Abstract
Several lines of evidence suggest that alterations of the ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) may be involved in cardiac diseases. Little is known, however, in hypertrophic cardiomyopathy (HCM). This study studied these pathways in two mouse models of HCM that mainly differ by the presence or absence of truncated mutant proteins. Analyses were performed in homozygous Mybpc3-targeted knock-in (KI) mice, carrying a HCM mutation and exhibiting low levels of mutant cardiac myosin-binding protein C (cMyBP-C), and in Mybpc3-targeted knock-out (KO) mice expressing no cMyBP-C, thus serving as a model of pure cMyBP-C insufficiency. In the early postnatal development of cardiac hypertrophy, both models showed higher levels of ubiquitinated proteins and greater proteasomal activities. To specifically monitor the degradation capacity of the UPS with age, mice were crossed with transgenic mice that overexpress UbG76V-GFP. UbG76V-GFP protein levels were fourfold higher in 1-year-old KI, but not KO mice, suggesting a specific UPS impairment in mice expressing truncated cMyBP-C. Whereas protein levels of key ALP markers were higher, suggesting ALP activation in both mutant mice, their mRNA levels did not differ between the groups, underlying rather defective ALP-mediated degradation. Analysis of key proteins regulated in heart failure did not reveal specific alterations in KI and KO mice. Our data suggest (1) UPS activation in early postnatal development of cardiac hypertrophy, (2) specific UPS impairment in old KI mice carrying a HCM mutation, and (3) defective ALP as a common mechanism in genetically engineered mice with cardiac hypertrophy.
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References
Bahrudin U, Morisaki H, Morisaki T, Ninomiya H, Higaki K, Nanba E, Igawa O, Takashima S, Mizuta E, Miake J, Yamamoto Y, Shirayoshi Y, Kitakaze M, Carrier L, Hisatome I (2008) Ubiquitin-proteasome system impairment caused by a missense cardiac myosin-binding protein C mutation and associated with cardiac dysfunction in hypertrophic cardiomyopathy. J Mol Biol 384:896–907. doi:10.1016/j.jmb.2008.09.070
Balasubramanian S, Mani S, Shiraishi H, Johnston RK, Yamane K, Willey CD, Cooper IV G, Tuxworth WJ, Kuppuswamy D (2006) Enhanced ubiquitination of cytoskeletal proteins in pressure overloaded myocardium is accompanied by changes in specific E3 ligases. J Mol Cell Cardiol 41:669–679 doi:10.1016/j.yjmcc.2006.04.022
Bardswell SC, Cuello F, Rowland AJ, Sadayappan S, Robbins J, Gautel M, Walker JW, Kentish JC, Avkiran M (2010) Distinct sarcomeric substrates are responsible for protein kinase D-mediated regulation of cardiac myofilament Ca2+ sensitivity and cross-bridge cycling. J Biol Chem 285:5674–5682. doi:10.1074/jbc.M109.066456
Birks EJ, Latif N, Enesa K, Folkvang T, le Luong A, Sarathchandra P, Khan M, Ovaa H, Terracciano CM, Barton PJ, Yacoub MH, Evans PC (2008) Elevated p53 expression is associated with dysregulation of the ubiquitin-proteasome system in dilated cardiomyopathy. Cardiovasc Res 79:472–480. doi:10.1093/cvr/cvn083
Breitschopf K, Zeiher AM, Dimmeler S (2000) Ubiquitin-mediated degradation of the proapoptotic active form of bid: a functional consequence on apoptosis induction. J Biol Chem 275:21648–21652. doi:10.1074/jbc.M001083200
Carrier L, Bonne G, Bahrend E, Yu B, Richard P, Niel F, Hainque B, Cruaud C, Gary F, Labeit S, Bouhour JB, Dubourg O, Desnos M, Hagege AA, Trent RJ, Komajda M, Fiszman M, Schwartz K (1997) Organization and sequence of human cardiac myosin binding protein C gene (MYBPC3) and identification of mutations predicted to produce truncated proteins in familial hypertrophic cardiomyopathy. Circ Res 80:427–434
Carrier L, Knoell R, Vignier N, Keller DI, Bausero P, Prudhon B, Isnard R, Ambroisine ML, Fiszman M, Ross J Jr, Schwartz K, Chien KR (2004) Asymmetric septal hypertrophy in heterozygous cMyBP-C null mice. Cardiovasc Res 63:293–304. doi:10.1016/j.cardiores.2004.04.009
Carrier L, Schlossarek S, Willis MS, Eschenhagen T (2010) The ubiquitin-proteasome system and nonsense-mediated mRNA decay in hypertrophic cardiomyopathy. Cardiovasc Res 85:330–338. doi:10.1093/cvr/cvp247
Chen Q, Liu JB, Horak KM, Zheng H, Kumarapeli AR, Li J, Li F, Gerdes AM, Wawrousek EF, Wang X (2005) Intrasarcoplasmic amyloidosis impairs proteolytic function of proteasomes in cardiomyocytes by compromising substrate uptake. Circ Res 97:1018–1026. doi:10.1161/01.RES.0000189262.92896.0b
Cuello F, Bardswell SC, Haworth RS, Ehler E, Sadayappan S, Kentish JC, Avkiran M (2011) Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation. J Biol Chem 286:5300–5310. doi:10.1074/jbc.M110.202713
Dantuma NP, Lindsten K, Glas R, Jellne M, Masucci MG (2000) Short-lived green fluorescent proteins for quantifying ubiquitin/proteasome-dependent proteolysis in living cells. Nat Biotechnol 18:538–543. doi:10.1038/75406
De Meyer GR, Martinet W (2009) Autophagy in the cardiovascular system. Biochim Biophys Acta 1793:1485–1495. doi:10.1016/j.bbamcr.2008.12.011
Depre C, Wang Q, Yan L, Hedhli N, Peter P, Chen L, Hong C, Hittinger L, Ghaleh B, Sadoshima J, Vatner DE, Vatner SF, Madura K (2006) Activation of the cardiac proteasome during pressure overload promotes ventricular hypertrophy. Circulation 114:1821–1828. doi:10.1161/CIRCULATIONAHA.106.637827
Ding WX, Ni HM, Gao W, Yoshimori T, Stolz DB, Ron D, Yin XM (2007) Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am J Pathol 171:513–524. doi:10.2353/ajpath.2007.070188
El-Armouche A, Pohlmann L, Schlossarek S, Starbatty J, Yeh YH, Nattel S, Dobrev D, Eschenhagen T, Carrier L (2007) Decreased phosphorylation levels of cardiac myosin-binding protein-C in human and experimental heart failure. J Mol Cell Cardiol 43:223–229. doi:10.1016/j.yjmcc.2007.05.003
Flavigny J, Souchet M, Sebillon P, Berrebi-Bertrand I, Hainque B, Mallet A, Bril A, Schwartz K, Carrier L (1999) COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes. J Mol Biol 294:443–456. doi:10.1006/jmbi.1999.3276S0022-2836(99)93276-X
Gautel M, Zuffardi O, Freiburg A, Labeit S (1995) Phosphorylation switches specific for the cardiac isoform of myosin binding protein-C: a modulator of cardiac contraction? EMBO J 14:1952–1960
Glickman MH, Ciechanover A (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82:373–428. doi:10.1152/physrev.00027.2001
Hedhli N, Wang L, Wang Q, Rashed E, Tian Y, Sui X, Madura K, Depre C (2008) Proteasome activation during cardiac hypertrophy by the chaperone H11 Kinase/Hsp22. Cardiovasc Res 77:497–505. doi:10.1093/cvr/cvm054
Helin K (1998) Regulation of cell proliferation by the E2F transcription factors. Curr Opin Genet Dev 8:28–35. doi:10.1016/S0959-437X(98)80058-0
King RW, Deshaies RJ, Peters JM, Kirschner MW (1996) How proteolysis drives the cell cycle. Science 274:1652–1659
Kirkin V, Dikic I (2007) Role of ubiquitin- and Ubl-binding proteins in cell signaling. Curr Opin Cell Biol 19:199–205. doi:10.1016/j.ceb.2007.02.002
Kirkin V, McEwan DG, Novak I, Dikic I (2009) A role for ubiquitin in selective autophagy. Mol Cell 34:259–269. doi:10.1016/j.molcel.2009.04.026
Kooij V, Boontje N, Zaremba R, Jaquet K, dos Remedios C, Stienen GJ, van der Velden J (2010) Protein kinase C alpha and epsilon phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium. Basic Res Cardiol 105:289–300. doi:10.1007/s00395-009-0053-z
Kostin S, Pool L, Elsasser A, Hein S, Drexler HC, Arnon E, Hayakawa Y, Zimmermann R, Bauer E, Klovekorn WP, Schaper J (2003) Myocytes die by multiple mechanisms in failing human hearts. Circ Res 92:715–724. doi:10.1161/01.RES.0000067471.95890.5C
Levine B, Kroemer G (2008) Autophagy in the pathogenesis of disease. Cell 132:27–42. doi:10.1016/j.cell.2007.12.018
Li B, Dou QP (2000) Bax degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci USA 97:3850–3855. doi:10.1073/pnas.070047997
Li HH, Kedar V, Zhang C, McDonough H, Arya R, Wang DZ, Patterson C (2004) Atrogin-1/muscle atrophy F-box inhibits calcineurin-dependent cardiac hypertrophy by participating in an SCF ubiquitin ligase complex. J Clin Invest 114:1058–1071
Li J, Horak KM, Su H, Sanbe A, Robbins J, Wang X (2011) Enhancement of proteasomal function protects against cardiac proteinopathy and ischemia/reperfusion injury in mice. J Clin Invest 121:3689–3700. doi:10.1172/JCI45709
Lindsten K, Menendez-Benito V, Masucci MG, Dantuma NP (2003) A transgenic mouse model of the ubiquitin/proteasome system. Nat Biotechnol 21:897–902
Marston S, Copeland O, Jacques A, Livesey K, Tsang V, McKenna WJ, Jalilzadeh S, Carballo S, Redwood C, Watkins H (2009) Evidence from human myectomy samples that MYBPC3 mutations cause hypertrophic cardiomyopathy through haploinsufficiency. Circ Res 105:219–222. doi:10.1161/CIRCRESAHA.109.202440
McClellan G, Kulikovskaya I, Winegrad S (2001) Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C. Biophys J 81:1083–1092
Mearini G, Gedicke C, Schlossarek S, Witt CC, Kramer E, Cao P, Gomes MD, Lecker SH, Labeit S, Willis MS, Eschenhagen T, Carrier L (2010) Atrogin-1 and MuRF1 regulate cardiac MyBP-C levels via different mechanisms. Cardiovasc Res 85:357–366. doi:10.1093/cvr/cvp348
Mearini G, Schlossarek S, Willis MS, Carrier L (2008) The ubiquitin-proteasome system in cardiac dysfunction. Biochim Biophys Acta 1782:749–763. doi:10.1016/j.bbadis.2008.06.009
Mizushima N, Levine B, Cuervo AM, Klionsky DJ (2008) Autophagy fights disease through cellular self-digestion. Nature 451:1069–1075. doi:10.1038/nature06639
Moolman JA, Reith S, Uhl K, Bailey S, Gautel M, Jeschke B, Fischer C, Ochs J, McKenna WJ, Klues H, Vosberg HP (2000) A newly created splice donor site in exon 25 of the MyBP-C gene is responsible for inherited hypertrophic cardiomyopathy with incomplete disease penetrance. Circulation 101:1396–1402
Palombella VJ, Rando OJ, Goldberg AL, Maniatis T (1994) The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. Cell 78:773–785 pii:S0092-8674(94)90482-0
Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G, Johansen T (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131–24145. doi:10.1074/jbc.M702824200
Predmore JM, Wang P, Davis F, Bartolone S, Westfall MV, Dyke DB, Pagani F, Powell SR, Day SM (2010) Ubiquitin proteasome dysfunction in human hypertrophic and dilated cardiomyopathies. Circulation 121:997–1004. doi:10.1161/CIRCULATIONAHA.109.904557
Razeghi P, Baskin KK, Sharma S, Young ME, Stepkowski S, Essop MF, Taegtmeyer H (2006) Atrophy, hypertrophy, and hypoxemia induce transcriptional regulators of the ubiquitin proteasome system in the rat heart. Biochem Biophys Res Commun 342:361–364. doi:10.1016/j.bbrc.2006.01.163
Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet JP, Millaire A, Desnos M, Schwartz K, Hainque B, Komajda M (2003) Hypertrophic Cardiomyopathy: Distribution of disease genes, spectrum of mutations and implications for molecular diagnosis strategy. Circulation 107:2227–2232. doi:10.1161/01.CIR.0000066323.15244.54
Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL (1994) Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761–771 pii:S0092-8674(94)90462-6
Rottbauer W, Gautel M, Zehelein J, Labeit S, Franz WM, Fischer C, Vollrath B, Mall G, Dietz R, Kubler W, Katus HA (1997) Novel splice donor site mutation in the cardiac myosin-binding protein-C gene in familial hypertrophic cardiomyopathy: characterization of cardiac transcript and protein. J Clin Invest 100:475–482. doi:10.1172/JCI119555
Sarikas A, Carrier L, Schenke C, Doll D, Flavigny J, Lindenberg KS, Eschenhagen T, Zolk O (2005) Impairment of the ubiquitin-proteasome system by truncated cardiac myosin binding protein C mutants. Cardiovasc Res 66:33–44. doi:10.1016/j.cardiores.2005.12.021
Schlossarek S, Carrier L (2011) The ubiquitin-proteasome system in cardiomyopathies. Curr Opin Cardiol 26:190–195. doi:10.1097/HCO.0b013e32834598fe
Schlossarek S, Mearini G, Carrier L (2011) Cardiac myosin-binding protein C in hypertrophic cardiomyopathy: mechanisms and therapeutic opportunities. J Mol Cell Cardiol 50:613–620. doi:10.1016/j.yjmcc.2011.01.014
Schlossarek S, Schuermann F, Mearini G, Geertz B, Eschenhagen T, Carrier L (2012) Adrenergic stress reveals septal hypertrophy and proteasome impairment in heterozygous Mybpc3-targeted knock-in mice. J Muscle Res Cell Motil doi:10.1007/s10974-011-9273-6 (in press)
Su H, Wang X (2010) The ubiquitin-proteasome system in cardiac proteinopathy: a quality control perspective. Cardiovasc Res 85:253–262. doi:10.1093/cvr/cvp287
Tanida I (2011) Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 14:2201–2214. doi:10.1089/ars.2010.3482
Tannous P, Zhu H, Nemchenko A, Berry JM, Johnstone JL, Shelton JM, Miller FJ Jr, Rothermel BA, Hill JA (2008) Intracellular protein aggregation is a proximal trigger of cardiomyocyte autophagy. Circulation 117:3070–3078. doi:10.1161/CIRCULATIONAHA.107.763870
Tsukamoto O, Minamino T, Okada K, Shintani Y, Takashima S, Kato H, Liao Y, Okazaki H, Asai M, Hirata A, Fujita M, Asano Y, Yamazaki S, Asanuma H, Hori M, Kitakaze M (2006) Depression of proteasome activities during the progression of cardiac dysfunction in pressure-overloaded heart of mice. Biochem Biophys Res Commun 340:1125–1133 doi:10.1016/j.bbrc.2005.12.120
van Dijk SJ, Dooijes D, Dos Remedios C, Michels M, Lamers JM, Winegrad S, Schlossarek S, Carrier L, Ten Cate FJ, Stienen GJ, van der Velden J (2009) Cardiac myosin-binding protein C mutations and hypertrophic cardiomyopathy haploinsufficiency, deranged phosphorylation, and cardiomyocyte dysfunction. Circulation 119:1473–1483. doi:10.1161/CIRCULATIONAHA.108.838672
Vignier N, Schlossarek S, Fraysse B, Mearini G, Kramer E, Pointu H, Mougenot N, Guiard J, Reimer R, Hohenberg H, Schwartz K, Vernet M, Eschenhagen T, Carrier L (2009) Nonsense-mediated mRNA decay and ubiquitin-proteasome system regulate cardiac myosin-binding protein C mutant levels in cardiomyopathic mice. Circ Res 105:239–248. doi:10.1161/CIRCRESAHA.109.201251
Voigt A, Bartel K, Egerer K, Trimpert C, Feist E, Gericke C, Kandolf R, Klingel K, Kuckelkorn U, Stangl K, Felix SB, Baumann G, Kloetzel PM, Staudt A (2010) Humoral anti-proteasomal autoimmunity in dilated cardiomyopathy. Basic Res Cardiol 105:9–18. doi:10.1007/s00395-009-0061-z
Voigt A, Trimpert C, Bartel K, Egerer K, Kuckelkorn U, Feist E, Gericke C, Klingel K, Kandolf R, Felix SB, Baumann G, Kloetzel PM, Stangl K, Staudt A (2010) Lack of evidence for a pathogenic role of proteasome-directed autoimmunity in dilated cardiomyopathy. Basic Res Cardiol 105:557–567. doi:10.1007/s00395-010-0096-1
Weekes J, Morrison K, Mullen A, Wait R, Barton P, Dunn MJ (2003) Hyperubiquitination of proteins in dilated cardiomyopathy. Proteomics 3:208–216. doi:10.1002/(SICI)1522-2683(19990101)20:4/5<898:AID-ELPS898>3.0.CO;2-B
Yang Q, Sanbe A, Osinska H, Hewett TE, Klevitsky R, Robbins J (1999) In vivo modeling of myosin binding protein C familial hypertrophic cardiomyopathy. Circ Res 85:841–847
Zheng Q, Li J, Wang X (2009) Interplay between the ubiquitin-proteasome system and autophagy in proteinopathies. Int J Physiol Pathophysiol Pharmacol 1:127–142
Zheng Q, Wang X (2010) Autophagy and the ubiquitin-proteasome system in cardiac dysfunction. Panminerva Med 52:9–25 pii:R41102476
Zhu H, Tannous P, Johnstone JL, Kong Y, Shelton JM, Richardson JA, Le V, Levine B, Rothermel BA, Hill JA (2007) Cardiac autophagy is a maladaptive response to hemodynamic stress. J Clin Invest 117:1782–1793. doi:10.1172/JCI27523
Zolk O, Schenke C, Sarikas A (2006) The ubiquitin-proteasome system: focus on the heart. Cardiovasc Res 70:410–421. doi:10.1016/j.cardiores.2005.12.021
Acknowledgments
We thank Nico Dantuma (Stockholm, Sweden) for providing the UbG76V-GFP transgenic mice, as well as Elisabeth Krämer and Birgit Geertz (Hamburg) for technical help. This work was supported by the sixth and seventh Framework Programs of the European Union (Marie Curie EXT-014051; Health-F2-2009-241577-Big-Heart project), the Deutsche Forschungsgemeinschaft (FOR-604-CA 618/1-1 and 1-2), and the Leducq Foundation (Research grant Nr. 11, CVD 04).
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Schlossarek, S., Englmann, D.R., Sultan, K.R. et al. Defective proteolytic systems in Mybpc3-targeted mice with cardiac hypertrophy. Basic Res Cardiol 107, 235 (2012). https://doi.org/10.1007/s00395-011-0235-3
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DOI: https://doi.org/10.1007/s00395-011-0235-3