Substrate stiffness modulates cardiac fibroblast activation, senescence, and proinflammatory secretory phenotype.

Publisher: American Physiological Society Country of Publication: United States NLM ID: 100901228 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1522-1539 (Electronic) Linking ISSN: 03636135 NLM ISO Abbreviation: Am J Physiol Heart Circ Physiol Subsets: MEDLINE

Original Publication: Bethesda, Md. : American Physiological Society,

Myocardium*/metabolism ; Myofibroblasts*; Phenotype ; Extracellular Matrix/metabolism ; Hydrogels/analysis ; Hydrogels/metabolism ; Fibroblasts/metabolism ; Cellular Senescence ; Cells, Cultured

In vitro cultures of primary cardiac fibroblasts (CFs), the major extracellular matrix (ECM)-producing cells of the heart, are used to determine molecular mechanisms of cardiac fibrosis. However, the supraphysiologic stiffness of tissue culture polystyrene (TCPS) triggers the conversion of CFs into an activated myofibroblast-like state, and serial passage of the cells results in the induction of replicative senescence. These phenotypic switches confound the interpretation of experimental data obtained with cultured CFs. In an attempt to circumvent TCPS-induced activation and senescence of CFs, we used poly(ethylene glycol) (PEG) hydrogels as cell culture platforms with low and high stiffness formulations to mimic healthy and fibrotic hearts, respectively. Low hydrogel stiffness converted activated CFs into a quiescent state with a reduced abundance of α-smooth muscle actin (α-SMA)-containing stress fibers. Unexpectedly, lower substrate stiffness concomitantly augmented CF senescence, marked by elevated senescence-associated β-galactosidase (SA-β-Gal) activity and increased expression of p16 and p21, which are antiproliferative markers of senescence. Using dynamically stiffening hydrogels with phototunable cross-linking capabilities, we demonstrate that premature, substrate-induced CF senescence is partially reversible. RNA-sequencing analysis revealed widespread transcriptional reprogramming of CFs cultured on low-stiffness hydrogels, with a reduction in the expression of profibrotic genes encoding ECM proteins, and an attendant increase in expression of NF-κB-responsive inflammatory genes that typify the senescence-associated secretory phenotype (SASP). Our findings demonstrate that alterations in matrix stiffness profoundly impact CF cell state transitions, and suggest mechanisms by which CFs change phenotype in vivo depending on the stiffness of the myocardial microenvironment in which they reside. NEW & NOTEWORTHY Our findings highlight the advantages and pitfalls associated with culturing cardiac fibroblasts on hydrogels of varying stiffness. The findings also define stiffness-dependent signaling and transcriptional networks in cardiac fibroblasts.

Science. 2005 Nov 18;310(5751):1139-43. (PMID: 16293750)
Proc Natl Acad Sci U S A. 2020 Sep 1;117(35):21258-21266. (PMID: 32817542)
Cell. 2019 Oct 31;179(4):813-827. (PMID: 31675495)
Circulation. 2022 Feb 15;145(7):513-530. (PMID: 35000411)
Circ Res. 2016 Mar 18;118(6):1021-40. (PMID: 26987915)
Mol Cell Biol. 2021 Jan 25;41(2):. (PMID: 33077499)
Adv Sci (Weinh). 2018 Dec 10;6(3):1801483. (PMID: 30775233)
Nat Rev Cardiol. 2017 Aug;14(8):484-491. (PMID: 28436487)
Am J Physiol Heart Circ Physiol. 2022 Aug 1;323(2):H285-H300. (PMID: 35714177)
Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9363-7. (PMID: 7568133)
Biophys J. 2007 Dec 15;93(12):4453-61. (PMID: 18045965)
Biomaterials. 2015 May;49:47-56. (PMID: 25725554)
Adv Mater. 2009 Dec 28;21(48):5005-5010. (PMID: 25377720)
Eur J Cancer. 1997 Apr;33(5):703-9. (PMID: 9282108)
Genome Biol. 2014;15(12):550. (PMID: 25516281)
Curr Biol. 2017 Sep 11;27(17):2652-2660.e4. (PMID: 28844647)
J Mol Cell Cardiol. 2016 Feb;91:1-5. (PMID: 26748307)
Am J Physiol Heart Circ Physiol. 2006 Jun;290(6):H2196-203. (PMID: 16473959)
Circulation. 2015 Apr 7;131(14):1247-59. (PMID: 25637629)
J Am Heart Assoc. 2020 Oct 20;9(19):e017025. (PMID: 32924724)
J Magn Reson Imaging. 2017 Nov;46(5):1361-1367. (PMID: 28236336)
Angew Chem Int Ed Engl. 2019 Jul 15;58(29):9912-9916. (PMID: 31119851)
Circ Res. 2016 Jun 10;118(12):1906-17. (PMID: 27140435)
Genes Dev. 2014 Jan 15;28(2):99-114. (PMID: 24449267)
Sci Adv. 2020 Apr 08;6(15):eaaw0158. (PMID: 32284989)
Nat Commun. 2016 Jul 22;7:12260. (PMID: 27447449)
Trends Biochem Sci. 2022 Dec;47(12):1009-1022. (PMID: 35835684)
Sci Transl Med. 2019 Sep 11;11(509):. (PMID: 31511425)
Bioeng Transl Med. 2022 Aug 22;7(3):e10394. (PMID: 36176599)
Mech Ageing Dev. 1976 Jan-Feb;5(1):45-56. (PMID: 1263608)
Bioinformatics. 2013 Jan 1;29(1):15-21. (PMID: 23104886)
J Mol Cell Cardiol. 2018 Jan;114:161-174. (PMID: 29158033)
EMBO J. 2003 Aug 15;22(16):4212-22. (PMID: 12912919)
Mol Cell Biol. 1995 May;15(5):2809-18. (PMID: 7739562)
Dev Dyn. 2010 Jun;239(6):1573-84. (PMID: 20503355)
Circ Res. 2019 Sep 13;125(7):662-677. (PMID: 31409188)
Nat Cell Biol. 2019 Jan;21(1):94-101. (PMID: 30602768)
Cell. 2003 Jun 13;113(6):703-16. (PMID: 12809602)

DP2 HL173948 United States HL NHLBI NIH HHS; R01 HL127240 United States HL NHLBI NIH HHS; R25 HL145817 United States HL NHLBI NIH HHS; T32 HL007444 United States HL NHLBI NIH HHS; R00 HL148542 United States HL NHLBI NIH HHS; K99 HL166708 United States HL NHLBI NIH HHS; K99 HL148542 United States HL NHLBI NIH HHS; R01 HL150225 United States HL NHLBI NIH HHS; R01 DK119594 United States DK NIDDK NIH HHS

Keywords: culture substrate; fibroblast; fibrosis; hydrogel; inflammation; senescence

0 (Hydrogels)

Date Created: 20231027 Date Completed: 20231206 Latest Revision: 20240830

20240830

PMC11213481

10.1152/ajpheart.00483.2023

37889253