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. 2023 Dec 7;51(1):68.
doi: 10.1186/s41182-023-00552-6.

Differential cardiomyocyte transcriptomic remodeling during in vitro Trypanosoma cruzi infection using laboratory strains provides implications on pathogenic host responses

Katherine-Sofia Candray-Medina  1   2   3 , Yu Nakagama  4   5 , Masamichi Ito  6   7 , Shun Nakagama  8 , Evariste Tshibangu-Kabamba  1   2   9 , Norihiko Takeda  10 , Yuki Sugiura  11 , Yuko Nitahara  1   2 , Yu Michimuko-Nagahara  1   2 , Natsuko Kaku  1   2 , Yoko Onizuka  12 , Carmen-Elena Arias  3 , Maricela Mejia  3 , Karla Alas  3 , Susana Peña  13 , Yasuhiro Maejima  8 , Issei Komuro  14   15 , Junko Nakajima-Shimada  12 , Yasutoshi Kido  1   2
Affiliations

Differential cardiomyocyte transcriptomic remodeling during in vitro Trypanosoma cruzi infection using laboratory strains provides implications on pathogenic host responses

Katherine-Sofia Candray-Medina et al. Trop Med Health. .

Abstract

Background: Chagas disease can lead to life-threatening cardiac manifestations. Regional factors, including genetic characteristics of circulating Trypanosoma cruzi (T. cruzi), have attracted attention as likely determinants of Chagas disease phenotypic expression and Chagas cardiomyopathy (CCM) progression. Our objective was to elucidate the differential transcriptomic signatures of cardiomyocytes resulting from infection with genetically discrete T. cruzi strains and explore their relationships with CCM pathogenesis and progression.

Methods: HL-1 rodent cardiomyocytes were infected with T. cruzi trypomastigotes of the Colombian, Y, or Tulahuen strain. RNA was serially isolated post-infection for microarray analysis. Enrichment analyses of differentially expressed genes (fold-change ≥ 2 or ≤ 0.5) highlighted over-represented biological pathways. Intracellular levels of reactive oxygen species (ROS) were compared between T. cruzi-infected and non-infected HL-1 cardiomyocytes.

Results: We found that oxidative stress-related gene ontology terms (GO terms), 'Hypertrophy model', 'Apoptosis', and 'MAPK signaling' pathways (all with P < 0.01) were upregulated. 'Glutathione and one-carbon metabolism' pathway, and 'Cellular nitrogen compound metabolic process' GO term (all with P < 0.001) were upregulated exclusively in the cardiomyocytes infected with the Colombian/Y strains. Mean intracellular levels of ROS were significantly higher in the T. cruzi-infected cardiomyocytes compared to the non-infected (P < 0.0001).

Conclusions: The upregulation of oxidative stress-related and hypertrophic pathways constitutes the universal hallmarks of the cardiomyocyte response elicited by T. cruzi infection. Nitrogen metabolism upregulation and glutathione metabolism imbalance may implicate a relationship between nitrosative stress and poor oxygen radicals scavenging in the unique pathophysiology of Chagas cardiomyopathy.

Keywords: Chagas disease; Dilated cardiomyopathy; Glutathione metabolism; HL-1; In vitro modeling; Neglected tropical diseases; Oxidative stress; Transcriptome; Trypanosoma cruzi.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Difference in cardiomyocyte infectivity among Trypanosoma cruzi infection conditions. Percentage of T. cruzi-infected cells (containing intracellular amastigotes) at 24- and 48-h post-infection. hpi hours post-infection
Fig. 2
Fig. 2
Cardiomyocyte infectivity among different Trypanosoma cruzi infection conditions. Intracellular amastigote counts per infected cell are plotted at A 24- and B 48-h post-infection. Pairwise comparisons were performed with Mann–Whitney test. Black horizontal bars indicate the mean and SD
Fig. 3
Fig. 3
Remodeling of gene expression profiles in cardiomyocytes infected with three different Trypanosoma cruzi strains at two time points post-infection. Y axes indicate the gene expression level of the non-infected control cardiomyocyte, while X-axes indicate gene expression levels of Trypanosoma cruzi-infected cardiomyocytes. The black dashed line, and upper and lower red dashed lines show the line of identity, and the 0.5- and twofold change thresholds, respectively. Each plot represents a differentially expressed gene (DEG). Blue and pink shaded areas represent down- and up-regulated DEGs, respectively. hpi hours post-infection
Fig. 4
Fig. 4
Identification of differentially expressed genes (DEGs) of the host cardiomyocyte upon Trypanosoma cruzi infection. Numbers of up- (left column) and down-regulated (right column) DEGs at 24- (upper row) and 48-h post-infection (lower row) are shown in the respective Venn diagrams. hpi hours post-infection
Fig. 5
Fig. 5
Principal component analysis plot of the transcriptional profile of the in vitro cardiomyocyte infection models. Principal component analysis plot, based on the fold-change (vs control) values of the 10,784 valid genes. Each dot represents a biological condition at 24- or 48-h post-infection. The principal components 1 and 2 together describe 58.9% of the total variance in the dataset. Dim dimension
Fig. 6
Fig. 6
Top differentially expressed genes of the cardiomyocyte upon Trypanosoma cruzi infection. Extensively A upregulated (fold-change > 5) and B downregulated (fold-change < 0.2) genes of the cardiomyocyte at 24-h post-infection are listed. Δ Gene expression, the fold-change or ratio between gene expression level of the infected cardiomyocytes and the non-infected negative control. Arrdc4 arrestin domain containing 4, Txnip thioredoxin interacting protein, Gdf15 growth differentiation factor 15, Atf3 activating transcription factor 3, Soat2 sterol o-acyltransferase 2, Dusp8 dual specificity phosphatase 8, Chac1 cation transport regulator 1, Gtpbp2 GTP binding protein 2, Capn5 Calpain 5, Dhcr24 24-dehydrocholesterol reductase, Lgals9 lectin galactose binding soluble 9, Acat2 acetyl-coenzyme A acetyltransferase 2, Hmgcs1 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1, Mvd mevalonate (diphospho) decarboxylase, Tpcn1 two pore channel 1, Medag mesenteric estrogen dependent adipogenesis, Sulf2 sulfatase 2, Fam64a family with sequence similarity 64 member A, Midn midnolin, Ldlr low density lipoprotein receptor, Acat3 acetyl-coenzyme A acetyltransferase 3, Triobp TRIO and F-actin binding protein
Fig. 7
Fig. 7
Signatures of the common cardiomyocyte response to Trypanosoma cruzi infection. A, B ‘WikiPathways’ and C, D ‘Biological Process gene ontology Terms’ enriched by commonly A, C upregulated and B, D downregulated genes of the host cardiomyocyte at 24-h post-infection are sorted based on P-value. Bar lengths represent enrichment Z-scores
Fig. 8
Fig. 8
Heatmap of expression levels of inflammation-related genes in HL-1 cells infected with T. cruzi at 24-h post-infection. Genes are represented in rows and infection conditions are represented in columns. Color legend represents the logarithm 2 of gene expression level. P-values were calculated by paired T-test. Gdf-15 growth differentiation factor 15, Cxcl10 C-X-C motif chemokine ligand 10, Cxcl2 C-X-C motif chemokine ligand 2, Il-6 interleukin-6, Tnf tumor necrosis factor-alpha
Fig. 9
Fig. 9
Signatures of transcriptomic remodeling unique to Colombian- and Y-infections. ‘WikiPathways’ (A) and 'Biological Process gene ontology Terms’ (B), enriched by the genes uniquely upregulated at 24-h post-infection in Colombian- and Y-infected cardiomyocytes are sorted based on P-value. Bar lengths represent enrichment Z-scores
Fig. 10
Fig. 10
Elevated intracellular levels of reactive oxygen species in Trypanosoma cruzi-infected cardiomyocytes. A Signal intensity of reactive oxygen species-specific fluorescence was higher in the cardiomyocytes infected with the Y strain at 24-h post-infection, compared with non-infected control cells. Statistical comparison was performed with Mann–Whitney test. Bars represent mean fluorescence intensity and error bars represent standard deviation. B Representative fluorescence microscopy images from reactive oxygen species-specific CellRox green staining. Comparison with the non-infected (upper) control cells shows higher intensity of reactive oxygen species fluorescence signal in the cardiomyocytes 24-h post-infection with Trypanosoma cruzi Y strain (lower). Scale bars represent 100 µm
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