negative control, experiments were performed with ultrasound treated (20 s, amplitude 50 %) mitochondria to exclude interference of dye and membrane fragments, as well as with IFM to confirm Cx43 hemichannel-specific effects.

Experiments for measuring velocity of mitochondrial K⁺ influx were modified from Miro-Casas et al. [36]. Freshly isolated mitochondria were resuspended at a concentration of 400 µg/ml in isolation buffer. Either 1, 10, or 25 µmol CBX, 0.5 mmol SNAP, the combination of both, or 5 µl DMSO was added to mitochondria and incubated for 20 min at 25 °C and mixed at 650 rpm. The experiments were repeated with the use of 1 mmol physiologically relevant NO donor GSNO instead of SNAP. Subsequently, mitochondria were loaded with 10 µmol/l acetoxymethyl of potassium-binding benzofuran isophthalate (PBFI; Life Technologies, Darmstadt, Germany) for 10 min at 25 °C and 650 rpm. K⁺ depletion from the mitochondrial matrix was achieved by adding 3 volumes of tetraethylammonium (TEA) buffer (120 mmol TEA-Cl, 10 mmol HEPES, 10 mmol succinate, 5 mmol Na₂HPO₄, 0.1 mmol EGTA, 0.5 mmol MgCl₂, 5 µmol rotenone, and 0.67 µmol oligomycin, pH 7.2), which replaces K⁺ in the mitochondrial matrix. Subsequently, mitochondria were washed, sedimented at 10,200g and 4 °C for 3 min, and resuspended in 30 µl isolation buffer. The kinetics of mitochondrial K⁺ uptake were measured after a KCl pulse of 140 mmol at alternated excitations at 340/380 nm and emission at 500 nm by a fluorometer (Cary Eclipse spectrophometer; Varian, Mulgrave, Australia) in 2 ml isolation buffer at medium sensitivity. To measure the K⁺ permeability for the Cx43 hemichannel only, during measurements, mitochondrial permeability transition pore (MPTP) opening was blocked by adding 1 µmol cyclosporine A (CsA; Sigma-Aldrich, Heidenheim, Germany) [22], the proton channel of the ATP-synthase was blocked with 1 µg/ml oligomycin (Sigma-Aldrich, Heidenheim, Germany), and opening of ATP-dependent potassium channels was blocked by 5 µmol glibenclamide (Sigma-Aldrich, Heidenheim, Germany) [62, 72]. The experiments were repeated without these inhibitors to investigate the influence of NO donors on the K⁺ influx under more physiological conditions. The K⁺ influx, the first 2 s after the KCl-pulse, was determined by the increase of the PBFI fluorescence ratio of 340/380 nm per second of the six different treated groups. As a positive control, K⁺ influx was determined after addition of 5 nmol valinomycin, which is highly sensitive to sodium (Na⁺) and K⁺ and functions as a K⁺-specific transporter across membranes. To confirm a Cx43 specific effect, experiments were also performed with IFM instead of SSM as Cx43 negative controls.

Mitochondria were treated either with 25 µmol CBX, 0.5 mmol SNAP, 1 mmol GSNO, the combination of NO donor and CBX, or 5 µl DMSO and then loaded with 10 µmol/l acetoxymethyl of sodium-binding benzofur (SBFI; Life Technologies, Darmstadt, Germany) instead of using PBFI. Sodium depletion was then performed with tetramethylammonium (TMA) buffer (120 mmol TMA-Cl, 10 mmol HEPES, 10 mmol succinate, 5 mmol KH₂PO₄, 0.1 mmol EGTA, 0.5 mmol MgCl₂, 5 µmol rotenone, and 0.67 µmol oligomycin, pH 7.2). After subsequent washing, the kinetics of mitochondrial Na⁺ uptake were measured after a NaCl pulse of 10 and 140 mmol at alternated excitations at 340/380 nm and emission at 500 nm by a fluorometer (Cary Eclipse spectrophotometer; Varian, Mulgrave, Australia). This was performed in 2 ml isolation buffer as well as in 2 ml succinate buffer supplemented with inhibitors for MPTP, ATP-synthase, and ATP-dependent potassium channels as described above at high sensitivity. Na⁺ influx of the first 2 s after the NaCl-pulses was determined by the increase of the SBFI fluorescence ratio of 340/380 nm per second of the six different treated groups. As a positive control, Na⁺ influx was determined after addition of 5 nmol gramicidin, which has a similar function as valinomycin and forms a channel for K⁺ and Na⁺, but does not interfere with SBFI dye as does valinomycin.

One mg of freshly isolated SSM or IFM were added to 2 ml incubation buffer (150 mmol KCl, 7 mmol NaCl, 2 mmol KH₂PO₄, 1 mmol MgCl₂, and 6 mmol MOPS, pH 7.4) containing 5 mmol glutamate and 2.5 mmol malate as a substrate, 1 U/ml horseradish peroxidase (HRP; Roche Diagnostics, Grenzach, Germany), and 50 µmol Amplex UltraRed reagent (Thermo Fisher Scientific Inc., Darmstadt, Germany). Amplex UltraRed is a fluorogenic substrate for HRP that reacts with hydrogen peroxide (H₂O₂). For estimating the NO-mediated influence of mtCx43-dependent ROS production either 1 or 25 µmol CBX, 0.5 mmol SNAP, 1 mmol, 1 µmol, or 48 nmol GSNO, a combination of NO donor and CBX, or 20 µl dH₂O was added 30 s before the measurement was started. In previous studies, NO donors—like SNAP or GSNO—were given at concentrations from 10 μmol to 1 mmol. Therefore, in the present study we used mmol concentrations but lowered them down to nmol concentrations. Additional experiments were performed with Cx43 inhibition by the connexin mimetic peptide Gap26 [12] either in the presence of 1 mmol GSNO or not. Experiments were repeated under respiratory chain uncoupling conditions induced by 30 nmol carbonyl cyanide p-(tri-fluromethoxy)phenyl-hydrazone (FCCP) and with 2 µmol of the respiratory chain complex 1 inhibitor rotenone. Analysis of ROS formation was performed by a fluorometer (Cary Eclipse spectrophometer; Varian, Mulgrave, Australia) at an extinction and emission wavelength of 568/581 nm. Mitochondrial H₂O₂ production was measured for 4 min and the increase of H₂O₂ was expressed in nmol/min/mg protein by comparing the data to a standard curve. After 4 min of measurement, 2 µg/ml of mitochondrial complex III inhibitor antimycin A (Sigma-Aldrich, Heidenheim, Germany) was added for inducing mitochondrial ROS overproduction, which served as a positive control.

A modified biotin switch method was used for labeling and quantification of SNO residues as previously described [27]. SSM protein samples (250 µg) and controls (250 µg IFM, and 250 µg right ventricle of rodent hearts) were diluted in HEN buffer (250 mmol HEPES-NaOH pH 7.7, 1 mmol EDTA, and 0.1 mmol neocuproine), supplemented with EDTA free complete protease inhibitor tablet (Roche Diagnostics, Indianapolis, IN, USA), phosphatase inhibitor (Roche Diagnostics, Grenzach, Germany or Indianapolis, IN, USA) and 2.5 % SDS (wt/vol). To block free thiols, 50 mmol N-ethylmaleimide (NEM; Sigma-Aldrich, St. Louis, MO, USA or Heidenheim, Germany) was used. After incubation for 20 min at 50 °C with gentle mixing every 5 min, free thiols were labeled with NEM and could not be modified. This procedure was stopped by removing NEM via cold acetone precipitation (−20 °C). The samples were then resuspended in HEN buffer with 1 % SDS (wt/vol) containing 1 mmol ascorbate (Sigma-Aldrich, St. Louis, MO, USA) for reduction of SNO modified cysteine residues. Reduced SNO groups were labeled with N-(biotinoyl)-N-(iodoacetyl)ethylenediamine (BIAM; Sigma-Aldrich, St. Louis, MO, USA or Heidenheim, Germany). Prior to incubating samples with streptavidin-agarose beads (Sigma-Aldrich, St. Louis, MO, USA) for precipitation of SNO modified proteins, 2 µl of loading control was taken. Precipitation was performed overnight with rotation at 4 °C in the dark. Samples were washed three times with HEN buffer, eluted in 30 µl sample buffer with 10 M urea and heated at 95 °C for 5 min. Specificity of the biotin switch method was proven by adding 10 or 100 mmol DTT before eluting the sample, which breaks the disulfide bound between the thiol group and biotin. Western blot analysis was subsequently performed.

The influence of NO donors on the phosphorylation status of mtCx43 was analyzed. Mitochondria were incubated either with 1 µmol CBX, 0.5 mmol SNAP, 1 mmol GSNO, the combination of a NO donor and CBX, or 5 µl DMSO in isolation buffer. Subsequent mitochondrial purity was achieved by Percoll gradient centrifugation as described above. Phosphorylation of Cx43 was then analyzed and quantified by using a phospho-Cx43 specific antibody and Western Blot analyses.

The following antibodies were used in this study: rabbit polyclonal anti-rat total connexin43 (Invitrogen, Carlsbad, CA, USA), mouse monoclonal anti-rat sodium/potassium (Na⁺/K⁺)-ATPase (Upstate, Waltham, MA, USA), mouse monoclonal anti-dog sarcoplasmic calcium (SERCA2)-ATPase (Sigma-Aldrich, St. Louis, MO, USA), rabbit monoclonal anti-human histone deacetylase 2 (HDAC2; Abcam, Cambridge, UK), mouse monoclonal anti-rabbit GAPDH (Hytest, Turku, Finland), rabbit polyclonal anti-human translocase of the outer membrane 20 (Tom20; Santa Cruz, CA, USA), rabbit polyclonal anti-human voltage-dependent anion channel (VDAC) (Abcam, Cambridge, UK), and rabbit polyclonal anti-human manganese superoxide dismutase (MnSOD, Upstate, Lake Placid, NY, USA). The phospho-Cx43 antibody Cx43-pS368 (#3511, Cell Signaling, Leiden, The Netherlands) was used for analyses of Cx43 phosphorylation.

Data are presented as mean ± SEM. Normal distribution of data was analyzed using a non-parametric Kolmogorov–Smirnov test. Unpaired Student’s t test was used between the two groups of IPC and control mitochondria of Western blot data and dye permeation data to determine differences in mean values. Data of LY, K⁺ uptake and ROS formation were compared by two-way repeated measures ANOVA and Fisher’s LSD. Statistical significance was determined at p < 0.05.

Rat mitochondria were treated separately either with 5 µl DMSO, 1 µmol or 25 µmol carbenoxolone (CBX), 0.5 mmol SNAP, 1 mmol GSNO, or a combination of a NO donor and CBX. Fluorescence analyses were performed after exposure to 50 µmol LY and 25 µg/ml RITC-dextran (Fig. 1a). Mitochondrial LY fluorescence intensity was significantly increased after application of NO donor SNAP compared to DMSO control by 38.4 ± 7.1 % (n = 12, p < 0.05). The NO-mediated increased mitochondrial permeability for LY was verified by application of NO donor GSNO instead of SNAP. GSNO application increased LY uptake by 28.1 ± 7.4 % (n = 12, p < 0.05) compared to control. The NO-mediated increase in LY fluorescence was abolished in SSM incubated with the Cx43 hemichannel blocker, CBX. CBX at a concentration of 1 µmol decreased LY uptake by 17.7 ± 0.8 % compared to DMSO control (n = 12, p < 0.05), by 36.7 ± 4 % (SNAP + CBX versus SNAP; n = 12, p < 0.05) compared to SNAP, and by 33.4 ± 5.6 % (GSNO + CBX versus GSNO; n = 12, p < 0.05) compared to GSNO-treated mitochondria. Increasing the CBX concentration up to 25 µmol had a similar effect on mitochondrial permeability. On the other hand, the fluorescence intensity of the hemichannel impermeant dye RITC-dextran showed no difference throughout the different protocols (n = 12, p = ns) (Fig. 1b).

Additionally, the experiments were repeated with IFM, a population of mitochondria which do not contain Cx43, to validate that the effect of increased permeability caused by SNO is specific for Cx43 hemichannels containing mitochondria. IFM did not show a difference in dye uptake between all treatment groups (n = 7, p = ns) (Fig. 1c). Cross reactions of LY with membrane fragments were excluded by repeating the experiments following ultrasound treatment, to rupture the mitochondria. These studies with ultrasound showed no significant alteration in LY uptake by any of the treatments (Supplemental Figure 1, n = 7, p = ns). Furthermore, fluorescence intensity of RITC-dextran did not differ with the different protocols (Supplemental Figure 1, n = 7, p = ns).

The permeability for LY and RITC-dextran was also analyzed in preconditioned mitochondria and mitochondria from control perfused rat hearts. The mitochondria from IPC rat hearts had a 13.1 ± 4.6 % (Fig. 2, left panel, n = 4, p < 0.05) higher LY dye uptake compared to mitochondria from control perfused hearts. However, the RITC-dextran fluorescence intensity did not differ between the groups (n = 4, p < 0.05). In addition, after exposure to UV light there was no detectable difference in dye uptake between IPC and control mitochondria (Fig. 2, right panel, n = 4, p < 0.05).

Mitochondria were incubated separately either with 1, 10, or 25 µmol CBX, 0.5 mmol SNAP, 1 mmol GSNO, a combination of NO donor and CBX, or 5 µl DMSO used as a vehicle. The velocity of K⁺ influx into the matrix of mitochondria from rat left ventricles was estimated by measuring the increase of the 340/380 nm fluorescence intensity ratio for 2 s after addition of a 140 mmol KCl pulse. To measure the K⁺ permeability through Cx43 hemichannels, other mitochondrial channels including ATP-dependent potassium channels, ATP-synthase, and MPTP were blocked with glibenclamide, oligomycin, and cyclosporine A. Mitochondria treated with NO donors showed a significant higher refilling rate of K⁺. The velocity of K⁺ uptake was 227.9 ± 30.1 % higher in SNAP-treated mitochondria compared to DMSO control (n = 10, p < 0.05). Application of the NO donor GSNO increased the velocity of K⁺ influx by 122.6 ± 28.1 % (n = 7, p < 0.05) compared to control. The NO-mediated increases of mitochondrial K⁺ influx by SNAP and GSNO were significantly blocked by CBX at a concentration of 1 µmol. With CBX application, velocity of K⁺ influx decreased by 153.2 ± 24.4 % (n = 17, p < 0.05) compared to DMSO control, decreased by 112.1 ± 4.5 % in SNAP (SNAP + CBX versus SNAP; n = 7, p < 0.05), and decreased by 170 ± 4.3 % (GSNO + CBX versus GSNO; n = 7, p < 0.05) compared to GSNO treated mitochondria (Fig. 3a). Again, IFM did not show alterations of K⁺ influx in response to NO donors or CBX (n = 7, p = ns) (Fig. 3b).

SNO-mediated K⁺ influx through Cx43 hemichannels was also measured without additional mitochondrial channel blockers. The data showed a NO-mediated increase of K⁺ influx, which was somewhat weaker with SNAP versus DMSO (138.9 ± 30.7 %, n = 7, p < 0.05) compared with SNAP versus DMSO with additional mitochondrial channel blockers. GSNO also increased K⁺ permeability compared to control (119.7 ± 16 %, n = 7, p < 0.05) as was the case in experiments where the K⁺ permeability for Cx43 hemichannels was measured in the presence of inhibitors. The NO-mediated increase of K⁺ influx was blocked by CBX (154.6 ± 10.1 % for 0.5 mmol SNAP with 1 µmol CBX and 103.8 ± 25.2 % for 1 mmol GSNO with 1 µmol CBX; n = 7, p < 0.05) (Fig. 4).

Permeability for Na⁺ was measured using the Na⁺ binding benzoflur SBFI. Only small, non-significant changes in Na⁺ fluxes were measured (Supplemental Figure 2, n = 6, p = ns) suggesting that mitochondrial Cx43 hemichannels have certain ion selectivity. Experimental set up function was proven by application of gramicidin, which forms a channel for Na⁺ in the mitochondrial membrane. Application of gramicidin leads to Na⁺ influx, which did not differ between groups.

Studies suggest that endothelial NOS docks to the mitochondrial outer membrane producing local NO [16]. Therefore, whether or not SNO of mitochondrial Cx43 influences mitochondrial ROS formation is of interest. ROS production of mitochondria was analyzed using 1, or 25 µmol CBX, Gap26, 0.5 mmol SNAP, 1 mmol, 1 µmol, or 50 nmol GSNO, a combination of a NO donor and CBX or Gap26, or 20 µl dH₂O. During ADP stimulated complex 1 respiration, ROS production was increased by application of the NO donor SNAP (22.9 ± 1.8 %, n = 9, p < 0.05) compared to control and by using GSNO (40.6 ± 7.1 %, n = 9, p < 0.05) compared to control. These increases were abolished by 25 µmol CBX (Fig. 5a). Concentrations of 1 µmol and 48 nmol GSNO increased ROS production by 20 ± 3.7 % (n = 16, p < 0.05) and 14.3 ± 2.8 % (n = 13, p < 0.05) respectively compared to control (Fig. 5b). The Cx43 mimetic peptide, Gap26 is known to inhibit GJs and non-junctional hemichannels [79]. Application of the Cx43 mimetic peptide Gap26 decreased ROS formation by 16.5 ± 2.6 % (n = 19, p < 0.05) compared to GSNO treated mitochondria, but ROS formation did not differ compared to control treated mitochondria (Fig. 5c). In IFM, the NO donors decreased ROS production. SNAP decreased ROS formation (14.4 ± 4 %, n = 9, p < 0.05) compared to control as did GSNO (13.8 ± 4 %, n = 9, p < 0.05) compared to control. CBX had no significant inhibitory effect on ROS production with ADP stimulated complex 1 respiration (n = 9, p = ns) (Fig. 5d).

Additionally, ROS measurements were performed using the respiratory chain uncoupling agent FCCP and the respiratory chain inhibitor rotenone. FCCP significantly decreased ROS formation by 45.2 ± 3.9 % (control vs. control FCCP; n = 10, p < 0.05), whereas ROS generation did not differ between the different treatment groups (Fig. 5e, f). In contrast, the respiratory chain complex 1 inhibitor rotenone increased ROS formation by 436.3 ± 113.6 % (control vs. control rotenone, n = 8, p < 0.05). However, ROS formation in the presence of rotenone did not differ between groups (Fig. 5g, h).

A modified biotin switch method was utilized for labeling SNO modified proteins with BIAM in mitochondria isolated by Percoll gradient centrifugation (Fig. 6a). Purity of mitochondria was determined by the absence of immunoreactivity for antibodies directed against markers of the plasma membrane (Na⁺/K⁺-ATPase), sarcoplasmic reticulum (SERCA2 ATPase), nucleus (HDAC), and cytosol (GAPDH) (Fig. 6b). Following precipitation and Western blot analysis, intensity of the 43 kDa bands representing SNO-modified mtCx43 was significantly increased in mitochondria incubated with NO donors. In total, samples which were treated with GSNO, SNAP, or a combination of NO donor and Cx43 hemichannel blocker CBX showed an increase (109.2 ± 21.5 %, n = 7, p < 0.05) in SNO modifications of mtCx43 compared to mitochondria not treated with a NO donor. A hemichannel blocker was also applied to exclude NO-interfering properties of CBX. The specificity of the biotin switch methods was proven by adding DTT to NO donor treated and already biotin-labeled samples leading to a breakdown of disulfide bounds. In samples treated with DTT and a NO donor (SNAP), the amount of SNO modified mtCx43 was significantly reduced by 59.7 ± 7.6 % compared to NO treated samples and reduced by 15.8 ± 7.8 % (n = 7, p < 0.05) compared to untreated samples (Fig. 6c, e). Application of 100 mmol DTT removed nearly completely the biotin label at Cx43 cysteine residues (Fig. 6d).

Also pharmacological preconditioning by injection of 48 nmol nitrite into the mouse left ventricular cavity in vivo led to an increased SNO modified mtCx43 level by 59.3 ± 18.2 % (n = 6, p < 0.05) compared to control injected mouse hearts (Fig. 6f, g).

To investigate if SNO increases also with IPC, rat hearts were assigned to a Langendorff perfusion protocol. One group of hearts was preconditioned, while the other group was control perfused and the amount of SNO-modified mtCx43 was quantified for each group by using the modified biotin switch method on pure mitochondria for labeling SNO-modified cysteines. First, Western blot quantification of mtCx43 per mitochondrion confirmed previously published data showing that the mitochondrial amount of Cx43 is increased by IPC (Fig. 7a, b). Second, the percentage of SNO-modified mtCx43 was increased by 41.6 ± 1.7 % (n = 17, p < 0.05) in preconditioned rat hearts compared to control perfused hearts (Fig. 7c, d). The increase in light sensitive SNO modification was lost by performing the Langendorff perfusion by daylight (n = 6, p = ns) or by adding 10 mmol DTT after SNO labeling (n = 5, p = ns) (Fig. 7e–g).

Changes in the extent of mtCx43 phosphorylation were analyzed after exposure to NO donors and the hemichannel blocker CBX. SSM were exposed either to 1 µmol CBX, 0.5 mmol SNAP, 1 mmol GSNO, a combination of a NO donor and CBX, or 5 µl DMSO used as a vehicle. Western blot analyses identified no changes in mtCx43 phosphorylation for serine residue 368 (S368) in response to the application of the NO donors or CBX (n = 4, p < 0.05) (Fig. 8).