Fig 2: Failing Rtf1 knockout hearts exhibit gene expression changes similar to those observed in dilated cardiomyopathy. (A) Plot of enriched Human Phenotype ontology terms among the genes that were differentially expressed between control and Rtf1CKO LVs after 21 days of tamoxifen feeding. (B) Plot of enriched KEGG gene sets among the genes that were differentially expressed between control and Rtf1CKO LVs after 21 days of tamoxifen feeding. Dot sizes in (A,B) indicate the number of genes associated with a given ontology term. Dot color in (A,B) indicates the significance level (adjusted p-value). (C) Quantitative PCR analysis of selected human dilated cardiomyopathy associated genes on LV samples from control and Rtf1CKO mice fed with tamoxifen chow for three weeks.

Fig 3: Myocardial knockout of Rtf1 causes dilated cardiomyopathy and LV systolic dysfunction. (A) Short-axis M-mode echocardiograms of control and Rtf1CKO ventricles prior to tamoxifen feeding and after 21 days of tamoxifen feeding. (B) Long-axis B-mode echocardiograms of control and Rtf1CKO hearts at the level of the left ventricle after 14 days of tamoxifen feeding. The inner wall of the LV is traced with a yellow dashed line. (C) Graph of ejection fraction values of control and Rtf1CKO hearts from 0 to 21 days of tamoxifen feeding. (D) Graph of fractional shortening values of control and Rtf1CKO hearts from 0 to 21 days of tamoxifen feeding. (E) Graph of LV volume at diastole values of control and Rtf1CKO hearts from 0 to 21 days of tamoxifen feeding. (F) Graph of LV volume at systole values of control and Rtf1CKO hearts from 0 to 21 days of tamoxifen feeding. Error bars in (C–F) represent standard deviations from the mean. *, **, *** denote p-values < 0.05, 0.01, and 0.001, respectively.

Fig 4: Rtf1 knockdown in NRVMs disrupts cell morphology. (A,B) Phase contrast microscopy of NRVMs treated with control and Rtf1 siRNAs. (A) Control siRNA-treated NRVMs have an elongated rod-like shape. (B) Rtf1 siRNA-treated NRVMs display filopodia-like protrusions. (C) Quantitative PCR analysis of Rtf1 expression in siRNA-treated NRVMs. * p-value < 0.05. (D–I) Fluorescent confocal microscopy of control and Rtf1 siRNA-treated NRVMs immunostained for a-actinin (green) and stained with phalloidin (red) and DAPI (blue). Yellow arrows point to representative Z-line sarcomere structures that are present in control siRNA-treated NRVMs (D) but not in Rtf1 siRNA-treated NRVMs (E).

Fig 5: Generation of a cardiomyocyte-specific inducible Rtf1 knockout mouse. (A) Diagram of generation of cardiomyocyte-specific inducible Rtf1 knockout mice. A cardiomyocyte-specific Myh6:MerCreMer insertion allele was bred into an Rtf1 exon 3 floxed background. Homozygous Rtf1 floxed mice with or without Myh6:MerCreMer were fed with tamoxifen chow, inducing recombination and deletion of Rtf1 exon 3 in MerCreMer expressing hearts. (B,C) Hematoxylin and eosin stained paraffin sections of the short axis of control (B) and Rtf1CKO (C) hearts after three weeks of tamoxifen feeding. Left (LV) and right (RV) ventricle chambers are labeled. (D) Western blot of Rtf1 protein and histone H3 protein (loading control) from left ventricle protein lysates isolated from control and Rtf1CKO hearts after three weeks of tamoxifen feeding. (E) Quantification of Rtf1 protein levels based on Western blotting of left ventricle protein lysates. ** p-value < 0.01.