Dissecting organoid-bacteria interaction highlights decreased contractile force as a key factor for heart infection

Reviewer #1 (Public review):

Summary:

In the study by Wang et al. entitled "Dissecting organoid-bacteria interaction highlights decreased contractile force as a key factor for heart infection", a simple cardiac organoid (CO) model was established, by combining a heterologous mixture of patient-specific human induced pluripotent stem cells (hiPSC)-derived cardiomyocytes (CMs) in combination with primary HUVECs (Human Umbilical Vein Endothelial Cells) and human mesenchymal stem cells (MSCs, representing stromal cells). This model was applied for investigating the interplay of COs' bacterial infections in vitro, aiming at revealing pathological mechanisms of bacterial infections of the heart in vivo, which may induce myocarditis and consequently heart failure in affected patients.

Strengths:

The paper is systematic, well written, and easy to follow.

Based on their results, the authors state that: "In this study, by developing quantitative tools for analyzing bacterial-cardiac organoid interactions in a 3D, dynamic, clinically relevant setting, we discovered the significant role of cardiac contractility in preventing bacterial infection."

In principle, the idea of establishing a simple yet functionally and physiologically relevant in vitro model and relevant analytical tools for enabling the study of complex pathological mechanisms of cardiovascular diseases is intriguing.

Weaknesses:

However, despite the combination of numerous analytical tools established and applied in the study, the work has substantial experimental limitations, indicating that the bold conclusions may represent a misinterpretation or overinterpretation of the findings.

Key limitations and questions:

(1) It seems that iPSCs from only one patient ("dilated cardiomyopathy (DCM) cells were derived from a 47-year-old Asian male with an LMNA gene mutation") were used in the study. Moreover, it seems that only one iPSC-line/clone from that DCM patient was used and compared to a single control iPSC line from a "healthy donor". Therefore, despite the different assays and experimental controls used in the study, there is a high risk that the observed phenomena reflect iPSC-line-/ clone-dependent effects, rather than revealing general pathophysiologic mechanisms. Thus, key experiments must be shown by cardiomyocytes/ cardiac organoids derived from additional independent iPSC-lines representing different patients and other non-diseased control lines as well. Moreover, it is established good experimental practice in the iPS cell field to generate and include isogenic iPSC controls i.e. iPSC lines of the same genetic background but with corrections of the hypothesised gene mutation underlying the respective e.g., cardiovascular disease.

(2) In Figure 1 (A) immunohistochemical staining for cardiomyocytes for the cardiac marker Troponin is shown, apparently indicating successful cardiomyogenic differentiation of the applied hiPSC lines. In supplemental Figure S1, a flow cytometry analysis specific to cTnT is shown to reveal the CMs content resulting from the monolayer differentiation of respective iPSC lines. Already, the exemplified plots indicate that the CMs' content/ purity for DCM-CMs was notably lower compared to healthy cardiomyocytes (CM; control). This is an important issue, since the non-CMs ("contaminating bystander cells") may have a substantial effect on the functional (including contractile) properties of the COs.

Interestingly, based on the method description, it seems that COs were generated from cryopreserved iPSC-CMs and iPSC-DCMs, including intermediate seeding and culture on Matrigel before COs formation. However, it remains unclear whether the CMs FACS analysis, which is apparently: "Representative FACS plots for analysis of the cell types in DCM monolayer culture after 33 days of differentiation" shows a CMs purity relevant to CO formation, or something different.

The lineage phenotype of non-CMs in respective differentiations should also be clarified. Moreover, it should be noted in the results that the CMs content in COs is lower than the 6:2:2 (CM:ECs:MSC) ratio indicated by the authors, since the CMs purity is not 100%, and is particularly reduced in the iPSC-DCMs.

Finally, to investigate the important latter questions of the "real CMs content" in COs, systematic technologies should be applied to quantify the lineage composition in COs (e.g. by IF staining for the 3 lineages plus DAPI, followed by COs clearance, confocal microscopy "3D stags" and automated, ImageJ-based quantitative cell counts for total cell number definition (see e.g. doi: 10.1038/s41596-024-00976-2) per CO, and quantification of respective lineage content as well.

These questions are of key importance since the presence of non-CMs and their phenotype has profound consequences on the cardiac organoid model, its contractile/ biophysical properties, and, in general, on models' sensitivity to bacterial infections as well.

(3) Figure 2: (F) Why is this figure (Confocal Observations) showing only healthy cardiac organoids (HCOs) but not DCM-COs?

The overall quality of these pictures is poor and not informative regarding the structural identity and tissue composition of the COs, which actually is an important topic in the frame of the paper, as the 3D structure and tissue composition - and differences between HCOs and DCM-COs - are of key importance to their contractile properties.

Moreover, the expective overlay of the cardiac markers alpha-actinin and MHC is not obvious from Figure 2F (see also comments on Figure 7, below).

In Figure 2E: COs at later stages/days should be shown, in particular at that stage, which was used for the functional assays i.e., bacteria infections and contraction pattern monitoring.

(4) Figure 7 (A) (B) - In the IF sections, it seems that there is no overlay between the expression of the cardiac marker MHC (seems to be expressed in the centre of COs only) and the cardiac markers alpha-actinin (which seems to be unexpectedly expressed in all cells on the sections) and Troponin (which seems to be vocally expressed on the outside, excluding the area of MHC expression).

(F) Quantification of the mean area of gene expression, e.g., for MHC indicates a larger area after MHC expression; this seems to entirely contradict the IF pictures (in Figures 7 A-D) of MHC expression before and after infection. This contraction is deemed very critical to this reviewer as it may indicate that the IF staining, data analysis, and/or data interpretation in this part of the manuscript is poor, misleading, or simply wrong.

(5) Overall, from the perspective of this reviewer, the CO-derived results do not reflect in a meaningful way the contractile and hydrodynamic conditions in the mouse heart or the human heart. Thus, it seems that the conclusions may rather represent a hypothesised outcome bias.

Reviewer #2 (Public review):

Summary:

The authors tried deconvoluting, for the first time, the effect of various components of heart contraction on initial bacterial adhesion, which increases the risk of infective endocarditis. The proposed organoid platform might be used to develop and test novel therapeutic agents for infective endocarditis.

Strengths:

(1) Use of a broad range of methods: finite element methods, -omics, particle tracking, animal experiments to investigate the connections between contractility and infective endocarditis.

(2) Detailed procedure and supportive information, which will allow other groups to replicate the results and extend the application of the proposed organoid platform.

(3) Despite the complexity of the work reported, the manuscript is rather readable and understandable by non-specialists.

Weaknesses:

There is a minor issue with some of the vocabulary (e.g., magnificent amount of bacteria).