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Reversal of cardiac fibrosis following LVAD implantation: the cardiac mast cell
The 7th Annual Scientific Meeting
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HFSA
S7
Moderated Posters 016
017
Muscarinic Receptor Stimulation Fails to Modify Beta-Adrenergic Activation in Left Ventricular Myocytes from Neuregulin-1 Knockout Mice Katashi Okoshi,1 Masaharu Nakayama,1 Xinhua Yan,1 Marina P. Okoshi,1 Adam J.T. Schuldt,1 Mark A. Marchionni,2 Beverly H. Lorell1—1Cardiovascular Division, Beth Israel Deaconess Medical Center - Harvard Medical School, Boston, MA; 2NRG Biotech, Arlington, MA
NRSF is a Transcriptional Upstream Regulator for Sudden Cardiac Death and Heart Failure Koichiro Kuwahara,1 Yoshihiko Saito,1 Makoto Takano,2 Yuji Arai,3 Shinji Yasuno,1 Nobuki Takahashi,1 Yasuaki Nakagawa,1 Genzo Takemura,4 Minoru Horie,5 Yoshihiro Miyamoto,6 Takayuki Morisaki,3 Kuratomi Shinobu,2 Akinori Noma,2 Hisayoshi Fujiwara,4 Hideyuki Kinoshita,1 Masaki Harada,1 Kazuwa Nakao1—1Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan; 2Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Kyoto, Japan; 3Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan; 4Second Internal Medicine, Gifu University, Gifu, Gifu, Japan; 5Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan; 6Atheroscrelosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, Japan
The suppression of neuregulin-erbB receptor signaling in injured hearts in humans is associated with an increased risk of heart failure. One of the major biologic effects of neuregulin signaling is stimulation of acetylcholine nicotinic receptor synthesis whereas effects on muscarinic receptors are unclear. If muscarinic receptors are modified, a possible contributing mechanism for development of heart failure is a hyper-adrenergic activation due to a reduction of cardiac parasympathetic signaling. We tested the hypothesis that parasympathetic modulation of β-adrenergic stimulation is depressed in adult myocytes from transgenic mice heterozygous for knockout of the neuregulin-1 gene (KO). Methods: We evaluated isolated left ventricular myocyte contraction and intracellular Ca2⫹ transients ([Ca2⫹]i) from KO (n ⫽ 48 experiments from 9 animals) and wildtype mice (WT; n ⫽ 38 experiments from 9 animals). Fractional cell shortening and intracellular Ca2⫹ were measured simultaneously in isolated myocytes loaded with the Ca2⫹ sensitive fluorescence indicator fluo-3. Under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca2⫹]o, 25ºC), characteristics of myocyte contraction/relengthening and systolic/diastolic [Ca2⫹]i were not different between WT and KO myocytes. β-adrenergic stimulation was then imposed by perfusion with 1 µmol/L isoproterenol. Modulation of the hyper-adrenergic state by the muscarinic agonist carbachol was then assessed. Results: The steady-state increases in fractional shortening (FS) and peak systolic [Ca2⫹]i in response to isoproterenol were similar in both groups (WT FS: 10.0 ⫾ 0.74% to 16.5 ⫾ 0.58%; KO FS: 9.85 ⫾ 0.64% to 17.5 ⫾ 0.54%; WT peak systolic [Ca2⫹]i: 416 ⫾ 20 to 623 ⫾ 28 nmol/L; KO peak systolic [Ca2⫹]i: 418 ⫾ 23 to 651 ⫾ 32 nmol/L). In WT myocytes stimulated with isoproterenol, 10 and 100 µmol/L carbachol caused a dose-dependent decrease in FS (from 16.4 ⫾ 0.82% to 14.5 ⫾ 0.78% and 13.5 ⫾ 0.81%, respectively) and peak systolic [Ca2⫹]i (from 604 ⫾ 35 to 564 ⫾ 32 and 542 ⫾ 34 nmol/L, respectively, both P ⫽ 0.001). In KO myocytes, 10 and 100 µmol/L carbachol failed to attenuate either FS (from 17.3 ⫾ 0.79% to 16.5 ⫾ 0.80% and 15.9 ⫾ 0.84%, respectively) or peak systolic [Ca2⫹]i (from 672 ⫾ 52 to 662 ⫾ 49 and 650 ⫾ 56 nmol/L, respectively, both P ⫽ NS). In absence of isoproterenol, carbachol did not modify contractile function. Conclusion: In myocytes from neuregulin-1 KO mice, muscarinic receptor stimulation does not attenuate the hyper-adrenergic state. These results suggest that hearts deficient in neuregulin signaling are unable to adequately counterbalance β-adrenergic activation by inhibitory parasympathetic activity. This may contribute to development of heart failure in injured hearts when neuregulin-erbB signaling is depressed.
Reactivation of a fetal cardiac gene program is a characteristic feature of failing hearts and correlates with impaired cardiac function and poor prognosis. However, the entire mechanisms governing the reversible expression of fetal cardiac genes remain unresolved. Recently we have shown that a repressor element, neuron-restrictive silencer element (NRSE) represses the promoter activities of atrial and brain natriuretic peptide (ANP and BNP, respectively) genes through the recruitment of a transcriptional repressor neuron-restrictive silencer factor (NRSF) - histone deacetylases (HDACs) complex in cultured ventricular myocytes. In addition, we have shown that the attenuation of NRSE-mediated repression contributes to the induction of their promoter activities in response to hypertrophic stimuli. NRSE also exists in α-skeletal actin gene, suggesting that NRSF regulates multiple fetal cardiac genes expression. However, roles of NRSF in the regulation of cardiac function have not been elucidated. To address this question, we made transgenic (Tg) mice expressing a dominant-negative mutant of NRSF (dnNRSF) in the heart. The inhibition of NRSF selectively upregulated the ventricular expression of ANP, BNP and α-skeletal actin genes. The dnNRSF Tg mice showed sudden death with 70–95% mortality by 40 weeks of age. Echocardiographical, hemodynamical and histological analysis demonstrated that 20-weeks-old Tg mice, but not 8-weeks old Tg mice, show the characteristic features of dilated cardiomyopathy. The electrocardiographic data obtained from Tg mice at the time of death showed VT/VF followed by asystole. An intracardiac electrophysiological study demonstrated high susceptibility to arrhythmias in the hearts of 12weeks-old Tg mice, indicating that the increased arrhythmogenicity precedes the establishment of cardiomyopathy. A cDNA micro array analysis revealed that the expression of genes that encodes two ion channels carrying fetal cardiac currents If and ICa,T is increased in 8-weeks-old Tg ventricles, and we confirmed the increase in If and ICa,T in Tg ventricles. Furthermore, we identified NRSE in these genes suggesting that NRSF regulates these fetal cardiac ion channels and that the alteration of these ionic currents contributes to the increased arrhythmogenicity in Tg mice. These currents have been reported to be induced in failing hearts as well as ANP and BNP. Taken together, our results indicate NRSF to be a key transcriptional regulator of the expression of multiple fetal cardiac genes including cardiac natriuretic peptides, a fetal isoform of contractile protein and fetal ionic currents. This study suggests an important role of NRSF in maintaining normal cardiac structure and function and in mediating common upstream signaling pathways leading to heart failure and sudden cardiac death.
018
019
Transgenic Mice Over-Expressing the g2 Subunit of AMP-Activated Protein Kinase: A Model of Hypertrophic Cardiomyopathy and Wolff-ParkinsonWhite Syndrome Joanna K. Davies,1 David Carling1—1Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College, London, United Kingdom
Reversal of Cardiac Fibrosis Following LVAD Implantation: The Cardiac Mast Cell Keith A. Youker,1 Javier A. Lafuente,1 Christian Skrabal,1 Larry O. Thompson,1 Matthias Loebe,1 George P. Noon1—1Surgery, Division of Transplantation, Baylor College of Medicine, Houston, TX
AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy metabolism. It is a heterotrimeric complex comprising 3 subunits, α, β and γ, of which there are two isofroms of α (1 & 2), two isoforms of β (1 & 2) and three isoforms of γ (1, 2 & 3). The α-subunit is the catalytic domain of the complex and, as yet, the main roles of the β and γ subunits remain unclear. In recent years, a number of missense mutations and the insertion of a leucine residue have been identified in the gene for the γ2 subunit of AMPK, PRKAG2, as being involved in some instances of familial hypertrophic cardiomyopathy (FHM) with Wolff-Parkinson-White syndrome (WPW). In 2001, Gollob et al identified a mutation in exon 15 of PRKAG2, Arg531Gly, in a family exhibiting WPW with early childhood onset. They observed that, although this particular family suffered ventricular preexcitation and atrial fibrillation, they were not presenting any indication of hypertrophy. Lines of transgenic mice have been produced using the human PRKAG2 cDNA, with and without the Arg531Gly mutation, which are currently under study. We have used the α-myosin heavy chain promoter which leads to cardiac-specific expression of the protein. Transgenic mice harbouring the Arg531Gly mutation display an obvious cardiac phenotype, including marked hypertrophy. Using γ2-specific antibodies, AMPK activity in heart was determined. Wild-type γ2 activity was similar to endogenous AMPK isolated from non-transgenic animals. In contrast, the Arg531Gly γ2 activity was significantly lower and was markedly less AMP-dependent which is consistent with our previous study examining the effect of the γ2 mutations in transiently transfected mammalian cells.
Hypertrophy and fibrosis represent structural changes in the myocardium which occur in heart failure and decrease the ability of the heart to contract. We have previously demonstrated that reduction of load by LVAD support induces a decrease in cardiac interstitial fibrosis as well as myocyte size. We also have reported that cardiac mast cells increase in heart failure and that a secondary increase occurs post LVAD. The secondary increase, however, is composed of primarily chymase negative mast cells and these mast cells show an altered (reduced) bFGF content. In this study we investigated the direct effects of cardiac mast cells isolated from myocardium from explanted hearts at the time of transplantation (heart failure) and from LVAD supported hearts at the time of device removal and transplantation (post LVAD). The isolated mast cells were co-cultured with cultured human lung fibroblasts which were sub optimally stimulated with 2% fetal calf serum. Following 12 hours co-culture, cardiac mast cells from heart failure induced a 2-fold increase in protein incorporation and proliferation in the cell cultures. The post-LVAD mast cells however induced a 50% decrease in protein incorporation and proliferation. Additional studies using supernatants from 2 hour stimulated mast cells, demonstrated the same degree of effect on the cultured proliferation and protein production, indicating the release of factors from the mast cells capable of altering fibroblast function. Additionally, immunohistochemical studies on myocardial tissue samples show a marked reduction in the expression of HSP47 (a chaperone protein required for collagen export from fibroblasts) following LVAD support. We propose that the fibrotic changes seen post LVAD represent a reduction in pro-fibrotic signals present in failing myocardium.
•
HFSA
S7
Moderated Posters 016
017
Muscarinic Receptor Stimulation Fails to Modify Beta-Adrenergic Activation in Left Ventricular Myocytes from Neuregulin-1 Knockout Mice Katashi Okoshi,1 Masaharu Nakayama,1 Xinhua Yan,1 Marina P. Okoshi,1 Adam J.T. Schuldt,1 Mark A. Marchionni,2 Beverly H. Lorell1—1Cardiovascular Division, Beth Israel Deaconess Medical Center - Harvard Medical School, Boston, MA; 2NRG Biotech, Arlington, MA
NRSF is a Transcriptional Upstream Regulator for Sudden Cardiac Death and Heart Failure Koichiro Kuwahara,1 Yoshihiko Saito,1 Makoto Takano,2 Yuji Arai,3 Shinji Yasuno,1 Nobuki Takahashi,1 Yasuaki Nakagawa,1 Genzo Takemura,4 Minoru Horie,5 Yoshihiro Miyamoto,6 Takayuki Morisaki,3 Kuratomi Shinobu,2 Akinori Noma,2 Hisayoshi Fujiwara,4 Hideyuki Kinoshita,1 Masaki Harada,1 Kazuwa Nakao1—1Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan; 2Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Kyoto, Japan; 3Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, Japan; 4Second Internal Medicine, Gifu University, Gifu, Gifu, Japan; 5Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, Kyoto, Japan; 6Atheroscrelosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, Japan
The suppression of neuregulin-erbB receptor signaling in injured hearts in humans is associated with an increased risk of heart failure. One of the major biologic effects of neuregulin signaling is stimulation of acetylcholine nicotinic receptor synthesis whereas effects on muscarinic receptors are unclear. If muscarinic receptors are modified, a possible contributing mechanism for development of heart failure is a hyper-adrenergic activation due to a reduction of cardiac parasympathetic signaling. We tested the hypothesis that parasympathetic modulation of β-adrenergic stimulation is depressed in adult myocytes from transgenic mice heterozygous for knockout of the neuregulin-1 gene (KO). Methods: We evaluated isolated left ventricular myocyte contraction and intracellular Ca2⫹ transients ([Ca2⫹]i) from KO (n ⫽ 48 experiments from 9 animals) and wildtype mice (WT; n ⫽ 38 experiments from 9 animals). Fractional cell shortening and intracellular Ca2⫹ were measured simultaneously in isolated myocytes loaded with the Ca2⫹ sensitive fluorescence indicator fluo-3. Under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca2⫹]o, 25ºC), characteristics of myocyte contraction/relengthening and systolic/diastolic [Ca2⫹]i were not different between WT and KO myocytes. β-adrenergic stimulation was then imposed by perfusion with 1 µmol/L isoproterenol. Modulation of the hyper-adrenergic state by the muscarinic agonist carbachol was then assessed. Results: The steady-state increases in fractional shortening (FS) and peak systolic [Ca2⫹]i in response to isoproterenol were similar in both groups (WT FS: 10.0 ⫾ 0.74% to 16.5 ⫾ 0.58%; KO FS: 9.85 ⫾ 0.64% to 17.5 ⫾ 0.54%; WT peak systolic [Ca2⫹]i: 416 ⫾ 20 to 623 ⫾ 28 nmol/L; KO peak systolic [Ca2⫹]i: 418 ⫾ 23 to 651 ⫾ 32 nmol/L). In WT myocytes stimulated with isoproterenol, 10 and 100 µmol/L carbachol caused a dose-dependent decrease in FS (from 16.4 ⫾ 0.82% to 14.5 ⫾ 0.78% and 13.5 ⫾ 0.81%, respectively) and peak systolic [Ca2⫹]i (from 604 ⫾ 35 to 564 ⫾ 32 and 542 ⫾ 34 nmol/L, respectively, both P ⫽ 0.001). In KO myocytes, 10 and 100 µmol/L carbachol failed to attenuate either FS (from 17.3 ⫾ 0.79% to 16.5 ⫾ 0.80% and 15.9 ⫾ 0.84%, respectively) or peak systolic [Ca2⫹]i (from 672 ⫾ 52 to 662 ⫾ 49 and 650 ⫾ 56 nmol/L, respectively, both P ⫽ NS). In absence of isoproterenol, carbachol did not modify contractile function. Conclusion: In myocytes from neuregulin-1 KO mice, muscarinic receptor stimulation does not attenuate the hyper-adrenergic state. These results suggest that hearts deficient in neuregulin signaling are unable to adequately counterbalance β-adrenergic activation by inhibitory parasympathetic activity. This may contribute to development of heart failure in injured hearts when neuregulin-erbB signaling is depressed.
Reactivation of a fetal cardiac gene program is a characteristic feature of failing hearts and correlates with impaired cardiac function and poor prognosis. However, the entire mechanisms governing the reversible expression of fetal cardiac genes remain unresolved. Recently we have shown that a repressor element, neuron-restrictive silencer element (NRSE) represses the promoter activities of atrial and brain natriuretic peptide (ANP and BNP, respectively) genes through the recruitment of a transcriptional repressor neuron-restrictive silencer factor (NRSF) - histone deacetylases (HDACs) complex in cultured ventricular myocytes. In addition, we have shown that the attenuation of NRSE-mediated repression contributes to the induction of their promoter activities in response to hypertrophic stimuli. NRSE also exists in α-skeletal actin gene, suggesting that NRSF regulates multiple fetal cardiac genes expression. However, roles of NRSF in the regulation of cardiac function have not been elucidated. To address this question, we made transgenic (Tg) mice expressing a dominant-negative mutant of NRSF (dnNRSF) in the heart. The inhibition of NRSF selectively upregulated the ventricular expression of ANP, BNP and α-skeletal actin genes. The dnNRSF Tg mice showed sudden death with 70–95% mortality by 40 weeks of age. Echocardiographical, hemodynamical and histological analysis demonstrated that 20-weeks-old Tg mice, but not 8-weeks old Tg mice, show the characteristic features of dilated cardiomyopathy. The electrocardiographic data obtained from Tg mice at the time of death showed VT/VF followed by asystole. An intracardiac electrophysiological study demonstrated high susceptibility to arrhythmias in the hearts of 12weeks-old Tg mice, indicating that the increased arrhythmogenicity precedes the establishment of cardiomyopathy. A cDNA micro array analysis revealed that the expression of genes that encodes two ion channels carrying fetal cardiac currents If and ICa,T is increased in 8-weeks-old Tg ventricles, and we confirmed the increase in If and ICa,T in Tg ventricles. Furthermore, we identified NRSE in these genes suggesting that NRSF regulates these fetal cardiac ion channels and that the alteration of these ionic currents contributes to the increased arrhythmogenicity in Tg mice. These currents have been reported to be induced in failing hearts as well as ANP and BNP. Taken together, our results indicate NRSF to be a key transcriptional regulator of the expression of multiple fetal cardiac genes including cardiac natriuretic peptides, a fetal isoform of contractile protein and fetal ionic currents. This study suggests an important role of NRSF in maintaining normal cardiac structure and function and in mediating common upstream signaling pathways leading to heart failure and sudden cardiac death.
018
019
Transgenic Mice Over-Expressing the g2 Subunit of AMP-Activated Protein Kinase: A Model of Hypertrophic Cardiomyopathy and Wolff-ParkinsonWhite Syndrome Joanna K. Davies,1 David Carling1—1Cellular Stress Group, MRC Clinical Sciences Centre, Imperial College, London, United Kingdom
Reversal of Cardiac Fibrosis Following LVAD Implantation: The Cardiac Mast Cell Keith A. Youker,1 Javier A. Lafuente,1 Christian Skrabal,1 Larry O. Thompson,1 Matthias Loebe,1 George P. Noon1—1Surgery, Division of Transplantation, Baylor College of Medicine, Houston, TX
AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy metabolism. It is a heterotrimeric complex comprising 3 subunits, α, β and γ, of which there are two isofroms of α (1 & 2), two isoforms of β (1 & 2) and three isoforms of γ (1, 2 & 3). The α-subunit is the catalytic domain of the complex and, as yet, the main roles of the β and γ subunits remain unclear. In recent years, a number of missense mutations and the insertion of a leucine residue have been identified in the gene for the γ2 subunit of AMPK, PRKAG2, as being involved in some instances of familial hypertrophic cardiomyopathy (FHM) with Wolff-Parkinson-White syndrome (WPW). In 2001, Gollob et al identified a mutation in exon 15 of PRKAG2, Arg531Gly, in a family exhibiting WPW with early childhood onset. They observed that, although this particular family suffered ventricular preexcitation and atrial fibrillation, they were not presenting any indication of hypertrophy. Lines of transgenic mice have been produced using the human PRKAG2 cDNA, with and without the Arg531Gly mutation, which are currently under study. We have used the α-myosin heavy chain promoter which leads to cardiac-specific expression of the protein. Transgenic mice harbouring the Arg531Gly mutation display an obvious cardiac phenotype, including marked hypertrophy. Using γ2-specific antibodies, AMPK activity in heart was determined. Wild-type γ2 activity was similar to endogenous AMPK isolated from non-transgenic animals. In contrast, the Arg531Gly γ2 activity was significantly lower and was markedly less AMP-dependent which is consistent with our previous study examining the effect of the γ2 mutations in transiently transfected mammalian cells.
Hypertrophy and fibrosis represent structural changes in the myocardium which occur in heart failure and decrease the ability of the heart to contract. We have previously demonstrated that reduction of load by LVAD support induces a decrease in cardiac interstitial fibrosis as well as myocyte size. We also have reported that cardiac mast cells increase in heart failure and that a secondary increase occurs post LVAD. The secondary increase, however, is composed of primarily chymase negative mast cells and these mast cells show an altered (reduced) bFGF content. In this study we investigated the direct effects of cardiac mast cells isolated from myocardium from explanted hearts at the time of transplantation (heart failure) and from LVAD supported hearts at the time of device removal and transplantation (post LVAD). The isolated mast cells were co-cultured with cultured human lung fibroblasts which were sub optimally stimulated with 2% fetal calf serum. Following 12 hours co-culture, cardiac mast cells from heart failure induced a 2-fold increase in protein incorporation and proliferation in the cell cultures. The post-LVAD mast cells however induced a 50% decrease in protein incorporation and proliferation. Additional studies using supernatants from 2 hour stimulated mast cells, demonstrated the same degree of effect on the cultured proliferation and protein production, indicating the release of factors from the mast cells capable of altering fibroblast function. Additionally, immunohistochemical studies on myocardial tissue samples show a marked reduction in the expression of HSP47 (a chaperone protein required for collagen export from fibroblasts) following LVAD support. We propose that the fibrotic changes seen post LVAD represent a reduction in pro-fibrotic signals present in failing myocardium.