Attenuating effects of inflammatory pathway by prolonged left ventricular unloading after myocardial infarction in male rats

Abstract

Background: Inflammatory response plays a pivotal role in myocardial injury and post-infarction remodeling after acute myocardial infarction (AMI). Mechanical unloading (UL) of the left ventricle (LV) has been proposed as a potential therapeutic strategy to preserve cardiac function; however, its effects on myocardial inflammation remain incompletely understood.

Methods: We employed a rat model of partial UL using heterotopic heart-lung transplantation following AMI. RNA sequencing (RNA-seq) was performed to evaluate transcriptomic changes, with a specific focus on inflammatory pathways in the non-infarcted remote area. Immune cell abundance was estimated using deconvolution analysis (QUANTISEQ). Quantitative PCR was performed to analyze some inflammatory cytokines, and macrophage polarization was evaluated by immunohistochemistry.

Results: AMI significantly impaired cardiac function, which was mitigated by UL. RNA-seq analysis revealed marked activation of inflammatory pathways and identified several hub genes involved in cytokine signaling following AMI, while these transcriptional changes were not significantly altered in UL groups after AMI. Immune cell profiling demonstrated an increase in M2 macrophages after AMI, while UL preserved M2 macrophage levels. Histological analysis further supported UL's modulatory effect on macrophage polarization. Pro-inflammatory cytokines TNFα and IL1β were upregulated after AMI but showed attenuation with UL.

Conclusion: Partial UL potentially attenuates cardiac functional deterioration after AMI while exerting substantial effects on inflammatory gene expression and macrophage polarization. These findings suggest that the cardioprotective effects of UL may be correlated with the modulation of inflammatory pathways in the remote area after AMI.

Fig 1. Study protocol (A) and left ventricular UL model (B).

White bars denote periods without intervention; black bars represent periods with intervention (LAD ligation or unloading). AMI, acute myocardial infarction; LAD, left anterior descending artery; UL, unloading.

Fig 2. Cardiac functional parameters (A) and myocyte size and fibrosis (B) 14 days after surgery.

Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. AMI, acute myocardial infarction; HR, heart rate; ns, not significant; RPP, rate pressure product; UL, unloading.

Fig 3. RNA-seq analysis of the LV myocardium in the non-infarcted area 14 days after surgery.

(A) Principal component analysis (PCA) of RNA-seq data (rlog-transformed counts). PC1 and PC2 explain 35.2% and 17.8% of total variance, respectively. Samples are color-coded by treatment group (non-AMI: light Blue; AMI; red; non-AMI with UL: purple; and AMI with UL: green). Each point represents an individual biological replicate (n = 5 or 6 per group). (B and C) Volcano plot of differentially expressed genes between non-AMI and AMI, non-AMI with UL and AMI with UL. Genes with adjusted p < 0.05 and |log₂ fold change| > 1.5 are considered significantly differentially expressed (red: upregulated; blue: downregulated; gray: not significant). Hierarchical clustering heatmap indicates the differentially expressed genes. Rows represent genes and columns represent individual samples. Color scale indicates relative expression levels (red: high; blue: low). AMI, acute myocardial infarction; LV, left ventricle; UL, unloading.

Fig 4. Gene clustering from k-means analysis.

(A) Heatmap of two gene expression clusters across the four groups (non-AMI, AMI, non-AMI/UL, and AMI/UL). (B) KEGG pathway enrichment for each cluster showing metabolism-related pathways in Cluster 1 and inflammation/immune-related pathways in Cluster 2. K-means analysis was used to cluster DEGs into distinct expression pattern groups, k = 2, according to the Silhouette method, and the 2000 most significant genes were selected using a fold change > 1.5 between non-AMI and AMI groups. (C) Gene expressions for Cpt1b, Cd36l1, and Fabp3. Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. *P < 0.05, ***P < 0.001, ****P < 0.0001. AMI, acute myocardial infarction; LV, left ventricle; UL, unloading.

Fig 5. Hub gene detection related to inflammation from the RNA-seq analysis.

(A) Classification of DEGs into distinct expression pattern groups using a fold change > 1.5 between the non-AMI and AMI groups as a cut-off. The genes in groups 1 and 9 were greatly elevated after AMI, and the elevation was suppressed by UL. (B) Pathway analysis using KEGG database and protein-protein interaction network to find hub genes from shared genes. (C) Comparison of the hub genes among the four groups.. Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. *P < 0.05, **P < 0.01. AMI, acute myocardial infarction; DEG, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; UL, unloading.

Fig 6. Deconvolved immune cell type expression from the raw RNA-seq data using QUANTISEQ.

(A) Comparison of the cell type fractions among the four groups. (B) Heatmap of the correlation between immune cell types and each rat of the four groups. (C) Association between the hub gene expressions and cell types. Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. ***P < 0.001. AMI, acute myocardial infarction; UL, unloading.

Fig 7. Gene expression levels of inflammatory cytokines in the LV myocardial samples of remote areas 14 days after surgery.

Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. **P < 0.01. AMI, acute myocardial infarction; IL, interleukin; LV, left ventricle; and TNF, tumor necrosis factor; UL, unloading.

Fig 8. M1, M2 and SPP1 positive macrophages from remote areas 14 days after surgery.

(A) Quantification of M1, M2, and M1/M2 macrophages across groups (non-AMI, AMI, non-AMI/UL, and AMI/UL). Data are shown as positive cell fractions with statistical comparisons indicated. (B) Representative immunofluorescence staining of iNOS and CD163 (red), SPP1 (green), and DAPI (blue) in the remote myocardium. No SPP1⁺ cells were detected in all groups. Scale bars, 100 μm. Data are presented as mean ± SEM. Statistical significance was assessed using two-way ANOVA, followed by Bonferroni post hoc test. Interaction p-values and effect sizes (partial η² values) are shown. **P < 0.01. AMI, acute myocardial infarction; UL, unloading.