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Human Molecular Genetics Advance Access originally published online on July 6, 2006
Human Molecular Genetics 2006 15(16):2479-2489; doi:10.1093/hmg/ddl170
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Pathology and nuclear abnormalities in hearts of transgenic mice expressing M371K lamin A encoded by an LMNA mutation causing Emery–Dreifuss muscular dystrophy

Yuexia Wang1,2, Alan J. Herron3,4,{dagger} and Howard J. Worman1,2,*

1 Department of Medicine, 2 Department of Anatomy and Cell Biology, 3 Institute of Comparative Medicine and 4 Department of Pathology, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA

* To whom correspondence should be addressed at: Department of Medicine, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, 10th Floor, Room 508, New York, NY 10032, USA. Tel: +1 2123058156; Fax: +1 2123056443; Email: hjw14{at}columbia.edu

Received May 22, 2006; Accepted July 2, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutations in LMNA, which encodes nuclear lamins A and C, cause a broad range of diseases, including autosomal dominant Emery–Dreifuss muscular dystrophy (EDMD) and related disorders with a predominant cardiomyopathy. Homozygous Lmna model ‘knock-in’ and null mice develop cardiomyopathy, whereas heterozygous mice do not. Overexpression of lamin A mutants that cause cardiomyopathy in cultured cells induces morphological abnormalities in the nuclear envelope and lamina; however, effects on tissue and organ pathology have not been determined. We used the heart-selective {alpha}-myosin heavy chain promoter to drive expression in transgenic mice of human wild-type and M371K lamin A, which causes EDMD. Mice expressing M371K lamin A were born at approximately 0.07 of the expected frequency and those born typically died at 2–7 weeks of age. Histological analysis showed increased eosinophilia and fragmentation of cardiomyofibrils, nuclear pyknosis and edema without fibrosis or significant inflammation, indicative of acute or subacute injury. Mice expressing human wild-type lamin A were born at only slightly less than the expected frequency and had normal life spans. Confocal immunofluorescence microscopy demonstrated abnormal nuclear envelopes with intranuclear foci of lamins in cardiac cells expressing M371K lamin A. Electron microscopy revealed extensively convoluted nuclear envelopes, intranuclear inclusions and chromatin clumps in cardiomyocyte nuclei. These results demonstrate that expression of a lamin A mutant that induces alterations in nuclear morphology can cause tissue and organ damage in mice with a normal complement of wild-type lamins.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Emery–Dreifuss muscular dystrophy (EDMD) is characterized by contractures of the elbows, Achilles’ tendons and posterior neck, slow progressive muscle wasting and a predominant cardiomyopathy (1–3). Clinically identical disorders are inherited in X-linked (OMIM no. 310300 [OMIM] ), autosomal dominant (OMIM no. 181350 [OMIM] ) and very rarely autosomal recessive (OMIM no. 604929 [OMIM] ) manners. X-linked EDMD was the first human disease found to result from mutation in a gene encoding a protein localized to the inner nuclear membrane (4–6). Subsequently, autosomal dominant EDMD was shown to be the first disease caused by mutations in LMNA (7). LMNA encodes A-type lamins, intermediate filament proteins that polymerize to form the nuclear lamina on the inner aspect of the inner nuclear membrane (8–12). LMNA mutations also cause dilated cardiomyopathy with conduction defect 1 (13), limb-girdle muscular dystrophy 1B (14) and autosomal recessive EDMD (15). Intriguingly, LMNA mutations are responsible for a diverse range of human diseases in addition to EDMD and related striated muscle disorders. These include Dunnigan-type familial partial lipodystrophy (16–18), which affects adipose tissue, mandibuloacral dysplasia (19), which affects adipose tissue as well as the skeleton, Charcot–Marie–Tooth disease type 2B1 (20), a peripheral neuropathy and the accelerated aging disorders Hutchison–Gilford progeria syndrome (21,22) and atypical Werner syndrome (23).

Three mouse models of EDMD have been generated using homologous recombination at the Lmna locus. These are Lmna ‘knock-out’ mice (24) and knock-in mice that express A-type lamins with the H222P (25) and N195K (26) amino acid substitutions. Mice homozygous for these mutations develop features of human EDMD, including cardiomyopathy. However, heterozygous mice are apparently normal, suggesting that mice must lose both copies of wild-type Lmna to develop significant disease. This is in contrast to humans, in which the vast majority of LMNA mutations that cause EDMD are autosomal dominantly inherited.

Although heterozygous Lmna knock-out and knock-in mice do not develop significant pathology, expression of mutant A-type lamins that cause EDMD can act in a ‘dominant’ manner to disrupt the nuclear lamina and alter the morphology of the nuclear envelope in transfected cultured cells (27–37). Although these findings in cultured cells have led to the hypothesis that dominantly induced alterations in nuclear morphology play a role in disease pathogenesis, striated muscle damage induced by expression of a mutant A-type lamin in vertebrate animals simultaneously expressing wild-type A-type lamins has not been demonstrated. To determine whether a mutant lamin A can act via a dominant mechanism in a model vertebrate animal and induce heart damage in the presence of wild-type A-type lamins, we generated transgenic mice that overexpressed human lamin A with the M371K amino acid substitution. This LMNA mutation causes autosomal dominant EDMD in humans (38), and overexpression of the protein in cultured cells leads to significant morphological alterations in the nuclear envelope and lamina (27,29). As a control, we generated transgenic mice that overexpressed wild-type human lamin A in heart.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Generation of minigenes encoding wild-type and M371K lamin A
To study the effects of expression of human lamin A with the M371K amino acid substitution, which is encoded by an LMNA mutation that causes EDMD in humans, we generated transgenic mice expressing this protein in heart. As a control, we generated transgenic mice expressing wild-type human lamin A. For cardiac-selective expression, we cloned cDNAs encoding wild-type and M371K human prelamin A downstream of the murine {alpha}-myosin heavy chain ({alpha}-MHC) promoter. The {alpha}-MHC promoter has been shown to drive cardiac muscle-selective expression in transgenic mice (39,40). To facilitate detection of the expressed proteins, we engineered the constructs such that they contained a FLAG epitope tags at their amino termini. The general structure of the minigene constructs is shown in Figure 1A.


Figure 1701
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  		Figure 1. Diagram showing minigene construction (A) and photomicrographs showing expressed proteins in transfected cells (B). (A) Minigene constructs contained an {alpha}-MHC promoter followed by DNA containing an ATG start codon and a FLAG epitope tag coding sequence (FLAG) fused in-frame to cDNAs encoding either prelamin A or M371K prelamin A (LmnA/M371K) followed by DNA encoding the 3'-untranslated sequence human growth hormone (Hgh). NotI and HindIII restriction endonuclease sites are indicated. The 8.1 kb fragment generated by digestion with NotI was used for microinjection of pronuclei of fertilized mouse oocytes. (B) Laser scanning confocal immunofluorescence micrographs of C2C12 cells transfected with plasmid constructs containing minigene constructs encoding FLAG-tagged wild-type lamin A (a–c) or FLAG-tagged M371K lamin A (d–f). Cells were transfected with plasmids containing the minigenes and processed for immunoflourescence with mouse anti-FLAG antibodies, rabbit anti-lamin B1 antibodies, fluorescein isothiocyanate-conjugated anti-mouse secondary antibodies and rhodamine-conjugated anti-rabbit secondary antibodies. (a and d) show the labeling by anti-FLAG antibodies (green) and (b and e) show the labeling of same cells by anti-lamin B1 antibodies (red). Overlapping signals from (a and b) are shown in (c) and from (d and e) in (f) (areas of overlap are yellow). Note that wild-type FLAG-lamin A is localized primarily to the nuclear rim in a smooth pattern, whereas FLAG-M371K lamin A gives a less pronounced rim labeling and transfected cells have nuclear blebs or ‘protrusions’ containing FLAG-tagged M371K lamin A and endogenous lamin B1 (arrows). Bar: 10 µm.

 
To examine the expression of FLAG-tagged wild-type and M371K lamin A prior to generating transgenic mice, we transiently transfected C2C12 mouse myoblastoid cells with plasmid constructs containing the minigenes. FLAG-wild-type lamin A localized to the nuclear periphery and smooth ring-like fluorescence of the nucleus overlapping that of endogenous lamin B1 was observed in ã80% of cells when examined by immunofluorescence confocal microscopy; ã20% had less pronounced rim-like fluorescence and ‘mild bleb-like’ structures were observed in the nuclear envelope in <20% of transfected cells (Fig. 1B). Expression of FLAG-M371K lamin A gave a less pronounced nuclear rim labeling with increased intranuclear fluorescence in all cells and induced formation of more prominent nuclear envelope ‘blebs’ or ‘protrusions’ in ã50% of transfected cells (Fig. 1B). The abnormal localization of M371K lamin A and its effects on nuclear envelope architecture were less pronounced than when expression of the same protein was driven by an SV40 early promoter in C2C12 cells (27). This is likely because of higher expression levels obtained with the SV40 early promoter. The normal localization of wild-type lamin A in the majority of transfected cells was also similar to that observed when expression was driven by an SV40 early promoter (27).

Generation of transgenic mice expressing wild-type and M371K lamin A in heart
The 8.1 kb sequences indicated in Figure 1A were separated from the plasmid backbones by restriction endonuclease digestion with NotI to generate liner DNA minigenes that encoded wild-type prelamin A (MHC-LmnA) and M371K prelamin A (MHC-M371K). These were microinjected separately into B6/CBA F1 fertilized oocytes that were subsequently transferred to pseudopregnant foster mothers to produce transgenic founders. Ten transgenic founder mice containing MHC-LmnA (designed 901–910) and six containing MHC-M371K (designated 911–916) were tentatively identified based on the results of polymerase chain reactions using primer pairs corresponding to portions of FLAG and human lamin A coding sequences and tail DNA as template (data not shown).

On backcrossing of the apparent founder mice to B6/CBA F1 mice, no transgenic progeny were identified from seven (901, 904, 906, 907, 912, 914, 915). Southern blot hybridization was then performed and confirmed the presence of the MHC-LmnA minigenes in only six founders (902, 903, 905, 908–910) and the MHC-M371K minigene in only three founders (911, 913, 916) (Fig. 2). Transgenic founder mice 902, 908, 909 and 916 died prior to reaching reproductive age. Although the exact copy numbers of the transgenes were not determined, these founders all appeared to have greater copy numbers than those that survived to reproduce, according to the results of Southern hybridizations (Fig. 2). Three founders containing the MHC-LmnA transgene (903, 905 and 910) and two containing the MHC-M371K transgene (911 and 913) produced transgenic offspring when backcrossed to wild-type B6/CBA F1 mice. This ultimately resulted in three separate transgenic lines expressing FLAG-wild-type lamin A and two separate transgenic lines expressing FLAG-M371K lamin A.


Figure 1702
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  		Figure 2. Autoradiogram showing Southern hybridization for minigenes in DNA from MHC-LmnA and MHC-M371K transgenic mice. Genomic DNA (10 µg) was digested by KpnI and separated on a 0.8% agar gel. A 595 bp probe encompassing the 5' end of the coding sequence, including the FLAG epitope tag coding sequence, was labeled using 32P-dCTP and hybridization carried out at 65°C. Lanes labeled 902, 903, 905 and 908–910 show results from founder mice containing the MHC-LmnA minigene. Lanes labeled 911, 913 and 916 show results from founder mice containing the MHC-M371K minigene. Lane labeled WT contains DNA from a wild-type mouse and lane labeled M contains molecular mass marker. The arrow at the right indicates a predicted minigene DNA fragment hybridizing to the probe of ~8 kb. Arrows at the top indicate mice that died prematurely without generating offspring. MHC-LmnA founders 903, 905 and 910 and MHC-M371K founders 911 and 913 were successfully backcrossed to wild-type mice to obtain F1 mice.

 
To detect the expression of the transgenes, we performed immunoblotting using anti-FLAG antibody and proteins isolated from heart, lung, liver and skeletal muscle from the transgenic mice. FLAG-tagged proteins encoded by MHC-LmnA and by MHC-M371K were not detected in lung and liver and detected at only minimal levels in skeletal muscle (data not shown). FLAG-tagged wild-type and M371K lamin A were readily detected by immunoblotting of proteins extracted from hearts of transgenic mice of all lines (Fig. 3). Immunoblotting with antibodies that recognized both lamin A and lamin C detected higher levels of lamin A in hearts of the transgenic mice compared with controls (Fig. 3). A ‘band’ recognized by anti-lamin A/C antibodies migrating between lamins A and C and slightly slower than lamin C was often observed in the mice expressing M371K lamin A (Fig. 3); its identity is unclear but may represent a lamin A degradation product. Scanning of several immunoblots of proteins extracted from hearts with antibodies that recognized lamins A and C showed approximately twice as much lamin A in each of the transgenic mice expressing wild-type lamin A (lines 903, 905 and 910) and M371K lamin A (lines 911 and 913) compared with non-transgenic littermates and other wild-type controls when normalized to actin expression. Scanning of immunoblots showed that lamin C levels were equal in mice from all lines, approximately the same as lamin A in non-transgenic mice and approximately half that of lamin A in the transgenic mice.


Figure 1703
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  		Figure 3. Immunoblot analysis of proteins encoded by minigenes in representative transgenic mice. Proteins were extracted from heart tissue, separated by SDS–PAGE on 10% polyacrylamide gels and transferred to nitrocellulose membranes. WT indicates lanes containing proteins from hearts of wild-type mice. MHC-LmnA+ indicates lane with proteins from a mouse containing the wild-type lamin A transgene and MHC-LmnA- a lane with proteins from a non-transgenic littermate. MHC-M371K+ indicates lane with proteins from a mouse containing the M371K lamin A transgene and MHC-M371K– indicates lane with proteins from a non-transgenic littermate. (A) Results of probing nitrocellulose blots with anti-FLAG antibodies (FLAG). (B) Results of the same blots probed with antibodies that recognize lamins A and C, and migrations of lamins A and C are indicated at the right. (C) Results of probing the same blots with anti-actin antibodies.

 
Cardiac-selective expression of M371K lamin A causes increased prenatal loss and post-natal cardiac pathology
Backcrossing of transgenic founders to wild-type mice suggested that expression of M371K lamin A resulted in a significantly increased risk of prenatal death (Table 1). F1 and subsequent generations of two different lines from founders that contained the MHC-LmnA minigene were born at the expected ratio of approximately 1:1 when compared with non-transgenic littermates. In a third line from the other founder with the MHC-LmnA transgene, this ratio was 1:4. In contrast, transgenic progeny of the two lines derived from founders containing the MHC-M371K minigene were born at significantly less than the expected ratio. In these lines, transgenic progeny were born approximately 0.07 times the frequency of non-transgenic littermates, suggesting that most of the transgenic mice died in utero.


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  		Table 1. Reproduction of transgenic mice with MHC-LmnA or MHC-M371K

 
Prenatal lethality of transgenic expression of M371K lamin A was also suggested by attempts to generate additional offspring by in vitro fertilization. We retrieved 160 unfertilized eggs from eight B6/CBA F2 female mice and fertilized them with sperm from a founder (913) that contained the MHC-M371K transgene. In the process, 126 eggs were fertilized, transferred into six foster mothers and 24 pups were born 19 days later. Genotyping by polymerase chain reaction showed that none of the progeny contained the transgene.

Transgenic mice expressing M371K lamin A typically died at 2–7 weeks of age. Necropsies of sacrificed mice revealed pulmonary and cardiac edema. Histological examination of hearts demonstrated increased cytoplasmic eosinophilia of cardiomyocytes, focal edema, fragmented cardiomyofibrils and pyknotic-appearing nuclei, which were not prominent in hearts of mice containing the MHC-LmnA transgene or hearts from non-transgenic littermates of the same age (Fig. 4). The cardiac lesions in mice expressing M371K lamin A were multifocal and did not include fibrosis or significant numbers of inflammatory cells, indicative of acute or subacute injury. Identical histological cardiac lesions were observed in lamin A M371K-expressing transgenic mice sacrificed at 2 weeks of age and in hearts obtained within a few hours postmortem of those that died naturally.


Figure 1704
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  		Figure 4. Representative histological sections of hearts from 2-week-old wild-type mice (WT), transgenic mice expressing FLAG-M371K lamin A (MHC-M371K) and transgenic mice expressing FLAG-lamin A (MHC-LmnA). Hearts were fixed in formalin, embedded in paraffin, sectioned and stained with H&E. Note increased cytoplasmic eosinophilia (arrows), focal edema (arrowheads), fragmented cardiomyofibrils and pyknotic-appearing nuclei in section from heart expressing FLAG-M371K lamin A. Bar: 20 µm.

 
All three lines of transgenic mice expressing wild-type lamin A had cumulative 75% survival out to 68 weeks of age, whereas both lines of transgenic mice expressing M371K lamin A typically died between 2 and 7 weeks of age (Fig. 5A). To further evaluate cardiac damage, which was likely responsible for the decreased survival of transgenic mice expressing M371K lamin A, we performed a systemic histological analysis on hearts obtained from the 2-week-old transgenic mice and age-matched littermates that did not contain the transgene. Hearts of 10 sacrificed mice from lines 903 and 910 (expressing wild-type lamin A) and of five mice from lines 911 and 913 (expressing M371K lamin A), three of which were sacrificed and two of which had died within a couple of hours, were used. A ‘blinded’ pathologist who was unaware of the genotypes performed the analysis. The pathologist scored tissue sections for lesions indicative of cardiac damage including increased cytoplasmic eosinophilia, fragmentation of cardiomyofibrils, pyknosis and focal edema using a range from 0 (no lesions) to 3 (severe lesions). Mean score for cardiac damage was significantly higher in mice containing the MHC-M371K transgene when compared with non-transgenic mice as well as mice containing the MHC-LmnA transgene (Fig. 5B). The lesions in the hearts from sacrificed MHC-M371K transgenic mice were similar in quality and degree to those observed in hearts removed within a couple of hours postmortem from MHC-M371K transgenic mice that had died naturally at 2 weeks of age. There was no significant difference in the cardiac damage scores between mice containing the MHC-LmnA transgene and non-transgenic mice (Fig. 5B). These results showed that transgenic expression of M371K lamin A in mice that did not die in utero led to acute or subacute cardiac damage, which typically caused death between 2 and 7 weeks after birth.


Figure 1705
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  		Figure 5. (A) Survival curves for transgenic mice overexpressing wild-type and M371K lamin A. Natural life spans were recorded up to 68 weeks and percentage survival versus time is shown for transgenic lines 911 (n=4) and 913 (n=7) overexpressing M371K lamin A and transgenic lines 903 (n=19), 905 (n=17) and 910 (n=23) overexpresing wild-type lamin A. (B) Scoring of cardiac damage assessed by histology. Fresh hearts were fixed in formalin, embedded in paraffin, sectioned and stained with H&E. A blinded pathologist, not aware of the genotypes, scored the sections for lesions indicative of cardiac damage including increased eosinophilia, fragmentation of cardiomyofibrils, pyknosis and focal edema using a scoring ranging from 0 (no lesions) to 3 (severe lesions). Mean scores±standard deviations are shown from transgenic mice expressing FLAG-lamin A (genotype MHC-LmnA+, n=10), non-transgenic littermates of mice expressing FLAG-lamin A (genotype MHC-LmnA–, n=10), transgenic mice expressing FLAG-M371K lamin A (genotype MHC-M371+, n=5) and non-transgenic littermates of mice expressing FLAG-M371 lamin A (genotype MHC-M371K–, n=5). Heart damage scores were significantly increased in MHC-371K+ mice expressing FLAG-M371K lamin A compared with their non-transgenic littermates (P<0.05) and compared with MHC-LmnA+ mice expressing FLAG-lamin A (P<0.05).

 
Nuclear morphology in hearts of transgenic mice expressing wild-type and M371K lamin A
To assess the effects of the M371K lamin A on nuclear morphology in heart tissue, we performed immunofluorescence microscopy on frozen embedded sections from sacrificed mice. We used antibodies that recognized the FLAG epitope tag of the proteins encoded by the transgenes and antibodies against endogenous lamin B1. Almost no normal nuclei were observed in cardiac cells of representative transgenic mice of line 911 expressing MHC-M371K lamin A. The cells contained nuclei of abnormal shape with intranuclear foci that were recognized by anti-FLAG and anti-lamin B1 antibodies (Fig. 6A–C). In contrast, nuclei in representative cardiac tissue sections from transgenic mice of line 903 overexpressing wild-type lamin A were grossly normal in shape and did not contain intranuclear foci (Fig. 6D–F). Morphology of the nuclei in cardiac cells of transgenic mice expressing wild-type human lamin A was similar to that in non-transgenic mice as assessed by anti-lamin B1 antibody labeling (Fig. 6G–I).


Figure 1706
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  		Figure 6. Laser scanning confocal immunofluorescence photomicrographs of tissue sections from hearts of transgenic and control mice. After mice were sacrificed, hearts were immediately embedded and frozen sections were cut at a thickness of 6 µm. Sections were processed for immunofluorescence with mouse anti-FLAG and rabbit anti-lamin B1 primary antibodies followed by fluorescein isothiocyanate-conjugated anti-mouse and rhodamine-conjugated anti-rabbit secondary antibodies. (A–C) Photomicrographs of heart section from transgenic mouse expressing FLAG-M371K lamin A. (A) Labeling with anti-FLAG antibodies (green), (B) labeling with anti-lamin B1 antibodies (red) and (C) overlay of these two signals with areas of overlap appearing yellow. (D–F) Photomicrographs of heart section from transgenic mouse expressing FLAG-lamin A. (D) Labeling with anti-FLAG antibodies (green), (E) labeling with anti-lamin B1 antibodies (red) and (F) overlay of these two signals with areas of overlap appearing yellow. (G–I) Photomicrographs of heart section from non-transgenic mouse. (G) Labeling with anti-FLAG antibodies (green), (H) labeling with anti-lamin B1 antibodies (red) and (I) overlay of these two signals with areas of overlap appearing yellow. Note foci of lamins in nuclei of mouse heart expressing FLAG-M371K lamin A (A–C). In contrast, lamins are in a peripheral rim-like distribution in nuclei of hearts from transgenic mice expressing FLAG-tagged wild-type lamin A (D–F). No signal with anti-FLAG antibodies is detected in heart from non-transgenic mouse (G). Nuclei labeled with anti-lamin B1 antibodies (E) but not with anti-FLAG antibodies (D) are likely those of cells other than cardiomyocytes in heart tissue. Bar: 10 µm.

 
We further examined the morphology of cardiomyocyte nuclei in hearts from the sacrificed mice examined by immunofluoresecne microscopy using transmission electron microscopy. In hearts of transgenic mice expressing M371K lamin A, almost all nuclei had extensively convoluted nuclear envelopes (Fig. 7A, D, G and J). About half contained nuclear inclusions possibly composed of lamins, chromatin clumps and lipid pseudoinclusions (representative examples shown in Fig. 7G and J). Nuclei in cardiomyocytes of hearts from transgenic mice overexpressing wild-type human lamin A had only slightly convoluted nuclear envelopes (Fig. 7B, E, H and K); however, this was much less dramatic than in mice expressing M371K lamin A. Only ã10% of nuclei examined from these mice had what appeared to be very small nuclear inclusion (representative examples shown in Fig. 7H and K). No nuclear inclusions, chromatin clumps or lipid pseudoinclusions were observed in cardiomyocyte nuclei of non-transgenic mice (Fig. 7C, F, I and L). These results showed that expression of M371K lamin A caused significant nuclear morphological abnormalities. However, fibers containing these abnormal nuclei were not all necrotic or apoptotic (Fig. 7A).


Figure 1707
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  		Figure 7. Electron microscopy analysis of mouse heart nuclei. Pieces of diced hearts were fixed immediately after removing from sacrificed mice, embedded and then ultra-thin sections were cut, stained and examined using a transmission electron microscope. (A–C) Representative lower magnification images of cardiomyocyte nuclei from hearts of mice expressing FLAG-M371K lamin A (A), FLAG-lamin A (B) and a wild-type control (C). (D–F) Representative images of more cardiomyocyte nuclei from hearts of mice expressing FLAG-M371K lamin A (D), FLAG-lamin A (E) and a wild-type control (F). (G) Micrograph showing severely abnormal nucleus in heart of mouse expressing FLAG-M371K lamin A with higher magnification of area indicated by tail of arrow shown in (J). CC indicates chromatin clump, IN indicates what appears to be an abnormal inclusion and LP a lipid pseudoinclusion within the nucleus. (H) Micrograph showing irregularly shaped nucleus in heart of mouse expressing FLAG-lamin A with higher magnification of area indicated by tail of arrow shown in (K). (I) Micrograph showing normal nucleus in heart of a non-transgenic wild-type mouse with higher magnification of area indicated by tail of arrow shown in (L). Bars: 1 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Despite several publications on the effects of expression of A-type lamin mutants that cause EDMD in transfected cultured cells (27–37), there have been no published studies reporting on the transgenic expression of these proteins in living vertebrate tissues. We have now shown that a human lamin A variant encoded by an LMNA mutation that causes EDMD induced severe nuclear morphological abnormalities and tissue damage when expressed in hearts of transgenic mice. In contrast, transgenic mice overexpressing wild-type human lamin A at approximately the same levels did not develop significant abnormalities. These results show that overexpression of a mutant lamin A in EDMD can act in a dominant manner to cause heart disease.

In human subjects, most LMNA mutations that cause autosomal dominant EDMD lead to expression of A-type lamins with amino acid substitutions or small in-frame deletions (7,13,38,41). The results of pulse chase experiments show that several of these mutant proteins have half-lives similar to that of wild-type lamin A (27), suggesting that they are ‘normally’ expressed in cells. Only a small minority of cases of autosomal dominant EDMD, sometimes with concurrent features of peripheral neuropathy, is caused by mutations that essentially lead to haploinsufficiency of A-type lamins (7,42). In contrast, in Lmna knock-in and knock-out mouse models of EDMD, heterozygous animals are not significantly affected and only homozygous mice with loss of both wild-type Lmna alleles clearly develop disease (24–26). This is also true for another Lmna knock-in mouse that develops a progeric phenotype (43). Our results demonstrate that lamin A containing an amino acid substitution found in humans with EDMD can cause striated muscle damage when expressed in mice that simultaneously express endogenous wild-type A-type lamins. Hence, loss of wild-type A-type lamins is not necessary for the development of striated muscle disease induced by A-type lamin mutants in mice. Other studies have shown that expression of the truncated prelamin A encoded by LMNA mutations that cause Hutchinson–Gilford progeria syndrome in humans causes pathology in tissues other than striated muscle in the presence of wild-type endogenous A-type lamins in mice (44,45).

The apparent bimodal pattern of death observed in the transgenic mice expressing M371K lamin A is consistent with the activity of the {alpha}-MHC promoter used to drive expression. In mouse embryos, {alpha}-MHC is expressed in newly formed cardiac tubes 7.5–8 days postcoitum; however, its ventricular expression subsequently decreases to low levels in fetal ventricular myocytes and then increases significantly after birth, replacing ß-MHC ~7 days later (46,47). Hence, the significantly lower than expected birth rate of transgenic mice containing the MHC-M371K minigene is consistent with detrimental effects of this protein when expressed early during embryonic heart development. The minority of embryos that apparently ‘survive’ expression of this protein early in development subsequently suffers from acute or subacute ventricular damage after the {alpha}-MHC promoter is reactivated after birth.

Variables not related to the direct effects of the M371K lamin A mutant could have potentially accounted for the pathological abnormalities observed in transgenic mice in this study. We feel that controls in our experimental design make these possibilities unlikely. First, the proteins encoded by the transgenes were expressed at approximately twice the level of endogenous lamin A in all of the transgenic line examined. For this reason, overexpression of lamins per se and not the pathological amino acid substitution could have been responsible for nuclear morphological abnormalities and tissue pathology. To control for this possibility, we expressed wild-type lamin A at similar levels in mouse hearts. Nuclei in hearts of transgenic mice overexpressing wild-type human lamin A did not have significant morphological abnormalities compared with nuclei from mice overexpressing M371K lamin A, which is consistent with what has been reported in transfected cultured cells for these two proteins (27,29). In addition, mice overexpressing wild-type lamin A were born at the expected frequency in two of three lines and at only slightly less than the expected frequency in one line. Transgenic mice overexpressing wild-type lamin A had normal life spans without significant cardiac pathology. Secondly, nuclear morphological alterations and pathology could have occurred secondary to disruption of an endogenous gene caused by insertion of transgenes. To control for this possibility, we examined two lines from different founders expressing M371K lamin A and both had the same phenotypes. In addition, the nuclear morphological alterations observed in cells of these mice were consistent with those previously reported in cells expressing mutant A-type lamins that cause EDMD and related striated muscle disorders (27–37,48–50) and cells from Lmna knock-in and knock-out mouse models of the disease (24–26).

The cardiac pathology observed in the M371K lamin A transgenic mice did not precisely recapitulate what is observed in the human subjects with autosomal dominant EDMD or knock-in and knock-out mouse models. Human subjects with LMNA mutations causing EDMD and related disorders generally have a progressive cardiomyopathy with a variable age of onset first characterized by atrioventricular conduction defects or sinus dysfunction followed by left ventricular chamber dilation and ventricular dysrhythmias (1–3,38,51–55). Histological analysis of hearts from a few affected human subjects has demonstrated non-specific cardiomyocyte damage and interstitial fibrosis (35,53,55,56). Homozygous Lmna knock-out mice develop dilated left ventricles at ~6 weeks after birth with interstitial fibrosis (24,57). Male homozygous Lmna H222P knock-in mice die between 4 and 9 months of age and females between 9 and 13 months of age and have ventricular chamber dilation and marked cardiac fibrosis (25). Homozygous Lmna N195K knock-in mice die between 12 and 14 weeks after birth and also have dilated left ventricles and interstitial fibrosis (26). Hence, the acute or subacute cardiac damage in the M371K transgenic mice is a major difference compared with the more gradual development of dilated cardiomyopathy in affected human subjects and the other mouse models. Nonetheless, one can speculate that the initial insult to cardiomyocytes, that of disruption of the nuclear lamina, is the same or similar in each of these instances. The variability may rather be in the severity of the insult, as the M371K transgenic mice overexpress the mutant protein at higher levels than heterozygous-affected humans and homozygous knock-out and knock-in mice. This could lead to more rapid initial damage to cardiomyocytes leading to death before the development of an adequate compensatory response.

Examination of the M371K transgenic mice demonstrates a correlation between the induction of morphological abnormalities in the nuclear envelope and tissue pathology. Notably, the nuclear morphological changes observed in these mice using transmission electron microscopy are similar to some that have been reported in cardiomyocytes from human subjects with LMNA mutations, which include chromatin disorganization, accumulation of glycogen and/or lipofuscin in the nucleoplasm, nuclear envelope blebs and loss of lamina integrity (35,53). Electron microscopic analysis of Lmna H222P and N195K knock-in mice and Lmna knock-out mice have also demonstrated abnormal nuclear envelope shape and abnormal chromatin condensation and clumping (24–26,57). Although nuclear alternations observed in affected tissue could be either a cause or an effect of tissue damage, we have shown that overexpression of M371K lamin A in transfected cultured cells leads to nuclear structural alterations. This suggests that structural abnormalities in the nuclear lamina and nuclear envelope may be a cause of, rather than a non-specific consequence of, cardiac tissue damage.

In conclusion, we have shown that transgenic expression of M371K lamin A, encoded by an LMNA mutation that causes EDMD in humans, results in cardiac pathology when overexpressed in mice in the presence of normal amounts of endogenous A-type lamins. The cardiac pathological damage correlates with marked nuclear structural abnormalities, which appear to be induced by overexpression of the protein. Similar overexpression of wild-type human lamin A does not cause significant heart pathology or nuclear morphological alterations. It remains to be established how the observed physical alterations in the nuclear lamina and nuclear envelope could be responsible for damaging myocardial tissue. Possibilities include direct cardiomyocyte death as a consequence of severe nuclear dysfunction or the abnormal activation of cellular ‘stress–response’ pathways that may be detrimental to normal tissue homeostasis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid construction
cDNAs of ~2 kb encoding FLAG-tagged prelamin A and M371K prelamin A were isolated from previously described constructs in pSVK3 (27) by restriction endonuclease digestion with EcoRI and SalI. The isolated cDNAs were cloned into the SalI restriction endonuclease site of pPSKII-MHC-GH (generous gift of Dr Ira Goldberg, Columbia University), downstream of the {alpha}-MHC promoter and upstream of a human growth hormone 3' untranslated sequence (Fig. 1A). Restriction endonuclease digestion analysis and DNA sequencing using an ABI 3100 capillary sequencer (Applied Biosystems) were carried out to characterize the constructs and ensure proper sequences.

Transfection and immunofluorescence microscopy of cultured cells
C2C12 mouse myoblasts were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (Life Technologies) and cultured in an incubator with 10% CO2 at 37°C as described (27). The cells were transfected and split in chamber slides using Lipofectamine PLUSTM (Invitrogen) following manufacturer's instructions. The cells were allowed in fresh medium for 24 h post-transfection before preparation for immunofluorescence microscopy. Fixation and labeling of cells were performed as described previously (27,58). Primary antibodies were mouse monoclonal anti-FLAG M-5 (Sigma-Aldrich) diluted 1:200 and rabbit polyclonal anti-lamin B1 (59) diluted 1:1000. Secondary antibodies were fluorescein isothocyanate-conjugated goat anti-mouse Immunoglobulin G and Rhodamine redTM-X-conjugated goat anti-rabbit Immunoglobulin G (Jackson ImmunoResearch Laboratories). Labeled and washed slides were dipped in methanol for a few seconds and air-dried. Cover slips were mounted using ProLong® Antifade Kit (Molecular Probes). Immunofluorescence microscopy was performed on a Zeiss LSM 510 META confocal laser scanning system attached to a Zeiss Axiovert 200 inverted microscope with spectral resolution of fluorescent labels and excitations at 488 and 543 nm (Carl Zeiss). Images were processed using Photoshop software (Adobe Systems) on a Macintosh G4 computer (Apple Computer).

Generation and breeding of transgenic mice
Transgenic mice were generated at the Herbert Irving Comprehensive Cancer Center Transgenic Mouse Facility at Columbia University Medical Center. The Institutional Animal Care and Use Committee at Columbia University Medical Center approved the protocol. Minigenes of ~8.1 kb encoding wild-type prelamin A and prelamin A with the M371K amino acid substitution were excised from plasmids generated in pPSKII-MHC-GH by restriction endonuclease digestion with NotI (Fig. 1A). The minigenes were microinjected separately into superovulated B6/CBA F1 fertilized oocytes in vitro and then the oocytes were transferred to pseudopregnant foster mothers to produce transgenic founders.

Founder transgenic mice were identified by polymerase chain reaction analysis of DNA from tail biopsy using primer pairs corresponding to sequences in FLAG and human lamin A. Polymerase chain reaction using primers corresponding to sequences in ß-globin and ß-actin were simultaneously performed as internal controls. Southern blot analysis was performed to confirm the presence of the transgene using DNA from tail biopsy using a 595 bp probe containing sequences corresponding to the FLAG-coding sequence and the 5' end of human prelamin A cDNA. Transgenic mice were backcrossed to wild-type B6/CBA F1 to obtain stable transgenic offspring and adequate numbers of individuals for further experiments. All the transgenic offspring were genotyped by polymerase chain reaction using tail DNA obtained prior to 2 weeks of age. Mice were fed a chow diet and autoclaved water and housed in a disease-free barrier facility with 12 h/12 h light/dark cycle at 25±2°C.

In vitro fertilization
Unfertilized eggs isolated from B6/CBA F2 female mice were fertilized in vitro with sperm from a 14-month-old male founder. The fertilized eggs were transferred to foster mothers to produce pups. Procedures were those of the Herbert Irving Comprehensive Cancer Center Transgenic Mouse Facility at Columbia University Medical Center.

Protein electrophoresis and immunoblotting
Proteins were extracted from diced whole organs in Laemmli sample buffer and separated by 10% sodium dodecyl sulfate–polyacrylamide slab gel electrophoresis (SDS–PAGE) at 125–200 V for 1.5–2.0 h at room temperature (60). For immunoblotting, proteins were transferred onto the nitrocellulose membrane by electroblotting at 70 V for 2.5 h at 4°C. Immunoblotting was performed using mouse anti-FLAG antibody M-5 at 1:500 dilution, mouse anti-lamin A/C antibody X67 (generous gift of Dr Georg Krohne, Universität Würzburg) at 1:500 dilution or goat anti-actin antibody (Santa Cruz Biotechnology) at 1:100 dilution. ECL-horseradish peroxidase-conjugated anti-mouse NA931versus was used at 1:5000 to 1:10 000 dilution to detect anti-FLAG and anti-lamin A/C primary mouse antibodies. Human anti-goat horseradish peroxidase-conjugated Immunoglobulin G was used as secondary antibody at 1:10 000 dilution to detect anti-actin primary goat antibodies. Signals were detected using SuperSignal® West Pico Chemiluminescent Substrate Kit (Pierce) and X-ray film (Kodak). Immunoblots were scanned and densities of the bands were quantified using Scion Image software. Expression levels of lamins A and C were normalized as percentages of the actin signals in each lane.

Pathological analysis of hearts from transgenic mice
Mice were scarified at 2 weeks of age and freshly removed hearts were fixed in formalin for at least 48 h, embedded in paraffin, sectioned at 6 µm and stained with hematoxylin and eosin (H&E). A ‘blinded’ comparative pathologist (A.J.H.), unaware of the genotype, scored the sections using a scoring system for cardiac lesions ranging from 0 (no lesion) to 3 (severe lesion). The lesions observed included increased eosinophilia and fragmentation of cardiomyofibrils, pyknosis of cardiomyocytes and focal edema. Resulting scores for each genotype of mice were compared using a Student's t-test for non-paired values at {alpha}=0.05 level using Excel software (Microsoft) on a Macintosh G4 computer (Apple Computer). Representative sections of heart stained with H&E were photographed using a light microscope (Nikon).

Immunofluorescence microscopy using tissue sections
Fresh hearts from sacrificed 2-week-old mice were immediately embedded in Tissue-Tek O.C.T. compound (VWR International) and frozen sections were cut at 6 µm and air-dried. Sections were fixed in cold acetone for 10 min at –20°C, then washed in phosphate-buffered saline three times at 2 min for each washing. Sections were incubated in 10% normal goat serum and 1% bovine serum albumin in phosphate-buffered saline at room temperature for 30 min in a dark box. Sections were next incubated with the primary antibodies (anti-FLAG M-5 at a dilution of 1:200 and anti-lamin B1 at a dilution of 1:1000) for 1.5 h at room temperature in the dark box. After three washes of 1 min each, the sections were incubated in secondary antibodies (fluorescein isothocyanate-conjugated goat anti-mouse Immunoglobulin G and Rhodamine red-X-conjugated goat anti-rabbit Immunoglobulin G) for 30 min. The remainder of the protocol was identical to that described under ‘Transfection and Immunofluorescence Microscopy of Cultured Cells’.

Electron microscopy
Hearts were removed from sacrificed 2-week-old mice, rapidly diced using razor blades and fixed with 2.5% glutaraldehyde in 0.1 M Sorenson's buffer (pH 7.2) for at least 1 h. Samples were then post-fixed with 1% OsO4 in Sorenson's buffer for 1 h. Enblock staining was performed using 1% tannic acid. After dehydration, specimens were embedded with a mixture of Lx-112 (Ladd Research Industries) and Embed-812 (EMS). Thin sections were cut on an MT-7000 ultramicrotome (RMC), stained with uranyl acetate and lead citrate and examined using a JEOL JEM-1200 EXII transmission electron microscope. Pictures were taken with an ORCA-HR digital camera (Hamamatsu) and recorded with an AMT Image Capture Engine.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from the National Institutes of Health (RO1-AR048997). We thank Chyuan-sheng (Victor) Lin (Columbia University) for microinjection of mouse oocytes and in vitro fertilization, Kristy Brown (Columbia University) for assistance with electron microscopy, Ira Goldberg (Columbia University) for supplying the plasmid containing the {alpha}-MHC promoter, Georg Krohne (Universität Würzburg) for antibodies and John F. Van Vleet (Purdue University) for reviewing electron micrographs.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Center for Comparative Medicine and Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. Back


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