Regular swim exercise improves cardiac function in a zebrafish model of TTNtv-related dilated cardiomyopathy

Background Truncating variants in the TTN gene (TTNtv) are the most common genetic cause of dilated cardiomyopathy (DCM). Patient-related clinical risk factors have been associated with earlier DCM onset but factors that protect against DCM have yet to be identified. Regular exercise is recommended for cardiovascular health but its safety and efficacy in titin-deficient hearts is uncertain. Purpose To determine the effects of moderate exercise training on cardiac structure and function in a zebrafish model of TTNtv-related DCM. Methods Adult male wildtype (WT) and heterozygous TTNtv mutant (TTNtv+) zebrafish (aged 17 months, n=8/genotype/treatment group) were exercised for 6 h/day, 5 days/week for 4 weeks using a 5L swim tunnel. Experiments were conducted at 70% of the group mean critical swim speed (Ucrit). Groups of non-exercised fish maintained under standard tank conditions comprised a control group. Cardiac function was assessed at baseline and after 4 weeks of swim training using high frequency echocardiography. Hearts were excised for histological analysis with haematoxylin & eosin and Masson's trichrome (n=4-5 group). Single ventricular cardiomyocytes were isolated using a zebrafish-tailored custom protocol and morphology assessed using confocal microscopy of cytospun cells followed by supervised single-cell segmentation and analysis. Statistical differences were determined using 2-way ANOVA with Fisher's LSD. Results All fish completed the exercise training protocol. Exercised fish had relatively lower heart rates when compared to controls (exercise effect P=0.003) with no significant changes in body weight. Exercised WT and TTNtv+ fish had higher end-diastolic volume (normalised to body surface area) when compared with non-exercised fish (exercise effect P<0.001). Ventricular ejection fraction was significantly lower in non-exercised TTNtv+ fish than in WT fish; following exercise ventricular ejection fraction improved in TTNtv+ fish and was not significantly different to WT (genotype effect P=0.003, exercise effect P<0.001). Improved ventricular contractile function in exercised fish was further demonstrated by higher values for global longitudinal strain (genotype effect P=0.021, exercise effect P<0.001). Hearts from exercised fish showed greater ventricular myocardial cross-sectional area (P=0.005) compared to controls with no change in collagen content. Isolated TTNtv+ ventricular cardiomyocytes demonstrated baseline hypertrophic remodelling with increased perimeter (P<0.001), thickness (P<0.001), and reduced circularity (P<0.001); exercise induced further cardiomyocyte remodelling in both genotypes. Conclusions Moderate exercise training improves ventricular contractile function and induces physiological hypertrophy in adult TTNtv+ zebrafish. These data provide a compelling rationale for investigating the potential benefits of exercise for disease treatment and prevention in human TTNtv patients.