Identification of Crucial Genes and Key Functions in Type 2 Diabetic Hearts by Bioinformatic Analysis

Figures

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  Figure 1. ∣ Identification of differentially expressed genes (DEGs) and overlapped homologous DEGs in T2D or non-T2D hearts with or without SARS-CoV-2 infection. (A) Scheme of the experimental procedure. The gene expression profiles in public datasets from NCBI-GEO were analyzed to identify DEGs in the heart tissues between T2D mice and control, as well as T2D patients who succumbed to SARS-CoV-2 infection and control. (B) Respective volcano plots of sifted out DEGs for datasets in the accordance with public database GSE161931(mice), GSE150316 vs GSE133054 (human) of NCBI-GEO. Blue and red plot represent up- and down-regulated genes, respectively. Gray plot represent the remaining genes with no significant difference. (C) The Venn diagrams of the overlapping homologous DEGs among the three datasets. Threshold was set to be FC > 1.5 (or <1/1.5) and P < 0.05.

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  Figure 2. GO functional enrichment and module analysis of DEGs in the three datasets. (A, B) Bubble maps showed the top-10 GO events associated with DEGs of molecular functions module and cellular components module. Significantly enriched functions of two modules were indicated in Y-axis. Rich factor in the X-axis represented the enrichment levels. The larger value of Rich factor represented the higher level of enrichment. Color of the dot stands for the different P-value and size of the dot reflected the number of target genes enriched in the corresponding functions. Green letters in both modules indicated the overlapping DEGs. (C) Frequency of crucial overlapping DEGs (≥ 2 times) associated with top-10 events in both modules through GO analysis. There are seven crucial genes in T2D hearts according to the gene abundance grades in single event among the top-10 GO events, including Capns1, F3, Mosp1, Tmpress5 in cellular components module, and CAPNS1, FECH, SFXN4, UCP3 in molecular function module. P < 0.05. Schematics of GO analysis of overlapping DEGs for datasets GSE150316, GSE133054, and GSE161931 from NCBI-GEO (left panel). The richest DEGs enriching in corresponding functions showed…

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  Figure 3. Transcription of the overlapping homologous DEGs with significance in T2D hearts. (A) Spectrum of the up- and down-regulation levels of the overlapping homologous DEGs in the datasets GSE161931 (mice), GSE150316 vs GSE133054 (human). (B) Distributions of the up- and down-regulated overlapping DEGs among the top-10 GO events in respective volcano plots.

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  Figure 4. Increased transcription of Capns1 in the hearts of db/db mice. (A) Cardiac Capns1 transcription enhanced in db/db mice (16-weeks-old). (B) The transcription levels of substrates Jp2, Tnni3, and Mybpc3 in cardiac calpain/CAPNS1 pathway were not reduced (n=4, each sample was repeated three times). (C) Representative blotting showing JP2 protein decreased in expression in T2D hearts (n=3, each sample was repeated three times, #3 and #17 were original lab codes of animals). *P < 0.05.

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  Figure 5. Mitochondrial cristae disruption and hydrolyzed JP2 nuclear translocation in myocardium of db/db mice. (A) Electron micrographs showed the decreased areas of cristae organization and density of mitochondria (M) in db/db mice compared with controls. Scale bar: 500 nm and 200 nm. (B) Representative images of echocardiography. Both left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) decreased. *P < 0.05, n=9 (BKS mice), n=7 (db/db mice). *P < 0.05, **P < 0.01. (C) The abundance and distribution of JP2 (red) changed in cardiomyocytes (green, cTnT) of T2D heart. The colocalization of JP2NT (red) with nuclei (blue, DAPI) appear in cardiomyocytes of T2D heart (arrow: nuclear JP2NT). (D) In the case of cardiac mitochondrial ultrastructure lesions, and cardiac dysfunction in T2D hearts, the hydrolyzed product JP2NT import into the nucleus of myocardium. Scale bar: 50 μm and 15 μm (left panel). Ratio of JP2NT existed in nuclei were showed in dot chart (right panel) (n=8~11, 4 random fields for each sample).

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  Figure 6. ∣ Hypothesis contributing to crucial gene and key functions in T2D hearts with and without SARS-CoV-2 infection. Capns1/CAPNS1 is the unique gene shared by molecular function module and cellular components module of overlapped DEGs, and involved in catalyzing JP2 hydrolysis, T2D hearts should possess intrinsic CAPNS1-dependent self-protective mechanism. The autopsy heart specimens of T2D patients display attenuated transcription of CAPNS1 in the case of SARS-CoV-2 infection. Attenuated cardiac CAPNS1 transcription in T2D patients who succumbed to SARS-CoV-2 infection must decrease in the ability to hydrolyze JP2 and weaken self-protective mechanism, thereby leading to adverse prognostics.