Connexin 43 acts as a cytoprotective mediator of signal transduction by stimulating mitochondrial KATP channels in mouse cardiomyocytes

Abstract

Potassium (K+) channels in the inner mitochondrial membrane influence cell function and survival. Increasing evidence indicates that multiple signaling pathways and pharmacological actions converge on mitochondrial ATP-sensitive K+ (mitoKATP) channels and PKC to confer cytoprotection against necrotic and apoptotic cell injury. However, the molecular structure of mitoKATP channels remains unresolved, and the mitochondrial phosphoprotein(s) that mediate cytoprotection by PKC remain to be determined. As mice deficient in the main sarcolemmal gap junction protein connexin 43 (Cx43) lack this cytoprotection, we set out to investigate a possible link among mitochondrial Cx43, mitoKATP channel function, and PKC activation. By patch-clamping the inner membrane of subsarcolemmal murine cardiac mitochondria, we found that genetic Cx43 deficiency, pharmacological connexin inhibition by carbenoxolone, and Cx43 blockade by the mimetic peptide 43GAP27 each substantially reduced diazoxide-mediated stimulation of mitoKATP channels. Suppression of mitochondrial Cx43 inhibited mitoKATP channel activation by PKC. MitoKATP channels of interfibrillar mitochondria, which do not contain any detectable Cx43, were insensitive to both PKC activation and diazoxide, further demonstrating the role of Cx43 in mitoKATP channel stimulation and the compartmentation of mitochondria in cell signaling. Our results define a role for mitochondrial Cx43 in protecting cardiac cells from death and provide a link between cytoprotective stimuli and mitoKATP channel opening, making Cx43 an attractive therapeutic target for protection against cell injury.

Figure 1. Cx43 is present in isolated subsarcolemmal mitoplasts.

Mitochondria were prepared by differential centrifugation as described in Methods. An equal amount of protein (30 μg) from distinct isolation fractions — i.e., crude homogenate of isolated myocytes (TP), first pellet with cell debris (P1, discarded), second pellet containing mitochondria (P2), second supernatant (S2, discarded), intact mitochondria (P3), mitoplasts (Mitopl) — was loaded in each lane, and proteins were analyzed by immunoblotting. Immunoreactivity to the following proteins served as markers for cellular compartments: GAPDH (cytosolic marker), Na⁺/K⁺-ATPase (plasma membrane marker), SERCA2A (endoplasmic reticulum marker), VDAC (outer mitochondrial membrane marker), UCP2 (inner mitochondrial membrane marker).

Figure 2. MitoK_ATP single-channel activation by diazoxide (100 μM) is independent of ROS.

(A) Baseline (left) and diazoxide-activated (middle) mitoK_ATP currents of wild-type mice in mitoplast-attached configuration at –60 mV, which was blocked by 10 μM glibenclamide (right). (B) Amplitude histogram of mitoK_ATP current under baseline conditions revealed a mean I_unitary of –0.83 ± 0.05 pA (n = 22). V_m, membrane voltage. (C) Single-channel amplitude (I) as a function of test potentials. Slope conductances determined by linear regression in individual experiments on wild-type mice were unaffected by the ROS scavenger L-NAC (5 mM) with or without diazoxide and with or without MgATP (100 μM) compared with control (see Figure 3B). (D) Baseline single-channel properties (left), the diazoxide effect on mitoK_ATP channel activity (middle), and channel inhibition by MgATP (right) (at –60 mV) were unaffected by L-NAC compared with control (A). (E) Slope of Amplex UltraRed fluorescence of intact mitochondria (10 μg protein) in incubation buffer was significantly increased by the addition of antimycin A; n = 6, *P < 0.05 versus control. (F) Slope of Amplex UltraRed fluorescence of mitoplasts (50 μg protein) in patch-clamp buffer was increased by antimycin A but remained unchanged upon addition of the solvent DMSO or diazoxide; n = 6, *P < 0.05 versus control.

Figure 3. MitoK_ATP single-channel activation by diazoxide (100 μM) is dependent on the presence of mitochondrial Cx43.

(A) The diazoxide effect on mitoK_ATP channel activity (at –60 mV) was reduced in wild-type mice by carbenoxolone (10 μM) (upper left) and ⁴³GAP27 (250 μM) (upper right); in Cx43^+/– mice (lower left); and in Cx43^Cre-ER(T)/fl + 4-OHT mice (lower right) compared with control (Figure 2A). (B) Single-channel amplitude as a function of test potentials in the absence (left) and presence (right) of diazoxide. Slope conductances were similar in all groups. (C) Mean values of the open probability (Po,total) in control, Cx43^+/– mice, wild-type + carbenoxolone, wild-type + ⁴³GAP27, and Cx43^+/– mice + ⁴³GAP27 in the absence and presence of diazoxide, as indicated; n values are shown in parentheses. *P < 0.05 versus control. (D) Mean values of the open probability (Po,total) in Cx43^fl/fl control mice (black) and Cx43^Cre-ER(T)/fl + 4-OHT mice (white) in the absence and presence of diazoxide and MgATP, as indicated; n values are shown in parentheses. *P < 0.05 versus Cx43^fl/fl control; ^§P < 0.05 versus Cx43^fl/fl control + diazoxide; ^#P < 0.05 versus Cx43Cre-ER(T)/f^l + 4-OHT. (E) Effect of various voltages on diazoxide-stimulated mitoK_ATP open probability (Po,total) in control, wild-type + carbenoxolone, and Cx43^+/– mice. (F) Western blot analysis of Cx43 and MN-SOD protein level in mitochondria from Cx43^+/+ control mice, Cx43^+/– mice, Cx43^fl/fl control mice, and Cx43^Cre-ER(T)/fl + 4-OHT mice. Bar graphs represent ratios of mitochondrial Cx43 level normalized to MN-SOD; n values are shown in parentheses. *P < 0.05 versus control.

Figure 4. IP increases mitochondrial Cx43 content in Cx43^+/– mice and rescues diazoxide sensitivity of mitoK_ATP channels (A–E).

(A) Western blots of Cx43 and MN-SOD in mitochondria from the AW subjected to IP and PW (no IP) of the same Cx43^+/– mouse (IP-induced Cx43 increase normalized to MN-SOD, 4.0 ± 0.9, n = 3). Mitochondrial proteins from wild-type left ventricle (WT LV): positive control; Na⁺/K⁺-ATPase: exclusion of contamination with sarcolemmal proteins. (B) The diazoxide effect on mitoK_ATP activity (at –60 mV) in Cx43^+/– mice was enhanced by IP (right) versus control (left). (C and E) Mean values of open probability (Po,total) (C) and mean open time (E) in PW (black) and AW (IP; white) of Cx43^+/– mice in the absence and presence of diazoxide, as indicated; n values are shown in parentheses. (D) Single-channel amplitude as a function of test potentials. Slope conductances were similar in PW and AW (subjected to IP) of Cx43^+/– mice and were unaffected by diazoxide.*P < 0.05 versus PW control; ^§P < 0.05 versus AW control; ^#P < 0.05 versus PW + diazoxide. (F–H) Mitochondrial PKCε phosphorylates mitochondrial Cx43 at Ser368. (F) Western blots for phospho-Cx43Ser368 and MN-SOD in mitochondria incubated for 30 minutes under control conditions or with ψεRACK (5 μM). Right ventricular protein (RV): positive control; Na⁺/K⁺-ATPase: exclusion of contamination with sarcolemmal proteins. (G) Ratios of mitochondrial phospho-Cx43Ser368 levels normalized to MN-SOD; n values are shown in parentheses. *P < 0.05 versus control. (H) Ratios of mitochondrial MN-SOD levels normalized to Ponceau S staining to demonstrate the suitability of MN-SOD as a housekeeping protein; n values are shown in parentheses.

Figure 5. PKC-mediated stimulation of mitoK_ATP channel activity is diminished in Cx43^+/– mice.

(A) MitoK_ATP single-channel activity is activated by PKC stimulation with PMA 2 μM (top left), which can be inhibited by glibenclamide 10 μM (top right). PMA-induced mitoK_ATP channel activation is reduced in Cx43^+/– mice (bottom). Voltages are indicated. (B) Mean values of mitoK_ATP channel open probability (Po,total; left) and mean open time (right) in control (black) and Cx43^+/– mice (white) in the absence and presence of PMA and glibenclamide, as indicated; n values are shown in parentheses. *P < 0.05 versus control; ^§P < 0.05 versus Cx43^+/–; ^#P < 0.05 versus control + PMA. (C) Proteins of mitochondria were immunoprecipitated (IMP) for Cx43 (top) or PKCε (bottom). Western blot analysis for coprecipitated Cx43 and PKCε revealed positive results. Precipitation of mitochondrial proteins with anti-rabbit IgGs was used as negative control. Input lysates and a total ventricular protein extract (TP) were analyzed as positive control. Immunoblotting against VDAC was performed to exclude any unspecific coimmunoprecipitation. (D) MitoK_ATP single-channel activity was activated by selective PKCε stimulation with ψεRACK (top left), which could be inhibited by MgATP (top right). ψεRACK-induced mitoK_ATP channel activation was reduced in Cx43^+/– mice (bottom). (E) Mean values of mitoK_ATP channel open probability in control (Po,total; black) and Cx43^+/– mice (white) in the absence and presence of the selective PKCε peptide agonist ψεRACK (0.5 μM) and MgATP (100 μM), as indicated; n values are shown in parentheses. *P < 0.05 versus control; ^§P < 0.05 versus Cx43^+/–; ^#P < 0.05 versus control + ψεRACK.

Figure 6. MitoK_ATP channel activity of interfibrillar mitochondria is insensitive to PKC and diazoxide (100 μM) stimulation.

(A) MitoK_ATP single-channel current (at –60 mV) of interfibrillar mitochondria (left), which was not activated by diazoxide (middle) but was inhibited by glibenclamide (right). (B) Mean values of mitoK_ATP channel open probability (Po,total; top left), I_peak (top right), mean open time (bottom left), and mean closed time (bottom, right) of interfibrillar mitochondria in the absence and presence of diazoxide, PMA, glibenclamide, and MgATP, as indicated; n values are shown in parentheses. *P < 0.05 versus control. Single-channel amplitude as a function of test potentials. (C) Slope conductance (14.1 ± 0.5 pS, n = 6) of interfibrillar mitoK_ATP channels determined by linear regression in individual experiments was similar to that of subsarcolemmal mitochondria (Figure 3B). (D) MitoK_ATP single-channel activity of interfibrillar mitochondria (IFM) was not activated by PKC stimulation with 2 μM PMA (top). Baseline activity was inhibited by 100 μM MgATP (bottom). Voltages are indicated. (E) Amplitude histogram of interfibrillar mitoK_ATP current under baseline conditions revealed a mean I_unitary of –0.85 ± 0.04 pA (n = 12).