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Increases in Ca Influx Induces Cardiomyocyte Hypertrophy In Vitro and In Vivo
The 11th Annual Scientific Meeting
HFSA
S71
Jay H. Cohn New Investigator Award: Basic Science 001
003
Increases in Ca Influx Induces Cardiomyocyte Hypertrophy In Vitro and In Vivo Xiongwen Chen1, Xiaoying Zhang2, Hongyu Zhang1, Hiroyuki Nakayama3, David Harris4, Rachel Wilson1, Jeffery Molkentin3, Steven Houser1; 1CVRC, Temple Univ, Philadelphia, PA; 2Fox Chase Cancer Center, Philadelphia, PA; 3Depart of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
A Novel Nuclear Function for GRK5 and Its Potential Role in the Hypertrophic Response Jeffrey S. Martini, Philip Raake, Leif E. Vinge, Brent R. DeGeorge, John K. Chuprun, David M. Harris, Andrea E. Eckhart, Julie A. Pitcher2, Walter J. Koch; 1 Center forTranslational Medicine, Thomas Jefferson University, Philadelphia, PA; 2 MRC Laboratory for Molecular Cell Biology and Department of Pharmacology, University College London, London, United Kingdom
Cardiac hypertrophy is a single independent risk factor for cardiovascular diseases. The mechanism for cardiac hypertrophy is still not well understood. A role of calcium (Ca) influx through the L-type calcium channel (LTCC) has been implicated in cardiomyocyte hypertrophy induced by adrenergic stimulation, angiotensin II and stretch in vitro. Here we tested if increased Ca influx through LTCC is sufficient to induce myocyte hypertrophy. Methods: Ca influx into cultured feline ventricular myocytes (VMs) and neonatal rat ventricular myocytes (NRVMs) was increased by infecting VMs with an adenovirus containing a fusion gene encoding the b2a subunit of the LTCC (b2a)-GFP. In vivo, we established transgenic mouse lines with inducible (Tet-off) overexpression of b2a gene (b2a TG). b2a could be turned on only in double transgenic mice (DTG) with both b2a and tTA genes and without deoxycycline offered. Results: (1) Overexpression the b2a subunit in cultured feline ventricular myocytes (FVM) increased cell volume (16%), protein abundance (25%) and ANF expression; (2) NFAT and HDAC translocations were increased in cultured b2a-VMs; (3) Blockade of LTCC (nifedipine), calcineurin (cyclosporine A and FK506) and CaMKII prevented the cell volume increase; (4) The surface area of NRVMs infected with Adb2a at 36 hours post infection was 105% larger than NRVMs infected with AdGFPs; (5) The LTCC current in DTG myocytes increased 77% (control: 13.7 6 0.7pA/pF, n 5 6 vs. TG: 24.3 6 2.6pA/pF, n 5 6; p ! 0.05); (6) The fractional shortening (control: 8.0 6 0.5%, n 5 16 vs. DTG: 11.9 6 1.1%, n 5 11) was also increased in VMs isolated from DTG mice; (7) Echocardigraphy showed significantly greater ejection fraction (control: 64.8 6 4.0%, n 5 7 vs. DTG: 79.1 6 2.4%, n 5 7, p ! 0.05), fractional shortening (control : 37.4 6 1.3%, n 5 7 vs. DTG: 46.9 6 2.3%, n 5 7, p ! 0.001)and posterior wall thickness (control: 1.03 6 0.05mm, n 5 7 vs. DTG: 1.32 6 0.03mm, n 5 7, p ! 0.001) in DTG mice; (8) The heart weight to body weight ratio in DTG mice (7.51 6 0.42mg/g, n 5 8) was significantly greater (p ! 0.05) than in control mice (6.25 6 0.21mg/g, n 5 23). Conclusion: The increase in Ca influx through the L-type calcium channel is sufficient to induce cardiomyocyte hypertrophy both in vitro and in vivo through a calcineurin/NFAT dependent mechanism.
002 Inhibition of Cardiomyocyte Apoptosis by Targeted Ablation of Bnip3 Restrains Post-Infarction Ventricular Remodeling Abhinav Diwan1, Maike Krenz2, Janaka Wansapura4, Xiaoping Ren3, R. Scott Dunn4, Amy Odley1, Jeffrey Robbins2, W. Keith Jones3, Gerald Dorn1; 1Molecular Cardiovascular Research, University of Cincinnati, Cincinnati, OH; 2Department of Pediatrics, Children’s Hospital, Cincinnati, OH; 3Department of Pharmacology, University of Cincinnati, Cincinnati, OH; 4Imaging Research Center, Children’s Hospital, Cincinnati, OH Introduction: Cardiomyocyte apoptosis contributes to infarct expansion and global ventricular dysfunction after myocardial infarction. Bnip3, a proapoptotic Bcl2 family protein, is transcriptionally upregulated in cardiomyocytes by hypoxia. We hypothesized that Bnip3 mediated cardiomyocyte apoptosis contributes to postinfarction left ventricular (LV) remodeling. Methods: We used gene targeting and conditional overexpression to evaluate the effects of Bnip3 on in vivo myocyte apoptosis, and determine the consequences of ablating murine Bnip3(KO) on LV remodeling after ischemia-reperfusion (IR) injury induced by reversible left anterior descending artery occlusion for 60 minutes. Results: Bnip3KO did not affect hearts of unstressed mice. After IR, Bnip3KO mice had no differences in mortality, early infarct size (by gadolinium enhanced MRI at 24 hours) or late infarct size (by MRI and pathology at 3 weeks) as compared to wild type (WT). Two days after IR, apoptosis was significantly diminished in Bnip3KO peri-infarct myocardium (6.6 6 0.5% vs 12.3 6 2.1% in WT; n 5 4, P 5 0.046) with a strong trend towards reduction in the remote myocardium (4.1 6 1.1% vs 7.1 6 1.2% in WT; P 5 0.075). Three weeks after IR, Bnip3KO mice exhibited preserved LV systolic performance as compared with a reduction in WT (change in LVEF from 24 hours to 3 weeks: 11 6 8% vs -20 6 7% in WT, n 5 8-9, P 5 0.015). This was accompanied by diminished LV dilation in Bnip3KO (change in LV end-diastolic volume: 46 6 11% vs 108 6 17% in WT, P 5 0.007; and change in LV end-systolic volume: 35 6 20% vs 181 6 41% in WT, P 5 0.005). Also, the myocardial scar was significantly thicker in Bnip3KO (0.91 6 0.12mm vs 0.61 6 0.55mm in WT, P 5 0.047). These observations suggest myocardial salvage by apoptosis inhibition following IR injury in Bnip3KO mice. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice (1.21 6 0.14% vs 0.17 6 0.02% in controls; n 5 4, P 5 0.029), causing progressive LV dilation and diminished systolic function recapitulating Bnip3-mediated post-IR remodeling. Conclusions: Postischemic cardiomyocyte apoptosis mediated by Bnip3 is an important determinant of LV remodeling in the infarcted heart.
G-protein coupled receptor kinases (GRKs) are critical regulators of adrenergic signaling in the heart. During heart failure (HF) GRK2 and GRK5 protein are elevated, leading to a diminished cardiac function. Mice with cardiac-specific overexpression of GRK5 have a unique phenotype in response to overload hypertrophy compared to control or GRK2 overexpressing mice. GRK5 mice but not GRK2 or control (NLC) mice rapidly decompensate within 4 weeks with signs of HF. GRK5, unlike GRK2 can reside in the nucleus and contains a nuclear localization (NLS) and export sequence (NES). Our hypothesis is that cardiac decompensation after pressure-overload in GRK5 mice is due to its unique activity in the nucleus. Histone deacetylases (HDAC) reside in the nucleus and act as transcriptional repressors of cardiac hypertrophy at the level of MEF2. Phosphorylation of HDACs results in activation of MEF2 and other hypertrophic genes. We have found that GRK5 can phosphorylate HDAC5 and associates with HDAC5 in myocytes. Further, we found nuclear export of HDAC5 when co-transfected with a nuclear form of GRK5 (GRK5DNES) while GRK5DNLS induced no HDAC translocation. Pressure-overload hypertrophy is triggered by Gq activation and we found that expression a constitutively active mutant Gaq in myocytes leads to GRK5 nuclear translocation and Gaq and GRK5 overexpression increases MEF2 activity. Our results indicate that GRK5 possesses nuclear HDAC kinase activity and this novel non-G protein-coupled receptor activity may play a key role in cardiac hypertrophy and HF.
004 Plasminogen Activator Inhibitor-1 Deficiency and Aging Contribute to Upregulation of Profibrotic Pathways, Cardiac Fibrosis, and Alterations of Myocardial Function in Mice William Bradham1, Linda Gleaves1, Mousumi Medda1, Douglas Vaughan1; 1 Medicine, Vanderbilt University Medical Center, Nashville, TN Cardiac fibrosis results from a variety of cardiac injury pathways. Perhaps underappreciated as a tissue diagnosis, cardiac fibrosis has deleterious functional consequences including systolic/diastolic abnormalities, and enhanced arrhythmia potential. The plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of cardiac fibrosis in mice. To investigate the interaction of PAI-1 and aging, this study assessed cardiac function by M-mode echocardiography, and analyzed tissue in four age groups (6, 12e14, 15e21, and 21.5e26 month old) of PAI-1 deficient (by gene knockout) (PKO)and wild-type (WT) control mice. The initial observed cardiac function alteration was reduced left ventricular (LV) diastolic internal dimension (p 5 0.0118) in the 15e21 month PKO group compared to WT. There was a trend, not yet statistically significant, toward increased LV posterior wall (LVPW) thickness in PKO compared to WT. In 21.5e26 month PKO, diastolic and systolic LVPW was thickened (p 5 0.0127, p 5 0.0212), diastolic and systolic LV internal dimension reduced (p 5 0.0486, p 5 0.0124), but LV fractional shortening preserved compared to WT. Myocardial tissue microscopy after animal sacrifice revealed fibrosis on the anterior epicardial surface of hearts in 18 month old PKO, while 26 month old PKO had extensive (10e17% by area) epicardial, perivascular, and interstitial fibrosis. No abnormal fibrosis was present in WT. Collagen immunohistochemistry (IHC) and electron microscopy revealed the fibrosis to consist of type I collagen fibrils. Real time polymerase chain reaction (RT-PCR) analyses of myocardial RNA revealed upregulation of TGF-B and fibroblast growth factor in PKO compared to WT (p 5 0.0234, p 5 0.037). Immunofluoresence confirmed this finding with bright TGF-B staining in intra-myocardial arterioles. As a surrogate for myocardial arteriole cells, thoracic aortic cells from aged (18e24 month) PKO and WT mice were grown in culture. Cell culture RT-PCR revealed 4 fold increased TGF-B and 17 fold increased SMAD3 (p ! 0.05 for both) RNA levels in PKO, indicating upregulation of a profibrotic TGF-B/SMAD tissue signaling pathway. Phosphorylated SMAD2/3, the downstream TGF-B signaling mediator, had extensive IHC staining in the fibrotic areas. Murine PAI-1 deficiency represents a new model of cardiac fibrosis, and the present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways.
HFSA
S71
Jay H. Cohn New Investigator Award: Basic Science 001
003
Increases in Ca Influx Induces Cardiomyocyte Hypertrophy In Vitro and In Vivo Xiongwen Chen1, Xiaoying Zhang2, Hongyu Zhang1, Hiroyuki Nakayama3, David Harris4, Rachel Wilson1, Jeffery Molkentin3, Steven Houser1; 1CVRC, Temple Univ, Philadelphia, PA; 2Fox Chase Cancer Center, Philadelphia, PA; 3Depart of Pediatrics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH
A Novel Nuclear Function for GRK5 and Its Potential Role in the Hypertrophic Response Jeffrey S. Martini, Philip Raake, Leif E. Vinge, Brent R. DeGeorge, John K. Chuprun, David M. Harris, Andrea E. Eckhart, Julie A. Pitcher2, Walter J. Koch; 1 Center forTranslational Medicine, Thomas Jefferson University, Philadelphia, PA; 2 MRC Laboratory for Molecular Cell Biology and Department of Pharmacology, University College London, London, United Kingdom
Cardiac hypertrophy is a single independent risk factor for cardiovascular diseases. The mechanism for cardiac hypertrophy is still not well understood. A role of calcium (Ca) influx through the L-type calcium channel (LTCC) has been implicated in cardiomyocyte hypertrophy induced by adrenergic stimulation, angiotensin II and stretch in vitro. Here we tested if increased Ca influx through LTCC is sufficient to induce myocyte hypertrophy. Methods: Ca influx into cultured feline ventricular myocytes (VMs) and neonatal rat ventricular myocytes (NRVMs) was increased by infecting VMs with an adenovirus containing a fusion gene encoding the b2a subunit of the LTCC (b2a)-GFP. In vivo, we established transgenic mouse lines with inducible (Tet-off) overexpression of b2a gene (b2a TG). b2a could be turned on only in double transgenic mice (DTG) with both b2a and tTA genes and without deoxycycline offered. Results: (1) Overexpression the b2a subunit in cultured feline ventricular myocytes (FVM) increased cell volume (16%), protein abundance (25%) and ANF expression; (2) NFAT and HDAC translocations were increased in cultured b2a-VMs; (3) Blockade of LTCC (nifedipine), calcineurin (cyclosporine A and FK506) and CaMKII prevented the cell volume increase; (4) The surface area of NRVMs infected with Adb2a at 36 hours post infection was 105% larger than NRVMs infected with AdGFPs; (5) The LTCC current in DTG myocytes increased 77% (control: 13.7 6 0.7pA/pF, n 5 6 vs. TG: 24.3 6 2.6pA/pF, n 5 6; p ! 0.05); (6) The fractional shortening (control: 8.0 6 0.5%, n 5 16 vs. DTG: 11.9 6 1.1%, n 5 11) was also increased in VMs isolated from DTG mice; (7) Echocardigraphy showed significantly greater ejection fraction (control: 64.8 6 4.0%, n 5 7 vs. DTG: 79.1 6 2.4%, n 5 7, p ! 0.05), fractional shortening (control : 37.4 6 1.3%, n 5 7 vs. DTG: 46.9 6 2.3%, n 5 7, p ! 0.001)and posterior wall thickness (control: 1.03 6 0.05mm, n 5 7 vs. DTG: 1.32 6 0.03mm, n 5 7, p ! 0.001) in DTG mice; (8) The heart weight to body weight ratio in DTG mice (7.51 6 0.42mg/g, n 5 8) was significantly greater (p ! 0.05) than in control mice (6.25 6 0.21mg/g, n 5 23). Conclusion: The increase in Ca influx through the L-type calcium channel is sufficient to induce cardiomyocyte hypertrophy both in vitro and in vivo through a calcineurin/NFAT dependent mechanism.
002 Inhibition of Cardiomyocyte Apoptosis by Targeted Ablation of Bnip3 Restrains Post-Infarction Ventricular Remodeling Abhinav Diwan1, Maike Krenz2, Janaka Wansapura4, Xiaoping Ren3, R. Scott Dunn4, Amy Odley1, Jeffrey Robbins2, W. Keith Jones3, Gerald Dorn1; 1Molecular Cardiovascular Research, University of Cincinnati, Cincinnati, OH; 2Department of Pediatrics, Children’s Hospital, Cincinnati, OH; 3Department of Pharmacology, University of Cincinnati, Cincinnati, OH; 4Imaging Research Center, Children’s Hospital, Cincinnati, OH Introduction: Cardiomyocyte apoptosis contributes to infarct expansion and global ventricular dysfunction after myocardial infarction. Bnip3, a proapoptotic Bcl2 family protein, is transcriptionally upregulated in cardiomyocytes by hypoxia. We hypothesized that Bnip3 mediated cardiomyocyte apoptosis contributes to postinfarction left ventricular (LV) remodeling. Methods: We used gene targeting and conditional overexpression to evaluate the effects of Bnip3 on in vivo myocyte apoptosis, and determine the consequences of ablating murine Bnip3(KO) on LV remodeling after ischemia-reperfusion (IR) injury induced by reversible left anterior descending artery occlusion for 60 minutes. Results: Bnip3KO did not affect hearts of unstressed mice. After IR, Bnip3KO mice had no differences in mortality, early infarct size (by gadolinium enhanced MRI at 24 hours) or late infarct size (by MRI and pathology at 3 weeks) as compared to wild type (WT). Two days after IR, apoptosis was significantly diminished in Bnip3KO peri-infarct myocardium (6.6 6 0.5% vs 12.3 6 2.1% in WT; n 5 4, P 5 0.046) with a strong trend towards reduction in the remote myocardium (4.1 6 1.1% vs 7.1 6 1.2% in WT; P 5 0.075). Three weeks after IR, Bnip3KO mice exhibited preserved LV systolic performance as compared with a reduction in WT (change in LVEF from 24 hours to 3 weeks: 11 6 8% vs -20 6 7% in WT, n 5 8-9, P 5 0.015). This was accompanied by diminished LV dilation in Bnip3KO (change in LV end-diastolic volume: 46 6 11% vs 108 6 17% in WT, P 5 0.007; and change in LV end-systolic volume: 35 6 20% vs 181 6 41% in WT, P 5 0.005). Also, the myocardial scar was significantly thicker in Bnip3KO (0.91 6 0.12mm vs 0.61 6 0.55mm in WT, P 5 0.047). These observations suggest myocardial salvage by apoptosis inhibition following IR injury in Bnip3KO mice. Forced cardiac expression of Bnip3 increased cardiomyocyte apoptosis in unstressed mice (1.21 6 0.14% vs 0.17 6 0.02% in controls; n 5 4, P 5 0.029), causing progressive LV dilation and diminished systolic function recapitulating Bnip3-mediated post-IR remodeling. Conclusions: Postischemic cardiomyocyte apoptosis mediated by Bnip3 is an important determinant of LV remodeling in the infarcted heart.
G-protein coupled receptor kinases (GRKs) are critical regulators of adrenergic signaling in the heart. During heart failure (HF) GRK2 and GRK5 protein are elevated, leading to a diminished cardiac function. Mice with cardiac-specific overexpression of GRK5 have a unique phenotype in response to overload hypertrophy compared to control or GRK2 overexpressing mice. GRK5 mice but not GRK2 or control (NLC) mice rapidly decompensate within 4 weeks with signs of HF. GRK5, unlike GRK2 can reside in the nucleus and contains a nuclear localization (NLS) and export sequence (NES). Our hypothesis is that cardiac decompensation after pressure-overload in GRK5 mice is due to its unique activity in the nucleus. Histone deacetylases (HDAC) reside in the nucleus and act as transcriptional repressors of cardiac hypertrophy at the level of MEF2. Phosphorylation of HDACs results in activation of MEF2 and other hypertrophic genes. We have found that GRK5 can phosphorylate HDAC5 and associates with HDAC5 in myocytes. Further, we found nuclear export of HDAC5 when co-transfected with a nuclear form of GRK5 (GRK5DNES) while GRK5DNLS induced no HDAC translocation. Pressure-overload hypertrophy is triggered by Gq activation and we found that expression a constitutively active mutant Gaq in myocytes leads to GRK5 nuclear translocation and Gaq and GRK5 overexpression increases MEF2 activity. Our results indicate that GRK5 possesses nuclear HDAC kinase activity and this novel non-G protein-coupled receptor activity may play a key role in cardiac hypertrophy and HF.
004 Plasminogen Activator Inhibitor-1 Deficiency and Aging Contribute to Upregulation of Profibrotic Pathways, Cardiac Fibrosis, and Alterations of Myocardial Function in Mice William Bradham1, Linda Gleaves1, Mousumi Medda1, Douglas Vaughan1; 1 Medicine, Vanderbilt University Medical Center, Nashville, TN Cardiac fibrosis results from a variety of cardiac injury pathways. Perhaps underappreciated as a tissue diagnosis, cardiac fibrosis has deleterious functional consequences including systolic/diastolic abnormalities, and enhanced arrhythmia potential. The plasminogen activator inhibitor-1 (PAI-1) has been implicated in the pathogenesis of cardiac fibrosis in mice. To investigate the interaction of PAI-1 and aging, this study assessed cardiac function by M-mode echocardiography, and analyzed tissue in four age groups (6, 12e14, 15e21, and 21.5e26 month old) of PAI-1 deficient (by gene knockout) (PKO)and wild-type (WT) control mice. The initial observed cardiac function alteration was reduced left ventricular (LV) diastolic internal dimension (p 5 0.0118) in the 15e21 month PKO group compared to WT. There was a trend, not yet statistically significant, toward increased LV posterior wall (LVPW) thickness in PKO compared to WT. In 21.5e26 month PKO, diastolic and systolic LVPW was thickened (p 5 0.0127, p 5 0.0212), diastolic and systolic LV internal dimension reduced (p 5 0.0486, p 5 0.0124), but LV fractional shortening preserved compared to WT. Myocardial tissue microscopy after animal sacrifice revealed fibrosis on the anterior epicardial surface of hearts in 18 month old PKO, while 26 month old PKO had extensive (10e17% by area) epicardial, perivascular, and interstitial fibrosis. No abnormal fibrosis was present in WT. Collagen immunohistochemistry (IHC) and electron microscopy revealed the fibrosis to consist of type I collagen fibrils. Real time polymerase chain reaction (RT-PCR) analyses of myocardial RNA revealed upregulation of TGF-B and fibroblast growth factor in PKO compared to WT (p 5 0.0234, p 5 0.037). Immunofluoresence confirmed this finding with bright TGF-B staining in intra-myocardial arterioles. As a surrogate for myocardial arteriole cells, thoracic aortic cells from aged (18e24 month) PKO and WT mice were grown in culture. Cell culture RT-PCR revealed 4 fold increased TGF-B and 17 fold increased SMAD3 (p ! 0.05 for both) RNA levels in PKO, indicating upregulation of a profibrotic TGF-B/SMAD tissue signaling pathway. Phosphorylated SMAD2/3, the downstream TGF-B signaling mediator, had extensive IHC staining in the fibrotic areas. Murine PAI-1 deficiency represents a new model of cardiac fibrosis, and the present study is one of the first to elucidate some of the functional consequences and relevant molecular signaling pathways.