Global Transcriptomic Profiling of Cardiac Hypertrophy and Fatty Heart Induced by Long-Term High-Energy Diet in Bama Miniature Pigs

Abstract

A long-term high-energy diet affects human health and leads to obesity and metabolic syndrome in addition to cardiac steatosis and hypertrophy. Ectopic fat accumulation in the heart has been demonstrated to be a risk factor for heart disorders, but the molecular mechanism of heart disease remains largely unknown. Bama miniature pigs were fed a high-fat, high-sucrose diet (HFHSD) for 23 months. These pigs developed symptoms of metabolic syndrome and showed cardiac steatosis and hypertrophy with a greatly increased body weight (2.73-fold, P<0.01), insulin level (4.60-fold, P<0.01), heart weight (1.82-fold, P<0.05) and heart volume (1.60-fold, P<0.05) compared with the control pigs. To understand the molecular mechanisms of cardiac steatosis and hypertrophy, nine pig heart cRNA samples were hybridized to porcine GeneChips. Microarray analyses revealed that 1,022 genes were significantly differentially expressed (P<0.05, ≥1.5-fold change), including 591 up-regulated and 431 down-regulated genes in the HFHSD group relative to the control group. KEGG analysis indicated that the observed heart disorder involved the signal transduction-related MAPK, cytokine, and PPAR signaling pathways, energy metabolism-related fatty acid and oxidative phosphorylation signaling pathways, heart function signaling-related focal adhesion, axon guidance, hypertrophic cardiomyopathy and actin cytoskeleton signaling pathways, inflammation and apoptosis pathways, and others. Quantitative RT-PCR assays identified several important differentially expressed heart-related genes, including STAT3, ACSL4, ATF4, FADD, PPP3CA, CD74, SLA-8, VCL, ACTN2 and FGFR1, which may be targets of further research. This study shows that a long-term, high-energy diet induces obesity, cardiac steatosis, and hypertrophy and provides insights into the molecular mechanisms of hypertrophy and fatty heart to facilitate further research.

Fig 1. Histopathological changes in miniature pig myocardial tissue (H&E staining).

The myocardial fibers from the control miniature pigs were neatly and tightly arranged (A, E, I, M, C, G, K, and O), but they were disorganized in the HFHSD group miniature pigs (arrows in N and P), showing inflammatory cell invasion (arrows in F and H) and cardiomyocyte hypertrophy (arrows in J, L). The tissue of the HFHSD group miniature pigs exhibited fat deposits (arrows in B and D), and some of the myocardial fibers were deformed by fat. A, E, I, M, C, G, K, and O: control group; and B, F, J, N, D, H, L, and P: high-fat and high-sucrose group; bar = 50 μm.

Fig 2. Mallory dye staining showing collagenous myocardial fibers in the two groups.

The myocardial fibers were separated by larger distances (arrows in B), and the collagen fibers around the blood vessels were increased (arrows in D) in the HFHSD group. A and C: control group; and B and D: high-fat and high-sucrose group; cross-section, bar = 100 μm.

Fig 3. Hierarchical clustering of genes and gene ontology (GO) analysis of biological processes in the HFHSD and control groups.

Genes with P values <0.05 were identified as clustered. Red indicates higher expression and green indicates lower expression in the HFHSD group pigs (n = 6) versus the control group pigs (n = 3). Black indicates no difference in expression. The smaller figure depicts the color scale used for clustering (A). Differentially expressed genes between the HFHSD and control groups were used to predict the biological processes involved (B). The GO terms and corresponding percentages are shown in different colors.

Fig 4. qRT-PCR for microarray validation.

Ten genes that were either up- or down-regulated in the hearts of the HFHSD miniature pigs and were associated with metabolism, actin cytoskeleton signaling, and immune responses were selected for qRT-PCR. The data are shown as the fold differences in the HFHSD group versus the control group.

Fig 5. The important proteins related to fatty heart and cardiac hypertrophy in HFHSD group and control group.

Fig 6. The genes related to the MAPK signaling pathway.

Fig 7. The genes related to the actin cytoskeleton pathway.

Fig 8. The genes related to the antigen processing and presentation pathway.