Abstract

   Septic shock with heart dysfunction is very common in intensive care
   units. However, whether long noncoding RNA (lncRNA) and mRNA profiles
   differ between patients with and without myocardial depression is
   unknown. We generated rat models of hypodynamic septic shock induced by
   lipopolysaccharide. A total of 12 rat models was constructed and heart
   tissue from each was collected. Whole genomic RNA sequencing was
   performed on left ventricular tissue; 6,508 novel lncRNAs and 432
   annotated lncRNAs were identified in heart samples, and 74 lncRNAs were
   expressed differently in the sepsis and control groups. Gene ontology
   term enrichment indicated apoptosis and its related pathways showed
   obvious enrichment, which suggested cell apoptosis could play a
   critical role in the process of myocardial depression. Furthermore, we
   focused on one lncRNA from the Pvt1 gene. By silencing this lncRNA, we
   demonstrated knockdown of Pvt1 expression could induce cell apoptosis
   in lipopolysaccharide-induced heart cells, through increasing the
   expression of c-Myc, Bid, Bax, and caspase-3 and decreasing the
   expression of Myd88 and Bcl-2, thereby proving its functional role
   in myocardial depression. These results demonstrate that lncRNAs both
   participate in and mediate the pathological process of myocardial
   depression. Our study improves the understanding of the basic molecular
   mechanisms underlying myocardial depression.

   Keywords: sepsis, lncRNA, myocardial depression, transcriptome, Pvt1

Introduction

   Sepsis is a syndrome of physiological, pathological, and biochemical
   abnormalities caused by the altered systemic host response to
   infection.[43]1, [44]2 Septic shock is a subset of sepsis, in which
   underlying circulatory and cellular abnormalities are profound enough
   to greatly increase mortality.[45]^1 Notably, the myocardial
   dysfunction during sepsis is usually called sepsis-induced myocardial
   depression or sepsis-induced heart dysfunction,[46]^3 characterized by
   impaired myocardial contractility and reduced ejection fraction.[47]3,
   [48]4 This phenomenon can lead to higher mortality, especially in
   pediatric septic shock patients.[49]^5 However, the underlying
   molecular mechanism of sepsis-induced myocardial depression remains
   unclear.

   Recent studies indicate that microRNAs (miRNAs) are involved in the
   process of sepsis-induced myocardial depression.[50]^6 Long noncoding
   RNAs (lncRNAs) are a type of noncoding RNA with a length exceeding 200
   nt. These lncRNAs contribute to transcriptional and
   post-transcriptional functions, and they can be broadly classified
   either as signaling molecules, decoy molecules, guide molecules, or
   scaffold molecules.[51]^7 Unlike miRNAs, which have been extensively
   studied, the lncRNAs present in sepsis-induced myocardial depression
   have never been sequenced. As is well known, next-generation sequencing
   has become a powerful approach that can reveal different expression
   profiles underlying phenotypic differences, as well as decipher
   non-annotated transcriptional activity by identifying various novel
   transcripts (protein-coding and noncoding) and additional alternative
   splice variants of known annotated transcripts.[52]8, [53]9 The limited
   number of identified lncRNAs in sepsis-induced myocardial depression
   may impede further study of its complex molecular mechanism.

   Therefore, we hypothesized that lncRNAs are involved in the
   pathological process of sepsis-induced myocardial depression. The
   present study aimed to describe the features of lncRNAs and mRNAs in
   sepsis-induced myocardial depression. Furthermore, we focused on one
   particular lncRNA (transcript ID: ENSRNOT00000092896, gene ID:
   ENSRNOG00000062170, and gene name: Pvt1) for further research, which
   was the transcript from the rat Pvt1 gene. Our study provides a useful
   resource to study the functional roles of lncRNA and link the
   characteristic lncRNAs and mRNAs to myocardial depression phenotypes.

Results

Model Identification of Myocardial Depression in Rats with Septic Shock

   Our previous study showed that there is a positive relationship between
   mean arterial pressure (MAP) and heart function.[54]^10 Therefore, we
   measured MAP and found that MAP started to decline approximately 1.5 h
   after the administration of LPS. Septic shock occurred approximately
   2 h after LPS administration, and it lasted for the rest of the
   observation period ([55]Figure 1A). In the control group, MAP appeared
   unchanged throughout the entire observation period. Furthermore, H&E
   staining showed that there was infiltration of inflammatory cells and
   rupture of myocardial cells in hearts of the septic rats compared to
   those in the control group ([56]Figure 1B). Terminal deoxynucleotidyl
   transferase-mediated dUTP nick-end labeling (TUNEL) staining of heart
   tissue showed a statistically significant increase in apoptosis rates
   in septic shock animals ([57]Figures 1C and 1D). Finally, we examined
   serum inflammatory biomarkers (tumor necrosis factor-α [TNF-α] and
   interleukin-6 [IL-6]) and cardiac enzymes (cardiac troponin I [cTnI]
   and creatine kinase MB form [CK-MB]) ([58]Figure 1E), and we found that
   all of these were significantly elevated in the septic animals.

Figure 1.

   [59]Figure 1
   [60]Open in a new tab

   Basic Characteristics of Animal Models between Septic Shock Group and
   Control Group

   (A) In vivo mean arterial pressure (MAP) hemodynamic studies. n = 6 in
   each group. (B) Pathological comparation in heart tissue by H&E
   staining (10×). (C and D) Apoptosis detection in heart tissue by TUNEL
   staining (400×). n = 3 in each group. (E) Serum inflammatory biomarker
   (TNF-α and IL-6) and cardiac enzyme (cTnI and CK-MB) levels were
   detected by ELISA. n = 6 in each group; *p < 0.05 compared to the
   control; **p < 0.01 compared to the control.

RNA Sequencing Identified the Features of lncRNA and mRNA in Myocardial
Depression of Septic Shock

   We analyzed RNA sequencing (RNA-seq) data from 12 rat heart samples, in
   which 82,000,000–115,000,000 raw data and 79,000,000–110,000,000 clean
   data were obtained ([61]Table 1). The classification of mapped reads in
   each heart tissue is shown in [62]Figure S1. After comparing the SNP,
   insertion or deletion (indel), and alternative splicing (AS) events in
   the sepsis and control groups, we found that the SNP and indel counts
   in sepsis were less than those in the control group, although there
   were no obvious differences in AS events ([63]Figure S2).

Table 1.

   The Detailed Information of RNA Sequencing
   Sample Raw Reads Clean Reads Clean Bases (G) Error Rate (%) Q20 (%) Q30
   (%) Total Mapped (%)
   Control 1 104,887,116 101,842,730 15.28 0.01 98.09 95.02 96.42
   Control 2 110,482,096 107,303,972 16.10 0.01 98.17 95.17 96.62
   Control 3 109,101,010 105,976,634 15.90 0.01 98.20 95.27 96.41
   Control 4 114,063,808 110,459,742 16.57 0.01 98.30 95.49 96.55
   Control 5 104,412,260 101,257,002 15.19 0.01 98.11 95.06 96.22
   Control 6 102,114,088 99,322,108 14.90 0.01 98.17 95.20 96.43
   Sepsis 1 85,541,078 82,755,500 12.41 0.02 97.22 92.88 95.85
   Sepsis 2 83,814,640 80,941,514 12.14 0.02 96.87 92.16 95.48
   Sepsis 3 92,375,116 90,576,722 13.59 0.01 97.21 93.01 95.09
   Sepsis 4 82,418,946 79,773,136 11.97 0.02 97.13 92.70 95.91
   Sepsis 5 101,060,828 98,158,896 14.72 0.01 98.08 94.99 96.43
   Sepsis 6 115,144,142 110,000,016 16.50 0.02 97.22 92.90 95.68
   [64]Open in a new tab

   To minimize the false-positive rate in identifying lncRNAs from 287,301
   assembled transcripts, we developed a filtering flowchart to discard
   transcripts without all characteristics of lncRNA ([65]Figure 2A). By
   this method, we identified 6,508 novel lncRNAs from an intersection of
   the analysis results of CPC, CNCI, and PFAM ([66]Figures 2B and 2C),
   which included 4,642 lncRNAs (71.3%), 979 antisense lncRNAs (15.0%),
   and 887 intronic lncRNAs (13.6%) ([67]Figure 2D). We compared the
   number of overlapping lncRNAs in the sepsis and control groups, which
   suggested that the lncRNAs that appeared in the sepsis group also
   appeared in the control group ([68]Figure 2E). There were 23,010 mRNAs
   included in our study, and we compared the number of overlapping mRNAs
   in the sepsis and control groups ([69]Figure 2F). In addition, our
   study found that annotated and novel lncRNAs had a smaller size, fewer
   exons, and fewer open reading frames than mRNAs ([70]Figures 2G–2I).
   However, there was no significant difference in transcript levels in
   heart tissue from the sepsis and control groups ([71]Figures 2J–2L).

Figure 2.

   [72]Figure 2
   [73]Open in a new tab

   Characterization of lncRNAs and mRNAs in Rat Heart Tissue

   (A and B) The five steps of novel lncRNA screening. (C) Coding
   potential analysis via CPC, PFAM, and CNCI. Those sequences
   simultaneously shared by the above three tools were selected as
   candidate lncRNAs. (D) The classification of identified lncRNAs. (E)
   Venn diagram showing the number of overlapping lncRNAs in sepsis group
   compared with control group. (F) Venn diagram showing the number of
   overlapping mRNAs in sepsis group compared with control group. (G–I)
   Density distribution diagram showing the expression features of exon
   number (G), length (H), and opening reading frame (ORF) (I) of
   annotated lncRNAs, novel lncRNAs, and mRNAs in rat heart tissue. (J–L)
   Boxplot (J), violin plot (K), and density distribution diagram (L)
   showing the expression features of rat heart tissue from the sepsis
   group and the control group. FPKM, fragments per kilobase million.

Differential Expression and Cluster Analysis of lncRNAs and mRNAs

   To check if lncRNAs were involved in the pathological process of
   sepsis-induced myocardial depression, the expression patterns of
   lncRNAs and mRNAs were analyzed. We analyzed differential expression
   (DE) lncRNAs and mRNAs using significance analysis following the
   criterion q < 0.05. As a result, there were 74 DE lncRNAs (33
   upregulated and 41 downregulated) and 4,011 DE mRNAs (1,918 upregulated
   and 2,093 downregulated) ([74]Figures 3A and 3B). The expression
   pattern of DE lncRNAs and mRNAs is shown using a cluster heatmap
   ([75]Figures 3C and 3D).

Figure 3.

   [76]Figure 3
   [77]Open in a new tab

   The Expression Profiling Changes of lncRNAs and mRNAs in Rat Heart
   Tissue

   (A and B) Volcano plot indicating up- and downregulated lncRNAs (A) and
   mRNAs (B) of rat heart tissue in the sepsis group compared with the
   control group; up- and downregulated genes are colored in red and blue,
   respectively. (C and D) Heatmap of lncRNAs (C) and mRNAs (D) showing
   hierarchical clustering of changed lncRNAs and mRNAs of rat heart
   tissue in the sepsis group compared with the control group; up- and
   downregulated genes are colored in red and blue, respectively. (E–G)
   Differences in the expressions of annotated lncRNAs (E), novel lncRNAs
   (F), and mRNAs (G) in rat heart tissue from the sepsis and normal
   groups. DE, differential expression.

   In addition, among all DE lncRNAs, there were 35 annotated lncRNAs and
   39 novel lncRNAs, and their relative levels of expression are shown in
   [78]Figures 3E and 3F. There was one novel downregulation lncRNA
   (LNC_006245) that was not detected in the sepsis group. The detailed
   information of the top 10 upregulated and 10 downregulated lncRNAs is
   summarized in [79]Table S1, and the top 20 upregulated and 20
   downregulated mRNAs are summarized in [80]Table S2. The relative
   expression levels of several prominent DE mRNAs are shown in
   [81]Figure 3G. These results reflect distinct lncRNA and mRNA
   expression profiles between sepsis-induced myocardial depression and
   the control group, implying different underlying pathophysiology in
   sepsis-induced heart dysfunction.

Systematic Functional Analysis of Differentially Expressed lncRNAs and mRNAs

   To elucidate the possible functional significance of observed changes
   in lncRNA and mRNA levels between sepsis-induced myocardial depression
   and the control group, we performed a Gene Ontology (GO) term
   enrichment analysis. There were 20,121 background genes in total. We
   summarized the significantly enriched GO terms of lncRNAs ([82]Figures
   4A–4C) and mRNAs ([83]Figures 4D–4F) regarding biological process,
   cellular component, and molecular function, respectively. Notably, the
   number of enriched genes at the lncRNA level was much less than that at
   the mRNA level regarding biological process. Interestingly, the
   differentially expressed lncRNAs and mRNAs were similar and
   significantly associated with regulation and metabolic processes in
   biological process term enrichment ([84]Figures 4A and 4D).
   Importantly, the GO term RNA metabolic process was a significant
   enrichment on both the lncRNA and mRNA levels, which indicates that
   noncoding RNAs and mRNAs could make sense in the pathological process
   of sepsis. It is noteworthy that cell apoptosis and cell death were
   enriched in both lncRNAs and mRNAs, suggesting that cell apoptosis and
   cell death might play a critical role in sepsis-induced myocardial
   depression.

Figure 4.

   [85]Figure 4
   [86]Open in a new tab

   GO and KEGG Analyses of Differentially Expressed lncRNA Target Genes
   and mRNAs in Rat Heart Tissue from the Sepsis Group and the Control
   Group

   (A–C) GO categories (biological process, A; cellular components, B; and
   molecular function, C) of differential lncRNA target genes in rat heart
   tissue from the sepsis group and the control group. (D–F) GO categories
   (biological process, D; cellular components, E; and molecular function,
   F) of differential mRNAs in rat heart tissue from the sepsis group and
   the control group. (G) KEGG analysis of differential lncRNA target
   genes in rat heart tissue from the sepsis group and the control group.
   (H) KEGG analysis of differential mRNAs in rat heart tissue from the
   sepsis group and the control group. (I–K) RCircos package analysis
   displaying chromosomal localization and the interactions of the
   co-expression of genes (STRING) regarding apoptosis-related pathways at
   the lncRNA level (I), lncRNA-mRNA level (J), and mRNA level (K).

   In addition, the enrichment of cellular components and molecular
   function also showed a similar pattern. For example, molecular-bounded
   organelle, intracellular part, and nucleus were all obviously enriched
   on the lncRNA and mRNA levels ([87]Figures 4B and 4E). For molecular
   function enrichment, terms regarding binding such as protein binding
   and enzyme binding were enriched, which suggested that complex
   pathological processes are involved in sepsis-induced myocardial
   depression.

   To determine if there were some specific pathways changed in sepsis, we
   performed the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis in lncRNA target genes and mRNAs. Interestingly, the apoptosis
   pathway was also enriched in both lncRNAs and mRNAs, which is
   consistent with the GO term enrichment results ([88]Figures 4G and 4H).
   Therefore, we performed further chromosomal localization, and we
   analyzed the interactions and co-expression of genes in
   apoptosis-related pathways, at the lncRNA, lncRNA-mRNA, and mRNA
   levels, respectively ([89]Figures 4I–4K). The TNF-signaling
   pathway,[90]11, [91]12 nuclear factor κB (NF-κB)-signaling
   pathway,[92]13, [93]14 and Jak-Stat pathway[94]15, [95]16 were
   enriched, which previous studies have implicated in sepsis-induced
   myocardial depression; our KEGG enrichment analysis also suggested
   their significance.

Validation of DE lncRNAs by qPCR

   According to the KEGG pathway enrichment analysis and previous sepsis
   studies, we concluded that the following pathways might be relevant in
   sepsis-induced myocardial depression: TNF-signaling pathway,[96]11,
   [97]12 NF-κB-signaling pathway,[98]13, [99]14 Jak-Stat pathway,[100]15,
   [101]16 and apoptosis pathway.[102]17, [103]18 To validate the
   reliability of the sequencing results and to provide the basis for
   further study, five annotated (ENSRNOT00000000787, ENSRNOT00000092896,
   ENSRNOT00000086178, ENSRNOT00000091713, and ENSRNOT00000076955) and
   four novel (LNC_001626, LNC_004326, LNC_004590, and LNC_004777) DE
   lncRNAs were chosen to analyze by qPCR, which included at least one
   target gene that appeared in the aforementioned four common pathways
   ([104]Figure 5).

Figure 5.

   [105]Figure 5
   [106]Open in a new tab

   qPCR Validations

   (A–I) qPCR validations of nine regulated lncRNAs in rat heart tissue
   from the sepsis group and the control group. n = 15 in each group;
   **p < 0.01 compared to the control.

   To detect widespread DE lncRNAs in sepsis-induced myocardial
   depression, we expanded the sample numbers (n = 15 in each group) for
   validation using our previous heart tissue samples. All lncRNAs
   selected for validation were statistically significant and consistent
   with the results obtained from next-generation sequencing, which
   indicated the high quality and validity of RNA-seq.

The Role of lncRNA rPvt1 in Sepsis-Induced Myocardial Depression

   By considering GO terms, KEGG pathway enrichment, and our findings
   using the septic shock model, we found that apoptosis could be a
   critical factor in myocardial depression. The Pvt1 gene and its related
   lncRNAs have been reported in research in humans, which suggested that
   this gene was related to cell apoptosis.[107]19, [108]20 We focused on
   an lncRNA from Pvt1, which was named lncRNA rPvt1 for further study.
   Surprisingly, the fold changes from qPCR and RNA-seq were almost the
   same and showed significant upregulation in the sepsis group. In
   addition, we realized that its target genes included several classical
   apoptosis-related genes, such as Myc, p53, Bid, and Myd88
   ([109]Figure S3), which is highly suggestive of a potential role in the
   apoptosis process.

   We first located its expression area by RNA fluorescence in situ
   hybridization (FISH) in the H9c2 cell line after LPS administration,
   and we noticed that lncRNA rPvt1 could express in both the nucleus and
   cytoplasm ([110]Figure 6A). In addition, we performed northern blot to
   evaluate its total sequence length, and we found that the expression
   bands were near the second shortest marker (representing the length of
   1,821 bp) ([111]Figure 6B). To validate the sequence accuracy and
   acquire its total length, we conducted rapid amplification of cDNA ends
   (RACE) clone experiments twice: the first time RACE clone was used to
   test the known sequence and acquire the 3′ end and 5′ end sequence, and
   the second RACE test aimed to demonstrate the acquired total sequence
   and show congruence. Similar to the results of the northern blot, we
   acquired an unknown 91-bp sequence from the 5′ end RACE and an unknown
   325-bp sequence from the 3′ end RACE ([112]Figure 6C).

Figure 6.

   [113]Figure 6
   [114]Open in a new tab

   Functional Study of lncRNA rPvt1

   (A) Expression location of lncRNA rPvt1 in LPS-induced H9c2 cell lines
   by fluorescence in situ hybridization (FISH) under confocal microscopy
   (600×). (B) The expression of lncRNA rPvt1 detected by northern blot
   (M, marker; C, positive control product; bands 1–3, heart tissue from
   the control group; bands 4–6, heart tissue from the sepsis group). (C)
   Rapid amplification of cDNA ends (RACE) to acquire the total sequence
   of lncRNA rPvt1 (i, 3′ end RACE; M, marker; 1, 3′-RACE inner PCR
   product (+); 2, 3′-RACE inner PCR product (−); ii, 5′ end RACE; M,
   marker; 1, PCR product; iii, second RACE for validation; M, marker; 1,
   validation PCR product [+]; 2, validation PCR product [−]). (D)
   Expression level of lncRNA rPvt1 in control and LPS-induced H9c2 cell
   line (i); detection of knock down rate by qPCR after lentiviral
   transfection for silencing lncRNA rPvt1 (ii). (E) Apoptosis detection
   in H9c2 cell line by flow cytometry. (F) Cell cycle detection in H9c2
   cell line by flow cytometry. (G) Proliferation detection in control and
   LPS-induced cell lines (i), LPS-induced NC cell line, and LPS-induced
   KD cell line (ii) by CCK-8. NC, negative control group; KD, lncRNA
   rPvt1 knockdown group; *p < 0.05 compared to the control; **p < 0.01
   compared to the control.

   To elucidate the potential role of lncRNA rPvt1 in apoptosis, we first
   detected that lncRNA rPvt1 showed upregulation in the LPS-induced H9c2
   cell line compared with the control group ([115]Figure 6Di), which was
   consistent with the result from animal models. We then performed
   lentivirus transfection to silence this lncRNA, and we observed that
   its expression level was clearly decreased ([116]Figure 6Dii).
   Furthermore, we performed the detection of cell apoptosis and cell
   cycle in four groups, including a control group, LPS-induced group,
   LPS-induced negative control (NC) group, and LPS-induced knockdown (KD)
   group. We found that the apoptosis rates increased significantly in the
   LPS-induced cells compared with the control group after 24 and 48 h. As
   for the LPS-induced NC and KD groups, the cell apoptosis rate increased
   in the silencing group compared with the NC group following LPS
   administration after 24 and 48 h, respectively ([117]Figure 6E).

   Regarding cell cycle analyses, we noticed that there was a decreasing
   trend of S phase cells in the LPS-induced group compared with normal
   cells, and there was also an obvious decrease of S phase cells in the
   LPS-induced KD group compared with the NC group after 48 h
   ([118]Figure 6F). We also performed cell counting kit-8 (CCK-8) to
   determine both the proliferation ability in the control and LPS
   administration groups and the influence of lncRNA rPvt1 on cell
   proliferation. This showed that there was a statistically significant
   difference between the control and LPS administration groups at 24, 36,
   and 48 h ([119]Figure 6Gi). Additionally, proliferation in the knockout
   (KO) group was decreased compared with the NC group after LPS
   administration at both 36 and 48 h ([120]Figure 6Gii). However, the
   absolute difference of estimated values in these two groups was less
   than 20%, which suggests that our results provide limited evidence that
   lncRNA rPvt1 can influence cell proliferation in LPS-induced H9c2
   cells.

The Potential Mechanism of lncRNA rPvt1 in the Regulation of Cell Apoptosis

   We identified that major target genes of lncRNA rPvt1 relating to
   apoptosis included Myd88, c-Myc, Bid, and p53. As such, we investigated
   the changes in these four proteins and other apoptosis-related
   proteins, such as Bax, Bcl-2, and caspase-3, in heart tissue from
   control and sepsis groups, using immunohistochemistry (IHC) and western
   blot. We found that the protein expressions of Myd88, c-Myc,
   cleaved-Bid, Bax, and cleaved-caspase-3 significantly increased and the
   expression of Bcl-2 obviously decreased in the sepsis group compared
   with the control group. However, there was no difference in the p53
   protein between the control and sepsis groups ([121]Figures 7A and 7B).
   Furthermore, to illustrate which genes were regulated and influenced by
   lncRNA rPvt1, we performed western blot of these proteins in a control
   cell line, an LPS-induced cell line, an LPS-induced NC group, and an
   LPS-induced KD group ([122]Figure 7C). We noticed that the Myd88 and
   Bcl-2 proteins decreased in the LPS-induced KD group but that the
   expressions of c-Myc, cleaved-Bid, Bax, and cleaved-caspase-3 increased
   compared with the LPS-induced NC group.

Figure 7.

   [123]Figure 7
   [124]Open in a new tab

   Mechanism Study of lncRNA rPvt1

   (A) Immunohistochemical staining of Myd88, c-Myc, cleaved-Bid, p53,
   Bax, Bcl-2, and cleaved-caspase-3 in rat heart tissue from the control
   and sepsis groups (400×). n = 3 in each group. (B) Western blot
   analysis of Myd88, c-Myc, cleaved-Bid, p53, Bax, Bcl-2, and
   cleaved-caspase-3 in rat heart tissue from control and sepsis rat
   models. n = 3 in each group. (C) Western blot analysis of Myd88, c-Myc,
   cleaved-Bid, Bax, Bcl-2, and cleaved-caspase-3 in the normal H9c2 cell
   line, LPS-induced cell line, LPS-induced NC cell line, and LPS-induced
   KD cell line. n = 3 in each group. (D) Silver staining of rat heart
   tissue during RNA pull-down in control and sepsis groups. n = 1 in each
   group. (E) Western blot analysis of Irak-2 and IL-1R in rat heart
   tissue (i) and H9c2 cell line (ii). n = 3 in each group; *p < 0.05
   compared to the control; **p < 0.01 compared to the control.

   Additionally, we performed RNA pull-down combined with mass
   spectrographic analysis to analyze whether the proteins were involved
   in the regulation process of lncRNA rPvt1 ([125]Figure 7D). We found
   that the Irak-2 protein, which participates in the IL-1R/Myd88 pathway,
   could bind directly with lncRNA rPvt1 in the sepsis group, but not in
   the control group. Furthermore, we proved that the expressions of
   Irak-2 and IL-1R were different in the control and sepsis groups
   ([126]Figure 7Ei) and that, after silencing lncRNA rPvt1, the
   expression of Irak-2 obviously decreased in the LPS-induced KD group
   compared with the LPS-induced NC group ([127]Figure 7Eii). These
   results suggest that the IL-1R/Myd88/Irak-2 pathway could be involved
   in septic myocardial depression and that lncRNA rPvt1 might influence
   Myd88 through binding with the Irak-2 protein.

Discussion

   Sepsis, as a clinical emergency, usually causes multi-organ dysfunction
   and can lead to high mortality.[128]^21 Despite improvements in
   intensive care treatments, septic shock, which is a subtype of sepsis
   with circulatory and cellular and/or metabolic dysfunction, is still
   associated with a higher risk of mortality. Unlike warm shock with
   highcardiac output and low peripheral vascular resistance occurring in
   adult septic shock, pediatric septic shock often occurs as cold shock
   and is characterized by low cardiac output and high peripheral vascular
   resistance. Therefore, it is necessary to understand the mechanisms of
   myocardial depression and aim to reduce the mortality in clinic. To
   reflect and simulate the pathological process of pediatric septic
   shock, which is usually modeled by cecal ligation and puncture, we used
   a rat model subjected to a high dose of LPS, which showed an obvious
   and rapid shock and accompanying myocardial dysfunction. Our model is
   relatively stable and has a high reproducibility rate; therefore, it is
   suitable for studying the mechanism of septic shock in children.

   Recently, with the development of next-generation high-throughput
   sequencing, multiple lncRNAs have been identified and shown to play
   important roles in many diseases.[129]22, [130]23 Compared with
   microarray, next-generation sequencing has many strengths, including
   high sensitivity and specificity, and the ability to discover important
   novel lncRNAs. However, there is no relevant study using whole genomic
   sequencing that reports and compares changes in lncRNAs and mRNAs in
   sepsis-induced myocardial depression.

   To realize the basic characteristics of lncRNAs and mRNAs and evaluate
   the potential role of lncRNAs in septic myocardial depression, our
   study identified entire lncRNAs and mRNAs changing significantly in
   left ventricular tissues compared with the non-sepsis group. In
   addition, to improve the reliability and validity of sequencing, we
   developed six septic shock animals compared with six control animals to
   find the ubiquitous DE lncRNAs and mRNAs. According to quality control
   and qPCR validation, our sequencing results had high reliability and
   quality. Through bioinformatics analysis, we predicted potential
   functions of lncRNAs and mRNAs by GO term enrichment and KEGG pathway
   enrichment, and we found important changes that help us to understand
   the molecular mechanisms of sepsis-induced myocardial depression more
   clearly. In addition, we focused on one specific lncRNA, lncRNA rPvt1,
   and we conducted several experiments to thoroughly investigate its
   characteristics and evaluate its potential role in cell function. Our
   study indicates that lncRNAs, including lncRNA rPvt1, play important
   roles in the pathological process of septic myocardial depression and
   provide a new direction toward understanding this pathological process.

   We identified 74 DE lncRNAs between sepsis and control groups; the
   underlying mechanisms of these lncRNAs are poorly understood. We also
   performed GO enrichment and found that the terms of biological
   processes occupied most of the significantly enriched terms. According
   to our KEGG enrichment results, we found that TNF-signaling pathway,
   NF-κB-signaling pathway, Jak-Stat pathway, and apoptosis pathway were
   the common pathways that appeared at both the lncRNA and mRNA levels.
   Although some published studies[131]11, [132]14, [133]16, [134]17
   stated the potential role of the aforementioned four common pathways in
   sepsis, the regulatory relationship between lncRNAs and these pathways
   is still unclear and should be studied in more depth in future basic
   studies.

   Although a recent study conducted by Takasu et al.[135]^24 showed that
   myocardial tissue from patients with sepsis lacked significant
   necrosis, apoptosis, or autophagy compared with control donor hearts,
   we detected a significant difference in both apoptosis rates and the
   levels of several proteins related to cell apoptosis, including Myd88,
   c-Myc, Bid, Bax, Bcl-2, and caspase-3, in heart tissue from the septic
   shock and control groups. These results are also consistent with the
   results of GO term and KEGG pathway enrichment. To realize the
   co-expression relationships of genes relating to the apoptosis pathway,
   we also performed gene analyses of chromosomal localization and
   regulatory association between lncRNA and mRNA. We detected an obvious
   rate of apoptosis, which is partly due to the fact that we performed
   the septic shock model with a large dose of LPS, producing a more
   serious subtype of sepsis characterized by severe heart dysfunction and
   damage. In the previous clinical study,[136]^24 the included patients
   were septic patients rather than those in septic shock, which might
   explain why they did not develop serious myocardial damage. Another
   potential explanation is that we used a pediatric septic shock model,
   which has a different pathological process compared to adult sepsis.
   The low cardiac output in pediatric sepsis may reflect a severely
   impaired heart function, whereas there is usually normal or high
   cardiac output in adult sepsis. Therefore, apoptosis could be an
   important mechanism of heart dysfunction in pediatric septic shock.

   We also focused on one particular lncRNA and estimated its role in a
   septic model in cell culture. From previous studies, we knew that the
   lncRNA from human Pvt1 could regulate the apoptosis process. For
   example, a study conducted by He et al.[137]^20 showed that silencing
   lncRNA Pvt1 could increase cell apoptosis in nasopharyngeal carcinomas,
   and other studies support this notion in different
   malignancies.[138]19, [139]25 Although the sequence of lncRNA rPvt1 is
   different from that lncRNA from the human Pvt1 gene, we speculated that
   our lncRNA could influence and regulate cellular function in heart
   tissue through bioinformatics analysis. Therefore, to evaluate the
   potential role of lncRNA rPvt1 in sepsis, we created a lentiviral
   vector and silenced lncRNA, after which we observed that knockdown of
   Pvt1 expression could induce cell apoptosis in the LPS-induced heart
   cell line. This result suggests that Pvt1 could play a role in
   sepsis-induced myocardial depression.

   Compared with the control group, lncRNA rPvt1 in the sepsis group had a
   tendency toward high expression levels, and therefore this lncRNA could
   have a protective effect in sepsis. Furthermore, we evaluated the
   influence of lncRNA rPvt1 on its potential target genes, and we found
   that lncRNA rPvt1 could regulate the expression of several genes,
   including Myd88, c-Myc, and Bid, and then further influence the
   expression of genes related to cell apoptosis, such as Bax, Bcl-2, and
   caspase-3. Notably, in cancer-related research, several studies[140]26,
   [141]27 had reported that Pvt1 controls levels of Myc through
   regulation of the protein’s stability. However, there was no prior
   study focusing on the role of lncRNA rPvt1 in heart tissue in sepsis.
   In our study, we found that silencing lncRNA rPvt1 could increase both
   the protein level of c-Myc and the expressions of Bax and caspase-3,
   therefore increasing cell apoptosis in rat heart tissue.

   Our results also showed that the expression of Myd88 decreased in the
   LPS-induced KD group compared with the LPS-induced NC group. After
   performing RNA pull-down, we hypothesized that this process might be
   mediated by Irak-2, which could bind with lncRNA rPvt1 directly, and
   that the IL-1R/Myd88/Irak-2 pathway might be involved in this process.
   Although silencing lncRNA rPvt1 could inhibit the expression of Myd88
   and Irak-2, which would decrease the inflammatory response to a certain
   extent, in fact we found that silencing lncRNA rPvt1 led to an increase
   in cell apoptosis. This effect may be explained by a greater relative
   increase in other apoptosis-related proteins, such as Bax and Bid, and
   a decrease in the anti-apoptotic protein Bcl-2. However, the mechanism
   by which lncRNA rPvt1 influences c-Myc and regulates the expressions of
   Bax, Bid, and caspase-3 still requires further research to fully
   understand.

   Our study is an investigation into the role of lncRNAs in the
   pathological process of myocardial depression in models of septic
   shock. By applying whole genomic next-generation sequencing, we
   compared the DE lncRNAs and mRNAs between sepsis and control groups.
   These abundant data prompted the proposal that lncRNAs may interact and
   regulate their related protein-gene expression and, thus, play a key
   role in the pathogenesis of sepsis-induced myocardial depression.
   Additionally, we investigated lncRNA rPvt1 and detected its role in the
   myocardial cell model of sepsis. lncRNAs may provide new insights into
   the pathophysiology of sepsis-induced myocardial depression, as well as
   the potential for development of lncRNA mediators in the future.

Materials and Methods

Animal Model of Septic Shock

   The study was approved by the Ethics Committee of Shengjing Hospital of
   China Medical University (2019PS073K). All experiments conformed to all
   relevant regulatory standards. The adolescent rat model of septic shock
   was generated by intraperitoneal injection of LPS, as in our previous
   description.[142]^10 Briefly, male pathogen-free Wistar rats from
   Changsheng Bio (Benxi, China) weighing from 170 to 190 g were
   anesthetized by 20% urethane (1 g/kg i.p.). We cannulated the left
   femoral artery to continue monitoring the MAP of the models (Biopac
   MP150 Biopac Systems, Goleta, CA, USA). After the MAP was stable, we
   challenged the rat with Escherichia coli 055:B5 (L-2880, Sigma-Aldrich,
   St. Louis, MO, USA; 20 mg/kg, 10 mg LPS dissolved in 1 mL 0.9% saline),
   and septic shock was established when the MAP decreased to 25%–30% of
   the baseline value. For the control group, 0.9% saline (2 mL/kg) was
   injected into the peritoneal cavity of rats. The left ventricle of the
   heart was excised after 12-h LPS or saline administration and
   immediately snap frozen in liquid nitrogen and stored at –80°C for
   further experiments. In addition, we collected blood from the abdominal
   aorta at 12 h after LPS or saline administration, which was centrifuged
   (3,000 rpm, 10 min) after being maintained at room temperature for
   15 min. The blood supernatant was collected and stored at –80°C for
   ELISA.

H&E Staining, TUNEL Assay for Apoptosis, and ELISA Determination of Rat Serum

   The heart samples were fixed in 10% formaldehyde for 48 h. The
   specimens were subjected to dehydration and permeabilization and then
   embedded in paraffin. The paraffin tissues were sliced into serial
   sections (0.4 μm) and stained with H&E. Pathological changes were
   observed under a microscope.

   We used the TUNEL technique to detect apoptosis of heart tissues. A
   TUNEL apoptosis assay kit was purchased from WanleiBio (Shenyang,
   China), and we performed the assay in accordance with the
   manufacturer’s instructions.

   We detected TNF-α, IL-6, CK-MB, and cTnI in blood serum according to
   the instructions of the ELISA kit (CusaBio, Wuhan, China). The
   concentration of each sample was calculated according to the optical
   density value and the standard curve.

RNA-Seq and Bioinformatics Analyses

   Sequencing data were deposited with the NCBI (Sequence Read Archive
   [SRA]: PRJNA527717). The procedures regarding sequencing, data
   processing, and further bioinformatics analyses can be found in detail
   in the [143]Supplemental Materials and Methods. Total RNAs from rats
   subjected to septic shock (n = 6) and controls (n = 6) were isolated
   and quality controlled. The preparation of whole-transcriptome
   libraries and next-generation sequencing were conducted by Novogene
   Bioinformatics Technology (Beijing, China). RNA-seq was performed on an
   Illumina Hiseq 4000 platform, and 150-bp-paired single-end reads were
   generated according to Illumina’s protocol. All the downstream
   bioinformatics analyses were based on the clean data with high quality.

Validation of Gene Expression in RNA-Seq by qPCR Analysis

   Total RNA was isolated from heart tissue or cultured cells, according
   to the manufacturer’s instructions supplied with the TRIzol Reagent
   (Invitrogen, Life Technologies, Carlsbad, CA, USA), and then reverse
   transcribed with a Thermo First Synthesis Kit. Primers of lncRNAs were
   designed and synthesized by GenechemBio (Shanghai, China). The specific
   quantitative primers for nine lncRNAs are listed in [144]Table S1. Each
   targeted cDNA was amplified using SYBR Green via the StepOnePLUS
   Real-Time PCR system (Applied Biosystems, Waltham, MA, USA). In
   addition, the primers of the GAPDH gene were designed as an endogenous
   control.

RACE Clone of lncRNA rPvt1

   The PCR and RACE primers were designed and synthesized by Takara Bio
   (Dalian, China). Total isolated RNA was converted into cDNA using
   PrimeScript 1st Strand cDNA Synthesis Kit (Takara, 6110A, Japan) with
   oligo dT primer. All kits used in this section were provided with
   SMARTer RACE 5′/3′ Kit component (Takara, 634858, Japan). We strictly
   conducted the 3′-RACE and 5′-RACE PCR reactions that generated the 3′
   and 5′ cDNA fragments according to the manufacturer’s instructions.
   Then, purified PCR products were cloned into the pUC-19 vector and
   sequenced by Takara Bio (Dalian, China).

Northern Blot Analysis

   The heart tissues from the septic shock and control groups were used
   for RNA isolation and detection, run on a 1% agarose gel, and
   transferred to positively charged nylon membranes. The membranes were
   subjected to hybridization with 10.0 mL DIG Easy Hyb-labeled probes
   overnight at 50°C. Then, the band lncRNA rPvt1 was detected with X-ray
   film in a dark room. The probe sequences for lncRNA rPvt1 gene were
   5′-AGATGTTACAAGTGGCCTAATG-3′ (forward) and 5′-AGAGGTCTATCCTTGCTCTTTA-3′
   (300 bp) (reverse), which were designed and synthesized by Sangon Bio
   (Shanghai, China).

Cell Culture and Treatment Protocol

   The rat cardiomyoblast H9c2 cell line, obtained from the Shanghai
   Institute of Biochemistry and Cell Biology (Shanghai, China), was
   cultured at 37°C under 5% CO[2] in DMEM containing 10% fetal bovine
   serum with 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were
   passaged regularly and subcultured to 80% confluence prior to
   experimental procedures. For a septic cell model,[145]^10 we treated
   the cell line with LPS (10 μg/mL) for 12, 24, 36, and 48 h.

FISH of lncRNA rPvt1

   RNA FISH was performed using a Fluorescent in situ Hybridization kit
   from RiboBio (Guangzhou, China), in accordance with the manufacturer’s
   instructions. We used U6 and 18 s as our endogenous controls, which
   were obtained from RiboBio. The stained samples were imaged under
   confocal microscopy.

Lentiviral Silencing of lncRNA rPvt1

   The development of a lentiviral vector system to knock down lncRNA
   rPvt1 expression was designed to express small interfering RNA (siRNA)
   sequences for targeting Pvt1. A lentiviral vector that carried a
   nontargeting sequence was used as the NC of siPVT1. To track targeted
   cells, we designed a co-expressed GFP marker. The sequence of the
   lncRNA rPvt1 targeting siRNA was as follows: 5′-CCTATGAGGTGATGATAAA-3′.
   The H9c2 cell line was seeded into 6-well plates for 24 h and
   subsequently transfected with lentivirus in both the KO and NC groups.
   The transfected cells were grown at 37°C with 5% CO[2] for 96 h and
   then used for further experiments.

Measurement of Cell Apoptosis and Cell Cycle

   The H9c2 cells were divided into four groups: normal cells, LPS-induced
   H9c2 cells, and LPS-induced H9c2 cells in the KO and NC groups. All
   cells were trypsinized and subsequently harvested and incubated using
   an Annexin V-APC apoptosis detection kit (eBioscience, Waltham, MA,
   USA) after regular centrifugation and washing. Cellular apoptosis was
   evaluated via flow cytometry (Phoenix Flow Systems, San Diego, CA, USA)
   within 5 min and analyzed using a flow cytometer (Coulter EPICS XL-MCL
   FACScan, BD Biosciences, Franklin Lakes, NJ, USA). The data were
   analyzed using the guava InCyte Software to determine the cell
   apoptosis rates.

   As for the measurement of cell cycle, cells were stained with a
   fluorescent dye (propidium iodide) that directly binds to the DNA in
   the nucleus. After ethanol fixation and centrifugation, cells were then
   resuspended and 100 μL RNase A (Sigma, St. Louis, MO, USA) was added
   into each sample. After 30-min incubation at 37°C, 500 μL propidium
   iodide was added to each tube for at least 30 min to provide the
   nuclear signal for fluorescence-activated cell sorting (flow
   cytometry). Finally, cell cycle was evaluated with a flow cytometer.

Cell Proliferation Assay

   The cells transfected with siRNA were seeded into 96-well plates (2,000
   cells per well), incubated for 24 h, and then treated with LPS.
   According to the manufacturer’s instructions, a CCK-8 (Sigma-Aldrich,
   St. Louis, MO, USA) was used to determine cell proliferation after LPS
   stimulation. The absorbance of each well was monitored using a
   spectrophotometer at a wavelength of 450 nm.

Immunohistochemical Staining

   Immunohistochemical staining for Myd88, c-Myc, cleaved-Bid, p53, Bax,
   Bcl-2, and cleaved-caspase-3 was conducted on the left ventricular
   tissue from control and sepsis group. The myocardial tissue was
   deparaffinized and rehydrated in a graded series of alcohol solutions.
   Sections were treated with 3% H[2]O[2] for 30 min to block endogenous
   peroxidase activity and then with 1% BSA in PBS for 30 min. Slides were
   incubated overnight at 4°C with primary antibody (Myd88, c-Myc, p53,
   Bax, and Bcl-2; 1:200; Abcam, Cambridge, MA, USA; cleaved Bid and
   cleaved-caspase-3; 1:200; WanleiBio, Shenyang, Liaoning Province,
   China). The secondary antibody with horseradish peroxidase was used for
   incubation for 1 h. Images were viewed by a bright-field microscope
   (Nikon).

Western Blot Analysis

   The rat heart tissue and cultured H9c2 cell line were homogenized in
   RIPA buffer. Proteins from the homogenate (20 μg/well) were separated
   on a one-dimensional 4%–10% SDS polyacrylamide gel and transferred to a
   polyvinylidene membrane (Bio-Rad, Hercules, CA, USA) with a semidry
   electrotransfer apparatus for 1.5 h at 300 mA. The expression levels of
   Myd88, c-Myc, cleaved-Bid, p53, Bax, Bcl-2, cleaved-caspase-3, Irak-2,
   and IL-1R were determined (Myd88, c-Myc, p53, Bax, Bcl-2, and IL-1R;
   1:1,000; Abcam, Cambridge, MA, USA; Irak-2; 1:1,000; Cell Signaling
   Technology, Danvers, MA, USA; cleaved Bid and cleaved-caspase-3; 1:500;
   WanleiBio, Shenyang, Liaoning Province, China). The immunoblots were
   visualized by enhanced chemiluminescence (ECL detection kit from GE
   Healthcare, Chicago, IL, USA).

RNA Pull-down Assay

   Briefly, biotin-labeled lncRNA rPvt1 and its antisense RNAs from heart
   tissue in control and sepsis groups were transcribed in vitro with the
   Biotin RNA labeling mix and T7 RNA polymerase, then treated with
   RNase-free DNase I. All the pull-down experiment was used and followed
   the protocol from Magnetic RNA-protein Pull-Down Kit (Thermo Fisher,
   Waltham, MA, USA). Then, the proteins were measured using mass
   spectrographic analysis.

Statistical Analysis

   All experiments were performed at least three times. Data of a normal
   distribution were presented as the mean ± SEM. The Student’s t test for
   unpaired data was used to compare between the two groups. Two-way ANOVA
   of repeated measures was used to analyze the data of MAP changes with
   LPS administration. The statistical analysis was done using SPSS 23.0
   software (IBM, Armonk, NY, USA). p < 0.05 was considered statistically
   significant.

Author Contributions

   T.-N.Z. and C.-F.L. conceived and designed the study. T.-N.Z performed
   most assays. T.-N.Z. and R.W. performed animal models and heart tissue
   collection. T.-N.Z., T.Z., and W.-L.S. performed qPCR and western blot.
   T.-N.Z., J.E.G., B.L., D.L., N.Y., J.X., H.Z., and C.-F.L performed
   RNA-seq data analysis and interpretation. T.-N.Z., J.E.G., B.L., D.L.,
   and C.-F.L. wrote the manuscript. All the authors read and approved the
   final manuscript.

Conflicts of Interest

   The authors declare no competing interests.

Acknowledgments