Functional and Molecular Characterisation of Heart Failure Progression in Mice and the Role of Myosin Regulatory Light Chains in the Recovery of Cardiac Muscle Function

Figures

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  Figure 4. Longitudinal sarcomeric protein phosphorylation and maximal isometric force measurements during HF progression. (Ai,Bi,Ci) Blot images of RLC, MyBP-C and TnI phosphorylation levels of post-MI and sham groups at different time points ranging between 2D and 28D post-MI, respectively. (Aii,Bii,Cii,Di) Bar graphs of RLC, MyBP-C and TnI phosphorylation and maximal isometric force of post-MI and sham groups at different time points ranging between 2D and 28D post-MI. Unpaired one-tailed t-test (*) was conducted on parametric and homogenous population. Unpaired one-tailed t-test with Welch’s correction (*) was conducted on parametric but inhomogeneous population. Unpaired one-tailed Mann–Whitney test (#) was conducted on non-parametric population. Number of mice used is summarised in Table S1. Note: ‘n’ refers to the number of technical replicates. (Aiii,Biii,Ciii,Dii) Bar graphs for relative RLC, MyBP-C and TnI phosphorylation and maximal isometric force values during HF progression, respectively. Kruskal-Wallis ANOVA test was performed on RLC phosphorylation populations and showed that the medians varied significantly (* p = 0.0102). Ordinary one-way ANOVA performed on MyBP-C…

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  Figure 1. LAD ligation optimisation. (A) Schematic diagram of the ligation location. H: ligation just above the bifurcation, L: ligation at the left branch just below the bifurcation. (B) Image of the mouse heart with ligation at H. (C) Longitudinal comparison of contractile force following LAD (H), LAD (L) surgery, control (no surgery) and sham surgery groups. Note: n = 3–13, where ‘n’ refers to the number of muscle preparations in each group from a total of 2–6 mice. Statistically insignificant differences among the means, verified by ordinary one-way ANOVA test.

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  Figure 2. Physiological, biological and structural markers verifying HF progression. (A) Raw hypertrophic indices of post-MI and sham groups between 2D and 28D post-MI. (B) Relative hypertrophic indices (post-MI/sham average) of post-MI groups between 2D and 28D post-MI. (C) Relative BNP gene expression levels at 7D and 14D post-MI and sham groups. (D) Fold changes in gene expression of post-MI populations with respect to their sham groups. Note: ‘n’ refers to the number of mice. Unpaired one-tailed t-test used to determine the differences between post-MI and sham groups. Note: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) No significant differences among the means were verified using ordinary one-way ANOVA. (E) Heart images and histology (Van Gieson stain) of longitudinal heart sections show that the heart displays features of hypertrophic cardiomyopathy at both 7D and 14D post-MI and dilated cardiomyopathy at 14D post-MI. Black arrows point to the site of thinned left ventricular walls. Images are taken at 10× magnification. Scale bar = 1 mm. Acronym: RV, right ventricle; LV, left ventricle; VS, ventricular septum. (F) H&E-stained, longitudinal heart sections at 7D and 14D…

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  Figure 3. Ca2+ sensitivity and cooperativity changes during MI progression. (A) Relative EC50 and (B) relative Hill slopes (post-MI/sham average) plotted by mouse. Each data point is the average of at least three muscle preparations from the cardiac papillary of one mouse. Note: n = 3–5, where ‘n’ refers to the number of mice used. One-tailed unpaired t-test was performed between timepoints. Note: * p < 0.05 Kruskal–Wallis test was used on EC50 of samples and ordinary one-way ANOVA was used on Hill slope of samples. There were no significant differences among the samples of the respective groups.

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  Figure 5. Potential for RLC phosphorylation in terms of muscle recovery. (Ai) Blot images of RLC extracted papillary muscle fibres under two treatments: 14D sham and post-MI permeabilized fibres (control) and 14D post-MI and sham permeabilized fibres exchanged with phosphorylated RLCs. Each lane contains protein samples extracted from five papillary fibre segments. (Aii) Bar graph representation of phosphorylation levels for the above-mentioned groups. Unpaired one-tailed Mann–Whitney test was performed for the respective groups, but no significant difference was detected between the ranks. Note: ‘n’ refers to the technical replicates. Two mice were used for post-MI groups and one mouse was used for sham group. (B) Bar graph of maximal isometric force of the above-mentioned groups. Unpaired one-tailed t-test was conducted to test for statistically significant differences. Three mice were used for each group. Note: ‘n’ refers to the number of papillary segments used for data collection; ** p < 0.01, **** p < 0.0001.

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  Figure 6. Longitudinal changes in enzymes in mice. (Ai,Bi,Ci) The blot images of MLCK, ZIPK and MYPT2, respectively. (Aii,Bii,Cii) Bar graph of normalised MLCK, ZIPK and MYPT2 amounts of post-MI and sham populations, respectively. All sample populations are normally distributed and validated by Shapiro–Wilk test. Differences in the standard deviations between two timepoints were tested by F-test. Unpaired one-tailed t-test with Welch’s correction (*) was performed on those with significant differences in the variances, while unpaired one-tailed t-test (*) was performed for all other samples. Note: n = 3–6, derived from at least two mice for post-MI and three mice for sham (except 7D sham, where one mouse was used). Note: ‘n’ refers to the number of technical replicates; * p < 0.05, ** p < 0.01. (D–F) Line graph plotted with normalised enzyme values and RLC phosphorylation to depict longitudinal changes in post-MI and sham populations.

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  Figure 7. Changes in the quantity of RLCs suggesting losses in diseased mouse sarcomere. (A) Blot image of RLC of post-MI and sham groups at different time points ranging between 2D and 28D post-MI. (B) Bar graph of normalised RLC amounts at different time points for post-MI groups. Unpaired one-tailed t-test with Welch’s correction (*) was used between 2D and 4D, 2D and 7D and 4D and 7D samples. Mann-Whitney one-tailed test (#) was performed between 7D and 28D samples. Kruskal-Wallis test indicated that the medians varied significantly among samples (p = 0.0194). All samples were normally distributed (except 28D post-MI), as validated by Shapiro-Wilk test. There were significant differences in the standard deviations, except between 4D and 7D post-MI and 7D and 28D post-MI, as validated by F-test. Results were obtained from at least two mice for post-MI samples. Note: n = 4-9, where ‘n’ refers to the number of technical replicates; * p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.05. (C) Bar graph of normalised RLC amounts at different time points for sham groups. Ordinary one-way ANOVA showed no significant differences among population means. Sample populations were normally distributed,…