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Rainer Schulz - One of the best experts on this subject based on the ideXlab platform.

  • Connexin 43 is an emerging therapeutic target in ischemia reperfusion injury cardioprotection and neuroprotection
    Pharmacology & Therapeutics, 2015
    Co-Authors: Rainer Schulz, Paul D Lampe, Philipp Maximilian Gorge, Aniko Gorbe, Peter Ferdinandy, Luc Leybaert
    Abstract:

    Connexins are widely distributed proteins in the body that are crucially important for heart and brain functions. Six Connexin subunits form a connexon or hemichannel in the plasma membrane. Interactions between two hemichannels in a head-to-head arrangement result in the formation of a gap junction channel. Gap junctions are necessary to coordinate cell function by passing electrical current flow between heart and nerve cells or by allowing exchange of chemical signals and energy substrates. Apart from its localization at the sarcolemma of cardiomyocytes and brain cells, Connexins are also found in the mitochondria where they are involved in the regulation of mitochondrial matrix ion fluxes and respiration. Connexin expression is affected by age and gender as well as several pathophysiological alterations such as hypertension, hypertrophy, diabetes, hypercholesterolemia, ischemia, post-myocardial infarction remodeling or heart failure, and post-translationally Connexins are modified by phosphorylation/de-phosphorylation and nitros(yl)ation which can modulate channel activity. Using knockout/knockin technology as well as pharmacological approaches, one of the Connexins, namely Connexin 43, has been identified to be important for cardiac and brain ischemia/reperfusion injuries as well as protection from it. Therefore, the current review will focus on the importance of Connexin 43 for irreversible injury of heart and brain tissues following ischemia/reperfusion and will highlight the importance of Connexin 43 as an emerging therapeutic target in cardio- and neuroprotection.

  • Connexin 43 impacts on mitochondrial potassium uptake
    Frontiers in Pharmacology, 2013
    Co-Authors: Kerstin Boengler, Luc Leybaert, Gerd Heusch, Elvira Ungefug, Rainer Schulz
    Abstract:

    In cardiomyocytes, Connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43(Cre-ER(T)/fl) + 4-OHT). The mitochondria of the Cx43(Cre-ER(T)/fl) + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  • Connexin 43 in ischemic pre and postconditioning
    Heart Failure Reviews, 2007
    Co-Authors: Rainer Schulz, Yukun Luo, Kerstin Boengler, David Garciadorado, Andreas Totzeck, Gerd Heusch
    Abstract:

    Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular myocardium, but Cx43 is also localized at the inner membrane of cardiomyocyte mitochondria. In cardiomyocytes, Cx43 is involved in the formation of reactive oxygen species, which are central to the signal transduction cascade of ischemic preconditioning’s protection. Accordingly, genetically-induced or age-related loss of Cx43 abolishes infarct size reduction by ischemic preconditioning. Similarly, mitochondrial import inhibition of Cx43 completely blocks infarct size reduction by pharmacological preconditioning with diazoxide. In contrast to its importance for preconditioning-induced cardioprotection, Cx43 is not important for infarct size reduction by ischemic postconditioning. In summary, Cx43––especially Cx43 localized in mitochondria––appears to be one key element of the signal transduction cascade of the protection by preconditioning.

  • impairment of diazoxide induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice
    Circulation Research, 2005
    Co-Authors: Frank R Heinzel, Rainer Schulz, Yukun Luo, Kerstin Boengler, Astrid Buechert, David Garciadorado, Fabio Di Lisa, Gerd Heusch
    Abstract:

    Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous Connexin 43 deficient (Cx43+/-) mice. Because Connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 micromol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 micromol/L) increased ROS formation by 43+/-10% versus vehicle in WT, but only by 18+/-4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7+/-1% versus 39+/-2%, (Cx43+/-): 8+/-1% versus 40+/-3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17+/-2%, P<0.01) in WT, but not in Cx43+/- (9+/-1%). Menadione (37 microg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24+/-4% versus 24+/-5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35+/-4% versus 55+/-3% of area at risk, P<0.01), but not in Cx43+/- mice (56+/-2% versus 54+/-3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

  • role of Connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions
    Journal of Molecular and Cellular Cardiology, 2004
    Co-Authors: Frank R Heinzel, Rainer Schulz, Kerstin Boengler, Gerd Heusch
    Abstract:

    Abstract Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type ( n  = 5) and heterozygous Cx 43-deficient mice ( n  = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 ± 5% at baseline vs. 46 ± 4 (mean ± S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 ± 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 ± 1%, P

Elissavet Kardami - One of the best experts on this subject based on the ideXlab platform.

  • ischemia induced dephosphorylation of cardiomyocyte Connexin 43 is reduced by okadaic acid and calyculin a but not fostriecin
    Molecular and Cellular Biochemistry, 2003
    Co-Authors: Madhumathy Jeyaraman, Stephane Tanguy, Robert R Fandrich, Anton Lukas, Elissavet Kardami
    Abstract:

    The gap junction protein Connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein Connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1-like phosphatase(s).

  • Ischemia-induced dephosphorylation of cardiomyocyte Connexin-43 is reduced by okadaic acid and calyculin A but not fostriecin
    Cardiac Cell Biology, 2003
    Co-Authors: Madhumathy Jeyaraman, Stephane Tanguy, Robert R Fandrich, Anton Lukas, Elissavet Kardami
    Abstract:

    The gap junction protein Connexin-43 (Cx43) exists mainly in the phosphorylated state in the normal heart, while ischemia induces dephosphorylation. Phosphatase(s) involved in cardiac Cx43 dephosphorylation have not as yet been identified. We examined the acute effects of ischemia on the dephosphorylation of the gap junction protein Connexin-43 in isolated adult cardiomyocytes and isolated perfused hearts. In addition we tested the effectiveness of protein phosphatase 1 and 2A (PP1/2A) inhibitors in preventing Cx43 dephosphorylation. In both models, significant accumulation of the 41 kDa non-phosphorylated Cx43, accompanied by decreased relative levels of the 43–46 kDa phosphorylated Cx43, was observed at 30 min of ischemia. Okadaic acid decreased ischemia-induced Cx43 dephosphorylation; it also decreased the accumulation of non-phosphorylated Cx43 at the intercalated discs of myocytes in the whole heart. Calyculin A, but not fostriecin, also decreased ischemia-induced Cx43 dephosphorylation in isolated cardiomyocytes. It is concluded that isolated adult myocytes respond to ischemia in a manner similar to whole hearts and that ischemia-induced dephosphorylation of Cx43 is mediated, at least in part, by PP1like phosphatase(s). (Mol Cell Biochem 242: 129–134, 2003)

  • the carboxy tail of Connexin 43 localizes to the nucleus and inhibits cell growth
    Molecular and Cellular Biochemistry, 2003
    Co-Authors: Xitong Dang, Bradley W Doble, Elissavet Kardami
    Abstract:

    Gap junctions are plasma membrane intercellular communication channels that in addition to ensuring electrical coupling and coordinated mechanical activity, can act as growth suppressors. To define the role of a non-channel forming domain of Connexin-43 (Cx43), the main constituent of cardiomyocyte gap junctions, on growth regulation, we expressed its C-terminal portion (CT-Cx43) in cardiomyocytes and HeLa cells. In addition to broad cytoplasmic localization, CT-Cx43 was also localized to the nucleus of both cell types, detected by immunofluorescence as well as immunoblotting of subcellular fractions. Furthermore, stable expression of CT-Cx43 in HeLa cells induced a significant decrease in proliferation. It is therefore suggested that plasma membrane localization and formation of channels are not required for growth inhibition by Cx43, and that nuclear localization of CT-Cx43 may exert effects on gene expression and growth.

Luc Leybaert - One of the best experts on this subject based on the ideXlab platform.

  • Connexin 43 is an emerging therapeutic target in ischemia reperfusion injury cardioprotection and neuroprotection
    Pharmacology & Therapeutics, 2015
    Co-Authors: Rainer Schulz, Paul D Lampe, Philipp Maximilian Gorge, Aniko Gorbe, Peter Ferdinandy, Luc Leybaert
    Abstract:

    Connexins are widely distributed proteins in the body that are crucially important for heart and brain functions. Six Connexin subunits form a connexon or hemichannel in the plasma membrane. Interactions between two hemichannels in a head-to-head arrangement result in the formation of a gap junction channel. Gap junctions are necessary to coordinate cell function by passing electrical current flow between heart and nerve cells or by allowing exchange of chemical signals and energy substrates. Apart from its localization at the sarcolemma of cardiomyocytes and brain cells, Connexins are also found in the mitochondria where they are involved in the regulation of mitochondrial matrix ion fluxes and respiration. Connexin expression is affected by age and gender as well as several pathophysiological alterations such as hypertension, hypertrophy, diabetes, hypercholesterolemia, ischemia, post-myocardial infarction remodeling or heart failure, and post-translationally Connexins are modified by phosphorylation/de-phosphorylation and nitros(yl)ation which can modulate channel activity. Using knockout/knockin technology as well as pharmacological approaches, one of the Connexins, namely Connexin 43, has been identified to be important for cardiac and brain ischemia/reperfusion injuries as well as protection from it. Therefore, the current review will focus on the importance of Connexin 43 for irreversible injury of heart and brain tissues following ischemia/reperfusion and will highlight the importance of Connexin 43 as an emerging therapeutic target in cardio- and neuroprotection.

  • Connexin 43 impacts on mitochondrial potassium uptake
    Frontiers in Pharmacology, 2013
    Co-Authors: Kerstin Boengler, Luc Leybaert, Gerd Heusch, Elvira Ungefug, Rainer Schulz
    Abstract:

    In cardiomyocytes, Connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43(Cre-ER(T)/fl) + 4-OHT). The mitochondria of the Cx43(Cre-ER(T)/fl) + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

Gerd Heusch - One of the best experts on this subject based on the ideXlab platform.

  • Connexin 43 impacts on mitochondrial potassium uptake
    Frontiers in Pharmacology, 2013
    Co-Authors: Kerstin Boengler, Luc Leybaert, Gerd Heusch, Elvira Ungefug, Rainer Schulz
    Abstract:

    In cardiomyocytes, Connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43(Cre-ER(T)/fl) + 4-OHT). The mitochondria of the Cx43(Cre-ER(T)/fl) + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  • Connexin 43 in ischemic pre and postconditioning
    Heart Failure Reviews, 2007
    Co-Authors: Rainer Schulz, Yukun Luo, Kerstin Boengler, David Garciadorado, Andreas Totzeck, Gerd Heusch
    Abstract:

    Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular myocardium, but Cx43 is also localized at the inner membrane of cardiomyocyte mitochondria. In cardiomyocytes, Cx43 is involved in the formation of reactive oxygen species, which are central to the signal transduction cascade of ischemic preconditioning’s protection. Accordingly, genetically-induced or age-related loss of Cx43 abolishes infarct size reduction by ischemic preconditioning. Similarly, mitochondrial import inhibition of Cx43 completely blocks infarct size reduction by pharmacological preconditioning with diazoxide. In contrast to its importance for preconditioning-induced cardioprotection, Cx43 is not important for infarct size reduction by ischemic postconditioning. In summary, Cx43––especially Cx43 localized in mitochondria––appears to be one key element of the signal transduction cascade of the protection by preconditioning.

  • impairment of diazoxide induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice
    Circulation Research, 2005
    Co-Authors: Frank R Heinzel, Rainer Schulz, Yukun Luo, Kerstin Boengler, Astrid Buechert, David Garciadorado, Fabio Di Lisa, Gerd Heusch
    Abstract:

    Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous Connexin 43 deficient (Cx43+/-) mice. Because Connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 micromol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 micromol/L) increased ROS formation by 43+/-10% versus vehicle in WT, but only by 18+/-4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7+/-1% versus 39+/-2%, (Cx43+/-): 8+/-1% versus 40+/-3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17+/-2%, P<0.01) in WT, but not in Cx43+/- (9+/-1%). Menadione (37 microg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24+/-4% versus 24+/-5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35+/-4% versus 55+/-3% of area at risk, P<0.01), but not in Cx43+/- mice (56+/-2% versus 54+/-3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

  • role of Connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions
    Journal of Molecular and Cellular Cardiology, 2004
    Co-Authors: Frank R Heinzel, Rainer Schulz, Kerstin Boengler, Gerd Heusch
    Abstract:

    Abstract Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type ( n  = 5) and heterozygous Cx 43-deficient mice ( n  = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 ± 5% at baseline vs. 46 ± 4 (mean ± S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 ± 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 ± 1%, P

Kerstin Boengler - One of the best experts on this subject based on the ideXlab platform.

  • Connexin 43 impacts on mitochondrial potassium uptake
    Frontiers in Pharmacology, 2013
    Co-Authors: Kerstin Boengler, Luc Leybaert, Gerd Heusch, Elvira Ungefug, Rainer Schulz
    Abstract:

    In cardiomyocytes, Connexin 43 (Cx43) forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria (SSM). Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake. SSM were isolated from rat left ventricular myocardium and loaded with the potassium-sensitive dye PBFI (potassium-binding benzofuran isophthalate). Intramitochondrial potassium was replaced by tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 μM Gap19, a peptide that specifically inhibits Cx43-based hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100 ± 11.6% in control mitochondria to 65.5 ± 10.7% (n = 6, p < 0.05). In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (4-OHT; Cx43(Cre-ER(T)/fl) + 4-OHT). The mitochondria of the Cx43(Cre-ER(T)/fl) + 4-OHT mice contained 3 ± 1% Cx43 (n = 6) of that in control mitochondria (100 ± 11%, n = 8, p < 0.05). The ablation of Cx43 (n = 5) reduced the velocity of the potassium influx from 100 ± 11.2% in control mitochondria (n = 9) to 66.6 ± 5.5% (p < 0.05). Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  • Connexin 43 in ischemic pre and postconditioning
    Heart Failure Reviews, 2007
    Co-Authors: Rainer Schulz, Yukun Luo, Kerstin Boengler, David Garciadorado, Andreas Totzeck, Gerd Heusch
    Abstract:

    Connexin 43 (Cx43) is the predominant protein forming gap junctions and non-junctional hemichannels in ventricular myocardium, but Cx43 is also localized at the inner membrane of cardiomyocyte mitochondria. In cardiomyocytes, Cx43 is involved in the formation of reactive oxygen species, which are central to the signal transduction cascade of ischemic preconditioning’s protection. Accordingly, genetically-induced or age-related loss of Cx43 abolishes infarct size reduction by ischemic preconditioning. Similarly, mitochondrial import inhibition of Cx43 completely blocks infarct size reduction by pharmacological preconditioning with diazoxide. In contrast to its importance for preconditioning-induced cardioprotection, Cx43 is not important for infarct size reduction by ischemic postconditioning. In summary, Cx43––especially Cx43 localized in mitochondria––appears to be one key element of the signal transduction cascade of the protection by preconditioning.

  • impairment of diazoxide induced formation of reactive oxygen species and loss of cardioprotection in Connexin 43 deficient mice
    Circulation Research, 2005
    Co-Authors: Frank R Heinzel, Rainer Schulz, Yukun Luo, Kerstin Boengler, Astrid Buechert, David Garciadorado, Fabio Di Lisa, Gerd Heusch
    Abstract:

    Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous Connexin 43 deficient (Cx43+/-) mice. Because Connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 micromol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 micromol/L) increased ROS formation by 43+/-10% versus vehicle in WT, but only by 18+/-4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7+/-1% versus 39+/-2%, (Cx43+/-): 8+/-1% versus 40+/-3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17+/-2%, P<0.01) in WT, but not in Cx43+/- (9+/-1%). Menadione (37 microg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24+/-4% versus 24+/-5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35+/-4% versus 55+/-3% of area at risk, P<0.01), but not in Cx43+/- mice (56+/-2% versus 54+/-3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.

  • Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning
    Cardiovascular Research, 2005
    Co-Authors: Kerstin Boengler, David Garciadorado, Giuliano Dodoni, Antonio Rodriguezsinovas, Alberto Cabestrero, Marisol Ruizmeana, Petra Gres, Ina Konietzka, Carmen Lopeziglesias, Fabio Di Lisa
    Abstract:

    Objective : Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. Methods and results : Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without ( n = 13) or with ( n = 13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4 ± 0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1 ± 0.2, p <0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262 ± 63% of baseline. Conclusion : These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.

  • role of Connexin 43 in ischemic preconditioning does not involve intercellular communication through gap junctions
    Journal of Molecular and Cellular Cardiology, 2004
    Co-Authors: Frank R Heinzel, Rainer Schulz, Kerstin Boengler, Gerd Heusch
    Abstract:

    Abstract Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type ( n  = 5) and heterozygous Cx 43-deficient mice ( n  = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 ± 5% at baseline vs. 46 ± 4 (mean ± S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 ± 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 ± 1%, P