A–C Bright-field images of isolated adult zebrafish hearts, 2 years of age: wild-type fish, remodeled plna R14del mutant heart and non-remodeled plna R14del heart. D–F Hematoxylin and eosin staining of the three conditions to identify nuclei, with zoom-in at indicated regions. G–I Picro-sirius red staining of collagen deposition for the three conditions, collagen fibers are shown as red staining. J–L Oil Red O staining for fat/lipid of the three conditions. All stainings were performed on WT n = 3, plna R14del n = 3, two experimental replicates. Zoom-in of each indicated region is included. Images were taken at a magnification of ×20. Scale bars are 200 µm for whole-heart tile scans and 50 µm for zoom-in regions. A atrium, V ventricle, BA bulbus arteriosus.

In situ hybridization on wild type and plna R14del mutant adult zebrafish hearts, 2 years of age. A, B Expression of tbx18 to indicate the epicardial cells. C, D Expression of myl7 to indicate the myocardial cells. E, F Expression of grn1 to indicate the immune cells. G, H Expression of postnb to indicate fibroblasts. All stainings were performed on WT n = 3, plna R14del n = 3, two experimental replicates. Zoom-in of each region is indicated. Images were taken at a magnification of ×20. Scale bars are 200 µm for whole-heart tile scans and 50 µm for zoom-in regions. A atrium, V ventricle, BA bulbus arteriosus.

A Graphical illustration of the zebrafish echocardiography imaging setup. B B-mode echocardiography imaging plane of the adult zebrafish heart, ventricular walls, and cardiac valves are depicted in yellow. C Heart rate of wild type (WT) and plna R14del mutant zebrafish, 6 and 10 months of age (mean ± SEM, WT n = 10, plna R14del n = 21, two-way ANOVA). D Cardiac output of WT and plna R14del zebrafish, 6 and 10 months of age (mean ± SEM, **p = 0.0013, ***p = 0.004, WT n = 10, plna R14del n = 21, one-way ANOVA). E Variation in outflow peak velocity in WT and plna R14del zebrafish, 6 and 10 months of age (mean ± SEM, **p = 0.01954, WT n = 10, plna R14del n = 21, one-way ANOVA). F Representative examples of color Doppler ventricular outflow measurements in 10-month-old WT and plna R14del zebrafish. Dashed white and red arrows indicate maximum and minimum peak velocities, respectively. All measurements were performed in two experimental replicates. G–L Representative images of in situ hybridization of these hearts with markers to observe immune/inflammation, epicardium, and fibroblasts (using grn1, tbx18, and postnb, respectively). All stainings were performed on WT n = 3, plna R14del n = 3, two experimental replicates. Images were taken at a magnification of ×20. Scale bars are 200 µm for whole-heart tile scans and 50 µm for zoom-in regions. Wild type are highlighted in black and plna R14del in red. A atrium, V ventricle, BA bulbus arteriosus, A–V valve atrial–ventricular valve, Vent. outflow ventricular outflow, variation outflow PV variation outflow peak velocity, bpm beats per minute, ml/min milliliter per minute, mm/s millimeter per second, VOT ventricular outflow tract. Source data are provided as a Source Data file.

A Typical action potentials (APs) of cardiomyocytes isolated from a 10-month-old wild type (WT) (top panel) and plna R14del mutant (bottom panel) zebrafish, paced at 1 Hz. B Average AP parameters at 1 Hz pacing (mean ± SEM, WT n = 12, plna R14del n = 13, unpaired Students t-test). C AP duration at 90% of repolarization (APD₉₀) at 0.2–4 Hz pacing (mean ± SEM, WT n = 10, plna R14del n = 13, unpaired Students t-test). D Two consecutive APs during 4 Hz pacing in a WT (top panel) and plna R14del (bottom panel) cardiomyocyte. The plna R14del cardiomyocyte showed an AP alternans where a long AP is followed by a short AP. E Average APD₉₀ difference between two consecutive APs (alternans) during 4 Hz pacing (mean ± SEM, *p = 0.044, WT n = 10, plna R14del n = 13, Mann–Whitney rank-sum test). Wild type are highlighted in black and plna R14del in red. WT wild type, Hz Hertz, mV millivolt, ms milliseconds, V/s Volts per second, RMP resting membrane potential, APA_max maximal action potential amplitude, APA_plat action potential amplitude at plateau phase, APD₂₀ AP duration at 20% of repolarization, APD₅₀ AP duration at 50% of repolarization, APD₉₀ AP duration at 90% of repolarization. Source data are provided as a Source Data file.

A Schematic representation of Ca²⁺ transient parameters showing Ca²⁺ transient upstroke time, Ca²⁺ transient recovery time, Ca²⁺ transient amplitude, and diastolic Ca²⁺ levels. B Representative image of an isolated ventricular cardiomyocyte and a Ca²⁺ trace from adult ventricular cardiomyocyte of wild type and plna R14del mutants. Intracellular Ca²⁺ parameters were measured in adult ventricular cardiomyocytes of wild type (WT) and plna R14del, including C diastolic Ca²⁺ levels (mean ± SEM, *p = 0.05, WT n = 4, plna R14del n = 5), D systolic Ca²⁺ levels, E Calcium transient duration at 20%, 50, and 80% (mean ± SEM, CaD20: p = 0.026 and CaD50: p = 0.005, WT n = 4, plna R14del n = 5), and F Tau of Ca²⁺ decay (mean ± SEM, *p = 0.041, WT n = 4, plna R14del n = 5). All measurements were performed in three experimental replicates. G DNA construct and sensor dynamics of GCaMP6f (left panel). GCaMP6f was placed under the control of the myl7 promoter to restrict its expression to the heart. The Gal4FF-UAS system amplifies its expression. GCaMP6f consists of a circularly permutated enhanced green fluorescence protein (cpEGFP) fused to calmodulin (CaM) and the M13 peptide. When intracellular calcium (Ca²⁺) rises, CaM binds to M13, causing increased brightness of cpEGFP. Using a high-speed epifluorescence microscope, movies of 3 dpf non-contracting GCaMP6f embryonic hearts were recorded. Several intracellular Ca²⁺ parameters were measured, including H Frequency of Ca²⁺ transients, I Ca²⁺ transient recovery time, J normalized Ca²⁺ transient amplitude (mean ± SEM, *p = 0.0129, WT n = 10, plna R14del n = 12), K normalized diastolic Ca²⁺ levels, and L Tau of Ca²⁺ decay in wild-type (WT) and plna R14del embryonic zebrafish (mean ± SEM, *p = 0.0498, WT n = 10, plna R14del n = 12). All measurements were performed in three experimental replicates. Wild type is highlighted in black and plna R14del in red. Statistical test: unpaired Student’s t-test. nM nanomolar, ms milliseconds, CaM calmodulin, UAS upstream activation sequence, Ca²⁺ calcium. Source data are provided as a Source Data file.

A Overview of the experimental setup. Embryos were embedded in agarose and incubated for 5 min at 28 °C. Baseline measurements were performed, and subsequently, embryos were incubated with 100 µM istaroxime for 30 min, and imaged again. B Representative calcium (Ca²⁺) transients from one wild type (WT) and one plna R14del embryonic zebrafish, before and after incubation with 100 µM istaroxime. C Ca²⁺ transient amplitude (mean ± SEM, *p = 0.0324, WT baseline n = 11, WT istaroxime n = 11, plna R14del baseline n = 12, plna R14del istaroxime n = 13, two-way ANOVA). D Diastolic Ca²⁺ levels and E Tau of Ca²⁺ decay in WT and plna R14del zebrafish, at baseline and after incubation with 100 µM istaroxime (mean ± SEM, *p = 0.0439, **p = 0.0049, WT baseline n = 11, WT istaroxime n = 11, plna R14del baseline n = 12, plna R14del istaroxime n = 13, two-way ANOVA). F–J Bar graphs of contractility parameters at baseline and after incubation with 100 µM istaroxime, including end-systolic volume F (mean ± SEM, **p = 0.005988 for WT and **p = 0.005588 plna R14del, WT baseline n = 12, WT istaroxime n = 12, plna R14del baseline n = 14, plna R14del istaroxime n = 15, two-way ANOVA), end-diastolic volume G, ejection fraction H (mean ± SEM, ***p = 0.0002, **p = 0.0015, WT baseline n = 11, WT istaroxime n = 11, plna R14del baseline n = 12, plna R14del istaroxime n = 13, two-way ANOVA), contractile cycle length contraction time I and contractile cycle length relaxation time J (mean ± SEM, *p = 0.0006, WT baseline n = 11, WT istaroxime n = 11, plna R14del baseline n = 12, plna R14del istaroxime n = 13, two-way ANOVA). All measurements were performed in three experimental replicates. Statistical test two-way ANOVA. Baseline condition is highlighted in black and treated in orange. Arb. units arbitrary units, ms milliseconds, pL picoliter. Source data are provided as a Source Data file.

A Typical action potentials (APs) of plna R14del isolated adult cardiomyocytes at baseline (black line) and after incubation with 5 µM istaroxime (orange line), paced at 1 Hertz (Hz). B Average AP parameters of plna R14del isolated adult cardiomyocytes at baseline (black bars) and after incubation with 5 µM istaroxime (orange bars), at 1 Hz pacing (mean ± SEM, APD₂₀: *p = 0.030, APD₅₀: *p = 0.022, APD₉₀: *p = 0.020, baseline n = 6, istaroxime n = 6, paired Student’s t-test). C Typical action potentials (APs) of plna R14del isolated adult cardiomyocytes at baseline (black line) and after incubation with 5 µM istaroxime (orange line), paced at 4 Hz. The plna R14del cardiomyocyte showed an AP alternans at baseline, where a long AP is followed by a short AP. D Average APD₉₀ difference between two consecutive APs (alternans) at baseline and after incubation with 5 µM istaroxime, during 4 Hz pacing (mean ± SEM, *p = 0.039, baseline n = 6, istaroxime n = 6, paired Student’s t-test). All measurements were performed in three experimental replicates. Baseline condition is highlighted in black and treated in orange. Hz Hertz, mV millivolt, APD₂₀ AP duration at 20% of repolarization, APD₅₀ AP duration at 50% of repolarization, APD₉₀ AP duration at 90% of repolarization. Source data are provided as a Source Data file.