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Improved recovery of ventricular contractility with injection of self-assembling peptide nanofiber scaffolds after myocardial infarction in a rat model
with other PBRT noted clinical cardiovascular associations, however direct physiological effects have not been investigated on cardiac cells. AIM: To assess the direct effects of PBRT’s, Phenyl acetic acid (PAA), m/p-Cresol (MC/PC) and their main circulating in vivo metabolite m/p-Cresylsulphate (MCS/PCS) on neonatal cardiac myocyte (NCM) hypertrophy and fibroblast (NCF) collagen synthesis. Materials and method: MC/PC were reacted with sulfur trioxide N,N-dimethylformamide complex to give the corresponding sulphates (PCS/MCS) as the sodium salts in moderate yields. Purity was confirmed by proton NMR spectroscopy and MS. NCM and NCF were isolated by enzymatic digestion, cultured and pretreated with increasing doses of PBRT. NCF collagen synthesis and NCM hypertrophy were determined by 3 H-proline and 3 H-leucine incorporation. Cell viability was assessed with MTT assays. Results: PC had minor inhibitory effect while PCS, MC, MCS, PAA, had no significant effect on NCF collagen synthesis and NCM hypertrophy at 100 m (100 m PBRT vs Control: PC: 112.4 ± 1.8, PCS: 118.9 ± 9.2, MC: 117.1 ± 4.4, MCS: 104.6 ± 7.6, PAA: 93.8 ± 4.3; All P > 0.05). With 100 nM Angiotensin II stimulation, 100 m PBRT also did not effect NCF collagen synthesis and NCM hypertrophy (100 m PBRT + 100 nM AngII vs Control: PC: 87.5 ± 5.8, PCS: 89.0 ± 0.7%, MC: 94.2 ± 4.8, MCS: 90.1 ± 7.2, PAA: 87.6 ± 4.9; all P > 0.05). Furthermore, PBRT’s tested did not affect NCF and NCM cell viability. Conclusion: Despite these compounds being elevated in clinical settings (chronic renal failure) we were unable to demonstrate direct effects on NCF and NCM. The clinical effects of these compounds may occur via other mechanisms which are worthy of further investigation. doi:10.1016/j.hlc.2009.05.618 573 HUMAN CARDIOSPHERE-DERIVED ADULT STEM CELLS ORIGINATE WITHIN THE HEART Anthony J. White, Satoshi Matsushita, Lawrence S. Czer, Tarun Chakravarty, Kevin Burton, Ernst R. Schwarz, Darryl R. Davis, Jason Lee, Qi Wang, Nancy L. Reinsmoen, James S. Forrester, Eduardo Marbán, Raj Makkar Cedars-Sinai Medical Center, Los Angeles, CA, United States Background: The aim of this study was to determine the origin of cardiosphere-derived cells (CDCs)—cardiac stem cells expanded in cell culture from endomyocardial biopsies. Tens of millions of CDCs can reliably be produced within weeks. Intra-myocardial injection of CDCs improves LV function in rodent models of myocardial infarction, and clinical trials are planned. A central unresolved issue is whether CDCs represent cells expanded from intrinsic cardiac cells, or cells seeded from an extracardiac site (or both). Methods: Cardiac biopsies were obtained from ten cardiac transplant recipients (57 ± 15 yr) and CDCs were cultured from each biopsy. The origin of the CDCs was
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investigated by short-tandem nucleotide repeat (STR) testing (“genetic fingerprinting”), performed on DNA from three sources - CDCs, donor heart, and recipient. In addition, in 2 cases in which the donor was of the opposite sex to the recipient, the presence of X and Y chromosomes in the CDCs was examined using fluorescence in situ hybridization (FISH). Results: Eight of the 10 subjects were male, with all transplanted hearts originating from male donors. In none of the 10 cases was any recipient DNA detected in the CDCs by STR analysis. In the two cases in which a female patient had received a male donor heart, FISH staining indicated that all CDCs had an X and a Y chromosome (511 of 511 cells, and 74 of 74 cells respectively), similarly indicating exclusively donor origin. Conclusions: CDCs are of endogenous cardiac origin, with no detectable contribution from extra-cardiac seeding. doi:10.1016/j.hlc.2009.05.619 574 IMPROVED RECOVERY OF VENTRICULAR CONTRACTILITY WITH INJECTION OF SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS AFTER MYOCARDIAL INFARCTION IN A RAT MODEL George T. Lau 1,2,3, , Fabrice Spiro 4 ,
James Roger Hajjar 2,3 1 Concord
Birkhead 5 ,
Prunier 3 , Zen Toby
Freyman 5 ,
Chu 4 , Lisa Ince Tan 6 ,
Repatriation General Hospital, Concord, Australia
2 Cardiology Division, Massachusetts General Hospital, Boston,
MA, USA 3 Cardiovascular
Research Center, Charlestown, MA, USA Inc., Cambridge, MA, USA 5 Boston Scientific Corporation, Natick, MA, USA 6 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA 4 3DM,
Background: Chronic heart failure is common after myocardial infarction despite current myocardial reperfusion strategies. We hypothesized that a recently developed injectable self-assembling peptide (SAP) that forms in vivo nanofiber scaffolds capable of myocyte preservation and recruitment may increase the recovery of cardiac contractility after infarction. Methods: Myocardial infarction was created in 27 rats by ligation of left anterior descending artery for 45 min, immediately followed by randomization to 50 L of 3% SAP, 1% SAP or vehicle control by injection into the infarcted area. Echocardiography was performed at 3 and 28 days after injection. Animals were sacrificed on day 28 and the hearts sliced into 5 cross-sections. Histopathological analysis of each cross-section was performed for extent of infarction, wall thinning and fibrosis (scored 0–4). Results: Mean left ventricular ejection fraction (LVEF) on day 3 was 69.4 ± 6.0%, with no difference between the groups. Improved recovery of left ventricular contractility between day 3 and day 28 was seen in the 3% SAP group compared with the vehicle control group
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286
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Heart, Lung and Circulation 2009;18S:S1–S286
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(LVEF +7.0 ± 7.2% versus −0.6 ± 6.3%, p = 0.04) with an intermediate response in the 1% SAP group (+2.2 ± 8.9%). In support of the functional results, while there was no difference in the area of infarction between the three groups (2.6 ± 1.4, 3.0 ± 1.3 and 3.1 ± 2.0 cross-sections/rat), there were trends for SAP injected rats to have less wall thinning ([3%]: 1.5 ± 1.2, [1.5%]: 1.4 ± 1.1 versus [controls]: 2.3 ± 1.7 cross-sections/rat) and severe infarctions ([3%]: 0.9 ± 0.8, [1%]: 0.8 ± 1.1 versus [controls]: 1.4 ± 1.5 cross-sections/rat) compared to control rats. Conclusion: Post-infarct injection of SAP has the potential to improve recovery of ventricular function after experimental myocardial infarction. doi:10.1016/j.hlc.2009.05.620 575 ISLET1, A TRANSCRIPTION FACTOR, PROMOTES CARDIAC DIFFERENTIATION Anthony J. White, Satoshi Matsushita, Tarun Chakravarty, James S. Forrester, Eduardo Marbán, Raj Makkar Cedars-Sinai Medical Center, Los Angeles, CA, United States Background: Islet1 (ISL1) is a transcription factor, essential for normal embryonic cardiac development. It is a marker of an embryonic cell population known as the second heart field, remnants of which persist in the adult heart. Methods: A recombinant nuclear-targeted form of the ISL1 protein was synthesised. The structure of the hybrid protein (ISL1-VP22) was a C-terminal herpes simplex protein (VP22) that conveyed nuclear localisation on the hybrid protein, and ISL1 at the N-terminal end. Mouse embryonic stem cells were maintained in a pluripotent state with leukemia inhibitory factor (LIF). LIF was then withdrawn to facilitate formation of embryoid bodies (EB). EBs were either treated with 10 g/mL of ISL1-VP22 or VP22 protein (daily days 1–10), or no additional protein. Each EB was examined for beating activity on days 7, 9, 13 and 17. Results: Exposure to the ISL1-VP22 protein markedly increased the proportion of beating EBs compared to control, or VP22 treated EBs (n = 9; see graph). The percentage of EBs with beating activity at day 17 post-LIF withdrawal in the ISL1-VP22 treated, VP22 treated, and untreated groups was 41.1 ± 18.5%, 18.8 ± 10.4% and 16.1 ± 4.2% respectively (mean ± SD. ANOVA p < 0.001. ISL1-VP22 vs. control, p < 0.001. ISL1-VP22 versus VP22, p < 0.001).
Conclusions: 1. ISL1 promotes cardiac differentiation of embryonic stem cells. 2. VP22 hybrid proteins can enable a transcription factor to localize to the nucleus while retaining functional activity. doi:10.1016/j.hlc.2009.05.621 576 NESPRIN/ACTIN CYTOSKELETAL NETWORK IS A CRITICAL DETERMINANT OF CARDIAC FUNCTION IN LAMIN A/C DEFICIENT MICE V. Nikolova-Krstevski 1 , C. Leimena 1 , A. Carlton 1 , C.L. Stewart 2 , C. Shanahan 3 , D. Fatkin 1 1 Molecular Cardiology Program, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia 2 National Cancer Institute-FCRDC, Frederick, MD, USA 3 Department of Medicine, Addenbrook’s Hospital, Cambridge, England
Mutations in the LMNA gene, encoding the nuclear lamina proteins, lamins A and C, cause familial dilated cardiomyopathy (DCM); the pathophysiological basis is unknown. Homozygous, lamin A/C-deficient (Lmna−/− ) mice develop severe DCM with altered cardiomyocyte nuclear morphology and disorganisation of desmin attachments at the nuclear surface. These findings implicated disrupted nuclear-cytoskeletal connections as a potential disease mechanism. Nesprins are novel spectrinrepeat-containing proteins with multiple intranuclear and cytoskeletal isoforms. Full-length nesprin-1 is a giant protein that forms a major intracellular scaffolding by linking the nucleus to the actin cytoskeleton. We hypothesised that lamin A/C-deficiency might alter nesprin anchoring and contribute to the cardiac phenotype in Lmna−/− mice. Myocardial sections from wildtype and Lmna−/− mice aged 4–6 weeks (severe DCM) were evaluated using immunogold-labelled nesprin-1 antibodies (N5, N3, C1) and electron microscopy. The density of gold-labelled epitopes was quantified using nearest neighbour analysis. Immunostaining and Western blot analysis of actin isoforms were also performed. Significant disruption of N5 (P = 0.016), N3 (P < 0.001) and C1 (P < 0.001) epitope was observed in Lmna−/− hearts. Smooth muscle actin (SMA) expression was also observed in 4-6 week Lmna−/− cardiomyocytes. These data suggest that perturbation of nesprin-1 contributes to changes in nuclear morphology and nuclear-cytoskeletal connections in Lmna−/− mice. The presence of the foetal form of actin (SMA) in adult Lmna−/− cardiomyocytes suggests a developmental defect resulting in smaller cardiomyocyte size. We propose that loss of nuclear-cytoskeletal integrity in lamin A/C-deficient hearts alters cytoskeletal tension and the mechanical efficiency of force transmission with subsequent impairment of contractile function. doi:10.1016/j.hlc.2009.05.622
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investigated by short-tandem nucleotide repeat (STR) testing (“genetic fingerprinting”), performed on DNA from three sources - CDCs, donor heart, and recipient. In addition, in 2 cases in which the donor was of the opposite sex to the recipient, the presence of X and Y chromosomes in the CDCs was examined using fluorescence in situ hybridization (FISH). Results: Eight of the 10 subjects were male, with all transplanted hearts originating from male donors. In none of the 10 cases was any recipient DNA detected in the CDCs by STR analysis. In the two cases in which a female patient had received a male donor heart, FISH staining indicated that all CDCs had an X and a Y chromosome (511 of 511 cells, and 74 of 74 cells respectively), similarly indicating exclusively donor origin. Conclusions: CDCs are of endogenous cardiac origin, with no detectable contribution from extra-cardiac seeding. doi:10.1016/j.hlc.2009.05.619 574 IMPROVED RECOVERY OF VENTRICULAR CONTRACTILITY WITH INJECTION OF SELFASSEMBLING PEPTIDE NANOFIBER SCAFFOLDS AFTER MYOCARDIAL INFARCTION IN A RAT MODEL George T. Lau 1,2,3, , Fabrice Spiro 4 ,
James Roger Hajjar 2,3 1 Concord
Birkhead 5 ,
Prunier 3 , Zen Toby
Freyman 5 ,
Chu 4 , Lisa Ince Tan 6 ,
Repatriation General Hospital, Concord, Australia
2 Cardiology Division, Massachusetts General Hospital, Boston,
MA, USA 3 Cardiovascular
Research Center, Charlestown, MA, USA Inc., Cambridge, MA, USA 5 Boston Scientific Corporation, Natick, MA, USA 6 Department of Pathology, Brigham and Women’s Hospital, Boston, MA, USA 4 3DM,
Background: Chronic heart failure is common after myocardial infarction despite current myocardial reperfusion strategies. We hypothesized that a recently developed injectable self-assembling peptide (SAP) that forms in vivo nanofiber scaffolds capable of myocyte preservation and recruitment may increase the recovery of cardiac contractility after infarction. Methods: Myocardial infarction was created in 27 rats by ligation of left anterior descending artery for 45 min, immediately followed by randomization to 50 L of 3% SAP, 1% SAP or vehicle control by injection into the infarcted area. Echocardiography was performed at 3 and 28 days after injection. Animals were sacrificed on day 28 and the hearts sliced into 5 cross-sections. Histopathological analysis of each cross-section was performed for extent of infarction, wall thinning and fibrosis (scored 0–4). Results: Mean left ventricular ejection fraction (LVEF) on day 3 was 69.4 ± 6.0%, with no difference between the groups. Improved recovery of left ventricular contractility between day 3 and day 28 was seen in the 3% SAP group compared with the vehicle control group
ABSTRACTS
Heart, Lung and Circulation 2009;18S:S1–S286
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Heart, Lung and Circulation 2009;18S:S1–S286
Abstracts
ABSTRACTS
(LVEF +7.0 ± 7.2% versus −0.6 ± 6.3%, p = 0.04) with an intermediate response in the 1% SAP group (+2.2 ± 8.9%). In support of the functional results, while there was no difference in the area of infarction between the three groups (2.6 ± 1.4, 3.0 ± 1.3 and 3.1 ± 2.0 cross-sections/rat), there were trends for SAP injected rats to have less wall thinning ([3%]: 1.5 ± 1.2, [1.5%]: 1.4 ± 1.1 versus [controls]: 2.3 ± 1.7 cross-sections/rat) and severe infarctions ([3%]: 0.9 ± 0.8, [1%]: 0.8 ± 1.1 versus [controls]: 1.4 ± 1.5 cross-sections/rat) compared to control rats. Conclusion: Post-infarct injection of SAP has the potential to improve recovery of ventricular function after experimental myocardial infarction. doi:10.1016/j.hlc.2009.05.620 575 ISLET1, A TRANSCRIPTION FACTOR, PROMOTES CARDIAC DIFFERENTIATION Anthony J. White, Satoshi Matsushita, Tarun Chakravarty, James S. Forrester, Eduardo Marbán, Raj Makkar Cedars-Sinai Medical Center, Los Angeles, CA, United States Background: Islet1 (ISL1) is a transcription factor, essential for normal embryonic cardiac development. It is a marker of an embryonic cell population known as the second heart field, remnants of which persist in the adult heart. Methods: A recombinant nuclear-targeted form of the ISL1 protein was synthesised. The structure of the hybrid protein (ISL1-VP22) was a C-terminal herpes simplex protein (VP22) that conveyed nuclear localisation on the hybrid protein, and ISL1 at the N-terminal end. Mouse embryonic stem cells were maintained in a pluripotent state with leukemia inhibitory factor (LIF). LIF was then withdrawn to facilitate formation of embryoid bodies (EB). EBs were either treated with 10 g/mL of ISL1-VP22 or VP22 protein (daily days 1–10), or no additional protein. Each EB was examined for beating activity on days 7, 9, 13 and 17. Results: Exposure to the ISL1-VP22 protein markedly increased the proportion of beating EBs compared to control, or VP22 treated EBs (n = 9; see graph). The percentage of EBs with beating activity at day 17 post-LIF withdrawal in the ISL1-VP22 treated, VP22 treated, and untreated groups was 41.1 ± 18.5%, 18.8 ± 10.4% and 16.1 ± 4.2% respectively (mean ± SD. ANOVA p < 0.001. ISL1-VP22 vs. control, p < 0.001. ISL1-VP22 versus VP22, p < 0.001).
Conclusions: 1. ISL1 promotes cardiac differentiation of embryonic stem cells. 2. VP22 hybrid proteins can enable a transcription factor to localize to the nucleus while retaining functional activity. doi:10.1016/j.hlc.2009.05.621 576 NESPRIN/ACTIN CYTOSKELETAL NETWORK IS A CRITICAL DETERMINANT OF CARDIAC FUNCTION IN LAMIN A/C DEFICIENT MICE V. Nikolova-Krstevski 1 , C. Leimena 1 , A. Carlton 1 , C.L. Stewart 2 , C. Shanahan 3 , D. Fatkin 1 1 Molecular Cardiology Program, Victor Chang Cardiac Research Institute, Darlinghurst, NSW, Australia 2 National Cancer Institute-FCRDC, Frederick, MD, USA 3 Department of Medicine, Addenbrook’s Hospital, Cambridge, England
Mutations in the LMNA gene, encoding the nuclear lamina proteins, lamins A and C, cause familial dilated cardiomyopathy (DCM); the pathophysiological basis is unknown. Homozygous, lamin A/C-deficient (Lmna−/− ) mice develop severe DCM with altered cardiomyocyte nuclear morphology and disorganisation of desmin attachments at the nuclear surface. These findings implicated disrupted nuclear-cytoskeletal connections as a potential disease mechanism. Nesprins are novel spectrinrepeat-containing proteins with multiple intranuclear and cytoskeletal isoforms. Full-length nesprin-1 is a giant protein that forms a major intracellular scaffolding by linking the nucleus to the actin cytoskeleton. We hypothesised that lamin A/C-deficiency might alter nesprin anchoring and contribute to the cardiac phenotype in Lmna−/− mice. Myocardial sections from wildtype and Lmna−/− mice aged 4–6 weeks (severe DCM) were evaluated using immunogold-labelled nesprin-1 antibodies (N5, N3, C1) and electron microscopy. The density of gold-labelled epitopes was quantified using nearest neighbour analysis. Immunostaining and Western blot analysis of actin isoforms were also performed. Significant disruption of N5 (P = 0.016), N3 (P < 0.001) and C1 (P < 0.001) epitope was observed in Lmna−/− hearts. Smooth muscle actin (SMA) expression was also observed in 4-6 week Lmna−/− cardiomyocytes. These data suggest that perturbation of nesprin-1 contributes to changes in nuclear morphology and nuclear-cytoskeletal connections in Lmna−/− mice. The presence of the foetal form of actin (SMA) in adult Lmna−/− cardiomyocytes suggests a developmental defect resulting in smaller cardiomyocyte size. We propose that loss of nuclear-cytoskeletal integrity in lamin A/C-deficient hearts alters cytoskeletal tension and the mechanical efficiency of force transmission with subsequent impairment of contractile function. doi:10.1016/j.hlc.2009.05.622