All experimental protocols were approved by Shengjing Hospital of China Medical University (2019PS073K). All methods were carried out in accordance with relevant guidelines and regulations. The study was carried out in compliance with the ARRIVE guidelines (http://​www.​nc3rs.​org.​uk/​page.​asp?​id=​1357). An adolescent rat model of septic shock was established by the intraperitoneal injection of lipopolysaccharide (LPS), as in our previous study [6, 7]. Briefly, male pathogen-free Wistar rats from Changsheng Bio Company (Benxi, China) weighing from 170 to 190 g were anesthetized with 20% urethane (1 g/kg i.p.). We cannulated the left femoral artery to continue monitoring the mean arterial pressure (MAP) of the animals (Biopac MP150; Biopac Systems, Goleta, CA, USA). To develop a model of septic shock with myocardial dysfunction, we challenged the rats with a large dose of Escherichia coli 055:B5 (L-2880; Sigma–Aldrich, St. Louis, MI, USA; 20 mg/kg, 10 mg LPS dissolved in 1 mL of 0.9% saline); septic shock was considered to have been established when MAP decreased to 25%–30% of the baseline value. The left ventricle of the heart was excised after 12 h of LPS or saline administration, immediately snap-frozen in liquid nitrogen, and stored at − 80℃ for further experiments.

Total RNAs from rats subjected to septic shock (n = 6) and controls (n = 6) were isolated and quality-controlled. All information regarding each relative kit and detailed procedures about the RNA extraction, RNA quality control and library preparation were displayed in our previous studeis [6, 7]. The preparation of whole-transcriptome libraries and next-generation sequencing were conducted by Novogene Bioinformatics Technology Corporation (Beijing, China). RNA-seq was performed on an Illumina Hiseq 4000 platform and 150-bp paired-end reads were generated in accordance with Illumina’s protocol. All of the downstream bioinformatic analyses were based on the clean data of high quality.

The Ballgown suite includes functions for interactive exploration of the transcriptome assembly, visualization of transcript structures and feature-specific abundances for each locus, and post hoc annotation of assembled features to annotated features. Cuffdiff provides statistical routines for determining the differential expression in digital transcript or gene expression data using a model based on the negative binomial distribution [11]. Transcripts with an corrected P value (also called as q valude) of < 0.05 were defined as differentially expressed, which was consistent with our previous study [6, 7].

The investigation of target gene prediction of TUCPs was acting on neighboring target genes (co-location analyses, cis role of TUCP). To investigate their possible functions, we searched the coding genes within 100 kb upstream and downstream of each TUCP [12, 13]. Trans role of target gene prediction is TUCP to identify each other by the expression level (co-expression analyses). We clustered the genes from different samples with WGCNA to search common expression modules and then analyzed their function through functional enrichment analysis [14]. We used co-localized and co-expressed mRNAs to predict the potential roles of TUCP transcripts during sepsis-induced myocardial depression by Gene Ontology (GO) enrichment and KEGG pathway analyses.

GO enrichment analysis of differentially expressed genes or lncRNA target genes was implemented using the GOseq R package, in which gene length bias was corrected [15]. GO terms with a corrected P value less than 0.05 were considered significantly enriched among the differentially expressed genes. Additionally, we used KOBAS software to test the statistical enrichment of differentially expressed genes or TUCP target genes in KEGG pathways (www.​kegg.​jp/​kegg/​kegg1.​html) [16, 17].

In our previous studies, we found that LPS-treated rats showed a gradual decline in cardiac function as evidenced by significant decreases in heart rate, LV peak rate of a pressure rise, and an LV peak rate of pressure decay, as well as a prolonged relaxation time constant, and there is a positive relationship between MAP and heart function [18]. Therefore, we used a rat sepsis model and measured the MAP to represent changes in heart function. We found that septic shock occurred approximately 2 h after LPS administration and lasted for the rest of the observation period [6, 7]. The left ventricle of the heart was then excised for RNA-seq and the determination of TUCPs. We identified 4,851 TUCPs from both the sepsis group and the control group. Detailed information regarding each TUCP including chromosome location, start and end locations, exon number, length, and open reading frame (ORF) is shown in Additional file 2: Table S1. The sequence of each TUCP is shown in Additional file 3: Table S2. To clarify the basic characteristics of TUCPs, we first compared their expression level with those of lncRNAs and mRNAs. We found that the expression level of TUCPs was similar to that of lncRNAs in these 12 samples, but was lower than that of mRNAs (Fig. 1a, b). In addition, we performed a comparison of the TUCPs with mRNAs in terms of length, exon number, and ORF. Our findings suggested that the TUCPs were longer than the mRNAs; however, the TUCPs had fewer number of exons and shorter ORFs than the mRNAs (Fig. 1c–e).

To determine whether TUCPs are involved in the pathogenesis of sepsis-induced myocardial depression, the expression pattern of TUCPs was analyzed and compared between the sepsis group and the control group. We analyzed DE using significance analysis with the threshold of q value < 0.05 (corrected P value). The results revealed 85 TUCPs that were differentially expressed between the sepsis group and the control group, including 38 that were upregulated and 47 that were downregulated in the sepsis group (Fig. 2a). The chromosome distribution of the DE TUCPs is shown in Additional file 1: Figure S1. In addition, the expression pattern of the DE TUCPs is shown using a cluster heatmap in Fig. 2b. Furthermore, the relative expression levels of the top 15 upregulated and downregulated TUCPs are shown in Fig. 2c. Detailed information on the 85 DE TUCPs including the relative expression levels in the two groups, fold change, and q value is displayed in Additional file 4: Table S3. Notably, three TUCPs (TUCP_000356, TUCP_002674, and TUCP_004817) were not detected in the control group but were expressed in the sepsis group. Besides, all the co-located and co-expressed genes for each DE TUCP were shown in Additional file 5: Table S4 and Additional file 6: Table S5, respectively. These results reflect distinct TUCP expression profiles between sepsis-induced myocardial depression and the control group, implying the critical role of TUCPs in the pathophysiology of septic myocardial depression.

To elucidate the possible functional significance of the observed changes in TUCPs between the sepsis group and the control group, we performed a GO term enrichment analysis based on co-location analyses (cis role of TUCPs) and co-expression analyses (trans role of TUCPs, Additional file 7: Table S6 and Additional file 8: Table S7). There were 20,121 background genes of GO terms in total. We summarized the GO terms significantly enriched for the TUCPs regarding co-location analyses (Fig. 3a–c) and co-expression analyses (Fig. 3d–f), for the categories of biological process, cellular component, and molecular function, respectively. For co-location, the GO terms “Regulation of the force of heart contraction,” “Regulation of ATPase activity,” and “Cardiac muscle contraction” were enriched, suggesting that several TUCPs participate in energy production and myocardial contraction as co-location analyses, highlighting the critical role of TUCPs in the pathogenesis of sepsis-induced myocardial depression. For co-expression, we found that TUCPs take part in metabolic processes in myocardial tissue. For example, the GO terms “Regulation of metabolic process,” “Regulation of primary metabolic process,” “Positive regulation of metabolic process,” and “Regulation of cellular metabolic process” were enriched, which showed that TUCPs have potential roles in regulating metabolism. Based on GO analysis, we found that complex pathological processes are involved in sepsis-induced myocardial depression and that TUCPs play key roles in this disease.

To determine whether some specific pathways changed in sepsis-induced myocardial depression, we performed KEGG enrichment analysis in TUCPs based on co-location analyses and co-expression analyses (Additional file 9: Table S8 and Additional file 10: Table S9). For co-location (Fig. 4a–c), we found that TUCPs could affect heart contraction by influencing some specific pathways. For example, “cGMP − PKG signaling pathway,” “Cardiac muscle contraction,” “Calcium signaling pathway,” and “Adrenergic signaling in cardiomyocytes” were downregulated, which was consistent with the pathophysiological changes of sepsis-induced myocardial depression and also similar to the GO enrichment results, indicating the critical role of TUCPs in regulating heart contraction. For co-expression (Fig. 4d–f), “TNF signaling pathway,” “NF-kappa B signaling pathway,” “Jak–STAT signaling pathway,” and “Apoptosis” were enriched, suggesting that TUCPs could function in these pathways in sepsis-induced myocardial depression. Our KEGG enrichment analysis also suggested the significance of TUCPs in sepsis-induced myocardial depression.