Characterization of human living myocardial slices culture-induced adaptations: a translational perspective

Figures

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  Fig. 1. Functional remodeling of LMS during culture. A) Representative contraction transient at different days of culture. Cultured LMS show a faster contraction-relaxation cycle (B), attributed to a reduction in both the contraction (C) and relaxation times (D) (A–D: Day 0 N = 9, n = 19; Day 1 N = 8, n = 16; Day 6 ± 1 N = 9, n = 19; N = 8, n = 17). Contraction force increases during culture (E), while diastolic tension decreases (F). The refractory period (G) decreases, while the post-pause potentiation (H) and stimulation threshold (I) increase. J) Cultured LMS lose the ability to develop more force when subjected to higher pacing frequencies while being more effective at lower frequencies (Tukey's multiple comparisons are provided in Supplemental Fig. S1D); (E–J: Day 0 N = 11, n = 126; Day 1 N = 11, n = 61; Day 6 ± 1 N = 11, n = 49; N = 10, n = 22). K) PCA displaying changes between LMS exposed to culture for 0, 1, 6 ± 1 and 10 ± 1 days based on five functional parameters (stimulation threshold, post-pause potentiation after a 30-second pause, contractile force, diastolic force, and refractory period) (N = 11). Ellipses indicate the standard deviation. In all experiments, N represents…

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  Fig. 2. LMS undergo cellular and molecular changes upon culture. A) Principal component analysis plot based on the 500-most differentially expressed genes in day 0 vs 10 ± 1 days cultured LMS. Ellipses indicate the standard deviation. B) Module score of the differentially upregulated genes between day 0 and day 10 ± 1. C) Mapping upregulated genes to cell markers specific to the different cell types in the heart (ventricular and atrial cardiomyocytes, fibroblasts, vascular cells, myeloid cells, and neurons). Pathway enrichment analysis of ventricular cardiomyocytes (D), myeloid cells (E), fibroblast (F), and vascular cells (G) on day 0 versus day 10 ± 1 cultured LMS. For all panels, N = 6 unique patients and n = 6 LMS were used.

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  Fig. 3. Electrophysiological remodeling of LMS during culture. Average calcium transient at 1 Hz (A) and 3 Hz (B) at day 0 and day 10 ± 1 of culture. At day 10 ± 1 of culture, LMS show an increased ability to follow pacing at higher frequencies (C), and increased calcium amplitude (D). Calcium transient decay time (CTD90) (E) is shorter and has a decreased short-term variability (STV) (F) at day 10 ± 1 of culture (C–F: day 0 N = 6, n = 16, day 10 ± 1 N = 6, n = 13). Pathway enrichment analysis on electrophysiological (G) and contractility (H) pathways on day 0 versus day 10 ± 1 cultured LMS (N = 6, n = 6). I) Heatmap representation of differentially expressed genes of key players associated with cardiac electrophysiology (N = 6, n = 6). Where N represents the number of patients of which LMS were derived and n indicates the individual LMS. Statistical analyses were performed using a mixed-effects model accounting for repeated measures within subjects, followed by Tukey's multiple comparisons test. Data are presented as mean ± SEM. Statistical significance was defined as p < 0.05.

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  Fig. 4. Structural remodeling of LMS during culture. A) Troponin-I release by LMS to the medium (N = 7, n = 15). Pathway enrichment analysis on cytoskeletal (B) and extracellular matrix (C) pathways on day 0 versus day 10 ± 1 cultured LMS (C & D: N = 6, n = 6). D) Representative confocal images of cardiac troponin-I (cTnI), gap junction alpha-1protein (Cx43), and mitochondrial ATP synthase F1 subunit alpha (ATP5a) immunolabelling of LMS at day 0 and day 10 ± 1 of culture (N = 3, n = 3), scale bars = 50 μm. N indicates the number of unique patients from whom the LMS were derived, n represents the individual LMS. Statistical analyses were performed using a mixed-effects model accounting for repeated measures within subjects, followed by Tukey's post hoc test. Significance was set at p < 0.05.

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  Fig. 5. Metabolic remodeling of LMS during culture. A) Volcano plot of differential pathways associated with metabolism. B) Pathway enrichment is subdivided into the four main metabolic pathways: amino acid, carbohydrate, fatty acid, and nucleotide metabolism, and pathways related to hormones and vitamins and gas exchange (A–B: N = 6, n = 6). Glucose consumption (C) and lactate production (D) were measured in the conditioned media of LMS (C–D: N = 8, n = 18). N refers to individual patient donors, and n indicates the number of LMS analyzed. Statistical analyses were performed using a mixed-effects model accounting for repeated measures within subjects with Tukey's multiple comparisons test and statistical significance was defined as p < 0.05.

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  Fig. 6. Developmental shift in LMS during culture. A) Pathway enrichment analysis of developmental pathways on day 0 versus day 10 ± 1 cultured LMS (N = 6 unique patients, n = 6 individual LMS). B) Heatmap representation of differentially expressed genes associated with cardiac development markers, cardiac maturation markers, and cell cycle markers in LMS at day 0 and day 10 ± 1 of culture (N = 6 unique patients, n = 6 individual LMS). C) Dot plot of the expression of markers associated with cardiac development markers, cardiac maturation markers, and cell cycle markers in the different evaluated cell types.