Duchenne muscular dystrophy (DMD) cardiomyocyte-secreted exosomes promote the pathogenesis of DMD-associated cardiomyopathy

Abstract

Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.

Keywords: Cardiomyocyte; Cardiomyopathy; Exosome; MicroRNA; Stress; Vesicle.

Fig. 1.

Differential paracrine effects exerted by DMD cardiac exosomes (DMD-exo) after 2, 24 and 48 h of exposure. Both DMD- and N-iCMs were exposed to exosomes isolated from DMD- and non-affected (N)-induced pluripotent stem cell-derived cardiomyocytes (iCMs) for 2, 24 or 48 h before stress assays. N-iCMs are represented by blue and purple bars and DMD-iCMs by red and pink bars. n=63-115 cells counted in each group. (A) Exposure to DMD- and N-exo for 2 h is protective, but 24 h and 48 h exposure reveals differences for DMD-exo in mitigating stress-induced cell death. n=91-1521 cells counted in each group. (B) Additional DMD-iCM lines demonstrate that DMD-exo no longer reduce cell death in DMD-iCMs after 48 h DMD-exo exposure. (C,D) Exposure to DMD-exo for 48 h did not reduce stress-induced ROS levels (C) or protect mitochondrial membrane potential in DMD-iCMs (D), unlike exposure to N-exo. Data represent the mean±s.e.m. Significance was determined using a two-way ANOVA. n=3-9 biological replicates per group; *P<0.05 stress versus no stress; **P<0.05 exosome versus vehicle; ^&P=0.08 exosome versus vehicle; ^#P<0.05 DMD-exo versus N-exo. A.U., arbitrary units; DHE, dihydroethidium; ND, not detected; PI, propidium iodide; TMRE, tetramethylrhodamine ethyl ester.

Fig. 2.

Inhibiting DMD-exo release is cardioprotective against stress in DMD-iCMs. Exosome release was inhibited in iCMs with 10 μM GW4869 for 24 h before stress and imaging assays. (A-C) Inhibition of DMD-exo release reduced ROS levels (A), preserved membrane potential (B) and reduced cell death (C) in DMD-iCMs. Data represent the mean±s.e.m. Significance was determined using a two-way ANOVA. In A,B, n=3-6 biological replicates per group; in C, n=52-721 cells counted in each group. *P<0.05 stress versus no stress; **P<0.05 versus (−) GW4869; ^#P<0.05 GW4869+DMD-exo versus (−) GW4869.

Fig. 3.

Inhibition of exosome secretion in mdx mice protects hearts from stress-induced injury. (A) Timeline of in vivo experiments in which mdx mice were subjected to isoproterenol-induced cardiac injury in conjunction with GW4869 exosome inhibition over 10 days, followed by histological assessment of cardiac damage. (B) Exosome quantitation assays confirm inhibition of mdx serum exosome levels. Data represent the mean±s.e.m. Statistical significance was determined by a one-way ANOVA. n=6 animals per group. *P<0.05 day 8 versus day 0. (C) Representative images from mdx heart sections subjected to Trichrome or Hematoxylin and Eosin (H&E) staining demonstrate isoproterenol-induced damage, which is mitigated by GW4869 exosome inhibition. (D) Quantitation of the percentage fibrotic area from H&E staining. Isoproterenol increases the percentage of cardiac fibrosis in mdx mouse hearts versus myocardial area, and blocking exosome secretion with GW4869 reduces fibrosis. Data represent the mean±s.e.m. Significance was determined using unpaired Student's t-test. n=6 animals per group; *P<0.05 versus vehicle+isoproterenol. Scale bar: 12.7 mm.

Fig. 4.

Differences in paracrine effects exerted by long-term DMD-exo exposure are not attributable to surface peptides. Surface peptides were stripped off exosomes with trypsin treatment, and trypsinized exosomes were used for exposure assays in DMD-iCMs. (A-C) Removal of DMD-exo surface peptides for 48 h exposure assays is associated with exacerbated stress-induced ROS levels (A), no change in mitochondrial membrane potential (B) and no alteration in cell death levels (C) in comparison to intact DMD-exo. (D-F) Trypsinization of N-exo surface peptides for exposure assays in DMD-iCMs is associated with increased stress-induced ROS levels (D), no change in mitochondrial membrane potential (E) and elevated stress-induced cell death levels (F), in comparison to intact N-exo. Data represent the mean±s.e.m. Significance was determined using a two-way ANOVA. In A,B, n=3-6 biological replicates per group; in C, n=74-165 cells counted in each group. *P<0.05 stress versus no stress; **P<0.05 exosome versus vehicle; ^#P<0.05 trypsinized exosomes versus intact exosomes; ^&P=0.06 trypsinized exosomes versus vehicle. Trypsinized peptides were collected from N- and DMD-exo and analyzed by mass spectrometry. (G) GO and KEGG pathway analysis of peptides expressed on DMD-exo. (H) GO and KEGG pathway of peptides expressed on N-exo. n=3 biological replicates per group.

Fig. 5.

Exosomal miR cargo contributes to altered response to stress in DMD-iCMs. Exosomal miR cargo was depleted by exposing iCMs to 5 µM acriflavine for 2 h, followed by collection of miR-depleted exosomes for exosome exposure assays. (A) N-exo and DMD-exo depleted of miR cargo reduce stress-induced ROS in DMD-iCMs. (B) miR-depleted N-exo and DMD-exo still offer some protection against loss of membrane potential in DMD-iCMs. (C) miR-depleted N-exo and DMD-exo reduce stress-induced cell death in DMD-iCMs. Data represent the mean±s.e.m. Significance was determined using a two-way ANOVA. In A,B, n=3-6 biological replicates per group; in C, n=74-142 cells counted in each group; *P<0.05 stress versus no stress; ^&P=0.07 exosome versus vehicle; **P<0.05 exosome versus vehicle; ^#P<0.05 miR-depleted exosomes versus intact exosomes. (D) Hierarchical clustering of miRs identified by small RNA-sequencing in DMD-exo and N-exo reveals differential expression of 894 miRs. n=3 biological replicates per group. (E) GO analysis of differentially expressed DMD-exo miRs shows their involvement in regulating processes such as cell death, gene expression and cytoskeleton protein binding. (F) KEGG analysis of DMD-exo miRs highlights their regulation of pathways including apoptosis, TGF-beta signaling and Hippo signaling.

Fig. 6.

Long-term exposure to DMD-exo leads to transcriptomic changes in DMD-iCMs attributable to altered miR cargo. (A) Hierarchical clustering shows expression patterns of differentially expressed genes among DMD-iCMs and N-iCMs, and exposure to exosomes for 48 h leads to transcriptional alterations that are unique between DMD-exo- and N-exo-exposed groups. Red color refers to higher expression and blue color to lower expression. n=2-3 biological replicates per group. (B) GO analysis of DEGs in DMD-iCMs reveals that biological processes resulting from exposure to either exosome type are similar. (C) KEGG pathway analysis shows differences in pathways stimulated by N- or DMD-exo. n=3 per group. Next, N-iCMs were exposed to miR-depleted N-exo for 48 h before 60 or 90 min stress. (D-F) miR-depleted N-exo exacerbate ROS levels in comparison to miR-enriched N-exo (D), do not protect mitochondrial membrane potential in comparison to N-exo (E) and exacerbate cell death in N-iCMs in comparison to N-exo (F). Data represent the mean±s.e.m. Significance was determined using a two-way ANOVA. In D,E, n=3-6 biological replicates per group; in F, n=31-142 cells counted in each group. *P<0.05 stress versus no stress; ^&P=0.07 exosome versus vehicle; **P<0.05 exosome versus vehicle; ^#P<0.05 miR-depleted exosomes versus intact exosomes. ND, not detected.