Cardiovascular Involvement in SYNE Variants: A Case Series and Narrative Review

Abstract

Cardiac laminopathies encompass a wide range of diseases caused by defects in nuclear envelope proteins, including cardiomyopathy, atrial and ventricular arrhythmias and conduction system abnormalities. Two genes, namely LMNA and EMD, are typically associated with these disorders and are part of the routine genetic panel performed in affected patients. Yet, there are other markedly fewer known proteins, the nesprins, encoded by SYNE genes, that play a pivotal role in connecting the nuclear envelope to cytoskeletal elements. So far, SYNE gene variants have been described in association with neurodegenerative diseases; their potential association with cardiac disorders, albeit anecdotally reported, is still largely unexplored. This review focuses on the role of nesprins in cardiomyocytes and explores the potential clinical implications of SYNE variants by presenting five unrelated patients with distinct cardiac manifestations and reviewing the literature. Emerging research suggests that SYNE-related cardiomyopathies involve disrupted nuclear–cytoskeletal coupling, leading to impaired cardiac function. Understanding these mechanisms is critical for furthering insights into the broader implications of nuclear envelope proteins in cardiac health and for potentially developing targeted therapeutic strategies. Additionally, our data support the inclusion of SYNE genes in the cardiac genetic panel for cardiomyopathies and cardiac conduction disorders.

1. Introduction

Laminopathies refer to a broad range of diseases caused by abnormalities in proteins that are part of the nuclear envelope, the structure that separates chromatin from the cytoplasm in eukaryotic cells [1]. These diseases may present with diverse clinical manifestations, including heart conduction disorders, ventricular and supraventricular arrhythmias, cardiomyopathy (more often with a dilated phenotype), neurodegenerative disorders and muscular dystrophy [2]. The term “laminopathies” originates from the discovery of the LMNA gene, the most common gene implicated in these disorders, which encodes lamin A and lamin C [3].

Over the past two decades, interest in the nuclear envelope and its various components has steadily increased. This paper focuses on two proteins, nesprin-1 and nesprin-2, which exist in numerous isoforms each, that are the results of the alternative initiation/termination of transcription and/or alternative splicing of the SYNE-1 and SYNE-2 genes. Nesprin-1 and -2, particularly the small isoforms, nesprin-1α2 and -2α1, are highly expressed in skeletal and cardiac muscles. To explore the potential clinical implications of alterations in these proteins, we describe five cases of unrelated patients with different clinical presentations, all of whom share the presence of a distinctive SYNE variant.

2. Case Reports

2.1. Patient 1

In 2019, a 50-year-old man presented to our hospital with sustained monomorphic ventricular tachycardia (VT) at 200 bpm (left bundle branch morphology, inferior axis, positive aVL) symptomatic for palpitations and chest pain which was terminated by pharmacological cardioversion with amiodarone; a similar episode had occurred a few months earlier with spontaneous resolution in 2 h. His non-cardiac medical history was characterized by autosomal dominant polycystic kidney disease leading to kidney transplant in 2020 and by multinodular thyroid struma in euthyroidism. Resting ECG showed regular atrioventricular (AV) conduction (PR 160 msec at 72 bpm) with left anterior fascicular block (LAFB) with associated ST segment and T wave abnormalities; cardiac ultrasound indicated mild left ventricular (LV) anterolateral apical hypokinesia confirmed by local strain analysis (−15%), with an overall preserved biventricular function (LVEF 60%, GLS −20%) and no valvular abnormalities. The patient refused cardiac magnetic resonance imaging (MRI) due to claustrophobia, while coronary angiography excluded significant coronary artery disease. The patient underwent a programmed electrophysiological study that failed to induce the clinical VT in a sustained way. Biventricular endocardial substrate mapping showed several areas of reduced voltages on a bipolar map that were targeted with ablation: the left side of the medio-basal interventricular septum at the junction with the LV anterior wall, the basal inferolateral portion of the right ventricle (RV) and the anterior infundibular region of the RV. The patient was implanted with a loop recorder. To rule out VT as a possible first manifestation of cardiac sarcoidosis, positron emission tomography with 18-fludeoxyglucose (18-FDG-PET) was performed and revealed a mild uptake of the tracer along the left ventricle free wall (atypical region for sarcoidosis, with no evidence of lymph node or pulmonary uptake [4]) (Figure 1a). Total Body Bone Scintigraphy ruled out amyloidosis (low probability). Before discharge, beta-blocker therapy with Nadolol was initiated. One year later, the same monomorphic VT recurred at 160 bpm, requiring interruption by intravenous amiodarone and prompting a second endocardial VT ablation. Once again, the clinical VT could not be induced, and a substrate-based procedure was performed, targeting the antero-apical and diaphragmatic portion of the RV; on top of ablation, an RV cardiac biopsy was performed that ruled out amyloidosis and local immune–inflammatory processes. Cardiac MRI, performed in July 2022, showed preserved biventricular geometry and function with no LV fibrosis other than a small area previously targeted with ablation. Genetic studies were performed in 2022 based on the Illumina True Sight One panel, with analysis restricted to a panel of genes involved in laminopathies and dilated and arrhythmogenic cardiomyopathies (list of genes reported in the Supplementary Materials S1). A missense single-nucleotide variant in exon 126 (c.22904C>T; p.Ala7635Val) of SYNE-1 was identified, classified as a variant of uncertain significance (VUS). This variant is reported twice in ClinVar, always as a VUS. Multiplex ligation-dependent probe amplification (MPLA) excluded deletions in the LMNA gene. After five years of follow-up, the patient remains asymptomatic on Nadolol therapy; the cardiac conduction disorder (LAFB) is stable.

2.2. Patient 2

In 2016, at the age of 33, the patient was implanted in a peripheral center with a permanent dual-chamber pacemaker due to the incidental finding of an asymptomatic advanced AV block (AVB; 2:1 alternating with 3:1 and with second-degree type 1 AVB) during a routine noncompetitive pre-participation sport screening (Figure 1b). Echocardiogram (without strain) and coronary angiography were normal. At the age of 40, he was referred for follow-up to our center, showing NYHA class I symptoms. Echocardiography revealed mild biventricular dysfunction: a left ventricle ejection fraction (LVEF) of 48% and a right ventricle fractional area change (RV-FAC) of 32% without significant valvopathies. Cardiac MRI confirmed these findings with no evidence of late gadolinium enhancement (LGE) or abnormal mapping. Pacemaker monitoring without ongoing cardiac drug use showed a few short runs of monomorphic non-sustained VT episodes (maximum 13 beats, 200 bpm) and multiple episodes of supraventricular tachycardia (some with sudden onset) at 160 bpm with 1:1 atrioventricular conduction. The percentage of ventricular pacing was 100%, but 1:1 spontaneous conduction, absent at rest, could still be elicited by prolonged handgrip, with a very prolonged first-degree AV block (400 msec). Accordingly, treadmill exercise testing revealed spontaneous sustained 1:1 AV conduction at the peak of effort (Figure 1c); no VT could be elicited during effort. The patient was then started on valsartan and Nadolol, and after one year of follow-up, he remains asymptomatic with an improved biventricular function (LVEF 54%, RV-FAC 45%). Genetic studies performed in 2022 were based on the Illumina True Sight One panel, with analysis restricted to a panel of genes involved in laminopathies and dilated and arrhythmogenic cardiomyopathies (list of genes reported in the Supplementary Materials S1). A single-nucleotide substitution in exon 18 of SYNE-1 causing a nonsense variant (c.2719C>T; p.Arg907*) was found. This variant is currently classified as a VUS, since most pathogenic variants have a dominant negative effect. This variant is reported only once in ClinVar, as likely benign, but with no evidence available. An MPLA genetic test excluded deletions in the LMNA gene.

2.3. Patient 3

In 2022, a 56-year-old man was referred to our center for genetic testing due to a history of conduction system disease (left bundle branch block, LBBB) since the age of 36 and of out-of-hospital cardiac arrest at the age of 55, with ventricular fibrillation [VF] upon presentation. Coronary angiography was negative, and cardiac MRI revealed normal biventricular function and no LGE (Figure 1d). AV conduction was only mildly prolonged (220 msec at 65 bpm). The patient was implanted with a subcutaneous implantable cardioverter defibrillator (s-ICD) and started on Bisoprolol. Genetic studies performed in 2022 were based on the Illumina True Sight One panel, with analysis restricted to a panel of genes involved in laminopathies and dilated and arrhythmogenic cardiomyopathies (list of genes reported in the Supplementary Materials S1). A missense single-nucleotide variant in exon 78 (c.14423C>T; p.Thr4808Met) of SYNE-1, classified as a VUS, was identified. This variant is reported five times in ClinVar, always as a VUS. An MPLA genetic test excluded deletions in the LMNA gene. One year later, the patient experienced multiple episodes of atrial fibrillation and atypical left atrial flutter, prompting a transcatheter ablation and the addition of Flecainide to his therapy. No more recurrences have occurred ever since.

2.4. Patient 4

In 2014, a 45-year-old man suffering from high blood pressure on ramipril therapy started to complain about fatigue during exertion. Cardiac ultrasound was normal, while cardiac MRI (2015) showed a slight reduction in LVEF (53%), without significant LGE. In 2016, he presented to our hospital due to the persistence of the symptom. Treadmill exercise testing revealed second-degree type 1 and 2:1 atrioventricular (AV) block at baseline and, for most of the effort, a recovery of 1:1 AV conduction over 150 bpm (Figure 1e,f); sporadic polymorphic premature ventricular complexes (PVCs) were observed during effort, including one couple. A loop recorder was initially implanted, which progressively documented an increase in the number and duration of the daily episodes of 2:1 AV blocks, up to an episode of complete AV block. A dual-chamber pacemaker was then implanted (05/2017). Preimplant cardiac MRI (03/2017) revealed stable LVEF (53%), without significant LGE; coronary CT was negative for epicardial lesions. Starting one year later, device monitoring detected multiple episodes of non-sustained monomorphic VT (maximum 13 beats, 200 bpm) and atrial ectopic tachycardia, prompting the initiation of beta-blocker therapy (initially with nebivolol, ineffective, then with Nadolol, effective). Valsartan (instead of ramipril) and spironolactone were subsequently added. Genetic studies performed in 2022 were based on the Illumina True Sight One panel, with analysis restricted to a panel of genes involved in laminopathies and dilated and arrhythmogenic cardiomyopathies (list of genes reported in the Supplementary Materials S1). A missense single-nucleotide variant in exon 66 (c.10584T>G; p.His3528Gln) of SYNE-1 (

Table 1. ACMG = American College of Medical Genetics and Genomics; AFL = atrial flutter; AVB = atrioventricular block; CMP = cardiomyopathy; LAFB = left anterior fascicular block; LBBB = left bundle branch block; LV EDVi = left ventricle end diastolic volume index; LV EF = left ventricle ejection fraction; NSVT = non-sustained ventricular tachycardia; RV FAC = right ventricular fractional area change; SYNE1 = synaptic nuclear envelope 1; VF = ventricular fibrillation; VT = ventricular tachycardia; VUS = variant of uncertain significance.

Case Index	Onset Age/First Manifestation	SYNE Variant	Genotype/ Classification (ACMG Criteria)	Protein Domain	Family History	Conduction Disease	Ventricular Arrythmias	LV EDVi LV EF/ RV FAC at Last FU (Years)	Skeletal Muscle Involvement
Patient 1	50/VT	SYNE 1: Exon 126, c.22904C>T p.Ala7635Val	Heterozygous VUS (PM2)	Spectrin repeats	2 mother’s brothers die for unknown CMP (40 and 55 years)	LAFB	VT	65 mL/mq 55%/38% (55 years)	No
Patient 2	33/AVB	SYNE 1: Exon 18, c.2719C>T p.Arg907*	Heterozygous VUS (PM2, BP6)	Spectrin repeats	Negative	Advanced AVB	NSVT	55 mL/mq 54%/53% (41 years)	No
Patient 3	36/LBBB	SYNE 1: Exon 78, c.14423C>T p.Thr4808Met	Heterozygous VUS (BP4)	Spectrin repeats	Negative	LBBB	VF	75 mL/mq 53%/39% (58 years)	No
Patient 4	46/AVB	SYNE 1: Exon 66, c.10584T>G p.His3528Gln	Heterozygous VUS (PM2, BP4)	Spectrin repeats	Negative	AVB II-degree type 1, 2:1 AVB	NSVT	86 mL/mq 46%/45% (55 years)	No
Patient 5	61/AFL	SYNE 2: Exon 48 c.9285del p.Lys3095Asnfs*7	Heterozygous C4 (PVS1, PM2)	Spectrin repeats	Negative	AVB I-degree	None	60 mL/mq 65%/42% (67 years)	No

), classified as a VUS, was identified. This variant is reported three times in ClinVar, always as a VUS. An MPLA genetic test excluded deletions in the LMNA gene. After seven years of follow-up, the patient remains asymptomatic, with no further arrhythmic events; valsartan was replaced with sacubitril/valsartan. The last cardiac ultrasound showed a mild LV ventricular dilation (left ventricular end diastolic volume index, LVEDVi, of 86 mL/mq) and dysfunction (LVEF 46% due to global hypokinesia), associated with mild left atrial dilation (left atrial volume index 36 mL/mq). AV conduction disorder progressed with no more elicitable spontaneous AV conduction and 100% ventricular pacing.

2.5. Patient 5

In 2022, a 65-year-old man with a negative family history was referred to the cardiology department to undergo transcatheter ablation for a recurrent typical atrial flutter. Past medical history was characterized by an acute type A aortic dissection in 2018 urgently treated with ascending aorta replacement with a valved tube including coronary ostia reimplantation. Resting ECG after flutter ablation showed normal AV and intraventricular conduction and elevated voltages in the precordial leads with anterolateral negative T waves. Cardiac ultrasound revealed a preserved biventricular systolic function along with increased LV wall thickness (maximum thickness 14 mm interventricular septum) and bi-atrial dilatation. Bone tracer cardiac scintigraphy (Perugini score 0) and hematological tests excluded cardiac amyloidosis. In February 2023, the patient started complaining about fatigue and palpitations; Holter ECG monitoring showed atrial fibrillation episodes with high ventricular response interspersed with periods of significant sinus bradycardia. The patient was therefore implanted with a permanent dual-chamber pacemaker. In the following months, while on chronic beta-blocker therapy (Metoprolol 150 mg/die), the device recorded several asymptomatic paroxysms of atrial fibrillation paired with a progressive increase in AV interval duration. Genetic studies performed in 2024 were based on the Illumina True Sight One panel, with analysis restricted to a panel of genes involved in laminopathies, hypertrophic cardiomyopathies and aortic disorders (list of genes reported in the Supplementary Materials S1). A deletion in exon 48 of SYNE-2 causing a frameshift variant (c.9285del; p.Lys3095Asnfn*7) was found. This variant is currently classified as likely pathogenic (C4). An MPLA genetic test excluded deletions in the LMNA gene.

2.6. The Familiar Segregation of the Identified SYNE Variants

Unfortunately, none of our patients had alive, reachable, first-degree family members older than 20 years old for segregating the variant within the families.

3. Review

3.1. Nesprins and the LINC Complex

Synaptic nuclear envelope 1 (SYNE-1) is a gene involved in the synthesis of proteins containing the Klarsicht/ANC-1/Syne Homology (KASH) domain. Specifically, SYNE-1 encodes nesprin-1 (nuclear envelope spectrin repeats) and several shorter isoforms through transcriptional and post-transcriptional mechanisms. These isoforms have distinct expression patterns, functions and localizations in various tissues [5]. Six KASH proteins have been identified: nesprin-1, -2, -3 and -4; KASH5 and lymphoid-restricted membrane protein (LRMP), encoded by the SYNE-1, -2, -3 and -4; KASH5 and LRMP genes, respectively [6].

Nesprin-1 is a key component of the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. This structure connects the nuclear envelope (NE) to cytoskeletal elements like actin filaments (F-actin), intermediate filaments (IFs, including desmin) and microtubules (MTs) [7]. In humans, full-length nesprin-1 and nesprin-2 are ubiquitously expressed giant nesprin isoforms, with a molecular weight of, respectively, 1 MDa (146 exons) and 800 kDa (116 exons), which makes them the second and third largest described proteins, after titin (4.2 MDa) [8].

Nesprin-1 comprises three main domains (Figure 2a–c): the N-terminal region with a Calponin Homology (CH) domain for interacting with actin filaments, also known as N-terminal actin-binding domain or ABD; a central domain with multiple spectrin repeats (SRs); and the KASH (C-terminal) domain, which interacts with Sad1p/UNC-84 proteins (SUN-1, SUN-2) to anchor nesprin-1 to the outer nuclear membrane (ONM) at the NE [9,10,11].

Like SYNE-1, SYNE-2 can generate multiple isoforms through alternative initiation and splicing, including the large nesprin-2 giant which is crucial for linking the nucleus to the cytoskeleton by means of actin filament interaction [12]. Nesprin-1 dominates the NE at first and then is partly replaced by nesprin-2 in mature myocytes. This is supported by the comparison between mature and immature muscle fibers; particularly in the latter, nesprin-2 is found to be undetectable in early myotubes [13]. Nuclear positioning in muscle appears to be related to nesprin-1 function, since it is not affected by nesprin-2 deletion [14].

Nesprin-3, which is ubiquitously expressed like nesprin-1 and -2 but is much smaller, links the nucleus to IFs and regulates cell morphology, perinuclear cytoskeletal architecture and cell polarization [15]. The nesprin-3 family lacks the N-terminal ABD and instead binds to the plakin family member plectin, which can than associate with IFs such as desmin.

Other proteins in the LINC complex include emerin (encoded by EMD gene) and lamin A/C (encoded by LMNA gene), both of which are located on the inner NE and interact with SUN proteins and a shorter isoform of nesprin-1 (nesprin-1α2), linking directly to chromatin proteins [7].

Finally, two proteins of the NE are of particular interest for their interactions with the LINC complex, namely luma, codified by the TMEM43 gene, and Lemd2 (LEM domain nuclear envelope protein 2), codified by the LEMD2 gene. Luma is a 45 kDa protein that contains four transmembrane helices and a domain of unknown function (DUF1625) located between the third and fourth helices. Luma is situated in the inner nuclear membrane (INM), where it interacts with emerin, lamin A/C, lamin B1, SUN2 and, indirectly, even with β-actin [16]. Lemd2 is a 56 kD protein with a Lap2/Emerin/Man1 (LEM) domain at its N terminus, followed by two transmembrane domains and a MAN1-Src1 C-terminal (MSC) domain. The LEM domain bridges chromatin to the NE through BAF (barrier-to-autointegration factor). Lemd2 is another INM protein involved in nuclear structure organization. Lemd2 requires binding to lamin A/C for proper retention in the nuclear envelope [17].

3.2. Role of Nuclear Envelope-Related Proteins in Cardiomyocytes

Cardiomyocytes are mononucleated cells that function as a syncytium through cell–cell coupling maintained by gap junctions [18]. The LINC complex is crucial for positioning myonuclei via its interactions with the cytoskeletal network. In SYNE-1 KASH single-knockout (KO) mice, synaptic and non-synaptic nuclei are unanchored [14]. Similarly, SUN-1 KO mice show defects in nuclear anchorage at the neuro-muscular junction, while SUN-1/SUN-2 double-KO mice have more severe nuclear positioning defects, leading to early postnatal death [9,19]. A CRISPR-modified mouse with a disrupted nesprin-1 KASH domain showed the mislocalization of several MT cytoskeleton proteins from the NE of striated muscle cells [20].

In cardiomyocytes, contractile force is mediated by myosin and sarcomeric actin, while F-actin, MT and intermediate filaments, through the LINC complex, connect the NE to the cellular membrane [21,22,23]. This cytoskeletal–nuclear coupling is essential for force transmission in tissues like the heart, where cardiomyocytes endure thousands of contractions over a lifetime [21,24]. To simulate mechanical stress transmission from the cytoskeleton to the nucleus, Guilluy et al. applied force directly on isolated nuclei through nesprin-1 using magnetic beads coated with anti-nesprin-1 antibodies. Successive force pulses increased nuclear stiffness, demonstrating the importance of LINC-mediated force transmission [25].

In nesprin-1 KO mice, cardiomyocytes exhibited altered heterochromatin density. Force application on nesprin-1 in vitro triggered nuclear stiffening through the tyrosine phosphorylation of emerin, a protein of the inner nuclear membrane linked to LINC [26]. In another model of isolated cardiomyocytes, biomechanical stress altered the expression of mechanically responsive genes, such as ERG-1, IEX-1, c-FOS, c-JUN and c-MYC, following nesprin-1 or nesprin-2 deletion [27].

Notably, like nesprins, the intermediate filament desmin has also been proven to play a pivotal role in nuclear anchorage and positioning. Global double-KO mice for nesprin-1 and desmin showed increased mortality and a worst dystrophic phenotype (including decreased nuclear deformation under biomechanical stretch) compared to single-KO mice [28], suggesting that nesprin-1 and desmin play partially redundant roles in nuclear anchorage in skeletal muscle. As already mentioned, desmin does not directly interact with nesprin-1: its binding partners in the LINC complex are nesprin-3 and plactin [23]. Accordingly, the N-terminal part of the 1A coil domain of desmin was suggested to be a hot spot for pathogenic desmin mutations, since genetic variants at this site affect filament assembly, leading to cytoplasmic desmin aggregation [29].

It was also demonstrated that the haploinsufficiency of TMEM43 in murine cardiac myocytes activates the DNA damage response pathway, leading to the upregulation of several senescence-associated secretary proteins, including transforming growth factor-β1 (TGFβ1) [30]. This led to the development of late-onset cardiomyopathy in mice, characterized by LV dilation, systolic dysfunction, increased cardiomyocyte size, fibrofatty infiltration and apoptosis. The pathogenesis of TMEM43-associated cardiomyopathy was also investigated in a zebrafish model, which confirmed the potential of two different TMEM43 genetic variants to induce an age-dependent cardiac phenotype characterized by ultrastructural remodeling and transcriptomic alterations, finally leading to cardiac enlargement in adulthood [31].

Finally, a homozygous LEMD2 knock-in mouse model was recently created to assess the effects of the p.L13R variant, which is located in the LEM domain, on cardiac structure and function [32]. The variant was proven to downregulate Lemd2 mRNA levels to 65%, to reduce Lemd2 protein levels and to disrupt the interaction between Lemd2 and BAF, which is essential for nuclear envelope repairing processes. Accordingly, mutant cells had an impaired nuclear envelope rupture repairing capacity, leading to DNA damage, premature senescence and a final fibrotic and inflammatory remodeling with LV dilation, dysfunction and increased arrhythmic susceptibility. Notably, conduction abnormalities including both PR and QRS prolongation were reported to occur at 6 months, before pronounced LV dilation (at 9 months). Specific data providing the mechanism underlying the conduction disorder were not provided. For instance, lamin A/C haploinsufficiency in mice was proven to cause an early-onset programmed cell death of AV nodal myocytes associated with both atrial and ventricular arrhythmias starting at 4 weeks of life, with impaired contractility developing much later (12 weeks) [33].

3.3. Nuclear Envelope-Related Proteins and Cardiomyopathy

SYNE variants contribute to a wide group of diseases called envelopathies, or more commonly laminopathies, caused by defects in proteins associated with the nuclear envelope [1]. In 1994, mutations in the X-linked gene EMD, encoding emerin, were linked to Emery–Dreifuss muscular dystrophy (EDMD) [34]. The same clinical phenotype, but with an autosomal dominant transmission, was later linked to variants in a second gene involved in the nuclear envelope: LMNA, encoding for lamin A and lamin C [3]. EDMD is a genetic disorder characterized by progressive muscle degeneration and weakness, affecting both skeletal and cardiac muscle, leading to atrioventricular conduction defects, arrhythmias and dilated cardiomyopathy (DCM) [35,36]. Genetic variants in LMNA and EMD can also be found in patients with isolated cardiac involvement, without the musculoskeletal features of EDMD [37,38]. Additionally, genetic variants in TMEM43, in most cases represented by the pathogenic p.Ser358Leu variant, have been associated with an autosomal dominant transmission, to both arrhythmogenic right ventricular cardiomyopathy with high penetrance and severe ventricular arrhythmias (ARVC-5, strong evidence) and to EMDM [39,40]. Rare cases of TMEM43 variants presenting with a prevalent/isolated LV phenotype with subepicardial scarring on MRI have also been described [41]. More recently, in 2019, a homozygous missense mutation in the LEMD2 gene c.38T>G (p.L13R) was reported for the first time in affected patients of the Hutterite population (a genetic isolate living in the US but originally from Europe) suffering from juvenile bilateral cataract and CMP with inferior and inferolateral LV scarring, a mild impairment of LV systolic function and a high risk of sudden cardiac death [42]. Affected cardiac myocytes and fibroblasts displayed irregularly shaped nuclei characterized by condensed peripheral heterochromatin. Finally, desmin, as already mentioned, interacts with LINC through SYNE-3. Desmin is well known and characterized for being involved in CMPs with all kinds of possible phenotypes, from HCM to DCM, restrictive CMP and ARVC. Notably, cardiac involvement may be associated with skeletal muscle manifestations, a phenotype better known as desminopathy characterized by bilateral skeletal muscle weakness that starts in distal leg muscles and spreads proximally, eventually leading to wheelchair dependence and respiratory insufficiency [43].

In 2007, Zhang et al. screened SYNE-1 and SYNE-2 genes in 190 EDMD and EDMD-like patients who lacked LMNA or EDM genetic variants. They identified six variants that were not present in a cohort of 384 control alleles; two were located in untranslated regions (5′-UTR), while the remaining four carried a missense variant within emerin- and lamin-binding domains: R257H, V572L, E646K in nesprin-1α and T89M in nesprin-2β. Most patients were heterozygous for one variant, with clinical presentations varying from elevated creatinine kinase levels to dilated cardiomyopathy requiring heart transplantation in a 26-year-old patient. Fibroblasts derived from these patients showed nuclear morphological changes and the mislocalization of LINC complex proteins (emerin and SUN-2), which were replicated by silencing nesprin-1 or nesprin-2 by siRNA in wild-type fibroblasts [44]. More than half of the identified and characterized SYNE-1 and SYNE-2 variants are in the C-terminus part of full-length nesprins-1 and -2 [12].

Similarly to LMNA, TMEM43 and EDM mutations, SYNE-1 variants can lead to isolated cardiac phenotypes. Puckelwartz et al. described a patient with non-ischemic hypokinetic dilated cardiomyopathy who required heart transplantation at a young age; the patient had a variant in a nesprin-1α isoform (arginine to histidine at amino acid 374 [R374H] in the spectrin repeat domain) not found in 300 control human chromosomes. Fibroblasts derived from this patient showed increased nesprin-1 and lamin A/C expression, suggesting that the variant disrupts LINC complex function [26]. A few years later, three other SYNE-1 variants (R8272Q, S8381C, N8406K, not present in a reference population of 420 alleles) in the nesprin-1 KASH domain were identified in seven unrelated DCM patients [45]. These variants led to a dysfunctional interaction with lamin A/C and emerin in fibroblasts derived from these individuals along with a modified regulation of extracellular signal-regulated kinase (Erk-1/2), involved in essential cardio-protective pathways [46].

A murine model of EDMD lacking the nesprin-1 KASH domain (Δ/ΔKASH) showed LINC complex disruption, resulting in muscle weakness and altered gait [47]. As for the heart, the model showed alterations characteristic of atrial disease such as prolonged electrocardiographic P wave and atrial refractoriness and exhibited progressive systolic dysfunction with age [26].

Interestingly, global nesprin-1 KO mice do not display cardiac abnormalities, but they do have severe skeletal muscle defects. Nesprin-2 KO mice, on the other hand, show neither cardiac nor skeletal alterations, indicating a functional overlap of functions between nesprin-1 and -2 [48]. This is also supported by evidence that double-KO mice lacking both nesprin-1 and -2 exhibit neonatal lethality due to respiratory failure [13]. In a double-KO model with a cardiomyocyte-specific deletion of nesprin-1 and -2, mice showed LV wall thinning, reduced fractional shortening, altered nuclear position and the mislocalization of emerin and lamin A/C [27]. Notably, very recently, a common SYNE2 genetic variant was found to be associated with lone atrial fibrillation [49,50]. The same variant was subsequently proven to lower the expression of nesprin-2α1 with remarkable downstream effects on nuclear and electrophysiological features, including shortened assessed action potential duration and decreased conduction velocity [51].

The connection between lamin A/C and nesprin-1 is further supported by findings that the disruption of the nesprin-1 KASH domain suppresses the pathological phenotype in a cardiac-specific LMNA KO mouse model. The authors proposed that mechanical forces transmitted via MTs to the NE may contribute to nuclear fragility in LMNA-mutant striated muscle cells and that displacing nesprin-1 from LINC could help preserve nuclear integrity [20].

3.4. Other Diseases Related to SYNE Variants

Two other diseases have been associated with SYNE variants, both inherited in an autosomal recessive manner. The first is autosomal recessive spinocerebellar ataxia type 8 (SCAR8), a neurodegenerative disorder characterized by cerebellar ataxia, affecting gait, speech and eye movements [52]. The second is part of a group of congenital non-progressive disorders known as Arthrogryposis Multiplex Congenita (AMC), which involves multiple joint contractures and muscle weakness present at birth [12,53].

3.5. SYNE Genotype–Phenotype Correlation

The importance of establishing genotype–phenotype associations is bidirectional: indeed, a genetic substrate could offer proof of a pathogenetic mechanism, and a clinical phenotype could refine a gene variant classification. Concerning SYNE-1, most genetic variants implicated in striate muscle diseases are located in the C-terminus of the gene, responsible for the transcription of the KASH domain [45]. In spite of that, over the years, some authors have reported clinical cases where the variant affecting SYNE-1 was located within the SR domain, as in the patients presented in this case series. Zhang et al. in 2007 described a man with a pure skeletal striated muscle phenotype, wheelchair-bound since the age of 26 (nesprin-1α isoform with substitution of arginine with histidine in position 257 [R257H, SYNE-1:c.24284G>A]) [44]. Three years later, Puckelwartz et al. showed the above-mentioned patient with non-ischemic hypokinetic dilated cardiomyopathy who required heart transplantation at the age of 26 (nesprin-1α with R374H substitution) [26].

3.6. Comparison to Patients with Cardiac Laminopathies

Cardiac conduction disorders (CCDs) in patients harboring LMNA and EMD genetic variants have the peculiar feature, compared to other forms of genetically determined bradyarrhythmias, of tending to start with a suprahisian AV involvement and to only subsequently progress to an infrahisian involvement [54]. Notably, the development and progression of the AV block in cardiolaminopathies are strictly associated with the risk of developing malignant arrhythmias, mostly reflecting, at advanced stages, a detectable, intramyocardial septal scar at imaging [55]. Two of the five cases of SYNE variants we described (patient 2 and patient 4) presented with a suprahisian disorder at onset, with preserved AV conduction during adrenergic activation; notably, both suffered several episodes of non-sustained VT recorded during device monitoring. Patient 1 and patient 3, on the other hand, despite presenting with major ventricular arrhythmias, including aborted cardiac arrest in one case, both suffered an infrahisian cardiac conduction disorder (LAFB in patient 1 and LBBB associated with mild first-degree AV block in patient 3). None of the patients we presented had a significant left atrial enlargement (a typical characteristic of cardiolaminopathies), yet four suffered from atrial arrhythmias, prompting ablation in two cases. Finally, none of our patients have developed overt heart failure symptoms so far, nor have any had a significant LV dysfunction, but it should be underlined that most (four out of five) of the patients are still below 60 years of age at present, while the youngest is 41. Actually, considering the only exception of a single patient with a hypertrophic phenotype (patient 5), they had a mildly depressed LVEF at the last follow-up (range 45–54%).

3.7. Implications for Genetic Investigation in Cardiomyopathies and Cardiac Conduction Disorders

The 2022 international position paper on genetic testing for cardiac diseases and the 2023 ESC guidelines on cardiomyopathies both emphasize the importance of searching relevant genes only to improve the diagnosis and clinical management in patients with suspected genetic conditions [56,57]. Recommended first-line genetic testing includes panel testing with next-genome sequencing (NGS) for genes with robust evidence for being disease-causing, and SYNE genes are currently not among them. Notably, in the case of early-onset (before the age of 60) and idiopathic cardiac CCDs, once sarcoidosis and an associated cardiomyopathy are ruled out, requiring more extensive genetic panels, the 2022 position paper recommends a minimum targeted gene panel including four genes (SCN5A, LMNA, GLA and PRKAG2). Recent case series, reporting the results of genetic testing performed with NGS but using a broader genetic panel (as many as 174 genes) without SYNE among patients under 50–60 years old undergoing pacemaker implantation due to an otherwise idiopathic CCD, report an extremely variable genetic yield ranging from 5% in studies not including VUSs up to 40–67% in studies including VUSs, with LMNA, TRPM4, SCN5A and MYH7 being the most frequently involved genes in decreasing order of frequency [58,59,60,61]. Additionally, it has to be underlined that a low but still detectable percentage of patients presenting with compatible clinical characteristics (e.g., cardiac conduction disorders with suprahisian onset/arrhythmias/early-onset non-ischemic cardiomyopathy) may be harboring a genetic hit represented by a copy variation number (cvn) in the LMNA/EMD genes that is extremely difficult to detect with NGS genetic analysis (other than a suspect in some but not all cases) and requires dedicated genetic tests (MLPA). The yield of cvns researched with MLPA within cardiomyopathies has been reported, according to the study and the type of cardiomyopathy, between 1% and 4% (the latter for dilated phenotypes), but this may be underestimated because MLPA is not routinely performed or recommended by current cardiological guidelines in the case of a negative NGS study [62,63,64]. There are no data concerning the yield of MLPA in the case of isolated early-onset CCDs leading to device implantation. In our case series, MLPA was performed in all patients; therefore, cvns affecting LMNA or EMD can be excluded. Notably, independently from the presence of an associated CCD, when the clinical presentation is a CMP with only a mildly depressed LVEF, there are few recognized genotypes associated with a high risk of sudden cardiac death, which should prompt a tailored arrhythmic risk stratification and eventually ICD implantation. According to the 2023 CMP guidelines [53], the list currently includes six genes, namely LMNA, FLNC (truncating variants), TMEM43, PLN, DSP and RBM20. Therefore, two out of six codify for nuclear envelope-related proteins.

Regarding the inclusion of SYNE in NGS genetic testing panels, while potentially beneficial for identifying certain conduction disorders/cardiomyopathies, it could also have negative implications. SYNE variants are often of uncertain significance, as those described in four out of five of our cases, given the gene’s large size and complex isoforms and the very limited number of studied variants. This could lead to an increase in ambiguous or incidental findings, complicating the interpretation of results. Furthermore, the identification of rare or poorly characterized SYNE variants may prompt unnecessary follow-up testing or interventions without clear clinical benefit. Nonetheless, the fact that 33% of the genes currently associated with a high risk of sudden cardiac death despite an only mildly depressed LVEF codify for nuclear envelope-related proteins mandates extreme caution before dismissing variants potentially associated with the dysfunction of these proteins.

The assessment of SYNE variants could be potentially considered in patients with specific clinical indications. These might include cases of juvenile (below 60 years old) non-ischemic cardiomyopathies with early-onset arrhythmias; cardiac conduction system disorders, particularly if suprahisian at onset; and unexplained ventricular arrhythmias, especially when there is no or very mild detectable structural heart disease or when conventional, commercially available, NGS testing panels without SYNE yield negative results. The indication to MLPA is still a matter of debate but could also be considered on a regular basis in such cases.

3.8. Potential Therapeutic Implications of Cardiomyopathy Due to Nuclear Envelope-Related Proteins

The identification of aberrant nuclear envelope-related proteins in association with the development of CMPs may have important implications not only from a diagnostic and prognostic point of view but also from a therapeutic one. So far, except for a few exceptions such as Fabry’s disease, for which enzyme replacement therapy is currently available, most CMPs are treated with the same currently available therapies, including drugs, implantable cardioverter defibrillators and electrical resynchronization [56]. Despite being effective in slowing disease progression and reducing the risk of death and ventricular arrhythmias, such therapies, most of which were originally developed in the setting of ischemic CMP, are not specifically aimed at counteracting the underlying disease mechanisms of genetically determined CMPs. Identifying and, if possible, specifically targeting the pathophysiological pathways involved might be more important for some subtypes of genetic CMP than others. For instance, patients harboring titin (TTN) variants were shown to have a significantly better left ventricular reverse remodeling under conventional optimized treatment than patients with LMNA pathogenetic variants, who are known for being poor responders to conventional treatments and in great need of new specific treatments [65,66]. Concerning LMNA-related cardiomyopathies, which are the most studied nuclear envelope-related cardiomyopathies so far, several pathophysiological hypotheses have been proposed [67]. The main three include a “structural hypothesis”, which attributes lamin-associated phenotypes to cellular morphological abnormalities; a “mechanotransduction hypothesis”, which relies on an abnormal response to mechanical stressors; and a “gene expression hypothesis”, which proposes that mutation-induced defects attributable to abnormal chromatin organization may lead to the abnormal control of gene expression and signaling pathways. Concerning the main signaling pathways identified so far in LMNA-related cardiomyopathies, these include the PI3K (Phosphoinositide 3-kinase) pathway, the MAPK pathway and the cGAS pathway [68]. The last one was very recently studied by Cheedipudi et al. who highlighted the crucial role of double-strand breaks in the pathogenesis of LMNA cardiomyopathy [69]. These double-strand breaks, which are released into the cytoplasm, are detected by cyclic guanosine monophosphate–adenosine monophosphate synthase (cGAS), thereby initiating DNA damage response (DDR) pathways. The findings of this study suggest that targeting the CGAS/DDR pathway may offer therapeutic potential for the treatment of LMNA cardiomyopathy. Regarding the other two pathways, several MAPK inhibitors have demonstrated positive effects in mouse models, including PD98059, selumetinib and SP600125 [68]. Notably, the effectiveness of the first human implant of a cardiac contractility modulation device in delaying HF progression in a patient with advanced LMNA-related dilated cardiomyopathy was recently reported and associated with MAPK inhibitions [70]. Finally, ARRY-371797, a small-molecule inhibitor targeting p38α of the MAPK pathway, was successfully used in an LMNA H222P mouse model, and a Phase 2 clinical trial was completed [71]. Unfortunately, a Phase 3 randomized, double-blind, placebo-controlled clinical trial (REALM-DCM) was recently terminated due to futility without any safety concerns, stressing once again the unmet treatment needs remaining among patients with LMNA-related dilated cardiomyopathy [72].

The expansion of our knowledge of SYNE-related CMPs might also lead to the development of targeted therapies.

4. Conclusions

Nesprin proteins, encoded by the SYNE-1 and SYNE-2 genes, play a pivotal role in maintaining nuclear integrity and linking the nucleus to the cytoskeleton in muscle cells, including cardiomyocytes. SYNE variants can lead to a variety of clinical presentations, from arrhythmias and conduction blocks to dilated and even hypertrophic cardiomyopathy, underscoring the importance of the LINC complex in cardiac function. The case series hereby presented, albeit mostly including VUSs, highlights the potential variability in the clinical expression of SYNE genetic variants. Further research is essential to better understand the mechanisms by which SYNE variants may contribute to cardiac disease to define the indication for SYNE genetic variant assessment in routine clinical practice and to potentially develop targeted therapeutic strategies.