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Electrophysiological Investigations of Human iPS Cell-Derived Engineered Heart Tissue in a Guinea Pig Infarction Model
S182
The Journal of Heart and Lung Transplantation, Vol 35, No 4S, April 2016
In Group 3, IL-6 levels were unchanged even after lungs were removed from circuit, suggesting that LF-trapped leukocytes did not contribute to increasing cytokine levels circulating in perfusate during EVLP. Conclusion: The potentially deleterious effects of LF-trapped leukocytes on lung grafts quality during EVLP appear to be limited while the cytokines storm stemming from lung grafts on EVLP can significantly impact their function. Our findings suggest the important role of leukocyte depletion or management in optimizing the lung grafts quality during EVLP.
4( 78) Development of an Unsupervised Classification with Multi-Level Analysis of the Heart Transplant Endomyocardial Biopsy E. Chang ,1 G. Fishbein,1 N. Jackson,1 M. Bakir,1 D. Liem,1 G. Bondar,1 S. Litovsky,2 J. Tallaj,2 C. Starling,1 P. Ping,1 E. Reed,1 M. Deng,1 E. Tabak,3 M. Cadeiras.1 1UCLA, Los Angeles, CA; 2University of Alabama at Birmingham, Birmingham, AL; 3New York University, New York, NY. Purpose: Endomyocardial Biopsy (EMB) have insufficient diagnostic accuracy which constitutes a limitation in management of HTx recipients. The aim is to develop a non-supervised evaluation of intramyocardial gene expression to improve EMB classification and understanding of underlying biology of cardiac allograft phenotypes. Methods: 64 tissue and 45 peripheral blood mononuclear cell (PBMC) samples from 47 patients subjected to mRNA sequencing. 21 tissues were used for microRNA analysis. An unsupervised algorithm using optimal transport to mitigate batch effects and to filter confounding sources of variability was developed to identify molecular signatures of rejection. Linear Mixed Model was applied to identify statistically significant genes. Weighed Gene Coexpression Network Analysis (WGCNA) established eigengene modules and module-trait correlation between 4 classes followed by histology (Fig 2&3). Results: Our algorithm classified EMBs into 4 groups solely based on gene expression independent of histopathological assessment. These categories are highly correlated with WGCNA modules and with clinical phenotypes. LMM identified top genes: CLNK, TNFRSF10A, TRADD, CD2, and RAB5A. GO Analysis (FDR< 0.05) showed enrichment in regulation of T-cell and B-cell proliferation and lymphocyte activation among many others. Top five miRNA probe sets filtered by highest intra-module correlation and statistical significance were hsa-miR-141-3p, hsa-miR-150-5p, hsa-miR-605, hsa-miR582-5p, hsa-miR-3150b-3p. Gene target prediction of miRNA resulted in 724 genes (184 GO categories). In mRNA dataset of same EMBs showed 685 overlapping genes. Conclusion: Development of machine learning algorithm, supervised by network and statistical analyses, myocardial mRNA-miRNA network transcript, and PBMC expression profiling may provide further insights into the pathophysiology of HTx rejection to reveal potential candidates for diagnostic or therapeutic applications.
4( 79) WITHDRAWN 4( 80) Electrophysiological Investigations of Human iPS Cell-Derived Engineered Heart Tissue in a Guinea Pig Infarction Model S. Pecha ,1 F. Weinberger,2 K. Breckwoldt,2 A. Hansen,2 H. Reichenspurner,1 T. Eschenhagen.2 1Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Experimental Pharmacology and Toxicology, University Medical Center Hamburg Eppendorf, Hamburg, Germany. Purpose: Engineered heart tissue (EHT) implantation might be a promising future cardiac repair approach. Besides cell survival, electrical coupling plays a major role to support failing hearts. In this study we transplanted EHTs from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) on cryo-injured guinea pig hearts and investigated their electrophysiological properties. Methods: Human iPSC were generated by retroviral reprogramming of dermal fibroblasts. Cardiac differentiation of hiPSC was performed by an embryoid body-based three-stage differentiation protocol. EHTs were created from hiPS-CM (5*106 cardiomyocytes and 2*106 hiPSC-endothelial cells per EHT). Left ventricular myocardial cryo-injury was induced in
Abstracts S183 adult guinea pigs (n= 32). 7 days after injury EHTs (2 per animal, n= 12) or cell-free constructs (n= 10) were implanted. In a group of animals (n= 9), electrophysiological investigations of Langendorff-perfused hearts were performed, using 2Photon Laser Scanning microscopy and voltage sensitive dye. Results: Cryo-injury resulted in large transmural infarctions of the left ventricle. Dystrophin and MLC2v positive cross-striated muscle tissue was observed in the scar area of EHT transplanted animals. The human origin was demonstrated by FISH analysis. Electrical activity within the transmural portion of the infarct region was investigated by line scan recordings. Evidence of electrical coupling was successfully demonstrated in 1 of 4 hEHT hearts, whereas no electrical activity could be measured in any of the 5 control hearts. Within the remote myocardium, hEHT- transplanted hearts demonstrated a significantly improved electrophysiological phenotype, with shorter AP duration (APD90 132 ms vs. 148 ms; p< 0.01) and faster transmural conduction velocity (23 m/s vs 31 m/s; p= 0.002) compared to control hearts. Conclusion: Evidence of electrical coupling in the infarction region was demonstrated in 1 of 4 EHT animals. Furthermore, within the remote myocardium, hEHT- transplanted hearts demonstrated a significantly shorter AP duration and faster transmural conduction velocity, compatible with a reversal of the heart failure phenotype in the hEHT transplanted hearts. 4( 81) WITHDRAWN 4( 83) 4( 82) aPC Pretreatment of hEPCR-Expressing PAECs Attenuates Endothelial Cell Damage and Thrombosis C.T. Laird , B. French, D.G. Harris, X. Cheng, D. Ayares, R.N. Pierson, A. Azimzadeh. University of Maryland School of Medicine, Baltimore, MD.
Control of Cardiomyocyte Proliferation Through p53/Mdm2-Regulated MicroRNAs S. Stanley-Hasnain ,1 L. Hauck,2 D. Grothe,2 F. Billia.2 1Toronto General Research Institute, Toronto, ON, Canada; 2Research, Toronto General Research Institute, Toronto, ON, Canada.
Purpose: Activated Protein C (aPC), acting primarily through the human endothelial protein C receptor (hEPCR), mediates anti-thrombotic and antiinflammatory cytoprotective effects in humans. We hypothesized that treatment of hEPCR expressing porcine endothelial cells (ECs) with recombinant aPC would attenuate xeno EC injury. Methods: Confluent GalKO.hCD46.hEPCR porcine aortic EC (PAEC) monolayers in microfluidic channels were pretreated with 0.02 µg/mL recombinant aPC (raPC) several hours prior to perfusion with heparinized human blood. Thrombosis, viability, and endothelial surface area coverage were calculated by fluorescent analysis using image processing software. Results: raPC pretreatment (Rx) of ECs reduced platelet adhesion (45.6±16.2 % surface area (SA) coverage in untreated hEPCR+ cells) to 27.3±10.2% after 2h exposure to aPC (p= .085 vs untreated), 6.6±1.6% after 4h (p= .003), and 5.3±1.8% after 6h (p= 0.0025). Relative to untreated monolayers (63.45±30 au), platelet aggregation also decreased with increasing duration of aPC Rx from 2h (34.6±16.1 au, p= 0.13) to 6h (26.1±8.23 au, p= 0.05). When perfused with human blood, untreated GalKO.hCD46. hEPCR PAEC monolayers showed cellular damage (7.37±1.81 PI+ cells/ hpf, vs none with medium) and reduced surface area coverage (75.5±3.5%, vs 100% with medium). Six hour Rx with raPC was associated with significantly fewer PI+ cells (3±1.23 cells/hpf, p= 0.0003 vs untreated EC), and significantly higher EC surface coverage (87.3±2.9 percent, p< 0.0001). Prevention of increased permeability of hEPCR+ PAEC after human thrombin exposure, measured by electrical impedance (xCELLigence), correlated directly with increasing duration of EC pretreatment with aPC. Effects of aPC pretreatment were only seen with pAECs that express hEPCR. Conclusion: aPC pretreatment of PAEC monolayers significantly reduced clot initiation, thrombus propagation, and endothelial cell injury in a timedependent and hEPCR-dependent manner.
Purpose: Heart failure is a leading cause of morbidity and mortality in Canada. As the quality of life and prognosis for these patients is poor, therapies to prevent and treat heart failure are urgently required. Adult cardiomyocytes (CM) are terminally differentiated cells with minimal regenerative capacity, making cardiac tissue particularly vulnerable to injury. Thus, defining the roadblocks responsible for adult CM cell cycle arrest lies at the core of developing regenerative therapies for cardiac diseases. We have previously demonstrated that inactivating the p53/ Mdm2 tumor suppressor circuitry, specifically in the heart, can allow differentiated CMs to regain proliferative capacity, through an upregulation of factors involved in cell cycle re-entry. These factors are repressed in quiescent CMs, in part through the action of microRNAs (miRNAs). Notably, knockout of either p53 or Mdm2 individually was insufficient to promote CM proliferation. Therefore, we hypothesized that inactivation of p53/Mdm2-regulated miRNAs could promote the expression of cell cycle activators and induce proliferation of adult murine CMs. Methods: A screen was performed to identify miRNAs regulated by both p53 and Mdm2. We compared total miRNA expression profiles from cardiac specific p53/Mdm2 double knock-out (DKO) mouse hearts with those from cardiac-specific single knockouts (p53KO and Mdm2KO), and wild-type controls, using a miRNA microarray. Results: The screen revealed a profile of 14 uniquely downregulated miRNAs in the “proliferative” DKO hearts versus the control that were not found in either of the single KOs. Of the 14 hits, 11 miRNAs were enriched for mRNA targets involved in cell cycle regulation. Conclusion: Ongoing in vitro studies will determine if knock-down of these miRNAs using antagomirs can upregulate neonatal rat CM proliferation. Ultimately, we aim to inject antagomirs targeting these miRNAs into animals post- myocardial infarction to determine the effect of p53/Mdm2-regulated miRNAs on heart function and CM proliferation in vivo.
The Journal of Heart and Lung Transplantation, Vol 35, No 4S, April 2016
In Group 3, IL-6 levels were unchanged even after lungs were removed from circuit, suggesting that LF-trapped leukocytes did not contribute to increasing cytokine levels circulating in perfusate during EVLP. Conclusion: The potentially deleterious effects of LF-trapped leukocytes on lung grafts quality during EVLP appear to be limited while the cytokines storm stemming from lung grafts on EVLP can significantly impact their function. Our findings suggest the important role of leukocyte depletion or management in optimizing the lung grafts quality during EVLP.
4( 78) Development of an Unsupervised Classification with Multi-Level Analysis of the Heart Transplant Endomyocardial Biopsy E. Chang ,1 G. Fishbein,1 N. Jackson,1 M. Bakir,1 D. Liem,1 G. Bondar,1 S. Litovsky,2 J. Tallaj,2 C. Starling,1 P. Ping,1 E. Reed,1 M. Deng,1 E. Tabak,3 M. Cadeiras.1 1UCLA, Los Angeles, CA; 2University of Alabama at Birmingham, Birmingham, AL; 3New York University, New York, NY. Purpose: Endomyocardial Biopsy (EMB) have insufficient diagnostic accuracy which constitutes a limitation in management of HTx recipients. The aim is to develop a non-supervised evaluation of intramyocardial gene expression to improve EMB classification and understanding of underlying biology of cardiac allograft phenotypes. Methods: 64 tissue and 45 peripheral blood mononuclear cell (PBMC) samples from 47 patients subjected to mRNA sequencing. 21 tissues were used for microRNA analysis. An unsupervised algorithm using optimal transport to mitigate batch effects and to filter confounding sources of variability was developed to identify molecular signatures of rejection. Linear Mixed Model was applied to identify statistically significant genes. Weighed Gene Coexpression Network Analysis (WGCNA) established eigengene modules and module-trait correlation between 4 classes followed by histology (Fig 2&3). Results: Our algorithm classified EMBs into 4 groups solely based on gene expression independent of histopathological assessment. These categories are highly correlated with WGCNA modules and with clinical phenotypes. LMM identified top genes: CLNK, TNFRSF10A, TRADD, CD2, and RAB5A. GO Analysis (FDR< 0.05) showed enrichment in regulation of T-cell and B-cell proliferation and lymphocyte activation among many others. Top five miRNA probe sets filtered by highest intra-module correlation and statistical significance were hsa-miR-141-3p, hsa-miR-150-5p, hsa-miR-605, hsa-miR582-5p, hsa-miR-3150b-3p. Gene target prediction of miRNA resulted in 724 genes (184 GO categories). In mRNA dataset of same EMBs showed 685 overlapping genes. Conclusion: Development of machine learning algorithm, supervised by network and statistical analyses, myocardial mRNA-miRNA network transcript, and PBMC expression profiling may provide further insights into the pathophysiology of HTx rejection to reveal potential candidates for diagnostic or therapeutic applications.
4( 79) WITHDRAWN 4( 80) Electrophysiological Investigations of Human iPS Cell-Derived Engineered Heart Tissue in a Guinea Pig Infarction Model S. Pecha ,1 F. Weinberger,2 K. Breckwoldt,2 A. Hansen,2 H. Reichenspurner,1 T. Eschenhagen.2 1Cardiovascular Surgery, University Heart Center Hamburg, Hamburg, Germany; 2Experimental Pharmacology and Toxicology, University Medical Center Hamburg Eppendorf, Hamburg, Germany. Purpose: Engineered heart tissue (EHT) implantation might be a promising future cardiac repair approach. Besides cell survival, electrical coupling plays a major role to support failing hearts. In this study we transplanted EHTs from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) on cryo-injured guinea pig hearts and investigated their electrophysiological properties. Methods: Human iPSC were generated by retroviral reprogramming of dermal fibroblasts. Cardiac differentiation of hiPSC was performed by an embryoid body-based three-stage differentiation protocol. EHTs were created from hiPS-CM (5*106 cardiomyocytes and 2*106 hiPSC-endothelial cells per EHT). Left ventricular myocardial cryo-injury was induced in
Abstracts S183 adult guinea pigs (n= 32). 7 days after injury EHTs (2 per animal, n= 12) or cell-free constructs (n= 10) were implanted. In a group of animals (n= 9), electrophysiological investigations of Langendorff-perfused hearts were performed, using 2Photon Laser Scanning microscopy and voltage sensitive dye. Results: Cryo-injury resulted in large transmural infarctions of the left ventricle. Dystrophin and MLC2v positive cross-striated muscle tissue was observed in the scar area of EHT transplanted animals. The human origin was demonstrated by FISH analysis. Electrical activity within the transmural portion of the infarct region was investigated by line scan recordings. Evidence of electrical coupling was successfully demonstrated in 1 of 4 hEHT hearts, whereas no electrical activity could be measured in any of the 5 control hearts. Within the remote myocardium, hEHT- transplanted hearts demonstrated a significantly improved electrophysiological phenotype, with shorter AP duration (APD90 132 ms vs. 148 ms; p< 0.01) and faster transmural conduction velocity (23 m/s vs 31 m/s; p= 0.002) compared to control hearts. Conclusion: Evidence of electrical coupling in the infarction region was demonstrated in 1 of 4 EHT animals. Furthermore, within the remote myocardium, hEHT- transplanted hearts demonstrated a significantly shorter AP duration and faster transmural conduction velocity, compatible with a reversal of the heart failure phenotype in the hEHT transplanted hearts. 4( 81) WITHDRAWN 4( 83) 4( 82) aPC Pretreatment of hEPCR-Expressing PAECs Attenuates Endothelial Cell Damage and Thrombosis C.T. Laird , B. French, D.G. Harris, X. Cheng, D. Ayares, R.N. Pierson, A. Azimzadeh. University of Maryland School of Medicine, Baltimore, MD.
Control of Cardiomyocyte Proliferation Through p53/Mdm2-Regulated MicroRNAs S. Stanley-Hasnain ,1 L. Hauck,2 D. Grothe,2 F. Billia.2 1Toronto General Research Institute, Toronto, ON, Canada; 2Research, Toronto General Research Institute, Toronto, ON, Canada.
Purpose: Activated Protein C (aPC), acting primarily through the human endothelial protein C receptor (hEPCR), mediates anti-thrombotic and antiinflammatory cytoprotective effects in humans. We hypothesized that treatment of hEPCR expressing porcine endothelial cells (ECs) with recombinant aPC would attenuate xeno EC injury. Methods: Confluent GalKO.hCD46.hEPCR porcine aortic EC (PAEC) monolayers in microfluidic channels were pretreated with 0.02 µg/mL recombinant aPC (raPC) several hours prior to perfusion with heparinized human blood. Thrombosis, viability, and endothelial surface area coverage were calculated by fluorescent analysis using image processing software. Results: raPC pretreatment (Rx) of ECs reduced platelet adhesion (45.6±16.2 % surface area (SA) coverage in untreated hEPCR+ cells) to 27.3±10.2% after 2h exposure to aPC (p= .085 vs untreated), 6.6±1.6% after 4h (p= .003), and 5.3±1.8% after 6h (p= 0.0025). Relative to untreated monolayers (63.45±30 au), platelet aggregation also decreased with increasing duration of aPC Rx from 2h (34.6±16.1 au, p= 0.13) to 6h (26.1±8.23 au, p= 0.05). When perfused with human blood, untreated GalKO.hCD46. hEPCR PAEC monolayers showed cellular damage (7.37±1.81 PI+ cells/ hpf, vs none with medium) and reduced surface area coverage (75.5±3.5%, vs 100% with medium). Six hour Rx with raPC was associated with significantly fewer PI+ cells (3±1.23 cells/hpf, p= 0.0003 vs untreated EC), and significantly higher EC surface coverage (87.3±2.9 percent, p< 0.0001). Prevention of increased permeability of hEPCR+ PAEC after human thrombin exposure, measured by electrical impedance (xCELLigence), correlated directly with increasing duration of EC pretreatment with aPC. Effects of aPC pretreatment were only seen with pAECs that express hEPCR. Conclusion: aPC pretreatment of PAEC monolayers significantly reduced clot initiation, thrombus propagation, and endothelial cell injury in a timedependent and hEPCR-dependent manner.
Purpose: Heart failure is a leading cause of morbidity and mortality in Canada. As the quality of life and prognosis for these patients is poor, therapies to prevent and treat heart failure are urgently required. Adult cardiomyocytes (CM) are terminally differentiated cells with minimal regenerative capacity, making cardiac tissue particularly vulnerable to injury. Thus, defining the roadblocks responsible for adult CM cell cycle arrest lies at the core of developing regenerative therapies for cardiac diseases. We have previously demonstrated that inactivating the p53/ Mdm2 tumor suppressor circuitry, specifically in the heart, can allow differentiated CMs to regain proliferative capacity, through an upregulation of factors involved in cell cycle re-entry. These factors are repressed in quiescent CMs, in part through the action of microRNAs (miRNAs). Notably, knockout of either p53 or Mdm2 individually was insufficient to promote CM proliferation. Therefore, we hypothesized that inactivation of p53/Mdm2-regulated miRNAs could promote the expression of cell cycle activators and induce proliferation of adult murine CMs. Methods: A screen was performed to identify miRNAs regulated by both p53 and Mdm2. We compared total miRNA expression profiles from cardiac specific p53/Mdm2 double knock-out (DKO) mouse hearts with those from cardiac-specific single knockouts (p53KO and Mdm2KO), and wild-type controls, using a miRNA microarray. Results: The screen revealed a profile of 14 uniquely downregulated miRNAs in the “proliferative” DKO hearts versus the control that were not found in either of the single KOs. Of the 14 hits, 11 miRNAs were enriched for mRNA targets involved in cell cycle regulation. Conclusion: Ongoing in vitro studies will determine if knock-down of these miRNAs using antagomirs can upregulate neonatal rat CM proliferation. Ultimately, we aim to inject antagomirs targeting these miRNAs into animals post- myocardial infarction to determine the effect of p53/Mdm2-regulated miRNAs on heart function and CM proliferation in vivo.