Canagliflozin combined with aerobic exercise protects against chronic heart failure in rats

Abstract

To determine the efficacy and potential protective mechanism of canagliflozin combined with aerobic exercise in treating chronic heart failure (CHF). Isoproterenol was injected into rats to create CHF models. The rats were then subsequently divided into saline, canagliflozin (3 mg/kg/d), aerobic exercise training, and canagliflozin combined with aerobic exercise training. Compared to the CHF group, the canagliflozin combined with the aerobic exercise group had superior ventricular remodeling and cardiac function. In rats treated with canagliflozin combined with aerobic exercise, the expression of cytochrome P450 (CYP) 4A3, CYP4A8, COL1A1, COL3A1, and FN1 was reduced, while the expression of CYP26B1, ALDH1A2, and CYP1A1 increased significantly. Additionally, canagliflozin combined with aerobic exercise decreased the phosphorylation of AKT and ERK1/2. Canagliflozin combined with aerobic exercise has a positive effect on the development of CHF via the regulation of retinol metabolism and the AKT/ERK signaling pathway.

Keywords: Biological sciences; Natural sciences; Pharmacology; Physiology.

Graphical abstract

Figure 1

Effects of canagliflozin combined with aerobic exercise on echocardiographic variables in rats induced by ISO (A) The representative images of echocardiography in different groups. (B) The quantitative results of echocardiography. (C) The expression level of NT-pro BNP. Data are shown as the mean ± SEM of n = 5 per group. Significance: ###p < 0.001 vs. control group; ####p < 0.0001 vs. control group; ∗p < 0.05 vs. ISO group; ∗∗p < 0.01 vs. ISO group. ISO, isoproterenol-treated group; ISO + AE, ISO + aerobic exercise group. ISO + CA, ISO + canagliflozin; ISO + AE + CA, ISO + aerobic exercise + canagliflozin; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; NT-pro BNP, N-terminal pro-B-type natriuretic peptide. Data are represented as mean ± SEM.

Figure 2

Effects of canagliflozin combined with aerobic exercise on ventricular remodeling in rat heart tissue (A) Representative images of HE staining of left ventricular tissue in different groups (Microscope magnification 10×, scale bar indicates 200 μm; Microscope magnification 40×, scale bar indicates 50 μm). (B) Representative images of Masson trichrome staining of left ventricular tissue in different groups, (Microscope magnification 10×, scale bar indicates 200 μm; Microscope magnification 40×, scale bar indicates 50 μm). (C–E) Immunohistochemical analysis of collagen I, collagen Ⅲ and fibronectin protein in heart cross sections of different groups (Microscope magnification 40×, scale bar indicates 50 μm). (F) The quantitative analysis of collagen deposition and percentage area of collagen I, collagen III and fibronectin. (G) The gene expression levels of COL1A1, COL3A1 and FN1 were detected by qPCR. The internal reference was β-actin. Data are shown as the mean ± SEM of n = 5–6 per group. Significance: ###p < 0.001 vs. control group; ####p < 0.0001 vs. control group; ∗∗∗p < 0.001 vs. ISO group; ∗∗∗∗p < 0.0001 vs. lSO group. ISO, isoproterenol-treated group; ISO + AE, ISO + aerobic exercise group; ISO + CA, ISO + canagliflozin; ISO + AE + CA, ISO + aerobic exercise + canagliflozin; HE, hematoxylin-eosin; COL1, Collagen I; COL3, Collagen III; FN1, fibronectin. Data are represented as mean ± SEM.

Figure 3

RNA-seq was performed to detect genome-wide transcriptomes and identify differentially expressed genes (A) Volcano plot of differential genes from the ISO group and the Control group identified through RNA-seq. The abscissa is log2 (fold change), and the vertical coordinate is the negative logarithm of the value of Q with a base of 10, i.e., -log10 (Q). The larger the value, the more significant the difference. (B) Scatterplot of differential genes. The horizontal and vertical coordinates represent the samples from the ISO group and the ISO+AE+CA group, respectively. The red indicates the up-regulated differential genes in the ISO+AE+CA group relative to the ISO group and the blue indicates the down-regulated differential genes in the AE + CA group relative to the ISO group, and the gray indicates genes that are not different between the two groups. (C) Venn diagram. The orange section indicates differential genes from the ISO group and the Control group targets, and the green section indicates differential the ISO group and the ISO+AE+CA group targets. One hundred and fifty-two targets in the middle overlapping section are common targets. (D) Histogram of differential genes. (E) Heatmap of differential genes. Each row represents a differential gene, each column represents the same rat sample, and each group has 3 replicates.

Figure 4

Enrichment analysis of the targets of canagliflozin combined with aerobic exercise in treating CHF (A) PPI network. A total of 120 target proteins and 258 interacting edges are in the network. The sizes and colors of the nodes are illustrated from big to small and blue to green in descending order of degree values. (B) GO functional analysis. Biological process items in GO analysis enriched for up-regulated and down-regulated genes respectively. (C) KEGG pathway enrichment analysis. The sizes of the bubbles were illustrated from big to small in descending order of the number of potential targets involved in the pathways. (D) Target-pathway network. A total of 36 nodes and 54 edges are in the network. The middle square nodes represent targets on the pathway, the red nodes represent key targets, and 12 blue V-shapes represent pathways. The sizes of the square node were illustrated from big to small in descending order of degree values. 54 edges represent the interaction relationship between components, targets, and pathways. CHF, chronic heart failure; PPI, protein-protein interaction; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 5

Effects of canagliflozin combined with aerobic exercise on retinol metabolic pathways and indicators of fibrosis (A) The gene expression levels of CYP4A3, CYP4A8 and CYP1A1 were detected by transcriptome sequencing and qPCR. The internal reference was β-actin. (B) The gene expression levels of CYP26B1, ALDH1A2 and AOX1 were detected by transcriptome sequencing and qPCR. The internal reference was β-actin. Data are shown as the mean ± SEM of n = 6 per group. Significance: #p < 0.05 vs. control group; ##p < 0.01 vs. control group; ####p < 0.0001 vs. control group; ∗p < 0.05 vs. ISO group; ∗∗p < 0.01 vs. ISO group; ∗∗∗p < 0.001 vs. ISO group; ∗∗∗∗p < 0.0001 vs. ISO group. ISO, isoproterenol-treated group; ISO + AE, ISO + aerobic exercise group; ISO + CA, ISO + canagliflozin; ISO + AE + CA, ISO + aerobic exercise + canagliflozin; ELISA, enzyme linked immunosorbent assay; qPCR, reverse transcription-polymerase chain reaction. Data are represented as mean ± SEM.

Figure 6

Effects of canagliflozin combined with aerobic exercise on the AKT/ERK signaling pathway in rats The protein expression levels of AKT, p-AKT, ERK1/2 and p-ERK1/2 were determined by western blot. (A) The protein expression levels of AKT, p-AKT, ERK1/2 and p-ERK1/2 in heart tissues, and β-tubulin was used as the internal standard. (B) The quantitative results were calculated by Image Lab software. Data are shown as the mean ± SEM of n = 3 per group. Significance: ##p < 0.01 vs. control group; ###p < 0.001 vs. control group; ∗p < 0.05 vs. ISO group; ∗∗p < 0.01 vs. ISO group; ∗∗∗p < 0.001 vs. lSO group; ∗∗∗∗p < 0.0001 vs. lSO group. ISO, isoproterenol-treated group; ISO + AE, ISO + aerobic exercise group; ISO + CA, ISO + canagliflozin; ISO + AE + CA, ISO + aerobic exercise + canagliflozin; p-AKT, phosphorylated AKT; p-ERK1/2, phosphorylated ERK1/2. Data are represented as mean ± SEM.

Figure 7

The mechanism of aerobic exercise combined with canagliflozin therapy against ISO-induced CHF Canagliflozin combined with aerobic exercise therapy modulates protein kinase activity. The combination therapy inhibited the activation of AKT/ERK signaling pathway, thereby improving myocardial fibrosis. Canagliflozin combined with aerobic exercise improved cardiac energy metabolism in CHF rats mainly through the retinol metabolic pathway. The combination significantly upregulated CYP1A1 levels to promote the conversion of retinol to retinoic acid, and it also improved myocardial fibrosis by regulating ALDH1A2 and CYP26B1 to maintain retinoic acid homeostasis. Meanwhile, the combination treatment reduced heart failure by downregulating CYP4A3 and CYP4A8 levels to reduce the production of 20-hydroxyeicosatetraenoic acid.