المؤلفون: Nivedhitha Velayutham, Jessica C. Garbern, Hannah L. T. Elwell, Zhu Zhuo, Laura Rüland, Farid Elcure Alvarez, Sara Frontini, Yago Rodriguez Carreras, Marie Eichholtz, Elisabeth Ricci‐Blair, Jeanna Y. Shaw, Aldric H. Bouffard, Morgan Sokol, Estela Mancheño Juncosa, Seth Rhoades, Daphne van den Berg, Alexander Kreymerman, Junya Aoyama, Jens Höfflin, Herb Ryan, Shannan Ho Sui, Richard T. Lee

المصدر: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, Vol 13, Iss 13 (2024)

مصطلحات موضوعية: cardiomyocytes, FOXM1, FOXO, maturation, p53, stem cells, Diseases of the circulatory (Cardiovascular) system, RC666-701

الوصف: Background Current protocols generate highly pure human induced pluripotent stem cell–derived cardiomyocytes (hiPSC‐CMs) in vitro that recapitulate characteristics of mature in vivo cardiomyocytes. Yet, a risk of arrhythmias exists when hiPSC‐CMs are injected into large animal models. Thus, understanding hiPSC‐CM maturational mechanisms is crucial for clinical translation. Forkhead box (FOX) transcription factors regulate postnatal cardiomyocyte maturation through a balance between FOXO and FOXM1. We also previously demonstrated that p53 activation enhances hiPSC‐CM maturation. Here, we investigate whether p53 activation modulates the FOXO/FOXM1 balance to promote hiPSC‐CM maturation in 3‐dimensional suspension culture. Methods and Results Three‐dimensional cultures of hiPSC‐CMs were treated with Nutlin‐3a (p53 activator, 10 μM), LOM612 (FOXO relocator, 5 μM), AS1842856 (FOXO inhibitor, 1 μM), or RCM‐1 (FOXM1 inhibitor, 1 μM), starting 2 days after onset of beating, with dimethyl sulfoxide (0.2% vehicle) as control. P53 activation promoted hiPSC‐CM metabolic and electrophysiological maturation alongside FOXO upregulation and FOXM1 downregulation, in n=3 to 6 per group for all assays. FOXO inhibition significantly decreased expression of cardiac‐specific markers such as TNNT2. In contrast, FOXO activation or FOXM1 inhibition promoted maturational characteristics such as increased contractility, oxygen consumption, and voltage peak maximum upstroke velocity, in n=3 to 6 per group for all assays. Further, by single‐cell RNA sequencing of n=2 LOM612‐treated cells compared with dimethyl sulfoxide, LOM612‐mediated FOXO activation promoted expression of cardiac maturational pathways. Conclusions We show that p53 activation promotes FOXO and suppresses FOXM1 during 3‐dimensional hiPSC‐CM maturation. These results expand our understanding of hiPSC‐CM maturational mechanisms in a clinically‐relevant 3‐dimensional culture system.

وصف الملف: electronic resource

العلاقة: https://doaj.org/toc/2047-9980Test

الوصول الحر: https://doaj.org/article/0f6ccd0ad57e4a3b9de0de5968e8b89eTest

المؤلفون: Nivedhitha Velayutham, Katherine E. Yutzey

المصدر: Journal of Cardiovascular Development and Disease, Vol 9, Iss 4, p 93 (2022)

مصطلحات موضوعية: cardiac regeneration, pig heart, large mammal, cardiac preclinical studies, Diseases of the circulatory (Cardiovascular) system, RC666-701

الوصف: Swine are popular large mammals for cardiac preclinical testing due to their similarities with humans in terms of organ size and physiology. Recent studies indicate an early neonatal regenerative capacity for swine hearts similar to small mammal laboratory models such as rodents, inspiring exciting possibilities for studying cardiac regeneration with the goal of improved clinical translation to humans. However, while swine hearts are anatomically similar to humans, fundamental differences exist in growth mechanisms, nucleation, and the maturation of pig cardiomyocytes, which could present difficulties for the translation of preclinical findings in swine to human therapeutics. In this review, we discuss the maturational dynamics of pig cardiomyocytes and their capacity for proliferative cardiac regeneration during early neonatal development to provide a perspective on swine as a preclinical model for developing cardiac gene- and cell-based regenerative therapeutics.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/2308-3425/9/4/93Test; https://doaj.org/toc/2308-3425Test

الوصول الحر: https://doaj.org/article/84253e0f1b1c44959f94c2f923674acbTest

المؤلفون: Stephanie L. Padula, Nivedhitha Velayutham, Katherine E. Yutzey

المصدر: International Journal of Molecular Sciences, Vol 22, Iss 6, p 3288 (2021)

مصطلحات موضوعية: cardiomyocyte, transcription factors, nucleation, polyploidization, hypertrophy, sarcomere, Biology (General), QH301-705.5, Chemistry, QD1-999

الوصف: During the postnatal period, mammalian cardiomyocytes undergo numerous maturational changes associated with increased cardiac function and output, including hypertrophic growth, cell cycle exit, sarcomeric protein isoform switching, and mitochondrial maturation. These changes come at the expense of loss of regenerative capacity of the heart, contributing to heart failure after cardiac injury in adults. While most studies focus on the transcriptional regulation of embryonic or adult cardiomyocytes, the transcriptional changes that occur during the postnatal period are relatively unknown. In this review, we focus on the transcriptional regulators responsible for these aspects of cardiomyocyte maturation during the postnatal period in mammals. By specifically highlighting this transitional period, we draw attention to critical processes in cardiomyocyte maturation with potential therapeutic implications in cardiovascular disease.

وصف الملف: electronic resource

العلاقة: https://www.mdpi.com/1422-0067/22/6/3288Test; https://doaj.org/toc/1661-6596Test; https://doaj.org/toc/1422-0067Test

الوصول الحر: https://doaj.org/article/eeaa7b65732d414e8eeab4b7b9d74ea9Test

المؤلفون: Blanca Scheijen, Nivedhitha Velayutham, Frank N. van Leeuwen, Katherine E. Yutzey

المصدر: Circulation Research. 129

مصطلحات موضوعية: BTG2, Cell cycle checkpoint, Physiology, Biology, Cardiology and Cardiovascular Medicine, BTG1, Cell biology

الوصف: Background: Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes p21 , Meis1 , and Btg2 . Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation. Methods and Results: By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs in vitro, siRNA-mediated loss of Btg2 leads to increased CM proliferation. In vivo , Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs. Conclusions: Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::a6718b9f853c0080ba263e41790bfdb5Test
https://doi.org/10.1161/res.129.suppl_1.p448Test

المؤلفون: Christina M. Alfieri, Emma J Agnew, Sithara Raju Ponny, Farhan Zafar, Kyle W. Riggs, Katherine E. Yutzey, Nivedhitha Velayutham, Richard Baker

المصدر: The FASEB Journal. 35

مصطلحات موضوعية: medicine.anatomical_structure, Cell, Genetics, medicine, Biology, Cycling, Molecular Biology, Biochemistry, Biotechnology, Cell biology

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::26ec53fa6149e7f52b0925e8d2e75cbaTest
https://doi.org/10.1096/fasebj.2021.35.s1.03211Test

المؤلفون: Farhan Zafar, Emma J Agnew, Christina M. Alfieri, Kyle W. Riggs, Nivedhitha Velayutham, Katherine E. Yutzey, Sithara Raju Ponny, Richard S Baker

المصدر: Circulation. 142

مصطلحات موضوعية: education.field_of_study, Rodent, biology, business.industry, Cell, Population, Mitotic arrest, Cell biology, Cardiac regeneration, medicine.anatomical_structure, Physiology (medical), biology.animal, medicine, Cardiology and Cardiovascular Medicine, education, business

الوصف: Background: Rodent cardiomyocytes (CM) undergo mitotic arrest and decline of mononucleated-diploid population post-birth, which are implicated in neonatal loss of heart regenerative potential. However, the dynamics of postnatal CM maturation are largely unknown in swine, despite a similar neonatal cardiac regenerative capacity as rodents. Here, we provide a comprehensive analysis of postnatal cardiac maturation in swine, including CM cell cycling, multinucleation and hypertrophic growth, as well as non-CM cardiac factors such as extracellular matrix (ECM), immune cells, capillaries, and neurons. Our study reveals discordance in postnatal pig heart maturational events compared to rodents. Methods and Results: Left-ventricular myocardium from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed. Mature cardiac sarcomeric characteristics, such as fetal TNNI1 repression and CX43 co-localization to cell junctions, were not evident until P30 in pigs. In CMs, appreciable binucleation is observed by P7, with extensive multinucleation (4-16 nuclei per CM) beyond P15. Individual CM nuclei remain predominantly diploid at all ages. CM mononucleation at ~50% incidence is observed at P7-P15, and CM mitotic activity is measurable up to 2mo. CM cross-sectional area does not increase until 2mo-6mo in pigs, though longitudinal CM growth proportional to multinucleation occurs after P15. RNAseq analysis of neonatal pig left ventricles showed increased expression of ECM maturation, immune signaling, neuronal remodeling, and reactive oxygen species response genes, highlighting significance of the non-CM milieu in postnatal mammalian heart maturation. Conclusions: CM maturational events such as decline of mononucleation and cell cycle arrest occur over a 2-month postnatal period in pigs, despite reported loss of heart regenerative potential by P3. Moreover, CMs grow primarily by multinucleation and longitudinal hypertrophy in older pigs, distinct from mice and humans. These differences are important to consider for preclinical testing of cardiovascular therapies using swine, and may offer opportunities to study aspects of heart regeneration unavailable in other models.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::01153abe24d55f66738137d9a9ac3fcbTest
https://doi.org/10.1161/circ.142.suppl_3.13391Test

المؤلفون: Emma J Agnew, Kyle W. Riggs, Sithara Raju Ponny, Katherine E. Yutzey, Christina M. Alfieri, R. Scott Baker, Farhan Zafar, Nivedhitha Velayutham

المصدر: J Mol Cell Cardiol

مصطلحات موضوعية: 0301 basic medicine, Cell cycle checkpoint, Swine, Heart growth, Cell, Carboxylic Acids, 030204 cardiovascular system & hematology, Cell junction, Muscle hypertrophy, Extracellular matrix, 0302 clinical medicine, Gene expression, Myocytes, Cardiac, Neurons, education.field_of_study, 0303 health sciences, Cell Cycle, Gap Junctions, Extracellular Matrix, Up-Regulation, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Cardiology and Cardiovascular Medicine, Signal Transduction, Sarcomeres, Heart Ventricles, Population, Down-Regulation, Mitosis, Biology, Models, Biological, Article, Andrology, 03 medical and health sciences, medicine, Animals, Weaning, education, Molecular Biology, Cell Proliferation, 030304 developmental biology, Cell Nucleus, Fetus, Regeneration (biology), Hypertrophy, Diploidy, 030104 developmental biology, Animals, Newborn, Reactive Oxygen Species, Transcriptome

الوصف: AimsCardiomyocyte (CM) cell cycle arrest, decline of mononucleated-diploid CMs, sarcomeric maturation, and extracellular matrix remodeling are implicated in loss of cardiac regenerative potential in mice after birth. Recent studies show a 3-day neonatal regenerative capacity in pig hearts similar to mice, but postnatal pig CM growth dynamics are unknown. We examined cardiac maturation in postnatal pigs and mice, to determine the relative timing of developmental events underlying heart growth and regenerative potential in large and small mammals.Methods and ResultsLeft ventricular tissue from White Yorkshire-Landrace pigs at postnatal day (P)0 to 6 months (6mo) was analyzed to span birth, weaning, and adolescence in pigs, compared to similar physiological timepoints in mice. Collagen remodeling increases by P7 in postnatal pigs, but sarcomeric and gap junctional maturation only occur at 2mo. Also, there is no postnatal transition to beta-oxidation metabolism in pig hearts. Mononucleated CMs, predominant at birth, persist to 2mo in swine, with over 50% incidence of mononucleated-diploid CMs at P7-P15. Extensive multinucleation with 4-16 nuclei per CM occurs beyond P30. Pigs also exhibit increased CM length relative to multinucleation, preceding increase in CM width at 2mo-6mo. Further, robust CM mitotic nuclear pHH3 activity and cardiac cell cycle gene expression is apparent in pig left ventricles up to 2mo. By contrast, in mice, these maturational events occur concurrently in the first two postnatal weeks alongside loss of cardiac regenerative capacity.ConclusionsCardiac maturation occurs over a 6mo postnatal period in pigs, despite a similar early-neonatal heart regenerative window as mice. Postnatal pig CM growth includes increase in CM length alongside multinucleation, with CM cell cycle arrest and loss of mononucleated-diploid CMs occurring at 2mo-6mo. These CM characteristics are important to consider for pig preclinical studies and may offer opportunities to study aspects of heart regeneration unavailable in other models.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::cecf4e2e92f39a00087c18f0c3d64899Test
https://doi.org/10.1101/773101Test

المؤلفون: Tarek Alsaied, Farhan Zafar, R. Scott Baker, Kyle W. Riggs, Sithara Raju Ponny, Katherine E. Yutzey, Victoria Moore, Emma J Agnew, Gabriela Matos Ortiz, Nivedhitha Velayutham, Aaron M. Gibson, Christina M. Alfieri

مصطلحات موضوعية: Cardiac function curve, medicine.medical_specialty, Ejection fraction, business.industry, Ischemia, Inflammation, Histology, medicine.disease, medicine.anatomical_structure, Internal medicine, Occlusion, medicine, Cardiology, medicine.symptom, business, Reperfusion injury, Artery

الوصف: Studies in mice show a brief neonatal period of cardiac regeneration with minimal scar formation. Less is known about reparative mechanisms in large mammals. A transient cardiac injury approach (ischemia/reperfusion, IR) was used in weaned postnatal day (P)30 pigs, to assess regenerative repair in young large mammals. Female and male P30 pigs were subjected to cardiac ischemia (1 hour) by occlusion of the left anterior descending artery followed by reperfusion, or to sham operation. Following IR, myocardial damage occurred, with cardiac ejection fraction significantly decreased 2 hours post-ischemia. No improvement or worsening of cardiac function to the 4 week study end-point was observed. Histology demonstrated cardiomyocyte (CM) cell cycling at 2-months-of-age in multinucleated CMs in both sham-operated and IR pigs. Regional scar formation and inflammation in the epicardial region proximal to injury were observed 4 weeks post-IR. Sex differences were found, suggestive of females creating a greater fibrotic response with worse cardiac function, highlighting the importance of representing both sexes in cardiac injury studies. Together, our results describe an effective novel cardiac injury model in P30 swine, at a time when CMs are still cycling. Pigs subjected to IR show myocardial damage with a prolonged decrease in cardiac function, formation of a small, regional scar with increased inflammation. These data demonstrate that P30 pigs do not regenerate myocardium, even in the presence of CM mitotic activity, but form a scar after transient IR injury.NEW & NOTEWORTHYHere, we report for the first time ischemia/reperfusion (IR) cardiac injury in 1-month-old (P30) pigs. This model of IR injury highlights lack of cardiac regeneration, even in the presence of cardiomyocyte (CM) cell cycling in young swine. An effective injury approach is described for use in large mammals to investigate cardiac function, CM cell cycling, extracellular matrix (ECM) remodeling, and gene expression changes, while highlighting the importance of studying both sexes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::3c36330df10b86932b9c3e3426ee6c2dTest
https://doi.org/10.1101/773218Test

المؤلفون: Emma J Agnew, Nivedhitha Velayutham, Victoria Moore, Richard S Baker, Tarek Alsaied, Farhan Zafar, Kyle W. Riggs, Katherine E. Yutzey

المصدر: Circulation Research. 125

مصطلحات موضوعية: Physiology, Cardiology and Cardiovascular Medicine

الوصف: Objective: Infants with severe congenital heart defects (CHD) typically require lifesaving cardiac surgery. Performing this surgery during a time when the myocardium can proliferate and repair could improve successful outcomes. The mouse heart can regenerate in the first week post-partum, but the mitotic activity of cardiomyocytes (CM) is extended to 1-2-months post-partum in the pig. Thus, the regulation of CM cell cycling ability and renewal in the hearts of young large mammals after injury was examined. Methods & Results: Pigs at postnatal day 30 (n=6) were subjected to cardiac ischemia (1-hour) by temporary occlusion of the left anterior descending (LAD) artery followed by reperfusion (IR), or to sham operation (n=6, no LAD occlusion). LAD occlusion, below the second diagonal branch, provided an effective injury as indicated by increased circulating cardiac troponin-I 2-hours after injury. In addition, ejection fraction (EF) decreased by 46% (57% to 31%) at 2 hours post-ischemia, which was then maintained to the 4-week study end point. Pigs were sacrificed 4 weeks after surgery and histology demonstrated evidence of scar formation in the area of injury. However, no change in number of proliferating CM or cell death (via pHH3 or TUNEL immunohistochemistry respectively) was detected in IR pigs vs sham, or between regions of the left ventricle. Conclusions: Here we report a successful ischemic injury method, at an age previously not reported in swine. Pigs did not continue to decline towards heart failure following an IR injury at 1 month of life, with preservation of EF up to 4-weeks post-injury. This is also without a change in CM cell cycling activity at 2 months of age. This study highlights that even in the presence of a scar, young, large mammals can adapt to cardiac injury to maintain cardiac function. The pathways regulating scar formation and CM cell cycling are being further investigated by RNA-seq studies. If similar mechanisms operate in humans, it may be beneficial to perform CHD surgeries at a younger age when the heart is able to better tolerate injury, to ultimately improve long term outcomes.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::0d9c820e529958c8e501f3753cbec984Test
https://doi.org/10.1161/res.125.suppl_1.131Test

المؤلفون: Christina M. Alfieri, Kyle W. Riggs, Katherine E. Yutzey, Emma J Agnew, Farhan Zafar, Nivedhitha Velayutham, Richard S Baker

المصدر: Circulation Research. 125

مصطلحات موضوعية: Physiology, Cardiology and Cardiovascular Medicine

الوصف: Objectives: Cardiomyocyte (CM) cell cycle arrest and decline of mononucleated diploid CMs have been implicated in loss of regenerative potential in postnatal mouse hearts. Sarcomeric and extracellular matrix (ECM) maturation also occur concurrently in mice, influencing CM proliferative arrest. Recent studies show a 3-day neonatal period of cardiac regeneration in pigs similar to mice, but the dynamics of postnatal pig CM growth are unknown. Our objective is to explore cardiac cell cycling, growth and maturation in postnatal pigs to understand the events guiding loss of cardiac regenerative capacity in large mammals. Methods & Results: Left-ventricular tissue from farm pigs (White Yorkshire-Landrace) at Postnatal day (P)0, P7, P15, P30, 2 months (2mo) and 6mo were utilized. CM dissociations revealed predominant CM mononucleation at birth in swine, with persistence of ~50% (1186/2537 cells, n=5) mononucleated CMs at P15, and ~10% (227/1785 cells, n=4) at 2mo. By 6mo, pig CMs are entirely multinucleated, exhibiting 4-16 nuclei per cell. Assessing hypertrophic growth in dissociated pig CMs revealed longitudinal CM growth relative to increased nucleation at all ages. However, onset of diametric hypertrophy only occurs beyond 2mo. When nuclear pHH3 and mRNA expression of cell cycle genes was assessed, pig hearts show robust cell-cycling up to 2mo. Also, fetal TNNI1 and MYH6 are active up to 2mo in pig hearts. Ongoing studies on collagen remodeling indicate ECM remodeling in swine occurs beyond P7. Future studies are designed to identify nuclear ploidy in postnatal pig CMs and measure cytokinesis. Conclusions: Cardiac maturational events are staggered over a 2-6 month postnatal window in pigs, with older pig hearts exhibiting extensive CM multinucleation and differences in longitudinal versus diametric CM growth. These fundamental variations in CM growth characteristics are important to consider when designing preclinical trials for cardiac regenerative strategies in pigs. Also, despite a similar period of regenerative capacity as mice, pig hearts do not undergo loss of CM cell cycling and mononucleation until 2mo after birth. Utilizing pigs may thus offer unique opportunities to study aspects of heart regeneration unavailable in other animal models.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::68abc842da4788e74746d0774efb6c74Test
https://doi.org/10.1161/res.125.suppl_1.423Test