Modeling Peripartum Cardiomyopathy With Human Induced Pluripotent Stem Cells Reveals Distinctive Abnormal Function of Cardiomyocytes
2018; Lippincott Williams & Wilkins; Volume: 138; Issue: 23 Linguagem: Inglês
10.1161/circulationaha.118.035950
ISSN1524-4539
AutoresNili Naftali‐Shani, Natali Molotski, Yael Nevo‐Caspi, Michael Arad, Rafael Kuperstein, Uri Amit, Irit Huber, Li‐at Zeltzer, Alina Levich, Haya Abbas, Lorenzo Monserrat, Gideon Paret, Jonathan Leor,
Tópico(s)Congenital Heart Disease Studies
ResumoHomeCirculationVol. 138, No. 23Modeling Peripartum Cardiomyopathy With Human Induced Pluripotent Stem Cells Reveals Distinctive Abnormal Function of Cardiomyocytes Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBModeling Peripartum Cardiomyopathy With Human Induced Pluripotent Stem Cells Reveals Distinctive Abnormal Function of Cardiomyocytes Nili Naftali-Shani, PhD, Natali Molotski, PhD, Yael Nevo-Caspi, PhD, Michael Arad, MD, Rafael Kuperstein, MD, Uri Amit, MD, MPH, PhD, Irit Huber, PhD, Li-at Zeltzer, BSc, Alina Levich, BSc, Haya Abbas, MSc, Lorenzo Monserrat, MD, PhD, Gideon Paret, MD and Jonathan Leor, MD Nili Naftali-ShaniNili Naftali-Shani Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Natali MolotskiNatali Molotski Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Yael Nevo-CaspiYael Nevo-Caspi Department of Pediatric Critical Care Medicine, Safra Children's Hospital, Tel-Hashomer, Israel (Y.N.-C., H.A., G.P.). , Michael AradMichael Arad SacklerSchool of Medicine (M.A., R.K., J.L.), Tel-Aviv University, Israel. Department of Cardiology (M.A., R.K., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Rafael KupersteinRafael Kuperstein SacklerSchool of Medicine (M.A., R.K., J.L.), Tel-Aviv University, Israel. Department of Cardiology (M.A., R.K., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Uri AmitUri Amit Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Irit HuberIrit Huber The Sohnis Family Laboratory for Cardiac Electrophysiology and Regenerative Medicine, Rappaport Faculty of Medicine, Technion-Institute of Technology, Haifa, Israel (I.H.). , Li-at ZeltzerLi-at Zeltzer Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Alina LevichAlina Levich Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. , Haya AbbasHaya Abbas Department of Pediatric Critical Care Medicine, Safra Children's Hospital, Tel-Hashomer, Israel (Y.N.-C., H.A., G.P.). , Lorenzo MonserratLorenzo Monserrat Cardiology Department, Health in Code, A Coruña, Spain (L.M.). , Gideon ParetGideon Paret Department of Pediatric Critical Care Medicine, Safra Children's Hospital, Tel-Hashomer, Israel (Y.N.-C., H.A., G.P.). and Jonathan LeorJonathan Leor Jonathan Leor, MD, Neufeld Cardiac Research Institute, Sheba Medical Center, Tel-Hashomer 52621, Israel. Email l E-mail Address: [email protected] Neufeld Cardiac Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Tel-Aviv University, Israel. SacklerSchool of Medicine (M.A., R.K., J.L.), Tel-Aviv University, Israel. Tamman Cardiovascular Research Institute (N.N.-S., N.M., U.A., L.-a.Z, A.L., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. Department of Cardiology (M.A., R.K., J.L.), Leviev Heart Center, Sheba Medical Center, Tel-Hashomer, Israel. Originally published3 Dec 2018https://doi.org/10.1161/CIRCULATIONAHA.118.035950Circulation. 2018;138:2721–2723Peripartum cardiomyopathy (PPCM) is a rare but significant heart failure disease of unknown cause.1 PPCM develops toward the end of pregnancy or in the first months after delivery.1 The mechanism of the disease is unclear but has been attributed to vascular dysfunction triggered by late gestational maternal hormones.1 The transcription factor signal transducer and activator of transcription 3 (STAT3) regulates a protective and reparative response in the heart. Low levels of STAT3 in cardiomyocytes and subsequent oxidative stress have been suggested to predispose endothelial cells to the toxic effects of prolactin metabolites and the development of PPCM.2 This theory is the rationale for the treatment of patients with PPCM with a prolactin antagonist. However, this theory has been questioned and debated. Thus, our aims were to model PPCM with induced pluripotent stem cell–derived cardiomyocytes (iCMs) and to determine whether low levels of STAT3 characterize PPCM iCMs.The study was approved by the Sheba Medical Center institutional review board, and the participants gave written informed consent. First, we generated induced pluripotent stem cells from skin biopsies (10 donors, successful in 8 patients) and then differentiated them toward cardiomyocytes (successful in 6 individuals: 3 with PPCM and 3 healthy control subjects; Figure [A]). We created iCMs from patients with a wide clinical spectrum (Figure [A]).Download figureDownload PowerPointFigure. Abnormal function of induced pluripotent stem cell–derived cardiomyocytes (iCMs) from patients with peripartum cardiomyopathy (PPCM).A, Characteristics of patients with PPCM and healthy control subjects. To determine whether low signal transducer and activator of transcription 3 (STAT3) characterized cardiomyocytes from patients with PPCM, we quantified STAT3 protein expression in iCMs from patients with PPCM and healthy control subjects before and after isoprenaline stimulation. Cardiomyocytes were treated with isoprenaline for 20 hours before protein extraction and analysis by Western blot. Representative Western blot for total STAT3α and STAT3β proteins in a patient with PPCM (No. 3) and a healthy control (No. 6) shows higher levels of STAT3α and STAT3β in PPCM cardiomyocytes (B). Relative quantification analysis shows that the basal levels of STAT3 isoforms (α and β) were higher in PPCM cardiomyocytes than in control subjects. The expression of STAT3 proteins remained higher after isoprenaline stimulation (C). To determine the paracrine profile of PPCM cardiomyocytes, we measured the levels of secreted cytokines in the conditioned medium of cultured iCMs (for 20 hours). Significantly, although incremental doses of isoprenaline stimulate vascular endothelial growth factor (VEGF) secretion from healthy cardiomyocytes, VEGF in PPCM medium was undetectable (D). Moreover, PPCM cardiomyocyte secreted higher levels of the VEGF inhibitor soluble fms-like tyrosine kinase-1 (sFlt-1; E) compared with healthy control subjects, with or without isoprenaline stimulation. Although increasing doses of isoprenaline stimulate cytokine secretion from healthy iCMs, they repressed cytokine secretion from PPCM iCMs (F through I). Variables are expressed as mean±SEM. Outliers were identified by the ROUT method (Q=1%). Differences between the groups for each isoprenaline concentration were analyzed by 2-way ANOVA and the Holm-Sidak multiple-comparison test. We used GraphPad Prism7 for Windows (GraphPad Software). IL indicates interleukin; LV, left ventricular; LVEF, left ventricular ejection fraction; NA, not applicable; ROUT, robust regression and outlier removal; and TNFα, tumor necrosis factor-α.To determine whether low expression of STAT3 characterizes cardiomyocytes from patients with PPCM, we quantified STAT3 protein levels in iCMs and compared them with healthy control subjects. To simulate the physiological and metabolic stress associated with late pregnancy and labor, we used the β1-adrenergic receptor stimulant isoprenaline. The rationale for this approach was the reported ability of isoprenaline to induce heart failure and high mortality in pregnant and nonpregnant mice carrying a cardiomyocyte-restricted deletion of STAT3 and allegedly to mimic PPCM.3We exposed iCMs to increasing doses of isoprenaline for 20 hours and measured STAT3 expression by Western blot analysis (Figure [B]). To our surprise, the basal levels of both STAT3 isoforms (α and β) were higher in PPCM iCMs than in the control subjects (Figure [C]). Furthermore, the amount of STAT3 proteins did not vary after isoprenaline stimulation and remained higher in PPCM than in control iCMs (Figure [C]). Of interest, the highest amounts of STAT3α and STAT3β, with and without isoprenaline stimulation, were encountered in a patient who developed end-stage heart failure who required a heart transplantation (patient 1; Figure [C]). The mean expression of STAT3β was similar with and without isoprenaline stimulation.PPCM is considered a disease of angiogenic imbalance.1,4 Thus, we measured the levels of the angiogenic vascular endothelial growth factor and its inhibitor, soluble fms-like tyrosine kinase-1, in the conditioned medium collected from the cardiomyocytes. Remarkably, although normal cardiomyocytes secreted increasing amounts of vascular endothelial growth factor in response to increasing doses of isoprenaline, the levels of vascular endothelial growth factor in the culture medium of PPCM iCMs were undetectable (Figure [D]). Furthermore, the levels of soluble fms-like tyrosine kinase-1 in the medium from PPCM iCMs were significantly greater than in normal iCMs (Figure [E]). Together, our findings suggest that the impaired angiogenic capacity in PPCM is partially mediated by cardiomyocyte soluble fms-like tyrosine kinase-1.PPCM is also considered a proinflammatory disease.1 Thus, we measured the amount of representative proinflammatory (interleukin [IL]-1α, tumor necrosis factor-α, IL-6) and profibrotic (IL-4) cytokines. The basal level of all tested cytokines was higher in PPCM iCMs than in control iCMs (Figure [F–I]), suggesting that PPCM iCMs are polarized toward a proinflammatory state. However, although isoprenaline significantly increased the secretion of IL-1α, tumor necrosis factor-α, IL-6, and IL-4 from normal cardiomyocytes, it suppressed the secretion of these cytokines from PPCM iCMs (Figure [F–I]). Thus, PPCM iCMs secrete more cytokines at the basal state but have an impaired cytokine response to stress.Finally, in our patient with PPCM, severe left ventricular dysfunction (patient 1; Figure [A]), and a family history of heart failure, we analyzed a gene panel for dilated cardiomyopathy that included 96 genes using a massively parallel sequencing method (Health in Code, A Coruña, Spain). We identified a mutation in the titin gene (TTN) (variant c.34681C>T, p.Arg11561*, heterozygosis) that causes protein truncation. Titin mutations are the most common genetic defect predisposing to the development of PPCM.5 Thus, our findings could be relevant to patients with PPCM of various severities and causes.Our findings should be interpreted with caution because of the small sample size, which may create a bias and overestimate the actual effect. A more extensive study is needed to confirm our findings. We believe that awareness of our findings is essential because our study challenges the low STAT3/prolactin theory about the pathogenesis of PPCM.2 This theory is the rationale for the potential harmful treatment with bromocriptine, a prolactin antagonist, for many women with PPCM worldwide. The doubt we raise about the validity of this theory is scientifically and clinically important. Our findings of inherent abnormalities in human PPCM cardiomyocytes should stimulate further research to determine the source of this abnormality.AcknowledgmentsThe authors thank Vivienne York for her skillful English-language editing.Sources of FundingThis project was supported by a research grant from Imperial Tobacco Ltd.DisclosuresNone.Footnotes*Drs Naftali-Shani and Molotski contributed equally.https://www.ahajournals.org/journal/circJonathan Leor, MD, Neufeld Cardiac Research Institute, Sheba Medical Center, Tel-Hashomer 52621, Israel. Email l[email protected]tau.ac.ilReferences1. Arany Z, Elkayam U. Peripartum cardiomyopathy.Circulation. 2016; 133:1397–1409. doi: 10.1161/CIRCULATIONAHA.115.020491LinkGoogle Scholar2. Hilfiker-Kleiner D, Kaminski K, Podewski E, Bonda T, Schaefer A, Sliwa K, Forster O, Quint A, Landmesser U, Doerries C, Luchtefeld M, Poli V, Schneider MD, Balligand JL, Desjardins F, Ansari A, Struman I, Nguyen NQ, Zschemisch NH, Klein G, Heusch G, Schulz R, Hilfiker A, Drexler H. A cathepsin D-cleaved 16 kDa form of prolactin mediates postpartum cardiomyopathy.Cell. 2007; 128:589–600. doi: 10.1016/j.cell.2006.12.036CrossrefMedlineGoogle Scholar3. Stapel B, Kohlhaas M, Ricke-Hoch M, Haghikia A, Erschow S, Knuuti J, Silvola JM, Roivainen A, Saraste A, Nickel AG, Saar JA, Sieve I, Pietzsch S, Müller M, Bogeski I, Kappl R, Jauhiainen M, Thackeray JT, Scherr M, Bengel FM, Hagl C, Tudorache I, Bauersachs J, Maack C, Hilfiker-Kleiner D. Low STAT3 expression sensitizes to toxic effects of β-adrenergic receptor stimulation in peripartum cardiomyopathy.Eur Heart J. 2017; 38:349–361. doi: 10.1093/eurheartj/ehw086MedlineGoogle Scholar4. Patten IS, Rana S, Shahul S, Rowe GC, Jang C, Liu L, Hacker MR, Rhee JS, Mitchell J, Mahmood F, Hess P, Farrell C, Koulisis N, Khankin EV, Burke SD, Tudorache I, Bauersachs J, del Monte F, Hilfiker-Kleiner D, Karumanchi SA, Arany Z. Cardiac angiogenic imbalance leads to peripartum cardiomyopathy.Nature. 2012; 485:333–338. doi: 10.1038/nature11040CrossrefMedlineGoogle Scholar5. Ware JS, Li J, Mazaika E, Yasso CM, DeSouza T, Cappola TP, Tsai EJ, Hilfiker-Kleiner D, Kamiya CA, Mazzarotto F, Cook SA, Halder I, Prasad SK, Pisarcik J, Hanley-Yanez K, Alharethi R, Damp J, Hsich E, Elkayam U, Sheppard R, Kealey A, Alexis J, Ramani G, Safirstein J, Boehmer J, Pauly DF, Wittstein IS, Thohan V, Zucker MJ, Liu P, Gorcsan J, McNamara DM, Seidman CE, Seidman JG, Arany Z; IMAC-2 and IPAC Investigators. Shared genetic predisposition in peripartum and dilated cardiomyopathies.N Engl J Med. 2016; 374:233–241. doi: 10.1056/NEJMoa1505517CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited ByHilfiker-Kleiner D, Sliwa K and Bauersachs J (2019) Letter by Hilfiker-Kleiner et al Regarding Article, "Modeling Peripartum Cardiomyopathy With Human Induced Pluripotent Stem Cells Reveals Distinctive Abnormal Function of Cardiomyocytes", Circulation, 139:21, (e990-e991), Online publication date: 21-May-2019.Naftali-Shani N, Arad M, Kuperstein R, Amit U and Leor J (2019) Response by Naftali-Shani et al to Letter Regarding Article, "Modeling Peripartum Cardiomyopathy With Human Induced Pluripotent Stem Cells Reveals Distinctive Abnormal Function of Cardiomyocytes", Circulation, 139:21, (e992-e993), Online publication date: 21-May-2019. Ballard L, Cois A and Kea B (2019) Peripartum Cardiomyopathy: a Review, Current Emergency and Hospital Medicine Reports, 10.1007/s40138-019-00192-3, 7:3, (127-134), Online publication date: 1-Sep-2019. Bekhite M and Schulze P (2021) Human Induced Pluripotent Stem Cell as a Disease Modeling and Drug Development Platform—A Cardiac Perspective, Cells, 10.3390/cells10123483, 10:12, (3483) Brodehl A, Ebbinghaus H, Deutsch M, Gummert J, Gärtner A, Ratnavadivel S and Milting H (2019) Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies, International Journal of Molecular Sciences, 10.3390/ijms20184381, 20:18, (4381) Martewicz S, Magnussen M and Elvassore N (2020) Beyond Family: Modeling Non-hereditary Heart Diseases With Human Pluripotent Stem Cell-Derived Cardiomyocytes, Frontiers in Physiology, 10.3389/fphys.2020.00384, 11 December 4, 2018Vol 138, Issue 23 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.118.035950PMID: 30571272 Originally publishedDecember 3, 2018 Keywordsinflammationvascular endothelial growth factorsSTAT3induced pluripotent stem cellscardiomyopathiesneovascularization, physiologicPDF download Advertisement SubjectsAngiogenesisCardiomyopathyCell Signaling/Signal TransductionCellular ReprogrammingGrowth Factors/Cytokines
Referência(s)