Mechanosensitive Molecular Mechanisms of Myocardial Fibrosis in Living Myocardial Slices

Figure 1. Electro‐mechanical culture of living myocardial slices (LMS). (A) Diagram of the experimental design. (B) Representative images of rat LMS showing YAP nuclear and cytoplasm expression. (C) YAP mean nuclear intensity after 48 h of culture (N = 5). (D) YAP nuclear/cytoplasm ratio after 48 h of culture (N = 5). (E) Fold change expression of genes activated by mechanical stress, FAK (N = 3) and ROCK2 after 48 h of culture (N = 5). (F) Cell viability analysed by live/dead staining. (G) Area quantification of living (Calcein) and dead (ethidium) cells on myocardial slices after 48 h of culture (N = 3). P value was calculated using a Student's t test. *, **, *** = P values <0.05, 0.01, and 0.001, respectively. Data shown as mean ± SEM. PL, physiological load; OL, overload; YAP, yes‐associated protein.

Figure 2. Rat living myocardial slices (LMS) for the study of mechanosensitive molecular mechanisms of myocardial fibrosis. (A) Fold change expression of pro‐fibrotic genes ACTA2, FN1, TGF‐β1, SMAD3, SMAD4, Collagen production gene COL3A1, and pro‐inflammatory gene IL‐6 in myocardial slices after 48 h of culture (N = 3–5). (B) Fold change expression of pro‐fibrotic, pro‐inflammatory and genes activated by mechanical load in fibroblasts isolated from 48 h cultured LMS (N = 9, each sample contains 3 LMS). (C) Representative images of rat LMS stained for Collagen I/Hoechst and (D) Vimentin/Hoechst after 48 h of culture with and without IL‐11 (N = 4). (E) Percentage quantifications of the LMS area covered by Collagen I and Vimentin. (F–I) Example images and quantifications of Western blots of CD45, COL1, αSMA, and GAPDH from 48 h cultured myocardial slices with and without IL‐11 (N = 4). P value was calculated using a Student's t test (gene expression) or two‐way ANOVA with Bonferroni's multiple comparison (IL‐11 experiments). *, **, ***, **** = P value <0.05, 0.01, 0.001, and 0.0001, respectively. Data shown as mean ± SEM. PL, physiological load; OL, overload.

Figure 3. Functional remodelling of rat living myocardial slices (LMS). (A) Representative force trace showing different parameters. (B) Representative traces of rat LMS contraction at 2.2 μm SL and 2.4 μm SL after 48 h of culture. (c) Contractility of LMS as measured by normalising the amplitude of force by the cross‐sectional area (0 h N = 10; 24 h PL N = 11; 24 h OL N = 10; 48 h PL N = 16; 48 h OL N = 16). (D–F) Contractility kinetics of rat LMS (0 h N = 8; 24 h PL N = 11; 24 h OL N = 8; 48 h PL N = 16; 48 h OL N = 16). (D) Time (in seconds) required to reach peak amplitude of force. (E) Time (in seconds) for the amplitude to decay from maximum force to 50%. (F) Time (in seconds) for the amplitude to decay from maximum force to 90%. (G–H) Functional remodelling of rat LMS after 48 h of IL‐11 treatment. (G) Contractility of LMS as measured by normalising the amplitude of force by the cross‐sectional area (−IL‐11 PL N = 8; −IL‐11 OL N = 7; +IL‐11 PL N = 13; +IL‐11 OL N = 12). (H) Time (in seconds) required to reach peak amplitude of force. (I) Time (in seconds) for the amplitude to decay from maximum force to 50%. (J) Time (in seconds) for the amplitude to decay from maximum force to 90%…

Figure 4. Human living myocardial slices (LMS) for the study of mechanosensitive molecular mechanisms of myocardial fibrosis. (A) Representative images and quantifications of human LMS after 48 h culture stained for Vimentin/Hoechst; (B) αSMA/Hoechst (C) Collagen I/Hoechst (non‐HF PL N = 4; non‐HF OL N = 4; HF PL N = 7; HF OL N = 6). (D) Contractility of LMS as measured by normalising the amplitude of force by the cross‐sectional area. (E) Time (in seconds) required to reach peak amplitude of force. (F) Time (in seconds) for the amplitude to decay from maximum force to 50%. (G) Time (in seconds) for the amplitude to decay from maximum force to 90% (non‐HF PL N = 4; non‐HF OL N = 4; HF PL N = 7; HF OL N = 6). Each point represents an individual slice. P value was calculated using two‐way ANOVA with Bonferroni's multiple comparison. *, **, *** = P value <0.05, 0.01, and 0.001, respectively. Data shown as mean ± SEM. HF, heart failure; PL, physiological load; OL, overload.

Figure 5. Anti‐fibrotic drug testing on living myocardial slices (LMS) cultured under physiological and pathological mechanical load. (A) TGFβ activation pathway. (B) Fold change expression of 48 h cultured rat LMS (− SB431542 PL N = 4–5; − SB431542 OL N = 4–5; +SB431542 PL N = 5; +SB431542 OL N = 4), TGF‐β with and without SB‐431542 treatment of pro‐fibrotic genes FN1, COL3A1, pro‐inflammatory gene IL‐6, genes activated by mechanical stress, ROCK2 and FAK. (C–F) Effects of SB‐431542 on contractility parameters of 48 h cultured rat LMS (−SB431542 PL N = 7–8; −SB431542 OL N = 9; +SB431542 PL N = 10; +SB431542 OL N = 7). (C) Contractility of LMS after 48 h culture determined by normalising the amplitude of force by the LMS cross‐sectional area. (D) Time (in seconds) required to reach peak amplitude of force. (E) Time (in seconds) for the amplitude to decay from maximum force to 50%. (F) Time (in seconds) for the amplitude to decay from maximum force to 90%. (G–H) Representative images and quantifications of rat LMS stained for Vimentin, Collagen I, α‐actinin and Hoechst after 48 h of culture with and without SB‐431542 (−SB431542 PL N = 4; −SB431542 OL N = 4; +SB431542 PL N = 4; +SB431542 OL…