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Aging and disease    2019, Vol. 10 Issue (1) : 102-115     DOI: 10.14336/AD.2018.0209
Orginal Article |
LMNA-mutated Rabbits: A Model of Premature Aging Syndrome with Muscular Dystrophy and Dilated Cardiomyopathy
Tingting Sui1, Di Liu1, Tingjun Liu1, Jichao Deng1, Mao Chen1, Yuanyuan Xu1, Yuning Song1, Hongsheng Ouyang1, Liangxue Lai1,2,*, Zhanjun Li1,*
1Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, Changchun 130062, China
2Key Laboratory of Regenerative Biology, and Guangdong Provincial Key Laboratory of Stem Cells and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong 510530, China
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Abstract  

Premature aging syndromes are rare genetic disorders mimicking clinical and molecular features of aging. Products of the LMNA gene, primarily lamin A and C, are major components of the nuclear lamina. A recently identified group of premature aging syndromes was related to mutations of the LMNA gene. Although LMNA disorders have been identified in premature aging syndromes, affect specifically the skeletal muscles, cardiac muscles, and lipodystrophy, understanding the pathogenic mechanisms still need to be elucidated. Here, to establish a rabbit knockout (KO) model of premature aging syndromes, we performed precise LMNA targeting in rabbits via co-injection of Cas9/sgRNA mRNA into zygotes. The LMNA-KO rabbits exhibited reduced locomotion activity with abnormal stiff walking posture and a shortened stature, all of them died within 22 days. In addition, cardiomyopathy, muscular dystrophy, bone and joint abnormalities, as well as lipodystrophy were observed in LMNA-KO rabbits. In conclusion, the novel rabbit LMNA-KO model, displayed typical features of histopathological defects that are observed in premature aging syndromes, and may be utilized as a valuable resource for understanding the pathophysiological mechanisms of premature aging syndromes and elucidating mysteries of the normal process of aging in humans.

Keywords CRISPR/Cas9      LMNA      premature aging syndrome      rabbit     
Corresponding Authors: Lai Liangxue,Li Zhanjun   
About author:

These authors contributed equally to this work

Issue Date: 04 January 2018
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Sui Tingting
Liu Di
Liu Tingjun
Deng Jichao
Chen Mao
Xu Yuanyuan
Song Yuning
Ouyang Hongsheng
Lai Liangxue
Li Zhanjun
Cite this article:   
Sui Tingting,Liu Di,Liu Tingjun, et al. LMNA-mutated Rabbits: A Model of Premature Aging Syndrome with Muscular Dystrophy and Dilated Cardiomyopathy[J]. Aging and disease, 2019, 10(1): 102-115.
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http://www.aginganddisease.org/EN/10.14336/AD.2018.0209     OR     http://www.aginganddisease.org/EN/Y2019/V10/I1/102
Figure 1.  Generation of LMNA-KO rabbits using CRISPR/Cas9 system. (A) Schematic diagram of the two sgRNA target sites located in exon 3 of the rabbit LMNA locus. LMNA exons are indicated by pink rectangles; target sites of the two sgRNA sequences, sgRNA1 and sgRNA2, are highlighted in red; protospacer-adjacent motif (PAM) sequence is highlighted in green (B) Mutation detection by T7E1 cleavage assay in rabbit pups 1-15. Gel images have been cropped. M, DL2000, has used to indicate band size. (C) Mutation detection by T7E1 cleavage assay in rabbit pups (n=16-32 pups). Gel images have been cropped. M, DL2000, has been used to indicate band size. Black line indicates the WT allele (490 bp). (D) T-cloning and Sanger sequencing of modified LMNA alleles in 1-15 pups. WT sequence is shown at the top of the targeting sequence. PAM sites are highlighted in green; target sequences are shown in red; deletions (-); insertions are shown in blue; WT, wild-type control.
ReplicationsNo. zygotes2-cell (%)Morula (%)Blastocyste (%)Blastocyst with
Mutantion (%)
Non-injection3128(95.3±1.36)a(87.5±0.77)a(79.3±1.25)a
Injection3130(96.0±0.89)a(86.7±0.99)a(80.4±1.42)a(95.3±0.97)b
Table 1.  Summary of embryo microinjections of Cas9/sgRNA in zygotes.
Figure 2.  Phenotype characterization of LMNA-KO rabbits. (A) The gross performance of 14-day-old LMNA-KO rabbits by photo, and joint stiffness (white arrows) by X-ray autoradiography examination. (B) Hind legs of WT and LMNA-KO rabbits showing stiff ankle joints in LMNA-KO rabbits (Red oval). (C) X-ray absorptiometry of hind legs from WT and LMNA-KO rabbits showing stiff ankle joints in LMNA-KO rabbits (Red oval). (D) H&E-staining of the skin, LMNA-KO rabbit showed decreased eccrine in skin (E) Behavioral photographs cut from video of the LMNA-KO-1, LMNA-KO-2, and WT control. (F) Body-weight comparison of LMNA-KO and WT rabbits from newborn to 22 days. (G) Survival curves of LMNA-KO and WT rabbits. Scale bar, 50 μm.
Figure 3.  Cardiomyopathy of LMNA-KO rabbits. (A) The heart from 18-day-old LMNA-KO rabbits and WT control. (B) The increased left ventricular diastolic diameter in LMNA-KO rabbits. (C) The decreased left ventricular ejection fraction (EF) in LMNA-KO rabbits. (D) The decreased fractional shortening in LMNA-KO rabbits. (E) The decreased heart rate in LMNA-KO rabbits. Data were presented as means ± SEM of at least three rabbits per group and analyzed by Student’s t-tests using GraphPad Prism software 7.0. * p < 0.05; ** p < 0.01; *** p < 0.001. Normalized LVDD, LV diastolic diameter-to-body weight ratio. (F) H&E-staining and Masson’ trichrome-staining of cardiac muscles from WT and LMNA-KO rabbits, showing significant fibrosis (Red arrows) and cardiomyocytes loss (Blue arrows) in LMNA-KO rabbits. (G) H&E-staining and Oil red-staining of the cardiac muscles from WT and LMNA-KO rabbits, showing significant fat infiltration (Green arrows) in LMNA-KO rabbits. (H) Aorta tissues of the 18-day-old LMNA-KO rabbits and WT controls. (I) H&E-staining of the aorta from WT and LMNA-KO rabbits. Scale bar, 50 μm.
RecipientsEmbryos transferredPregnancyPups obtained
(%transferred)
Pups with mutations
(% pups)
Pups with biallelic
mutations (%pups)
136YES15 (41.7%)14 (93.3%)13 (92.6%)
231YES7 (22.6%)7 (100.0%)7 (100.0%)
329YES7 (24.1%)6 (85.7%)6 (100.0%)
430YES3 (10.0%)3 (100.0%)3 (100.0%)
Table 2  Generation of LMNA-KO rabbits using CRISPR/Cas9 system.
Figure 4.  Muscular dystrophy in LMNA-KO rabbits. (A) Gross muscles of the 18-day-old LMNA-KO rabbits and WT control by photo. (B) Masson’s trichrome staining of the tongue, bladder and diaphragm muscles from WT and LMNA-KO rabbits, showing inflammatory cells infiltration in the tongue (Green arrows), and thinner bladder muscles of LMNA-KO rabbits (Black arrows). (C) Longitudinal section of the H&E-staining and Masson’s trichrome staining of the gastrocnemius muscles from WT and LMNA-KO rabbits, showing inflammatory cells infiltration (Green arrows) in LMNA-KO rabbits. (D) Cross section of the H&E-staining and Masson’s trichrome stained of the gastrocnemius muscles from WT and LMNA-KO rabbits, showing a wide variation in the fiber size (Black arrows), an increased number of atrophic fibers, hypertrophic fibers and lobulated fiber (Blue arrows) in LMNA-KO rabbits. (E) Statistical analysis of the mean fibers diameter of the muscle fibers from the gastrocnemius muscles of the 18-day-old WT and LMNA-KO rabbits. (F) Statistical analysis of the mean fibers area of the muscle fibers from the gastrocnemius muscles of the 18-day-old WT and LMNA-KO rabbits. Scale bar, 50 μm.
Figure 5.  Bone abnormalities of LMNA-KO rabbits. X-ray absorptiometry of the femur (A) and the tibia (B) from WT and LMNA-KO rabbits. Statistical analysis of the average femur length (C) and tibia length (D) in WT and LMNA-KO rabbits. Data are presented as means ± SEM of at least three rabbits per group and analyzed by Student’s t-test using Graphpad Prism software 7.0. * p < 0.05; ** p < 0.01; *** p < 0.001. (E) H&E-staining of the cortical bone and diaphysis bone from WT and LMNA-KO rabbits, showing decreased cortical bone width, significantly reduced numbers of the osteoblasts and osteocytes in LMNA-KO rabbits. (F) H&E-staining of the wrist and growth plates from WT and LMNA-KO rabbits, showing rough articular surface and irregular arrangement of the growth plate with more porous areas in LMNA-KO rabbits. Scale bar, 50 μm.
Figure 6.  Lipodystrophy of LMNA-KO rabbits. (A) Dysplasia of the adipose tissue in LMNA-KO rabbits by anatomy analysis. (B) The liver of LMNA-KO rabbits and WT controls. (C) H&E-staining and Oil red staining of the brown adipose tissue from LMNA-KO rabbits and WT rabbits. (D) H&E-staining of liver sections from WT and LMNA-KO rabbits. (E) Gene expression of PPARɡ, SREBF1, GLUT4, FABP4, and ADIPOQ was determined by qRT-PCR. WT, WT control; LMNA-KO, LMNA gene knockout rabbit. All experiments were repeated for three times for each gene. Data are presented as the mean ± SEM and analyzed by t-tests using Graphpad Prism software 6.0. * p < 0.05; ** p < 0.01; *** p < 0.001. Scale bar, 50 μm.
Biochemical indexes8-Day
16-Day
WT controlsLMNA-KOWT controlsLMNA-KO
T. Chol (mmol/l)1.90 ± 0.377.77 ± 0.39a2.72 ± 0.259.86± 1.73a
Triglycerides (mmol/L)2.37 ± 0.201.35 ± 0.12a1.97 ± 0.230.82±0.25a
HDL (mmol/L)0.65± 0.131.37 ± 0.05a0.71 ± 0.072.24± 0.17a
LDL (mmol/L)1.26 ± 0.314.66 ± 0.56a1.37 ± 0.154.94 ± 0.44a
Table 3  Serum biochemical analysis in WT and LMNA-KO rabbits.
[1] Lee DC, Welton KL, Smith ED, Kennedy BK (2009). A-type nuclear lamins act as transcriptional repressors when targeted to promoters. Exp Cell Res, 315:996-1007.
[2] Shumaker DK, Solimando L, Sengupta K, Shimi T, Adam SA, Grunwald A, et al. (2008). The highly conserved nuclear lamin Ig-fold binds to PCNA: its role in DNA replication. J Cell Biol, 181:269-280.
[3] Spann TP, Goldman AE, Wang C, Huang S, Goldman RD (2002). Alteration of nuclear lamin organization inhibits RNA polymerase II-dependent transcription. J Cell Biol, 156:603-608.
[4] Spann TP, Moir RD, Goldman AE, Stick R, Goldman RD (1997). Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis. J Cell Biol, 136:1201-1212.
[5] Moir RD, Spann TP, Goldman RD (1995). The dynamic properties and possible functions of nuclear lamins. Int Rev Cytol, 162B:141-182.
[6] Stuurman N, Heins S, Aebi U (1998). Nuclear lamins: their structure, assembly, and interactions. J Struct Biol, 122:42-66.
[7] Hutchison CJ (2002). Lamins: building blocks or regulators of gene expression? Nat Rev Mol Cell Biol, 3:848-858.
[8] Fatkin D, MacRae C, Sasaki T, Wolff MR, Porcu M, Frenneaux M, et al. (1999). Missense mutations in the rod domain of the lamin A/C gene as causes of dilated cardiomyopathy and conduction-system disease. N Engl J Med, 341:1715-1724.
[9] Muchir A, Bonne G, van der Kooi AJ, van Meegen M, Baas F, Bolhuis PA, et al. (2000). Identification of mutations in the gene encoding lamins A/C in autosomal dominant limb girdle muscular dystrophy with atrioventricular conduction disturbances (LGMD1B). Hum Mol Genet, 9:1453-1459.
[10] Shackleton S, Lloyd DJ, Jackson SN, Evans R, Niermeijer MF, Singh BM, et al. (2000). LMNA, encoding lamin A/C, is mutated in partial lipodystrophy. Nat Genet, 24:153-156.
[11] De Sandre-Giovannoli A, Chaouch M, Kozlov S, Vallat JM, Tazir M, Kassouri N, et al. (2002). Homozygous defects in LMNA, encoding lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth disorder type 2) and mouse. Am J Hum Genet, 70:726-736.
[12] Novelli G, Muchir A, Sangiuolo F, Helbling-Leclerc A, D’Apice MR, Massart C, et al. (2002). Mandibuloacral dysplasia is caused by a mutation in LMNA-encoding lamin A/C. Am J Hum Genet, 71:426-431.
[13] De Sandre-Giovannoli A, Bernard R, Cau P, Navarro C, Amiel J, Boccaccio I, et al. (2003). Lamin a truncation in Hutchinson-Gilford progeria. Science, 300:2055.
[14] Eriksson M, Brown WT, Gordon LB, Glynn MW, Singer J, Scott L, et al. (2003). Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature, 423:293-298.
[15] Chen L, Lee L, Kudlow BA, Dos Santos HG, Sletvold O, Shafeghati Y, et al. (2003). LMNA mutations in atypical Werner’s syndrome. Lancet, 362:440-445.
[16] Navarro CL, De Sandre-Giovannoli A, Bernard R, Boccaccio I, Boyer A, Genevieve D, et al. (2004). Lamin A and ZMPSTE24 (FACE-1) defects cause nuclear disorganization and identify restrictive dermopathy as a lethal neonatal laminopathy. Hum Mol Genet, 13:2493-2503.
[17] van der Kooi AJ, Bonne G, Eymard B, Duboc D, Talim B, Van der Valk M, et al. (2002). Lamin A/C mutations with lipodystrophy, cardiac abnormalities, and muscular dystrophy. Neurology, 59:620-623.
[18] Garg A, Speckman RA, Bowcock AM (2002). Multisystem dystrophy syndrome due to novel missense mutations in the amino-terminal head and alpha-helical rod domains of the lamin A/C gene. Am J Med, 112:549-555.
[19] Sullivan T, Escalante-Alcalde D, Bhatt H, Anver M, Bhat N, Nagashima K, et al. (1999). Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J Cell Biol, 147:913-920.
[20] Mounkes LC, Kozlov S, Hernandez L, Sullivan T, Stewart CL (2003). A progeroid syndrome in mice is caused by defects in A-type lamins. Nature, 423:298-301.
[21] Arimura T, Helbling-Leclerc A, Massart C, Varnous S, Niel F, Lacene E, et al. (2005). Mouse model carrying H222P-Lmna mutation develops muscular dystrophy and dilated cardiomyopathy similar to human striated muscle laminopathies. Hum Mol Genet, 14:155-169.
[22] Wang Y, Fan N, Song J, Zhong J, Guo X, Tian W, et al. (2014). Generation of knockout rabbits using transcription activator-like effector nucleases. Cell Regen (Lond), 3:3.
[23] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science, 339:819-823.
[24] Sui T, Yuan L, Liu H, Chen M, Deng J, Wang Y, et al. (2016). CRISPR/Cas9-mediated mutation of PHEX in rabbit recapitulates human X-linked hypophosphatemia (XLH). Hum Mol Genet, 25:2661-2671.
[25] Guschin DY, Waite AJ, Katibah GE, Miller JC, Holmes MC, Rebar EJ (2010). A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol, 649:247-256.
[26] Gutpell KM, Hrinivich WT, Hoffman LM (2015). Skeletal muscle fibrosis in the mdx/utrn+/- mouse validates its suitability as a murine model of Duchenne muscular dystrophy. PLoS One, 10:e0117306.
[27] Mu X, Tang Y, Lu A, Takayama K, Usas A, Wang B, et al. (2015). The role of Notch signaling in muscle progenitor cell depletion and the rapid onset of histopathology in muscular dystrophy. Hum Mol Genet, 24:2923-2937.
[28] Kane AM, DeFrancesco TC, Boyle MC, Malarkey DE, Ritchey JW, Atkins CE, et al. (2013). Cardiac structure and function in female carriers of a canine model of Duchenne muscular dystrophy. Res Vet Sci, 94:610-617.
[29] Ackerman J, Gilbert-Barness E (2002). Hutchinson-Gilford progeria syndrome: a pathologic study. Pediatr Pathol Mol Med, 21:1-13.
[30] Caux F, Dubosclard E, Lascols O, Buendia B, Chazouilleres O, Cohen A, et al. (2003). A new clinical condition linked to a novel mutation in lamins A and C with generalized lipoatrophy, insulin-resistant diabetes, disseminated leukomelanodermic papules, liver steatosis, and cardiomyopathy. J Clin Endocrinol Metab, 88:1006-1013.
[31] Song K, Dube MP, Lim J, Hwang I, Lee I, Kim JJ (2007). Lamin A/C mutations associated with familial and sporadic cases of dilated cardiomyopathy in Koreans. Exp Mol Med, 39:114-120.
[32] Bonne G, Di Barletta MR, Varnous S, Becane HM, Hammouda EH, Merlini L, et al. (1999). Mutations in the gene encoding lamin A/C cause autosomal dominant Emery-Dreifuss muscular dystrophy. Nat Genet, 21:285-288.
[33] Gordon CM, Gordon LB, Snyder BD, Nazarian A, Quinn N, Huh S, et al. (2011). Hutchinson-Gilford progeria is a skeletal dysplasia. J Bone Miner Res, 26:1670-1679.
[34] Gordon LB, Rothman FG, Lopez-Otin C, Misteli T (2014). Progeria: a paradigm for translational medicine. Cell, 156:400-407.
[35] Friesen M, Cowan CA (2018). FPLD2 LMNA mutation R482W dysregulates iPSC-derived adipocyte function and lipid metabolism. Biochem Biophys Res Commun, 495:254-260.
[36] Honda A, Hirose M, Sankai T, Yasmin L, Yuzawa K, Honsho K, et al. (2015). Single-step generation of rabbits carrying a targeted allele of the tyrosinase gene using CRISPR/Cas9. Exp Anim, 64:31-37.
[37] Rork JF, Huang JT, Gordon LB, Kleinman M, Kieran MW, Liang MG (2014). Initial cutaneous manifestations of Hutchinson-Gilford progeria syndrome. Pediatr Dermatol, 31:196-202.
[38] Ullrich NJ, Gordon LB (2015). Hutchinson-Gilford progeria syndrome. Handb Clin Neurol, 132:249-264.
[39] Hoorntje ET, Bollen IA, Barge-Schaapveld DQ, van Tienen FH, Te Meerman GJ, Jansweijer JA, et al. (2017). Lamin A/C-Related Cardiac Disease: Late Onset With a Variable and Mild Phenotype in a Large Cohort of Patients With the Lamin A/C p.(Arg331Gln) Founder Mutation. Circ Cardiovasc Genet, 10.
[40] Nikolova V, Leimena C, McMahon AC, Tan JC, Chandar S, Jogia D, et al. (2004). Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C-deficient mice. J Clin Invest, 113:357-369.
[41] Akinci B, Onay H, Demir T, Savas-Erdeve S, Gen R, Simsir IY, et al. (2017). Clinical presentations, metabolic abnormalities and end-organ complications in patients with familial partial lipodystrophy. Metabolism, 72:109-119.
[42] Sebastiani P, Thyagarajan B, Sun F, Schupf N, Newman AB, Montano M, et al. (2017). Biomarker signatures of aging. Aging Cell, 16:329-338.
[43] Hitomi Y, Yoshida A (1993). Nutritional significance of dietary cystine for maintaining the hepatic albumin mRNA level in rats fed on a soybean diet. Biosci Biotechnol Biochem, 57:1218-1219.
[44] Lloyd DJ, Trembath RC, Shackleton S (2002). A novel interaction between lamin A and SREBP1: implications for partial lipodystrophy and other laminopathies. Hum Mol Genet, 11:769-777.
[45] Muchir A, Pavlidis P, Decostre V, Herron AJ, Arimura T, Bonne G, et al. (2007). Activation of MAPK pathways links LMNA mutations to cardiomyopathy in Emery-Dreifuss muscular dystrophy. J Clin Invest, 117:1282-1293.
[46] DeBusk FL (1972). The Hutchinson-Gilford progeria syndrome. Report of 4 cases and review of the literature. J Pediatr, 80:697-724.
[47] Kieran MW, Gordon L, Kleinman M (2007). New approaches to progeria. Pediatrics, 120:834-841.
[48] Rober RA, Sauter H, Weber K, Osborn M (1990). Cells of the cellular immune and hemopoietic system of the mouse lack lamins A/C: distinction versus other somatic cells. J Cell Sci, 95(Pt 4):587-598.
[49] Kirkwood TB (2005). Understanding the odd science of aging. Cell, 120:437-447.
[50] Smith ED, Kudlow BA, Frock RL, Kennedy BK (2005). A-type nuclear lamins, progerias and other degenerative disorders. Mech Ageing Dev, 126:447-460.
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