In vivo elongation of thin filaments results in heart failure

Autoři: Lei Mi-Mi aff001;  Gerrie P. Farman aff001;  Rachel M. Mayfield aff001;  Joshua Strom aff001;  Miensheng Chu aff001;  Christopher T. Pappas aff001;  Carol C. Gregorio aff001
Působiště autorů: Department of Cellular and Molecular Medicine and Sarver Molecular Cardiovascular Research Program, The University of Arizona, Tucson, AZ, United States of America aff001
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0226138


A novel cardiac-specific transgenic mouse model was generated to identify the physiological consequences of elongated thin filaments during post-natal development in the heart. Remarkably, increasing the expression levels in vivo of just one sarcomeric protein, Lmod2, results in ~10% longer thin filaments (up to 26% longer in some individual sarcomeres) that produce up to 50% less contractile force. Increasing the levels of Lmod2 in vivo (Lmod2-TG) also allows us to probe the contribution of Lmod2 in the progression of cardiac myopathy because Lmod2-TG mice present with a unique cardiomyopathy involving enlarged atrial and ventricular lumens, increased heart mass, disorganized myofibrils and eventually, heart failure. Turning off of Lmod2 transgene expression at postnatal day 3 successfully prevents thin filament elongation, as well as gross morphological and functional disease progression. We show here that Lmod2 has an essential role in regulating cardiac contractile force and function.

Klíčová slova:

Cardiac muscles – Fibrosis – Hyperexpression techniques – Mouse models – Muscle contraction – Cardiomyocytes – Heart – Sarcomeres


1. Bang M-L, Li X, Littlefield R, Bremner S, Thor A, Knowlton KU, et al. Nebulin-deficient mice exhibit shorter thin filament lengths and reduced contractile function in skeletal muscle. J Cell Biol. 2006 Jun 19;173(6):905–16. doi: 10.1083/jcb.200603119 16769824

2. Ottenheijm CAC, Witt CC, Stienen GJ, Labeit S, Beggs AH, Granzier H. Thin filament length dysregulation contributes to muscle weakness in nemaline myopathy patients with nebulin deficiency. Hum Mol Genet. 2009 Jul 1;18(13):2359–69. doi: 10.1093/hmg/ddp168 19346529

3. Gokhin DS, Dubuc EA, Lian KQ, Peters LL, Fowler VM. Alterations in thin filament length during postnatal skeletal muscle development and aging in mice. Front Physiol. 2014 Sep 29;5:375. doi: 10.3389/fphys.2014.00375 25324783

4. Pappas CT, Mayfield RM, Henderson C, Jamilpour N, Cover C, Hernandez Z, et al. Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality. Proc Natl Acad Sci. 2015;112(44):13573–8. doi: 10.1073/pnas.1508273112 26487682

5. Winter JMD, Joureau B, Lee EJ, Kiss B, Yuen M, Gupta VA, et al. Mutation-specific effects on thin filament length in thin filament myopathy. Ann Neurol. 2016;79(6):959–69. doi: 10.1002/ana.24654 27074222

6. Sanger JW, Wang J, Fan Y, White J, Mi-Mi L, Dube DK, et al. Assembly and Maintenance of Myofibrils in Striated Muscle. In: Handbook of experimental pharmacology. 2016. p. 39–75.

7. Henderson CA, Gomez CG, Novak SM, Mi-Mi L, Gregorio CC. Overview of the Muscle Cytoskeleton. Compr Physiol. 2017 Jun 18;7(3):891–944. doi: 10.1002/cphy.c160033 28640448

8. Fowler VM, Dominguez R. Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles. Biophys J. 2017;112(9):1742–60. doi: 10.1016/j.bpj.2017.03.034 28494946

9. Conley CA, Fritz-Six KL, Almenar-Queralt A, Fowler VM. Leiomodins: Larger members of the tropomodulin (Tmod) gene family. Genomics. 2001;73(2):127–39. doi: 10.1006/geno.2000.6501 11318603

10. Chereau D, Boczkowska M, Skwarek-Maruszewska A, Fujiwara I, Hayes DB, Rebowski G, et al. Leiomodin is an actin filament nucleator in muscle cells. Science. 2008 Apr 11;320(5873):239–43. doi: 10.1126/science.1155313 18403713

11. Tsukada T, Pappas CT, Moroz N, Antin PB, Kostyukova AS, Gregorio CC. Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle. J Cell Sci. 2010;123(18):3136–45.

12. Chen X, Ni F, Kondrashkina E, Ma J, Wang Q. Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells. Proc Natl Acad Sci. 2015;112(41):12687–92. doi: 10.1073/pnas.1512464112 26417072

13. Boczkowska M, Rebowski G, Kremneva E, Lappalainen P, Dominguez R. How Leiomodin and Tropomodulin use a common fold for different actin assembly functions. Nat Commun. 2015;6(May):1–12.

14. Arslan B, Colpan M, Gray KT, Abu-Lail NI, Kostyukova AS. Characterizing interaction forces between actin and proteins of the tropomodulin family reveals the presence of the N-terminal actin-binding site in leiomodin. Arch Biochem Biophys. 2018;638(December 2017):18–26.

15. Nanda V, Miano JM. Leiomodin 1, a new serum response factor-dependent target gene expressed preferentially in differentiated smooth muscle cells. J Biol Chem. 2012;287(4):2459–67. doi: 10.1074/jbc.M111.302224 22157009

16. Garg A, O’Rourke J, Long C, Doering J, Ravenscroft G, Bezprozvannaya S, et al. KLHL40 deficiency destabilizes thin filament proteins and promotes nemaline myopathy. J Clin Invest. 2014 Aug;124(8):3529–39. doi: 10.1172/JCI74994 24960163

17. Nworu CU, Kraft R, Schnurr DC, Gregorio CC, Krieg PA. Leiomodin 3 and Tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis. J Cell Sci. 2014;jcs.152702:Advance Online Article November 27, 2014.

18. Li S, Mo K, Tian H, Chu C, Sun S, Tian L, et al. Lmod2 piggyBac mutant mice exhibit dilated cardiomyopathy. Cell Biosci. 2016;6(1):1–9.

19. Ahrens-Nicklas RC, Pappas CT, Farman GP, Mayfield RM, Larrinaga TM, Medne L, et al. Disruption of cardiac thin filament assembly arising from a mutation in LMOD2: A novel mechanism of neonatal dilated cardiomyopathy. Sci Adv. 2019;5(9):eaax2066. doi: 10.1126/sciadv.aax2066 31517052

20. Halim D, Wilson MP, Oliver D, Brosens E, Verheij JBGM, Han Y, et al. Loss of LMOD1 impairs smooth muscle cytocontractility and causes megacystis microcolon intestinal hypoperistalsis syndrome in humans and mice. Proc Natl Acad Sci. 2017 Mar 28;114(13):E2739–47. doi: 10.1073/pnas.1620507114 28292896

21. Yuen M, Sandaradura SA, Dowling JJ, Kostyukova AS, Moroz N, Quinlan KG, et al. Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. J Clin Invest. 2015 Jan 3;125(1):456–7. doi: 10.1172/JCI80057 25654555

22. Cenik BK, Garg A, McAnally JR, Shelton JM, Richardson JA, Bassel-Duby R, et al. Severe myopathy in mice lacking the MEF2/SRFdependent gene leiomodin-3. J Clin Invest. 2015;125(4):1569–78. doi: 10.1172/JCI80115 25774500

23. Tian L, Ding S, You Y, Li T -r., Liu Y, Wu X, et al. Leiomodin-3-deficient mice display nemaline myopathy with fast-myofiber atrophy. Dis Model Mech. 2015;8(6):635–41. doi: 10.1242/dmm.019430 26035871

24. Kozak M. An analysis of 5’-noncoding sequences from 699 vertebrate messenger rNAS. Nucleic Acids Res. 1987;15(20):8125–48. doi: 10.1093/nar/15.20.8125 3313277

25. Subramaniam A, Jones WK, Gulick J, Wert S, Neumann J, Robbins J. Tissue-specific regulation of the alpha-myosin heavy chain gene promoter in transgenic mice. J Biol Chem. 1991 Dec 25;266(36):24613–20. 1722208

26. Zhang JCL, Woo YJ, Chen JA, Swain JL, Lee Sweeney H. Efficient transmural cardiac gene transfer by intrapericardial injection in neonatal mice. J Mol Cell Cardiol. 1999;31(4):721–32. doi: 10.1006/jmcc.1998.0905 10329200

27. Konhilas JP, Irving TC, De Tombe PP. Frank-Starling law of the heart and the cellular mechanisms of length-dependent activation. Pflugers Arch Eur J Physiol. 2002;445(3):305–10.

28. Farman GP, Walker JS, de Tombe PP, Irving TC. Impact of osmotic compression on sarcomere structure and myofilament calcium sensitivity of isolated rat myocardium. Am J Physiol Heart Circ Physiol. 2006 Oct;291(4):H1847–55. doi: 10.1152/ajpheart.01237.2005 16751283

29. Kentish JC, Stienen GJ. Differential effects of length on maximum force production and myofibrillar ATPase activity in rat skinned cardiac muscle. J Physiol. 1994 Feb 15;475(1):175–84. doi: 10.1113/jphysiol.1994.sp020059 8189390

30. de Tombe PP, Mateja RD, Tachampa K, Mou YA, Farman GP, Irving TC. Myofilament length dependent activation. J Mol Cell Cardiol. 2010;48(5):851–8. doi: 10.1016/j.yjmcc.2009.12.017 20053351

31. Littlefield R, Fowler VM. Measurement of thin filament lengths by distributed deconvolution analysis of fluorescence images. Biophys J. 2002;82(5):2548–64. doi: 10.1016/S0006-3495(02)75598-7 11964243

32. Gokhin DS, Fowler VM. Software-based measurement of thin filament lengths: an open-source GUI for Distributed Deconvolution analysis of fluorescence images. J Microsc. 2017 Jan;265(1):11–20. doi: 10.1111/jmi.12456 27644080

33. Walker JM, Jackson M, Taylor a H, Jones E a, Forrester LM. Mouse Cell Culture. Vol. 633, Methods in Molecular Biology. 2010. 29–56 p.

34. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402–8. doi: 10.1006/meth.2001.1262 11846609

35. Kuwahara K, Saito Y, Takano M, Arai Y, Yasuno S, Nakagawa Y, et al. NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function. EMBO J. 2003 Dec 1;22(23):6310–21. doi: 10.1093/emboj/cdg601 14633990

36. Forbes MS, Sperelakis N. Intercalated discs of mammalian heart: A review of structure and function. Tissue Cell. 1985;17(5):605–48. doi: 10.1016/0040-8166(85)90001-1 3904080

37. Ferreira-Cornwell MC, Luo Y, Narula N, Lenox JM, Lieberman M, Radice GL. Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart. J Cell Sci. 2002 Apr 15;115(Pt 8):1623–34. 11950881

38. Kostetskii I, Li J, Xiong Y, Zhou R, Ferrari VA, Patel V V., et al. Induced deletion of the N-cadherin gene in the heart leads to dissolution of the intercalated disc structure. Circ Res. 2005;96(3):346–54. doi: 10.1161/01.RES.0000156274.72390.2c 15662031

39. Vite A, Radice GL. N-cadherin/catenin complex as a master regulator of intercalated disc function. Cell Commun Adhes. 2014;21(3):169–79. doi: 10.3109/15419061.2014.908853 24766605

40. Mayosi BM, Fish M, Shaboodien G, Mastantuono E, Kraus S, Wieland T, et al. Identification of Cadherin 2 (CDH2) Mutations in Arrhythmogenic Right Ventricular Cardiomyopathy. Circ Cardiovasc Genet. 2017;10(2).

41. Perriard JC, Hirschy A, Ehler E. Dilated cardiomyopathy: A disease of the intercalated disc? Trends Cardiovasc Med. 2003;13(1):30–8. doi: 10.1016/s1050-1738(02)00209-8 12554098

42. Pinsky WW, Lewis RM, Hartley CJ, Entman ML. Permanent changes of ventricular contractility and compliance in chronic volume overload. Am J Physiol. 1979 Nov;237(5):H575–83. doi: 10.1152/ajpheart.1979.237.5.H575 40438

43. Lehtonen LA, Antila S, Pentikäinen PJ. Pharmacokinetics and pharmacodynamics of intravenous inotropic agents. Clin Pharmacokinet. 2004;43(3):187–203. doi: 10.2165/00003088-200443030-00003 14871156

44. Suga H, Sagawa K, Shoukas AA. Load independence of the instantaneous pressure-volume ratio of the canine left ventricle and effects of epinephrine and heart rate on the ratio. Circ Res. 1973 Mar;32(3):314–22. doi: 10.1161/01.res.32.3.314 4691336

45. Pacher P, Nagayama T, Mukhopadhyay P, Bátkai S, Kass DA. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc. 2008;3(9):1422–34. doi: 10.1038/nprot.2008.138 18772869

46. Brenner B, Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci. 1986 May;83(10):3542–6. doi: 10.1073/pnas.83.10.3542 2939452

47. Granzier HL, Akster HA, Ter Keurs HE. Effect of thin filament length on the force-sarcomere length relation of skeletal muscle. Am J Physiol Physiol. 1991 May;260(5):C1060–70.

48. De Tombe PP. Cardiac myofilaments: Mechanics and regulation. J Biomech. 2003;36(5):721–30. doi: 10.1016/s0021-9290(02)00450-5 12695002

49. Szatmári D, Bugyi B, Ujfalusi Z, Grama L, Dudás R, Nyitrai M. Cardiac leiomodin2 binds to the sides of actin filaments and regulates the ATPase activity of myosin. PLoS One. 2017;12(10):1–21.

50. Pappas CT, Farman GP, Mayfield RM, Konhilas JP, Gregorio CC. Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure. J Mol Cell Cardiol. 2018 Sep 10;122(August):88–97.

Článek vyšel v časopise


2020 Číslo 1