Human cardiomyopathy mutations induce myocyte hyperplasia and activate hypertrophic pathways during cardiogenesis in zebrafish

Abstract

To assess the effects during cardiac development of mutations that cause human cardiomyopathy, we modeled a sarcomeric gene mutation in the embryonic zebrafish. We designed morpholino antisense oligonucleotides targeting the exon 13 splice donor site in the zebrafish cardiac troponin T (tnnt2) gene, in order to precisely recapitulate a human TNNT2 mutation that causes hypertrophic cardiomyopathy (HCM). HCM is a disease characterized by myocardial hypertrophy, myocyte and myofibrillar disarray, as well as an increased risk of sudden death. Similar to humans with HCM, the morphant zebrafish embryos displayed sarcomere disarray and there was a robust induction of myocardial hypertrophic pathways. Microarray analysis uncovered a number of shared transcriptional responses between this zebrafish model and a well-characterized mouse model of HCM. However, in contrast to adult hearts, these embryonic hearts developed cardiomyocyte hyperplasia in response to this genetic perturbation. The re-creation of a human disease-causing TNNT2 splice variant demonstrates that sarcomeric mutations can alter cardiomyocyte biology at the earliest stages of heart development with distinct effects from those observed in adult hearts despite shared transcriptional responses.

Fig. 1.

A human TNNT2 splice mutation is copied in zebrafish by using a splice targeting morpholino. (A) A human TNNT2 intron 15 mutation that causes two different mutant splice products of TNNT2. (B) Morpholino targeting of zebrafish tnnt2 exon 13 causes mis-splicing of tnnt2 mRNA, resulting in a smaller gene product (smaller band on agarose gel). (C) Comparison of human and zebrafish TNNT2 wild-type and mutant protein sequences (box around exon 15 human/exon 13 zebrafish).

Fig. 2.

TNNT2sp morphants have reduced ventricular dimensions and impaired ventricular function. (A) Comparison of control-injected (top) and TNNT2sp morphant (bottom) embryos at 96 hpf. (B) Representative high-magnification image of control (left) and TNNT2sp morphant (right) heart (A, atria; V, ventricle; atria not visible in control). (C) Chamber dimensions in control (black bar) and TNNT2sp morphants (gray bar). (D) FS in control (black bar) and TNNT2sp morphants (gray bar). *P<0.05; n=5 for all measurements. All data expressed as mean + s.e.m.

Fig. 3.

Disruption of sarcomere structure and induction of myocardial hyperplasia in TNNT2sp morphants. (A) Representative electron micrographs of 96 hpf (top), 8 dpf (middle) and 21 dpf (bottom) ventricular cardiomyocytes. (B) Total cardiomyocytes at 48 hpf and 96 hpf. (C) Cardiomyocyte area at 96 hpf. *P<0.05; n=3–5 hearts per time point. All data expressed as mean + s.e.m.

Fig. 4.

Distinct alterations in Ca²⁺ handling induced by altered TNNT2 splicing. Diastolic (A) and transient amplitude (B) Ca²⁺ measurements in specific heart regions in controls, TNNT2sp morphants and TNNTatg (null) morphants. (C) CTD50 measured in atrium, atrioventricular canal (AVC), outer ventricular curvature (OC), mid-ventricle (MV) and inner ventricular curvature (IC). Asterisk donates P<0.05 for comparison of TNNT2sp morphant with control sample, or TNNT2atg morphant with control. All data expressed as mean ± s.e.m.

Fig. 5.

Altered tnnt2 splicing induces significant changes in the expression of sarcomeric genes, neurohormonal genes and markers of hypertrophy. (A) Expression profiling identifies upregulation of myh6 (black bar) and confirms efficacy of TNNT2sp MO [white bar labeled ‘tnnt2 (Exon 13)’. (B) Genes involved in cardiomyocyte Ca²⁺ handling that are differentially regulated in TNNT2sp morphants. (C) Pathway analysis identifies significant downregulation of expression of genes encoding specific hormones – ghrh, pth1a, pomca and uts2a. (D) qPCR confirmation of select up- and downregulated genes. All data expressed as mean + s.e.m.

Fig. 6.

Conservation of gene expression between the zebrafish and mouse models of sarcomeric protein mutations. The zebrafish genes that were concordantly upregulated (blue) or downregulated (brown) compared with a mouse model of HCM are displayed in a heatmap. Top – most upregulated gene; bottom – most downregulated gene. Each column corresponds to a unique biological replicate.