Stressing on the Nucleolus in Cardiovascular Disease

Abstract

The nucleolus is a multifunctional organelle with multiple roles involving cell proliferation, growth, survival ribosome biogenesis and stress response signaling. Alteration of nucleolar morphology and architecture signifies an early response to increased cellular stress. This review briefly summarizes nucleolar response to cardiac stress signals and details the role played by nucleolar proteins in cardiovascular pathophysiology.

Introduction

The nucleolus is a suborganelle within the nucleus and was one of the first intracellular structures identified by simple light microscopy back in the mid-1800s [1]. Nucleoli are disassembled during cell division and re-formed at the end of mitosis around chromosomal regions termed nucleolar organizing regions consisting of tandem repeats of ribosomal DNA (rDNA) [2, 3]. With the identification of ribosomal RNA (rRNA) and proteins within the nucleolus, the organelle was determined as the site for nascent ribosomal biogenesis and assembly, nearly a century after its initial discovery[1, 2]. Over the past few decades however, the nucleolus has been associated with many diverse cellular functions. Of 4500 nucleolar proteins discovered, approximately 30% are linked to ribosomal assembly and processing [3, 4] and a predominant population of nucleolar proteins play vital roles in cell cycle control, signal recognition particle assembly, cell growth, microRNA biogenesis, viral replication, cell death, nuclear transport, stem cell fate and commitment, cellular senescence, and stress response signaling in eukaryotic cells[1, 2, 5–10]. This review discusses the role of the nucleolus and nucleolar proteins in regulating cardiovascular pathophysiology.

Nucleolus as a stress sensor in cardiac disease

The mammalian nucleolus is normally tripartite having three morphologically distinct subcompartments, the fibrillar center, the dense fibrillar component and the granular component (Figure 1) [1, 2, 11]. The nucleolus functions as a sensor of cellular stress and responds by undergoing morphological and molecular reorganizations of its architecture (Figure 2) [3, 12]. Nucleolar enlargement is indicative of increased protein synthesis and growth and is one of the early changes observed in hypertrophied human hearts [13, 14]. Neonatal rat cardiac myocytes treated with phenylephrine, an α1-adrenergic receptor agonist and stressed cardiac myocytes in the border zone of a myocardial infarction (MI) also display enlarged, irregularly shaped nucleoli [15]. An increase in nucleolar size concomitantly associated with alterations in the ultrastructure is observed during ischemic and dilated cardiomyopathies in humans, wherein nucleoli become less granular and more fibrillar, suggestive of increased ribosomal activity [16]. Nucleolar hypertrophy also results from polyploidy and endoreduplication of chromosomes [13]. Disruption of the nucleolus is common in response to DNA damage, genotoxic stress, change in temperature, hypoxia, and treatment with chemotherapeutic drugs that inhibit transcription and ribosomal subunit processing [3, 12, 17, 18]. The nucleolus shrinks, segregates (separation of granular component and fibrillar center upon compaction of the nucleolus) [19] or fragments (decondensation and unraveling of rDNA) [12] upon receiving cues for increased cellular stress. Treatment with chemotherapeutic drugs Actinomycin D and doxorubicin in cardiac myocytes causes decondensation and shrinkage of the nucleolus with delocalization of nucleolar stress sensor proteins [15]. Nucleolar and ribosomal stress have been demonstrated to elicit p53 dependent and independent signaling pathways to activate stress response signaling, which in turn leads to increased apoptosis, cell cycle arrest or senescence in a cell type and stimulus dependent manner [3, 17, 20, 21].

Figure 1.

The nucleolus is tripartite and has 3 subcompartments – Fibrillar center (FC, black), dense fibrillar component (DFC, red) and granular compartment (GC, green)

Figure 2.

The nucleolus functions as a stress sensor and responds by undergoing enlargement or disruption leading to further ultra structural modifications. The characteristics and sources of nucleolar stress are listed based on the effect seen (see text for details). Nucleolar subcompartments are not shown.

Nucleolar organizing region as a determinant of cardiac pathology

Nucleolar organizing regions (NORs) are tightly aggregated chromosomal DNA involved in transcription of rRNA and present in the nucleoli of cells in interphase [22–24]. Since NORs contain acidic, non-histone proteins which have increased affinity for silver ions, staining with silver (Ag) acetate enables easy visualization of NORs (termed Ag-NOR) [24]. Ag-NORs which are identified as black dots under the light microscope correspond to the proteins in the fibrillar center and dense fibrillar component and correlate with cellular proliferation rate in cancer cells [24, 25]. Activity of cardiac NORs correlates positively with myocardial weight, left ventricular wall thickness and maximal diastolic pressure in hypertensive hearts [26], suggestive of increased NOR and nucleolar activity during hypertrophy [13]. Ag-NORs are decreased in cardiomyocytes of hearts with severe ischemia and heart failure, owing to decreased metabolic activity and diminished rRNA synthesis [26, 27]. Cardioplegia or temporary cardiac arrest also decreases Ag-NORs, while reperfusion increases them [27, 28]. Doxorubicin induced cardiotoxcity increases number of Ag-NORs, but also causes them to be morphologically altered (enlarged and rod shaped compared to small round dots) [22]. Collectively, these observations imply that NORs function as highly sensitive indicators of cardiac function and disease.

The following section will detail the role played by evolutionarily conserved nucleolar proteins in regulation of cardiovascular diseases. Although a myriad of proteins translocate into the nucleolus in response to stress signaling and either activate or attenuate different signal transduction pathways, for purposes of this review, we will focus only on four proteins which are predominantly localized to and function mainly within the nucleolus.

Nucleolar proteins associated with cardiac pathophysiology

Nucleolin

Nucleolin (Ncl or C23) is a multifunctional, nucleolar phosphoprotein with important roles in ribosomal biogenesis, cell cycle regulation, growth, cell death and signal transduction [29–31]. Ncl is mainly associated with cell growth and pro-survival responses in the heart, consistent with loss of proliferation and increased apoptosis observed upon Ncl downregulation in C2C12 skeletal myoblasts [32]. Transcriptional regulation of Ncl occurs in rat cardiac myocytes during development and early postnatal growth, when cardiomyocytes retain their limited proliferative potential. However, translation but not transcription is increased in cardiac myocytes during hypertrophic growth indicating differential regulation of Ncl in response to hyperplastic and hypertrophic stimuli [33]. Ncl is required for cell survival and mediates the antiapoptotic effects downstream of heat shock protein 70 upon oxidative stress in cardiac myocytes [34]. Transgenic mice with cardiac specific overexpression of Ncl are resistant to ischemia-reperfusion injury and have reduced infarction size and cell death [35]. The important role played by Ncl in regulating cell proliferation and survival also has implications in heart diseases in humans. Patients receiving heart transplant show 9 fold higher levels of antibodies against Ncl, suggesting that Ncl inhibition is associated with coronary artery disease and allograft failure in heart transplant patients owing to increased apoptosis and decreased endothelial cell proliferation [36]. Although predominantly nucleolar, Ncl translocates to the nucleoplasm, cytoplasm and cell surface in proliferating endothelial cells [37, 38]. Antibodies against cell surface Ncl inhibit angiogenesis in vitro and in vivo [38, 39]. Increases in Ncl protein expression and nucleolar localization are observed during ischemic and dilated cardiomyopathies, which also correlate with altered left ventricular dimensions and cardiac function [16]. Therefore as a pro-survival and pro-growth molecule, Ncl is involved in regulating the structure and function of the heart.

Nucleostemin

Nucleostemin (NS) also known as guanine nucleotide-binding like 3 (gnl3) is highly enriched in cancer and stem cells and has a conserved role in regulating proliferation and cell cycle progression [5, 40–43]. NS has been implicated in pre-rRNA processing and ribosomal biogenesis [44–46], although an initial study identified NS to be predominantly localized to the granular component of the nucleolus, away from the site of rRNA biogenesis (which occurs in the fibrillar center and dense fibrillar compartment) [47]. Maintenance of stem cell pluripotency, regulation of telomere length, inhibition of stem cell differentiation, senescence and cell death are among the pivotal roles played by NS [5, 48–52]. Our group has been actively studying the role of NS in the heart and cardiac cells for the past few years [15, 53]. NS expression is increased during cardiac development and declines rapidly after birth with age. Cultured neonatal cardiac myocytes and c-kit+ cardiac progenitor cells (CPCs) with increased proliferative potential express high levels of NS. NS expression is lost upon commitment of CPCs, but is reinduced upon cardiac injury in the nucleoli of CPCs and cardiac myocytes in the border zone of the ischemic heart [53]. Pressure overload induced cardiac hypertrophy and mouse models of chronic dilated cardiomyopathy also display elevated NS levels, suggesting a role for NS in proliferative and survival signaling in the heart [15, 53]. Consistently, loss of NS increases cell death in response to DNA damage [15]. NS has been linked with maintenance of nucleolar architecture in cancer cells [54], but our findings demonstrate a more compelling role for an interacting partner of NS in the maintenance of nucleolar integrity in cardiac cells [15] (described in detail below). NS also preserves telomere length in CPCs by increasing expression of telomerase reverse transcriptase and proteins that positively regulate telomere length [53]. NS is required for cell cycle progression, maintenance of CPC stemness and antagonizing cardiac senescence (Hariharan N and Sussman MA, unpublished results). Multifunctional roles in stem cell biology make NS an ideal candidate to enhance myocardial regeneration and antagonize aging.

Nucleophosmin

Nucleophosmin (NPM or B23) also is a nucleolar phosphoprotein that functions as a histone chaperone [55, 56]. NPM regulates cell survival and proliferation and is required for maintenance of DNA integrity and chromosome stability [55, 57]. Mutations in NPM gene affect growth characteristics in large mammals [58, 59]. Importantly, NPM functions as a nucleolar stress sensor in noncardiac [60, 61] and cardiac cells [15], as demonstrated previously by our group. Delocalization of NPM and NS is an early response to transcriptional and genotoxic stress stimuli in cardiac myocytes and in the adult mouse heart [15]. Interestingly however NPM, but not NS, is required for maintenance of nucleolar architecture in CPCs [15]. Loss of NPM causes nucleolar fragmentation, inhibits pre-rRNA synthesis and induces apoptosis in cardiac progenitor cells and myocytes, while activating p53 mediated stress response signaling [15]. NPM expression increases in mouse hearts after myocardial infarction and pressure overload-induced hypertrophy, however a select group of myocytes in the border zone of MI and hypoxic myocytes in vitro undergoing nucleolar stress exhibit delocalization of NPM and NS [15]. NPM is also identified as one of the genes that is upregulated 2 fold in the atria of dogs fed with high fat diet to induce obesity-related hypertension [62, 63]. The important role of NPM in regulating cell proliferation and growth suggests that NPM could be contributory to early remodeling during atrial hypertension [62]. NPM also regulates activity of nuclear factor kappa-light chain enhancer of activated B cells (NF-kB) in endothelial cells during aging [64, 65]. Expression of NPM mRNA increases in carotid arteries and hearts of aged rats, concomitantly associated with increased NF-kB activity, implying a role for NPM in regulating oxidative stress response and proinflammatory pathways associated with cardiovascular aging [64]. Studies also link NPM to the onset of atherosclerosis. Human vascular endothelial cells treated with oxidized low density lipoprotein show dephosphorylated levels of NPM correlating with decreased proliferation and increased cellular dysfunction [66], consistent with studies demonstrating enhanced endothelial cell proliferation correlating to increased NPM phosphorylation upon treatment with cardioprotective drugs [67].

Fibrillarin

Fibrillarin (Fbl) is a methyl transferase enzyme that is a component of different ribonucleoproteins, including nucleolar small nuclear ribonucleoprotein (snRNP) and small nucleolar ribonucleoproteins (snoRNP) [68–70]. Located in the dense fibrillar compartment of the nucleolus, Fbl is involved in the initial steps of pre-rRNA processing and is essential for embryogenesis and small nucleolar RNA (snoRNA) synthesis [68, 69, 71]. Fbl is associated with NCl in the formation of NORs, and in the rapid change in NOR numbers during conditions like cardioplegia and reperfusion in humans undergoing heart surgery [28]. Autoantibodies against Fbl are generated during systemic sclerosis, an autoimmune disease characterized by increased fibrosis in the skin and visceral organs like the heart. Presence of Fbl antibodies predicts the extent of the disease and identifies patients with renal disease, pulmonary hypertension and myositis as a consequence of systemic sclerosis [72].

Conclusion

The nucleolus is at the hub of proliferative, prosurvival and growth reponse signaling. However, the effects played by nucleolar proteins have been largely overlooked in the cardiac context. Cardiac-specific conditional knockout mice models of nucleolar proteins are yet to be created and characterized. Detailed studies on the role and function of nucleolar proteins and the nucleolus will provide mechanistic insights into several cardiovascular diseases and may hold the key to therapeutic options in the future.

Highlights.

• The nucleolus functions as a stress sensor in cardiovascular disease.

• Nucleolar enlargement and disruption are consequences of nucleolar stress.

• Nucleolar proteins are important for cardiac growth and survival.