Progressive Dystrophic Pathology in Diaphragm and Impairment of Cardiac Function in FKRP P448L Mutant Mice

Abstract

Mutations in the gene for fukutin-related protein represent a subset of muscular dystrophies known as dystroglycanopathies characterized by loss of functionally-glycosylated-alpha-dystroglycan and a wide range of dystrophic phenotypes. Mice generated by our lab containing the P448L mutation in the fukutin-related protein gene demonstrate the dystrophic phenotype similar to that of LGMD2I. Here we examined the morphology of the heart and diaphragm, focusing on pathology of diaphragm and cardiac function of the mutant mice for up to 12 months. Both diaphragm and heart lack clear expression of functionally-glycosylated-alpha-dystroglycan throughout the observed period. The diaphragm undergoes progressive deterioration in histology with increasing amount of centranucleation and inflammation. Large areas of mononuclear cell infiltration and fibrosis of up to 60% of tissue area were detected as early as 6 months of age. Despite a less severe morphology with only patches of mononuclear cell infiltration and fibrosis of ~5% by 12 months of age in the heart, cardiac function is clearly affected. High frequency ultrasound reveals a smaller heart size up to 10 months of age. There are significant increases in myocardial thickness and decrease in cardiac output through 12 months. Dysfunction in the heart represents a key marker for evaluating experimental therapies aimed at cardiac muscle.

Fig 1. Histology of P448Lneo- and C57 diaphragm at the ages of 6–8 weeks, 6 months, 8 months, 10 months and 12 months.

(A) H&E staining. Of note is the increasing amount of extracellular matrix and infiltration of mononuclear cells at 6 months of age in the P448Lneo- mutant mice. (B) Masson’s trichrome staining. Blue staining indicates increasing amount of collagen connective tissue. Significant collagen connective tissue replacement can be seen at 6 months of age in P448Lneo- mutant diaphragm and increasing through 12 months of age. Little to no blue staining is present in C57 diaphragm up through 12 months of age. Representative of a minimum of 3 mice per age group. All samples shown at 20X magnification. Yellow bar equal to 200μm.

Fig 2. Percentage of central nucleation and fibrosis of diaphragm and heart.

(A) Diaphragm central nucleation. Control samples show a slight increase in percentage of centrally nucleated fibers, up to 5%. There is a large increase in centrally nucleated fibers of P448Lneo- animals at 6 months of age and reaching approximately 60% by 8 months. (B) Diaphragm fibrosis. Control samples maintain less than 10% fibrosis throughout the 12 month period. P448Lneo- samples show a marked increase in fibrosis at 6 months and reaching 60% by 10 months. (C) Heart fibrosis. Control samples maintain approximately 1% fibrosis throughout the 12 month period however P448Lneo- samples show a marked increase at around 6 months of age. Heart fibrosis area does not increase more than 6%. Averages are taken using data collected from 3 separate images from 3 animals for each age point (total of 9 images). Error bars represent mean ± SEM.

Fig 3. Immunohistochemistry with IIH6C4 antibody for the detection of glycosylated α-DG in diaphragm and heart muscle of P448Lneo- mutant and C57/Bl6 normal control.

(A) Diaphragm. Control samples show expression of glycosylated α-DG in muscle fibers throughout a 12 month period. There is an increasing level of background staining, and almost all fibers lackingclear membrane signal for glycosylated α-DG in P448Lneo- diaphragm. (B) Heart. Control samples show expression of glycosylated α-DG in muscle fibers throughout a 12 month period. P448Lneo- cardiac muscle shows a complete lack of expression of glcyosylated α-DG. Representative of a minimum of 3 mice per age group. All samples shown at 20X magnification. Yellow bar equal to 200μm.

Fig 4. Histology of P448Lneo- and C57 heart at 6–8 weeks, 6 months, 8 months, 10 months and 12 months.

(A) H&E staining. Of note is the slight increase in areas containing mononuclear cell infiltration beginning at 10 months of age in P448Lneo- mutant heart. The number and size of areas of mononuclear cell infiltration increases by 12 months of age. However the overall amount of affected area in the heart remains limited. (B) Masson’s trichrome staining. Blue staining indicates increasing amount of collagen connective tissue. Areas of P448Lneo- mutant heart begin to show increase of connective tissue beginning at 6 months of age up to 12 months of age. No clear replacement is seen in hearts of control C57 mice up to 12 months of age. Black arrows denote streaks of fibrosis. Representative of a minimum of 3 mice per age group. Samples in A are shown at 10X magnifcation while samples in B are at 20X magnification. Yellow bar equal to 200μm for both.

Fig 5. Cardiac function markers in C57 and P448Lneo- mutant mice at 6–8 weeks, 6 months, 8 months, 10 months and 12 months.

Values for myocardial thickness, stroke volume, cardiac output, end diastolic volume (EDV), end systolic volume (ESV), endocardial area, epicardial (epi) area, weight and cardiac index (CI) for C57 and P448Lneo- mutant mice were taken at 6–8 weeks, 6 months, 8 months, 10 months (minimum n = 15), and 12 months (minimum n = 6). Statistically significant differences (p<0.05) were observed starting at 8 months for myocardial thickness and 6 months for cardiac output. Stoke volume became statistically significant at 6 months however no significant difference was seen at 12 months. Statistically significant differences (p<0.05) were observed in EDV, ESV as well as endocardial and epicardial area starting at 6 months and remaining until 12 months of age. A larger body weight was observed in C57 mice throughout the 12 month period with a reduced CI starting at 6 months of age and remaining until 12 months. Error bars represent mean ± SEM.