Myosins and MyomiR Network in Patients with Obstructive Hypertrophic Cardiomyopathy

Figures

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  Figure 1. Myosin protein analysis by MS. PRM quantification of myosin isoforms in HCM (n = 14) and CTRL (n = 8) is presented as MS1 and MS2 total area of representative selected peptides. The HCM patients without HCM-associated mutations are presented in violet, and those bearing mutations are presented in pink. Two additional morphologically normal human samples, from atrium and skeletal muscle biopsies, serve as comparators (dotted lines). Mean values from technical replicates/samples are presented as dots and boxes (min to max). Unpaired t-test was applied; significant differences with p < 0.0001 are indicated by ****.

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  Figure 2. MyomiR network gene expression in myectomies: myosin genes, Mhrt and miR-499 targets. The expression levels of MYH6, MYH7, and MYH7B mRNA (A), of MYLC2 mRNA (B) and of Mhrt (C) evaluated by RT-qPCR in the myocardium of HCM vs. CTRL are shown. Values are presented as boxes (min to max), and dots indicate single sample values. The expression levels of miR-499-target genes coding for transcription factors, SOX6 ((D), left and middle panels) and PTBP3 ((D), right panel) by RT-qPCR are compared in the myocardium of HCM and CTRL. Values are presented as boxes (min to max), and dots indicate single sample values. The inverse relationship between miR-499 and SOX6 is plotted ((D), middle, panel). The HCM patients without HCM-associated mutations are presented in violet; those bearing mutations are in pink. Unpaired t-test is applied and significant differences are shown as * p < 0.05; ** p < 0.01 and *** p < 0.0005, **** p < 0.0001. Significance within HCM group between individuals with or without mutations is indicated as §§ p < 0.01.

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  Figure 3. MYH7B, miR-499, and SOX6 gene expression in cardiomyocytes. The levels of expressed miR-499 (A), MYH7B (B), and SOX6 (C) are evaluated by RT-qPCR in cardiomyocyte samples isolated from a subset of myectomies using LCM. Values are presented as boxes (min to max), and dots indicate single sample values. The distribution of miR and gene levels in HCM patients without HCM-associated mutations are presented in violet; those bearing mutations are in pink. The t-test was applied, and significant differences between HCM and CTRL are shown as *** p < 0.001. Significant difference between HCM with or without mutation is evaluated by the Mann–Whitney test, §§ p < 0.01.

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  Figure 4. Circulating miR-499. The difference between hsa-miR-499-5p levels in plasma samples from HCM (n = 16) and healthy volunteers (CTRL, n = 11) is shown ((A), left plot). The comparison between expression levels of hsa-miR-499-5p in myocardial and plasmatic samples from the same HCM patients is presented ((A), right plot). The difference between hsa-miR-499-5p levels in plasma EV from subsets of HCM (n = 12) vs. CTRL (n = 8) is plotted (B). Mann–Whitney test and paired and unpaired t-tests are applied, respectively. p-value < 0.05 is considered significant and indicated by *; p < 0.01 is indicated by **.

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  Figure 5. MYHs, MyomiRs, and SOX6 expression in transfected cardiomyocytes from iPSC. The levels of expressed miR-499, MYH7B, MYH7, Mhrt, miR-208b, and SOX6 by RT-qPCR in cardiomyocytes differentiated from iPSC and transfected with miR-208a are shown at different time points (A). The expression of MYH7, Mhrt, miR-208b, and SOX6 determined in cardiomyocytes after transfection with miR-499 is plotted (B). The gene expression is presented as the ratio between 2^−ΔCt in treated vs. untreated cells (ratio control value =1 is indicated by dotted line where applicable). Amount of miR-499 released from transfected cardiomyocytes into culture supernatants is also shown (C). Values are presented as boxes (min to max), and dots indicate single sample values. ANOVA and Friedman tests are applied. p-value < 0.05 is considered significant and shown as *; p < 0.01 is indicated by **, and p < 0.001 by ***. Significant difference (p-value < 0.05 ) vs. untreated cells is indicated by §.