Cardiac-specific deletion of the microtubule-binding protein CENP-F causes dilated cardiomyopathy

Abstract

CENP-F is a large multifunctional protein with demonstrated regulatory roles in cell proliferation, vesicular transport and cell shape through its association with the microtubule (MT) network. Until now, analysis of CENP-F has been limited to in vitro analysis. Here, using a Cre-loxP system, we report the in vivo disruption of CENP-F gene function in murine cardiomyocytes, a cell type displaying high levels of CENP-F expression. Loss of CENP-F function in developing myocytes leads to decreased cell division, blunting of trabeculation and an initially smaller, thin-walled heart. Still, embryos are born at predicted mendelian ratios on an outbred background. After birth, hearts lacking CENP-F display disruption of their intercalated discs and loss of MT integrity particularly at the costamere; these two structures are essential for cell coupling/electrical conduction and force transduction in the heart. Inhibition of myocyte proliferation and cell coupling as well as loss of MT maintenance is consistent with previous reports of generalized CENP-F function in isolated cells. One hundred percent of these animals develop progressive dilated cardiomyopathy with heart block and scarring, and there is a 20% mortality rate. Importantly, although it has long been postulated that the MT cytoskeleton plays a role in the development of heart disease, this study is the first to reveal a direct genetic link between disruption of this network and cardiomyopathy. Finally, this study has broad implications for development and disease because CENP-F loss of function affects a diverse array of cell-type-specific activities in other organs.

Fig. 1.

Cardiomyocyte-specific disruption of the CENP-F gene. (A) In situ hybridization of an E11.5 mouse embryo with an antisense CENP-F probe demonstrates the distribution of CENP-F transcripts. CENP-F is ubiquitous in the developing embryo but the highest levels of expression are observed in the heart (arrow) and regions of the brain. (B) A schematic presentation of the targeted CENP-F gene with the location of loxP sites marked by yellow arrowheads. Gray boxes indicate CENP-F exons. The first five exons of CENP-F are removed when crossed with the cTNT-Cre mouse line expressing Cre recombinase in cardiomyocytes beginning at E7.5. (C,D) Genomic PCR confirms Cre-mediated excision of the floxed CENP-F gene segment. Lanes are as labeled. In panel C, primers flank the 5′ loxP site and show different sized transcripts for WT and loxP-containing genes. In panel D, primers flank both loxP sites and amplify a product only when recombination has occurred (only in the heart). (E–H) CENP-F protein expression in WT (E,F) and CENP-F^−/− (G,H) heart. Anti-CENP-F antibody staining is absent in CENP-F^−/− cardiomyocytes, but retained in the overlying epicardium, where cTNT does not recombine (G,H; arrow).

Fig. 2.

Embryonic and neonatal CENP-F^−/− hearts display morphological differences in size and chamber wall thickness. (A) Hearts from E12.5 WT and KO littermates. CENP-F^−/− hearts have a distinctly angular appearance of the ventricle when compared with WT hearts. (B) Hearts from P4 mice. CENP-F^−/− hearts are 30–40% smaller by weight than WT (n=12) yet no difference in animal size is observed. (C) Cross-sections through the widest point in the left ventricle show that the WT ventricle has thicker walls and is more trabeculated than the CENP-F^−/− ventricle. The right ventricles of CENP-F^−/− hearts were similarly affected (not shown). Arrows demonstrate regions where variation in wall thickness is readily observed. (D) Non-trabeculated regions of WT and CENP-F^−/− ventricular wall were measured in μm (n=38). Significant differences in thickness were observed (CENP-F^−/−: 28.8±4.8 μm; WT: 18.4±5.2 μm (mean ± s.d.; P<0.05).

Fig. 3.

BrdU and phosphohistone-H3 labeling of neonatal hearts demonstrates decreased proliferation in CENP-F^−/− hearts. (A–F) Cross-sections through WT (A–C) and CENP-F^−/− (D–F) hearts showing anti-BrdU (green) with nuclear staining (DAPI, blue) after 90 minutes of in vivo BrdU labeling. Panels A and D show an overview of sections at neonatal day (N)4 at 10× magnification; others are days 2 and 4 as labeled at 20× magnification. Significantly fewer BrdU-positive cells are seen in the CENP-F^−/− hearts. (G,H) Graphs showing the percentage of positive BrdU (G) and phosphohistone-H3 (H) labeling from P0-P7 in WT and CENP-F^−/− hearts. All counts were performed on 40× sections; 6–16 fields are included for each data point. WT numbers are shown in blue/circles and CENP-F^−/− in pink/squares. There is significantly less BrdU and phosphohistone-H3 labeling in the CENP-F^−/− hearts until day 5, when WT and CENP-F^−/− labeling equalizes for the remaining days assayed. *P≤0.001.

Fig. 4.

CENP-F^−/− hearts contain fewer intercalated discs. (A,B) Cross-section through WT (A) and CENP-F^−/− (B) hearts at 12 months. Connexin-43 staining (green) shows a significant decrease in disc number in CENP-F^−/− hearts as compared with WT. (C) Quantification of disc numbers shown as a fold increase. *P≤0.005.

Fig. 5.

MT network and costamere structure is abnormal in CENP-F^−/− hearts. (A–D) Low-power views of MTs (green) in cross-section from 12-month-old WT (A,B) and CENP-F^−/− (C,D) hearts. (B,D) Merged images of MTs (green), actin (red) and TOPRO (blue). (E–G) Serial confocal sections (0.38 μm) of a WT MT network (green) at high power. (H) Composite reconstruction of three successive confocal slices showing MTs (green), actin (red) and TOPRO (blue) in a WT heart. (I–K) Serial confocal sections (0.38 μm) of a CENP-F^−/− MT network (green) at high power. (L) Composite reconstruction of three successive confocal slices showing MTs (green), actin (red) and TOPRO (blue) in a CENP-F^−/− heart. Scale bars: 10 μm.

Fig. 6.

Mice with CENP-F^−/− hearts develop dilated cardiomyopathy and dysrhythmias. (A,B) M-mode ECG images from unanesthetized WT and CENP-F^−/− mice, showing measurement of left ventricular internal dimension in diastole (LVIDd; red) and in systole (LVIDs; yellow). (C) Graph of LVIDd from WT (blue/circles) and CENP-F^−/− (pink/squares) mice at ages as shown. CENP-F^−/−mice show mild but progressive dilation over time, compared with WT. (D) Graph of cardiac function calculated as shortening fraction, or (LVIDd-LVIDs)/LVIDd, in WT (blue/circles) and CENP-F^−/− (pink/squares) mice. CENP-F^−/−mice show progressive functional impairment over time, compared with WT. (E,F) Examples of rhythm disturbances observed in CENP-F^−/− mice. In E, there is a P wave (‘j’) that does not conduct, whereas the others do (‘N’), representing an episode of second degree heart block. In F, there is slowing of the sinus rate with preserved AV conduction, representing sinus node dysfunction.

Fig. 7.

Adult CENP-F^−/− hearts are fibrotic and enlarged. (A–F) Masson’s trichrome stain of 12-month-old WT (A,C,E) and CENP-F^−/− (B,D,F) hearts in cross-section. Low-power magnification shows coronary enlargement in CENP-F^−/− mice (B) as compared with WT (A). High-power magnification reveals significant fibrosis (blue) in CENP-F^−/− hearts (D,F) as compared with WT (C,E). (G,H) Two whole-mount views of WT and CENP-F^−/− hearts. CENP-F^−/− hearts have nodular surfaces (G; right) and are significantly enlarged compared with WT. Scale bars: 200 μm.

Fig. 8.

Principal component analysis of signal values for 528 probes identified as significantly differential between 12-month-old CENP-F^−/− and WT mouse hearts. (A) The colors represent the different sample types (red: CENP-F^−/− samples; blue: WT samples), and lines mark the centroids for each group. The x-axis, y-axis, and z-axis components represent 79.7%, 6.8% and 5.2%, respectively, of the total variability between experimental replicates. Ideally, experimental replicates cluster together and are apart from other samples, which was indeed the case. (B) Hierarchical clustering of 528 probes detected as significantly differential (at least 1.5-fold; P<0.05) between CENP-F^−/− and WT mouse hearts. Values shown are log base 2, and bright red, bright blue and gray indicate the highest, lowest and median normalized signal values, respectively. Vertical dendrograms represent the individual samples, of which there are three replicates for each sample type. CENP-F^−/− samples are lanes 1–3 from the left and WT samples are lanes 4–6. 192 probes were significantly upregulated and 336 were significantly downregulated as compared with WT. (C) Heat map generated by hierarchical clustering of standardized fold-changes of 125 genes found as having differential expression in CENP-F^−/− mice and also in dilated cardiomyopathy (DCM; in mouse or dog models of DCM and/or in human DCM). Color indicates relative levels of expression replicates, with bright red indicating upregulation and bright blue representing downregulation) in CENP-F^−/− mice and DCM, relative to respective controls. Each row represents one gene, found as up- or downregulated in this study and also in DCM. Columns represent the three mouse CENP-F^−/− replicates (compared with WT animal controls) and DCM. All genes shown were found to have statistically significant (P<0.05; fold-change>1.5) expression in our study and also in public repository data that were analyzed similarly to our data where possible (see supplementary material Table S2 for a list of genes).