Fig. 2: Tmem65 KD leads to eccentric hypertrophic cardiomyopathy and slowed cardiac conduction 3 weeks post injection.

a Morphology of Tmem65 KD and controls hearts. Tmem65 KD hearts were qualitatively larger than control hearts, especially the left atrium (black arrow). (scale bar = 5 mm). b Measurement of heart weight-to-tibia length ratio in Tmem65 KD and controls hearts. **P < 0.01, n = 6 per group. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of the means. c H&E staining of cardiac transverse sections. Scale bar = 500 µm. d Phalloidin (F-actin) staining of adult mouse cardiomyocytes isolated from Tmem65 KD or control hearts. Scale bar = 20 µm. e Quantifications of cell dimensions of isolated adult mouse cardiomyocytes. Both lengths (left panel) and width (right panel) significantly increased in Tmem65 KD cardiomyocytes. **p < 0.01, n > 90 cells. Three mice per group. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of the means. f Immunoblots of multiple hypertrophic markers including α-ACTININ, cTnT, FHL1, MYH7, and phosphorylated ERK1/2 with densitometry quantifications. **P < 0.01; *P < 0.05. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of the means. g qPCR showing increased transcript levels of natriuretic peptide A and B (**P < 0.01) in Tmem65 KD hearts. Experiments were performed in more than 3 mice of both sexes. Statistical analyses were performed by one-way ANOVA with Tukey’s post-hoc test. Data were expressed as mean ± standard error of the means. h Representative images of cardiac optical mapping. Transfer of voltage-sensitive dye di-4-ANEPPS was monitored in control and Tmem65 KD hearts at the Sinus rhythm (top images) or paced at 11 Hz (bottom images). i The conduction velocity of the right ventricular free wall of Tmem65 KD hearts (n = 6 hearts, 4 females and 2 males) was significantly lower than that of control hearts at the Sinus rhythm or paced at 11 Hz (*P < 0.05). All cells were isolated from male mouse hearts. For optical mapping, data were expressed as mean ± SEM. Differences between scrambled shRNA and Tmem65 shRNA groups at sinus rhythm and during pacing were assessed with a two-way ANOVA with Sidak’s multiple comparison test. Data were presented as mean ± standard deviation.