Altered expression of cyclin A 1 in muscle of patients with facioscapulohumeral muscle dystrophy (FSHD-1)

Abstract

Objectives: Cyclin A1 regulates cell cycle activity and proliferation in somatic and germ-line cells. Its expression increases in G1/S phase and reaches a maximum in G2 and M phases. Altered cyclin A1 expression might contribute to clinical symptoms in facioscapulohumeral muscular dystrophy (FSHD).

Methods: Muscle biopsies were taken from the Vastus lateralis muscle for cDNA microarray, RT-PCR, immunohistochemistry and Western blot analyses to assess RNA and protein expression of cyclin A1 in human muscle cell lines and muscle tissue. Muscle fibers diameter was calculated on cryosections to test for hypertrophy.

Results: cDNA microarray data showed specifically elevated cyclin A1 levels in FSHD vs. other muscular disorders such as caveolinopathy, dysferlinopathy, four and a half LIM domains protein 1 deficiency and healthy controls. Data could be confirmed with RT-PCR and Western blot analysis showing up-regulated cyclin A1 levels also at protein level. We found also clear signs of hypertrophy within the Vastus lateralis muscle in FSHD-1 patients.

Conclusions: In most somatic human cell lines, cyclin A1 levels are low. Overexpression of cyclin A1 in FSHD indicates cell cycle dysregulation in FSHD and might contribute to clinical symptoms of this disease.

Figure 1. Cyclin A1 expression levels in FSHD and other myopathies (microarrays).

CCNA1 Human Exon 1.0 ST Array signal levels in FSHD (n = 4), healthy controls (n = 7), CAV3 (n = 4), DYSF (n = 4) and FHL1 (n = 3).

Figure 2. Cyclin A1 expression levels in FSHD (RT-PCR).

(A) CCNA1 ΔCt-18S in myotubes derived from FSHD patients (n = 7) and age-matched healthy controls (n = 7), ΔΔCt = −3.2, n-fold change: 2^{−ΔΔCt =}9.2. Data are given as median, 25^th and 75^th percentiles and range. *) p<0.05; Mann-Whitney U-test; (B) CCNA1 ΔCt-18S in muscle tissue derived from FSHD patients (n = 3) and age-matched healthy controls (n = 3), ΔΔCt = −1.9, n-fold change: 2^{−ΔΔCt =}3.7. Data are given as single values; (C) CCNA1 ΔCt-18S in myoblasts (MB) and myotues (MT) from healthy controls (n = 14), ΔΔCt = 0.3. Data are given as median; (D) CCNA1 ΔCt-18S in MB and MT from FSHD patients (n = 7), ΔΔCt = 2.0. Data are given as median.

Figure 3. Immunostaining of myoblasts (MB), and myoblasts and myotubes (MB-MT).

(40×magnification) from one FSHD patient and one healthy control. Desmin (red), cyclin A1 (green), Hoechst (blue) staining. Cyclin A1 was detected in nuclei of myoblasts and myotubes derived from both FSHD patients and healthy controls.

Figure 4. (A) Cyclin A1 protein expression (representative Western blot) in myotubes from FSHD patients (n = 4) and age-matched healthy controls (n = 4).

(B) Cyclin A1 protein expression (relative changes) in the Vastus lateralis muscle from FSHD patients and healthy controls (data derived from A).

Figure 5. Immunostaining of cryosections of muscle tissue (40×magnification) from one FSHD patient and one age-matched control.

Dystrophin (red), cyclin A1 (green), Hoechst (blue) staining.

Figure 6. (A) Cyclin A1 protein expression (representative Western blot) in muscle tissue from FSHD patients (n = 3) and age-matched healthy controls (n = 3).

(B) Cyclin A1 protein expression (relative changes) in the Vastus lateralis muscle from FSHD patients and healthy controls (data derived from A)

Figure 7. Distribution patterns of myofiber diameters derived from FSHD patients (n = 5) and age-matched healthy controls (n = 5).

Diameters (µm) from 1000 myofibers each were measured in H&E-stained cross sections under a light microscope by using the Image J software. Cumulative data are presented (FSHD: mean 71 µm, SD 23 µm; Control: mean 59 µm, SD 17 µm), p<0.001, Wilcoxon test.

Figure 8. Cross sections of muscle fibers of (A) one FSHD patient and (B) one healthy control (20×magnification, H&E-stained).

Myofiber diameter size difference indicates hypertrophy in the Vastus lateralis muscle of the patient.