Fig 2: Pathological characterization of skeletal muscle (gastrocnemius) in compound heterozygous (CHet) mice. (A) Acid α-glucosidase (GAA) activity in skeletal muscle homogenates, showing significantly reduced enzymatic activity in CHet mice compared to wild-type (WT) (****p < 0.0001). (B) Quantification of skeletal muscle glycogen content, revealing elevated levels in CHet mice (***p < 0.001). (C) Hematoxylin and eosin (H&E) staining of gastrocnemius sections, demonstrating vacuolar degeneration (black arrows) and disrupted muscle fiber architecture in CHet mice. Scale bar: 50 μm. (D-G) Transmission electron microscopy (TEM) analysis: (D) Representative TEM images of skeletal muscle fibers, showing excessive glycogen granules (black arrows) and abnormal mitochondrial morphology (white arrows) in CHet mice. scale bar: 2 μm (left), 1 μm (right). (E) Quantification of glycogen granule area (%) (*p < 0.05); (F) Mitochondrial density (number/100 µm²) (*p < 0.05); (G) Proportion of mitochondria with structural abnormalities (e.g., cristae disruption, swelling) (*p < 0.0). Data are presented as mean ± SEM (n = 6). Statistical significance was determined by Student’s t-test (*p < 0.05, ****p < 0.0001)

Fig 3: Hepatic pathology and ultrastructural abnormalities in compound heterozygous (CHet) mice. (A) Acid α-glucosidase (GAA) activity in liver homogenates, demonstrating significantly reduced enzymatic activity in CHet mice compared to wild-type (WT) (****p < 0.0001). (B) Hepatic glycogen content quantification, showing marked accumulation in CHet mice (****p < 0.0001). (C) Hematoxylin and eosin (H&E) staining of liver sections, revealing hepatocyte swelling (black arrows) and cytoplasmic vacuolation (white arrows) in CHet mice. Scale bar:100 μm (left), 50 μm (right). (D-G) Transmission electron microscopy (TEM) analysis: (D) Representative TEM images of hepatocytes, showing excessive glycogen granules (black arrows) and mitochondria with disrupted cristae (right arrows) in CHet mice. Scale bar: 2 μm (left), 1 μm (right). (E) Quantification of glycogen granule area (%) (*p < 0.05); (F) Mitochondrial density (number/100 µm²) (*p < 0.05); (G) Proportion of mitochondria exhibiting structural abnormalities (e.g., cristae loss, matrix swelling) (***p < 0.05) (n = 6). Data are presented as mean ± SEM (n = 6. Statistical significance was determined by Student’s t-test (*p < 0.05 ****p < 0.0001)

Fig 4: Generation and phenotypic validation of Gaa compound heterozygous mice (Gaa R668*/K889*) via CRISPR/Cas9-mediated gene editing. (A) Schematic of the knock-in (KI) allele design targeting the R608* mutation. The exon (asterisk) harboring the mutation is shown, with gRNA binding site (red) and genotyping primers (F, R). (B) Sanger sequencing chromatogram of a heterozygous Gaa+/p.R608* F1 mouse, with the mutant allele (red arrow) and wild-type (WT) sequence labeled. (C) KI allele design for the K889* mutation, indicating the exon (asterisk), gRNA target (red), and primers (F, R). (D) Sequencing confirmation of the Gaa+/p.K889* heterozygous mutation (red arrow) in F1 progeny. (E) Breeding scheme to generate compound heterozygous (CHet: R608*/K889*) mice through crossing single heterozygous parents. (F) Sanger sequencing of CHet mice validates both R608* and K889* mutations (highlighted in red). (G) Dry blood spot (DBS) assay reveals significantly reduced acid α-glucosidase (GAA) activity in CHet mice compared to WT (***p < 0.001, Student’s t-test). (H) Plasma glycogen levels are elevated in CHet mice (**p < 0.01), indicating impaired glycogen metabolism. Data are presented as mean ± SEM (n = 6). Statistical significance: **p < 0.01, ***p < 0.001

Fig 5: Characterization of hypertrophic cardiomyopathy in compound heterozygous (CHet) mice. (A) Acid α-glucosidase (GAA activity in cardiac tissues of wild-type (WT) and CHet mice, measured by colorimetric assay (****p < 0.0001). (B) Quantification of glycogen content in cardiac tissues via enzymatic assay, showing elevated levels in CHet mice (***p < 0.001). (C) Periodic acid–Schiff (PAS) staining of cardiac sections, highlighting glycogen accumulation (pink) in CHet myocardium. Scale bar: 50 μm. (D) Quantitative analysis of PAS-positive glycogen area (%) (***p < 0.001). (E-G) Cardiac hypertrophy assessment: (E) Hematoxylin and eosin (H&E) staining of whole-heart longitudinal sections (scale bar: 2 mm); (F) Heart weight-to-body weight (HW/BW) ratio (*p < 0.05); (G) Heart weight-to-tibia length (HW/TL) ratio (*p < 0.05). (H-J) Wheat germ agglutinin (WGA) staining: (H) Representative fluorescence images of cardiomyocyte cross-sectional area (scale bar: 20 μm); (I) Quantification of cardiomyocyte area (**p < 0.01); (J) Cytoplasmic WGA signal intensity (***p < 0.01). (K-M) Histopathological analysis: (K) H&E staining of myocardial sections (scale bar: 100 μm); (L) Vacuolation area (%) (****p < 0.001); (M) Inflammatory cell infiltration area (%) (**p < 0.01). (N-Q) Transmission electron microscopy (TEM) analysis: (N) Representative TEM images of cardiomyocytes (scale bar: 2 μm (left), 1 μm (right)); (O) Glycogen granule area (%) (*p < 0.05); (P) Mitochondrial density (number/100 µm²) (*p < 0.05); (Q) Abnormal mitochondria ratio (%) (*p < 0.05). Data are presented as mean ± SEM (n = 6). Statistical significance determined by Student’s t-test or one-way ANOVA