Nucleoporins in Cardiovascular Disease

Abstract

Cardiovascular disease is a pressing health problem with significant global health, societal, and financial burdens. Understanding the molecular basis of polygenic cardiac pathology is thus essential to devising novel approaches for management and treatment. Recent identification of uncharacterized regulatory functions for a class of nuclear envelope proteins called nucleoporins offers the opportunity to understand novel putative mechanisms of cardiac disease development and progression. Consistent reports of nucleoporin deregulation associated with ischemic and dilated cardiomyopathies, arrhythmias and valvular disorders suggests that nucleoporin impairment may be a significant but understudied variable in cardiopathologic disorders. This review discusses and converges existing literature regarding nuclear pore complex proteins and their association with cardiac pathologies, and proposes a role for nucleoporins as facilitators of cardiac disease

1. Introduction

New light is being shed on the etiology of cardiac pathologies by studies focused on the role of discrete nuclear envelope components [1, 2]. Specifically, the role of proteins within the nuclear pore complex (NPC) called nucleoporins (NUPs) have been noted as potential causes for a variety of cardiac diseases ranging from atrial fibrillation to cardiomyopathy [3–6]. NPCs, first identified in the 1950s by Callan and Tomlin [7], are comprised of multiple subcomplexes that form a physical channel between the nucleus and cytoplasm to regulate macromolecular transport across the nuclear envelope [8, 9]. The canonical role of NPCs in nucleocytoplasmic trafficking is well characterized, in which cargoes bound for either nuclear or cytoplasmic compartments are respectively dependent on specific nuclear localization or export signaling sequences [10]. This requires coordination of multiple soluble transport receptors, cofactors, and nuclear envelope bound components that work synergistically to facilitate transport [11], in addition to employing a GTP gradient across the nuclear envelope that provides the driving force for nucleocytoplasmic translocation [11, 12]. Furthermore, phosphorylating enzymes such as MAP kinases, CaM kinases, and lipid dependent kinases have been reported to regulate transport processes and nuclear pore expression [13].

Recent high resolution molecular mapping studies of NPC anatomy have advanced the knowledge of how subcomplexes that comprise the NPC are oriented in three dimensional space as well as their intra- and inter- modular interactions [14]. Each of these subcomplexes are composed of discrete NUPs localized to specific regions of the NPC [15, 16] (Figure 1). In the mammalian NPC, NUP45/58, NUP54, and NUP62 are part of a trimeric complex that make up the central channel of the NPC [17]. POM121, NDC1, and NUP210 are integral membrane bound NUPs that anchor the NPC to the nuclear membrane. TPR, NUP153, and NUP50 comprise the nuclear basket found on the nucleoplasmic face, with NUP358, NUP214, NUP88, ALADIN and NUP42/hCG1 located on the cytoplasmic side. The inner ring (NUP35/53, NUP93, NUP155, NUP188, NUP205) [18] and Y-subcomplex (NUP37, NUP43, NUP75/85, NUP96, NUP107, NUP133, NUP160, SEC13, SEH1L and ELYS [8] provide structural support to the NPC, while NUP98 is found on both cytoplasmic and nucleoplasmic sides of the NPC [16, 19, 20]. In addition, each of these subcomplexes possess distinct prioritized functions (Table 1). For example, the cytoplasmic, central channel, and nuclear basket regions mediate nucleocytoplasmic trafficking and harbor phenylalanineglycine (FG) rich NUPs that define the transport dynamics of the NPC [8, 21]. Considering the nature of the molecular cargo moving in and out of the nucleus, it is noteworthy that the cytoplasmic regions contains NUPs that specialize in finalizing mRNA export [22], while the nuclear face of the NPC possesses NUPs optimized for chromatin binding and remodeling [3, 23–26]. The Y-complex and inner ring complexes are composed of scaffold NUPs that do not possess these FG repeats [27], while the membrane bound NUPs are characterized by transmembrane domains that define their localization within the NPC [28–30]. In total, ~30 different NUPs are accepted as building blocks of the contemporary NPC [8, 31].

Figure 1.

Schematic illustration of the Nuclear Pore Complex (NPC) depicting the location of known nucleoporins, and their subcomplexes, within the NPC. Highlighted (*) are the mobile nucleoporins. CCR = central channel region, IR = inner ring, NB = nuclear basket, OR = outer rings.

Table 1.

Nucleoporin functions.

NPC region	Protein Symbol	Description	Function	References
Cytoplasmic	NUP358	Nucleoporin 358; RAN binding protein 2 (RANBP2)	SUMOylation enhancer	[60–62]
	NUP214	Nucleoporin 214	Regulation of protein activity	[63]
	NUP88	Nucleoporin 88	Chromatin binding	[20]
	ALADIN	ALADIN/ADRACALIN	Spindle formation and chromosomal alignment	[64]
	NUP42	Nucleoporin 42; NUPL2; HCG1; NLP1/2	RNA export/enhances RNA helicase activity	[65]
	NUP98*	Nucleoporin 98*	Chromatin binding, mRNA export	[20, 66, 67]
Y-complex	NUP37	Nucleoporin 37	NPC stabilization and assembly during mitosis	[68, 69]
	NUP43	Nucleoporin 43	NPC assembly during mitosis	[70, 71]
	NUP85	Nucleoporin 85	NPC assembly during mitosis	[70, 71]
	NUP96*	Nucleoporin 96*	mRNA export, role in immune response mediated by interferons	[72–75]
	NUP107	Nucleoporin 107	NPC assembly during mitosis	[70, 71]
	NUP133	Nucleoporin 133	mRNA export, NPC assembly during mitosis	[71, 76]
	NUP160	Nucleoporin 160	mRNA export, NPC assembly during mitosis	[70, 71, 76]
	SEC13	SEC13 Homolog, Nuclear Pore and COPII Complex Component	NPC assembly	[77]
	SEH1L	SEH1 Like Nucleoporin	Activation of mTOR signaling, NPC assembly during mitosis	[70, 78]
	ELYS	AHCTF1 gene, AT-Hook Containing Transcription Factor 1	NPC stabilization and assembly during mitosis	[68]
Inner Ring	NUP35	Nucleoporin 53	Post-transcriptional regulation; oocyte meiotic spindle formation	[79, 80]
	NUP93	Nucleoporin 93	Gene repression	[47, 81]
	NUP155	Nucleoporin 155	Chromatin binding/tethering and accessibility	[43, 51, 82, 83]
	NUP188	Nucleoporin 188	Gene repression	[81]
	NUP205	Nucleoporin 205	Gene repression	[81]
Central channel	NUP45/58	Nucleoporin 58; NUPL1	Centrosome regulation	[84]
	NUP54	Nucleoporin 54	Double strand break repair	[85]
	NUP62	Nucleoporin 62	Imprinted domain regulation; chromatin tethering; centrosome homeostasis	[51, 86, 87]
Transmembrane	POM121	Nuclear pore membrane protein 121 kDa	Transcriptional regulation; NPC assembly	[88–90]
	NUP210/GP210	Nuclear pore membrane glycoprotein 210	NE/ER homeostasis; gene regulation	[91–93]
	NDC1/TMEM48	Nuclear Division Cycle 1 homolog; Transmembrane protein 48	NPC assembly	[94]
Nucleoplasmic	NUP50	Nucleoporin 50	Chromatin binding	[19, 92]
	NUP153	Nucleoporin 153	Chromatin binding, RNA export	[3, 44, 45, 92, 95]
	TPR	Translocated Promoter Region	Heterochromatin exclusion	[48]

Provided here are functions/biological contexts documented for NUPs. In bold are NUPs with reported high mobility (NUP98, NUP210, NUP50, and NUP153) that can be found distal from the NPC within the nuclear envelope and/or the nucleoplasm.

^*NUP96 and NUP98 are translated from the same transcript, and post-translational cleavage of the protein product generates both.

In addition to the canonical transport function of the NPC, other constitutive roles include NPC biogenesis, nuclear envelope assembly, as well as functional roles in cell cycling and mitosis that are the subject of excellent studies (Table 1). Individual NUPs have other cellular roles including chromatin organization, gene expression regulation, and DNA repair, among others [32–35]. Furthermore, NUPs may temporally recruit a diversity of protein-binding partners to form distinct signaling complexes, and this may occur at the nuclear envelope or within the nucleoplasm [36–40]. In tandem with these individual functions for NUPs, a consistent and recognized role for NUPs in development is emerging [9, 41]. Indeed, evidence for NUPs directly and indirectly binding to chromatin to regulate gene expression has been demonstrated [42–47].

The ability of NUPs to spatially organize chromatin underlies their capacity as gene regulators, and several modes of dynamic chromatin regulation have been reported for specific NUPs. For example, the maintenance of heterochromatin exclusion zones within the nucleoplasm proximal to the nuclear basket of the NPC is a function of TPR [48], and was proposed to exist as a means of maintaining a transcriptionally permissive microenvironment. This notion is supported by work in the yeast homologue of TPR, Mlp1, that maintains chromatin loops as a form of transcriptional memory. This was elegantly demonstrated by work in which Mlp1 was found to bind the 5’ and 3’ ends of the HXK1 gene to promote rapid metabolic responses to substrate switching [49]. The ability for NUPs to dynamically interact with chromatin appears to be broadly conserved [50], and is not exclusive to TPR. In a model of stem cell-derived neurogenesis, the identification of NUP153 as a regulator of superenhancer activity confirmed its function as a developmental regulator with clear demonstration of its ability to mediate chromatin interactions between distal gene elements [44, 50]. Complementary to loop formation, regulation of chromatin tethering in somatic and female germline Drosophila cells has been reported for the insect homologues of NUP155, NUP93, and NUP62 [51]. In their proposed model, chromatin is constitutively tethered to the nuclear periphery via interactions with NUP155; this interaction is fully or partially released upon interaction with NUP62 or NUP93, respectively. The area of chromatin regulation and NUP function is an exciting field, yet the large-scale chromatin anchoring and release dynamics reported for NUPs is unsurprising given their ability to effect individual gene regulation.

NUP153 was shown to regulate the cardiopathogenic expression of several genes in the setting of muscular dystrophy [3]. Specifically, NUP153 was localized to promoters of specific genes in cardiac tissue to facilitate recruitment of the activating lysine acetylase CBP/p300 [3]. This was confirmed by gene NUP153 overexpression and knockdown in which respective increases and decreases of p300 and p300/CBP loci interaction and enrichment was observed. In support of a broader gene regulatory function for NUP153, recent independent work demonstrated that NUP153 also acts in concert with SOX2 to regulate superenhancer dynamics during neurogenesis [44]. Importantly, this latest evidence in cardiac and neural contexts has emerged after investigation of NUP153 chromatin binding in other metazoan systems [52], thus highlighting the prevalence and conservation of this mode of gene regulation which may serve as a paradigm for understanding other NUPs with similar mobility and DNA interaction profiles.

NUP98 was shown to recruit methylating SET1-containing complexes to promoter regions as a paradigm of gene activation in the setting of hematopoiesis [53], and was further supported by independent demonstration of NUP98-mediated enhancer-promoter looping occurring at activated genes [54]. Indirect modes of chromatin interaction by NUPs have also been reported. Earlier work by Kehat et al sought to identify binding partners of HDAC4, given its role in cardiac hypertrophy [55]. In this manner, NUP155 was identified as a direct binding partner of HDAC4, and demonstrated the ability to control chromatin localization and regulate both activation and repression of key genes associated with cardiac hypertrophy [43].

While it is recognized that disruption of homeostatic nuclear transport underlies a diversity of pathophysiological conditions [56], recognition of the expanded functional repertoire for other transport machinery like NUPs is critical for understanding their potential pathognomonic role. This is of particular importance for polygenic disorders such as cardiovascular pathologies, as improved knowledge of disease etiology may provide novel opportunities for therapeutic intervention [57–59]. Here we provide an overview regarding the role of NUPs in normal cardiac development, and summarize dysregulated dynamics for NUPs that have been associated with diverse cardiopathologies.

2. NUPs in Cardiovascular Development

The first data suggesting a role for NUPs in cardiac development was provided by reports of congenital heart defects associated with specific NUP expression disorders. Specifically, NUPs can act distal and independent of the nuclear pore affecting the establishment of cardiac asymmetry during embryonic development [96]. In a study focused on examining copy number variations in patients with heterotaxia and cardiac asymmetry disorders, a gene duplication of NUP188 was discovered that demonstrated significant heart looping defects when modeled in Xenopus [97]. Subsequent work identified impairment of ciliogenesis dependent on NUP188 and its binding partner NUP93. As cardiac looping is critically dependent on proper ciliogenic dynamics [98], the role of the NUP93/NUP188 complex in determining vertebrate laterality and proper asymmetric morphogenesis through its effects on cilia regulation established a precedent for consideration of NUPs in cardiac development. This was followed by the identification and investigation of other NUPs, such as NUP98, NUP205, and NUP210 in establishing proper cardiac asymmetry [96, 99–101].

Among these NUPs, mechanisms have been elucidated in part for NUP188, its binding partner NUP93, and NUP98. For the former, genetic and super resolution imaging studies identified NUP93/NUP188 organization into barrel structures comprising the pericentriolar material that encases the centriolar core at the ciliary base [96]. Furthermore, the observation that knockdown of NUP93 and/or NUP188 led to embryo-wide cilia loss provided evidence in support of a role for both NUPs in ciliogenesis and ultimately on cardiac laterality [96]. NUP98 has an active role in controlling ciliary length by actively gating soluble tubulin efflux out of the ciliary base [101]. In addition, localization of NUP98 to the ciliary base is controlled by NEK2-mediated phosphorylation, such that phosphorylation insensitive NUP98 mutants were found with higher frequency in the ciliary base [100]. Altered NUP98 concentrations at this location ultimately affects tubulin transport dynamics into and out of the ciliary matrix that regulates cilia length [101]. These data are currently the only evidence for a specific function for NUPs in developmental cardiac phenotypes, and remains an exciting and emerging field. Intriguingly, the bulk of existing and contemporary data has pointed to potential roles for NUPs in cardiac diseases (Figure 2), allowing for studies of a novel etiology for cardiopathogenesis.

Figure 2.

Nucleoporins associated with cardiac disorders. Representative illustration of nucleoporins associated with cardiac disorders and their localization within the nuclear pore complex, with each group highlighted in different colors. Nucleoporins associated with dilated and ischemic cardiomyopathy are highlighted in pink (NUP358, NUP62, NDC1, NUP93, NUP160, NUP153), valve disorders in blue (NUP43, NUP188), congestive heart failure (HF) in green (NUP85), heterotaxy and situs inversus in purple (NUP188, NUP205, NUP210) and arrhythmias in orange (NUP155, NUP107, NUP37, NUP153)

3. Cardiovascular Diseases and Nucleoporin Dynamics

3.1. Nucleoporins in Arrhythmias

The role of nucleoporins in arrhythmogenesis has been implicated by clinical as well as basic research studies, as shown in Figure 2 and Table 2 [5, 102, 103]. The first evidence of nucleoporins being involved in arrhythmogenesis was suggested by linkage mapping studies on an isolated South American community in which sudden cardiac death associated with atrial fibrillation (AF) was observed for pediatric cohorts from several generations within the same family [5]. Analysis revealed mutation in the NUP155 gene that resulted in an Arg to His substitution at amino acid position 391 (R391H). While heterozygous expression of this mutation in carriers were reported to have normal phenotypes without any cardiac complications, offspring with homozygous expression died perinatally from arrhythmogenic impairment [5, 108]. Follow up studies in mouse models demonstrated comparable electrophysiological abnormalities heterozygously expressing a truncated form of NUP155 [5]. Furthermore, cursory examination of cellular phenotypes revealed dysregulated NUP155 localization, suggesting that its function as part of the nuclear transport machinery could be compromised. Nuclear import was impaired in these cells, thus it was concluded that deficiencies in the nuclear transport of HSP70 and its cognate mRNA could be responsible for the electrophysiological deficits observed both in the experimental model and in the patient cohort [5]. In another clinical report investigating the molecular pathology underlying sudden unexplained nocturnal death syndrome (SUNDS), a rare NUP155 variant of uncertain significance resulting in a Leu to Phe change (L503F) was discovered in a 26 year old male reported to have died from atrial fibrillation associated with SUNDS [102]. Postmortem autopsy revealed no significant morphological differences between case and control hearts, aside from a slight but statistically relevant increase in myocardial size for SUNDS patients. Of note, for both studies that reported AF-associated NUP155 gene disruption, no evidence of abnormal myocardial geometry or deficits in cardiomyocyte ultrastructure were reported.

Table 2.

List of nucleoporins associated with cardiovascular disorders. Each nucleoporin is depicted with their respective gene and/or protein alteration associated with the respective cardiopathology and related reference.

Cardiopathology	Gene Name	Description	Gene/protein alteration	Reference
Arrhythmias	NUP155	Nucleoporin 155	missense mutation (R391H, L503F)	[5, 42, 102]
	NUP107	Nucleoporin 107	disrupted export of scn5a	[103]
	NUP37	Nucleoporin 37	nonsense mutation	[2]
	NUP153	Nucleoporin 153	protein overexpression	[3]
Dilated Cardiomyopathy	NDC1	NDC1 transmembrane nucleoporin	protein overexpression	[4]
	NUP160	Nucleoporin 160	protein overexpression	[4]
	NUP93	Nucleoporin 93	protein overexpression	[4]
	NUP153	Nucleoporin 153	protein overexpression	[3, 4, 104]
	NUP358/RANBP2	RAN binding protein 2	gene down regulation	[104]
Ischemic Cardiomyopathy	NDC1	NDC1 transmembrane nucleoporin	protein overexpression	[4]
	NUP160	Nucleoporin 160	protein overexpression	[4]
	NUP153	Nucleoporin 153	protein overexpression	[3, 4, 104]
	NUP358/RANBP2	RAN binding protein 2	gene down regulation	[104]
	NUP62	Nucleoporin 62	protein changes	[105, 106]
Congestive Heart Failure	NUP85/FROUNT	Nucleoporin 85	protein overexpression and cytosolic mislocalization	[107]
Valve, vascular disorders	NUP188	Nucleoporin 188	Splice-donor site mutation, gene duplication	[2, 96]
	NUP43	Nucleoporin 43	nonsense mutation	[2]
Heterotaxy/Situs inversus	NUP205	Nucleoporin 205	missense mutation	[99]
	NUP210	Nucleoporin 210	missense mutation	[99]
	NUP188	Nucleoporin 188	gene duplication	[97]

Systems biology analysis of a heterozygous nup155 exon-trapped mouse embryonic stem (mES) cell line revealed potential developmental susceptibility to developing AF [42]. In this study, transcriptome remodeling occurred in mES cells that expressed truncated NUP155 as a result of the heterozygous nup155 exon trap and significant re-prioritization of Gene Ontology Consortium (GOC)-defined cell membrane interactions and functions occurred in compromised cells. Furthermore, differentiation of the NUP155 truncated mES cells into beating embryoid bodies revealed cardiomyocyte-containing contractile foci that recapitulated the electrophysiological deficits previously reported [5, 42]. Significantly, NUP155-deficient cells exhibited severely dysregulated Ca²⁺-handling, and failed to mount a proper physiological response to β-agonist treatment [42]. This systems biology analysis, together with previous clinical reports as well as independent mechanistic evidence implicating the role of NUP155 in regulating cardiac hypertrophy [43], provide intriguing evidence in support of future work to discern the potential role of NUP155 in cardiac biology. Identifying other cardiopathological variants of NUP155, as well as recapitulating these in translational human induced pluripotent stem cell models would provide clear evidence of a role for NUP155 in electrical cardiac disorders. Broadly, this would have implications for understanding idiopathic cases of AF as well as defining a novel etiology for arrhythmogenesis.

A recent animal model study by Guan and colleagues showed that increased mRNA expression of Nup107 was associated with an enhanced susceptibility to postmyocardial infarction cardiac arrhythmias [103]. They demonstrated that Nup107 regulates cardiac electrical activity by facilitating the posttranscriptional transport of Scn5a (sodium voltage-gated channel alpha subunit 5) mRNA to increase Nav1.5 protein after cardiac injury, without any effect seen in other ion channels. The increase in expression of Nup107 in diseased cardiac tissue was also reported by a previous study documenting significant Y-complex overexpression in infarcted rat myocardial tissue [80]. The Y-complex consists of NUP160, NUP133, NUP107, NUP98/96, NUP85, NUP43, NUP37, SEC13, and SEH1, and is essential for NPC assembly and mRNA transport as well as regulating microtubule polymerization at kinetochores [9, 109–112].

Whole exome sequencing (WES) analyses in patients with severe cardiovascular disease identified novel disease-causing variants in NUP37, NUP43 and NUP188 [2]. Namely, this study associated clinical atrial fibrillation and sudden cardiac death with a heterozygous nonsense variant of NUP37. Functional examination of this mutation in a zebrafish model revealed development of arrhythmias, heart failure and heart chamber abnormalities including an abnormal atrium. The other two identified variants, NUP43 and NUP188, were associated with a strong family history of valve and vascular disorders that included mitral regurgitation, mitral valve prolapse and thoracic aortic dilation [2].

3.2. Nucleoporins in Cardiomyopathy

Evidence also indicates potential associations of several NUPs with inherited cardiomyopathies (Figure 2 and Table 2). Tarazon and colleagues published work that reported patients with heart failure (HF) with specific NUP expression changes [4]. In their study, they reported on NUPs localized to distinct regions within the NPC, i.e. TPR and NUP153 for the nucleoplasmic region, NUP93 and NUP155 that represented the inner ring subcomplex, NUP160 from the Y-subcomplex, and NDC1 from the integral membrane proteins [4], and identified increased protein expression of NDC1, NUP160, NUP153, and NUP93 in HF patients. In addition to significant increases in NUP protein expression, NDC1 was observed to distribute within the nucleoplasm under cardiomyopathic conditions [4], in contrast to its well-established localization as a transmembrane NUP anchoring the NPC to the nuclear envelope [113].

In recent studies, a novel correlation between increased expression of NUP153 has been associated with compromised ventricular function in ischemic cardiomyopathy [114]. Other studies have identified NUP153 transcripts as deregulated in dilated cardiomyopathy [104] though the function of up regulated NUP153 in cardiac ischemia is not well understood [4]. Some mechanistic clues have been provided by a study carried out by Nanni et al in which NUP153 and its potential role as a mediator of nitric oxide (NO) altered signing in the setting of cardiac dysfunction was examined [3]. The authors hypothesized that NUP153-regulated NO signaling would drive epigenetic alterations that contribute to development of cardiac dysfunction. The authors discovered that post NO deregulation, acetylated regulation of NUP153 overexpression occurred in dystrophic cardiac muscle. Furthermore, this process controlled a set of genes involved in cardiac dysfunction that included: nexilin (nexn), versican (vcan), adrenergic receptor alpha 2a (adra2), and cAMP responsive element modulator (crem). This study was the first to identify novel roles for NUP153 in dystrophic cardiac pathology. Mechanistically, the authors reported several novel findings in support of their conclusion that post-translational acetylation of NUP153 is regulated due to NO and oxidative stress. First, NUP153 modification significantly reduced P300/CBP-associated factor (PCAF) and p300 specific activities [3]. This stress-mediated regulation of NUP153, and its role as an epigenetic regulator, was a novel characteristic of the dystrophic heart. Second, the study reported lysine acetyltransferases (KATs) and NUP153 as binding partners in hearts of Dmd^mdx mice, an established model of Duchenne’s muscular dystrophy (DMD). In comparison, the expression of vcan, adra2, nexn, and crem were all deregulated in the presence of acetylated NUP153 in 5-month old Dmd^mdx mice. This finding suggests that these conditions could be associated with a pathological program of cardiac remodeling. Third, Nanni et al used cardiomyocytes derived from induced pluripotent stem cells (iPSC) isolated from DMD patients. Their finding showed that cardiomyocytes derived iPSC had increased NUP153 protein expression and acetylation, plus abnormal expression and distribution levels of nexilin. With this, Nanni et al provided evidence for NUP153 and its role in altered NO signaling, with important epigenetic implications for gene regulation in dystrophic hearts. Importantly, this study identifies a potential therapeutic target for cardiomyopathy and other cardiac disease related to NO dysfunction [3].

3.3. Nucleoporins in other cardiovascular diseases

3.3.1. Cardiac Disorders

Other cardiovascular disorders, besides arrhythmias and cardiomyopathies, have been linked to nucleoporin dysregulation. Congestive heart failure (CHF) has been clinically associated with nucleoporin 85 (FROUNT) [107]. NUP85 was thought to be correlated with C-C chemokine receptor 2 (CCR2) and was associated with a function in the chemokine system, which is involved in the development of CHF [107]. In the study presented by Satoh and colleagues, they examined biopsied tissues obtained from CHF and non-HF patients. The team found that the levels of CCR2 and CCL2 mRNA were closely correlated to NUP85 mRNA levels in patients with CHF. The levels of NUP85 mRNA in relation to CCR2 and CCL2 were also found to be related to left ventricle systolic dysfunction [107]. They concluded that the levels of NUP85, CCR2, and CCL2 were higher in patients with severe, compared to mild, CHF.

Recently, clinical mutations in NUP205 and NUP210 were identified as potential etiological determinants underlying heterotaxy and situs inversus totalis (SIT) [99]. In this study, whole exome and genome sequencing was performed using samples from patients with heterotaxy and SIT in which the authors reported several bi-allelic missense mutations for NUP205 and NUP210 [99]. Generation of induced pluripotent stem cells for functional analysis from the patient expressing the bi-allelic NUP205 T1044M/P1610R mutation revealed that NUP205 did not localize to the nuclear envelope. Furthermore, mutant NUP205 demonstrated impaired binding with its cognate inner ring subcomplex partners as well as a diminished interaction with the nuclear export factor XPO1 [99].

Left-right asymmetry deficits have been associated with dysregulated nucleoporin-driven mechanisms. In this regard, NUP98 interacts with NEK2 kinase to regulate cilia synthesis and resorption dynamics that ultimately affects proper cardiac looping [100, 101]. In a departure from nuclear transport-related modes of action, NUP98 located at the ciliary base stabilizes the cilium and prevents NEK2-mediated resorption. Phosphorylation of NUP98 by NEK2 is proposed to be the main protein modification of NUP98 that regulates both cilia resorption and nuclear envelope breakdown at the G2/M transition that ultimately affects cardiac looping.

Other NUPs at the nuclear envelope also contribute to proper myocardial asymmetry, with a number of clinical instances underscoring the role of NUPs in normal cardiac development. Within the context of cardiopathology, a duplication of NUP188 was reported for patients who presented with heterotaxy sampled from a population that presented phenotypes of right atrial isomerism, left atrial isomerism, and transposition of the great arteries [97]. When modeled in Xenopus, NUP188 depletion, or knockdown of its binding partner NUP93, led to impaired cilia formation and abnormal heart patterning in the embryos downstream of aberrant cardiac looping [96]. Though the experimental approach diminishing Xenopus NUP188 expression did not recapitulate clinical NUP188 overexpression, the authors concluded that abnormal NUP188 expression in general impaired ciliogenesis that disrupted cardiac looping and proper establishment of left-right asymmetry [96].

The association of cardiac asymmetry defects with NUP205, NUP188, and NUP93 is unsurprising given the interaction of these proteins in the NPC. [18, 81] Along with NUP155 and NUP35, these five nucleoporins form the inner ring subcomplex of the NPC. [18] Interestingly, although no cardiac developmental symmetry problems have been reported for NUP155 or NUP35, there have been cardiac phenotypes associated with these NUPs. Arrhythmogenic deficits are associated with NUP155 as noted in the present review, while cardiac ischemia reperfusion injury has been reported to involve dysregulation of NUP35 via its ability to regulate expression of the sodium-hydrogen exchanger NHE1 in cardiomyocytes [80].

NHE1 is critical to the pathogenesis of cardiac ischemia-reperfusion injury [115]. In brief, increased cytosolic acidification during cardiac ischemia stimulates sodium hydrogen exchange, bringing in one Na⁺ while extruding one H⁺, with acute increases in activity triggered by internal pH levels less than 7.2 [116]. This sodium influx is functionally coupled to the electrogenic Na⁺/Ca²⁺ exchanger that moves three Na⁺ out of the cell for uptake of one Ca²⁺ and whose different isoforms can confer variable susceptibilities to calcium overload in the subsequent reperfusion phase [117, 118]. This can lead to paradoxical increases in diastolic length of the cardiomyocyte, impaired contractility, and cardiomyocyte death [119–122]. Within the molecular sequelae of ischemia reperfusion injury, it was recently discovered that NUP35 post-transcriptionally regulates NHE1 expression [80]. Mechanistically this occurs by interaction of an Nterminal RNA recognition motif (RRM) within NUP35 with the 5’-UTR of NHE1 mRNA to promote its nuclear export efficiency and availability for translation. It was also observed that in an animal model of acute myocardial infarction, concomitant down regulation of NUP35 and NHE1 was observed 3 days following following ischemic injury. Overexpression of NUP35 rescued ischemia-induced downregulation, in further support of a potential role for NUP35 in regulating cardiomyocyte pH homeostasis. Indeed, other work has identified NUP35 as a bona fide cardiac RNA binding protein defined by a classic RNA binding domain within its RRM [123]. Given this, it is likely that NUP35 has the ability to bind a variety of isoforms and RNAs other than NHE1, and may function in this capacity in other tissues.

Cardiac disorders that manifest as secondary phenotypes within a syndrome related to nucleoporin expression disorders have been reported. For example, dysregulation or mutation in AAAS leads to Triple A syndrome in which patients present with poor heart rate variability that is a result of overall dysautonomia in affected individuals [124]. In another example, myocarditis associated with Coxsackievirus B3 (CVB3) infection was proposed to occur as a result of virus-mediated inhibition of NUP98 expression that stimulated Th17 cell differentiation [125]. It is clear from these examples that the causal relationship between heart disease and nucleoporin biology is more complicated than a source-to-sink model of disease etiology. With recognition of the expanded functional repertoire of individual NUPs, it is thus critical to include investigations of non-transport modes of regulation as potential mechanisms that when disrupted give rise to heart disease.

3.3.2. Vascular Disorders

A role for NUPs in vascular disorders has been reported, where they may function to drive vascular smooth muscle cell proliferation (VSMC) in the setting of neointimal hyperplasia [126]. Similar to nucleoporin dynamics in cases of clinical cardiac disease, overexpression of NUP62 and NUP153 were observed in an animal model of stress-induced VSMC proliferation. This is corrobrated by independent work showing that inhibition of NUP153 inhibits angiogenesis in a HUVEC culture model [127]. Additional supporting evidence demonstrates that increases in VSMC growth, import dynamics and NUP expression occurs in response to mechanical stimulation [13] and oxidized LDL exposure [128, 129]. Furthermore, mislocalized NUP153 is associated with aging VSMCs [130] that may be facilitated in part by NUP-mediated effects on the RAN gradient critical for nucleocytoplasmic transport [131]. In these aging cells, NUP153 was observed to relocalize away from the nuclear envelope into the nucleoplasmic interior. It is unclear if the loss of critical interactions at the nuclear envelope or binding to chromatin or protein within the nuclear interior is the main consequence of increased nucleoplasmic localization. Given that intranuclear mobility is a normal function and characteristic of NUP153, it may reflect a dysregulation in what NUP153 interacts with. These emerging data point to potential and specific functional roles for nucleoporins in vascular development and disease that remains to be studied further.

4. NUPs, -omics, and cardiac systems biology

Pioneering DamID studies were among the first high throughput approaches to identify chromatin wide occupation of model genomes by various NUPs. Early work in Drosophila revealed that NUPs bound genes discretely and distally from the nuclear periphery, indicating a genomic occupancy function for each of these NUPs [20, 52]. As an example, SEC13 and NUP98 enriched at transcriptionally active sites in accordance with developmental timing, while NUP88 occupied silent loci, supporting the notion of NUP-specific functions in the context of genome dynamics [20]. Since these initial reports, independent work has identified chromatin looping mechanisms [49, 51], nucleoporin mobility [23, 53, 132], and NUP-mediated complex recruitment and formation [44, 53] related to development and disease signaling. It is conceivable that NUPs engage a diversity of regulatory epigenomic and transcriptomic modalities, thus offering researchers the opportunity to leverage the power of high throughput technology to understand the global effects of NUP function on gene and protein expression.

In this regard, present work has examined NUPs in the context of epigenomic regulation, where NUPs bind to and regulate chromatin access via direct or indirect interactions. ChIP-seq, or variations thereof, are widely used in these studies, which has helped to identify genomic enrichment of a variety of NUPs in different developmental contexts [23, 43–45, 53, 88]. Importantly, technical and analytical caveats associated with earlier approaches can be addressed and refined with advancements in sample preparation and bioinformatic algorithms [133–135]. In one approach, to circumvent limitations associated with a lack of antibody specificity for myocardin, CRISPR-based CETCh-seq was used to identify myocardin genome occupancy profiles [135]. This is particularly powerful when applied to non-canonical genomic regulators such as NUPs that may not have optimized antibodies for ChIP-seq. Aligned with these emerging epigenomic studies are transcriptome remodeling investigations, in which differential transcriptome signatures can be profiled to detect systemic RNA expression differences. RNA based approaches, e.g. RNA-seq, PAR-CLIP, and GRO-seq, have been used to provide insight into cardiac specific transcriptome signatures, as well as sub-transcriptome composition and dynamics [136–139]. In light of recent studies that identify a subset of NUPs with defined RNA binding domains, these approaches are relevant as they may further define the role of NUPs in differential RNA metabolism [123, 140].

Complementing these genomic and transcriptomic approaches are proteomic studies that inform on the cellular machinery coded by the DNA/RNA milieu. In this regard, governing proteomic composition is in line with the canonical functions of NUPs as regulators of nucleocytoplasmic transport. Changes in NUP expression or functionality directly impact the nuclear pore complex and its function as a macromolecular transporter, and by extension molecular cargo dynamics within the cell. In support of this, the discovery of cell- and tissue- specific NPC composition and NUP isoforms argues in favor of differential transport that can facilitate proteomic remodeling [141, 142].

Integrating these multi-omic datasets is fundamental to understanding the global regulatory roles of candidate molecules in the context of cardiac development and disease. The application of network theory in this context offers opportunities to consolidate disparate (epi)genomic, transcriptomic, and proteomic datasets for a comprehensive understanding of cardiac health and disease systems biology [143–146]. Network, or graph theory, facilitates identification of molecules key to the function of the overall system, with the potential to prioritize therapeutically tractable targets [147–151]. This is particularly relevant for individualized medicine approaches, where patient variability can render standard treatments less effective [152]. In the context of investigating potential substrates for idiopathic cardiovascular disease such as NUPs, systems biology approaches identify global nuances that cannot be detected by traditional single pathway analyses. Ultimately, understanding these molecular processes from an integrated perspective unifies existing reductionist knowledge that facilitates the identification of potentially powerful interventional targets and advances overall understanding of the system as a whole [153, 154].

5. Conclusion

The NPC regulates development and disease of multiple systems via a variety of potential mechanisms. Novel studies are improving our knowledge of these processes in the context of cardiovascular disease, although many aspects of how the NPC does so, and to what extent, remain uncharacterized. To date, the dozen or so clinically reported NUP dysregulations associated with cardiac disorders may act through canonical transport dysfunction. However, evidence suggests that other epigenomic mechanisms may facilitate the development and progression of disease and understanding these will be critical to devising novel therapeutic measures that address the polygenic etiology of cardiovascular disorders. Furthermore, NUP dynamics and their association with regulatory networks is facilitated by the ongoing development of increasingly sophisticated approaches for high throughput molecular profiling. This will be key to understanding the intricate molecular foundation that predisposes and facilitates cardiopathogenesis.

Cardiovascular disease is a confounding disorder with a multigenic basis. While a number of clinical studies have demonstrated consistent nucleoporin derangement associated with disease, it remains uncharacterized whether or not nucleoporin dysregulation occurs as a cause or effect of cardiovascular pathology. Evidence for NUP malfunction as the underlying cause for cardiac disease is strongly supported by current data. However, considering the spectrum of inherited and acquired cardiac disorders associated with nucleoporin dysfunction, the possibility that NUP derangements follow cardiac insult cannot be ruled out. NUPs may act within a feedback loop as both effector and marker of cardiac disease, with an increased susceptibility to cardiopathology imposed by NUPs harboring genetic lesions. More studies in this regard, with experiments designed to specifically target NUP function in an exclusive cardiac or vascular context will be required to move forward in this field. Ultimately, identification and characterization of NUPs as regulators of CVD will provide insights into the intricate mechanisms that gives rise to cardiac and vascular pathologies, and will provide novel targets with which to treat cardiovascular disorders. Understanding NUP-driven mechanisms that contribute to disease development is paramount to developing advanced therapeutic strategies for improvement of patient longevity and quality of life, with concomitant mitigation of the social and financial burden of pervasive cardiovascular disease.

Highlights.

• The nuclear pore complex regulates development and disease of multiple systems via a variety of mechanisms.

• Identification and consistent association of nucleoporin dysfunction with clinical cardiac diseases parallels nucleoporin roles in cardiac disease etiology.

• Nucleoporins have potential roles in cardiomyopathy, arrhythmogensis, and congenital heart disease.

• High throughput molecular profiling is paramount to understanding nucleoporin association substrates that cause cardiopathogenesis and other disorders.