Schematic of experiments with patient-derived cardiac organoids for bacterial infection study.

Preparation of human cardiac organoids (COs) of healthy and dilated cardiomyopathy (DCM) characteristics

(A) Immunohistochemical staining for cardiomyocyte biomarker (Troponin). Scale bars, 100 µm.

(B) Electrophysiological data of DCM and CM measured using patch clamp.

(C) Membrane patch-clamp measurements of the potential and heartbeat frequency data in DCM and CM.

(D) Fluorescence microscope observation of the formation of HCOs with different cell ratios. The ECs were red (Celltracker-far red), and MSC was green (CFSE) in the Fluorescence Observation. The bar is 50µm.

(E) Fluorescence microscope observation of the formation of DCM-COs with different cell ratios. The ECs were red (Celltracker-far red), and MSC was green (CFSE) in the Fluorescence Observation. The bar is 50µm.

(F) Confocal Observation of Calponin / α-actinin / MHC / Troponin localisation within the HCOs. The Calponin / MHC were labelled green (Alexaflour 488), and Troponin / α-actinin were labelled red (Alexaflour 647). The bar is 50 µm.

(G) Immunohistochemical staining for classic endocarditis biomarker (vWF, CD31 and Collagen Type 1) on Mice heart valves section. The vWF waslabelled green (Alexaflour 488), CD31 Collagen Type 1 and was labelled red (Alexaflour 647). Scale bars, 10µm.

(H) Immunohistochemical staining for classic endocarditis biomarker (vWF, CD31 and Collagen Type 1) on Mice heart ventricle section. The vWF waslabelled green (Alexaflour 488), CD31 Collagen Type 1 and was labelled red (Alexaflour 647). Scale bars, 10µm.

(I) Immunohistochemical staining for classic endocarditis biomarker (vWF, CD31 and Collagen Type 1) on Cardiac organoids section. The vWF waslabelled green (Alexaflour 488), CD31 Collagen Type 1 and was labelled red (Alexaflour 647). Scale bars, 10µm.

Dissection of COs-bacteria interactions in the contraction-induced fluidic system

(A) Tracking process of the COs movement. The first step was to check the tracking area, the second was to confirm the partition size, and the third was to select the appropriate threshold for tracking.

(B) Mean speed for different tracked IDs obtained from HCOs. Each CO was divided into 100-200 15-μm circles.

(C) Mean displacement heatmap of the HCOs and DCM-COs. Each CO was divided into 100-200 15-μm circles. Tested on two group of 6 independent COs in total (n=6).

(D) The total displacement produced by the HCOs and DCM-COs in 3 seconds.

(E) Hotelling’s Test examines the correlation between HCOs and DCM-COs moving in 3 seconds.

(F) Displacement of HCOs within a single systolic-diastolic cycle for contraction and circulation, respectively. Tested on two groups of 6 independent HCOs in total (n=6).

(G) Displacement of DCM-COs within a single systolic-diastolic cycle for contraction and circulation, respectively. Tested on two groups of 6 independent DCM-COs in total (n=6).

(H) Two-dimensional hotelling’s test analysis of motion correlations between HCOs and DCM-COs for systolic and diastolic displacement processes.

(I) Finite element analysis of the forces exerted by HCOs on a 1-µm2 water field. The analysed velocities of the acting forces were taken from the maximum and minimum velocities of the HCOs obtained from the tracking procedure.

(J) Finite element analysis of the forces exerted by DCM-CO on a 1-µm2 water field. The analysed velocities of the acting forces were taken from the maximum and minimum velocities of the DCM-COs obtained from the tracking procedure.

Dynamic analysis of bacterial infection of cardiac organoids

(A) Heatmap of the total displacement and track path of S. A around HCOs. The bar is 50 μm.

(B) Heatmap of the total displacement and track path of S. A around DCM-COs. The bar is 10 μm.

(C) Heatmap of S. A total displacement and track path of the planktonic S. A in the medium. For (A-C), the tracing lasting for two diastolic-systolic cycles was 3 seconds. The bar is 10 μm

(D) Analysis of the movement routes of S. A in the systole-diastolic cycle near the fringe around HCOs. The bar is 10 μm

(E) Analysis of the movement routes of S. A in the systole-diastolic cycle near the fringe around DCM-COs. The bar is 10 μm

(F) Z-stacked live observation of S. A infection after a 24-hour co-culture with HCOs (left) or DCM-COs (right). Red: HUVEC (CellTrace Far-red); blue: CM (Cytotell Blue); Green: S. A (CFSE). The bar is 50 μm

(G) Fluorescence observation of S. A infection after a 24-hour co-culture with HCOs (left) or DCM-COs (right). Green: S. A (CFSE). The bar is 50 μm

(H) Fluorescence Observation of the infection outcome of S. A infection after 48h co-culture with HCOs and DCM-COs, respectively. Green: S. A (CFSE). The bar is 50 μm

(I) CLSM Observation MHC/DAPI/S. A of the infection outcome of S. A infection after 24h incubation in the vicinity of HCOs.The bar is 10 μm

(J) CLSM Observation MHC / DAPI / S. A of the infection outcome of S. A infection after 24h incubation in the vicinity of DCM-COs.The bar is 10 μm

(K) Quantification of the bacterial attachment on HCOs and DCM-COs after 24h incubation using fluorescence intensity measurement.

Quantifying the influence of force on bacteria adhesion

(A) Mean displacement of S. A near HCOs (HCO-S. A) and correlated HCOs compared to planktonic S. A.

(B) Mean displacement of S. A-near DCMCOs (DCMCO-S. A) and correlated DCMCOs compared to planktonic S. A.

(C) Two-dimensional hotelling’s test analysis of motion correlations between S. A-near HCOs (HCO-S. A), S. A near DCM-COs (DCMCO-S. A) and planktonic S. A.

(D) Two-dimensional hotelling’s test analysis of motion correlations between S. A-near HCOs (S. A-CM) and correlated HCOs (HCOs), S. A near DCM-COs (S.A-DCM) and correlated DCM-COs (DCM-COs)

(E) Quantitative analysis of HCOs contraction strength (total displacement during a systole-diastolic cycle) after treatment with different concentrations of isoproterenol (1 or 50μM).

(F) The infection status of HCOs organoids after 24-hour co-culture with S. A (105/mL) under different 3 treatment groups (PBS as control, 1 or 5 μM isoproterenol as drug) compared to the S. A in medium (Blank Control) determined by the Colony forming unit (CFU) method.

(G) The counting results in the CFU assay obtained from Fig. 5F.

Dissecting the influence of toxins on CO contractility and bacterial infection

(A) Mean displacement of the S. A infected HCOs (24-hour inoculation) and the healthy control HCOs, the track length is 3s. n= 6 biologically independent samples (Two groups for HCOs/Infected HCOs each).

(B) Heat map of DE genes in the metabolic pathway Reactome ECM organisation Pathway (R-HSA-1474244) in the organoid model showing different trends in the changes in gene expression after S. A infection compared to healthy organoid control after 24 hours. The colour scale shows the row z score. Organoid samples, n= three biologically independent samples per group (each sample containing one well of 10-20 organoids);

(C) Heat map of DE genes in the metabolic pathway Reactome Muscle Contraction Pathway (R-HSA-397014) in the organoid model showing different trends in the changes in gene expression after S. A infection compared to healthy organoid control after 24 hours. The colour scale shows the row z score. Organoid samples, n= three biologically independent samples per group (each sample containing one well of 10-20 organoids);

(D) Distribution of protein secretion and fold change; non-DE (yellow) and DE genes (Red for downregulation and blue for up-regulation) of S. A isolated from S. A HCOs infection model were compared with the control S. A.

(E) Heat map of DE protein secretion in the metabolic pathway KEGG bacterial infection Pathway (H02076) in the organoid model shows different trends in gene expression changes after S. A infection compared to healthy organoid control after 48 hours. The colour scale shows the row z score. Organoid samples, n= three biologically independent samples per group (each sample containing one well of 10-20 organoids);

(F) Immunohistochemical staining for S. A infection site on H-COs after 24h incubation. The bar is 10 μm.

(G) Immunohistochemical staining for S. A infection site on HCOs after 24h incubation. The bar is 10 μm.

(H) Immunohistochemical staining for S. A infection site on HCOs after 24h incubation. The bar is 4 μm.

Bacterial infection reduced COs contraction by influencing myosin structure

(A) CLSM Observation HCO’s DAPI/ Calponin/ α-actinin localisation. The bar is 50μm.

(B) CLSM Observation HCO’s DAPI/ MHC/ Troponin localisation. The bar is 50μm.

(C) CLSM Observation HCO’s DAPI/ Calponin/ α-actinin localisation after infection after 24h co-culture with S. A. The bar is 50μm.

(D) CLSM Observation HCO’s DAPI/ MHC/ Troponin localisation after infection after 24h co-culture with S. A. The bar is 50μm.

(E) The distribution pattern of Calponin and α-actinin structures in HCOs before and after S. A infection, obtained through Stardist analysis.

(F) Quantitative analysis of the formed areas of MHC, Troponin, Calponin, and α-actinin before and after infection to demonstrate the extent of structural disruption caused by the infection.

Dissecting the influence of contractility on S. A infection in vivo using mice model.

(A) Experimental set-up: Mice were assigned into groups (Sham, Mock, Amoxicillin and Enalapril; n=6). All C57Bl/6 mice except the Sham group first received TAC surgery to induce reduced heart function. Different drugs were injected into mice for further experiments.

(B) Electrocardiography of the mice from different experimental and Sham groups was measured two hours after drug administration.

(C) Fraction shortening (%) of the mice from different experimental groups and Sham group measured by Electrocardiography at 2h after drug administration.***P<0.005 ****p<0.001

(D) The heart specimens collected post-experimentation from different groups.

(E) The number of deaths, IE on ventricle, and non-IE on ventricle among mice in different experimental groups. The infection status of decimals was confirmed through observation of cardiac bacteria vegetations and Gram staining under a microscope.

(F) Aortic ventricle endocarditis in different experiment groups. Staining: haematoxylin and eosin (H&E) and Gram staining. The bar is 50 μm

(G) Comparative analysis of the Fractal Dimension in mice with or without endocarditis in the cardiac ventricles in Enalapril, Mock and Sham groups. Significance:****p<0.001

(H) Comparative analysis of the Fractal Dimension in mice with or without endocarditis in the cardiac valves in Enalapril, Mock and Sham group. Significance:****p<0.001