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Introduction
A large body of evidence recently highlighted the involvement of long non-coding RNAs (lncRNAs) in cardiovascular disease [1] and some dysregulated lncRNAs have been associated with diabetic cardiomyopathy [2,3,4,5]. Among them, a higher expression of the lncRNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) has been observed in diabetic cardiomyopathy [6, 7]. However, a clear understanding of the molecular mechanisms leading to pathological regulation of lncRNAs in diabetic cardiomyopathy is still missing. Our prior work by Barbati et al. [8], established that, in the presence of high glucose, nitric oxide (NO) signaling derangement might alter the epigenetic landscape of cardiac cells, both in vitro and in vivo, via transcription factor CREM activation.
Aim
The present study is aimed at investigating the role of high glucose (HG) and NO pathway in the regulation of MALAT1 in the heart of mice after 6 months of prolonged hyperglycemia and in two cellular models of cardiomyocytes exposed to HG.
Methods
Cell lines, treatments, and RNA interference
Mouse HL-1 cells and rat H9C2 differentiated into cardiomyocytes were cultured and treated with high glucose (30 mM) for 72 h, DETANO and Sildenafil as in [8]. CREM silencing was performed with siRNA (TriFECTa and DsiDNA duplex, Integrated DNA Technologies) according manufacture’s instruction as in [8].
Animal care and treatment
All experiments were performed in accordance with European Community guidelines and upon Approval of the Italian National Institute of Health (DGSAF0005330 n° 202/2016-PR) and ethical committee of Università Cattolica as described in [8]. Male CD1 mice were made hyperglycaemic by streptozotocin (STZ) injection and analyzed at 6 months after STZ treatment. Sildenafil treatment were as in [8].
RNA extraction and qRT
Analysis was performed as in [8] using the following primers designed on (NR_002847) and (XR_350899) sequences, respectively:
mMALAT1 5′-GTAGGTTAAGTTGACGGCCGTTA-3′ and 5′-ATCTTCCCTGTTTCCAACTCATG-3′;
rMALAT1 5′-CCTTTTTAATTACTTCAGTTGTAGCTTTGAC-3′ and 5′-TGATGGAGCCCAGCAGTTTAG-3′.
Gene expression by qPCR
Analysis was performed as in [9] using LncRNA Profiler qPCR Array Kit (SBI System Biosciences) according manufacturer’s instruction.
Chromatin immunoprecipitation (ChIP)
ChIP was performed as in [8] using the following primers:
mCDR1as promoter 5′-ATATGTCCACGGGTGTACAATGAT-3′ and 5′-CGGTCTATGGATGAGGCTCTTG-3′;
mMALAT1 promoter 5′-CCTTTCCCCTCCGTCGTAGT-3′ and 5′-CCGTGGCGGCAAGGT-3′;
rMALAT1 promoter 5′-TGCGAAGGGACACGTCACT-3′ and 5′-GGCCCACGCACCATCA-3′.
Statistical analysis
Data are expressed as mean ± SEM as indicated in figure legends. Statistical analyses were performed by using Sigma Plot 13. Significance was calculated using a two-tailed t-test or one-way ANOVA. Differences between groups were calculated with ANOVA and post-hoc Tukey HSD test with Bonferroni correction. A p-value of <0.05 was considered significant.
Results
Nitric oxide counteracts MALAT1 upregulation in cardiomyocytes exposed to high glucose and in heart of hyperglycemic mice
Mouse HL-1 and rat cardiomyocytes obtained from differentiated H9C2 cells were treated with HG for 72 h in the presence or absence of the NO donor, DETA/NO, or the PDE5 inhibitor Sildenafil. In addition, STZ injected CD1 mice, treated or not with Sildenafil, have been used as an in vivo model of cardiac damage determined by 6 months of prolonged hyperglycemia. First, we analyzed the expression of 90 lncRNAs in HL-1 cells by qPCR profiling. Among those, a subset of 30 lncRNAs was found expressed in HL-1 (Supplemental Fig. 1a). Seven of them were significantly up-regulated and one down-regulated (p < 0.05) by HG compared to control (Fig. 1a). In this condition, treatment with DETA/NO normalized expression of the lncRNAs modulated by HG (Fig. 1a). To molecularly investigate the mechanism involved in this effect we performed a series of Chromatin immunoprecipitations (ChIPs) on lncRNAs up or down-regulated by HG including CDR1as and MALAT1 (Fig. 1b). Specifically, we studied the enrichment modulation of epigenetic marks on histone H3 such as tri-methyl lysine 4 (H3K4me3), tri-methyl lysine 36 (H3K36me3) and the lysine 9 acetylation (H3K9ac) [10]. In HG, H3K4me3 and H3K36me3 were significantly downregulated on CDR1as promoter whereas H3K4me3 and H3K9Ac were upregulated on that of MALAT1 suggesting that the modulation observed at RNA level (Fig. 1a) may rely on transcriptional regulation. To further confirm the key role of NO in the HG-dependent lncRNA alteration, we used a different cellular model the H9C2 cells differentiated into cardiomyocytes and exposed to HG in the presence/absence of DETA/NO or Sildenafil that elevated intracellular level of NO and cGMP, respectively. As expected, MALAT1 upregulation induced by HG was significantly counteracted by the co-treatment with DETA/NO or Sildenafil (Fig. 1c). Of note, Sildenafil was effective in counteracting the negative consequences of hyperglycemia also in vivo. Indeed, the heart of STZ-injected mice showed a significant increase of MALAT1 in STZ-treated mice compared to controls (Fig. 1d). In this condition, Sildenafil administered daily for 12 weeks, starting 3 months after STZ injection, rescued the effect of hyperglycemia reducing the level of MALAT1 (Fig. 1d). To gain further insights about the molecular mechanism controlling the expression of MALAT1 in the presence of HG, we analyzed the potential involvement in its transcription of transcriptional factor CREM previously identified as involved in gene expression determined by hyperglycemia [8]. Of interest, mouse and rat MALAT1 promoters have a similarly structured regulatory region at −700 bp from the transcriptional start site enriched in CREM binding sites as determined by transcription factor database (TRANSFAC 8.3) analysis. Remarkably, silencing of CREM in H9C2 cells completely abrogated the induction of MALAT1 mediated by HG treatment (Fig. 1e). ChIP analysis of MALAT1 promoter (Fig. 1f) revealed a significant CREM recruitment in the presence of HG. A condition which was inhibited by DETA/NO or Sildenafil. In parallel, the enrichment of H3K9Ac in the presence of HG was completely rescued by treatment with DETA/NO or Sildenafil. On the opposite, the recruitment of the Histone Deacetylase 1 (HDAC1), reduced by HG, was normalized in the presence of DETA/NO or Sildenafil. Accordingly, ChIP performed on cardiac chromatin, isolated 6 months after STZ treatment, showed a significant increase of CREM binding on MALAT1 promoter in STZ vs. saline-treated mice paralleled by an induction of H3K9ac level and a reduction of HDAC1 occupancy (Supplemental Fig. 1b) thus further confirming CREM as a possible mediator of hyperglycemia-dependent MALAT1 upregulation in vivo.
Discussion
Taken altogether, these data suggest that Sildenafil counteracts the increase of MALAT1 occurring in cardiomyocytes as a consequence of elevated glucose levels in vitro and in vivo. In light of the present data we reasoned that the following considerations might be extrapolated: (i) reduction of nitric oxide or cGMP bio-availability caused by prolonged hyperglycemia impairs lncRNA expression and (ii) Sildenafil, restoring function of nitric oxide signaling normalized MALAT1 expression levels. To our knowledge, this is the first observation about the involvement of nitric oxide signaling in the transcriptional regulation of MALAT1, a lncRNA putatively implicated in glucose sensing and diabetic cardiomyopathy.
Reduction of nitic oxide bio-availability is an early and key determinant in diabetic cardiomyopathy impairing both endothelial and cardiac function ([11] and references therein). The present study reveals that restoring the intracellular impact of nitric oxide signaling might be important to reverse the effect of hyperglicemia also controlling transcription of lncRNAs involved in cardiac injury and in the development of cardiomyopathy associated to dysmetabolic conditions including diabetes. Indeed, this mechanism might be part of a more complex process determining the cardioprotective effect of Sildenafil in heart failure [12, 13] as well as in diabetic cardiomyopathy [14, 15] suggesting for novel transcriptionally-based therapeutics strategies in the presence of prolonged hyperglycemia.
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Funding
The present study was funded by: Italian Ministry of Education, University and Research (FIRB-MIUR RBFR10URHP_002) to SN, (PRIN2015ZHKFTA) to AF, (PRIN2015HPMLFY_004) to AP; Italian Ministry of Health (GR 2011-02351557) to SN, (RF 2010-2318330) to AF and (Ricerca Corrente Ministero della Salute) to CG.
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All procedures performed in studies involving animals were in accordance with the ethical standards of the institutional and/or national research committee.
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Bacci, L., Barbati, S.A., Colussi, C. et al. Sildenafil normalizes MALAT1 level in diabetic cardiomyopathy. Endocrine 62, 259–262 (2018). https://doi.org/10.1007/s12020-018-1599-z
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DOI: https://doi.org/10.1007/s12020-018-1599-z