An Unbiased Screen Identified the Hsp70-BAG3 Complex as a Regulator of Myosin-Binding Protein C3

Figures

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  Figure 1. Alpha-LISA MyBP-C/MYH Assay Development (A) Schematic of the Alpha-LISA testing platform. (B) Inducible pluripotent stem cell–derived cardiomyocyte (iPSC-CM) models used in the screen. (C) Myosin-binding protein C3 (MyBP-C), the protein product made by gene MYBPC3, and myosin heavy chain (MYH) Alpha-LISA raw results under screening assay conditions for positive control (MYBPC3 Patient1) iPSC-CMs, negative control (MYBPC3 [-/-]) iPSC-CMs, and negative control 293T cells. (D and E) Serial 2-fold dilution of MYBPC3 Patient1 iPSC-CM lysate tested in triplicate shows that assay conditions fall within the linear range for MyBP-C (D) with R2 = 0.98 and MYH (E) with R2 = 0.99.

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  Figure 2. Compound Screen Results (A) Normalized MyBP-C/MYH results from 2 representative screening plates. Overall, 100% is defined by MYBPC3 Patient1 iPSC-CMs treated with vehicle (dimethyl sulfoxide) control (green circles); 0% is defined by MYBPC3 (–/–) iPSC-CMs (gray squares). Hit compounds exhibited a MyBP-C/MYH value >3 SDs above (red open triangles) or below (red closed triangles) the control. Most compounds did not significantly change MyBP-C/MYH levels (blue triangles). (B) Hits subsequently went through a validation process detailed in the flowsheets herein. CRC = concentration-response curve; other abbreviations as in Figure 1.

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  Figure 3. Validated Hits: JG98 and PTL (A) JG98 and parthenolide (PTL) retained activity when fresh powder was tested (N = 4). (B) Compounds were also tested across a variety of cardiomyocyte cell lines and exhibited activity throughout. Graphs denote the concentration that results in a 50% reduction (EC50) of MyBP-C or MYH, and error bars represent the 95% CI (Table 1). (C) Structural analogues of JG98 and PTL were tested to explore structure activity relationships. The concentration-response curves of the 2 compounds with the lowest EC50(orange triangles, red squares) and a representative inactive structurally related compound (black circles) are shown (Supplemental Table 11). Abbreviations as in Figure 1.

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  Figure 4. JG98 Reduces MyBP-C Protein Levels Without Inducing Toxicity or Disrupting Myofilament Structure (A) Representative images of Ctrl-3 iPSC-CMs with JG98 (10 μM), JG258 (10 μM), or vehicle control are shown with α-actinin (green) and MyBP-C (red) (Supplemental Figure 12). Blinded analysis (N = 24) reveals reduced levels of MyBP-C/α-actinin in JG98 but not in JG258 samples and no significant change in myofibrillar disorder. (B) Cellular toxicity of JG98 and analogues did not exhibit significant toxicity at the concentrations that lead to MyBP-C reductions (Supplemental Tables 9 and 12). ∗∗∗P < 0.001. Conc = concentration; DMSO = dimethyl sulfoxide; neg = negative; Veh = vehicle; other abbreviations as in Figure 1.

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  Figure 5. Disruption of the BAG3-Hsp70 Complex by BAG3 Knockdown or JG98 Treatment Reduces MyBP-C Protein Levels (A) Proposed model: JG98 induces MyBP-C degradation by inhibiting heat shock protein 70 (Hsp70) and Bcl-2–associated athanogene 3 (BAG3) binding. (B) Micropatterned Ctrl-3 iPSC-CMs were treated with JG98 (10 μM) or vehicle control in duplicate. Representative images of immunofluorescence for Hsp70 (green) and BAG3 (red) and merged images are shown. The extent of co-localization within the cytosol between Hsp70 (green) and BAG3 (red) was measured by using Pearson’s correlation for each cell tested. There is less Hsp70 and BAG3 co-localization (lower Pearson’s correlation) in cells treated with JG98 than in vehicle control (N = 20). (C) A representative Western blot is shown. Ctrl-2 iPSC-CMs were cells treated with an adenovirus at a multiplicity of infection 5 carrying scrambled small hairpin RNA (shRNA) (gray bars, black squares) or BAG3 shRNA (blue bars, black triangles) for 18 hours. Cellular lysates were analyzed 4 days after shRNA treatment (n = 27; 5 biologic replicates). Quantification of Western blots shows significant reduction of BAG3 and MyBP-C.…