Advertisement

Therapeutic potential of AAV9-S15D-RLC gene delivery in humanized MYL2 mouse model of HCM

  • Sunil Yadav
  • Chen-Ching Yuan
  • Katarzyna Kazmierczak
  • Jingsheng Liang
  • Wenrui Huang
  • Lauro M. Takeuchi
  • Rosemeire M. Kanashiro-Takeuchi
  • Danuta Szczesna-CordaryEmail author
Original Article

Abstract

Familial hypertrophic cardiomyopathy (HCM) is an autosomal dominant disorder characterized by ventricular hypertrophy, myofibrillar disarray, and fibrosis, and is primarily caused by mutations in sarcomeric genes. With no definitive cure for HCM, there is an urgent need for the development of novel preventive and reparative therapies. This study is focused on aspartic acid-to-valine (D166V) mutation in the myosin regulatory light chain, RLC (MYL2 gene), associated with a malignant form of HCM. Since myosin RLC phosphorylation is critical for normal cardiac function, we aimed to exploit this post-translational modification via phosphomimetic-RLC gene therapy. We hypothesized that mimicking/modulating cardiac RLC phosphorylation in non-phosphorylatable D166V myocardium would improve heart function of HCM-D166V mice. Adeno-associated virus, serotype-9 (AAV9) was used to deliver phosphomimetic human RLC variant with serine-to-aspartic acid substitution at Ser15-RLC phosphorylation site (S15D-RLC) into the hearts of humanized HCM-D166V mice. Improvement of heart function was monitored by echocardiography, invasive hemodynamics (PV-loops) and muscle contractile mechanics. A significant increase in cardiac output and stroke work and a decrease in relaxation constant, Tau, shown to be prolonged in HCM mice, were observed in AAV- vs. PBS-injected HCM mice. Strain analysis showed enhanced myocardial longitudinal shortening in AAV-treated vs. control mice. In addition, increased maximal contractile force was observed in skinned papillary muscles from AAV-injected HCM hearts. Our data suggest that myosin RLC phosphorylation may have important translational implications for the treatment of RLC mutations-induced HCM and possibly play a role in other disease settings accompanied by depressed Ser15-RLC phosphorylation.

Key messages

  • HCM-D166V mice show decreased RLC phosphorylation and decompensated function.

  • AAV9-S15D-RLC gene therapy in HCM-D166V mice, but not in WT-RLC, results in improved heart performance.

  • Global longitudinal strain analysis shows enhanced contractility in AAV vs controls.

  • Increased systolic and diastolic function is paralleled by higher contractile force.

  • Phosphomimic S15D-RLC has a therapeutic potential for HCM.

Keywords

Adeno-associated virus D166V-mutation In vivo rescue of function Regulatory light chain (RLC) S15D-phosphorylation mimic 

Notes

Authors’ contributions

SY, CCY, WH, KK, and DSC conceived research; SY, CCY, WH, KK, JL, LT, and RKT performed experiments; SY, CCY, KK, RKT, and DS-C analyzed data; SY and DS-C wrote the paper.

Funding information

This work was supported by the National Institutes of Health R01-HL123255 (DSC) and the American Heart Association: 17PRE33650085 (SY), 15PRE23020006 (CCY) and 12PRE12030412 (WH).

References

  1. 1.
    Ho CY (2010) Hypertrophic cardiomyopathy. Heart Fail Clin 6:141–159CrossRefGoogle Scholar
  2. 2.
    Alcalai R, Seidman JG, Seidman CE (2008) Genetic basis of hypertrophic cardiomyopathy: from bench to the clinics. J Cardiovasc Electrophysiol 19:104–110Google Scholar
  3. 3.
    Maron BJ (2002) Hypertrophic cardiomyopathy: a systematic review. JAMA 287:1308–1320Google Scholar
  4. 4.
    Szczesna-Cordary D (2003) Regulatory light chains of striated muscle myosin. Structure, function and malfunction. Curr Drug Targets Cardiovasc Haematol Disord 3:187–197CrossRefGoogle Scholar
  5. 5.
    Alfares AA, Kelly MA, McDermott G, Funke BH, Lebo MS, Baxter SB, Shen J, McLaughlin HM, Clark EH, Babb LJ et al (2015) CORRIGENDUM: results of clinical genetic testing of 2,912 probands with hypertrophic cardiomyopathy: expanded panels offer limited additional sensitivity. Genet Med 17:319CrossRefGoogle Scholar
  6. 6.
    Gaita F, Di Donna P, Olivotto I, Scaglione M, Ferrero I, Montefusco A, Caponi D, Conte MR, Nistri S, Cecchi F (2007) Usefulness and safety of transcatheter ablation of atrial fibrillation in patients with hypertrophic cardiomyopathy. Am J Cardiol 99:1575–1581CrossRefGoogle Scholar
  7. 7.
    Szczesna-Cordary D, Guzman G, Ng SS, Zhao J (2004) Familial hypertrophic cardiomyopathy-linked alterations in Ca2+ binding of human cardiac myosin regulatory light chain affect cardiac muscle contraction. J Biol Chem 279:3535–3542CrossRefGoogle Scholar
  8. 8.
    Rayment I, Rypniewski WR, Schmidt-Base K, Smith R, Tomchick DR, Benning MM, Winkelmann DA, Wesenberg G, Holden HM (1993) Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261:50–58CrossRefGoogle Scholar
  9. 9.
    Szczesna D, Ghosh D, Li Q, Gomes AV, Guzman G, Arana C, Zhi G, Stull JT, Potter JD (2001) Familial hypertrophic cardiomyopathy mutations in the regulatory light chains of myosin affect their structure, Ca2+ binding, and phosphorylation. J Biol Chem 276:7086–7092CrossRefGoogle Scholar
  10. 10.
    Yadav S, Szczesna-Cordary D (2017) Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy. Biophys Rev 9:57–64CrossRefGoogle Scholar
  11. 11.
    van der Velden J, Papp Z, Boontje NM, Zaremba R, de Jong JW, Janssen PM, Hasenfuss G, Stienen GJ (2003) Myosin light chain composition in non-failing donor and end-stage failing human ventricular myocardium. Adv Exp Med Biol 538:3–15CrossRefGoogle Scholar
  12. 12.
    van der Velden J, Papp Z, Boontje NM, Zaremba R, de Jong JW, Janssen PML, Hasenfuss G, Stienen GJM (2003) The effect of myosin light chain 2 dephosphorylation on Ca2+-sensitivity of force is enhanced in failing human hearts. Cardiovasc Res 57:505–514CrossRefGoogle Scholar
  13. 13.
    Kerrick WGL, Kazmierczak K, Xu Y, Wang Y, Szczesna-Cordary D (2009) Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice. FASEB J 23:855–865CrossRefGoogle Scholar
  14. 14.
    Abraham TP, Jones M, Kazmierczak K, Liang H-Y, Pinheiro AC, Wagg CS, Lopaschuk GD, Szczesna-Cordary D (2009) Diastolic dysfunction in familial hypertrophic cardiomyopathy transgenic model mice. Cardiovasc Res 82:84–92CrossRefGoogle Scholar
  15. 15.
    Scruggs SB, Hinken AC, Thawornkaiwong A, Robbins J, Walker LA, de Tombe PP, Geenen DL, Buttrick PM, Solaro RJ (2009) Ablation of ventricular myosin regulatory light chain phosphorylation in mice causes cardiac dysfunction in situ and affects neighboring myofilament protein phosphorylation. J Biol Chem 284:5097–5106CrossRefGoogle Scholar
  16. 16.
    Sheikh F, Ouyang K, Campbell SG, Lyon RC, Chuang J, Fitzsimons D, Tangney J, Hidalgo CG, Chung CS, Cheng H, Dalton ND, Gu Y, Kasahara H, Ghassemian M, Omens JH, Peterson KL, Granzier HL, Moss RL, McCulloch AD, Chen J (2012) Mouse and computational models link Mlc2v dephosphorylation to altered myosin kinetics in early cardiac disease. J Clin Invest 122:1209–1221CrossRefGoogle Scholar
  17. 17.
    Huang J, Shelton JM, Richardson JA, Kamm KE, Stull JT (2008) Myosin regulatory light chain phosphorylation attenuates cardiac hypertrophy. J Biol Chem 283:19748–19756CrossRefGoogle Scholar
  18. 18.
    Warren SA, Briggs LE, Zeng H, Chuang J, Chang EI, Terada R, Li M, Swanson MS, Lecker SH, Willis MS, Spinale FG, Maupin-Furlowe J, McMullen JR, Moss RL, Kasahara H (2012) Myosin light chain phosphorylation is critical for adaptation to cardiac stress. Circulation 126:2575–2588CrossRefGoogle Scholar
  19. 19.
    Yuan CC, Muthu P, Kazmierczak K, Liang J, Huang W, Irving TC, Kanashiro-Takeuchi RM, Hare JM, Szczesna-Cordary D (2015) Constitutive phosphorylation of cardiac myosin regulatory light chain prevents development of hypertrophic cardiomyopathy in mice. Proc Natl Acad Sci U S A 112:E4138–E4146CrossRefGoogle Scholar
  20. 20.
    Muthu P, Huang W, Kazmierczak K, Szczesna-Cordary D (2012) Functional consequences of mutations in the myosin regulatory light chain associated with hypertrophic cardiomyopathy. In: Veselka J (ed) Cardiomyopathies – from basic research to clinical management. Ch. 17. InTech, Croatia, pp 383–408Google Scholar
  21. 21.
    Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M et al (2003) Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 107:2227–2232 and erratum (2004), 2109(2225), p.3258CrossRefGoogle Scholar
  22. 22.
    Muthu P, Kazmierczak K, Jones M, Szczesna-Cordary D (2012) The effect of myosin RLC phosphorylation in normal and cardiomyopathic mouse hearts. J Cell Mol Med 16:911–919CrossRefGoogle Scholar
  23. 23.
    Muthu P, Liang J, Schmidt W, Moore JR, Szczesna-Cordary D (2014) In vitro rescue study of a malignant familial hypertrophic cardiomyopathy phenotype by pseudo-phosphorylation of myosin regulatory light chain. Arch Biochem Biophys 552-553:29–39CrossRefGoogle Scholar
  24. 24.
    Bish LT, Morine K, Sleeper MM, Sanmiguel J, Wu D, Gao G, Wilson JM, Sweeney HL (2008) Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Hum Gene Ther 19:1359–1368CrossRefGoogle Scholar
  25. 25.
    Konkalmatt PR, Wang F, Piras BA, Xu Y, O’Connor DM, Beyers RJ, Epstein FH, Annex BH, Hossack JA, French BA (2012) Adeno-associated virus serotype 9 administered systemically after reperfusion preferentially targets cardiomyocytes in the infarct border zone with pharmacodynamics suitable for the attenuation of left ventricular remodeling. J Gene Med 14:609–620CrossRefGoogle Scholar
  26. 26.
    Palomeque J, Chemaly ER, Colosi P, Wellman JA, Zhou S, Del Monte F, Hajjar RJ (2007) Efficiency of eight different AAV serotypes in transducing rat myocardium in vivo. Gene Ther 14:989–997CrossRefGoogle Scholar
  27. 27.
    Yuan CC, Kazmierczak K, Liang J, Zhou Z, Yadav S, Gomes AV, Irving TC, Szczesna-Cordary D (2018) Sarcomeric perturbations of myosin motors lead to dilated cardiomyopathy in genetically modified MYL2 mice. Proc Natl Acad Sci U S A 115:E2338–E2347CrossRefGoogle Scholar
  28. 28.
    Wang Y, Xu Y, Kerrick WGL, Wang Y, Guzman G, Diaz-Perez Z, Szczesna-Cordary D (2006) Prolonged Ca2+ and force transients in myosin RLC transgenic mouse fibers expressing malignant and benign FHC mutations. J Mol Biol 361:286–299CrossRefGoogle Scholar
  29. 29.
    Yuan CC, Kazmierczak K, Liang J, Kanashiro-Takeuchi R, Irving TC, Gomes AV, Wang Y, Burghardt TP, Szczesna-Cordary D (2017) Hypercontractile mutant of ventricular myosin essential light chain leads to disruption of sarcomeric structure and function and results in restrictive cardiomyopathy in mice. Cardiovasc Res 113:1124–1136CrossRefGoogle Scholar
  30. 30.
    Tei C, Ling LH, Hodge DO, Bailey KR, Oh JK, Rodeheffer RJ, Tajik AJ, Seward JB (1995) New index of combined systolic and diastolic myocardial performance: a simple and reproducible measure of cardiac function--a study in normals and dilated cardiomyopathy. J Cardiol 26:357–366Google Scholar
  31. 31.
    Chang AN, Battiprolu PK, Cowley PM, Chen G, Gerard RD, Pinto JR, Hill JA, Baker AJ, Kamm KE, Stull JT (2015) Constitutive phosphorylation of cardiac myosin regulatory light chain in vivo. J Biol Chem 290:10703–10716CrossRefGoogle Scholar
  32. 32.
    Perrie WT, Smillie LB, Perry SB (1973) A phosphorylated light-chain component of myosin from skeletal muscle. Biochem J 135:151–164CrossRefGoogle Scholar
  33. 33.
    Huang W, Liang J, Kazmierczak K, Muthu P, Duggal D, Farman GP, Sorensen L, Pozios I, Abraham T, Moore JR et al (2014) Hypertrophic cardiomyopathy associated Lys104Glu mutation in the myosin regulatory light chain causes diastolic disturbance in mice. J Mol Cell Cardiol 74:318–329CrossRefGoogle Scholar
  34. 34.
    van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, Burton PBJ, Goldmann P, Jaquet K, Stienen GJM (2003) Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res 57:37–47CrossRefGoogle Scholar
  35. 35.
    Chang AN, Mahajan P, Knapp S, Barton H, Sweeney HL, Kamm KE, Stull JT (2016) Cardiac myosin light chain is phosphorylated by Ca2+/calmodulin-dependent and -independent kinase activities. Proc Natl Acad Sci U S A 113:E3824–E3833CrossRefGoogle Scholar
  36. 36.
    Granzier HL, de Tombe PP (2015) Myosin light chain phosphorylation to the rescue. Proc Natl Acad Sci U S A 112:9148–9149CrossRefGoogle Scholar
  37. 37.
    Prasad K-MR, Xu Y, Yang Z, Acton ST, French BA (2011) Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Gene Ther 18:43–52CrossRefGoogle Scholar
  38. 38.
    Bruch C, Schmermund A, Marin D, Katz M, Bartel T, Schaar J, Erbel R (2000) Tei-index in patients with mild-to-moderate congestive heart failure. Eur Heart J 21:1888–1895CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Sunil Yadav
    • 1
  • Chen-Ching Yuan
    • 1
  • Katarzyna Kazmierczak
    • 1
  • Jingsheng Liang
    • 1
  • Wenrui Huang
    • 1
  • Lauro M. Takeuchi
    • 2
  • Rosemeire M. Kanashiro-Takeuchi
    • 1
    • 2
  • Danuta Szczesna-Cordary
    • 1
    Email author
  1. 1.Department of Molecular and Cellular PharmacologyUniversity of Miami Miller School of MedicineMiamiUSA
  2. 2.Interdisciplinary Stem Cell InstituteUniversity of Miami Miller School of MedicineMiamiUSA

Personalised recommendations