Elevated LMNA mRNA levels in the LMNA transgenic zebrafish. LMNA mRNA expression levels in (A) 7-day-old F1 larvae (N = 20 larvae), each dot represents the mRNA extracted from 20 larva. (B) 9-month-old F1 adult of AB(WT) and various LMNA transgenic fish (N = 3 adult fish), each dot represents the mRNA extracted from one adult fish. Statistical analysis was performed using ordinary One-way ANOVA with Dunnett’s test, and the level of statistical significance is indicated by the following notation: *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001.

Swim speed analysis of F1 adult zebrafish expressing mutant human lamin A/C with those expressing WT human lamin A/C. (A–C) Swim speed analysis of F1 adult fish using T-maze behavior. The red plot represents AB(WT) fish, orange plot represents LMNA(WT) fish, green plot represents LMNA(L35P) fish, brown plot for LMNA(E358K) fish, blue plot for LMNA(R453W) fish, purple plot for LMNA(W520G) fish, and black plot for LMNA(A539V) fish. Each dot represents one fish. AB(WT): n = 6; LMNA(WT): n = 6; LMNA(L35P): n = 6; LMNA(E358K): n = 5; LMNA(R453W): n = 3; LMNA(A539V): n = 4. Statistical analysis was performed using ordinary One-way ANOVA comparing the mean of each column with the mean of control column (LMNA(WT)), and corrected for multiple comparisons using Dunnett’s test. The presented p-values have been appropriately adjusted, and the level of statistical significance is indicated by the following notation: *0.01 < P ≤ 0.05; **0.001 < P ≤ 0.01; ***0.0001 < P ≤ 0.001; ****P ≤ 0.0001.

The muscle endurance and heartbeat measurements of F1 adult LMNA transgenic zebrafish. (A) Muscle endurance was assessed using the critical swim speed (U_crit) measured in a Brett-type swimming tunnel. AB(WT): n = 6; LMNA(WT): n = 4; LMNA(L35P): n = 7; LMNA(E358K): n = 5; LMNA(R453W): n = 3; LMNA(A539V): n = 6. (B) The length of the adult fish were shown. AB(WT): n = 8; LMNA(WT): n = 7; LMNA(L35P): n = 8; LMNA(E358K): n = 7; LMNA(R453W): n = 3; LMNA(A539V): n = 8. (C) body weight of the adult fish were recorded. AB(WT): n = 8; LMNA(WT): n = 7; LMNA(L35P): n = 8; LMNA(E358K): n = 6; LMNA(R453W): n = 3; LMNA(A539V): n = 8. (D) The heartbeat of 4dpf F1 larval fish with different LMNA transgenic variations was measured. AB(WT): n = 8; LMNA(WT): n = 8; LMNA(L35P): n = 8; LMNA(E358K): n = 8; LMNA(R453W): n = 8; LMNA(A539V): n = 8. The red plot represents AB(WT) fish, the orange plot for LMNA(WT) fish, the green plot for LMNA(L35P) fish, the brown plot for LMNA(E358K) fish, the blue plot for LMNA(R453W) fish, and the black plot for LMNA(A539V) fish. Each dot represents one fish. Statistical analysis was conducted using ordinary One-way ANOVA with Dunnett’s test. The presented P-values have been appropriately adjusted, and the level of statistical significance is denoted as follows: *for 0.01 < P ≤ 0.05, **for 0.001 < P ≤ 0.01, ***for 0.0001 < P ≤ 0.001, and ****for P ≤ 0.0001.

Histopathological analysis of muscle specimens from F1 adult LMNA transgenic zebrafish. (A) The histopathological characteristics of muscle tissues in F1 adult LMNA transgenic zebrafish were conducted by histological staining using H&E stain, Gömöri trichrome stain, and NADH-TR stain. The blue arrowheads indicated the decreased fiber size. The images were captured at a magnification of × 400, and the scale bar represents 20 μm. (B) The statistical analysis of muscle fiber size from LMNA transgenic zebrafish. The muscle fiber diameter of F1 adult from AB(WT) and LMNA transgenic zebrafish was measured. Each dot represents one muscle fiber. AB(WT): n = 198; LMNA(WT): n = 141; LMNA(L35P): n = 212; LMNA(E358K): n = 226; LMNA(R453W): n = 92; LMNA(A539V): n = 175. The red plot represents AB(WT) fish, the orange plot for LMNA(WT) fish, the green plot for LMNA(L35P) fish, the brown plot for LMNA(E358K) fish, the blue plot for LMNA(R453W) fish, and the black plot for LMNA(A539V) fish. Statistical analysis was comparing the mean of each column with the mean of no drug control using ordinary One-way ANOVA comparing the mean of each column with the mean of control column (LMNA(WT)), and the multiple testing with Dunnett’s test. The presented P-values have been appropriately adjusted, and the level of statistical significance is denoted as follows: *for 0.01 < P ≤ 0.05, **for 0.001 < P ≤ 0.01, ***for 0.0001 < P ≤ 0.001, and ****for P ≤ 0.0001.

The effects of chemical treatment on F2 heterozygous larvae of LMNA transgenic zebrafish. The larvae fish were subjected to chemical treatment, and their swim speed was assessed using DanioVision from 5 to 8 dpf. (A) l-carnitine 10 μM, (B) Creatine 100 μM, (C) Terazosin 2.5 μM, (D) Taurine 1 mM, (E) Tyrosine 10 μM treatments were administered to AB(WT), LMNA (WT), LMNA(A539V), LMNA(L35P), LMNA(R453W) larvae fish. Statistical analysis was conducted using a One-way ANOVA with Dunnett’s test comparing the swim speed of the chemically treated larvae to those with no drug treatment. Each dot represents one fish. AB(WT), LMNA(WT), LMNA(L35P), LMNA(E358K), LMNA(R453W), LMNA(A539V), n = 8 for each group. The presented P-values have been appropriately adjusted, and the level of statistical significance is expressed as a P-value, where indicated as following: *represents 0.01 < P ≤ 0.05, **represents 0.001 < P ≤ 0.01.

The muscle endurance of 1-month-old F2 heterozygous LMNA transgenic zebrafish after treated with chemicals. (A) Muscle endurance of AB(WT): n = 8; LMNA(L35P): n = 5; LMNA(L35P) + l-carnitine: n = 8. (B) Muscle endurance of AB(WT): n = 8; LMNA(R453W); n = 6; LMNA(R453W) + tyrosine: n = 8; LMNA(R453W) + taurine: n = 6; LMNA(R453W) + creatine: n = 7. (C) Muscle endurance of AB(WT): n = 9; LMNA(A539V): n = 7, LMNA(A539V) + l-carnitine: n = 7. Adult fish were treated with chemical for 1-month. Each dot represents one fish. Statistical analysis was conducted using a One-way ANONVA with Dunnett’s test, comparing the treated groups to either AB(WT) or the no drug treatment group. The presented p-values have been appropriately adjusted, and the level of statistical significance is indicated as following *represents 0.01 < P ≤ 0.05, **represents 0.001 < P ≤ 0.01, and ****represents P ≤ 0.0001. (D) Ingenuity Pathway Analysis (IPA) network pathway analysis revealed dysregulated genes in LMNA(R453W) mutant transgenic fish, resulting in decreased levels of creatine showing in blue. TGFB1 transforming growth factor-beta1, CCR2 C–C chemokine receptor type 2, C3 complement component 3, EDNRB endothelial receptor type B, CASP1 Caspase 1.

The expression patterns of the AMPK and mTOR pathways in chemical treated 7-day-old larvae and 1-month-old F2 heterozygous LMNA transgenic zebrafish. (A,Aʹ) Muscle endurance in the 1-month-old LMNA(L35P) and LMNA(R453W) fish, as well as swimming speed in the 7-day-old larvae with or without chemical treatment. The expression levels of key genes related to the AMPK and mTOR pathways were assessed using qPCR in both 7-day-old larvae and 1-month-old LMNA transgenic zebrafish. LMNA(L35P): n = 5; LMNA(L35P) + l-carnitine: n = 8; LMNA(R453W); n = 6; LMNA(R453W) + creatine: n = 7. (B,Bʹ) Expression of ampk gene. (C,Cʹ) Expression of foxo1a gene. (D,Dʹ) Expression of ppargc1a gene. (E,Eʹ) Expression of mTOR gene. (F,Fʹ) Expression of eif4ebp3l gene. For larvae: n = 20; for adult: n = 10. Statistical analysis was performed using ordinary One-way ANOVA, comparing the expression levels to the no drug treatment group. The presented P-values have been appropriately adjusted, and the level of statistical significance is indicated by the P-value, where denoted as following *represents 0.01 < P ≤ 0.05, **represents 0.001 < P ≤ 0.01, ***represents 0.0001 < P ≤ 0.001, and ****represents P ≤ 0.0001.

Ingenuity Pathway Analysis (IPA) network pathway analysis uncovers dysregulated genes in LMNA mutant transgenic fish. (A) F2 LMNA(L35P) mutants exhibit dysregulation in genes associated with behavior deficits, movement disorders, and cycling of the centrosome. (B) Dysregulation of genes related to movement disorders is observed in F2 LMNA(R453W) mutants. (C) LMNA(A539V) mutants demonstrate dysregulation in genes associated with myocardial dysfunction. These findings obtained through IPA provide insights into the dysregulated pathways and potential molecular mechanisms underlying the observed phenotypic changes in LMNA mutant transgenic fish.