Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin

Abstract

Nucleolar stress, characterized by loss of nucleolar integrity, has not been described in the cardiac context. In addition to ribosome biogenesis, nucleoli are critical for control of cell proliferation and stress responses. Our group previously demonstrated induction of the nucleolar protein nucleostemin (NS) in response to cardiac pathological insult. NS interacts with nucleophosmin (NPM), a marker of nucleolar stress with cytoprotective properties. The dynamic behavior of NS and NPM reveal that nucleolar disruption is an early event associated with stress response in cardiac cells. Rapid translocation of NS and NPM to the nucleoplasm and suppression of new preribosomal RNA synthesis occurs in both neonatal rat cardiomyocytes (NRCM) and cardiac progenitor cells (CPC) upon exposure to doxorubicin or actinomycin D. Silencing of NS significantly increases cell death resulting from doxorubicin treatment in CPC, whereas NPM knockdown alone induces cell death. Overexpression of either NS or NPM significantly decreases caspase 8 activity in cultured cardiomyocytes challenged with doxorubicin. The presence of altered nucleolar structures resulting from myocardial infarction in mice supports the model of nucleolar stress as a general response to pathological injury. Collectively, these findings serve as the initial description of myocardial nucleolar stress and establish the postulate that nucleoli acts as sensors of stress, regulating the cellular response to pathological insults.

Fig. 1.

Nucleolar disruption is a common response to stress in neonatal rat cardiomyocytes and cardiac progenitor cells. (A and B) Single channel confocal scanning and merge showing NRCMs after 4 h (A) and CPCs after 8 h (B) of treatment with DMSO (Top), 1 μM DOX (Middle) or 0.05 μg/mL ActD (Bottom). NS (green) and NPM (red) colocalize within nucleoli of DMSO-treated cells and delocalize into the nucleoplasm of DOX- and ActD-treated cells. Phalloidin (Phal, purple) for cell structure and sytox blue (Nuc, Blue) for nuclei are shown as a merged image (Mer). (Scale bars: 10 μm.)

Fig. 2.

Nucleolar disruption is associated with impairment of new rRNA synthesis and precedes apoptosis. DOX and ActD inhibit new rRNA synthesis in NRMCs (A) and CPCs (B). Levels of pre-rRNA were measured by qRT-PCR and normalized to 18S expression (n = 3 for each group, *P < 0.05 vs. 0). (C) Phosphorylation of histone H2AX (γH2AX), assessed by FACS analysis, significantly increases at early time points and in parallel with nucleolar stress in NRCMs (n = 3 for each group, *P < 0.05 vs. 0). (D) γH2AX levels increased similarly in CPCs and in parallel to p53 as assessed by measuring γH2AX⁺/p53⁺ cells (n = 3 for each group, *P < 0.05 vs. 0). (E and F) Cell death, estimated by FACS analysis, increases with time in DOX-treated NRMCs (E) and CPCs (F; n = 3 for each group, *P < 0.05 vs. DMSO).

Fig. 3.

Gain and loss of function studies. (A) Cell death is increased in CPCs silenced for NS (ShNS) and then challenged with DOX for 16 h. NPM silencing (ShNPM) increases cells death even under basal conditions. CPCs overexpressing a scramble shRNA (ShSc) were used as control (n = 4, *P < 0.05). (B) Confocal images showing CPCs infected with ShSc, ShNS ShNPM with a cell showing fragmented nucleoli (arrow), and a nearby cell infected with low efficiency showing regular nucleoli and normal levels of NS and NPM (arrowhead). NS (green), NPM (red), Phalloidin (Phal, purple), nuclei (Nuc, blue). (Scale bars: 10 μm.) (C) Increased cell death in NRCMs challenged for 16 h with the NPM-specific inhibitor NSC348884 (NPMi). Cotreatment with DOX does not further increase cell death (n = 3, *P < 0.05). (D) NRCMs overexpressing NS or NPM show decreased caspase 8 activation compared with GFP-overexpressing cells after 4 and 8 h of DOX treatment (n = 3,*P < 0.05).

Fig. 4.

Doxorubicin induces nucleolar stress in vivo. (A) Confocal images of heart sections from mice at postnatal day 7 injected with DMSO (Upper) or 15 mg/kg DOX (Lower) and killed 24 h later. NPM (green), desmin (red). Cardiomyocytes from DOX-treated mice display delocalization of NPM (arrows). (Scale bars: 40 μm.) (B) Representative immunoblot showing p53 accumulation in heart from DOX-treated mice. (C) pre-rRNA expression was down-regulated in DOX-treated mice (n = 3; *P < 0.05 vs. DMSO). (D) Up-regulation of mdm2 and p21 genes confirming the activation of p53 pathway in DOX-treated hearts (n = 3; *P < 0.05 vs. DMSO).

Fig. 5.

NS and Nucleophosmin are induced after pathological challenge in the heart. (A and D) Confocal images showing adult cardiomyocytes in infarcted (A, MI) and in TAC (D, TAC) hearts 4 d after surgery. NS (green), NPM (red), and desmin (Des, blue) are shown. (Scale bars: 10 μm.) (B and E) Immunoblot quantifications of MI (B) and TAC (E) heart lysates showing significant increase of NS and NPM levels over time. (n = 3 mice per group for each experiment, *P < 0.05). (C) qRT-PCR showing increase in pre-rRNA level in LV from MI hearts in comparison with time matching LV from sham operated mice (CON; n = 3,*P < 0.05). (F) qRT-PCR showing increase in pre-rRNA level in LV from TAC hearts in comparison with LV from control mice (0; n = 3,*P < 0.05).