Exosomally derived Y RNA fragment alleviates hypertrophic cardiomyopathy in transgenic mice

Figures

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  Figure 1. Biodistribution of YF1 (A) Levels of YF1 30 min post-injection expressed as fold change over background in each tissue. (B) Levels of YF1 30 min post-injection expressed as copies per mg in each of the solid organs sampled. (C) Persistent measurable levels of YF1 in heart 4 h post-injection.

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  Figure 2. YF1 treatment improves mobility and cardiac function in animals with hypertrophic cardiomyopathy (A) Schematic describing the experimental protocol for cTnI146Gly mice with vehicle or YF1 treatment by retro-orbital (r.o.) injection. (B) Change in body weight (BW) over time in male and female mice. (C) Representative echocardiography images of the left ventricle in diastole. (D) Change in diastolic interventricular septal wall diameter (IVSd) and left ventricular posterior wall diameter (LVPWd) in male and female mice over time. (E) Representative images depicting grooming behavior in male and female mice in each experimental group. (F) Distance covered by treadmill in male and female mice, presented as a percentage of distance covered by WT mice. (G) Heart weight (HW)-to-tibial length (TL) ratio and lung weight (LW)-to-TL at endpoint (5 months) in male mice. Graphs depict mean ± SEM with n = 4–8/group. Statistical significance was determined using two-tailed, unpaired, Student’s t test or one-way ANOVA followed by Tukey’s multiple corrections test. ∗p < 0.05.

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  Figure 3. Cardiac fibrosis and hypertrophy are attenuated following YF1 treatment (A) Representative images and quantification of interstitial fibrosis as determined by Picrosirius red staining. (B) Representative images and quantification of cardiomyocyte cross-sectional diameter using cell borders defined by wheat germ agglutinin (WGA), α-sarcomeric actinin (αSA), and DAPI stains. (C) Western blot images of fibrosis and hypertrophy signaling proteins. (D) Quantification of immunoblots in (C). Graphs depict mean ± SEM with n = 4–5/group. Statistical significance was determined using two-tailed, unpaired, Student’s t test or one-way ANOVA followed by Tukey’s multiple corrections test. ∗p < 0.05.

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  Figure 4. YF1 suppresses granulocyte and monocyte peripheral blood mobilization (A) Representative flow plots depicting gating strategy. (B) Quantification of changes in cell populations in (A). (C) Representative flow plots and quantification of myeloid cell populations. (D) Representative image of protein array and quantification. Boxes depict proteins with significant changes in expression; CXCL1 denoted by solid border, TNF R1 denoted with dotted border. (E) Gene-expression analysis of Cxcl1 expression in heart tissue. (F) Gene-expression analysis of Cxcl1 in angiotensin II (AngII) treated and untreated cardiomyocytes (NRVM, neonatal rat ventricular cardiomyocytes) and macrophages (BMDM, bone marrow-derived macrophages). Graphs depict mean ± SEM with n = 4–5/group. Statistical significance was determined using two-tailed, unpaired, Student’s t test or one-way ANOVA followed by Tukey’s multiple corrections test. ∗p < 0.05.

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  Figure 5. YF1 suppresses inflammatory cytokines and myeloid cell infiltration in the heart (A) Gene expression of proinflammatory cytokines by qPCR. (B) Gene expression of myeloid cell marker Cd14 by qPCR. (C) Representative images of CD68+ macrophage infiltration in the heart. (D) Quantification of immunohistochemistry in (C). Graphs depict mean ± SEM with n = 4–5/group. Statistical significance was determined using two-tailed, unpaired, Student’s t test or one-way ANOVA followed by Tukey’s multiple corrections test. ∗p < 0.05.

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  Figure 6. YF1 modifies macrophage gene profile to elicit cardioprotection (A) Gene expression of NRVM treated with AngII studied as per the diagram of experimental workflow. (B) All groups were stimulated with AngII and then treated with vehicle (saline), YF1, or conditioned media from YF1-treated macrophages (CM YF1 BMDM). (C) Heatmap of differentially expressed genes in macrophages (left, vehicle or YF1; right, vehicle or YF1 followed by AngII treatment). (D) Volcano plot of the top differentially expressed genes in vehicle- and YF1-treated macrophages. (E) Volcano plot of the top differentially expressed genes in vehicle- and YF1-treated macrophages stimulated with AngII. Graphs depict mean ± SEM with n = 3–5/group. Statistical significance was determined using one-way ANOVA followed by Tukey’s multiple corrections test. ∗p < 0.05.