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FIBROBLAST GROWTH FACTOR (FGF-2) PREVENTS HUMAN CARDIAC FIBROBLAST-MEDIATED EXTRACELLULAR MATRIX REMODELING
Abstracts CONCLUSION:
In this pilot study there were no observable histological differences in human right atrial tissue from individuals at high- and low-risk for OSA. Further investigation would be required for more definitive results.
Canadian Cardiovascular Society (CCS) Oral THE HUMAN MYOCYTE Saturday, October 25, 2014
S153
myofibrotic phenotype. In contrast, FGF-2 treatment resulted in shorter cell extensions with a phenotype more consistent with quiescent fibroblasts. In addition, FGF-2 attenuated fibroblast-mediated matrix compaction relative to the TGF-b group. CONCLUSION: FGF-2 attenuates TGF- b1 mediated human cardiac fibroblast activation and restores ECM homeostasis and regulation. These data provide proof-of-concept evidence for FGF-2’s therapeutic potential in preventing adverse cardiac remodeling and subsequently heart failure.
Trainee Research Award Finalist e Basic Science 196 FIBROBLAST GROWTH FACTOR (FGF-2) PREVENTS HUMAN CARDIAC FIBROBLAST-MEDIATED EXTRACELLULAR MATRIX REMODELING DA Svystonyuk, JM Ngu, HE Mewhort, DG Guzzardi, BD Lipon, D Park, G Teng, DD Belke, PW Fedak Calgary, Alberta BACKGROUND: After myocardial injury, elevation of profibrotic cytokine transforming growth factor-b1 (TGF-b1) results in myofibroblast differentiation leading to matrix remodeling and progression of heart failure. Fibroblast growth factor (FGF-2) has the potential to antagonize TGF-b1’s profibrotic actions. In this study, we examine the effects of FGF-2 on human cardiac fibroblast mediated extracellular matrix remodeling. METHODS AND RESULTS: Human cardiac fibroblasts were isolated from atrial or ventricular heart biopsies and seeded into 3D collagen matrices. Myofibroblast activation was functionally assessed by the extent of matrix contraction. As compared to baseline myofibroblast activity, FGF-2 attenuated TGF-b1 mediated myofibroblast activation (1.06 versus 1.17, P<0.0001) using cells isolated from atrial tissue. FGF2 similarly attenuated TGF-b1 mediated myofibroblast activation in cells isolated from ventricular core biopsies (1.51 vs. 1.12, P<0.05). New collagen synthesis was assessed by 3H-proline incorporation assay. Fibroblasts treated with TGF-b1 demonstrated an increase in collagen synthesis while addition of FGF-2 reduced collagen synthesis (P<0.05). ECM dysregulation was investigated by in situ zymography using embedded Gelatin-FITC to measure matrix proteolysis. TGF-b1 increased total protease activity in the matrix microenvironment while FGF-2 attenuated this response (2.81 x 109 1.07 x 109 vs. 1.4 x 109 4.49 x108 total florescence units, P<0.01). Quantitative RT-PCR and multiplex analysis assessed mRNA and protein profiles for TIMP-1 and TIMP-2. FGF-2 treatment restored TIMP1 and TIMP-2 gene and protein expression relative to TGFb1 treated cells (P<0.05 and P<0.01, respectively). ECM remodeling adjacent to activated fibroblasts was examined within thin nylon-based collagen matrices using confocal microscopy. Figure 1 shows TGF-b1 activated fibroblasts with long cell extensions (stellate) representing an activated
197 SWI/SNF CHROMATIN REMODELING ENZYMES IN TRANSCRIPTIONAL REGULATION OF PATHOLOGICAL CARDIAC HYPERTROPHY G Mehta, J Wu, I de la Serna Toledo, Ohio BACKGROUND:
Pathological hypertrophy of adult hearts characterized by increased cell size and reactivation of fetal genes, frequently leads to heart failure and sudden death of an individual. Previous studies report that, the transcriptional regulator, Microphthalmia-associated transcription factor (MITF) regulates cardiac hypertrophy in mice. However, the molecular mechanism(s) that determine MITF activity in the hypertrophic response are unknown. The interaction of transcription factors with chromatin remodeling enzymes such as SWI/SNF is critical for the regulation of cardiac functioning. BRG1, a component of SWI/SNF complex is reported to be upregulated in hypertrophic hearts; however a functional link between BRG1 and transcription factors involved in hypertrophy has not been identified and the main aim of this study is to elucidate that unknown functional link. METHODS: Transverse Aortic constriction (TAC) was carried out for 4 weeks in 3 month old mice. The hearts excised and left ventricles from both SHAM and TAC animals were used for protein, RNA and Chromatin Immunoprecipitation (ChIP) analysis. H9c2 cells were used as an in vitro model of cardiac hypertrophy. The cells were treated with 10mM Isoproterenol for 4 hours and harvested. Knockdown experiments were
In this pilot study there were no observable histological differences in human right atrial tissue from individuals at high- and low-risk for OSA. Further investigation would be required for more definitive results.
Canadian Cardiovascular Society (CCS) Oral THE HUMAN MYOCYTE Saturday, October 25, 2014
S153
myofibrotic phenotype. In contrast, FGF-2 treatment resulted in shorter cell extensions with a phenotype more consistent with quiescent fibroblasts. In addition, FGF-2 attenuated fibroblast-mediated matrix compaction relative to the TGF-b group. CONCLUSION: FGF-2 attenuates TGF- b1 mediated human cardiac fibroblast activation and restores ECM homeostasis and regulation. These data provide proof-of-concept evidence for FGF-2’s therapeutic potential in preventing adverse cardiac remodeling and subsequently heart failure.
Trainee Research Award Finalist e Basic Science 196 FIBROBLAST GROWTH FACTOR (FGF-2) PREVENTS HUMAN CARDIAC FIBROBLAST-MEDIATED EXTRACELLULAR MATRIX REMODELING DA Svystonyuk, JM Ngu, HE Mewhort, DG Guzzardi, BD Lipon, D Park, G Teng, DD Belke, PW Fedak Calgary, Alberta BACKGROUND: After myocardial injury, elevation of profibrotic cytokine transforming growth factor-b1 (TGF-b1) results in myofibroblast differentiation leading to matrix remodeling and progression of heart failure. Fibroblast growth factor (FGF-2) has the potential to antagonize TGF-b1’s profibrotic actions. In this study, we examine the effects of FGF-2 on human cardiac fibroblast mediated extracellular matrix remodeling. METHODS AND RESULTS: Human cardiac fibroblasts were isolated from atrial or ventricular heart biopsies and seeded into 3D collagen matrices. Myofibroblast activation was functionally assessed by the extent of matrix contraction. As compared to baseline myofibroblast activity, FGF-2 attenuated TGF-b1 mediated myofibroblast activation (1.06 versus 1.17, P<0.0001) using cells isolated from atrial tissue. FGF2 similarly attenuated TGF-b1 mediated myofibroblast activation in cells isolated from ventricular core biopsies (1.51 vs. 1.12, P<0.05). New collagen synthesis was assessed by 3H-proline incorporation assay. Fibroblasts treated with TGF-b1 demonstrated an increase in collagen synthesis while addition of FGF-2 reduced collagen synthesis (P<0.05). ECM dysregulation was investigated by in situ zymography using embedded Gelatin-FITC to measure matrix proteolysis. TGF-b1 increased total protease activity in the matrix microenvironment while FGF-2 attenuated this response (2.81 x 109 1.07 x 109 vs. 1.4 x 109 4.49 x108 total florescence units, P<0.01). Quantitative RT-PCR and multiplex analysis assessed mRNA and protein profiles for TIMP-1 and TIMP-2. FGF-2 treatment restored TIMP1 and TIMP-2 gene and protein expression relative to TGFb1 treated cells (P<0.05 and P<0.01, respectively). ECM remodeling adjacent to activated fibroblasts was examined within thin nylon-based collagen matrices using confocal microscopy. Figure 1 shows TGF-b1 activated fibroblasts with long cell extensions (stellate) representing an activated
197 SWI/SNF CHROMATIN REMODELING ENZYMES IN TRANSCRIPTIONAL REGULATION OF PATHOLOGICAL CARDIAC HYPERTROPHY G Mehta, J Wu, I de la Serna Toledo, Ohio BACKGROUND:
Pathological hypertrophy of adult hearts characterized by increased cell size and reactivation of fetal genes, frequently leads to heart failure and sudden death of an individual. Previous studies report that, the transcriptional regulator, Microphthalmia-associated transcription factor (MITF) regulates cardiac hypertrophy in mice. However, the molecular mechanism(s) that determine MITF activity in the hypertrophic response are unknown. The interaction of transcription factors with chromatin remodeling enzymes such as SWI/SNF is critical for the regulation of cardiac functioning. BRG1, a component of SWI/SNF complex is reported to be upregulated in hypertrophic hearts; however a functional link between BRG1 and transcription factors involved in hypertrophy has not been identified and the main aim of this study is to elucidate that unknown functional link. METHODS: Transverse Aortic constriction (TAC) was carried out for 4 weeks in 3 month old mice. The hearts excised and left ventricles from both SHAM and TAC animals were used for protein, RNA and Chromatin Immunoprecipitation (ChIP) analysis. H9c2 cells were used as an in vitro model of cardiac hypertrophy. The cells were treated with 10mM Isoproterenol for 4 hours and harvested. Knockdown experiments were