Fig. 1: Tmem65 KD leads to dilated cardiomyopathy, fibrosis, and increased mortality.

a Schematic illustration for establishing a Tmem65 KD mouse model. b Kaplan–Meier survival curve showing all Tmem65 KD mice (red line, n = 30 total mice; blue dotted line, n = 16 males; green dotted line, n = 14 female) died within 7 weeks, while scrambled control mice (blue line, n = 29) remained healthy. Greater than 50% of Tmem65 KD mice died 3 weeks post injection. Experiments were performed in mice of both sexes. c M-mode recordings of mouse left ventricles showed depressed cardiac contractility in a Tmem65 KD mouse heart compared to a scrambled control heart. d Pulsed wave Doppler measurements at aortic orifice measuring blood velocity in a Tmem65 KD mouse heart and control mouse heart. The velocity of blood flow was reduced, or flow waveform was irregular or missing (yellow arrows) in Tmem65 KD mouse hearts. Echocardiography was performed in 6 female mice per group. e Morphology of Tmem65 KD and controls hearts. Tmem65 KD hearts were grossly larger compared to control hearts, including both atria and ventricles. Blood clots were always found in the left atrium (black arrow). Scale bar = 5 mm. f H&E staining of atria and ventricles of Tmem65 KD and control hearts. g Higher magnification of H&E-stained hearts showing many eosin-negative lesions, haematoxylin-positive nuclei. h Masson trichrome staining showing collagen deposits (blue color). Scale bar = 50 µm. i Quantification of cardiac fibrosis of cardiac tissues stained with Masson Trichrome dye. A nearly tenfold increase in fibrosis in Tmem65 KD hearts compared to control hearts. **P < 0.01. Histopathology was performed in 6 female mouse hearts. j Immunoblots demonstrating changes of Tmem65 protein levels in Tmem65 KD hearts. β-actin was included as a loading control. k Densitometry quantification of Tmem65 levels in j. Both long (25 kDa) and short (17 kDa) forms of Tmem65 were compared to the control samples. **P < 0.01, n = 4. All statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of the means.