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Figures
- Fig. 1.
MYOM2 mutations and expression in patients. (A) Schematic of MYOM2, with its domain structure and mutations found in TOF (blue) and HCM (red) patients. Mutation positions are based on the human reference genome hg38. Nucleotide changes are based on transcript ENST00000262113. Amino acid changes are based on protein ENSP00000262113. Damage prediction by PolyPhen2, SIFT or MutationTaster are indicated by ‘#’. The minor allele frequency (MAF) is based on 71,702 genomes from unrelated individuals of the gnomAD (v3). The binding sites of two interaction partners, MYH7 and creatine kinase (muscle isoform), are indicated below. (B) RNA-seq expression level of MYOM2 in RV tissue of TOF patients and normal hearts (NH). P-value is derived from an unpaired two-sided Student's t-test. Boxplots depict the median and interquartile range, and whiskers show minimum and maximum values. FPKM, fragments per kilobase million; het, heterozygous variation; hom, homozygous variation Schematic based on Agarkova et al. (2005).
- Fig. 2.
Reduced passive force in CMs derived from a HCM-patient with a MYOM2 mutation. (A) Light and electron micrographs of control (upper panel) and patient HCM-02 (lower panel) myocardial samples. In comparison to the control myocardium, the patient sample showed disoriented CMs (disarray) with great variations in cellular diameter. Irregularly formed connections between CMs (asterisks) and widened interstitial spaces (white arrows) were present. Within CMs, myofibrils (Mf) of the patient frequently showed disarray, with sarcomeres running in various directions, whereas the sarcomeres of control tissue were mostly parallel. Mi, mitochondria; Tt, T-tubule. (B) Example of gel electrophoretic analysis of the phosphorylation status of native myocardial tissue from HCM-02 and controls. For detailed analysis see Fig. S2A. (C) Isometric force generation at increasing calcium concentrations (pCa) normalized to maximum force. Lines were fitted according to a modified Hill equation (Kraft et al., 2013). (D) Calcium concentration at 50% of maximum force generation (pCa50) derived from fitted curves in C. (E) Isometric force at maximum calcium activation (pCa 4.63) appeared not to be altered in HCM-02 CMs carrying the mutation. (F) Passive force at increasing sarcomere length showed a significant reduction for HCM-02 CMs carrying a MYOM2 mutation, compared to controls (P<0.01 for all sarcomere lengths). All functional analyses of CMs from HCM-02 and the control in C-F occurred after adjustment of PKA-dependent phosphorylation (n=16 control CMs; n=10 HCM-02 CMs). Data are mean±s.e.m.
- Fig. 3.
Interaction network and expression of CG14964 – a putative ortholog of MYOM2. (A) Interaction network of MYOM2, TTN and MYH7 with Drosophila orthologs CG14964, bt and sls. Physical interactions are based on GeneMania v.3.6.0 (Warde-Farley et al., 2010) and other studies (Blandin et al., 2013; Brulé et al., 2010; Hornemann et al., 2003; Obermann et al., 1998). Drosophila orthologs are based on the DIOPT database (Hu et al., 2011). (B) Expression of CG14964 in adult fly hearts (arrows/arrowheads mark perinuclear space with CG14964 (red) and GAPDH1/2 (green) transcript localization). Gapdh1/2 is used as a reference. (C) Expression of CG14964 and GAPDH1/2 in the heart (encircled), and in body wall muscles (arrows).
- Fig. 4.
Cardiac-specific knockdown of CD14964 leads to dosage-dependent heart defects in the adult fly. (A-D). End-diastolic diameter (EDD) of 3-week-old flies harboring a knockdown of CG14964 by using (A) CG14964-GDi crossed with Hand4.2-Gal4, (B) CG14964-GDi crossed with Mef2-Gal4, (C) CG14964 deficiency or (D) transheterozygous CG14964CRIMIC/deficiency. We observed heart dilation (A-C, mild knockdown) or constriction (D, strong knockdown). All raw data are shown (mean±s.d.), as well as effect size and 95% c.i. below the data. In addition, Phalloidin-stained cardiac myofibrils show altered heart diameters in representative examples (measurements taken at the green lines). Scale bars: 50 μm.
- Fig. 5.
Muscle-specific knockdown of CG14964 causes locomotion defects and reduced lifespan in adult flies. (A) Locomotion test performed using the RING assay on adult flies expressing CG14964i-GD (left) and CG14964i-T3 (right) in muscles (Mef2-Gal4) showed reduced locomotion ability at 3 weeks in Mef2>CG14964i-T3 flies only. Graph shows percentage of fly population in a defined section of the vial after 20 s. (B) A survival assay performed on Mef2>CG14964i-GD and Mef2>CG14964i-T3 with appropriate controls revealed a decreased survival for Mef2>CG14964i-T3, with fewer than half of the flies surviving 28 days versus more than 90% in the other tested lines.
- Fig. 6.
Interaction between CG14964 and Mhc. (A) End-diastolic diameters (EDD) are decreased in Mhc1 heterozygous flies (orange) and become further constricted upon strong CG14964 knockdown (purple). (B) Mild CG14964 knockdown causes enlarged hearts (green), which is restricted by Mhc1 heterozygosity (purple). All raw data are shown (mean±s.d.), as well as effect size and 95% c.i. below the data. For Mhc-CG14964 interaction, significance was tested using an unpaired two-sided Student's t-test (*P<0.05; **P<0.01; ***P<0.001).
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