Deletion of the Lmna gene in fibroblasts causes senescence-associated dilated cardiomyopathy by activating the double-stranded DNA damage response and induction of senescence-associated secretory phenotype

Abstract

Introduction: Mutations in the LMNA gene, encoding Lamin A/C (LMNA), are established causes of dilated cardiomyopathy (DCM). The phenotype is typically characterized by progressive cardiac conduction defects, arrhythmias, heart failure, and premature death. DCM is primarily considered a disease of cardiac myocytes. However, LMNA is also expressed in other cardiac cell types, including fibroblasts.

Aim: The purpose of the study was to determine the contribution of the fibroblasts to DCM caused by LMNA deficiency.

Methods and Results: The Lmna gene was deleted by crossing the platelet-derived growth factor receptor α-Cre recombinase (Pdgfra-Cre) and floxed Lmna (Lmna^F/F) mice. The LMNA protein was nearly absent in ~80% of the cardiac fibroblasts and ~25% of cardiac myocytes in the Pdgfra-Cre:Lmna^F/F mice. The Pdgfra-Cre:Lmna^F/F mice showed an early phenotype characterized by cardiac conduction defects, arrhythmias, cardiac dysfunction, myocardial fibrosis, apoptosis, and premature death within the first six weeks of life. The Pdgfra-Cre:Lmna^{Wild type/F} (Lmna^W/F) mice also showed a similar but slowly evolving phenotype that was expressed within one year of age. RNA sequencing of LMNA-deficient and wild-type cardiac fibroblasts identified differential expression of ~410 genes, which predicted activation of the TP53 and TNFA/NFκB and suppression of the cell cycle pathways. In agreement with these findings, levels of phospho-H2AFX, ATM, phospho-TP53, and CDKN1A, markers of the DNA damage response (DDR) pathway, were increased in the Pdgfra-Cre:Lmna^F/F mouse hearts. Moreover, expression of senescence-associated beta-galactosidase was induced and levels of the senescence-associated secretory phenotype (SASP) proteins TGFβ1, CTGF (CCN2), and LGLAS3 were increased as well as the transcript levels of additional genes encoding SASP proteins in the Pdgfra-Cre:Lmna^F/F mouse hearts. Finally, expression of pH2AFX, a bonafide marker of the double-stranded DNA breaks, was increased in cardiac fibroblasts isolated from the Pdgfra-Cre:Lmna^F/F mouse hearts.

Conclusion: Deletion of the Lmna gene in fibroblasts partially recapitulates the phenotype of the LMNA-associated DCM, likely through induction of double-stranded DNA breaks, activation of the DDR pathway, and induction of expression of the SASP proteins. The findings indicate that the phenotype in the LMNA-associated DCM is the aggregate consequence of the LMNA deficiency in multiple cardiac cells, including cardiac fibroblasts.

One sentence summary: Cardiac fibroblasts contribute to the pathogenesis of DCM - associated with LMNA deficiency through activation of the senescence-associated secretory phenotype.

Keywords

Fibroblasts / senescence / fibrosis / apoptosis / lamin A/C / heart failure / cardiomyopathy

References

[1] Emmons-Bell S,Roth G.Prevalence, incidence and survival of heart failure: a systematic review.Heart2022:heartjnl-2021

[2] James SL,Abate KH.Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017.Lancet2018;392:1789-858. PMCID:PMC6227754

[3] Khush KK,Chambers DC.The international thoracic organ transplant registry of the international society for heart and lung transplantation: thirty-sixth adult heart transplantation report - 2019; focus theme: donor and recipient size match.J Heart Lung Transplant2019;38:1056-1066. PMCID:PMC6816343

[4] Hershberger RE,Jordan E.The complex and diverse genetic architecture of dilated cardiomyopathy.Circ Res2021;128:1514-1532. PMCID:PMC8158434

[5] McNally EM.Dilated cardiomyopathy: genetic determinants and mechanisms.Circ Res2017;121:731-748. PMCID:PMC5626020

[6] Fatkin D,Sasaki T.Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease.N Engl J Med1999;341:1715-1724.

[7] Taylor MR,Sinagra G.Familial dilated cardiomyopathy registry research g. natural history of dilated cardiomyopathy due to lamin A/C gene mutations.J Am Coll Cardiol2003;41:771-780.

[8] Rober RA,Osborn M.Differential timing of nuclear lamin A/C expression in the various organs of the mouse embryo and the young animal: a developmental study.Development1989;105:365-378.

[9] Schreiber KH.When lamins go bad: nuclear structure and disease.Cell2013;152:1365-1375. PMCID:PMC3706202

[10] Vidak S.Molecular insights into the premature aging disease progeria.Histochem Cell Biol2016;145:401-417. PMCID:PMC4796323

[11] Corrado D,Judge DP.Arrhythmogenic cardiomyopathy.Circ Res2017;121:784-802.

[12] Anselme F,Savoure A.Implantable cardioverter-defibrillators in lamin A/C mutation carriers with cardiac conduction disorders.Heart Rhythm2013;10:1492-1498.

[13] Van Berlo JH,van der Kooi AJ.Meta-analysis of clinical characteristics of 299 carriers of LMNA gene mutations: do lamin A/C mutations portend a high risk of sudden death?.J Mol Med (Berl)2005;83:79-83.

[14] Kim Y.Generation and characterization of a conditional deletion allele for Lmna in mice.Biochem Biophys Res Commun2013;440:8-13. PMCID:PMC3840542

[15] Roesch K,Trimarchi JM.The transcriptome of retinal Muller glial cells.J Comp Neurol2008;509:225-238. PMCID:PMC2665263

[16] Lombardi R,Ruggiero A.Cardiac Fibro-adipocyte progenitors express desmosome proteins and preferentially differentiate to adipocytes upon deletion of the desmoplakin gene.Circ Res2016;119:41-54. PMCID:PMC4920717

[17] Rouhi L,Chen SN.Haploinsufficiency of Tmem43 in cardiac myocytes activates the DNA damage response pathway leading to a late-onset senescence-associated pro-fibrotic cardiomyopathy.Cardiovasc Res2021;117:2377-2394. PMCID:PMC8861264

[18] Cheedipudi SM,Fan S.Exercise restores dysregulated gene expression in a mouse model of arrhythmogenic cardiomyopathy.Cardiovasc Res2020;116:1199-1213. PMCID:PMC7177479

[19] Auguste G,Matkovich SJ.BET bromodomain inhibition attenuates cardiac phenotype in myocyte-specific lamin A/C-deficient mice.J Clin Invest2020;130:4740-4758. PMCID:PMC7456228

[20] Devereux RB,Lutas EM.Echocardiographic assessment of left ventricular hypertrophy: comparison to necropsy findings.Am J Cardiol1986;57:450-458.

[21] Chen SN,Karmouch J.DNA Damage Response/TP53 Pathway Is Activated and Contributes to the Pathogenesis of Dilated Cardiomyopathy Associated With LMNA (Lamin A/C) Mutations.Circ Res2019;124:856-873. PMCID:PMC6460911

[22] Olcum M,Rouhi L.The WNT/beta-catenin pathway regulates expression of the genes involved in cell cycle progression and mitochondrial oxidative phosphorylation in the postmitotic cardiac myocytes.J Cardiovasc Aging2022;2:15 PMCID:PMC8874274

[23] Auguste G,Lombardi R,Willerson JT.Suppression of activated FOXO transcription factors in the heart prolongs survival in a mouse model of laminopathies.Circ Res2018;122:678-692. PMCID:PMC5834384

[24] Kim D,Park C,Salzberg SL.Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype.Nat Biotechnol2019;37:907-915. PMCID:PMC7605509

[25] Liao Y,Shi W.Featurecounts: an efficient general purpose program for assigning sequence reads to genomic features.Bioinformatics2014;30:923-930.

[26] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550. PMCID:PMC4302049

[27] Hochberg Y.More powerful procedures for multiple significance testing.Stat Med1990;9:811-818.

[28] Rouhi L,Cheedipudi SM.Effects of tamoxifen inducible MerCreMer on gene expression in cardiac myocytes in mice.J Cardiovasc Aging2022;2:8 PMCID:PMC8874274

[29] Accornero F,Benard MJ,Carmeliet P.Placental growth factor regulates cardiac adaptation and hypertrophy through a paracrine mechanism.Circ Res2011;109:272-280 PMCID:PMC3146170