Protein 2, which contains the LEM domain, is essential for heart development

The integrity of the nuclear envelope is essential for the compartmentalization of the nucleus and the cytoplasm. Importantly, mutations in genes coding for the nuclear envelope and related proteins are the second most common cause of familial dilated cardiomyopathy. One such NE protein that causes cardiomyopathy in humans and influences heart development in mice is Lem2.

(red) The nuclear envelope (NE) in eukaryotic cells separates the nuclear and cytoplasmic compartments from each other. Beneath the NE is the nuclear lamina, which consists of lamins A/C, B1 and B2 [2]. The nuclear lamina is thought to provide structural rigidity to the nucleus and play a role in chromatin binding and the regulation of gene expression [3,4]. The NE is interspersed with the LINC complex (Linker of Nucleoskeleton and Cytoskeleton), which spans the double membrane [5–7]. In striated muscles such as the heart, the LINC complex and its associated proteins have been shown to be essential for normal function [8–13]. Associated with the LINC complex are the proteins containing the domains lamina-associated peptide 2 (Lap2), emerin and MAN1 (LEM), which also play an important role in the heart. In fact, mutations in Emerin, Lap2 and LEM domain-containing 2 (Lem2) are associated with Emery-Dreifuss muscular dystrophy with cardiac conduction disturbances, dilated cardiomyopathy and arrhythmogenic cardiomyopathy (AC), respectively [14–18]. In particular, a leucine-13-to-arginine mutation (L13R) in Lem2 leads to AC and sudden death [16,17]. In addition, a C-terminal mutation that changes serine 479 to phenylalanine (S479F) leads to right bundle branch block and septal hypertrophy [18].

Lem2 is thought to play a role in vitro and in lower organisms. These range from NE repair and recapping after mitosis to maintenance of nuclear shape, regulation of heterochromatin and gene expression, and regulation of mitogen-activated protein kinase (MAPK) signaling [19–27]. In mice, global loss of Lem2 resulted in embryonic lethality between E10.5 and E11.5, with significantly smaller total body size and most tissues [28]. The more specific developmental defects included impaired neurogenesis in neuronal tissues and thinner myocardial walls in the heart. However, the specific role that Lem2 plays in the heart is still insufficiently understood.

Lem2 is essential for normal heart development

To investigate the role of Lem2 in cardiac function, a recent study [1] generated cardiomyocyte-specific knockout mice using a Cre-LoxP approach, referred to as Lem2 cKO. Lem2 ablation was confirmed by Western blotting (WB) and immunofluorescence analysis. The majority of Lem2 cKO mice did not survive to embryonic day 18.5 (E18.5), indicating lethality at late fetal stages. Tissue sampling at E18.5 revealed that the Lem2 cKO hearts were smaller and had enlarged atria in which blood was pooling, indicating abnormal heart function. To investigate this further, high-resolution episcopic microscopy (HREM) was performed for 3D organ reconstruction and morphological analysis [29]. Surprisingly, no obvious congenital defects involving abnormal ventricular development could be observed, which explained the fetal demise in Lem2 cKO mice. However, the wall thickness of the ventricles was slightly reduced at E14.5, which was even more pronounced at E16.5 and indicates a developmental delay. However, no changes were detected in the trabecular organization, which was performed using fractal analyses on HREM data at E16.5 to quantify morphological complexity.

Lem2 ablation activates cell death and inhibits cardiac development pathways

To further evaluate the changes in heart development, RNA sequencing analysis was performed at E14.5 on control and Lem2 cKO whole hearts. Differential gene expression showed slight but significant changes in gene expression, with 92 and 49 genes significantly upregulated and downregulated, respectively (Fig. 1A) [1]. Gene Ontology (GO) analysis revealed a significant enrichment of apoptotic cell death and a reduction in processes important for proper cardiac development, including those involved in morphogenesis, contraction and conduction (Fig. 1B, C and F) [1]. The upregulated genes included Gadd45b and Gadd45g, which are associated with cell death [30,31], as well as Fos, Spp1 and Arrdc3. Downregulated genes included Rnf207, Kcnq1 and Cacng6, which are associated with cardiac conduction and contraction, and Adamts6, which regulates cardiac morphogenesis [32–35] (Fig. 1G and H) [1]. These changes were detected at both the mRNA and protein levels (Fig. 1E and H) [1], demonstrating that Lem2 is required for cell viability and essential for normal gene expression during heart development.

Loss of Lem2 leads to cardiomyocyte apoptosis, accumulation of DNA damage and micronuclei damage.

To test whether the upregulated cell death pathway leads to apoptosis, hearts were labeled for cleaved caspase 3, which confirmed a high level of apoptosis in Lem2 cKO hearts between E13.5 and E16.5, consistent with results in neural tubes from global Lem2 KO mice [28]. Accordingly, a ~41% reduction in the number of cardiomyocytes isolated from E14.5 hearts of cKO compared to controls was routinely observed (mean ± SD: 94 500 ± 25 500 vs. 159 900 ± 34 700 cardiomyocytes).

These results were confirmed by an independent model in which Cre expression was driven by the cardiac troponin T (cTnT) promoter to generate Lem2f/f; cTnT-Cre/+ cKO hearts [36]. Given that Lem2 is a regulator of MAPK signaling [28], these pathways were examined and found that Lem2 cKO hearts had elevated ERK1/2 but not p38 activation levels. Lem2 cKO hearts showed increased levels of γH2AX (which marks DNA double-strand breaks) [37] and micronuclei at E14.5 and E16.5.

The development of the heart is strictly regulated and requires a precise balance between proliferation and apoptosis. Therefore, EdU and anti-phospho-histone H3 staining was used to mark the cells undergoing DNA synthesis and mitosis, respectively. The proliferation rates between the genotypes were comparable.

Cardiomyocyte nuclei lacking Lem2 are abnormally shaped and more susceptible to mechanical rupture

After increased apoptosis, DNA damage and micronuclei were observed in vivo, cardiomyocytes were plated on soft PDMS hydrogels at a pressure of 6 kPa to mimic the stiffness of the heart in vivo [38–40]. A different nuclear shape and an enlarged nuclear area were observed in E14.5 Lem2 cKO cardiomyocytes compared to control cells (Fig. 2A-C) [1].

To assess NE integrity and observe whether a similar process occurs in Lem2 cKO cardiomyocytes, the catalytically inactive cyclic guanosine monophosphate adenosine monophosphate synthase (cGAS) fused to an mScarlet fluorescent tag was transiently expressed in Lem2 cKO cardiomyocytes to mark the exposure of cytoplasmic chromatin and thus nuclear breakage events [41,42]. A significantly increased incidence of nuclear rupture was observed in Lem2 cKO cells compared to control cells, demonstrating an important role of Lem2 in maintaining nuclear integrity (Fig. 2D) [1]. Similar observations were made when the cells were plated on glass, which is much stiffer than hydrogel (Fig. 2B-D) [1].

cGAS has previously been shown to be associated with chromatin after NE was collapsed during mitosis and NE was not resealed after mitosis [43-45], which may contribute to the amounts of cGAS observed at NE. To determine whether cGAS accumulation at the NE is due to rupture during interphase (NERDI) or to defective NE resealing after mitosis [46]live cell imaging was performed overnight on cardiomyocytes expressing cGAS and the frequency of cGAS accumulation at the NE was monitored in cardiomyocytes that did not undergo mitosis. High NERDI values (~8 and ~1%) were observed for Lem2 cKO over 16 hours compared to controls (Fig. 2E) [1]. As further evidence that cGAS accumulation at the NE is largely the result of NERDIs, cardiomyocytes were transduced with BAF-GFP as a marker for NERDI scars [46]. In Lem2 cKO, a significantly increased BAF localization at the NE was observed compared to the control, which is reminiscent of the cGAS data. In line with this, almost 100% agreement of BAF localization with cGAS to NE foci was observed.

Inhibition of muscle contraction weakens shape defects and ruptures of the nuclei

Since the nuclei of the cardiomyocytes are subjected to constant mechanical stress via the LINC complex, which transmits force from the sarcomeres to the nuclear skeleton, the cardiomyocytes were treated with para-nitroblebbistatin, which inhibits myosin and stops muscle contraction [47]. This agent was able to completely attenuate the defects of the nuclear shape and the ruptures (Fig. 2F-J) [1].

Since para-nitroblebbistatin is a general inhibitor of the myosin class II family, muscle contraction was specifically inhibited by a different mechanism using verapamil, a clinically used drug that inhibits voltage-gated calcium channels to affect excitation-contraction coupling. Similarly, verapamil was also able to completely rescue aberrant shapes and fissures in the cardiomyocyte nuclei (Fig. 2F-J) [1] [48,49]. To further test whether the integrity of the nuclei is affected by muscle contraction, a cardiac myosin activator (Omecamtiv mecarbil) was used to stimulate muscle contraction. After stimulation of muscle contraction, increased nuclear rupture was observed in Lem2 cKO nuclei (Fig. 2G, H, J) [1]. Indeed, increased DNA damage was detected in cKO cardiomyocytes compared to controls, which could be reversed by inhibiting muscle contraction (Fig. 2K) [1]. These results indicate that Lem2 in cardiomyocytes is essential for the maintenance of NE and genome integrity under mechanical stress caused by muscle contraction forces. However, detailed WB and immunofluorescence analyses showed no detectable differences in the majority of NE and lamina proteins between the genotypes.

No obvious defects in heart function are observed in Lem2-iCKO mice

Following the finding that Lem2 is essential in fetal cardiomyocytes, the question arose as to whether it also plays a similar role in adults. Therefore, after the expression of Lem2 in adult cardiomyocytes was detected using isolated cells and heart slices (Fig. 3A and B) [1], Lem2 inducible conditional knockout (iCKO) mice were generated using a tamoxifen-inducible cardiomyocyte-specific Cre mouse line Tnnt2MerCreMer/+ [50].

Echocardiographies were performed at eight weeks of age and before Lem2 ablation at nine weeks of age, followed by serial echocardiographies at 33, 48 and 83 weeks of age. The cardiac function and wall thickness of the Lem2 iCKO mice were comparable to those of the control mice in all measurements and at all ages (Fig. 3C-E) [1]. No changes were observed in the expression levels of the fetal gene program and pro-fibrotic markers by quantitative RT-PCR analysis, indicating that there was no adverse remodeling or fibrosis.

In addition to the echocardiography and gene expression analyses, histologic analysis revealed no evidence of morphologic defects, changes in extracellular matrix deposition or changes in myocyte size (Fig. 3F) [1]. In addition, there were also no changes in the ratio of heart weight to rib length and heart weight to body weight in Lem2 iCKO mice compared to control mice. These data suggest that removal of Lem2 does not affect cardiac function up to 83 weeks of age, once the adult mouse heart has fully developed.

Most NE protein levels are unchanged in Lem2 iCKO hearts and isolated cardiomyocytes

Given the lack of basic phenotype in the Lem2 iCKO mice, it was hypothesized that other NE proteins might compensate for Lem2, as previously shown in other systems [51,52]. To test this, WBs were performed on whole hearts from control and Lem2 iCKO mice and analyzed for a range of NE and lamina proteins (Fig. 3G) [1]. As expected, the Lem2 concentration in Lem2 iCKO was reduced to 46% of the control concentration. Other proteins were unchanged, with the exception of the LINC complex protein SUN2, which was increased 1.5-fold compared to the control (p<0.01).

Immunofluorescence levels and localization of most NE proteins were unchanged, with the exception of Lem2, which was significantly reduced in iCKO cardiomyocytes, and a slight increase in SUN2, which was consistent with WBs. The mRNA transcript levels of Sun2 (as well as other components of the LINC complex) were similar between genotypes, suggesting that the increase in SUN2 observed in Lem2 iCKO cardiomyocytes is likely regulated at the protein rather than gene expression level.

Nuclear integrity and stiffness are not impaired in adult Lem2 iCKO cardiomyocytes

Comprehensive 2D and 3D analyses of the cardiomyocyte nuclei revealed no differences in the shape parameters between the genotypes. Interestingly, however, invaginations of the NE/lamina were observed in both the controls and the iCKO mice, but to a similar extent in both genotypes.

Previous work has shown that mutations in the interaction partners of Lem2, lamins A and C, influence core stiffness and make the cores more deformable under mechanical stress [4]. To test whether this is also the case with Lem2 iCKO, nanoindentation was performed on adult cardiomyocytes from Lem2 iCKO and control mice. The calculated modulus of elasticity showed comparable core stiffnesses between Lem2 iCKO and the control mice (1.01 and 1.26 kPa, respectively). Lem2 is therefore not required for normal nuclear morphology and mechanics in adult mouse cardiomyocytes.

Nuclear shape is maintained in response to increased pressure in adult Lem2 iCKO cardiomyocytes

Given the lack of basic phenotype in Lem2 iCKO cardiomyocytes, nuclei were next stressed in living Lem2 iCKO cardiomyocytes. For this purpose, adult cardiomyocytes were isolated from Lem2 iCKO or littermate controls and exposed to hydrodynamic pressures. These corresponded to those observed in either healthy, normal or pressure-overloaded hearts in vivo using a pressure stimulator (CellScale MechanoCulture TR; modified for a low pressure range [53]) [54].

Adult cardiomyocytes from Lem2 iCKO and oscillatory pacing controls were subjected to normal (120/15 mmHg) or high pressure (200/30 mmHg) and nuclear shape analyses were performed. Interestingly, the cardiomyocyte nuclei of both genotypes responded similarly to high pressure and became more irregular and less circular, as measured by the circularity, firmness and aspect ratio of the nuclei. However, no changes in nuclear shape were observed between Lem2 iCKO and control cells.

In adult cardiomyocytes that have adapted to the physical demands of NE, removal of Lem2 appears to have no effect on cardiac function or nuclear shape. Lem2 is not required to maintain nuclear shape under high pressure in adult cardiomyocytes. Nevertheless, it cannot be excluded that the remaining Lem2 in Lem2 iCKO hearts is sufficient to maintain the function of Lem2 in this role.

Insight into the cardiomyopathy of Lem2 mutations and cardio-
Laminopathies

The results of the study suggest that Lem2 is crucial for the integrity of the developing NE in the fetal heart and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by partial Lem2 depletion, possibly due to better established NE and increased adaptation to mechanical stress. These data provide insights into mechanisms underlying cardiomyopathy in patients with Lem2 mutations and cardio-laminopathies.

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