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Human Molecular Genetics Advance Access originally published online on June 22, 2005
Human Molecular Genetics 2005 14(15):2167-2180; doi:10.1093/hmg/ddi221
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Published by Oxford University Press 2005

Expression of an LMNA-N195K variant of A-type lamins results in cardiac conduction defects and death in mice

Leslie C. Mounkes1, Serguei V. Kozlov1, Jeffrey N. Rottman2 and Colin L. Stewart1,*

1National Cancer Institute, Cancer and Developmental Biology Laboratory, Frederick, PO Box B, Building 539, Room 121A, MD 21702, USA and 2Department of Internal Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6300, USA

* To whom correspondence should be addressed. Tel: +1 3018461755; Fax: +1 3018467117; Email: stewartc{at}ncifcrf.gov

Received May 2, 2005; Revised May 24, 2005; Accepted June 16, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear lamina is an ~10 nm thick proteinaceous layer underlying the inner nuclear membrane. The A-type lamins, nuclear intermediate filament proteins encoded by the LMNA gene, are basic components of the nuclear lamina. Mutations in LMNA are associated with the laminopathies, congenital diseases affecting tissue regeneration and homeostasis. One of these laminopathies associated with missense mutations in LMNA is dilated cardiomyopathy with conduction system disease (DCM-CD1). To understand how the laminopathies arise from different mutations in a single gene, we derived a mouse line by homologous recombination expressing the Lmna-N195K variant of the A-type lamins with an asparagine-to-lysine substitution at amino acid 195, which causes DCM in humans. This mouse line shows characteristics consistent with DCM-CD1. Continuous electrocardiographic monitoring of cardiac activity demonstrated that LmnaN195K/N195K mice die at an early age due to arrhythmia. By immunofluorescence and western analysis, the transcription factor Hf1b/Sp4 and the gap junction proteins connexin 40 and connexin 43 were misexpressed and/or mislocalized in LmnaN195K/N195K hearts. Desmin staining revealed a loss of organization at sarcomeres and intercalated disks. Mutations within the LMNA gene may therefore cause cardiomyopathy by disrupting the internal organization of the cardiomyocyte and/or altering the expression of transcription factors essential to normal cardiac development, aging or function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A structural matrix of type V intermediate filaments, the nuclear lamina, is juxtaposed to the inner nuclear membrane and provides shape and structural integrity to the nucleus. The nuclear lamina is composed primarily of two types of lamins, the A-type and B-type lamins. Lamins B1 and B2 are encoded by two separate genes, and at least one B-type lamin is expressed in all somatic cells. The A-type lamins are generated by alternative splicing of the RNA encoded by the single LMNA gene, and include lamin A and lamin C and two minor isoforms, lamin A{Delta}10 and lamin C2. The major isoforms, lamin A and lamin C, are expressed in virtually all terminally differentiated cell types in the adult. Expression of some mutant isoforms of A-type lamins in vitro results in abnormal nuclear shapes and altered distribution of subnuclear compartments, separation of the outer and inner nuclear membranes and alterations in the distribution of heterochromatin (1Go–3Go). In addition, there is evidence that lamins function in chromatin organization, DNA repair, transcriptional regulation, intracellular signaling pathways, cell cycle checkpoints and development (4Go).

The A-type lamins are developmentally regulated, being expressed in differentiated cell types (5Go). Cells lacking A-type lamins include undifferentiated hematopoietic cells, early neuroendocrine cells (5Go,6Go), preimplantation mouse embryos, embryonic stem (ES) cells and undifferentiated embryonal carcinoma cells (6Go,7Go). The importance of lamins in the normal function of many postnatal tissues is underscored by the fact that mutations in the LMNA gene are associated with at least nine clinically distinct human diseases, collectively known as the laminopathies (8Go). The laminopathies are varied and affect muscle regeneration, cardiac function, white fat distribution, neuronal degeneration and aging. Just how so many different diseases arise from mutations in a single gene that is widely expressed in the nuclei of most adult tissues is a particularly intriguing problem in the study of laminopathies (4Go).

One of the laminopathies linked to mutations in the LMNA gene is dilated cardiomyopathy with conduction system disease [DCM-CD1 (9Go)], and a similar form of conduction disease is the primary cause of death in another laminopathy, autosomal dominant Emery–Dreifuss muscular dystrophy (AD-EDMD) (10Go,11Go). DCM is characterized by dilation of the heart chambers and thinning of the ventricular walls. Hypertrophy, or enlargement of cardiomyocytes, represents a putative compensatory mechanism to counteract the loss of myocytes and decreased conductive output of the heart muscle in cardiomyopathy. Hypertrophy of myocytes increases the stress load on individual cells, leading to loss of myocytes, which process further increases conductive and contractile stress on remaining cells (12Go,13Go). In addition to the restructuring of heart myocytes, interstitial fibrosis increases, contributing to the changes in cardiac structure (14Go,15Go). Many forms of hypertrophic and dilated cardiomyopathies are caused by mutations affecting sarcomeric proteins and the cytoskeleton, such as myosin heavy chain (16Go), actin (17Go), dystrophin (18Go) and desmin (19Go). LMNA mutations identify another subcellular component important for cardiac development and maintenance (9Go).

Arrhythmia defects in patients carrying an LMNA point mutation encoding a LaminA-N195K variant included sinus bradycardia, atrioventricular conduction block and atrial arrhythmias (9Go). However, patients carrying the LMNA-N195K variation did not show signs of muscular dystrophy (9Go). Over half of the known point mutations in LMNA (64%) are associated with AD-EDMD, characterized by progressive muscle wasting of the lower legs, upper arms and shoulders and contractures of the tendons of the Achilles, neck and elbows (20Go,21Go). Cardiac arrhythmias leading to sudden heart block are the most common cause of death in AD-EDMD (11Go). In contrast, only 18% of LMNA mutations are specifically associated with DCM-CD1. To undertake a study of the mechanistic relationship between mutations in a nuclear lamina protein and a terminal cardiac-specific pathology, we have created a mouse model of DCM-CD1 using homologous recombination to introduce the mutation encoding the Lmna-N195K variant.

Mutant mice homozygous for the Lmna-N195K variation have defects in multiple components of the conduction system and show signs of dilated cardiomyopathy by 9 weeks of age. The conduction defects may be mediated, in part, by abnormal expression and distribution of connexins (Cnx) in the heart, the major gap junction proteins responsible for conducting impulses through the myocardium. In addition, an Sp family transcription factor, HF1b/Sp4 (22Go,23Go), is misregulated in hearts of LmnaN195K/N195K mice, suggesting that defects in the nuclear lamina can result in altered expression patterns of heterologous genes important in tissue-specific development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Introduction of a lysine at the N195 position of Lmna results in shortened lifespan
A missense mutation encoding an N195K alteration was introduced into the murine Lmna gene by homologous recombination to create a mouse model of DCM-CD1 (Fig. 1A and B). Mutant mice carrying the missense mutation and lacking the neomycin resistance marker were identified by polymerase chain reaction (PCR) using primers specific for either the mutated or the wild-type (WT) alleles (Fig. 1C). Northern analysis indicated that introduction of the point mutation encoding the Lmna-N195K variation did not affect the expression levels or the stability of mutant Lmna transcripts (Fig. 1D). In the hearts of mutant mice, a mutant lamin C protein was detected by western analysis. However, the levels of mutant protein appeared to be lower than that of WT lamin C, suggesting that the mutant protein was less stable than WT protein (Fig. 1E). Similar decreased levels of both lamin A and C proteins were observed in western analysis of primary mouse embryonic fibroblasts (PMEFs) using an antibody recognizing both lamins A and C (Fig. 1F).



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  		Figure 1. A point mutation introduced into Lmna encodes mutant A-type lamins with an N195K variation. (A) The targeting strategy for introducing point mutations into Lmna (see Materials and Methods). (B) The mutant locus contained three nucleotide changes, resulting in a single amino acid change when compared with the WT locus. (C) Allele-specific PCR genotyping after neo-cassette excision. The mutant-specific PCR does not amplify the WT allele, and the WT-specific PCR does not amplify the mutant band. Heterozygotes show both a WT and a mutant band. (D) Northern analysis showed unaltered transcript levels of Lmna A (LaA) and Lmna C (LaC). (E) Mutant lamin C (LaC) protein was detected by western analysis in homozygous mutant animals. (F) Mutant lamins A and C were present at lower levels in homozygous mutant PMEFs.

 
Although early development of homozygous LmnaN195K/N195K mice appeared to be normal, postnatal growth and longevity of mutant mice were altered when compared with WT mice. Postnatal growth measured during the first 7 weeks of life showed that LmnaN195K/N195K mutants decreased in weight gain starting at ~4 weeks of age (Fig. 2A); however, the growth retardation observed in LmnaN195K/N195K mice was not nearly as severe as that seen in either Lmna deficient mice (24Go) or progeric mice with a Lmna mutation (25Go). Mice carrying one copy of the Lmna-N195K variation grew at rates indistinguishable from WT littermates. Despite the minor growth defect, LmnaN195K/N195K mice were otherwise healthy, with no diminished activity levels or behavioral defects through most of their lives. Often homozygous LmnaN195K/N195K mice were difficult to distinguish from littermates until just before their demise, which generally followed an acute period of deterioration during which mutant mice appeared slightly disheveled and exhibited reduced activity. LmnaN195K/N195K mice had increased mortality rates and lived until 12–14 weeks of age when compared with heterozygous and WT littermates (Fig. 2B). No homozygous mutants have lived past 16 weeks of age, and most mutants die by 12 weeks. Heterozygous LmnaN195k/+ animals have lived as long as WT littermates (up to 1.5 years). On the basis of the lifespans of 22 female and 38 male homozygous mutant mice, male homozygous mutant mice exhibited an altered survival curve in a Kaplan–Meier analysis (Fig. 2C). LmnaN195K/N195K males died significantly earlier by ~1 week than homozygous mutant females; however, there was no statistically significant difference in the overall lifespan of mutant males when compared with females.



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  		Figure 2. LmnaN195K/N195K mice showed slight growth retardation and died at an early age. (A) WT (n=8), Lmna+/N195K (n=8) and LmnaN195K/N195K (n=4) mice were weighed to determine growth rates. Statistical significance was determined using a Student's t-test. (B) Survival of 60 homozygous mutant mice was compared with 25 WT and 25 heterozygous mice in Kaplan–Meier analysis. (C) Survival of homozygous mutant males (n=38) was compared with females (n=22). Statistical significance was determined using a log rank test.

 
Tissue-specific pathology is associated with Lmna-N195K expression
Although LmnaN195K/N195K animals developed normally and appeared healthy through most of their lives, their shortened lifespan suggested that the expression of the N195K lamin variant resulted in pathogenesis. Necropsy of the mice revealed no overt pathology, but histological examination of mutant hearts revealed moderate degeneration of cardiac muscle and a very mild degeneration of smaller skeletal muscles, such as the paravertebral and pharyngeal muscles. Cardiac degeneration consisted of decreased fiber size with fragmentation, increased granular cytoplasm with loss of striation and occasional vacuolation (Fig. 3A). Most muscle groups were normal (Fig. 3B), showing none of the hallmark signs of muscular dystrophy, such as centrally localized nuclei and varied muscle fiber sizes. Homozygous mutant mice had slightly dilated heart chambers, accompanied by thinning of the chamber walls (Fig. 3C), and mutant heart weights normalized to body weight were increased when compared with the hearts of WT, age-matched littermates (Fig. 3D), consistent with a diagnosis of dilated cardiomyopathy. Electron microscopy (EM) of heart tissue from LmnaN195K/N195K mice showed a loss of sarcomere organization in cardiac myocytes, which appeared to lack continuity compared with WT cardiac muscle (Fig. 3E). In addition, nuclear defects were apparent by EM: heterochromatin was frequently hypercondensed and failed to localize to the normal peripheral position seen in WT nuclei, suggesting a loss of contact between the nuclear lamina and the heterochromatin in mutant cells. However, computer-based analysis of the sizes and shapes of mutant heart nuclei by confocal reconstructions of thick heart sections stained with YoPro-1 revealed no statistically significant differences in mutant nuclei size or shape when compared with WT nuclei (data not shown). Animals heterozygous for the Lmna-N195K mutation were indistinguishable from their WT littermates by histological and EM examination.



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  		Figure 3. Histological pathology was limited to the hearts in LmnaN195K/N195K animals. (A) H&E stained ventricles of mutant (right panel) animals compared with WT (left panel) hearts. (B) Skeletal muscle was unaffected in mutants (right panel) when compared with WT animals (left panel). (C) Ventricles and atria of 10-week-old mutant hearts (Lmna-N195K) showed mild dilation when compared with WT hearts. From top to bottom, sections were atrial, midventricular and tip of the ventricles. (D) Heart weights of eight animals of each genotype were normalized to body weights. Error bars represent standard deviation. BW, body weight. (E) WT (+/+), heterozygous (+/–) and homozygous mutant (–/–) heart tissues were examined by EM. The scale bar represents 5 µm. (F) Fractional shortening, a measure of ventricular size and function, by genotype over time. Mortality precluded later measurements of homozygous ventricular function. **P<0.001, difference from control and *P<0.05, different from prior measurement.

 
Ventricular size and function
Ventricular size and function were assessed using conscious echocardiography (26Go). LmnaN195K/N195K mice showed significantly decreased fractional shortening (FS) when compared with WT age-matched controls at 8 weeks of age (Fig. 3F, 35±1.6 versus 58±1.6%, LmnaN195K/N195K versus WT, P<0.001). FS decreased further with increasing age, with a decline from 35±1.6 to 25±1.3% in LmnaN195K/N195K mice surviving to 11 weeks (P<0.05). Heart rates did not differ significantly among groups during echocardiography, and heart rates all exceeded 550 b.p.m.; therefore, bradycardia did not contribute to this substantial difference in contractile function (26Go). Left ventricular end-diastolic dimension tended to be larger in LmnaN195K/N195K mice than that in control littermates: 0.303+0.008 versus 0.279+0.005 cm. This difference was highly significant when corrected for the smaller body weight of the homozygous mutants (P<0.001). Ventricular size, function and heart rate (HR) did not differ between controls and LmnaN195K/+ heterozygotes.

LmnaN195K/N195K mutants upregulate a fetal gene expression profile
Dilated cardiomyopathy is a disease characterized by the progressive restructuring of cardiac tissue in an attempt to compensate for decreased conduction efficiency, and is accompanied by the upregulation of a fetal gene expression profile preceding reorganization of the heart. To test whether such a fetal expression profile was activated in hearts expressing mutant A-type lamins, the expression levels of two indicators of a restructuring heart, atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), were compared in mutant versus WT animals (27Go–29Go). Semi-quantitative RT–PCR analysis showed increased levels of ANP and BNP transcripts in the hearts of LmnaN195K/N195K animals (Fig. 4). The upregulation of these genes suggested an etiology in LmnaN195K/N195K mice similar to that seen in human patients with DCM.



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  		Figure 4. LmnaN195K/N195K mice show altered expression levels of genes important in cardiac development. RT–PCR analysis showed increased levels of ANP and BNP in mutant hearts (lanes 3 and 4) when compared with WT hearts (lanes 1, and 2). HF1b/Sp4 expression was lower in whole mutant hearts than that in WT hearts. Lanes 5–8 (–RT, no reverse transcriptase) show PCR amplification of RNA only. Size markers (1 kb-Plus, Invitrogen) on the far right range from 1 kb at the top to 100 nt at the bottom.

 
LmnaN195K/N195K hearts have increased interstitial fibrosis
Interstitial fibrosis often accompanies loss of myocytes and restructuring of the cardiomyopathic heart, and increased production of extracellular matrix is observed in both human patients and other mouse heart disease models. Staining with Masson's trichrome to visualize collagen in the heart revealed increased interstitial fibrosis in the mutant hearts of 9-week-old animals (Fig. 5A and B), further supporting the model of DCM in the LmnaN195K/N195K mice. Although loss of myocardial tissue due to apoptosis, especially in the ventricles, is a frequent observation in cardiomyopathic hearts, LmnaN195K/N195K mice showed little evidence of apoptosis in the ventricles or atria of mutant hearts (data not shown).



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  		Figure 5. Increased interstitial fibrosis was observed in LmnaN195K/N195K heart tissue. Sections of ventricular tissue from a WT (A) and mutant (B) heart were stained with Masson's trichrome for collagen. Magnification is the same for the two panels. (C and D) Desmin localization in hearts of WT (C) and LmnaN195K/N195K animals (D). Desmin staining appears green and tissue was counterstained blue with DAPI to visualize nuclei.

 
Desmin localization is abnormal in LmnaN195K/N195K hearts
To determine whether cytoskeletal intermediate filament proteins might be affected by the expression of mutant A-type lamins, the localization of desmin in cardiac muscle of homozygous mutant animals was investigated. Desmin is expressed in human and chick hearts in the Z-lines of the sarcomeres and in the intercalated disks forming the cell-to-cell junctions between cardiomyctes (30Go,31Go). Z-lines were clearly stained with anti-desmin in WT animals, but the staining was less defined and was discontinuous in the hearts of LmnaN195K/N195K animals (Fig. 5C and D), suggesting defects in the organization of sarcomeres in mutant hearts. These results were consistent with the discontinuous appearance of sarcomeres observed by EM (Fig. 3E). Similarly, desmin staining at intercalated disks was observed at a lower frequency in mutant hearts when compared with WT hearts (Fig. 5C and D).

Conduction defects lead to early death in LmnaN195K/N195K mice
To determine the cause of death in LmnaN195K/N195K animals which appeared healthy throughout most of their lives, we implanted transmitters capable of continuously monitoring heart activity over time in mutant and WT mice at the age of 8 weeks. Continuous electrocardiographic (EKG) monitoring of heart rates and conductive activity showed periods of extreme bradycardia and a variety of conduction abnormalities. Excessive diurnal variation in HR with profound sinus slowing during the daytime was observed in four mutant mice (Fig. 6A), even while the mouse was awake and moving. The terminal events for three mice were recorded and were all bradyarrhythmic. Two of these mice showed progressive prolongations of the PR interval during the final 3 days of life, ultimately leading to high grade and then complete heart block (Fig. 6B–D). The third mouse showed profound and progressive sinus bradycardia as the terminal rhythm, without evidence of antecedent sustained atrial-to-ventricular block (Fig. 6E). Within the monitoring time-frame, periods of abrupt, ~2-fold, changes in atrial rate were intermittently observed in LmnaN195K/N195K mice, suggestive of sinoatrial exit block (Fig. 6F), as well as profound sinus pauses, exceeding 1000 ms, not terminated by an escape beat (Fig. 6G). Although prolonged pauses and block can be seen as normal findings in some mice (32Go), these finding were in marked contrast to >400 h of continuous EKG monitoring in the WT and heterozygote mice, where no RR intervals exceeding 400 ms were observed, and RR intervals exceeding 200 ms averaged <1 h. No significant sustained ventricular tachyarrhythmias were observed in LmnaN195K/N195K mice, but consistent and progressive increases in the PR interval were also observed in the terminal 72 h of monitoring in LmnaN195K/N195K mice (Fig. 7). These conduction abnormalities and the absence of a compensatory escape mechanism are consistent with defects in multiple components of the conductive system of mutant mice.



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  		Figure 6. Heart rate and EKG abnormalities in LmnaN195K/N195K mice. (A) Mean 3 min heart rates during the terminal 18 days of EKG monitoring showed progressive marked diurnal variation corresponding to profound sinus slowing in a LmnaN195K/N195K mouse. (B) Terminal rhythm from the mouse in (A), showing complete heart block with underlying sinus bradycardia and slow escape mechanism. (C and D) Terminal rhythms of another LmnaN195K/N195K mouse showing 2:1 and then complete heart block. (E) Terminal rhythm of a third LmnaN195K/N195K mouse, showing profound sinus/atrial slowing, without high-grade A–V block. (F) Abrupt 2:1 change in atrial rate suggestive of sino-atrial exit block, during a period of sinus rhythm, 4 days prior to death. (G) Profound sinus pauses without evidence of supraventricular escape, 5 days prior to death.

 


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  		Figure 7. Changes in atrioventricular conduction during terminal monitoring in three LmnaN195K/N195K mice, showing the relationship between PR and PP intervals. Measurements were made using electronic calipers every 3 h during the final 72 h of EKG monitoring prior to death in mutant mice. Small blue diamonds indicate values in WT mice: changes in autonomic tone roughly balance the decremental conduction properties of the AV node, resulting in the nearly flag relationship (linear). Values in three LmnaN195K/N195K mice (triangles, red diamonds and open squares) progressively diverge from this line, indicating changes in both sinus node and AV nodal/His-Purkinje function. The final measurements prior to death are indicated by the larger symbols.

 
Gap junction proteins show abnormal localization in mutant hearts
Gap junctions relay the conductive pulses of a beating heart, and mislocalization of two of the most important Cnx in the mouse heart was observed in LmnaN195K/N195K hearts, consistent with the observed conduction defects demonstrated electrophysiologically. Cnx 43 RNA levels were not altered in mutant LmnaN195K/N195K hearts by RT–PCR (data not shown); however, the protein was mislocalized in heart tissues of mutant mice in both the atria and the ventricles. Mutant myocytes showed diffuse cnx43 staining, rather than the sharply defined junctions found between cells in WT hearts (Fig. 8A and B). The gap junction protein more restricted to atria, and conductive tissue, cnx 40, was virtually undetected in the ventricles of both WT and mutant hearts as expected. However, cnx40 appeared to be downregulated in mutant atrial sections by immunofluorescence when compared with WT staining of similar sections (Fig. 8C and D). Western analysis confirmed that cnx43 protein levels were not altered in mutant hearts when compared with WT (Fig. 8E), and cnx40 protein levels were lower in mutant atria than in WT atria (Fig. 8F), consistent with the pattern of cnx40 found by immunofluorescence of heart sections. Interestingly, cnx40 protein expression was upregulated in mutant livers, but downregulated in a mutant skeletal muscle, tongue (Fig. 8F), suggesting that defects in A-type lamins can alter the levels of heterologous proteins in a tissue-specific manner.



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  		Figure 8. Gap junction proteins are mislocalized in mutant heart tissues of LmnaN195K/N195K animals. Connexin 43 (A and B) and connexin 40 (C and D) (red signals) are shown in WT (A and C) and mutant (B and D) hearts. Nuclei are counterstained blue with DAPI. All micrographs were taken at the same magnification. (E and F) Tissue lysates from WT and LmnaN195K/N195K (mutant) tongue (T), liver (L), heart atria (A) and ventricles (V) were probed with an antibody to connexin 43 (E) (Cnx43, 43 kDa) and connexin 40 (F) (Cnx40, 40 kDa).

 
A transcription factor, Hf1b, shows altered expression patterns in LmnaN195K/N195K hearts
Several cardiac proteins have been identified by transgenic or gene deletion strategies as having important functions in the heart, including serca-2 (33Go), phospholamban (34Go), Cnx (35Go,36Go), Nkx2–5 (37Go,38Go) and Hf1b/Sp4 (23Go). Serca-2 and phospholamban function in calcium homeostasis of the contracting heart, but expression levels of these genes were normal in LmnaN195K/N195K mice by conventional RT–PCR (data not shown). Nkx2–5, a homeobox transcription factor, which functions in specifying the conduction system of the heart during development, was also unaffected by the expression of the mutant A-type lamins. However, Hf1b/Sp4 was expressed at overall lower levels in whole mutant hearts when compared with WT hearts (Fig. 4). Hf1b/Sp4 is an Sp family transcription factor, which when deleted in mice results in death at ~6–8 months of age due to conduction defects in the heart (23Go).

Because Hf1b/Sp4, which is important in the development of the cardiac conduction system, was found by RT–PCR to be downregulated in LmnaN195K/N195K whole hearts, the expression and localization of this protein by western and immunofluorescence were also examined. In heart sections stained with an antibody to Hf1b/Sp4, mutant ventricles showed a few nuclei positive for the transcription factor, and WT ventricles were also sporadically positive for the protein (Fig. 9A and B). In contrast, Hf1b/Sp4 strongly stained many nuclei of LmnaN195K/N195K atria; whereas, WT atria showed modest Hf1b/Sp4 staining (Fig. 9C and D). Similar immunofluorescence results were obtained from sections of three different mutant hearts and two WT hearts. Western analysis showed somewhat variable expression of Hf1b in mutant atria and lower but detectable signal in mutant ventricles (Fig. 8F). It is not clear whether the misexpression of Hf1b/Sp4 in the atria of adult mutant animals is a cause or effect of the pathology associated with expression of the N195K variant of A-type lamins.



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  		Figure 9. Hf1b localization is altered in LmnaN195K/N195K hearts. Immunofluorescent signal for Hf1b is shown in homozygous mutant auricle (A) and WT auricle (B). Ventricular HF1b staining in mutants (E and F) was less intense than that in WT (C and D). Nuclei are counterstained with DAPI (D and F). (G) HF1b was expressed at slightly lower levels mutant ventricles (3Go,4Go) when compared with WT (1Go,2Go) by western analysis.

 
Expression of Lmna-N195K causes nuclear envelope defects
The ectopic expression of mutant variants of A-type lamins in cells results in a variety of defects at the level of the nuclear envelope, and specifically, the expression of the Lmna-N195K mutant protein results in extremely disorganized and fragile nuclei (1Go–3Go). PMEFs made from homozygous LmnaN195K/N195K mice showed similar nuclear herniations in 89% of mutant cells (Fig. 10A and B). Staining of PMEFs demonstrated that mutant protein was found at the nuclear periphery, although this mutant protein appeared to be less stable than the WT protein based on western visualization of the mutant lamins A and C (Fig. 1). In LmnaN195K/N195K PMEFs, the expression of mutant lamins caused the loss of emerin from the nuclear envelope (Fig. 10C), as well as nuclear pore clustering (Fig. 10D), similar to nuclear alterations found when the Lmna gene is knocked out (24Go). EM analysis of PMEFs also demonstrated that the loss of nuclear envelope integrity due to homozygous expression of Lmna-N195K lamins resulted in profound alterations in cytoplasmic organization. Rough endoplasmic reticulum and mitochondria appeared enlarged in homozygous mutant cells (Fig. 10E). In addition, mutant cells exhibited highly condensed heterochromatin disorganized into large aggregates, which were no longer associated with the nuclear periphery. The highly specialized pathology restricted to the heart in LmnaN195K/N195K mice suggests that many of the nuclear alterations routinely found associated with different Lmna mutations in cultured cells may not be related to or predictive of the tissue-specific pathology found in laminopathies.



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  		Figure 10. Endogenous expression of lamin-N195K results in multiple nuclear abnormalities in homozygous mutant PMEFs. (A) Immunofluorescent staining of WT (1Go) and mutant PMEFs (2Go) using an antibody to lamin A, lamin C (B), emerin (C) and nuclear pore protein Nup154 (D). (E) EM showed nuclear envelope disruptions and cytoplasmic disarray in LmnaN195/N195K PMEFs (–/–) and to a lesser degree in Lmna195K/+ (+/–) PMEFs. WT PMEFs (+/+) showed no abnormalities. The bar represents 0.5 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The introduction of a missense mutation in the Lmna gene resulted in the expression of a mutant protein, Lmna-N195K, which caused death in homozygous mutant mice due to a heart-specific pathology reminiscent of DCM-CD1. Phenotypes observed in the LmnaN195K/N195K mice consistent with DCM included dilation of heart chambers, increased heart weights, increased interstitial fibrosis, the upregulation of a fetal gene expression profile and conduction defects. However, two features observed in LmnaN195K/N195K mice, not consistent with other models of dilated cardiomyopathy, were the noticeable lack of apoptosis in the ventricles of the heart and the apparent lack of ventricular myocyte hypertrophy. The overall levels of heart dilation and fibrosis are mild when compared with other mouse models of DCM or with the pathology observed in human hearts from terminal DCM patients. Indeed, the increased normalized heart weights in mutant animals (Fig. 3D) may be explained, in part, by the slight decrease in body weights in mutants (Fig. 2A) at the end of life.

The findings in these mice are potentially consistent with abnormal development or aberrant aging of the conduction system. Although impulse propagation initially appears normal, the conduction system may form with a decreased ‘safety margin’ or may develop this deficit with increasing age. The same age-dependent phenotypic progression is observed in the human disorder. Because the QRS duration corresponding only to depolarization in mouse is not readily defined or measured, and there may be temporal overlap of depolarization and repolarization (32Go), the electrocardiogram itself cannot accurately indicate whether dyssynchronous activation of the ventricle in the mutant hearts might result in reduced efficiency of the contractile apparatus. To offset this reduced cardiac output, the mutant hearts may initiate the steps of growth and reorganization observed in dilated cardiomyopathy. Long-term telemetric recordings in this study defined a rapidly progressive change in cardiac conduction within individual mice with clear delineation of the terminal events. The small size, early mortality and rapid progression of conduction abnormalities and debilitated condition of the homozygous mice are currently limiting for in vivo electrophysiologic study. However, as techniques for murine electrophysiologic study and direct mapping are refined, more detailed information can be obtained about the sites and progression of conduction abnormalities (39Go,40Go). If the primary defect in LmnaN195K/N195K mice is indeed a failure to develop the conduction system properly, it is also possible that compensatory restructuring of the heart might introduce further stresses, surpassing a threshold for survival.

Two other DCM models associated with defects in Lmna have been reported. Mice lacking the Lmna gene show signs of DCM (41Go); however, the LmnaN195K/N195K mice live twice as long as Lmna–/– mice and do not show the multiple tissue pathologies of the Lmna null mice. The heart defects of the Lmna deficient mice consistent with LmnaN195K/N195K mice include conduction defects, lack of hypertrophy, dilation of heart chambers and nuclear shape defects. Increased levels of apoptosis were reported in the ventricles of Lmna–/– mice (41Go), but no evidence of such ventricular cell death was found in the present model. However, the levels of apoptosis previously reported were low and only 2-fold higher in knockout animals than that in WT animals. Bonne and colleagues have created a different cardiomyopathic laminopathy by introducing an H222P variation into Lmna: these mice exhibit conduction defects, chamber dilation, increased fibrosis and lack of hypertrophy (42Go). However, the Lmna-H222P model also shows evidence of muscular dystrophy and death at 4–9 months (compared with 3 months for the LmnaN195/N195K mice). Partial emerin mislocalization to the cytoplasm and lower levels of mutant Lmna-N195K protein are the two further distinguishing features seen in the LmnaN195K/N195K mice when compared with LmnaH222P/H222P mice.

To date, no instance of autosomal dominance has been demonstrated with Lmna point mutations in mouse models. In humans, a single copy of the Lmna-N195K allele is sufficient to cause DCM (9Go). However, only homozygous mutant mice that displayed the heart pathology are described here; heterozygous mice show no overt signs of pathology. In contrast, although rare, autosomal recessive mutations in the human LMNA gene have been reported to cause EDMD (43Go) and familial partial lipodystrophy (44Go), suggesting autosomal dominance is not the only genetic mechanism possible in the etiology of laminopathies. In mice, autosomal dominance has not been recapitulated in many mouse models of human diseases, including the Lmna knockout mouse and a progeric mouse with a Lmna mutation (24Go,25Go) The extreme variability in both phenotype and penetrance in human patients with laminopathies suggests that the genetic basis may be complex.

Because the LmnaN195K/N195K mice live longer and are healthier than Lmna–/– mice (24Go), the mutant N195K protein is suggested to at least partly replicate the function of the WT protein. The nuclear distortion observed in PMEFs from LmnaN195K/N195K mice was more severe than the degree of herniation observed in either Lmna–/– mice (Sullivan, unpublished data) or progeric mice (25Go). Both the Lmna–/– mice and the progeric mice show pleiotropic effects in multiple tissues, and both model systems have shorter lifespans than LmnaN195K/N195K mice. In addition, the overall good health of LmnaN195K/N195K mice through the first 10 weeks of life is contrary to the extreme weakness and decreased growth exhibited by the previous two models. These comparisons suggest that the degree of architectural nuclear disruption observed in cell culture is not a reliable indicator of the severity, specificity or pleiotropy of pathology, which will be observed in adult tissues.

The lack of tissue pleiotropy in phenotypes of LmnaN195K/N195K animals also argues that fewer functional pathways are affected in the point mutant mice than in Lmna–/– mice. Such tissue specificity in phenotypes depending on the point mutation introduced is consistent with a proposed role of A-type lamins in determining cell fate and/or maintenance of the differentiated state. It is especially intriguing that another protein proposed to act as an early determinant of cell fates in the heart, Hf1b/Sp4 (23Go), is also altered in the LmnaN195K/N195K mutant hearts. The present characterization of LmnaN195K/N195K mice suggests a mechanistic role for the nuclear lamina in determining the cell fates of conductive cells and/or cardiomyocytes in the developing heart. One possible means of achieving this end is for A-type lamins to regulate the differentiation of cardiac conduction cells. Alternatively, loss of certain aspects of lamin function could allow more developmental plasticity in the myocytes of mutant hearts, leading to the repopulation of the auricles with more conductive cells, especially in response to arrhythmia defects.

Genetic manipulation of Hf1b/Sp4 and Cnx43 results in mouse models characterized by dilated cardiomyopathy and/or sudden death. Hf1b/Sp4 and cardiac restricted Cnx43 knockouts display few symptoms of cardiomyopathy, and the cause of death in these mice is described as sudden death due to arrhythmias. Mice ablated for Hf1b/Sp4 live 6–8 months before dying suddenly of conduction arrhythmias (23Go), whereas mice with a cardiac restricted knockout of Cnx43 die by 2 months of age (36Go). The moderately impaired ventricular function and the profound conduction abnormalities of LmnaN195K/N195K mice are consistent with the histological and biochemical changes observed in the mutant mice. The conduction changes are also suggestive of the findings in the related human disorder, where initial manifestations of conduction abnormality may progress rapidly and unpredictably to lethal bradyarrhythmias. The conduction abnormalities in LmnaN195K/N195K mice suggest diffuse involvement of impulse generation and propagation, as sinus rate, AV conduction and escape mechanisms are all abnormal. Some, but not all, of these changes could also be due to differences in cardiac innervation, neurohormonal milieu or the cardiac response to these factors. Finally, in contrast to mice conditionally lacking Cnx43 (36Go), significant ventricular arrhythmias were not observed, again suggesting a more generalized problem in impulse generation and propagation in LmnaN195K/N195K animals.

The diffuse Cnx43 and Cnx40 staining at the borders of cardiomyocytes in LmnaN195K/N195K mutants (Fig. 8) is consistent with the cardiac conduction defects observed in our animals. Such a pattern of staining indicates that many of the gap junctions formed by Cnx43 and 40 may be non-functional, which might compromise the conduction of impulses in the hearts of mutant animals. Because few functional gap junctions would be expected to fulfill conductive requirements between myocytes of the heart, it is unclear how much of the mislocalization of Cnx can explain the electrophysiological abnormalities. However, a similar diffuse staining pattern for Cnx40 protein has been described in the Hf1b/Sp4 knockout mouse (23Go). However, the Hf1b/Sp4 deficient animals live 2–4 months longer than LmnaN195K/N195K mutants on average, suggesting that the defects in Lmna functions are more severe or more varied than the effects of an Hf1b/Sp4 knockout, and that the expression of defective A-type lamins may adversely affect more components of heart development than Hf1b/Sp4. For example, the altered expression of ANP and BNP (Fig. 4) could also contribute to the electrophysiological changes of the heart.

Abnormal patterns of Hf1b/Sp4, Cnx40 and 43 proteins in the hearts of LmnaN195K/N195K animals (Figs 8 and 9) are consistent with a model, in which the expression of the lamin-N195K protein has a downstream effect on other gene products. The altered localization of Hf1b/Sp4 in the various compartments of LmnaN195K/N195K hearts, in addition to the overall transcriptional downregulation of Hf1b/Sp4 (Fig. 4), is especially intriguing in this respect. The altered expression of Hf1b/Sp4 mRNA suggests that A-type lamin malfunction can affect gene regulation in a tissue-specific manner. Hf1b/Sp4 is normally expressed in ventricular myocytes and cells of the developing conduction system during embryogenesis (23Go). In WT adult hearts, Hf1b/Sp4 protein was found in the ventricles but not in the atria (Fig. 9), whereas in the LmnaN195K/N195K animals, Hf1b/Sp4 protein was not found in the ventricles and, by immunofluorescence, was strongly expressed in the atria. These observations would be consistent with a model in which the Hf1b/Sp4 gene product might repress Cnx40 expression in the atria of mutant animals.

One explanation that could account for the abnormal expression of Hf1b/Sp4 in the atria and ventricles of mutant hearts fits well with the postulated role of the lamins in gene regulation (21Go), often proposed to account for the tissue-specific nature of laminopathies. Mutant A-type lamins and subsequent defects in the nuclear lamina might alter chromatin organization and subsequent expression of Hf1b/Sp4 at critical points in heart development, which could result in a deficiency of conduction-specific cells in the developing heart. Because the A-type lamins are first expressed at the time of early heart development at approximately embryonic day 9 (Sullivan and Stewart, unpublished data), this possibility is particularly interesting. Downstream effects on genes controlled by the Hf1b/Sp4 transcription factor could account for further anomalous gene expression in the developing heart. It will be interesting to test whether Hf1b/Sp4, or its targets, is misexpressed or localized in developing embryonic hearts of LmnaN195K/N195K animals.

The diffuse staining pattern of the gap junction protein Cnx43 in LmnaN195K/N195K mutant hearts further corroborates an increasing interest in models, suggesting that lamins function as intracellular sensors relaying mechanosignaling information between the cytoskeleton and the nucleus (45Go). It is intriguing that the expression of mutant A-type lamins juxtaposed to the inner nuclear membrane can result in the abnormal configuration of protein complexes residing at the plasma membrane, without altering the overall expression level of that protein. Such an effect is evidence that not only the nuclear lamina is in communication with cytoplasmic components, but also it can influence the performance of proteins functioning at the plasma membrane. Such an influence implies the existence of a communication link among the nuclear lamina, the cytoskeletal network and the cell membrane. One such link may be mediated by the Sun domain proteins connecting the lamina with the Nesprins/synes at the nuclear envelope; the Nesprins in turn connect to the actin cytoskeleton (46Go,47Go). The response of cardiomyocytes to abnormal conduction may be exacerbated by an overall decreased ability of LmnaN195K/N195K cells to institute an adequate stress response (45Go) to the injuries caused by continuous contraction cycles. The timing of the onset of conduction defects may be influenced by a superimposition of age-related changes in cell–cell coupling in the mutant hearts with an inability to maintain an appropriate safety margin for conduction due to the alterations in Cnx function (48Go). Taken together, these results suggest that the DCM aspects of the heart pathology displayed in LmnaN195K/N195K mice are a secondary effect of an intrinsic failure of the heart either to develop properly, maintain function postnatally, or to respond to the load of rigorous contractile activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of recombinant mice
All mice were handled in accordance with ACUC guidelines, under the protocol number 02-032. A genomic Lmna fragment containing part of intron 2 and exons 3 through 5 was altered using the QuickChange XL Mutagenesis Kit (Stratagene, La Jolla, CA, USA). Three nucleotides were changed: one introduced the N195K variation in the Lmna gene and the other two nucleotide changes created a HaeII restriction site by altering the nucleotides, which did not further change the amino acid sequence (Fig. 1B). A PGK–neomycin resistance selectable marker flanked by loxP sites was introduced into intron 2 at a unique StuI restriction site. The linearized targeting construct was electroporated into W9.5 ES cells (129S1 genetic background), selected with neomycin, and two recombinant ES clones were injected into C57Bl day 4 blastocysts, as previously described (49Go). Recombinant ES clones were identified by Southern analysis of EcoRI-digested genomic DNA probed with a 1.3 kb genomic fragment containing Lmna exons 9 and 10 by standard methods (Sambrook et al., 1989). Chimeric mice established two independent recombinant mouse lines harboring the Lmna N195K variation and neomycin cassette. F1 pups derived from chimeras were genotyped by Southern analysis, which identified mutants carrying a neomycin resistance cassette (Fig. 1A and C). The neomycin marker was removed using Cre–loxP recombination by crossing Neo+ mice to a constitutive, general Cre-deletor line, R26Cre with a C57Bl genetic background (50Go), leaving only the point mutations (Fig. 1B) and a remnant loxP site at the StuI site. All phenotypic characterization was performed on Neo-deleted lines. No differences in phenotype were found between the two recombinant lines of mice. After the neomycin marker had been deleted, mutant mice were genotyped using PCR with an anchor primer in combination with either WT or mutant-specific primers as follows: WT-specific, 5'-GCGAGTGGATGCTGAGAACAGGG-3'; mutant-specific, 5'-GCGTAGTGGATGCTGAGAAGCGC-3'; anchor primer, 5'-CTCACGCAGTTCCTGAAAGG-3'. Thirty-five PCR cycles of (95°Cx 30 s; 56°Cx 30 s; 72°Cx 45 s) resulted in a 261 nucletoide band.

Pathological characterization of LmnaN195K/N195K mice
Litters from heterozygous intercrosses were weighed three to four times a week from day 2 through 7 weeks of age. Survival was plotted for 60 homozygous mutant and 25 heterozygous and WT mice. LmnaN195K/N195K mice and age-matched littermates were submitted for necropsy at 9 weeks of age. Tissues were dehydrated, cleared, embedded in paraffin, sectioned at 5 µm and stained with hematoxylin and eosin (H&E). Organs harvested included thymus, thyroid, parathyroid, trachea, esophagus, heart, liver, gall bladder, spleen, testis, epididymis, seminal vesicle, prostate, urinary bladder, vertebrae, spinal cord, interscapular brown fat, gonadal fat, shoulder, hip tibia and radius. To view dilation of heart chambers, hearts were sectioned transversely into three parts. Sections of heart and skeletal muscle were stained with Masson's trichrome for extracellular matrix. The hearts of eight mice of each genotype (WT, heterozygous and homozygous mutant) were weighed and normalized for corresponding body weights. The standard deviations are shown in error bars, and statistical significance was derived using a two-sided Student's t-test.

Echocardiography
Transthoracic echocardiography in conscious mice was performed, as described (26Go), in nine to 11 mice of each genotype. Briefly, images were acquired using a Sonos 5500 (Agilent, Andover, MA, USA) with a 15 MHz high frequency linear transducer. Prior to initiation of the study, the mice were trained on two separate occasions over 1–2 days. Two-dimensional targeted M-mode echocardiographic images were obtained at the level of the papillary muscles from the parasternal short-axis view. Measurements were made on screen using the digitally recorded M-mode tracings using the leading-edge technique. The FS (%), a measure of left ventricle (LV) systolic performance, was calculated from M-mode-derived LV. HR was determined from the cardiac cycles recorded on the M-mode tracing, using at least three consecutive beats.

Continuous electrocardiography
EKG transmitters (Data Sciences) were implanted surgically in four mice of each genotype. ECG signals were recorded for 14–18 days or until death and analyzed using custom software (J.N.R.) designed for the analysis of murine ECGs, on the basis of the Physionet suite (51Go). All QRS complexes were annotated and reviewed, with direct review of all unusually long or short RR intervals or abrupt changes in heart period.

Expression analysis
Total RNA was isolated using either Trizol (Invitrogen, Carlsbad, CA, USA) or RNEasy Fibrous Tissue MiniKit (Qiagen, Valencia, CA, USA), according to the manufacturer's instructions. Polyadenylated RNA from PMEFs was purified and northern analysis was performed, according to standard procedures (54Go). Two micrograms of polyA+RNA was run on a 1x MOPS/formaldehyde gel, and the blot was hybridized with a human LMNA cDNA fragment corresponding to the rod domain of A-type lamins. The blot was stripped and reprobed with a human {alpha}-actin fragment (Clontech, Palo Alto, CA, USA) to control for loading of wells.

Western analyses were performed using standard 10% SDS–PAGE electrophoresis and blotting methods (54Go). Gels were blotted onto PVDF (BioRad, Hercules, CA, USA) and blocked in TTBS/3% milk. A rabbit anti-laminC antibody was used at 1:4000 (25Go), and in all cases, appropriate secondary anti-host-horseradish peroxidase (HRP) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at 1:2000. Blots were developed employing enhanced chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). Additional primary antibodies were as follows: {alpha}-connexin40, {alpha}-connexin43 from Zymed (South San Francisco, CA, USA) and {alpha}-HF1b/SP4, {alpha}-actin and {alpha}-desmin from Santa Cruz.

RT–PCR was performed on total tissue RNA isolated from heart and skeletal muscle (quadriceps) of WT and mutant littermates. First strand synthesis on 0.5 µg total RNA was carried out according to the manufacturer's protocol (First Strand cDNA Synthesis Kit, Roche, Indianapolis, IN, USA). Eighteen to 25 cycles of PCR were performed using appropriate annealing temperatures for the following primers: Cnx40, 5'-TGGTTTCAACTTCGACCTCA-3' and 5'-AACCAGGCTGAATGGTATCG-3'; Cnx43, 5'-GGACTGCTTCCTCTCACGTC-3' and 5'-TTTGGAGATCCGCAGTCTTT-3'; HF1b/SP4, 5'-GGGCAGACAATTCAGACCAT-3' and 5'-CACAGGAGCAATCTGAGCAA-3'; ANP, 5'-GAGAAGATGCCGGTAGAAGA-3' and 5'-AAGCACTGCCGTCTCTCAGA-3'; BNP, 5'-CTGCTGGAGCTGATAAGAGA-3' and 5'-TGCCCAAAGCAGCTTGAGAT-3'; GAPDH, 5'-TGCTGAGTATGTCGTGGAGTCTA-3' and 5'-AGTGGGAGTTGCTGTTGAAGTCG-3'.

Immunofluorescence
Hearts were harvested into OCT compound (Tissue-Tek, Torrance, CA, USA) and placed into isopentane chilled on dry ice until soupy. Eight micron cryosections were fixed 1 min in chilled acetone, air-dried and stained with primary antibodies as described (52Go). Primary antibodies were used at 1:200 dilutions and secondary antibody was used at 1:200 (Molecular Probes, A11036 [GenBank] or A11078 [GenBank] , Eugene, OR, USA). Sections were counterstained with 300 nM DAPI in phosphate-buffered saline (PBS). Slides were mounted in Mowiol (Calbiochem, San Diego, CA, USA) and viewed under ultraviolet in a Zeiss Axiophot.

Electron microscopy
Tissues and PMEFs were harvested and washed with PBS and then fixed overnight at 4°C in glutaraldehyde and osmium tetroxide. Processing of samples was essentially as previously described (53Go). Epon sections (50 nm) were stained with uranyl acetate and lead acetate and viewed at 75 kV.


   ACKNOWLEDGEMENTS
 
We thank L. Sewell for technical assistance in our animal facility; C.D. Smith and D.V.M. for help in pathological examination of mice; K. Nagashima for assistance with electron microscopy; R. Frederickson of the NCI-Frederick Publications Department for assistance in preparation of figures and R. Lee and B. Burke for helpful discussions and critical reading of this manuscript.

Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Raharjo, W.H., Enarson, P., Sullivan, T., Stewart, C.L. and Burke, B. (2001) Nuclear envelope defects associated with LMNA mutations cause dilated cardiomyopathy and Emery–Dreifuss muscular dystrophy. J. Cell Sci., 114, 4447–4457.

  2. Ostlund, C., Bonne, G., Schwartz, K. and Worman, H.J. (2001) Properties of lamin A mutants found in Emery–Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy. J. Cell Sci., 114, 4435–4445.

  3. Holt, I., Ostlund, C., Stewart, C.L., Man, N., Worman, H.J. and Morris, G.E. (2003) Effect of pathogenic mis-sense mutations in lamin A on its interaction with emerin in vivo. J. Cell Sci., 116, 3027–3035.[Abstract/Free Full Text]

  4. Mounkes, L. and Stewart, C.L. (2004) Structural organization and functions of the nucleus in development, aging, and disease. Curr. Top. Dev. Biol., 61, 191–228.[Web of Science][Medline]

  5. Broers, J.L., Machiels, B.M., Kuijpers, H.J., Smedts, F., van den Kieboom, R., Raymond, Y. and Ramaekers, F.C. (1997) A- and B-type lamins are differentially expressed in normal human tissues. Histochem. Cell Biol., 107, 505–517.[CrossRef][Web of Science][Medline]

  6. Rober, R.A., Sauter, H., Weber, K. and 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, 587–598.[Abstract/Free Full Text]

  7. Stewart, C. and Burke, B. (1987) Teratocarcinoma stem cells and early mouse embryos contain only a single major lamin polypeptide closely resembling lamin B. Cell, 51, 383–392.[CrossRef][Web of Science][Medline]

  8. Mounkes, L., Kozlov, S., Burke, B. and Stewart, C.L. (2003) The laminopathies: nuclear structure meets disease. Curr. Opin. Genet. Dev., 13, 223–230.[CrossRef][Web of Science][Medline]

  9. Fatkin, D., MacRae, C., Sasaki, T., Wolff, M.R., Porcu, M., Frenneaux, M., Atherton, J., Vidaillet, H.J., Jr, Spudich, S., De Girolami, U. 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.[Abstract/Free Full Text]

  10. Bonne, G., Di Barletta, M.R., Varnous, S., Becane, H.M., Hammouda, E.H., Merlini, L., Muntoni, F., Greenberg, C.R., Gary, F., Urtizberea, J.A. et al. (1999) Mutations in the gene encoding lamin A/C cause autosomal dominant Emery–Dreifuss muscular dystrophy. Nat. Genet., 21, 285–288.[CrossRef][Web of Science][Medline]

  11. Morris, G.E. (2001) The role of the nuclear envelope in Emery–Dreifuss muscular dystrophy. Trends Mol. Med., 7, 572–577.[CrossRef][Web of Science][Medline]

  12. Olson, E.N. (2004) A decade of discoveries in cardiac biology. Nat. Med., 10, 467–474.[CrossRef][Web of Science][Medline]

  13. MacLellan, W.R. and Schneider, M.D. (1997) Death by design. Programmed cell death in cardiovascular biology and disease. Circ. Res., 81, 137–144.[Abstract/Free Full Text]

  14. Francis, G.S. (1998) Changing the remodeling process in heart failure: basic mechanisms and laboratory results. Curr. Opin. Cardiol., 13, 156–161.[Web of Science][Medline]

  15. Weber, K.T. (2000) Fibrosis and hypertensive heart disease. Curr. Opin. Cardiol., 15, 264–272.[CrossRef][Web of Science][Medline]

  16. Seidman, C.E. and Seidman, J.G. (1991) Mutations in cardiac myosin heavy chain genes cause familial hypertrophic cardiomyopathy. Mol. Biol. Med., 8, 159–166.[Web of Science][Medline]

  17. Olson, T.M., Michels, V.V., Thibodeau, S.N., Tai, Y.S. and Keating, M.T. (1998) Actin mutations in dilated cardiomyopathy, a heritable form of heart failure. Science, 280, 750–752.[Abstract/Free Full Text]

  18. Muntoni, F., Di Lenarda, A., Porcu, M., Sinagra, G., Mateddu, A., Marrosu, G., Ferlini, A., Cau, M., Milasin, J., Melis, M.A. et al. (1997) Dystrophin gene abnormalities in two patients with idiopathic dilated cardiomyopathy. Heart, 78, 608–612.[Abstract/Free Full Text]

  19. Li, D., Tapscoft, T., Gonzalez, O., Burch, P.E., Quinones, M.A., Zoghbi, W.A., Hill, R., Bachinski, L.L., Mann, D.L. and Roberts, R. (1999) Desmin mutation responsible for idiopathic dilated cardiomyopathy. Circulation, 100, 461–464.[Abstract/Free Full Text]

  20. Emery, A.E. (1989) Emery–Dreifuss syndrome. J. Med. Genet., 26, 637–641.[Abstract/Free Full Text]

  21. Goldman, R.D., Gruenbaum, Y., Moir, R.D., Shumaker, D.K. and Spann, T.P. (2002) Nuclear lamins: building blocks of nuclear architecture. Genes Dev., 16, 533–547.[Free Full Text]

  22. Supp, D.M., Witte, D.P., Branford, W.W., Smith, E.P. and Potter, S.S. (1996) Sp4, a member of the Sp1-family of zinc finger transcription factors, is required for normal murine growth, viability, and male fertility. Dev. Biol., 176, 284–299.[CrossRef][Web of Science][Medline]

  23. Nguyen-Tran, V.T., Kubalak, S.W., Minamisawa, S., Fiset, C., Wollert, K.C., Brown, A.B., Ruiz-Lozano, P., Barrere-Lemaire, S., Kondo, R., Norman, L.W. et al. (2000) A novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages. Cell, 102, 671–682.[CrossRef][Web of Science][Medline]

  24. Sullivan, T., Escalante-Alcalde, D., Bhatt, H., Anver, M., Bhat, N., Nagashima, K., Stewart, C.L. and Burke, B. (1999) Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy. J. Cell Biol., 147, 913–920.[Abstract/Free Full Text]

  25. Mounkes, L.C., Kozlov, S., Hernandez, L., Sullivan, T. and Stewart, C.L. (2003) A progeroid syndrome in mice is caused by defects in A-type lamins. Nature, 423, 298–301.[CrossRef][Medline]

  26. Rottman, J.N., Ni, G., Khoo, M., Wang, Z., Zhang, W., Anderson, M.E. and Madu, E.C. (2003) Temporal changes in ventricular function assessed echocardiographically in conscious and anesthetized mice. J. Am. Soc. Echocardiogr., 16, 1150–1157.[CrossRef][Web of Science][Medline]

  27. Altemose, G.T., Gritsus, V., Jeevanandam, V., Goldman, B. and Margulies, K.B. (1997) Altered myocardial phenotype after mechanical support in human beings with advanced cardiomyopathy. J. Heart Lung Transplant., 16, 765–773.[Web of Science][Medline]

  28. Cameron, V.A. and Ellmers, L.J. (2003) Minireview: natriuretic peptides during development of the fetal heart and circulation. Endocrinology, 144, 2191–2194.[Abstract/Free Full Text]

  29. Mizuno, Y., Yoshimura, M., Harada, E., Nakayama, M., Sakamoto, T., Shimasaki, Y., Ogawa, H., Kugiyama, K., Saito, Y., Nakao, K. et al. (2000) Plasma levels of A- and B-type natriuretic peptides in patients with hypertrophic cardiomyopathy or idiopathic dilated cardiomyopathy. Am. J. Cardiol., 86, 1036–1040, A11.

  30. Wang, S.M., Huang, Y.S., Wu, J.C. and Tseng, Y.Z. (2000) Role of desmin filaments in chicken cardiac myofibrillogenesis. J. Cell Biochem., 77, 635–644.[CrossRef][Web of Science][Medline]

  31. Thornell, L.E., Johansson, B., Eriksson, A., Lehto, V.P. and Virtanen, I. (1984) Intermediate filament and associated proteins in the human heart: an immunofluorescence study of normal and pathological hearts. Eur. Heart J., 5 (Suppl. F), 231–241.

  32. London, B. (2001) Cardiac arrhythmias: from (transgenic) mice to men. J. Cardiovasc. Electrophysiol., 12, 1089–1091.[CrossRef][Web of Science][Medline]

  33. Schultz Jel, J., Glascock, B.J., Witt, S.A., Nieman, M.L., Nattamai, K.J., Liu, L.H., Lorenz, J.N., Shull, G.E., Kimball, T.R. and Periasamy, M. (2004) Accelerated onset of heart failure in mice during pressure overload with chronically decreased SERCA2 calcium pump activity. Am. J. Physiol. Heart Circ. Physiol., 286, H1146–H1153.[Abstract/Free Full Text]

  34. Schmitt, J.P., Kamisago, M., Asahi, M., Li, G.H., Ahmad, F., Mende, U., Kranias, E.G., MacLennan, D.H., Seidman, J.G. and Seidman, C.E. (2003) Dilated cardiomyopathy and heart failure caused by a mutation in phospholamban. Science, 299, 1410–1413.[Abstract/Free Full Text]

  35. Kirchhoff, S., Kim, J.S., Hagendorff, A., Thonnissen, E., Kruger, O., Lamers, W.H. and Willecke, K. (2000) Abnormal cardiac conduction and morphogenesis in connexin40 and connexin43 double-deficient mice. Circ. Res., 87, 399–405.[Abstract/Free Full Text]

  36. Gutstein, D.E., Morley, G.E., Tamaddon, H., Vaidya, D., Schneider, M.D., Chen, J., Chien, K.R., Stuhlmann, H. and Fishman, G.I. (2001) Conduction slowing and sudden arrhythmic death in mice with cardiac-restricted inactivation of connexin43. Circ. Res., 88, 333–339.[Abstract/Free Full Text]

  37. Lyons, I., Parsons, L.M., Hartley, L., Li, R., Andrews, J.E., Robb, L. and Harvey, R.P. (1995) Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeo box gene Nkx2–5. Genes Dev., 9, 1654–1666.[Abstract/Free Full Text]

  38. Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N. and Izumo, S. (1999) The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development, 126, 1269–1280.[Abstract]

  39. Gutstein, D.E., Danik, S.B., Sereysky, J.B., Morley, G.E. and Fishman, G.I. (2003) Subdiaphragmatic murine electrophysiological studies: sequential determination of ventricular refractoriness and arrhythmia induction. Am. J. Physiol. Heart Circ. Physiol., 285, H1091–H1096.[Abstract/Free Full Text]

  40. Maguire, C.T., Wakimoto, H., Patel, V.V., Hammer, P.E., Gauvreau, K. and Berul, C.I. (2003) Implications of ventricular arrhythmia vulnerability during murine electrophysiology studies. Physiol. Genomics, 15, 84–91.[Abstract/Free Full Text]

  41. Nikolova, V., Leimena, C., McMahon, A.C., Tan, J.C., Chandar, S., Jogia, D., Kesteven, S.H., Michalicek, J., Otway, R., Verheyen, F. et al. (2004) Defects in nuclear structure and function promote dilated cardiomyopathy in lamin A/C-deficient mice. J. Clin. Invest., 113, 357–369.[CrossRef][Web of Science][Medline]

  42. Arimura, T., Helbling-Leclerc, A., Massart, C., Varnous, S., Niel, F., Lacene, E., Fromes, Y., Toussaint, M., Mura, A.M., Keller, D.I. 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.[Abstract/Free Full Text]

  43. Brown, C.A., Lanning, R.W., McKinney, K.Q., Salvino, A.R., Cherniske, E., Crowe, C.A., Darras, B.T., Gominak, S., Greenberg, C.R., Grosmann, C. et al. (2001) Novel and recurrent mutations in lamin A/C in patients with Emery–Dreifuss muscular dystrophy. Am. J. Med. Genet., 102, 359–367.[CrossRef][Web of Science][Medline]

  44. Hegele, R.A., Cao, H., Anderson, C.M. and Hramiak, I.M. (2000) Heterogeneity of nuclear lamin A mutations in Dunnigan-type familial partial lipodystrophy. J. Clin. Endocrinol. Metab., 85, 3431–3435.[Abstract/Free Full Text]

  45. Lammerding, J., Schulze, P.C., Takahashi, T., Kozlov, S., Sullivan, T., Kamm, R.D., Stewart, C.L. and Lee, R.T. (2004) Lamin A/C deficiency causes defective nuclear mechanics and mechanotransduction. J. Clin. Invest., 113, 370–378.[CrossRef][Web of Science][Medline]

  46. Burke, B. and Stewart, C.L. (2002) Life at the edge: the nuclear envelope and human disease. Nat. Rev. Mol. Cell Biol., 3, 575–585.[CrossRef][Web of Science][Medline]

  47. Starr, D.A. and Han, M. (2003) ANChors away: an actin based mechanism of nuclear positioning. J. Cell Sci., 116, 211–216.[Abstract/Free Full Text]

  48. Spach, M.S., Heidlage, J.F., Dolber, P.C. and Barr, R.C. (2000) Electrophysiological effects of remodeling cardiac gap junctions and cell size: experimental and model studies of normal cardiac growth. Circ. Res., 86, 302–311.[Abstract/Free Full Text]

  49. Abbondanzo, S.J., Gadi, I. and Stewart, C.L. (1993) Derivation of embryonic stem cell lines. Methods Enzymol., 225, 803–823.[Web of Science][Medline]

  50. Soriano, P. (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat. Genet., 21, 70–71.[CrossRef][Web of Science][Medline]

  51. Goldberger, A.L., Amaral, L.A., Glass, L., Hausdorff, J.M., Ivanov, P.C., Mark, R.G., Mietus, J.E., Moody, G.B., Peng, C.K. and Stanley, H.E. (2000) PhysioBank, PhysioToolkit, and PhysioNet: components of a new research resource for complex physiologic signals. Circulation, 101, E215–E220.

  52. Pashmforoush, M., Lu, J.T., Chen, H., Amand, T.S., Kondo, R., Pradervand, S., Evans, S.M., Clark, B., Feramisco, J.R., Giles, W. et al. (2004) Nkx2–5 pathways and congenital heart disease; loss of ventricular myocyte lineage specification leads to progressive cardiomyopathy and complete heart block. Cell, 117, 3733–3786.

  53. Gonda, M.A., Aaronson, S.A., Ellmore, N., Zeve, V.H. and Nagashima, K. (1976) Ultrastructural studies of surface features of human normal and tumor cells in tissue culture by scanning and transmission electron microscopy. J. Natl Cancer Inst., 56, 245–263.[Web of Science][Medline]

  54. Sambrook, J. and Russell, D. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. CSHL Press, Cold Spring Harbor, New York.


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