Insulin plays a critical role in heart development and function, particularly in glucose metabolism [11, 12], as well as cardiac function and contractibility [13–15, 29–32]. In previous studies, we found that CE accumulation impairs insulin action by decreasing insulin intracellular pathway activation and glucose uptake in HL-1 cardiomyocytes and in rabbit myocardium [5, 6]. This impairment could be similar to what occurs in macrophages that uptake oxidized LDL via CD36 and scavenger receptor class A (SR-A), leading to foam cell formation and altered cholesterol homeostasis [33–35]. Additionally, aggLDL uptake promotes CE accumulation in lysosomes by inhibiting hydrolysis in macrophages [36]. Therefore, we investigated the impact of lipid accumulation on insulin response, the calcium cycle, and cardiac function at the transcriptomic level. To evaluate changes in gene expression due to lipid accumulation, we performed bulk RNA-sequencing analysis on HL-1 cardiomyocytes treated with aggLDL (100 µg/mL) for 8 h or vehicle (control). Additionally, to compare the gene expression profile modified by aggLDL to that regulated by insulin, transcriptome analysis was performed on HL-1 cardiomyocytes treated with insulin (100 nM) for 2 h or vehicle (control). We validated the transcriptome profiles through visual inspection of the data, including PCA analysis, Pearson correlation and unsupervised hierarchical clustering with heatmap representation (Fig. 1). Our analysis confirmed methodological repeatability and segregations, with similarity of biological replicates and no outlier samples within each cluster, and the robust segregation between aggLDL, insulin and control conditions (Fig. 1a). The complete expression matrix-based Pearson’s correlation analysis (Fig. 1b) supports these conclusions by showing a positive correlation between all datasets, typically exceeding R = 0.98 among samples belonging to the same cluster. We also performed unsupervised hierarchical clustering on the top100 most expressed genes within the transcriptome profiles, shown by the dendrogram and heatmap (Fig. 1c). The generation of gene unsupervised clusters suggests that genes may be regulated similarly or serve biological functions. The insulin clustering pattern was similar to the control condition (likely due to the short stimulus time), while the aggLDL condition pattern was completely different, as visually apparent in the heatmap. Interestingly, our RNA-Seq dataset showed a completely opposing expression pattern of genes between insulin and aggLDL conditions. Treatment with aggLDL resulted in the upregulation of 286 genes and downregulation of 859 genes relative to the control (Fig. 2a-left panel), while insulin resulted in the upregulation of 65 genes and downregulation of 85 genes relative to control (Fig. 2a-right panel).
The results of the gene ontology analysis comparing aggLDL to control are displayed in the left panel of Fig. 2b. We found upregulated genes belonging to processes such as ‘antibacterial innate immune response’ (related to lipoprotein-induced training immunity in macrophages, monocytes and endothelial cells by oxidized LDL [33, 34], and ‘activation of innate and humoral immune response’, as seen in other modified LDLs [37, 38]). We also observed upregulation of genes involved in ‘cellular oxidative stress’ (increasing gene expression related to reactive oxygen species production, similar to that generated by oxidized and acetylated LDL in macrophages [39]), ‘fatty acid metabolism’, ‘lipid uptake transport’ (related to lipid uptake, transport, and cholesterol esterification for storage in agreement with evidence found in macrophage related to aggLDL effect on CE accumulation in lysosomes by inhibition of its hydrolysis [40], and plasma lipoproteins particle remodelling), and ‘triglyceride catabolism’ (increasing gene expression of lipoprotein and triglyceride lipase activity). We also found downregulated processes mainly involved in ‘cell development and morphogenesis’ (related to muscle cell differentiation and proliferation and tissue development), ‘lipid biosynthesis’ (decreasing gene expression related to cholesterol and fatty acid biosynthesis, and cholesterol efflux), ‘phosphorylation activity’ (related to insulin and Wnt signalling pathways, and regulation of phosphate metabolic process), ‘positive cell regulation’ (including processes such as cell homeostasis, proliferation, apoptosis, muscle differentiation, and calcium cycle, similar to that described for human vascular smooth muscle cells (hVSMC) exposed to oxLDL [41]), ‘cell migration’ (decreasing gene expression involved in cell motility and locomotion), ’cell adhesion molecules’ (related to expression of cell adhesion molecules), ‘phospholipid homeostasis’ (involving process such as cell junction organization and regulation of cell structure size), and ‘nucleoside metabolic process’. It is notable that the number of biological processes affected by downregulated genes was higher compared to upregulated genes. This may be because the number of downregulated genes was three times higher than the number of upregulated genes (as seen in Fig. 2a-left panel). Furthermore, we confirmed the impairment in insulin signalling gene expression in agreement with our previous evidence in HL-1 cardiomyocytes [5]. We also evaluated our hypothesis-driven by mapping the gene expression levels into mouse WikiPathways [42]. We selected insulin signalling, (WP65, https://www.wikipathways.org/index.php/Pathway:WP65), calcium regulations in cardiac cells (WP553, https://www.wikipathways.org/index.php/Pathway:WP553), striated muscle contraction (WP216, https://www.wikipathways.org/index.php/Pathway:WP216), and cholesterol metabolism (WP4346, https://www.wikipathways.org/index.php/Pathway:WP4346). The logFC mapped to the different WikiPathways can be found in Supplementary Table 1. Despite the majority of downregulated genes in certain pathways, such as insulin signalling, having a logFC value greater than − 2, we were able to identify several downregulated genes within the same pathway. Thus, the cumulative effects of those genes could explain the impairment of the pathway.
In the comparison between insulin and control, we found significant upregulation of genes mainly involved in ‘absorption and transport of lipids’ (increasing gene expression for lipogenic enzymes, fatty acid transport, and intestinal lipid absorption and lipid efflux), ‘regulating signalling pathways’ (insulin and Wnt pathways gene expression are also promoted) and ‘defence response to the bacteria’ (increasing gene expression related to innate immune response to bacteria, in agreement with evidence about insulin-enhanced immune response and trained immunity in macrophage [40]). On the other hand, downregulated genes were involved in processes such as ‘leukocyte cell adhesion’ (decreasing gene expression involved in cell adhesion, T cell differentiation and activation), ‘response to external biotic’ (related to the biological process involved in interspecies interaction between the organism and chemokine production), ‘cellular response to hormone’ (decreasing gene expression involved in cellular response to cAMP, cellular response to glucocorticoid, corticosteroids and steroid hormones), and ‘collagen metabolism’ (by decreasing the expression of metalloproteinase and genes related to collagen biosynthetic pro-cess) (Fig. 2b-right panel).
We also evaluated the combined effect of incubating HL-1 cells with 100 µg/mL of aggLDL for 8 h followed by 100 nM insulin for 2 h. Compared to untreated cells (control), this combined treatment resulted in an upregulation of several processes including ‘lipid biosynthesis’, ‘leukocyte adhesion’, ‘innate immune response’, ‘growth signalling pathway’, ‘lipid esterification’, ‘cholesterol absorption’, and ‘negative regulation of NF-κB signalling’, which could be related to alteration in apoptosis and cell survival. Among the downregulated processes were ‘processing of fatty acids’, ‘calcium homeostasis and cardiac contractibility’, ‘Wnt and insulin signalling of the pathway’, and ‘processing of extracellular matrix or cell motility’ (Supplementary Fig. 1 and Supplementary Table 1). All these up- and downregulated processes were similar to the aggLDL-induced condition, which it may be due to the short duration of the insulin stimulus being insufficient to reverse the effect of lipid accumulation in these cells. Further studies are needed to determine whether insulin can counteract the detrimental of lipid accumulation with longer stimuli.
Based on the unsupervised GO enrichment and WikiPathways analysis, a network diagram with dysregulated genes of biological processes of interest and logFC values of aggLDL or insulin treatments compared to control is shown in Fig. 3. The focused was on processes related to insulin signalling and glucose uptake (Insr, Ins1, Pik3ip1, Slc2a4 gene expression), calcium cycle (Cacna1s and Gjc2 gene expression) and calcium-dependent cardiac contractility (Myh3), and cholesterol efflux (Abca1), which are well-known processes in the heart.
We found that aggLDL downregulated the expression of these genes, while insulin upregulated them. To validate the diagram in Fig. 3, we performed qPCR validations on fresh samples of HL-1 cardiomyocytes treated with aggLDL (100 µg/mL for 8 h) or insulin (100nM for 2h), same conditions as the transcriptome was done. Our analysis revealed that mRNA expression of the insulin signalling pathway Insr, Ins1; and Pik3ip1 (encoding the insulin receptor (IR), insulin receptor substrate-1 (INS1) and phospho-inositide-3-kinase interacting protein 1 (PIK3IP1), respectively), glucose uptake Slc2a4 (encoding for glucose transporter type 4 (GLUT4)), calcium cycle Cacna1s and Gjc2 (encoding for calcium voltage-gated channel subunit alpha1 S (CACNA1S) and connexin-47, respectively), calcium-dependent cardiac con-tractility Myh3 (encoding miosina-3), as well as cholesterol efflux Abca1 (encoding the ABCA1 transporter) showed a differential expression with a significant reduction by aggLDL and increased expression induced by insulin (Fig. 4). This is in agreement with our previous data that showed that aggLDL effectively increases intracellular lipid accumulation in HL-1 cardiomyocytes (Supplementary Fig. 2) [6]. These results align with previous evidence suggesting that insulin promotes gene expression involved in its own signalling pathway and GLUT4 [29–31], as well as calcium cycling and contractibility [32]. In the present study, the downregulatory effect of aggLDL on these same genes in cardiomyocytes suggests that the intracellular CE accumulation has a deleterious effect on these insulin-regulated processes.
Finally, in previous studies we found that ApoE-KO mice had higher levels of circulating cholesterol, triglycerides, LDL-cholesterol, and HDL-cholesterol, and increased fasting plasma insulin levels [28]. The increase in fasting insulin was detected at 6 months of age, while the alteration in the lipid profile (high levels of total cholesterol, HDL and LDL cholesterol) was found at 4 months of age and lasted until 6 months of age [28]. In the present study, we found that ApoE-KO mice (6 months of age) had increased lipid accumulation in the myocardium detected by BODIPY-stained lipid droplet (Supplementary Fig. 3). This suggests that alterations in lipid metabolism precede and, therefore, could be the cause of impaired insulin response. Using qPCR, we characterized the gene expression levels associated with insulin signalling, glucose uptake, calcium cycle, calcium-dependent cardiac contractibility, and lipid efflux in hearts of ApoE-KO mice. Compared to wild-type (Wt) mice, we found that the hearts of ApoE-KO mice had decreased expression of Insr, Ins1, and Pik3ip1(insulin signalling pathway), Slc2a4 (glucose uptake), Cacna1s and Gjc2 (calcium cycle), Myh3 (calcium-dependent cardiac contractility), and Abca1 (cholesterol efflux) genes (Fig. 5). The decreased gene expression of Abca1 in hearts of ApoE-KO mice is consistent with the increased lipid accumulation in the myocardium (Supplementary Fig. 3).