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. 2024 Apr 5;27(5):109672.
doi: 10.1016/j.isci.2024.109672. eCollection 2024 May 17.

Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation

Hariprasad Thangavel  1 , Dhanya Dhanyalayam  1 , Michelle Kim  1 , Kezia Lizardo  1 , Tabinda Sidrat  1 , John Gomezcoello Lopez  1 , Xiang Wang  2 , Shivani Bansal  3 , Jyothi F Nagajyothi  1
Affiliations

Adipocyte-released adipomes in Chagas cardiomyopathy: Impact on cardiac metabolic and immune regulation

Hariprasad Thangavel et al. iScience. .

Abstract

Chronic Trypanosoma cruzi infection leads to Chagas cardiomyopathy (CCM), with varying manifestations such as inflammatory hypertrophic cardiomyopathy, arrhythmias, and dilated cardiomyopathy. The factors responsible for the increasing risk of progression to CCM are not fully understood. Previous studies link adipocyte loss to CCM progression, but the mechanism triggering CCM pathogenesis remains unexplored. Our study uncovers that T. cruzi infection triggers adipocyte apoptosis, leading to the release of extracellular vesicles named "adipomes". We developed an innovative method to isolate intact adipomes from infected mice's adipose tissue and plasma, showing they carry unique lipid cargoes. Large and Small adipomes, particularly plasma-derived infection-associated L-adipomes (P-ILA), regulate immunometabolic signaling and induce cardiomyopathy. P-ILA treatment induces hypertrophic cardiomyopathy in wild-type mice and worsens cardiomyopathy severity in post-acute-infected mice by regulating adipogenic/lipogenic and mitochondrial functions. These findings highlight adipomes' pivotal role in promoting inflammation and impairing myocardial function during cardiac remodeling in CD.

Keywords: Biology experimental methods; Cell biology; Disease; Microbiology parasite.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
T. cruzi infection induces adipocyte apoptosis, releasing adipomes that regulate adipogenic signaling (A) Immunoblot analysis of cleaved Caspase 7 expression in the lysates of 3T3-L1 adipocytes (uninfected, TNFα-treated, and T. cruzi infected) (n = 3/group). Bar graph values were derived from densitometry analysis and by normalizing target protein expression to β-Actin. (B) Fold change expression of CASP3, RIPK3, and MLKL mRNA transcripts (normalized to HPRT) in cultured human adipocytes (uninfected, TNFα-treated and T. cruzi infected) (n = 3). (C and D) Adipome size distribution and absolute concentration as determined by Spectradyne nCS1 with a C-900 microfluidic cartridge. Both mouse and human adipomes were derived from cultured 3T3-L1 and human adipocyte-conditioned media, respectively. Inset: A (blue) = uninfected, B (green) = TNFα-treated, C (red) = T. cruzi infected. (E) PCR analysis of adipogenic (Adipoq, Fabp4, and Pparg), apoptotic (Chop) and extracellular vesicle (Tsg101 and Cd13) marker genes in 3T3-L1-derived adipomes. Lane 1, 2, and 3 represent 3T3-L1-derived adipomes from three independent cultures. Product size: Adipoq, 192bp; Fabp4, 133bp; Pparg, 132bp; Chop, 118bp; Tsg101, 103bp; and Cd13, 121bp. (F and G) Fold change expression of Adipoq mRNA transcripts (normalized to Hprt) in RAW macrophages (F) and human fibroblasts (G) treated with 3T3-L1- and human adipocyte-derived adipomes, respectively, at 1:1 and 1:50 cell-to-adipome ratio for 48 h (n = 3). All treatment groups (Treated - A, - B and - C) were compared to untreated groups. The # symbol indicates comparison between Treated - B and - C. Treated - A = adipomes from uninfected adipocytes; Treated - B = adipomes from TNFα-treated adipocytes; and Treated - C = adipomes from T. cruzi-infected adipocytes. Data are represented as mean ± SEM. (∗/#p < 0.05, ∗∗p ≤ 0.01, ∗∗∗/###p ≤ 0.001 and ∗∗∗∗p ≤ 0.0001).
Figure 2
Figure 2
Adiponectin enables selective capture of adipomes from murine white adipose tissue (WAT) (A) Immunoblot analysis of phospho-Perilipin, cleaved Caspase 7 and Annexin V expression in the WAT lysates of infected (20 DPI) and uninfected C57BL/6J mice (n = 4–8 per group). Bar graph values were derived from densitometry analysis and by normalizing target protein expression to GDI. Error bars indicate the standard error of the mean (∗p < 0.05). (B) Immunoblot analysis of adiponectin in WAT-derived large-EVs (L-EV) and small-EVs (S-EV). Black arrow points to the 25 kDa marker band on the pre-stained protein ladder. Adiponectin band appears at 26/30 kDa. Lanes: C, control WAT lysate; L Ev, large-EV; and S Ev, small-EV. (C) Surface immunofluorescence staining of 3T3-L1 adipocytes show the co-localization of Adiponectin (green) with Annexin V (red) (top panel) and FABP4 (red) with Annexin V (green) (bottom panel) on budding apoptotic bodies. Scale bar, 50 μm. (D) Immunoblot analysis of adiponectin in WAT-derived large-adipomes and small-adipomes. Black arrow points to the 25 kDa marker band on pre-stained protein ladder. Adiponectin band appears at 26/30 kDa. Lanes: C, control WAT lysate; L, large-adipomes; and S, small-adipomes. (E) Immunofluorescence assay (IFA) of bead-bound adipomes stained with Adiponectin-AF488 (top panel) and FABP4-AF488 (bottom panel) imaged on the FITC channel along with their corresponding bright field (BF) images. Scale bar, 5 μm. (F) WAT-derived adipome size distribution and absolute concentration as determined by Spectradyne nCS1 with a C-2000 microfluidic cartridge. Inset: L (blue), large-adipomes; S (green), small-adipomes. Gate: G1, particle concentration at size range of 250–400 nm diameter showing more small-adipomes than large-adipomes; and G2, particle concentration at size range of 700–1100 nm diameter showing mostly large-adipome distribution. (G) Transmission Electron Microscopy (TEM) images of small-adipomes (left, scale bar 80 nm) and large-adipomes (right, scale bar 100 nm) with a direct magnification of 20,000×. Black arrow indicates the budding scars on L-adipomes.
Figure 3
Figure 3
The lipidome of L-adipomes is distinct from that of S-adipomes (A and G) Principal component analysis (PCA) score plot of the lipid profiles from CLA and ILA (A), and ILA and ISA (G) showing clusters of two groups. (B and H) Heatmap representation of the clustering analysis of top 25 differentially abundant lipid molecular species (with lowest FDR-adjusted p-values) based on z-scores for the normalized, transformed, and scaled data between CLA and ILA (B), and ILA and ISA (H). For class names, red represents CLA or ILA and green represents ILA or ISA. For the abundance of each lipid, red represents high, and blue represents low. (C and I) Volcano plots represent significantly altered lipid metabolites in ILA compared to CLA (C), and ISA compared to ILA (I). Red dots indicate upregulated lipids and blue dots indicate downregulated lipids. (D) KEGG pathway analysis revealed seven dysregulated metabolic pathways enriched in ILA compared to CLA. All matched pathways were plotted according to p-value and pathway impact score from pathway enrichment analysis and pathway topology analysis, respectively. Color gradient: yellow, higher p-value; and red, lower p-value. Circle size: large, higher impact score; and small, lower impact score. (E and F) Pathway representation of Sphingolipid metabolism (E) and Glycerophospholipid metabolism (F) with lipid metabolites (highlighted in red) enriched in ILA. Data represents the mean (n = 3 per group). Abbreviations: CLA, control large-adipomes; ILA, infected large-adipomes; and ISA, infected small-adipomes.
Figure 4
Figure 4
Infected L-adipomes alter immunometabolic signaling and macrophage polarization (A–D) Changes in the expression of genes involved in adipogenesis (A), lipid metabolism (B), and immune and inflammatory signaling (C and D) based on mRNA fold change values (normalized to Hprt) in macrophages treated with CLA and ILA for 48 h as demonstrated by custom RT2 Profiler qPCR array analysis. Fold change was calculated using Qiagen data analysis spreadsheet. (E and F) Fold change expression of Arg1 mRNA transcript (E) and mRNA levels of genes involved in mitochondrial oxidative phosphorylation (F) (normalized to Gapdh) in macrophages treated with CLA and ILA for 48 h as demonstrated by qPCR analysis (n = 3). Data are represented as mean ± SEM. (∗p < 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001).
Figure 5
Figure 5
Adipomes play a major regulatory role in cardiomyocytes functioning (A–C) Fold change in mRNA transcript levels of genes involved in adipogenic and lipogenic signaling (A), mitochondrial oxidative phosphorylation (B), and inflammatory signaling (C) (normalized to Gapdh) in murine primary cardiomyocytes treated with CLA, ILA, CSA, or ISA for 48 h and compared to untreated cardiomyocytes as demonstrated by qPCR analysis (n = 3). All adipome-treated groups (CLA, ILA, CSA, and ISA) were compared to untreated groups. Data are represented as mean ± SEM. (∗p < 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001).
Figure 6
Figure 6
Circulatory adipomes induce ER stress in cardiomyocytes Immunoblot analysis of β1-AR and CHOP expression in murine primary cardiomyocytes treated with plasma-derived adipomes (P-CLA, P-ILA, P-CSA, and P-ISA) and naive cells (n = 3/group). Bar graph values were derived from densitometry analysis and by normalizing target protein expression to GDI. The “&” symbol denotes significance calculated between different adipome-treated groups. Data are represented as mean ± SEM. (∗p < 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001).
Figure 7
Figure 7
P-ILA cause mitochondrial dysfunction in primary cardiomyocytes (A) Seahorse Mito Stress assay showing the oxygen consumption rate (OCR) from plasma-adipome treated (P-CLA and P-ILA) and untreated mouse cardiomyocytes (n ≥ 6). Data normalized to nuclear content by staining and fluorescence cell counting. (B–F) Representative graphs depicting the different mitochondrial respiration parameters evaluated, such as basal respiration (B), maximal respiration (C), proton leak (D), spare respiratory capacity (E), and ATP production (F). Data are represented as mean ± SEM. (∗p < 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗p ≤ 0.0001).
Figure 8
Figure 8
P-ILA elevate the risk for cardiomyopathy in murine CD model during post-acute phase (A–E) Immunoblot analysis of inflammation (TNFα, IFNγ, IL6), immune cell infiltration (F4/80), lipogenesis (SREBP1), lipid droplet formation (Perilipin-1), lipid hydrolysis (phospho-Perilipin), lipid oxidation (PPARα), mitochondrial oxidative phosphorylation (SDHA, Cytochrome c, COX IV, HSP60), ER stress (BiP, phospho-eIF2α, PDI), apoptosis (CHOP, Caspase 7, Cleaved-Caspase 7), and necrosis (BNIP3) markers in heart lysates of uninfected and post-acute T. cruzi-infected mice treated with intracardiac injections of either P-ILA or vehicle. Bar graph values were derived from densitometry analysis and by normalizing target protein expression to GDI. Lanes: 1, uninfected vehicle-injected; 2, uninfected P-ILA-injected; 3, T. cruzi-infected vehicle-injected; and 4, T. cruzi-infected P-ILA-injected murine heart tissue lysates. “&” symbol indicates significance calculated between uninfected and T. cruzi-infected P-ILA injections. Data are represented as mean ± SEM. (∗p < 0.05, ∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001).
Figure 9
Figure 9
Intracardiac P-ILA treatment increases cardiac adiponectin (APN) and Atrial natriuretic peptide (ANP) in murine CD model (A) Immunoblot analysis of adiponectin and β1-AR expression in heart lysates of uninfected and post-acute T. cruzi-infected mice treated with intracardiac injections of either P-ILA or vehicle. Bar graph values were derived from densitometry analysis and by normalizing target protein expression to GDI. Lanes: 1, uninfected vehicle-injected; 2, uninfected P-ILA-injected; 3, T. cruzi-infected vehicle-injected; and 4, T. cruzi-infected P-ILA-injected murine heart tissue lysate. The “&” symbol indicates significance calculated between uninfected and T. cruzi-infected P-ILA injections. (B) Representative immunohistochemistry (IHC) images of vehicle- and P-ILA-injected heart tissue from uninfected and T. cruzi-infected mice showing ANP immunostaining visualized with DAB and counterstained with hematoxylin. Dot plot shows staining intensity in O.D. (mean gray value) obtained by color deconvolution analysis.Data are represented as mean ± SEM. (∗∗p ≤ 0.01, and ∗∗∗p ≤ 0.001).
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