Zebrafish dspb-/- mutant as a model for non-dilated left ventricular cardiomyopathy: exploring cardiac dysfunction and exercise modulation

Figures

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  Fig. 1. Age-dependent penetrance and event free survival in DSP truncating variant carriers according to physical activity. Kaplan–Meier curves show earlier age at diagnosis in individuals with moderate/intense physical activity compared with sedentary/light individuals a, while no differences were observed in event-free survival b Adjusted hazard ratios were obtained using multivariable Cox regression. Numbers at risk are shown below

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  Fig. 2. Impaired survival and development in dspb−/− larvae. a Survival analysis of dspb-/- vs. wt group (HR 4.1 [95% CI: 2.4-6.9]; **** p < 0.0001). Test: Simple Survival Analysis (Kaplan-Meier) with Log-rank (Mantel-Cox test) and Hazard Ratio (Mantel-Haenszel). Comparison of larvae at 3 dpf. b Body measurements showed significant differences in body lengths between wt and dspb-/- group (* p < 0.05), with dspb-/- larvae being generally shorter than the control group. However, eye size and cardiac area data, normalized with each embryo’s body length, showed no statistically significant differences. While wt group showed 100% normal embryos (c, d), dspb-/- group consisted of 80% normal embryos (c, e), 10% embryos showing an abnormal yolk sac (c, f), 4% unhatched eggs (c, g), and 6% dead larvae (*** p < 0.0002, ns – not significant) Sample size: n=100. Test: Student’s t-test

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  Fig. 3. Gene expression analysis in wt and dspb−/−. a larvae and b adults at different stages, to assess the expression levels of key genes in fundamental signaling pathways. To determine the enrichment of each gene in each age group, constitutive gene rps11 was used as control to calculate the fold change (**p < 0.01, ***p < 0.001, ****p < 0.0001) Error bars: SEM. Number of experiments: 2 replicates for larvae stages; 3 replicates for 1 year-old; 2 replicates for 2.5-year-old. Test: Two-way ANOVA. Sample size: Pool of 20 larvae for 6 dpf, 10 larvae for 20 dpf, pool of 3 hearts for each adult group

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  Fig. 4. Cardiac function in larvae and adults. a Heart rate in dsp mutant larvae between 3–6 dpf, measured by daily counting heart rates of larvae and comparing dspb−/− mean to the wt mean (****p < 0.0001) (bpm: Beats per minute) Sample size: 3 dpf n = 100; 4 dpf n = 70; 5 dpf n = 50; 6 dpf = 50 Error bars: SEM. Test: Student’s t-test. b-k Cardiac function in wt and dspb−/− adults, pre-training, measured by echocardiography. Significant differences were seen between the two groups in (b) higher heart rate and respiratory frequency for dspb−/− mutants, c smaller systolic and diastolic areas for dspb−/−,d smaller systolic and diastolic volumes for dspb−/−, e smaller systolic and diastolic ventricular entry for dspb−/−, f reduced stroke volume, g reduced cardiac output, h reduced ejection fraction for dspb−/−, i reduced fractional area change for dspb−/− and k lower values for atrial contraction in dspb−/− (*p < 0.05, **p < 0.01, ****p < 0.0001) Sample size: n = 11. Error bars: SEM. Test: Unpaired Multiple T-test with Bonferroni correction

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  Fig. 5. Macroscopical and histological study of zebrafish heart at different ages—Comparison between wt and dspb−/−. a Images of hearts after explantation were acquired with a Zeiss Stemi 305 Stereo Microscope and hearts were measured in length, width, perimeter and area. b Measurements show wt heart becoming smaller throughout time, while dspb−/− heart being significantly smaller than wt heart at young age, with a tendency to a size increase at old age (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Sample size: n = 5/genotype and age group. Error bars: SEM. Test: Two-way ANOVA with Bonferroni correction. BMI: Body mass index. c Semi-thin sections stained with toluidine blue of adult zebrafish hearts from wt and dspb−/− mutants, shown at low (vertical panels) and high magnification (horizontal panels). Mutant hearts show increased cellular density with numerous small, darkly stained nuclei and disorganized myocardial structure compared to wt controls

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  Fig. 6. Structural differences in gap junctions and sarcomeres in cardiac tissue between wt and dspb−/− mutants, observed by TEM. a, b Representative TEM images of gap junctions in wt (a) and dspb−/−b cardiac tissue. Insets highlight the ultrastructural differences. c Quantification of junction length (left) and intermembrane gap distance (right) in wt and dspb−/− samples. While the total junction length remains comparable between genotypes, the intermembrane distance is significantly increased in dspb−/− hearts, indicating defective cell–cell adhesion. Sample size: n = 47 for dspb−/− and n = 29 for wt; Error bars: SEM. (****p < 0.0001, ns – not significant). d, e TEM images of wt cardiac tissue, showing well-aligned sarcomeres, clearly defined Z lines and homogeneously distributed mitochondria. The presence of intact I bands and regular intercalated discs reflects a preserved cardiac ultrastructure. f, g TEM images of dspb−/− mutant cardiac tissue, highlighting sarcomere disorganization, loss of I band definition, and disrupted Z alignment, with thicker Z lines. Mitochondrial clustering and irregular intercalated discs are also evident, suggesting severe structural remodeling and…

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  Fig. 7. The impact of physical activity. a-h Results before and after long-time moderate training in dspb−/− mutants, with significant differences, as mutants after one month show: a lower heart rate, normalization in b diastolic area, c systolic/diastolic volumes, d stroke volume, e cardiac output, f ejection fraction, g. fractional area change, but also in (h) aortic outflow (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Error bars: SEM. Sample size wt and dspb−/− pre: n = 11; Sample size wt and dspb−/− post: n = 10. Test: Two-way ANOVA with Bonferroni correction. i-l Endurance assays pre and post moderate training. Results show that dspb−/− mutants were less able to cope with high intensity training compared to the wt group, but gradually increased endurance after a previous period of moderate training, as demonstrated by some parameters measured by EthoVision XT software (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Error bars: SEM. Sample size: n = 4. Test: Two-way ANOVA with Bonferroni correction. (l-top) Heat maps generated by EthoVision XT software that analyse movement during the endurance assays, showing how dspb−/− mutants that have been previously trained…

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  Fig. 8. Swimming protocols explained. a Moderate one-month training schedule, with echocardiography pre and post training protocol. b High-intensity endurance training protocol for previously trained and untrained fish. c Moderate one-month training schedule from day 2 to day 30, showing daily variations in flow intensity as part of the exercise regimen. d The fish were divided into two groups and placed in the swim tunnels for one month. All the fish underwent the same training simultaneously with groups of 5 individuals per tunnel. Figure created in BioRender