A novel mutation in human EMD gene and mitochondrial dysfunction in emerin knockdown cardiomyocytes

Abstract Emerin is an inner nuclear envelope protein encoded by the EMD gene, mutations in which cause Emery–Dreifuss muscular dystrophy type 1 (EDMD1). Cardiac involvement has become a major threat to patients with EDMD1; however, the cardiovascular phenotype spectrums of emerinopathy and the mechanisms by which emerin regulates cardiac pathophysiology remain unclear. Here, we identified a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene in a Han Chinese family through high‐throughput sequencing. Two family members were found to have EDMD1 with muscle weakness and cardiac arrhythmia. Mechanistically, we first discovered that knockdown of emerin in HL‐1 or H9C2 cardiomyocytes lead to impaired mitochondrial oxidative phosphorylation capacity with downregulation of electron transport chain complex I and IV and upregulation of complex III and V. Moreover, loss of emerin in HL‐1 cells resulted in collapsed mitochondrial membrane potential, altered mitochondrial networks and downregulated multiple factors in RNA and protein level, such as PGC1α, DRP1, MFF, MFN2, which are involved in regulation of mitochondrial biogenesis, fission and fusion. Our findings suggest that targeting mitochondrial bioenergetics might be an effective strategy against cardiac disorders caused by EMD mutations.


| INTRODUC TI ON
Emerin is an integral membrane protein that localizes predominantly at the inner nuclear envelope. 1,2 In 1994, human EMD gene encoding for emerin was first identified as the causative gene for X-linked Emery-Dreifuss muscular dystrophy (X-EDMD), now assigned to the subtype EDMD type 1 (EDMD1), which is classically characterized by progressive muscle dystrophy, early joint contractures and cardiomyopathy with conduction abnormalities. [3][4][5] The incidence of EDMD1 is estimated as 1 in 100,000. 6 The characteristics include a high rate of cardiac involvement like atrioventricular conduction block, atrial flutter and fibrillation, atrial standstill, or even sudden cardiac death (SCD), which may appear before left ventricle systolic dysfunction. [7][8][9] Until recently, more than 100 mutations, mostly nonsense or frameshift, have been reported, 95% of which result in loss of emerin protein. 10 However, how emerin affects the pathophysiology of cardiac disorders remains elusive.
Mitochondria are the powerhouse of cardiomyocytes and must constantly produce large amounts of ATP to maintain contractile function of the heart. 11 To meet substantial energy requirements, the heart metabolizes various fuels to generate ATP through oxidative phosphorylation (OXPHOS), which provides approximately 95% of the cellular energy demand. 12,13 Compromised energy production efficiency has been considered as a key mechanism of pump failure, and cardiac rhythm disruption occurs due to insufficient energy supply to ion channels and transporters. 14,15 Mitochondria are also dynamic, semiautonomous organelles continuously undergoing processes essential for maintaining mitochondrial homeostasis, such as biogenesis, fission and fusion. 16 Mitochondrial biogenesis, a type of symmetric fission that generates two healthy mitochondria, is controlled by an important transcription factor, peroxisome proliferator activator receptor γ coactivator-1α (PGC1α). 17 In failing hearts, PGC1α is downregulated, which contributes to mitochondrial dysfunction. 18 Mitochondrial fusion and fission are crucial to mitochondrial damage repair through recombination of organelles and segregation of injured components for lysosomal degradation. 13 There are multiple proteins regulating this antagonistic but related process, such as dynamin-related protein 1 (DRP1), mitochondrial fission 1 protein (FIS1), mitochondrial fission factor (MFF), which promote fission, and mitofusin 1 (MFN1), MFN2, optic atrophy 1 (OPA1), which promote fusion. 19 Mitochondrial fusion, fission and trafficking are generally termed as mitochondrial dynamics, and they are paramount not only for mitochondrial network remodelling but also for optimal mitochondrial function in physiological conditions. 20 Thus, we sought to test whether loss of emerin leads to defective mitochondrial biogenesis or dynamics in cardiomyocytes.
Here, we investigated a family containing 11 subjects. Two family members have EDMD1 with muscle weakness and cardiac arrhythmia, along with right atrium and ventricle enlargement in one and joint contracture in the other. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the genetic background of this family and a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene was identified in both affected patients. To further characterize the function of emerin in cardiomyocytes, we silenced the EMD gene in HL-1 and H9C2 cells. We found that loss of emerin caused impaired oxidative phosphorylation, aberrant electron transport chain (ETC) protein expression, collapsed mitochondrial membrane potential, altered mitochondrial networks, and decreased RNA and protein level of multiple factors involved in mitochondrial biogenesis, fusion and fission. Our findings unveil that emerin deficiency contributes to mitochondrial dysfunction in cardiomyocytes.

| Human blood samples
We enrolled a Han Chinese family spanning three generations with 11 members ( Figure 1A) from the Department of Cardiovascular Medicine in Shanghai Ruijin Hospital. Peripheral blood was collected from the affected proband and three other family members (II-3, II-5, III-1, III-3). Peripheral blood mononuclear cells were isolated and stored in liquid nitrogen until use.

| Genetic analysis
Genomic DNA was extracted from specimens of the whole blood using QIAamp® DNA Micro Kit (Qiagen, #56304) according to the manufacturer's instructions. Sample purity was detected by Nanodrop8000 (Thermo) before database construction. Double-terminal 2x150bp whole-exome sequencing (WES) was performing using Illumina Novoseq 6000 (S4). The sequencing results were referenced to 1000 Genomes, gnomAD, ESP, ExAC and Kaviar databases. High-frequency mutations were removed at a mutation frequency cut-off of 1%, and homozygous mutations as well as synonymous mutations were also excluded.
SIFT_score and Polyphen2_score were used to analyse the possible effects of mutation sites on protein sequence structure and function.
Suspicious mutations were obtained according to SIFT_score ≤0.05, and Polyphen2_score ≥0.447 and ≥0.453 with reference to HumanVar database and HumanDiv database, respectively. The DAVID (https:// david.ncifc rf.gov/) website was then used to annotate suspicious mutations in Disease Ontology, Gene Ontology Biological Processes, tissue distribution and KEGG pathway to further narrow down the range of suspected pathogenic genes in the family. Sanger sequencing was used to verify the suspected pathogenic EMD gene obtained by the above screening process. A primer pair was designed to amplify the EMD variant c.C57G, p.Y19X (Forward, 5'-GACAACGATTCGGCTGTGAC-3′; Reverse, 5′-CCTCTGGGTCTCGTACTCGAAG-3′).

| Small interfering RNA transfection
The rat-specific small interfering RNA (siRNA) targeting EMD was synthesized by GenePharma and used according to the manufacturer's instructions. Briefly, H9C2 cells were plated in six-well plates.

| RNA extraction and Quantitative real-time PCR analysis
Total RNA was extracted using TRIzol (Invitrogen, #15596026) and was reverse-transcribed to cDNA using Hifair® II 1st Strand cDNA Synthesis SuperMix (Yeason, #11123ES10). cDNA transcripts were then quantified by ABI QuantStudio™ 6 Fluorescence Quantitative PCR system with Hieff™ qPCR SYBR® Green Master Mix (Yeason, #11203ES03). The mRNA levels were normalized to 18S rRNA levels. All primer sequences were listed in Table S2.

| Protein extraction and Western blot
Proteins were extracted from cells in RIPA buffer (Beyotime, #P0013B) with protease and phosphatase inhibitor. Cell lysates were centrifuged at 12,000 g for 30 min at 4°C, and the protein concentration was measured by BCA Protein Assay Kit (EpiZyme, #ZJ102). Proteins were separated by SDS-PAGE gel and transferred to 0.45 μm PVDF membranes (Millipore, #IPVH00010), which were then blocked with 5% skim milk for 1 h at room temperature. Imaging System (Syngene). Intensity of bands was quantified using ImageJ software.

| Mitochondrial oxygen consumption rate measurement
Mitochondrial oxygen consumption rate (OCR) was meas-
Quantification of TMRM fluorescence intensity was performed using ImageJ software.

| Mitochondrial network analysis
Images obtained from cultured HL-1 cells were processed using the Mitochondrial Network Analysis (MiNA) ImageJ macro. 22 In short, images were pre-processed for sharpness and contrast enhancement, followed by converted to binary, and then skeletonized as a wireframe of lines one pixel wide. After that, all pixels within a skeleton were divided into three categories: end point pixels, slab pixels and junction pixels. Four distinct morphologies were recognized: puncta, rods, networks and 'large and round' structures. MiNA computes nine parameters, four of which were used: individuals (puncta, rods and large/round), networks (structures with at least one junction), mitochondrial footprint (total area of mitochondrial structures calculated prior to skeletonization), and mean branches per network (mean number of branches per network).

| Statistical analysis
All data are shown as mean ± SD. Statistical analysis was performed using GraphPad Prism 8 software. Pairwise comparisons were calculated using unpaired two-tailed student's t-test. p < 0.05 was considered significantly different.

| Pedigree, clinical feature and a novel EMD mutation
A family containing 11 subjects spanning three generations was studied. As the pedigree chart shows in Figure 1A Due to low prevalence in the general healthy population and absence of hereditary heart disease in II-6 parents, we speculated that the genetic pattern of this family is X chromosome recessive inheritance, and the mother of the proband (II-5) is a mutation-carrier.
Next, we used WES to investigate the pathogenic gene in this family ( Figure 1A, II-3, II-5, III-1, III-3). Forty eight mutation loci were obtained by screening common mutations in patients II-3, III-3 and carrier II-5, but not in healthy member III-1. After removing synonymous mutations, there were 43 candidate mutation loci in the remaining 38 genes (Table S1). Single nucleotide polymorphism (SNP) frequencies were then selected by 1000 Genomes, gnomAD, ESP, ExAC and Kaviar databases to screen out the mutation sites with a mutation frequency less than 1%. Finally, the only suspected mutant gene EMD was obtained. In addition, Sanger sequencing was performed to reconfirm the validity of the variant and a nonsense mutation c.C57G, p.Y19X was found in the EMD gene on the X chromosome ( Figure 1D). This mutation led to premature translation termination at the N-terminus of the 19th amino acid (aa) in the EMD sequence.
The mutated nucleotide and aa sites were shown in Figure S1. This mutation was identified in two male patients ( Figure 1A, II-3, III-3) and one female asymptomatic carrier (II-5), which has not been reported in existing databases. Therefore, combined with clinical features, we discovered a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene in an EDMD1 family.

| Knockdown of emerin in HL-1 and H9C2 cardiomyocytes leads to defective mitochondrial oxidative phosphorylation
Previous studies have suggested that mitochondrial dysfunction may be involved in the pathophysiologic mechanism of cardiomyopathy and conduction disorders. 15 In keeping with this data, spare respiration capacity (%) was markedly reduced in eKD HL-1 cells, while non-mitochondrial oxygen consumption and coupling efficiency remained unchanged ( Figure S3A). In the same way, the OXPHOS levels were tested in eKD H9C2 cells ( Figure 2C). Similarly, decreased ATP production, maximal respiration level, spare respiration capacity and coupling efficiency were observed in eKD H9C2 cells, whereas other indicators were comparable to control cells ( Figure 2D and Figure S3B). Taken together, these results suggested that emerin deficiency in HL-1 and H9C2 cells impaired mitochondrial respiration capacity.

| Knockdown of emerin in HL-1 cells impairs mitochondrial respiratory chain integrity and membrane potential
Given that healthy cardiomyocytes drive ATP production via the integrated ETC, we deduced that the defective mitochondrial respiration function shown above is due to impaired ETC complexes. 16 With this in mind, we interrogated expression levels of protein components of ETC complexes in eKD HL-1 cells. As predicted, significant decreased protein levels of complex I, IV components (NDUFB8, MTCO1) and increased complex III, V components (UQCRC2, ATP5A) were found in eKD HL-1 cells compared to control cells ( Figure 3A,B). As impaired ETC integrity and ATP deprivation were associated with mitochondrial membrane potential depolarization, a critical characteristic of dysfunctional mitochondria, 24 we detected mitochondrial membrane potential in emerin-deficient HL-1 cells using the OMX ultra-high-resolution microscopic imaging system. Some of the mitochondria in eKD cells that lost their membrane potential were detected when stained with etramethylrhodamine methyl ester (TMRM) (Figure 3C,D). Collectively, these data demonstrated that emerin plays a vital role in regulating ETC complexes' expression and loss of emerin significantly decreased mitochondrial membrane potential in cardiomyocytes.

| Mitochondrial network alterations in eKD HL-1 cells
In addition to mitochondrial function, mitochondrial morphology alternations have become more apparent in the pathophysiology of cardiomyopathy in recent years. 23  To further explore the molecular mechanism underlying mitochondrial fragmentation, the expression levels of multiple genes involved in regulation of mitochondrial biogenesis, fission and fusion were assessed. As shown in Figure 5A Together, these observations indicated that emerin deficiency leads to reduced mitochondrial mass and altered mitochondrial morphology, which may result from impaired biogenesis and fusion.

| DISCUSS ION
Our study discovered a novel mutation in human EMD gene and revealed significant mitochondrial dysfunction in cultured cardiomyocytes downregulated for emerin, as shown in Figure 6. Our genetic studies identified a novel nonsense mutation (c.C57G, p.Y19X) in the EMD gene in a Han Chinese family, which has not been reported in existing databases. Cardiac performance of the proband characterized by conduction defect associated with right atrium and ventricle enlargement has never been reported to our knowledge, which reflects the heterogeneity of cardiac emerinopathy. These findings expand the mutation and clinical phenotypic spectrum of EDMD1 even though the genotype-phenotype correlations remain to be investigated.
The precise molecular mechanism by which emerin mutations get involved in the pathophysiology of cardiac damage remains elusive to date. Emerin null mice show mild cardiac dysfunction; however, when under pressure overload, they present with more compromised cardiac function compared with wild-type. 25,26 Given that mitochondria play a central role in the cardiovascular system and impaired mitochondrial bioenergetics increases the propensity to heart failure and cardiac arrhythmias, we focused on the role of mitochondrial function in cardiac emerinopathy. 15,27 In support of this idea, we observed appreciably decreased mitochondrial OXPHOS in emerin-deficient H9C2 and HL-1 cells, which strongly suggests the regulation of emerin on cardiac energy metabolism. Furthermore, data analysis indicated reduced ATP production, maximal respiration level and spare respiration capacity in eKD cells. These may reflect lower substrate availability, ETC integrity, and ability of cells to meet energy demands under certain stressful conditions without emerin. At the molecular level, we found a marked downregulation of ETC complex I and IV, consistent with previous research showing that disrupted complex I and IV was associated with heart failure and atrial fibrillation. [28][29][30][31] Otherwise, we also detected mild upregulation of complex III and V, which may serve as compensatory mechanism to sustain cellular basal oxygen consumption. Thus, we concluded that loss of emerin in cardiomyocytes leads to ETC remodelling and impaired mitochondrial bioenergetics. Combined with past in vivo data, our in vitro experiments suggested that emerin might play an important role in regulating the energy metabolism of cardiomyocytes.
Mitochondrial energy depletion induces loss of membrane potential, which subsequently causes voltage-dependent anion channels' (VDACs) activation, Ca 2+ overload, permeability transition pore (PTP) opening, and eventually triggering apoptosis and necrosis. 24,32 Collapsed mitochondrial membrane potential is a critical feature of various cardiovascular diseases such as heart failure, ischemia reperfusion (I/R) injury, lethal ventricular arrhythmias and SCD. 24,33,34 In accordance with this, we observed decreased mitochondrial membrane potential in eKD HL-1 cells, which may act as an early cell death signal implicated in the pathological process of cardiac emerinopathy.
Of note, cardiac mitochondrial biogenesis is an essential step in mitochondrial quality control. 35  The findings above strongly suggest that emerin has a mitochondrial protective role in cardiomyocytes; however, a few limitations could be further explored in the future. Firstly, we do not have direct evidence indicating the emerin nonsense mutant (c.C57G, p.Y19X) leads to emerin deficiency, even if the truncated forms of emerin (aa 3-41 and 3-44) had been proved undetectable in cells, 39 and the nonsense mutation site we found giving rise to premature translation termination (located at the codon for residue 19, ahead of the 41th aa). Secondly, our experiments in vitro were based on two cardiac cell lines, which may not be able to fully retain metabolic characteristics in vivo. 40 Neonatal or adult mouse cardiomyocytes of emerin lacking or mutant mice with mature mitochondrial networks and dynamics could be a better choice. Thirdly, our findings did not uncover the potential mechanism linking emerin and mitochondrial function. Previous studies have shown that emerin regulates multiple signal pathways such as MAPK/ERK, MKL1-SRF, wnt/β-catenin, notch signalling, which may underlie cardiac defects observed in EDMD1. 26,[41][42][43] These pathways correlate closely to mitochondrial dysfunction in cardiovascular diseases. [44][45][46][47] Thus, a comprehensive understanding of the upstream mechanisms by which emerin regulate mitochondrial function would be a future direction of research favourable for investigating therapeutic strategies and genetic manipulations aimed to treat cardiac emerinopathy.

| CON CLUS IONS
We were the first to identify a novel EMD nonsense mutation (c.C57G, p.Y19X) in a family associated with EDMD1. We observed that the proband manifested with distinct right atrium and ventricle enlargement accompanied with third-degree atrioventricular block.

ACK N OWLED G EM ENTS
We are grateful to the patients who participated in this study. This

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Zunhui Du
https://orcid.org/0000-0003-2832-2880 Tong Yin https://orcid.org/0000-0003-3037-7629 F I G U R E 6 A novel mutation in human EMD gene and significant mitochondrial dysfunction in emerin knockdown cardiomyocytes. eKD cardiomyocytes show mitochondrial dysfunction including impaired oxidative phosphorylation, aberrant ETC protein expression, collapsed mitochondrial membrane potential, altered mitochondrial networks, decreased RNA and protein level of multiple factors involved in mitochondrial biogenesis, fusion and fission, suggesting mitochondrial bioenergetics as a potential target against cardiac disorders caused by EMD mutations. ETC, electron transport chain; PMNCs, peripheral mononuclear cells.