Induction of cardiac dysfunction in developing and adult zebrafish by chronic isoproterenol stimulation.

Zebrafish is a widely used model to evaluate genetic variants and modifiers that can cause heart muscle diseases. Surprisingly, the ß-adrenergic receptor (ß-AR) pathway in zebrafish is not well characterized, although abnormal ß-AR signaling is a major contributor to human heart failure (HF). Chronic ß-AR activation in the attempt to normalize heart function in the failing heart results in a reduction of the ß-ARs expression and receptor desensitization, largely mediated through G-protein coupled receptor kinase 2 (GRK2) upregulation. This in turn leads to further deterioration of heart function and progression towards HF. This study seeks to systematically characterize the function of the ß-AR signaling in developing and adult zebrafish to ultimately assess the ability to induce HF through chronic ß-AR activation by isoproterenol (ISO) as established in the mouse model. Larval hearts first responded to ISO by 3dpf, in concordance with robust expression of key components of the ß-AR signaling pathway. Although ISO-induced ß1-AR and ß2-AR isoform upregulation persisted, chronic ISO stimulation for 5d caused systolic cardiac dysfunction concurrently with maximal expression of G-protein-coupled receptor kinase-2 (GRK2). More consistent to mammalians, adult zebrafish developed significant heart failure in concert with ß1-AR downregulation, and GRK2 and brain natriuretic peptide (BNP) upregulation in response to prolonged, 14d ISO-stimulation. This was accompanied by significant cell death and inflammation without detectable fibrosis. Our study unveils important characteristics of larvae and adult zebrafish hearts pertaining to ß-AR signaling. A lack of ß-AR responsiveness and atypical ß-AR/GRK2 ratios in larval zebrafish should be considered. Adult zebrafish resembled the mammalian situation on the functional and molecular level more closely, but also revealed differences to dysfunctional mammalian hearts, i.e. lack of fibrosis. Our study establishes the first ISO-inducible HF model in adult zebrafish and present critical characteristics of the zebrafish heart essential to be considered when utilizing the zebrafish as a human disease and future drug discovery model.

miR-19b Regulates Ventricular Action Potential Duration in Zebrafish.

Sudden cardiac death due to ventricular arrhythmias often caused by action potential duration (APD) prolongation is a common mode of death in heart failure (HF). microRNAs, noncoding RNAs that fine tune gene expression, are frequently dysregulated during HF, suggesting a potential involvement in the electrical remodeling process accompanying HF progression. Here, we identified miR-19b as an important regulator of heart function. Zebrafish lacking miR-19b developed severe bradycardia and reduced cardiac contractility. miR-19b deficient fish displayed increased sensitivity to AV-block, a characteristic feature of long QT syndrome in zebrafish. Patch clamp experiments from whole hearts showed that miR-19b deficient zebrafish exhibit significantly prolonged ventricular APD caused by impaired repolarization. We found that miR-19b directly and indirectly regulates the expression of crucial modulatory subunits of cardiac ion channels, and thereby modulates AP duration and shape. Interestingly, miR-19b knockdown mediated APD prolongation can rescue a genetically induced short QT phenotype. Thus, miR-19b might represent a crucial modifier of the cardiac electrical activity, and our work establishes miR-19b as a potential candidate for human long QT syndrome.

Essential light chain S195 phosphorylation is required for cardiac adaptation under physical stress.

AIMS: Regulatory proteins of the sarcomere are pivotal for normal heart function and when affected by mutations are frequently causing cardiomyopathy. The exact function of these regulatory proteins and how mutations in these translate into distinct cardiomyopathy phenotypes remains poorly understood. Mutations in the essential myosin light chain (ELC) are linked to human cardiomyopathy characterized by a marked variability in disease phenotypes and high incidences of sudden death. Here we studied the role of the highly conserved S195 phosphorylation site of ELC using heterozygous adult zebrafish lazy susan (laz(m647)) in regulating contractile function in normal physiology and disease. METHODS AND RESULTS: Echocardiography revealed signs of systolic dysfunction in otherwise phenotypically unremarkable heterozygote mutants. However, after physical stress, heart function of laz heterozygous zebrafish severely deteriorated causing heart failure and sudden death. Mechanistically, we show that upon physical stress, ELCs become phosphorylated and lack of S195 dominant-negatively impairs ELC phosphorylation. In vitro motility analysis with native myosin from adult heterozygous hearts demonstrates that S195 loss, specifically following physical stress, results in altered acto-myosin sliding velocities and myosin binding cooperativity, causing reduced force generation and organ dysfunction. CONCLUSION: Using adult heterozygous zebrafish, we show that ELC S195 phosphorylation is pivotal for adaptation of cardiac function to augmented physical stress and we provide novel mechanistic insights into the pathogenesis of ELC-linked cardiomyopathy.

Advanced echocardiography in adult zebrafish reveals delayed recovery of heart function after myocardial cryoinjury.

Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage.