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Signal transducer and activator of transcription 3 is located in the matrix of cardiomyocyte mitochondria
ABSTRACTS / Journal of Molecular and Cellular Cardiology 44 (2008) 711–825
762
Increased tolerance to ischemia/reperfusion (I/R) injury (infarct size limitation) in the diabetic (D) heart is considered to involve similar protective pathways as ischemic preconditioning (P) in the non-diabetic (ND) heart. Since both adaptive phenomena are associated with enhanced production of reactive oxygen species (ROS) and activation of pro-survival protein kinases, we explored the role of ROS and PI3K/Akt in susceptibility to ischemic arrhythmias in Langendorff-perfused hearts of 1-week D rats (STZ 65 mg/kg, i.p.) and in ND hearts preconditioned (ND-P) by 1 cycle of I/R (5 min each) before 30-min LAD occlusion with or without prior 15-min perfusion with PI3K/Akt inhibitor LY294002 (LY; 5 μM) or antioxidant N-acetylcysteine (NAC; 4 mM) that both abrogate infarct size limiting effect and suppression of reperfusioninduced arrhythmias. Total number of ventricular premature beats (VPB) during ischemia was significantly lower in D and ND-P groups as compared with ND controls (C). In addition, duration of ventricular tachycardia (VT) was shorter than in C. Both, NAC and LY were antiarrhythmic in ND hearts. On the other hand, reduced number of VPB, as well as decreased VT duration in D and ND-P hearts was reversed by neither NAC nor LY. Table 1 Groups
VPB (total number) C
ND 551 ± 61 ND-P 195 ± 40⁎ D 224 ± 53⁎ ⁎ P b 0.05 vs. ND control hearts.
NAC
LY
290 ± 52⁎ 151 ± 39⁎ 207 ± 51⁎
155 ± 15⁎ 77 ± 19⁎ 251 ± 73⁎
The signal transducer and activator of transcription 3 (STAT3) transduces signals from the plasma membrane to the nucleus and is central for the cardioprotection by ischemic preand postconditioning. However, preliminary data suggest that STAT3 is also located in mitochondria. The aim of the present study was to confirm the presence of STAT3 in cardiomyocyte mitochondria, to elucidate its submitochondrial localization and to identify STAT3-interacting proteins within this cell organelle. Western blot analysis and confocal laser scan microscopy showed the presence of STAT3 in mitochondria isolated from mouse left ventricles (LV, n = 4). In contrast, no STAT3 was detected in mitochondria isolated from the LV of mice with a cardiomyocyte-specific deletion of STAT3 (n = 3). The analysis of subfractionated mitochondria by Western blot demonstrated STAT3 predominantly in the fraction negative for marker proteins of the outer membrane (voltage dependent anion channel), inner membrane (ATP synthase alpha), and the intermembrane space (cytochrome c), but positive for marker proteins of the matrix such as manganese superoxide dismutase (MnSOD) and cyclophilin D. Immunoprecipitation of STAT3 from right ventricular and mitochondrial proteins showed a co-precipitation of connexin 43 (Cx43), which is present both at the sarcolemma and at the inner mitochondrial membrane and is also involved in cardioprotection, but not with GSK3β, MnSOD or cytochrome c. Taken together, STAT3 is present in the matrix of cardiomyocyte mitochondria. STAT3 may be involved in cardioprotection via an interaction with mitochondrial Cx43. Keywords: Signal transducer and activator of transcription 3; Mitochondria; Connexin 43 doi:10.1016/j.yjmcc.2008.02.122
In conclusion, reduced arrhythmogenesis during ischemia in acutely D heart is similar to preconditioning in ND hearts. Different from I/R injury, antiarrhythmic mechanisms do not require activation of PI3K/Akt and/or ROS signaling. Grants VEGA SR 2/0173/08, APVV SK-CZ-0049-07, and GACR 305/07/0875. Keywords: Cardioprotection; Ischemic arrhythmias; Cell signaling doi:10.1016/j.yjmcc.2008.02.121
Abstract No. 121 Signal transducer and activator of transcription 3 is located in the matrix of cardiomyocyte mitochondria Kerstin Boengler b,⁎, Ina Konietzka b, Anita van de Sand b, Denise Hilfiker-Kleiner a, Gerd Heusch b, Rainer Schulz b. a Universitätsklinikum Essen, Germany. b Medizinische Hochschule Hannover, Germany ⁎ Corresponding author. Institut für Pathophysiologie, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel.: +49 201 7234488; fax: +49 201 7234481. E-mail address: [email protected]
Abstract No. 122 p66shc and oxidative stress induced by post-ischemic reperfusion injury A. Carpia,⁎, M. Giorgiob, P.G. Peliccib, P. Bernardia,c, F. Di Lisaa,c, R. Menabòc. aInstitute of Molecular Oncology (IFOM), Milano, Italy. bDepartment of Biomedical Sciences, University of Padova, Viale G. Colombo, 3, 35131 Padova, Italy. cCNR Institute of Neurosciences, University of Padova, Italy ⁎ Corresponding author. Tel.: +39 0498276131; fax: +39 0498276040. E-mail address: [email protected] p66shc is a member of a family of adaptor proteins involved in growth factor and stress signalling. Surprisingly, recent studies demonstrated that p66shc catalyzes the formation of reactive oxygen species (ROS) within the mitochondria. Although it is accepted that mitochondrial ROS formation contributes to ischemia–reperfusion (I/R) injury, sites and mechanisms are far from being elucidated. The relationship among p66shc, ROS production and cardiac damage was investigated by comparing hearts from p66shc knockout mice (KO) and wild type (WT)
762
Increased tolerance to ischemia/reperfusion (I/R) injury (infarct size limitation) in the diabetic (D) heart is considered to involve similar protective pathways as ischemic preconditioning (P) in the non-diabetic (ND) heart. Since both adaptive phenomena are associated with enhanced production of reactive oxygen species (ROS) and activation of pro-survival protein kinases, we explored the role of ROS and PI3K/Akt in susceptibility to ischemic arrhythmias in Langendorff-perfused hearts of 1-week D rats (STZ 65 mg/kg, i.p.) and in ND hearts preconditioned (ND-P) by 1 cycle of I/R (5 min each) before 30-min LAD occlusion with or without prior 15-min perfusion with PI3K/Akt inhibitor LY294002 (LY; 5 μM) or antioxidant N-acetylcysteine (NAC; 4 mM) that both abrogate infarct size limiting effect and suppression of reperfusioninduced arrhythmias. Total number of ventricular premature beats (VPB) during ischemia was significantly lower in D and ND-P groups as compared with ND controls (C). In addition, duration of ventricular tachycardia (VT) was shorter than in C. Both, NAC and LY were antiarrhythmic in ND hearts. On the other hand, reduced number of VPB, as well as decreased VT duration in D and ND-P hearts was reversed by neither NAC nor LY. Table 1 Groups
VPB (total number) C
ND 551 ± 61 ND-P 195 ± 40⁎ D 224 ± 53⁎ ⁎ P b 0.05 vs. ND control hearts.
NAC
LY
290 ± 52⁎ 151 ± 39⁎ 207 ± 51⁎
155 ± 15⁎ 77 ± 19⁎ 251 ± 73⁎
The signal transducer and activator of transcription 3 (STAT3) transduces signals from the plasma membrane to the nucleus and is central for the cardioprotection by ischemic preand postconditioning. However, preliminary data suggest that STAT3 is also located in mitochondria. The aim of the present study was to confirm the presence of STAT3 in cardiomyocyte mitochondria, to elucidate its submitochondrial localization and to identify STAT3-interacting proteins within this cell organelle. Western blot analysis and confocal laser scan microscopy showed the presence of STAT3 in mitochondria isolated from mouse left ventricles (LV, n = 4). In contrast, no STAT3 was detected in mitochondria isolated from the LV of mice with a cardiomyocyte-specific deletion of STAT3 (n = 3). The analysis of subfractionated mitochondria by Western blot demonstrated STAT3 predominantly in the fraction negative for marker proteins of the outer membrane (voltage dependent anion channel), inner membrane (ATP synthase alpha), and the intermembrane space (cytochrome c), but positive for marker proteins of the matrix such as manganese superoxide dismutase (MnSOD) and cyclophilin D. Immunoprecipitation of STAT3 from right ventricular and mitochondrial proteins showed a co-precipitation of connexin 43 (Cx43), which is present both at the sarcolemma and at the inner mitochondrial membrane and is also involved in cardioprotection, but not with GSK3β, MnSOD or cytochrome c. Taken together, STAT3 is present in the matrix of cardiomyocyte mitochondria. STAT3 may be involved in cardioprotection via an interaction with mitochondrial Cx43. Keywords: Signal transducer and activator of transcription 3; Mitochondria; Connexin 43 doi:10.1016/j.yjmcc.2008.02.122
In conclusion, reduced arrhythmogenesis during ischemia in acutely D heart is similar to preconditioning in ND hearts. Different from I/R injury, antiarrhythmic mechanisms do not require activation of PI3K/Akt and/or ROS signaling. Grants VEGA SR 2/0173/08, APVV SK-CZ-0049-07, and GACR 305/07/0875. Keywords: Cardioprotection; Ischemic arrhythmias; Cell signaling doi:10.1016/j.yjmcc.2008.02.121
Abstract No. 121 Signal transducer and activator of transcription 3 is located in the matrix of cardiomyocyte mitochondria Kerstin Boengler b,⁎, Ina Konietzka b, Anita van de Sand b, Denise Hilfiker-Kleiner a, Gerd Heusch b, Rainer Schulz b. a Universitätsklinikum Essen, Germany. b Medizinische Hochschule Hannover, Germany ⁎ Corresponding author. Institut für Pathophysiologie, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany. Tel.: +49 201 7234488; fax: +49 201 7234481. E-mail address: [email protected]
Abstract No. 122 p66shc and oxidative stress induced by post-ischemic reperfusion injury A. Carpia,⁎, M. Giorgiob, P.G. Peliccib, P. Bernardia,c, F. Di Lisaa,c, R. Menabòc. aInstitute of Molecular Oncology (IFOM), Milano, Italy. bDepartment of Biomedical Sciences, University of Padova, Viale G. Colombo, 3, 35131 Padova, Italy. cCNR Institute of Neurosciences, University of Padova, Italy ⁎ Corresponding author. Tel.: +39 0498276131; fax: +39 0498276040. E-mail address: [email protected] p66shc is a member of a family of adaptor proteins involved in growth factor and stress signalling. Surprisingly, recent studies demonstrated that p66shc catalyzes the formation of reactive oxygen species (ROS) within the mitochondria. Although it is accepted that mitochondrial ROS formation contributes to ischemia–reperfusion (I/R) injury, sites and mechanisms are far from being elucidated. The relationship among p66shc, ROS production and cardiac damage was investigated by comparing hearts from p66shc knockout mice (KO) and wild type (WT)