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Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure-Overload in Mice
The 19th Annual Scientific Meeting
Young Investigators’ Award 2 YA2-1 Paradigm Shift of PKG1a Redox Modulation in the Stressed Heart TAISHI NAKAMURA1,2, HISAO OGAWA1 1 Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2Devision of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, USA PKG1a disulfide dimerization between cysteine 42 residues enhances oxidantinduced vasorelaxation. However, its impact on myocardial regulation remains unknown. Here, we reveal that disulfide PKG1a oxidation occurs in human and animal heart disease, results in contributing to adverse remodeling, and enables to change the therapeutic efficacy. Hearts and myocytes expressing a cysteine redox-insensitive PKG1a (PKG1aC42S) better adapted to sustained pressure overload and Gq-coupled stimulation, by suppressing fetal genes, calcineurin (Cn)/ NFAT, calcium/calmodulindependent kinase, Erk1/2, and PI3K/Akt. Importantly, PKG activity was similar despite the form. We instead found redox-dependent changes in PKG1a translocation to sarcolemmal-membrane which enhances suppression of transient receptor potential cation channel type-6 (TRPC6) via PKG phosphorylation sites. Conversely, PKG1aC42S diminished the capacity of phosphodiesterase 5 (PDE5) inhibitor, canceling its phosphorylation at S92 and further activation, both of which were normally observed with oxidized PKG activation. Mice harboring PKG1aC42S showed no further improvement from PDE5a inhibitor in functional, histological and molecular levels despite persisting significant dysfunction by pressure overload. Intriguingly, this therapeutic disparity was specific to PDE5, unlike alternative cGMP approaches, proposing the significance of PKG redox modulation in PDE5 kinetics, which potentially explains negative result of RELAX clinical trial in patients with HFpEF. PKG1a redox modulation changes its localization and targeting, by which PKG1aC42S counters pathological stress, while uniquely blunts its intrinsic benefit from PDE5 inhibitor to treat heart disease.
YA2-2 Cardiac Sirt1 Mediates the Cardioprotective Effect of Caloric Restriction by Suppressing Local Complement System Activation after Ischemia/Reperfusion TSUNEHISA YAMAMOTO1, KEN SHINMURA2, MOTOAKI SANO1, JIN ENDO1, KEIICHI FUKUDA1 1 Department of Cardiology School of Medicine, Keio University; 2Division of General Medicine, Department of Internal Medicine, Hyogo College of Medicine Caloric restriction (CR) confers cardioprotection against ischemia/reperfusion (I/ R) injury. However, the mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout mice (CM-Sirt1-/-) and the corresponding control mice to either 3-month ad libitum (AL) feeding or CR (-40%). Isolated perfused hearts were subjected to global ischemia, followed by reperfusion. The recovery of left ventricle function after I/R improved and LDH release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but the cardioprotective effect of CR was not observed in the CMSirt1-/- mice. Quantitative real-time polymerase chain reaction analysis demonstrated that CR up-regulated antioxidant genes in a Sirt1-dependent manner. In addition, the expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1-/- mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/ R injury in conventional C3 knockout mice (C3-/-) treated with CR was similar to that in AL-fed C3-/- mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression.
YA2-3 Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure-Overload in Mice KOTA SUZUKI, KIMIO SATOH, JUNICHI OMURA, TAIJYU SATOH, SHIN KUDO, TOMOHIRO OTSUKI, HIROAKI SHIMOKAWA Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine Background: Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinase and promotes inflammation. Methods and Results: We performed transverse aortic constriction (TAC) in Bsg+/ and wild-type (WT) mice, and thereafter examined the time-course for 4 weeks. Bsg+/+ mice showed significantly less cardiac fibrosis compared with WT mice after TAC. Both metalloproteinase activities and oxidative stress in loaded LV were significantly less in Bsg+/ mice compared
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with WT mice. Echocardiography revealed that Bsg+/ mice showed less hypertrophy and preserved LV function compared with WT mice. Consistently, Bsg+/ mice showed better survival compared with Bsg+/+ mice, regardless of the source of bone marrow (Bsg+/+ or Bsg+/ ). Conversely, cardiac-specific Bsg overexpression mice showed significantly poor survival as compared with littermate controls. Next, we isolated cardiac fibroblasts (CFs) and examined their responses to angiotensin-II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion and ERK/Akt/JNK activities in Bsg+/+ CFs, which were significantly less in Bsg+/ CFs. Recombinant Bsg promoted ERK/Akt activities in CFs. Consistently, Bsg+/ CFs were significantly less proliferative compared with Bsg+/+ CFs. In the clinical study, serum levels of soluble Bsg (sBsg) were significantly elevated in patients with heart failure compared with healthy controls, significantly associated with poor prognosis. Conclusions: Bsg plays a crucial role in the pathogenesis of cardiac hypertrophy, fibrosis and failure in mice and humans.
YA2-4 The Novel Mitophagic Receptor Protein, Bcl2-like Protein 13: New Insights for the Molecular Mechanisms of the Pathogenesis of Heart Disease TOMOKAZU MURAKAWA1, OSAMU YAMAGUCHI1, KOJI OKAMOTO2, YASUSHI SAKATA1, KINYA OTSU3 1 Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan; 2Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan; 3Cardiovascular Division, King’s College London British Heart Foundation Centre of Excellence, London, United Kingdom Cardiac function highly depends on energy generated by mitochondria, which are injured by various stresses such as pressure overload or aging. Damaged mitochondria in failing hearts are removed by a mitochondria-specific autophagy, called mitophagy. Dysregulation of mitophagy is implicated in the pathogenesis of heart disease such as heart failure. Mitophagy is closely associated with mitochondrial fission to make mitochondria engulfable size by autophagosomes. Atg32 is an essential protein for mitophagy in yeast. Some molecules have been reported to be involved in mitophagy, such as Parkin, FUNDC1 and Bnip3l. However, no Atg32 homologue has been identified in mammalian cells. By screening a public protein database for Atg32 homologues, we identified Bcl-2like protein 13 (Bcl2-L-13). Bcl2-L-13 induced mitochondrial fragmentation when overexpressed in neonatal rat cardiomyocyte. We carried out further investigation into functions of Bcl2-L-13 using HEK293 cells. Bcl2-L-13 is localized at the mitochondrial outer membrane and bound to LC3 through the WXXI motif and induced mitochondrial fragmentation and mitophagy. In Bcl2-L-13, the BH domains are important for mitochondrial fragmentation, while the WXXI motif facilitates mitophagy. Knockdown of Bcl2-L-13 attenuated mitochondrial damage-induced fragmentation and mitophagy. Furthermore, Bcl2-L-13 induced mitophagy in Atg32-deficient yeast. Induction and/or phosphorylation of this protein may regulate its activity. Our findings thus offer novel insights into molecular mechanisms of the pathogenesis of heart disease.
YA2-5 The Akt-mTOR Axis is a Pivotal Regulator of Eccentric Hypertrophy during Volume Overload MASATAKA IKEDA1, TOMOMI IDE1, TAKEO FUJINO1, KENJI SUNAGAWA2 1 Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Therapeutic Regulation of Cardiovascular Homeostasis, Center for Disruptive Cardiovascular Medicine, Kyushu University, Fukuoka, Japan Background: The heart has two major modalities of hypertrophy in response to hemodynamic stress: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains to be fully elucidated. Methods and Results: We created an arteriovenous fistula in the abdominal aorta, inducing VO in mice. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)dependent manner, mTOR inhibition not only suppressed eccentric hypertrophy but also induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation during VO. Various growth factors including insulin-like growth factor, neuregulin-1, and connective tissue growth factor were upregulated, which contributed to AktmTOR activation under VO. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time integration of mTOR activity during VO. Conclusion: These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart in heart failure.
Young Investigators’ Award 2 YA2-1 Paradigm Shift of PKG1a Redox Modulation in the Stressed Heart TAISHI NAKAMURA1,2, HISAO OGAWA1 1 Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 2Devision of Cardiology, Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, USA PKG1a disulfide dimerization between cysteine 42 residues enhances oxidantinduced vasorelaxation. However, its impact on myocardial regulation remains unknown. Here, we reveal that disulfide PKG1a oxidation occurs in human and animal heart disease, results in contributing to adverse remodeling, and enables to change the therapeutic efficacy. Hearts and myocytes expressing a cysteine redox-insensitive PKG1a (PKG1aC42S) better adapted to sustained pressure overload and Gq-coupled stimulation, by suppressing fetal genes, calcineurin (Cn)/ NFAT, calcium/calmodulindependent kinase, Erk1/2, and PI3K/Akt. Importantly, PKG activity was similar despite the form. We instead found redox-dependent changes in PKG1a translocation to sarcolemmal-membrane which enhances suppression of transient receptor potential cation channel type-6 (TRPC6) via PKG phosphorylation sites. Conversely, PKG1aC42S diminished the capacity of phosphodiesterase 5 (PDE5) inhibitor, canceling its phosphorylation at S92 and further activation, both of which were normally observed with oxidized PKG activation. Mice harboring PKG1aC42S showed no further improvement from PDE5a inhibitor in functional, histological and molecular levels despite persisting significant dysfunction by pressure overload. Intriguingly, this therapeutic disparity was specific to PDE5, unlike alternative cGMP approaches, proposing the significance of PKG redox modulation in PDE5 kinetics, which potentially explains negative result of RELAX clinical trial in patients with HFpEF. PKG1a redox modulation changes its localization and targeting, by which PKG1aC42S counters pathological stress, while uniquely blunts its intrinsic benefit from PDE5 inhibitor to treat heart disease.
YA2-2 Cardiac Sirt1 Mediates the Cardioprotective Effect of Caloric Restriction by Suppressing Local Complement System Activation after Ischemia/Reperfusion TSUNEHISA YAMAMOTO1, KEN SHINMURA2, MOTOAKI SANO1, JIN ENDO1, KEIICHI FUKUDA1 1 Department of Cardiology School of Medicine, Keio University; 2Division of General Medicine, Department of Internal Medicine, Hyogo College of Medicine Caloric restriction (CR) confers cardioprotection against ischemia/reperfusion (I/ R) injury. However, the mechanism by which Sirt1 in cardiomyocytes mediates the cardioprotective effect of CR remains undetermined. We subjected cardiomyocyte-specific Sirt1 knockout mice (CM-Sirt1-/-) and the corresponding control mice to either 3-month ad libitum (AL) feeding or CR (-40%). Isolated perfused hearts were subjected to global ischemia, followed by reperfusion. The recovery of left ventricle function after I/R improved and LDH release into the perfusate during reperfusion was attenuated in the control mice treated with CR, but the cardioprotective effect of CR was not observed in the CMSirt1-/- mice. Quantitative real-time polymerase chain reaction analysis demonstrated that CR up-regulated antioxidant genes in a Sirt1-dependent manner. In addition, the expression levels of cardiac complement component 3 (C3) at baseline and the accumulation of C3 in the ischemia-reperfused myocardium were attenuated by CR in the control mice, but not in the CM-Sirt1-/- mice. Resveratrol treatment also attenuated the expression levels of C3 protein in cultured neonatal rat ventricular cardiomyocytes. Moreover, the degree of myocardial I/ R injury in conventional C3 knockout mice (C3-/-) treated with CR was similar to that in AL-fed C3-/- mice, although the expression levels of Sirt1 were enhanced by CR. These results demonstrate that cardiac Sirt1 plays an essential role in CR-induced cardioprotection against I/R injury by suppressing cardiac C3 expression.
YA2-3 Basigin Promotes Cardiac Fibrosis and Failure in Response to Chronic Pressure-Overload in Mice KOTA SUZUKI, KIMIO SATOH, JUNICHI OMURA, TAIJYU SATOH, SHIN KUDO, TOMOHIRO OTSUKI, HIROAKI SHIMOKAWA Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine Background: Basigin (Bsg) is a transmembrane glycoprotein that activates matrix metalloproteinase and promotes inflammation. Methods and Results: We performed transverse aortic constriction (TAC) in Bsg+/ and wild-type (WT) mice, and thereafter examined the time-course for 4 weeks. Bsg+/+ mice showed significantly less cardiac fibrosis compared with WT mice after TAC. Both metalloproteinase activities and oxidative stress in loaded LV were significantly less in Bsg+/ mice compared
JHFS
S147
with WT mice. Echocardiography revealed that Bsg+/ mice showed less hypertrophy and preserved LV function compared with WT mice. Consistently, Bsg+/ mice showed better survival compared with Bsg+/+ mice, regardless of the source of bone marrow (Bsg+/+ or Bsg+/ ). Conversely, cardiac-specific Bsg overexpression mice showed significantly poor survival as compared with littermate controls. Next, we isolated cardiac fibroblasts (CFs) and examined their responses to angiotensin-II or mechanical stretch. Both stimuli significantly increased Bsg expression, cytokines/chemokines secretion and ERK/Akt/JNK activities in Bsg+/+ CFs, which were significantly less in Bsg+/ CFs. Recombinant Bsg promoted ERK/Akt activities in CFs. Consistently, Bsg+/ CFs were significantly less proliferative compared with Bsg+/+ CFs. In the clinical study, serum levels of soluble Bsg (sBsg) were significantly elevated in patients with heart failure compared with healthy controls, significantly associated with poor prognosis. Conclusions: Bsg plays a crucial role in the pathogenesis of cardiac hypertrophy, fibrosis and failure in mice and humans.
YA2-4 The Novel Mitophagic Receptor Protein, Bcl2-like Protein 13: New Insights for the Molecular Mechanisms of the Pathogenesis of Heart Disease TOMOKAZU MURAKAWA1, OSAMU YAMAGUCHI1, KOJI OKAMOTO2, YASUSHI SAKATA1, KINYA OTSU3 1 Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine, Suita, Japan; 2Laboratory of Mitochondrial Dynamics, Graduate School of Frontier Biosciences, Osaka University, Suita, Japan; 3Cardiovascular Division, King’s College London British Heart Foundation Centre of Excellence, London, United Kingdom Cardiac function highly depends on energy generated by mitochondria, which are injured by various stresses such as pressure overload or aging. Damaged mitochondria in failing hearts are removed by a mitochondria-specific autophagy, called mitophagy. Dysregulation of mitophagy is implicated in the pathogenesis of heart disease such as heart failure. Mitophagy is closely associated with mitochondrial fission to make mitochondria engulfable size by autophagosomes. Atg32 is an essential protein for mitophagy in yeast. Some molecules have been reported to be involved in mitophagy, such as Parkin, FUNDC1 and Bnip3l. However, no Atg32 homologue has been identified in mammalian cells. By screening a public protein database for Atg32 homologues, we identified Bcl-2like protein 13 (Bcl2-L-13). Bcl2-L-13 induced mitochondrial fragmentation when overexpressed in neonatal rat cardiomyocyte. We carried out further investigation into functions of Bcl2-L-13 using HEK293 cells. Bcl2-L-13 is localized at the mitochondrial outer membrane and bound to LC3 through the WXXI motif and induced mitochondrial fragmentation and mitophagy. In Bcl2-L-13, the BH domains are important for mitochondrial fragmentation, while the WXXI motif facilitates mitophagy. Knockdown of Bcl2-L-13 attenuated mitochondrial damage-induced fragmentation and mitophagy. Furthermore, Bcl2-L-13 induced mitophagy in Atg32-deficient yeast. Induction and/or phosphorylation of this protein may regulate its activity. Our findings thus offer novel insights into molecular mechanisms of the pathogenesis of heart disease.
YA2-5 The Akt-mTOR Axis is a Pivotal Regulator of Eccentric Hypertrophy during Volume Overload MASATAKA IKEDA1, TOMOMI IDE1, TAKEO FUJINO1, KENJI SUNAGAWA2 1 Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan; 2Department of Therapeutic Regulation of Cardiovascular Homeostasis, Center for Disruptive Cardiovascular Medicine, Kyushu University, Fukuoka, Japan Background: The heart has two major modalities of hypertrophy in response to hemodynamic stress: concentric and eccentric hypertrophy caused by pressure and volume overload (VO), respectively. However, the molecular mechanism of eccentric hypertrophy remains to be fully elucidated. Methods and Results: We created an arteriovenous fistula in the abdominal aorta, inducing VO in mice. While mTOR in the heart was activated in a left ventricular end-diastolic pressure (LVEDP)dependent manner, mTOR inhibition not only suppressed eccentric hypertrophy but also induced cardiac atrophy even under VO. Notably, Akt was ubiquitinated and phosphorylated in response to VO, and blocking the recruitment of Akt to the membrane completely abolished mTOR activation during VO. Various growth factors including insulin-like growth factor, neuregulin-1, and connective tissue growth factor were upregulated, which contributed to AktmTOR activation under VO. Furthermore, the rate of eccentric hypertrophy progression was proportional to mTOR activity, which allowed accurate estimation of eccentric hypertrophy by time integration of mTOR activity during VO. Conclusion: These results suggested that the Akt-mTOR axis plays a pivotal role in eccentric hypertrophy, and mTOR activity quantitatively determines the rate of eccentric hypertrophy progression. As eccentric hypertrophy is an inherent system of the heart for regulating cardiac output and LVEDP, our findings provide a new mechanistic insight into the adaptive mechanism of the heart in heart failure.