Dilated cardiomyopathy (DCM), characterized by left ventricular (LV) dilation and dysfunction, is believed to be of genetic origin in approximately 35% or more of all cases (1). Endocardial fibroelastosis (EFE), associated with diffuse LV endocardial thickening due to proliferation of fibrous and elastic tissue, is a rare cardiac disorder with poor prognosis that occurs in infants and young children (2). Although virus-induced fetal myocarditis or X-linked Barth or autosomal recessive inherited genes have been reported to be causative for EFE, most cases are of unknown etiology (3). Various molecular mechanisms are involved in the development of these disorders because of the overlapping functions of cytoskeletal proteins, beginning from their involvement in sarcomeric structure, to their functions in cardiac differentiation, myofibrillogenesis, and transcriptional regulation (4- 5).

The nebulin family is a group of actin-binding proteins whose universal feature is the presence of “nebulin-repeats” that bind a single actin subunit and is involved in tropomyosin-troponin assembly (6). Moreover, expression of specific nebulin isoforms with specific numbers of “nebulin repeats” correlates thin filament length in a striated muscle-specific manner (7). The only cardiac-specific member, nebulette (NEBL), a 107 kDa protein appears to be involved in early myogenesis and myofibrillar organization in cardiomyocytes (8- 9). The nebulin-repeat domain of nebulette tethers it to the I-band, while the C-terminus is embedded inside the Z-lines, interacting with α-actinin2 (9), the titin-PEVK (7), myopalladin (10), desmin (11), and filamin C (12). Additionally, nebulette significantly increases the affinity of tropomyosin-troponin for F-actin (13). Involvement in these key protein-protein interactions suggests the functional importance of nebulette in the heart.

Nebulette polymorphism (Asn654Lys) in the actin-binding motif has been previously associated with nonfamilial DCM (14). To determine whether NEBL plays roles in the genetic and cellular basis of cardiomyopathies, we screened NEBL in patients with DCM. Transgenic (Tg) mice with cardiac-specific overexpression of NEBL were created, and functional analysis was pursued to explore the molecular mechanisms responsible for the development and regulation of the clinical phenotypes seen in humans. We investigated the effect of the mutations under mechanical stretch by applying cyclic mechanical strain in transient nebulette-expressing H9C2 cardiomyoblasts.

We assessed genomic deoxyribonucleic acid (DNA) from 260 patients with DCM recruited after informed consent using direct sequencing methods (ABI 3730). Cytoskeletal and sarcomeric genes were investigated for mutations as previously described (15). For each variant identified, available family members and 300 unrelated ethnic-matched persons were screened.

A paraffin-embedded sample of the explanted heart from patient S84-5162 carrying the Q128R NEBL variant was available for analysis after informed consent. As a control, heart tissue was obtained from a normal donor subject (head trauma victim) after informed consent of the victim's family. The samples were dewaxed and stained with nebulette, desmin, myopalladin, and α-actinin2 antibodies using standard techniques (15).

Full-length 3.1 kb human nebulette wild-type (WT) clone DNA (cDNA [NM-006393]) was amplified by reverse transcription–polymerase chain reaction from human heart ribonucleic acid (Ambion, Houston, Texas) and ligated into vector containing the murine α-myosin heavy chain (α-MyHC) promoter and downstream human growth hormone gene (Dr. Jeffrey Robbins, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio). Mutants (K60N, Q128R, G202R, and A592E) were created using the QuikChange Site-directed mutagenesis kits (Stratagene, La Jolla, California). The transgenic (Tg) mice were created according to the Baylor College of Medicine animal regulations. Offspring were genotyped using polymerase chain reaction of genomic DNA. Transcription and protein expression levels were confirmed by real-time quantitative reverse transcription–polymerase chain reaction and Western blotting. Embryos were studied at E7.5, 9.5, 12.5, and 16.5 stages (n >3).

To determine latent exercise intolerance, Tg and age- and sex-matched non-Tg mice underwent graded treadmill testing at 4 and 6 months of age (n ≥5). The treadmill (Exer-6M, Columbus Instruments, Columbus, Ohio) was set at a slope of 15° with a starting speed of 16 m/min; the speed was increased by 2 m/min every 5 min until the mouse exhibited signs of exhaustion, and total running distance was recorded. Echocardiography and cardiac magnetic resonance imaging was performed (n = 5) as described previously (15).

Mouse hearts were studied by histology, immunohistochemistry, Masson trichrome, or transmission electron microscope, as described previously (15). Antibodies against nebulette and myopalladin (11), α-actinin2 (Upstate [Millipore], Billerica, Massachusetts), desmin (Abcam, Cambridge, Massachusetts), Z-band alternatively spliced PDZ-motif protein (ZASP; Dr. Georgine Faulkner, ICGEB, Trieste, Italy), tropomyosin, cardiac troponin I, cardiac troponin T, and filamin C (Santa Cruz Biotechnology, Santa Cruz, California) were used as recommended by the manufacturers. Multiple sections from at least 5 mice per group were analyzed. Protein expression was quantified using Western blotting, and relative intensity (RI) was carried out using a Kodak Image system (Kodak, Rochester, New York). Beta-actin was chosen as a control, since nebulette only interacts with the muscle elements (α- and F-actin) and not the nonmuscle elements (β-actin) in cardiac background (16).

Rat H9C2 cardiomyoblasts were used to study the effects of nebulette mutations on myofibrillar assembly during differentiation under mechanical stress. Human WT and mutant nebulette cDNA was ligated into a pcDNA3.1NT-GFP-TOPO vector (Invitrogen, Carlsbad, California) and transfected into H9C2 cells plated on BioFlex plates using Effectine (Qiagen, Valencia, California). After 48 h, cells were cultured in Dulbecco's Modified Eagle Medium containing 2% of heat-inactivated horse serum, and subjected to 10% 1-h per day cyclic mechanical stretch for 3 days, as previously described (17). The H9C2 cells were fixed, then stained with anti-GFP and Alexa Fluor Phalloidin-594 (Invitrogen, Carlsbad, California), and visualized using an Olympus confocal microscope (Olympus, San Jose, California).

Statistical analysis was performed using Student t test and analysis of variance with post-hoc Tukey testing. Data were reported as the mean with standard error of the mean (SEM). Values of p < 0.05 were considered significant.

We identified 4 variants in NEBL: c.180G>C (exon 3; p.K60N), c.383A>G (exon 5; p.Q128R), c.604G>A (exon 7; p.G202R), and c.1775C>A (exon 17; p.A592E) (Figure 1A). Variant K60N has previously been reported in the dbSNP database (rs41277374), but neither the population frequency nor the effect on function has been previously reported. Variants K60N, Q128R, and G202R are located in the nebulin-repeat domain, which binds F-actin and tropomyosin-troponin complex, whereas A592E is located in the Z-disk binding region. None of these variants was identified in 300 race- and ethnicity- matched control subjects (600 alleles). Family analysis was available only for K60N variant, which confirmed the de novo origin of the variant.

The clinical features of the affected subjects hosting these variants were heterogeneous (Figure 1B). The newborn patient (S84-5162) carrying the Q128R variant had DCM and EFE with a clinical phenotype demonstrating endocardial thickening, deposition of elastic tissue and collagen (Figure 2A), along with a severely dilated left ventricle and severely depressed systolic function by echocardiography, with a fractional shortening of 10% (Figure 1B). The child underwent heart transplantation at 8 months of age. The K60N and G202R variants, however, were identified in patients who had clinical manifestations of DCM in adulthood. The patient carrying the A592E variant displayed the clinical features of DCM as a newborn; this patient also carried a digenic mutation in ACTN2 (Figure 1B).

Transgenic mouse lines expressing either WT or mutant nebulette under the control of the α-MyHC promoter were made to assess the impact of the NEBL mutations in vivo. The WT, G202R, and A592E mice showed 45%, 40%, and 35% transmission frequencies, respectively. We were unable to identify positive live offspring from K60N and Q128R founders, however. No mutant embryos were harvested from the K60N line at E7.5 or later, whereas mutant Q128R embryos were harvested up to age E12.5. Since K60N Tg embryos numbers were 50% to 70% less than expected, the possibility of embryonic lethality at an earlier stage of development could not be excluded. Histology of the Q128R embryonic hearts revealed severe cardiac changes, including biventricular dilation (Figure 2B, b; ×2 magnification) and wall thinning (×40 magnification). Such cardiac structural changes are considered to be potential causes of embryonic death.

Founders of the K60N and Q128R lines died at 1 year of age with severe enlargement of the heart (Figure 2C) and heart failure. Cardiac magnetic resonance imaging of chimeric mice revealed remarkable dilation (p < 0.05) (Figure 2D) compared with control mice. Transmission electron microscopy of hearts revealed enlarged and deformed mitochondria (Figure 2E, asterisks), abnormal lysosomes, and mitochondrial remnants (Figure 2E, arrowheads). Localized intercalated disk disruption and accumulation of lipids in cardiomyocytes were noted in Q128R (Figure 2E, arrows).

Comparative immunohistochemistry of human (control and S84-5162) and transgenic mouse hearts (non-Tg and Q128R) revealed that nebulette was partially disassociated with Z-lines, probably depositing as aggregates throughout the sarcoplasm in the S84-5162 heart (Figure 3A, e, arrowheads); the Q128R mutant exhibited punctate foci and more diffuse fluorescence for nebulette than the controls (Figure 3B, e, arrows). Myopalladin (Figure 3B, f) and α-actinin2 (Figure 3B, g) were smeared in S84-5162 (Figure 3B, asterisks), whereas loss of desmin association with Z-disks in both S84-5162 and Q128R-Tg hearts was notable (Figure 3B, h, double arrows). In Q128R-Tg hearts, myopalladin (Figure 3B, f) and α-actinin2 (Figure 3B, g) were not altered.

The WT, G202R, and A592E Tg mice were born and initially developed normally. Protein analysis (Figure 4A) revealed overexpressed nebulette in N219-WT-Tg, N136-G202R-Tg, and N211-A592E-Tg mouse hearts (RI = 3.4 to 1.9) compared with non-Tg animals (RI = 1.0). This was considered as evidence of overexpressed transgene, and these lines were then bred for further study. Disorganization and disarray of overexpressed nebulette (Figure 4B, asterisks) along with diffuse expression patterns in F-actin staining in G202R-Tg and A592E-Tg hearts (arrowheads) were detected by immunochemical analysis, compared with WT-Tg hearts (double arrows).

By 6 months of age (Figure 5A), markedly reduced (p < 0.01) tolerance against acute stress, as evidenced by decreased running distances, was observed in G202R mice (270.7 ± 89.0 m) and A592E mice (273.4 ± 55.77 m). On echocardiography (Figure 5A, lower panels), decreased fractional shortening was noted in mutant mice: 50.37 ± 0.7% and 55.24 ± 1.5% in non-Tg and WT-Tg mice, respectively, versus 43.36 ± 3.1% in G202R-Tg mice and 43.14 ± 3.1% in A592E-Tg mice (p < 0.05). Significant dilation of the left ventricle was notable, with increased LV end-systolic dimensions in G202R-Tg mice (1.66 ± 0.04 mm) and A592E-Tg mice (1.67 ± 0.02 mm) compared with non-Tg mice (1.47 ± 0.04 mm) and WT-Tg mice (1.51 ± 0.07 mm). No changes in LV end-diastolic dimensions and LV mass-body weight ratio were detected (data not shown).

Cardiac enlargement due to LV dilation (Figure 5B, arrows), myocyte disarray, and interstitial cell infiltration was evident in G202R and A592E hearts (Figure 5C). Transmission electron microscopy revealed focal separation of intercalated disks (Figure 5D, arrow), increased residual body liposome, and mild variation in mitochondrial shape in the G202R mutants (asterisks), whereas the A592E-Tg hearts had mild accumulation of lipids (arrowheads) and abnormalities of mitochondrial shape and size.

Proteins expressed mainly in the I-band, such as cardiac troponin I (RI = 0.5), cardiac troponin T (RI = 0.3), and tropomyosin (RI = 0.2) were significantly down-regulated (Figure 6B, arrows) and disrupted in G202R-Tg mutants (Figure 6A, i to l, asterisks). The mobility of filamin C was abnormal in Western blotting, suggesting cleavage (Figure 6B, arrowhead). In contrast, Z-disk-associated proteins, such as α-actinin-associated LIM protein (RI = 0.2), ZASP (RI = 0.7), α-actinin2 (RI = 0.4), and myopalladin (RI = 0.3) appeared to be down-regulated (Figure 7B, arrows) and more disrupted (Figure 7A, m–p, asterisks) in the A592E-Tg hearts. Myopalladin localization in Z-disks in G202R-Tg and A592E-Tg hearts appeared normal; however, Western blotting revealed a second lower band in the G202R mice, suggesting cleavage (Figure 7B, double arrows). Interestingly, α-actinin-associated LIM protein expression was down-regulated in WT hearts (RI = 0.2, arrowhead) compared with non-Tg hearts (RI = 1.0) and G202R-Tg hearts.

As a model broadly used in mechanical stretch experiments, rat H9C2 embryonic cardiomyoblasts (ATCC, Manassas, Virginia) was chosen to study the nebulette expression pattern during differentiation. Transfection efficiency of WT- and mutant-nebulette-GFP in H9C2 cells was approximately 10%. Perinuclear cytoplasmic localization of nebulette-GFP was observed in differentiating WT and mutant cells (data not shown).

We hypothesized that nebulette acts as a stretch-induced mechanosensor in cardiomyoblasts (16). To test this, as well as to investigate expression of mutated nebulette in differentiating cardiomyoblasts during mechanical stretch, we performed cyclic mechanical strain of H9C2 cardiomyoblasts. On day 1 of cyclic mechanical stretch, both WT and mutant nebulette-GFP were localized uniformly (Figure 8A, d, g, and j; higher resolution, inset) to the perinuclear region in differentiating cells (asterisks). By day 3, WT nebulette-GFP was distributed throughout the cytoplasm along with F-actin filaments reaching the periphery of cells (Figure 8B, d to f, arrows) and tended to form lines, presumably assembling into the maturating Z-lines (higher resolution, right corner). In contrast, despite being normally extended to the periphery of the sarcoplasm F-actin filaments (Figure 8B, g to l, arrowheads), sustained perinuclear nebulette-GFP localization in all mutants was evident (asterisks; Q128R and A592E mutants representing I-band and Z-disk mutations, respectively, are shown).

Over the past decade, variants in a many genes encoding structural proteins have been reported in patients with cardiomyopathies (1). In particular, genes encoding cytoskeletal and sarcomeric proteins, including Z-disk proteins, have been found to be the “final common pathway” of DCM (18). The events leading to EFE are not well understood, but have been reported to result from an environmental insult, including virus or chemical exposure, in a genetically susceptible person (2- 3). The goal of this study was to uncover the molecular basis of cardiac dysfunction in subjects with DCM, focusing on genes encoding proteins of the sarcomere and Z-disk. Four NEBL gene variants (K60N, Q128R, G202R, and A592E) were identified in patients with DCM and EFE. We demonstrated that these mutations lead to a variety of cardiac phenotypes and levels of severity in vivo, from embryonic lethality due to a severe cardiac abnormalities (K60N and Q128R), to postnatal DCM with clinical signs of impairment in cardiac function (G202R and A592E) as seen in humans recapitulating the human disease phenotype, with or without EFE. Interestingly, G202R and A592E mutations were found to result in cardiac dysfunction despite causing different ultrastructural changes. The I-band proteins were significantly disrupted in G202R mouse hearts, whereas the Z-disk associated proteins appeared to be more disrupted in the A592E hearts. These findings led us to hypothesize that NEBL mutations have different effects on protein function depending on the location of the mutation in specific portions of the nebulin repeats.

Nebulette is a component of the early dense body structures involved in myofibrillogenesis of developing cardiac muscle (8- 9). We demonstrated that cyclic mechanical strain is a factor that initiates the distribution of nebulette to the sarcomere, being an important determinant in stimulating formation of Z-lines in differentiating H9C2 cardiomyoblasts. To support this hypothesis, we evaluated NEBL mutations to determine whether the mutations affect nebulette distribution and differentiation in cardiomyoblasts, and found no abnormalities occurred. When H9C2 cells were subjected to cyclic strain, however, we demonstrated that NEBL mutations perturbed the codistribution of nebulette-GFP throughout the sarcomere with the actin filaments and delayed expression of nebulette-GFP in the maturing Z-lines.

Taken together, these data support our hypothesis that nebulette functions as a mechanosensor and may have signaling properties in stretch-induced activation of specific mechanotransduction pathways in cardiomyoblasts during myogenesis. These data also support our in vivo results that demonstrate that expression of mutant Q128R nebulette leads to severe structural abnormalities in the embryonic heart, and thus, ultimately to embryonic lethality. Moreover, an abnormal expression pattern for nebulette, myopalladin, and desmin was discovered in the heart of a child carrying the Q128R mutation. It suggests that interruption of these proteins may cause specific EFE phenotype in humans. We speculate that the nebulette-desmin protein-protein interaction transmits information from the Z-disks to the intermediate filaments, maintaining the structural and functional integrity of myocytes (12). On the basis of its timing during embryogenesis, as well as its functional effects on key binding partners, it appears likely that the Q128R mutation alters cardiac development, specifically affecting myofibrillogenesis through the early nebulette-actin interactions and later nebulette-desmin association. We speculate that disruption in nebulette-actin codistribution and/or delay in nebulette expression in the maturating Z-disks at the earliest stages of myogenesis may be present in K60N mice.

In contrast to K60N and Q128R, the fact of down-regulated tropomyosin and troponins in the mutant mice as an effect of G202R mutation suggests that this mutation may cause changes in force generation in mature cardiomyocytes (19). Our data are consistent with the data reported by Bonzo et al. (7) in which overexpression of nebulette fragments was associated with loss of endogenous nebulette, tropomyosin, and troponins accompanied with a decrease in thin filament length and concomitant loss of F-actin. In addition, nebulin repeats were found to contribute to the assembly and the allosteric properties of the striated muscle thin filament (14). Although we did not measure cardiac actin level and actin-filament length in the G202R mice, it is likely that dysregulated tropomyosin, troponins, and filamin C cause the development of LV dilation in our model.

The A592E mutation, in contrast, led to cytoarchitectural changes with different patterns of expression of Z-disk proteins, and dysregulation of Z-disk proteins suggests that A592E may disturb force transmission. Myofibrillar functional integrity is regulated by the Z-disk network linking the sarcomere, cytoskeleton, sarcoplasmic reticulum, and sarcolemma (4). Mature cardiomyocytes respond to mechanical stretch with an immediate increase in contractility as well as long-term changes in gene expression resulting in myocyte hypertrophy (5). Orchestrated cross-talking between multiple independent and mechanosensitive signaling pathways such as MAPK, PI3K/Akt, Ras, JAK/STAT, and Ca²⁺ signaling determines the final phenotype of the cardiomyopathies. The desmin-based unit is one of the major contributors in mechanotransduction (12,16), and it appears that the I-band–nebulette–Z-disk connection may play a crucial role in transmitting information from the Z-disks to the intermediate filaments.

In summary, we identified novel nebulette mutations (K60N, Q128R, G202R, and A592E) in patients with DCM, EFE, and cardiac failure. We propose that NEBL is a new susceptibility gene for EFE and DCM and that these mutations lead to cardiac remodeling and dysfunction. This work suggests that disruption of the proposed signaling pathways leads to unfavorable cardiac remodeling and, consequently, cardiac dysfunction. Furthermore, mutations in certain domains of nebulette alter the function of its interacting proteins in a distinct, domain-defined manner and ultimately will result in activation and/or disruption of signaling cascades, culminating in structural remodeling of cardiomyocytes.