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 could result from an environmental insult, such as a virus or chemical exposure, in a genetically susceptible individual.2,3
The goal of this study was to uncover the molecular basis of cardiac dysfunction in subjects with DCM. We focused in the sarcomere and Z-disk and identified NEBL
gene variants (K60N, Q128R, G202R and A592E) in patients with DCM and EFE. We demonstrated that the mutations lead to a variety of cardiac phenotypes and levels of severity in vivo
, from embryonic lethality due to a lethal cardiac abnormality (K60N and Q128R), to 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. I-band proteins were significantly disrupted in G202R mouse hearts, whereas Z-disk associated proteins appeared to be more disrupted in the A592E hearts. These facts 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 appeared to be the factor which initiated the distribution of nebulette through the sarcomere and, probably, could be important determinant in stimulating formation of Z-lines in differentiating H9C2 cardiomyoblasts. To support this, we found that nebulette mutations did not affect nebulette distribution and differentiation of cardiomyoblasts. When H9C2 cells subjected to cyclic strain, we demonstrated that nebulette mutations perturbed the co-distribution of nebulette-GFP throughout the sarcomere with the actin filaments and delayed expression of nebulette-GFP in the maturating Z-lines under cyclic mechanical strain. Taken together, these data support our hypothesis that nebulette may function 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 embryonic lethality due to severe structural abnormalities in the embryonic heart. Moreover, an abnormal expression pattern for nebulette, myopalladin and desmin was discovered in the heart of patient carrying the Q128R mutation. It is possible that interruption of these proteins could cause the specific phenotype of EFE in humans. We speculate that nebulette-desmin chain transmits information from the Z-disks to the intermediate filaments maintaining the structural and functional integrity of myocytes.12
Based on its timing during embryogenesis, as well as its functional effects on key binding partners, it appears likely that Q128R mutation alters cardiac development, specifically affecting myofibrillogenesis through the early nebulette-actin interactions, later nebulette-desmin association. We assume that disruption in nebulette-actin co-distribution 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 effect of the G202R mutation, which resulted in downregulated tropomyosin and troponins in the mutant mice, suggests that this mutation may cause changes in force generation in mature cardiomyocytes.19
Our data was consistent with Bonzo et al
reported that overexpression of nebulette fragments was associated with loss of endogenous nebulette tropomyosin and troponins accompanied with decrease in thin filament length and concomitant loss of F-actin, but not overall actin staining. 7
In addition, “nebulin-repeats” was found to contribute to the assembly and the allosteric property of the striated muscle thin filament.14
Although, we did not measure actin-filaments length in G202R mice, it is likelihood dysregulated tropomyosin, troponins and filamin C may cause development of LV dilation.
On the other hand, the A592E mutation led to cytoarchitectural changes with different patterns in expression of Z-disk proteins. It is believed that myofibrillar functional integrity is regulated by the Z-disk, network linking the sarcomere, cytoskeleton, sarcoplasmic reticulum and sarcolemma.4
Dysregulation of Z-disk proteins in A592E-Tg hearts suggests that A592E may disturb force transmission. 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 mechanotransduction12,16
and it appears that I-band-nebulette-Z-disk connection may play a crucial role in transmitting information from the Z-disks to the intermediate filaments.
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 these mutations lead to cardiac remodeling and dysfunction. In summary, we suggest that disruption of the proposed signaling pathways leads to unfavorable cardiac remodeling and consequently cardiac dysfunction. Furthermore, mutations in certain domains of nebulette will alter the function of it’s interacting proteins in distinct, domain-defined manner and ultimately will result in activation and/or disruption of signaling cascades, culminating in structural remodeling of cardiomyocytes.