After injury, the heart inevitably undergoes fibrosis which leads to heart failure. The development of injury-induced myocardial fibrosis is resulted from the interaction of multiple factors, including the matrix structural remodeling and the cell functional change [12]. As cardiomyocytes plays an important role in the heart functioning, a further study on cardiomyocytes can lead to better understanding of the mechanism of the heart disease development. Besides, it also provides an important target molecule for clinical gene therapy of heart diseases.

Recently, non-coding RNAs have become a major focus of biological research. Longer than 200 nucleotides and without protein-coding potential, the long noncoding RNAs (lncRNAs) are highly abundant in the human body [13]. As a newly found epigenetic regulatory molecule, the lncRNAs attract a lot of attention in the field of cardiovascular disease research [14]. It shows that certain lncRNAs are regulated in acute myocardial infarction (e.g. Novlnc6 [4]) and heart failure (e.g. Mhrt [6]). In addition, lncRNAs demonstrate an ability to control the cardiomyocyte hypertrophy, mitochondrial function and apoptosis. In the vascular system, the endothelin-expressing lncRNAs (e.g.MALAT1 [15]) shows to regulate growth and function of the vascular, while the smooth muscle-expressing lncRNAs and the migration/differentiation-related lncRNAs enriching in endothelial cells prove to control the contraction of the smooth muscle cells [16]. These findings suggest that lncRNAs play a significant role in regulating the development of cardiovascular disease.

The Ftx gene is a non-protein-coding gene located on the human X chromosome. Its DNA sequence encodes nine introns, seven of which are transcribed to RNA fragments and then linked to be lncRNA Ftx (long non-coding RNA Ftx, Ftx) [7]. LncRNA Ftx consists of approximately 2300 nucleotides. In the past, studies on the function of Ftx focused on its involvement in the development of congenital diseases [17]. Recently, studies turn to the regulatory role of Ftx in acquired diseases. Studies demonstrate that lncRNA Ftx induces the progression of liver cancer, colon cancer, kidney cancer, and liver fibrosis [18, 19]. For cardiovascular diseases, Long B et al. shows that the lncRNA Ftx is significantly downregulated in the ischemia–reperfusion injured and hydrogen peroxide-treated myocardial tissue, and the overexpression of lncRNA Ftx attenuates the apoptosis induced by hydrogen peroxide in cardiomyocyte. This function may be associated with the regulation of Bcl212 expression by lncRNA Ftx-mediated miR-29b-1-5p [20]. Recent studies find that overexpression of lncRNA Ftx can upregulate Fmr1 by sponging miR-410-3p (fragile X mental retardation 1) to induce cell proliferation, inhibit apoptosis and oxidative stress, which alleviates the cardiomyocyte injury induced by hypoxia/reoxygenation [10]. Yang X et al. uses arginine II to induce hypertrophy of neonatal mouse cardiomyocyte in vitro and demonstrates that the expression of lncRNA Ftx is significantly downregulated. While the overexpression of lncRNA Ftx significantly reduces the apoptosis, myocardial contractility, and the expression of some key molecules such as c-Jun, A-type natriuretic peptide (ANP) and B-type natriuretic peptide (B). A further study confirms that lncRNA Ftx played a role in reducing myocardial hypertrophy by sponging miRNA-22 to regulate the PTEN/PI3K/Akt signaling pathway [11].

Previous findings confirm the differential expression levels of lncRNA Ftx in cardiac diseases, suggesting its regulatory role in the cardiac disease development, but the specific regulatory effects and mechanisms have not been fully studied. To investigate the effects and potential molecular mechanisms of lncRNA Ftx on cardiomyocytes, this work establish the lncRNA Ftx function gain-and-loss model in the cardiomyocyte AC16 cell line. The overall proteome of cardiomyocyte in response to lncRNA Ftx knockdown and overexpression is systematically characterize with the quantitative proteomics and bioinformatics analysis.

Combined with the functional clustering analysis of the selected Hub genes and differential proteins, we find that knockdown of lncRNA Ftx upregulates the proteins associated with cell aging, fibrogenic differentiation, and apoptosis, while downregulates the proteins associated with cell cycle, cell proliferation, and anti-apoptosis to a certain extend. These alterations are dominated by the Hub genes ITGB1, TFRC, and RB1. In terms of pathways, the downregulation of the lncRNA Ftx upregulates the activation of the CAMs, PI3K-Akt signaling pathway, and PPAR signaling pathway, while inhibits the activation of JAK-STAT signaling pathway. On the other hand, the overexpression of the lncRNA Ftx upregulates the proteins associated with positive regulation of growth and cell cycle, and downregulates the iron death-related pathways, evidenced by the altered expression levels of several Hub genes, including VCP, PSMD1, 4,7, GSS and LPCAT3. The difference in protein expression of AC16 cells is associated with the LncRNA Ftx overexpression and knockdown, indicating the lncRNA Ftx is involved in altering myocardial function, and making it a potential target for disease occurrence, development and treatment.

The normal regulation of the cell cycle is essential for cell proliferation, differentiation, and apoptosis. Lack of regenerative capacity, cardiomyocytes are replaced by collagen fibers after dying due to inflammation or injury, which eventually leads to myocardial remodeling. Therefore, apoptosis and ageing of the cardiomyocyte plays an essential role in the myocardial disease development, and the inhibition of apoptosis in myocardial ischemic disease shows to mitigate this process [21, 22]. In the lncRNA Ftx dysfunction model, we identify multiple proteins involved in apoptosis, evidenced by the upregulation of ITGB1, HMGA2, KRAS and the downregulation of STAT3, RB1, LGALS3.

ITGB1 (Integrin β1), a member of the Integrin family, acts as an extracellular matrix receptor that regulate cell–matrix interactions, cell proliferation, and epithelial-mesenchymal transformation [23]. ITGB1 have impacts on the cardiovascular system in several aspects, including myocardial function and differentiation [24]. Transient episodes of myocardial ischemia promote the proliferation of endothelial cells and the formation of small arteries throughout the myocardium in response to the myocardial infarction, where the ITGB1 is involved [25]. Some study shows that the cardiomyocyte-specific knockdown of ITGB1 leads to the development of myocardial fibrosis and heart failure [26]. However, the proteomic analysis in this study show that the knockdown of lncRNA Ftx resulted in the upregulation of ITGB1 expression, and more studies will be needed to reveal the effects of the lncRNA Ftx on the myocardium.

HMGA2 (High mobility group protein AT-hook 2) belongs to the high mobility A genome. As a structural transcription factor, HMGA2 is important for cell growth and differentiation and is involved in the epithelial mesenchymal transformation [27]. A study confirms that HMGA2 plays a crucial role in cardiogenesis and remodeling [28]. Wong, L. L. et al. shows that targeting the 3'-UTR of HMGA2 could inhibit apoptosis and protect cardiomyocyte from ischemic injury [29]. HMGA2 induces the apoptosis by upregulating cleaved Caspase 3 through the DNA damage pathway, associated with the upregulation of cleaved Caspase 9, p53, Bax, and the downregulation of Bcl2, Apaf1. However, another study in a mouse model of myocardial remodeling demonstrates that the cardiac-specific expression of HMGA2 reduces myocardial fibrosis and improves cardiac function by activating the PPAR pathway [30]. Our results also show the concomitant activation of the PPAR pathway in the presence of upregulated HMGA2. Therefore, the specific effects of the upregulated HMGA2 protein after lncRNA Ftx knockdown need to be further investigated.

STAT3 is an important factor of the signaling pathway, and its downstream target genes are involved in the regulation of cell differentiation, proliferation, apoptosis, angiogenesis, metabolism and immune response, etc. The protective effects of STAT3 on cardiomyocytes are reflected by the anti-apoptosis and the energy generation. On the one hand, STAT3 helps the cardiomyocyte survive by upregulating the expression of anti-apoptotic genes Bcl-xL and Bcl2[31], and block the TNF-α pro-apoptotic channel [32]. On the other hand, STAT3 demonstrates to present in the mitochondria of cardiomyocyte, which regulates the activity of the type I complexes and oxygen consumption, and participates in energy production [33]. In addition, recent studies demonstrates that the JAK/STAT3 signaling pathway plays a crucial role in the induction, maintenance, and differentiation of the multipotential stem cells [34]. Further studies on STAT3 will benefits the cardiac regenerative therapy.

In addition, we notice a programmed cell death modulated by lncRNA Ftx which is distinct from apoptosis—ferroptosis. Overexpression of the lncRNA Ftx in cardiomyocyte shows to downregulates the ferroptosis pathway-related proteins (GSS and LPCAT3), indicating that the overexpression of lncRNA Ftx may play a protective role in cardiac disease by inhibiting ferroptosis in the cardiomyocyte.

Ferroptosis is a newly found mode of programmed cell death characterized by iron overload, reactive oxygen species (ROS) accumulation, or lipid peroxidation. Distinct from apoptosis, pyroptosis, autophagy and necrosis, it is first identified by Brent R. Stockwell’s laboratory [35, 36]. The cells with ferroptosis exhibit morphological loss of membrane integrity, accompanied by nuclei swelling, mitochondria crinkling, cristae reduction or absence, and outer membranes fragmentation [37]. Ferroptosis shows to be associated with a variety of diseases such as tumor, degenerative disease (Alzheimer's disease, Huntington's chorea, Parkinson's syndrome), and renal failure [38]. Ferroptosis is also involved in the development of many cardiac diseases, including myocardial ischemia–reperfusion injury, myocardial hypertrophy, diabetic heart disease, and doxorubicin-induced cardiotoxicity [39, 40].

Ferroptosis is closely related to many biological processes such as iron metabolism, glutathione (GSH) metabolism and lipid peroxidation. Therefore, the molecules involved in these metabolic pathways can affect the level of ferroptosis in cells [41]. GSS (Glutathione synthetase) is involved in the GSH anabolism and responds rapidly to the increased GSH demand. Glutathione peroxidase 4 (CPX4), an important negative regulator of ferroptosis, shows to play a biological role by regulating the GSS/GSR complexes [42]. LPCAT3 (lyso-phosphatidylcholine acyltransferase-3) is a major lyso-PL acyltransferases (LPLAT) isomer exhibiting a strong specificity to the polyunsaturated fatty acid (PUFA) [43]. Both of the proteins shows to be negatively regulated by the lncRNA Ftx, though further experimental evidence is needed to correlate them to the ferroptosis inhibition.

In summary, this study, for the first time, provides the data on the comprehensive changes in the quantitative proteomic profile of cardiomyocytes after lncRNA Ftx knockdown and overexpression by constructing the lncRNA Ftx function gain-and-loss model for the AC16 cells, with quantitative proteomics and bioinformatics analysis. The protein annotation, enrichment and clustering analysis reveal the properties of the differentially quantified proteins identified from the quantitative proteomic data. Potential Hub genes are selected by the protein interaction network analysis. Our results show that the lncRNA Ftx regulates the apoptosis and ferroptosis in cardiomyocytes and improves the cellular energy metabolism. LncRNA Ftx is involved in expression changes of several proteins such as ITGB1, HMGA2, STAT3, GSS and LPCAT3. It demonstrates to play a vital role in the occurrence and progression of myocardial diseases such as ischemia–reperfusion injury, myocardial hypertrophy, and myocardial fibrosis, thus provides a promising target for the protection of the myocardium and the reversal of myocardial fibrosis.