Recessive TMOD1 mutation causes childhood cardiomyopathy

Figures

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  Fig. 1. Cardiac magnetic resonance (CMR) imaging. The linked videos, as indicated in each separate panel, are available as Supplementary Videos 1–6. a, b Patient 1 findings at the time of the diagnosis showed dilated cardiomyopathy (left ventricle end-diastolic volume 105 mL/m2, Z+2.4, end-systolic volume 65 mL/m2, Z+4.5) and poor left ventricular systolic function (ejection fraction EF 38%). The right ventricular size and the systolic function were normal (right ventricular end-diastolic volume 87 mL/m2, Z-0.5 end-systolic volume 47 mL/m2, Z+1, EF 46%) in four-chamber and short-axis cine. Both right and left atria were mildly dilated, as a marker of diastolic dysfunction. c Gadolinium-enhanced T1 fat-saturated images showed mild late enhancement in the mid-myocardium of the intraventricular septum (arrows). d, e Patient 2 findings at the time when she was listed for heart transplantation showed pericardial effusion (stars), small left and right ventricles, and bi-atrial dilatation (left ventricular end-diastolic volume 43 mL/m2, Z–3 and left ventricular end-systolic volume 22 mL/m2, Z–1.2). The left ventricular systolic function was moderately decreased (ejection fraction EF 37%).…

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  Fig. 2. TMOD1 is identified as a disease gene for cardiomyopathy. a Pedigrees of Family 1 and 2. b Multiple sequence alignment of TMOD1 homologs from different species. c Diagram of the protein with domains. d Modeling of patient variant R189W into the crystal structure of human actin-binding site 2 of TMOD1 (blue) in complex with an actin monomer (white) (Protein Data Bank ID: 4PKI). e Western blotting of TMOD1 and TMOD4 using protein lysates from skeletal muscle biopsies of Patient 1 and three controls. Quantification of TMOD1 and TMOD4 blots relative to vinculin. Data shown are mean ± standard deviation (n = 1 or 3 biologically independent samples). Patient 1 and control samples are shown in black and white bars, respectively.

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  Fig. 3. TMOD1 localizes correctly in patient cardiomyocytes. a Sarcomeric structure in Patient 1 and three control iPSC-cardiomyocytes (Scale bars = 50 µm and 10 µm). cTnT: cardiac troponin T (green) and alpha-actinin (red): component of the Z-disk. b TMOD1 (green) localization in the patient and control iPSC-cardiomyocytes (Scale bars = 25 µm and 5 µm). Phalloidin (red) staining was used for visualization of filamentous actin. The regions in white boxes in left panels of (a) and (b) are shown at higher magnification in the right panels of (a) and (b). Note that the sporadic appearance of TMOD1 in both control and patient samples as a broader band (doublet) is linked to the precise relaxation of the sarcomere and/or the angle of the myofibril that is being photographed.

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  Fig. 4. The p.R189W variant weakens TMOD1’s ability to cap the pointed ends of actin filaments. a Representative curves of pyrene-actin fluorescence (A.U.: arbitrary units) from pointed ends of gelsolin-capped actin filaments (1:10 gelsolin-to-actin molar ratio) over time (min) in the presence of 0.8 μM TMOD1wt or TMOD1R189W (control: spontaneous actin pointed end polymerization) and in the absence of TPM1 (−TPM1). b Concentration-dependent inhibition of actin polymerization in the presence of TMOD1wt or TMOD1R189W (p-value = 0.0299, 0.0480, 0.0146, 0.8498, respectively). c Representative curves of pyrene-actin fluorescence (A.U.) from pointed ends of gelsolin-capped actin filaments (1:100 gelsolin-to-actin molar ratio) over time (min) in the presence of TPM1 (+TPM1), with 25 nM TMOD1wt or TMOD1R189W. d Concentration-dependent inhibition of actin polymerization in the presence of TPM1 and TMOD1wt or TMOD1R189W (p-value = 0.0153, 0.0474, 0.0485, 0.0161, 0.9125, respectively). e Representative curves of decrease in pyrene-actin fluorescence (A.U.) resulting from depolymerization from the pointed ends of gelsolin-capped actin filaments (1:10 gelsolin-to-actin molar ratio) over time (min) in…