Ryanodine receptor dysfunction causes senescence and fibrosis in Duchenne dilated cardiomyopathy

Abstract

Background: Duchenne muscular dystrophy (DMD) is an X-linked disorder characterized by progressive muscle weakness due to the absence of functional dystrophin. DMD patients also develop dilated cardiomyopathy (DCM). We have previously shown that DMD (mdx) mice and a canine DMD model (GRMD) exhibit abnormal intracellular calcium (Ca²⁺) cycling related to early-stage pathological remodelling of the ryanodine receptor intracellular calcium release channel (RyR2) on the sarcoplasmic reticulum (SR) contributing to age-dependent DCM.

Methods: Here, we used hiPSC-CMs from DMD patients selected by Speckle-tracking echocardiography and canine DMD cardiac biopsies to assess key early-stage Duchenne DCM features.

Results: Dystrophin deficiency was associated with RyR2 remodelling and SR Ca²⁺ leak (RyR2 Po of 0.03 ± 0.01 for HC vs. 0.16 ± 0.01 for DMD, P < 0.01), which led to early-stage defects including senescence. We observed higher levels of senescence markers including p15 (2.03 ± 0.75 for HC vs. 13.67 ± 5.49 for DMD, P < 0.05) and p16 (1.86 ± 0.83 for HC vs. 10.71 ± 3.00 for DMD, P < 0.01) in DMD hiPSC-CMs and in the canine DMD model. The fibrosis was increased in DMD hiPSC-CMs. We observed cardiac hypocontractility in DMD hiPSC-CMs. Stabilizing RyR2 pharmacologically by S107 prevented most of these pathological features, including the rescue of the contraction amplitude (1.65 ± 0.06 μm for DMD vs. 2.26 ± 0.08 μm for DMD + S107, P < 0.01). These data were confirmed by proteomic analyses, in particular ECM remodelling and fibrosis.

Conclusions: We identified key cellular damages that are established earlier than cardiac clinical pathology in DMD patients, with major perturbation of the cardiac ECC. Our results demonstrated that cardiac fibrosis and premature senescence are induced by RyR2 mediated SR Ca²⁺ leak in DMD cardiomyocytes. We revealed that RyR2 is an early biomarker of DMD-associated cardiac damages in DMD patients. The progressive and later DCM onset could be linked with the RyR2-mediated increased fibrosis and premature senescence, eventually causing cell death and further cardiac fibrosis in a vicious cycle leading to further hypocontractility as a major feature of DCM. The present study provides a novel understanding of the pathophysiological mechanisms of the DMD-induced DCM. By targeting RyR2 channels, it provides a potential pharmacological treatment.

Keywords: Calcium; DMD; Ryanodine receptor; Senescence; hiPSC‐derived cardiomyocytes.

Figure 1

S107 prevents the stress‐induced RyR2 Ca²⁺ leak and post‐translational remodelling in the three DMD patient hiPSC‐CMs. (A) Representative illustrations of RyR2 single channel recordings under 150 nmol/L free cis [Ca²⁺] in HC, DMD hiPSC‐CMs. DMD hiPSC‐CMs treated with S107 (5 μM overnight). The « c‐ » represents the RyR2 channel closed state. (B) Bar chart with each data point superimposed showing the single channel open probability (Po) of RyR2 channels in healthy control (HC) and DMD ± S107 SR microsomes. (C) Frequency of openings (Fo in events/min) of RyR2 channels in HC and DMD ± S107 SR microsomes. (D) Single channel Po in each DMD (1, 2 and 3) treated or not with S107. Data are presented as mean ± SEM. **P < 0.01 (Mann–Whitney test). (E) Representative immunoblot bands of the three HC, three DMD and three DMD + S107 lysates under stress (1 μM of isoproterenol) for total RyR2, PKA‐phosphorylated RyR2 at 2809 site (P2809), CaMKII‐phosphorylated RyR2 at 2815 site (P2815), S‐nitrosylation of cysteines (Cys‐NO), RyR2 oxidation (DNP) and Castabin2 (FKBP12.6) binding. (F) Relative calstabin2 amount bound to RyR2 in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. The number of experiments varies from three to nine in HC and DMD hiPSC‐CM lysates. Data are presented as mean ± SEM. **P < 0.01 (Kruskal–Wallis test).

Figure 2

SR Ca²⁺ leak leads to signalling pathways involving fibrosis and senescence in DMD hiPSC‐CMs. (A) Overlap of proteins above cut‐off (P‐value < 0.05 and |fold‐change| ≥ 1.5) for HC versus DMD versus DMD + S107 comparisons. For S107 application, DMD hiPSC‐CMs were treated with 5 μM S107 for 10 days in culture medium. (B) Heatmap of the differentially expressed proteins (DEPs) that are common to both HC versus DMD and DMD versus DMD treated with S107 comparisons. (C) Most representative dysregulated pathways in Gene Ontology analysis. The data are three biological replicates from the three HC and three DMD lines ± S107 treatment.

Figure 3

SR Ca²⁺ leak causes senescence in DMD hiPSC‐CMs. (A) Representative images of HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107 showing SA‐β‐gal blue staining (as represented by the white arrows). The acquisition was performed using EVOS XL Core imaging system microscopy (magnification ×40). For S107 application, DMD hiPSC‐CMs were treated with 5 μM S107 for 10 days in culture medium. (B) Bar chart with each data point showing the percentage of SA‐β‐Gal‐positive senescent cells in HC hiPSC‐CMs (ratio of 47/322 cells, white bars), DMD hiPSC‐CMs (ratio of 189/283 cells, black bars) and DMD hiPSC‐CMs treated with S107 for 10 days (ratio of 432/1434 cells, grey bars). (C) Bar graphs summarizing the percentage of SA‐β‐gal‐positive senescent cells in each DMD (1, 2 and 3) treated or not with S107 for 10 days. (D) Representative immunostaining for α actinin (in red) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107 for 10 days. The nuclei are stained in blue (as represented by the white arrows). Scale bar: 10 μm. (E) Bar graphs summarizing the nucleus area (in pixels) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107 for 10 days. (F) Nucleus area in each DMD (1, 2 and 3) treated or not with S107 for 10 days. The number of experiments varies from 99 to 240 for each graph. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Kruskal–Wallis test).

Figure 4

GRMD aged of 12 months exhibit senescence. Bar graphs summarizing the fold change in gene expression analysis for p21 (cyclin‐dependent kinase inhibitor 1) (A), p16 (cyclin‐dependent kinase inhibitor 2A) (B), TGFβ2 (transforming growth factor‐beta 2) (C) IL6 (interleukin 6) (D) and IL1β (interleukin 1 beta) (E) in WT (white dot plots) and GRMD (black dot plots) cardiac biopsies at 6‐ and 12‐month‐old. Data are represented as mean ± SEM. *P < 0.05, **P < 0.01 (Kruskal–Wallis test).

Figure 5

S107 prevents the fibrotic deposit in hypertrophic DMD hiPSC‐CMs. (A) Immunostaining for α actinin (in red) and collagen type 1 α 1 (COL1A1) (in green) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. The nuclei are stained in blue. For S107 application, DMD hiPSC‐CMs were treated with 5 μM S107 for 10 days in culture medium. Scale bar: 10 μm. (B) COL1A1 fluorescence intensity (AU) in HC hiPSC‐CMs (white dot plots), DMD hiPSC‐CMs (black dot plots) and DMD hiPSC‐CMs treated with S107 (diamond plots). The number of experiments varies from 66 to 134 for each graph. (C) COL1A1 fluorescence intensity (AU) in each DMD (1, 2 and 3) treated or not with S107. (D) Cell area (μm²) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. (E) Cell area in each DMD (1, 2 and 3) treated or not with S107. The number of experiments varies from 71 to 242 for each graph. Data are represented as mean ± SEM, **P < 0.01 (Kruskal–Wallis test).

Figure 6

S107 prevents the stress‐induced aberrant release of Ca²⁺ in DMD hiPSC‐CMs. (A) Representative original line‐scan of Ca²⁺ and corresponding Ca²⁺ transients in HC, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107 (5 μM overnight) under stress conditions (1 μM isoproterenol and 1 Hz pacing at 20 V for 5 ms duration). (B) Normalized Ca²⁺ amplitude (AU) in HC hiPSC‐CMs (white dot plots), DMD hiPSC‐CMs (black dot plots) and DMD hiPSC‐CMs treated with S107 (diamond plots). (C) Rate of RyR2 Ca²⁺ release (dF/dtmax in ΔF/s) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. (D) Frequency of diastolic leaky events (Hz) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. (E) Decay time (ms) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. The number of experiments varies from 178 to 370 cells for each graph. Data are represented as mean ± SEM. **P < 0.01 (Kruskal–Wallis test).

Figure 7

S107 prevents the stress‐induced hypocontractility in DMD hiPSC‐CMs. (A) Representative traces of video‐edge capture recordings in HC hiPSC‐CMs, DMD hiPSC‐CMs ± S107 (5 μM overnight) in presence of 1 μM isoproterenol (ISO). (B) Beat rate (bpm) in HC hiPSC‐CMs (white dot plots), DMD hiPSC‐CMs (black dot plots) and DMD hiPSC‐CMs + S107 (diamond plots). (C) Average amplitude (μm) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs + S107. (D) Contraction time (ms) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs treated with S107. (E) Relaxation time (ms) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs + S107. (F) Resting time (ms) in HC hiPSC‐CMs, DMD hiPSC‐CMs and DMD hiPSC‐CMs + S107. The number of experiments varies from 130 to 630 for each dot plot. Data are represented as mean ± SEM. **P < 0.01 (Kruskal–Wallis test).