Spatial metabolomics identifies localized chemical changes in heart tissue during chronic cardiac Chagas Disease

Abstract

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is one of nineteen neglected tropical diseases. CD is a vector-borne disease transmitted by triatomines, but CD can also be transmitted through blood transfusions, organ transplants, T. cruzi-contaminated food and drinks, and congenital transmission. While endemic to the Americas, T. cruzi infects 7-8 million people worldwide and can induce severe cardiac symptoms including apical aneurysms, thromboembolisms and arrhythmias during the chronic stage of CD. However, these cardiac clinical manifestations and CD pathogenesis are not fully understood. Using spatial metabolomics (chemical cartography), we sought to understand the localized impact of chronic CD on the cardiac metabolome of mice infected with two divergent T. cruzi strains. Our data showed chemical differences in localized cardiac regions upon chronic T. cruzi infection, indicating that parasite infection changes the host metabolome at specific sites in chronic CD. These sites were distinct from the sites of highest parasite burden. In addition, we identified acylcarnitines and glycerophosphocholines as discriminatory chemical families within each heart region, comparing infected and uninfected samples. Overall, our study indicated global and positional metabolic differences common to infection with different T. cruzi strains and identified select infection-modulated pathways. These results provide further insight into CD pathogenesis and demonstrate the advantage of a systematic spatial perspective to understand infectious disease tropism.

Fig 1. Disconnect between sites of parasite persistence and metabolic alterations in chronic cardiac CD.

(A) Median cardiac parasite burden, as determined by qPCR. Parasite burden was highest at the heart base (position A) for strain CL and central heart segments (position C) for strain Sylvio X10/4, indicating parasite strain-specific differences in parasite tropism. (B) Statistically significant perturbations in the overall metabolite profile between uninfected and strain CL-infected mice (left), and between uninfected and strain Sylvio X10/4-infected mice (right). The highest significant metabolite perturbation was at central heart segments (position C) for strain CL (***, p < 0.001 by PERMANOVA) and at the heart apex (position D) for strain Sylvio X10/4 (**, p < 0.01 by PERMANOVA).

Fig 2. Limited overlap of specific differential metabolites between strains.

Yellow and red circles represent differential metabolites between strain Sylvio X10/4-infected and matched uninfected controls, and between strain CL-infected and matched uninfected controls, respectively. Intersect are metabolites impacted by infection in both strains. (A-D) Differential metabolites for each strain, at given heart positions, as determined by random forest classifier, with variable importance score cutoff as described in Methods. (E) Metabolites impacted by infection with each strain, irrespective of position, as determined by random forest classifier, with variable importance score cutoff as described in Methods. (F) Metabolites impacted by infection with each strain, irrespective of heart position, using FDR-corrected Mann Whitney p<0.05 cutoff.

Fig 3. Spatial impact of chronic T. cruzi infection on cardiac acylcarnitines.

Normal levels and distribution of acylcarnitines are represented by uninfected samples. (A, B) Differential total acylcarnitine distribution between uninfected and infected heart sections for both CL and Sylvio X10/4 strains. CL-infected mice showed statistically significant decreases in total acylcarnitine levels at heart base when compared to uninfected mice (*, p<0.05 by Mann-Whitney test). (C, D) CL-infected mice showed statistically significant decreases (*, p<0.05, by Mann-Whitney test) in short-chain acylcarnitine (≤ C4) at heart base. (E, F) Sylvio X10/4-infected mice showed statistically significant decreases in mid-chain acylcarnitines at all positions compared to uninfected mice. (*, p<0.05 by Mann-Whitney test). (G, H) Sylvio X10/4-infected mice showed statistically significant decreases in long-chain acylcarnitines (≥C12) at most heart positions compared to uninfected mice (*, p<0.05 by Mann-Whitney test).

Fig 4. Spatial impact of chronic T. cruzi infection on cardiac glycerophosphocholines.

(A, B) Differential total glycerophosphocholine distribution between uninfected and infected heart sections for both CL and Sylvio X10/4 strains. CL-infected mice showed statistically significant increases in total glycerophosphocholine levels at central heart positions when compared to uninfected mice (*, p<0.05 by Mann-Whitney test). (C,D) Both infected strains showed statistically significant increases (*, p<0.05, by Mann-Whitney test) for short glycerophosphocholines (m/z 400–599.99) at central and apical positions for strain CL and apical positions for strain Sylvio X10/4. (E, F) Mid-sized glycerophosphocholines (m/z 600–799.99) were not significantly affected by infection for both strains. (G, H) Sylvio X10/4-infected mice showed a statistically significant increase (*, p<0.05 by Mann-Whitney test) in long glycerophosphocholines (m/z> 800) at apical positions.