Nucleostemin dysregulation contributes to ischemic vulnerability of diabetic hearts: Role of ribosomal biogenesis

Diabetes is a major health problem worldwide. As well-known, diabetes greatly increases cardiac vulnerability to ischemia/reperfusion (I/R) injury, but the underlying mechanisms remain elusive. Nucleostemin (NS) is a nucleolar protein that controls ribosomal biogenesis and exerts cardioprotective effects against I/R injury. However, whether NS-mediated ribosomal biogenesis regulates ischemic vulnerability of diabetic hearts remains unanswered. Utilizing myocardial I/R mouse models, we found that cardiac NS expression significantly increased in response to I/R in normal diet (ND)-fed mice. Surprisingly, cardiac NS failed to be upregulated in high fat diet (HFD)-induced diabetic mice, accompanied by obvious ribosomal dysfunction. Compared with ND group, cardiac specific overexpression of NS by adenovirus (AV) injection significantly restored I/R-induced ribosomal function enhancement, reduced cardiomyocyte apoptosis, improved cardiac function, and decreased infarct sizes in diabetic mice. Notably, co-treatment of homoharringtonine ( HHT), a selective inhibitor of ribosomal function, totally blocked NS-mediated cardioprotective effects against I/R injury. Furthermore, in cultured cardiomyocytes, saturated fatty acids treatment, but not high glucose exposure, significantly inhibited simulated I/R-induced NS upregulation and r ibosomal function improvement. In conclusion, these data for the first time demonstrate that NS dysregulation induced by saturated fatty acids exposure might be an important cause of increased ischemic vulnerability to I/R injury in diabetic hearts. Targeting NS dysregulation and subsequent ribosomal dysfunction could be a promising therapeutic strategy for diabetic I/R injury management.

Diabetes is rapidly growing in the world due to the prevalence of unhealthy lifestyles. As well-known, ischemic heart disease (IHD) is the major cause of death in diabetic individuals. Diabetes significantly increases the mortality of IHD and exacerbates cardiac injury induced by ischemia, which is termed as ischemic vulnerability of diabetic hearts [1-3]. However, the cellular and molecular mechanisms underlying ischemic vulnerability of diabetic hearts remain largely unknown. It is of great significance to explore the underlying mechanisms of increased cardiac ischemic vulnerability in diabetic individuals.Nucleostemin (NS) is a nucleolar protein initially identified in the nucleoli of rat neural stem cells and is subsequently found highly expressed in various stem cells and cancer cells [4-7]. It is well-known that NS protein critically regulates blastocyst formation, embryogenesis, postnatal tissue regeneration, cancer development, and reprogramming to pluripotency [8]. Recently, it is reported that NS is also expressed in cardiac tissues. Interestingly, NS can be rapidly upregulated by multiple myocardial injur ies, such as myocardial infarction (MI) and trans-aortic constriction (TAC). Upregulation of NS plays an anti-apoptotic role during myocardial ischemia injury [9-11]. These results demonstrate that stress-induced NS upregulation in the cardiomyocyte may represent as a novel protective or adaptive response to myocardial injuries. Notably, cardiac NS expression is also upregulated during myocardial ischemia/reperfusion (I/R) injury. Knockdown of NS expression exacerbates cardiomyocyte apoptosis induced by hypoxia/reoxygenation ( H/R) [12]. These results suggest that NS might exert cardioprotection against myocardial I/R injury. However, whether NS plays a role in increased ischemic vulnerability of diabetic hearts remains totally unknown.

The ribosome is essential for the adaptation of a cell to multiple environmental stresses [13, 14]. Critical processes of ribosomal biogenesis include ribosomal DNA transcription, precursor rRNA (pre-rRNA) processing, and assembly with ribosomal proteins. The perturbation of any major step in the ribosomal biogenesis process triggers ribosomal stress and leads to cell death [15-18]. Accumulating evidence show that NS is an important endogenous regulator of ribosomal biogenesis by promoting pre-rRNA processing. Specially, NS can form a large protein complex that co-fractionates with the pre-60S ribosomal subunit, therefore leading to an enhancement of pre-rRNA processing. NS knockdown delays the processing of 32S pre-rRNA into 28S rRNA and decreases cellular protein abundance. In contrast, NS overexpression significantly promotes the processing of 32S pre-rRNA and enhances ribosomal biogenesis [19]. In NS mutant zebrafish models, ribosomal biogenesis is obviously disturbed [20]. As well-recognized, ribosomal biogenesis plays a regulatory role in multiple pathophysiological processes. However, the role of ribosomal biogenesis in ischemic heart injury is still unrevealed. Given the fact that NS critically regulates ribosomal biogenesis in mammalian cells, we hypothesized that NS-mediated ribosomal biogenesis might regulate cardiac ischemic vulnerability in diabetic individuals.Therefore, the present study specifically aimed (1) to determine whether or not NS-mediated ribosomal biogenesis is dysregulated in diabetic hearts during I/R; (2) if so, to determine whether NS dysregulation contributes to increased ischemic vulnerability of diabetic hearts; (3) to clarify the mechanisms underlying diabetes-induced cardiac NS dysregulation.

2.Materials and Methods:
All study protocols were approved by the Animal Care and Use Committee of the Fourth Military Medical University, and strictly followed the National Institutes of Health Guidelines on the Use of Laboratory Animals (NIH publication No. 85-23, revised 1996). Adult male C57BL/6j mice were purchased from the Laboratory Animal Center of the Fourth Military Medical University. For the study duration, all mice had unrestricted access to food and water. Mice were randomly divided into normal diet (ND) group or high fat diet (HFD) group. The HFD (60% kcal fat, D12492) and ND (10% kcal fat, D12450) were purchased from Research Diets Inc. (USA). Food intake were monitored for 12 weeks. After 12 weeks, myocardial I/R mouse models were induced as described in our previous studies [21]. Brief ly, a slipknot was tied around the left descending coronary artery. After 30 min of ischemia, the slipknot was released and the myocardium was reperfused for 3 h, 6 h or 24 h. Homoharringtonine (HHT, 1.3mg/kg, Abcam, USA), a selective 60S r ibosome inhibitor, was administered by intraperitoneal (IP) injection before I/R operation [22]. At the end of reperfusion, animals were sacrificed. Then left ventricle (LV) was dissected, frozen in liquid nitrogen and stored at -80℃. Other mice were used to perform the Evans blue/TTC double staining.

2.2Determination of cardiomyocyte apoptosis
Cardiomyocyte apoptosis was determined by TdT-mediated dUTP nick end labeling (TUNEL, Roche) and cleaved caspase-3 examination, which includes the entire I/R area commonly termed as area at risk as described in our previous study [23].At 24 h post-reperfusion, mice were re-anesthetized and cardiac function was determined using echocardiographic imaging system (Vevo 2100, VisualSonic, Toronto, Canada). Briefly, mice were anesthetized with 1.5% isoflurane and two-dimensional echocardiographic views of the mid-ventricular short axis were obtained at the level of the papillary muscle tips below the mitral valve. LV wall thickness and internal dimensions were measured and the LV ejection fraction (LVEF) was calculated [21]. After completion of functional determination, the ligature around the coronary artery was retied, and the myocardial infarct size was determined by the Evans blue/TTC double staining method as described previously [23].Total RNA was extracted from frozen hearts or cultured cells by using MiniBEST Universal RNA Extraction Kit (9767, Takara), and reverse-transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (DRR047A, Takara). RT-PCR was performed on all samples in tr iplicate using PCR detection kit (DRR081A, TaKaRa) and CFX96 system (Bio-rad). All primer sequences are shown in Supplementary Table 1. Levels of pre-rRNA were measured by RT-PCR and normalized to 18S expression as previously reported [10].

Lysates were collected in RIPA buffer (P0013B, Beyotime) in the presence of protease and phosphatase inhibitor cocktail (5872, Cell Signaling Technology). Then proteins were separated on SDS-PAGE gels, transferred to PVDF (polyvinylidenedifluoride, Millipore), and incubated overnight at 4℃ with antibodies directed against NS (1:1,000, R&D Systems), caspase-3 (1:1,000, Abgent), cleaved caspase-3 (1:1,000, Cell Signaling Technology), and β-actin (1:250, Santa Cruz Biotechnology). After washing blots to remove excessive primary antibodies, blots were incubated for 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:2,000). Antibody binding was detected via enhanced chemiluminescence (Millipore). Film was scanned with ChemiDocXRS ( Bio-Rad Laboratory, Hercules, CA). The immunoblot band intensity wasanalyzed with Lab Image software.After a 16 h fast, alert mice were challenged with a glucose load of 1.5 g/kg administered via IP injection. Tail blood was taken 0, 30, 60, 90, and 120 min after glucose administration, and blood glucose levels were determined using an OneTouch II glucose meter (Lifescan, USA).Adenovirus expressing mouse full length GNL3 (NS) was constructed by Hanbio Co., Ltd (Shanghai, China). Br iefly, the pHBAd-MCMV-GFP-GNL3 was constructed by cloning the target gene GNL3 into pHBAd-MCMV-GFP. After sequence confirmation, pHBAd- MCMV-GFP-GNL3 was transferred into the recombinant adenovirus frame vector of pHBAd- BHG. The adenovirus vector was then amplified in HEK293 cells and viral titer was measured.

Control vectors (AV-GFP) were constructed and produced concomitantly. Either AV-NS or AV-GFP were delivered as previously described with minor changes [21]. Brief ly, mice were anesthetized with 2% isoflurane inhalation. A skin cut (1.2 cm) was made over the left chest and a purse suture was made. After dissection of pectoral muscles and exposure of the ribs, the heart was smoothly and gently ‘popped out’ through a small hole made at the 4th intercostal space. Each adenovirus was diluted to 2.5×1011 particles and 25 μL was then injected directly into LV free wall with a Hamilton syringe (Hamilton Co. Reno, Nevada) with the needle size of 30.5. Intramyocardial injections were performed: 1) starting from apex and moving toward to the base in LV anterior wall; 2) at the upper part of LV anterior wall; and 3) starting at the apex and moving toward to base in LV posterior wall. After gene delivery, the heart was immediately placed back into the intrathoracic space followed by manual evacuation of pneumothoraces, closure of muscle, and the skin suture.

After 72 h, I/R operation was performed as described above.H9c2 cardiomyocytes were plated in DMEM (Hyclone, USA) with 10% fetal bovine serum (FBS, Gbico, USA) and 1% antibiotics (Hyclone, USA). When reached 50%-60% intensity, cells were incubated with AV-NS (MOI=100) or AV-GFP (MOI=100), together with 5 μg/mL polybrene for 3 h. Then 200 μM palmitate (Pal) and 500 nM HHT were used to incubate H9c2 cells for 24 h [24]. After incubation, the green fluorescence was examined. Then the medium was replaced with DMEM lacking FBS before the cells were placed into a hypoxic incubator (95% N2,5% CO2, 37℃). After 30 min or 1 h in the hypoxic incubator, cells were transferred to a normal incubator (95% O2, 5% CO2, 37℃) for 2 h to reoxygenate. Then total RNA or proteins were obtained.Protein synthesis was determined by using a protein synthesis assay kit (Cayman, USA). Br iefly, cells were incubated in a 96-well cell culture plate. Then O-Propargyl-puromycin (OPP) was used to incubate cells. After fixation, cells were stained and ready for examination by a fluorescent plate reader (Victor X, PerkinElmer).All data were presented as mean±SEM (standard error of the mean) and were analyzed using Graphpad Prism software (Graphpad Software, USA). For two groups, unpaired student’s t-test was conducted. For more than two groups, two-way ANOVA with a post-hoc analysis was performed. A P value<0.05 was considered as statistically significant. 3.Results Ribosomal biogenesis is tightly associated with cell metabolism and is essential for the adaptation of a cell to multiple environmental stresses [13, 14]. Given the important role NS plays in ribosomal biogenesis, we hypothesized that NS-regulated ribosomal biogenesis may be dysfunctional in diabetic hearts in response to myocardial I/R injury. 12-week HFD successfully induced diabetic mouse models, as evidenced by elevated body weight, fasten blood glucose level, and impaired glucose tolerance (Fig. S1). In ND mice, cardiac NS expression was significantly induced after 24 h of reperfusion, accompanied by a pre-rRNA upregulation. In contrast, HFD mice did not show statistic significance in NS expression or pre-rRNA levels between I/R 24 h group and sham group (Fig. 1). These results demonstrated that NS was dysregulated specifically in diabetic hearts, other than metabolically normal hearts during I/R injury.Because NS is rapidly induced following myocardial injury and plays an anti-apoptotic role in cardiomyocytes [9, 10], we then myocardial-specifically overexpressed NS usingadenovirus to investigate whether NS dysregulation is a reason for the increased cardiac vulnerability to I/R injury or just an epiphenomenon. AV-NS delivery successfully increased NS and pre-rRNA expression in LV after 24 h of reperfusion (Fig. 2), but had no effect on fasten blood glucose or glucose tolerance (Fig. S1). NS overexpression significantly reduced myocardial infarct sizes and improved cardiac function in both ND group and HFD group. Of note, AV-NS administration abolished the statistic differences in LVEF(%) and myocardial infarct sizes between ND+AV-GFP group and HFD+AV-GFP group (Fig. 2). These data suggested that NS dysregulation is causative for increased ischemic vulnerability of diabetic hearts and restoring NS expression exerted cardioprotection against myocardial I/R injury. The reservation of cardiac function by NS overexpression may be due to pre-rRNA upregulation and myocardial infarct size reduction.NS is an important regulator of cell cycle and plays an anti-apoptotic role in cardiomyocytes [10, 12]. Therefore, to further study how did cardiac overexpression of NS restore cardiac function and reduce myocardial infarct sizes, cleaved caspase-3 expression and TUNEL were examined to determine cardiomyocyte apoptosis. We found that AV-NS administration reduced cardiomyocyte apoptosis and cleaved caspase-3 expression in both ND and HFD group at 6 h post-reperfusion. Notably, NS overexpression decreased cardiomyocyte apoptosis by a larger degree in HFD mice than in ND mice and abrogated the difference in cardiomyocyte apoptosis between ND group and HFD group (Fig. 3). These results demonstrated that NS overexpression protected the cardiomyocyte against apoptosis in response to I/R injury. This effect is especially potent in diabetic hearts.Recent studies have demonstrated that NS plays an essential role in ribosomal biogenesisprocess, especially in the processing of pre-60S ribosomal subunit to 60S subunit [19, 20]. NS is critical for ribosomal function as knockdown of NS can significantly reduce total protein content [25]. As our results showed that NS overexpression elevated the pre-rRNA level (Fig. 2), we then investigated the role of ribosomal biogenesis and function in NS-mediated cardioprotection. By using HHT, a selective 60S ribosome inhibitor, we blocked the effect ofNS overexpression on ribosomal function. Both AV-NS and HHT had no effect on NS expression, pre-rRNA level, fasten blood glucose or glucose tolerance (Fig. 4 and Fig. S2). Notably, HHT administration abolished the improvement of LVEF(%) and reduction of myocardial infarct sizes caused by NS overexpression. Compared with ND+HHT+AV-NS group, HFD+HHT+AV-NS group showed significantly lower LVEF(%) and larger myocardial infarct sizes (Fig. 4). These data directly suggested that NS exerted cardioprotection against I/R injury by restoring ribosomal biogenesis and function.The perturbation of any key step in the ribosomal biogenesis process can cause ribosomal stress and lead to cell apoptosis [15, 26-30]. To further investigate whether NS overexpression-induced ribosomal biogenesis exerts cardioprotection against I/R injury by reducing cardiomyocyte apoptosis, we used HHT to inhibit ribosomal function and observed cardiomyocyte apoptosis. HHT administration induced cardiomyocyte apoptosis in both ND group and HFD group as evidenced by increased cleaved caspase-3 expression and TUNEL positive cardiomyocytes (Fig. 5). Notably, compared with ND+HHT+AV-NS group, HFD+HHT+AV-NS group showed more cell apoptosis, while there is no significant difference in cardiomyocyte apoptosis between ND+AV-NS group and HFD+AV-NS group (Fig. 5). These results demonstrated that NS-mediated ribosomal biogenesis protected diabetic hearts against I/R injury by reducing cardiomyocyte apoptosis.3.6Saturated fatty acid is the pathopysiological mechanism underlying NS dysregulation.Glucotoxicity and lipotoxicity are two major pathological factors contributing to cardiac injury in response to I/R in diabetic hearts [31-33]. To investigate whether excess saturatedfatty acids or high glucose is the cause of NS dysregulation, we used high glucose (25 mM)and high fatty acids (palmitate) to incubate cardiomyocytes. In consistent with in vivo studies,NS was significantly upregulated after H/R (1 h/2 h) in the control group, accompanied by anobvious pre-rRNA elevation. Palmitate exposure, but not high glucose treatment, significantlyinduced NS dysregulation in cardiomyocytes in response to H/R (Fig. 6). AV-NSadministration markedly upregulated NS expression, elevated pre-rRNA levels and increased newly synthesized protein content (Fig. 7). NS overexpression abrogated the differences in cleaved caspase-3 expression and TUNEL positive cell counts between control group andpalmitate group. Moreover, the effects of NS overexpression on protein synthesis and cardiomyocyte apoptosis were abolished by HHT administration (Fig. 7). These data provided direct evidence suggesting that high fatty acids (such as palmitate) exposure causes cardiac NS dysregulation and subsequent ribosomal biogenesis dysfunction in diabetic hearts. Meanwhile, high glucose treatment had limited effect on cardiac NS expression. Furthermore, NS protected cadiomyocytes against apoptosis by promoting ribosomal biogenesis and thus stimulating protein synthesis. 4.Discussion In the present study, we found that cardiac NS expression was obviously upregulated in response to myocardial I/R injury, accompanied by highly increased pre-rRNA in ND mice. In contrast, cardiac NS and pre-rRNA were dysregulated during I/R in HFD mice. Restoring NS expression by AV-NS delivery significantly restored the pre-rRNA level, promoted protein synthesis, and protected hearts against I/R injury. Furthermore, inhibition of ribosomal function by HHT abrogated the NS-mediated cardioprotection against myocardial I/R injury. These data reveal that NS dysregulation- induced ribosomal biogenesis dysfunction plays a causative role in the increased ischemic vulnerability of diabetic hearts. Of note, we identified that hyperlipidemia, a major metabolic signature of diabetic individuals, might be the cause of NS dysregulation in diabetic hearts. These data suggest that NS may be a novel and promising therapeutic target for IHD treatment, especially for the diabetic heart which is more vulnerable to ischemia injury.NS is a protein expressed in nucleolus and is involved in ribosomal biogenesis [19, 20]. Under physiologic conditions, NS is expressed at a low level in myocardium [9, 11]. Interestingly, NS is induced rapidly after MI, I/R, and TAC, playing an anti-apoptotic role in cardiomyocytes [10, 12]. These data strongly suggest that NS may have a role in the regulation of cardiac function/structure under stressful conditions. However, the role of NS in diabetes and diabetes complications remains completely unknown. In our present study, we observed a remarkable upregulation of cardiac NS expression in ND mice during myocardial I/R injury, which is consistent with the previous study [12]. Notably, for the first time, we found that cardiac NS was dysregulated in diabetic hearts in response to I/R. By adenovirus injection, cardiac NS expression was significantly upregulated in both ND mice and HFD mice during myocardial I/R injury, accompanied by an amelioration of cardiac dysfunction and structural impairment. However, wehave also administrated the same dose of AV-NS into sham-operated mice and we did not observeany functional or structural alteration. These data suggest that cardiac NS dysregulation acts as adirect contributor to increased ischemic vulnerability of diabetic hearts. Therefore, improving cardiac NS dysregulation may be a novel strategy to ameliorate myocardial I/R injury in diabetic subjects.As well-known, hyperglycemia and hyperlipidemia, two major metabolic signatures ofdiabetic individuals, contribute critically to increased myocardial ischemic injury in diabetes[31-33]. Therefore, we hypothesized that hyperglycemia or hyperlipidemia may be the cause ofcardiac NS dysregulation and subsequent ribosomal biogenesis disorders. We found that palmitate exposure, but not high glucose treatment, obviously induced NS dysregulation in culturedcardiomyocytes in response to simulated I/R. These data for the first time provided direct evidencesuggesting that high fatty acids (such as palmitate) exposure causes cardiac NS dysregulation inthe diabetic heart. High glucose treatment have limited effects on cardiac NS expression. Webelieve that these data have revealed an important mechanistic link between diabetes and NS dysregulation. However, how saturated fatty acids cause NS dysregulation remains largelyunknown and the underlying mechanisms should be further investigated.Ribosomal biogenesis and protein synthesis are tightly related to metabolic conditions. Experimental type 1 diabetes results in a pronounced fall in the rate of protein synthesis in the heart and skeletal muscle [34-36]. Ribosomal biogenesis is acknowledged as a critical biological process which is very sensitive to stresses. Stresses usually inhibit total protein translation which is often accompanied by the selective translation of proteins involved in ribosomal biogenesis and assembly, which are required for cell survival under stresses [13, 14]. Ribosomal biogenesis also plays important roles in the heart in physiological and pathophysiological conditions [37, 38]. It is recently reported that cardiac pre-rRNA is increased after acute MI, which may represent a compensative protective response [10]. These data strongly suggest that ribosomal biogenesis may play a regulatory role in the ischemic vulnerability of diabetic hearts. In the current study, for what we think is the first time, we found that HHT, a ribosomal function inhibitor, successfully blocked the enhancement effects of NS overexpression on ribosomal function and abolished the cardioprotection of NS overexpression against I/R injury in both non-diabetic and diabeticsubjects. Collectively, these data provided experimental evidences that ribosomal biogenesis is indispensible in NS-mediated cardioprotection and suggested that NS dysregulation- induced ribosomal biogenesis dysfunction mediates a causative effect in increased ischemic vulnerability of diabetic hearts. In conclusion, the present study has built up a novel mechanism underlying increased ischemic vulnerability of diabetic hearts. Specifically, in diabetic hearts, I/R-induced cardiac NS expression is obviously impaired, resulting in a subsequent ribosomal biogenesis dysfunction. Furthermore, NS dysregulation-induced ribosomal biogenesis dysfunction is a direct contributor to the ischemic vulnerability of diabetic hearts. Importantly, these data suggest that targeting cardiac NS dysregulation or ribosomal biogenesis disorder might be novel therapeutic strategies for ischemic heart injury management in diabetic Homoharringtonine patients.