High-Frequency Ultrasound Echocardiography to Assess Zebrafish Cardiac Function.

The zebrafish (Danio rerio) has become a very popular model organism in cardiovascular research, including human cardiac diseases, largely due to its embryonic transparency, genetic tractability, and amenity to rapid, high-throughput studies. However, the loss of transparency limits heart function analysis at the adult stage, which complicates modeling of age-related heart conditions. To overcome such limitations, high-frequency ultrasound echocardiography in zebrafish is emerging as a viable option. Here, we present a detailed protocol to assess cardiac function in adult zebrafish by non-invasive echocardiography using high-frequency ultrasound. The method allows visualization and analysis of zebrafish heart dimension and quantification of important functional parameters, including heart rate, stroke volume, cardiac output, and ejection fraction. In this method, the fish are anesthetized and kept underwater and can be recovered after the procedure. Although high-frequency ultrasound is an expensive technology, the same imaging platform can be used for different species (e.g., murine and zebrafish) by adapting different transducers. Zebrafish echocardiography is a robust method for cardiac phenotyping, useful in the validation and characterization of disease models, particularly late-onset diseases; drug screens; and studies of heart injury, recovery, and regenerative capacity.

Harnessing Cardiac Regeneration as a Potential Therapeutic Strategy for AL Cardiac Amyloidosis.

PURPOSE OF REVIEW: Cardiac regeneration has received much attention as a possible means to treat various forms of cardiac injury. This review will explore the field of cardiac regeneration by highlighting the existing animal models, describing the involved molecular pathways, and discussing attempts to harness cardiac regeneration to treat cardiomyopathies. RECENT FINDINGS: Light chain cardiac amyloidosis is a degenerative disease characterized by progressive heart failure due to amyloid fibril deposition and light chain-mediated cardiotoxicity. Recent findings in a zebrafish model of light chain amyloidosis suggest that cardiac regenerative confers a protective effect against this disease. Cardiac regeneration remains an intriguing potential tool for treating cardiovascular disease. Degenerative diseases, such as light chain cardiac amyloidosis, may be particularly suited for therapeutic interventions that target cardiac regeneration. Further studies are needed to translate preclinical findings for cardiac regeneration into effective therapies.

Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

Reversal of pathological cardiac hypertrophy via the MEF2-coregulator interface.

Cardiac hypertrophy, as a response to hemodynamic stress, is associated with cardiac dysfunction and death, but whether hypertrophy itself represents a pathological process remains unclear. Hypertrophy is driven by changes in myocardial gene expression that require the MEF2 family of DNA-binding transcription factors, as well as the nuclear lysine acetyltransferase p300. Here we used genetic and small-molecule probes to determine the effects of preventing MEF2 acetylation on cardiac adaptation to stress. Both nonacetylatable MEF2 mutants and 8MI, a molecule designed to interfere with MEF2-coregulator binding, prevented hypertrophy in cultured cardiac myocytes. 8MI prevented cardiac hypertrophy in 3 distinct stress models, and reversed established hypertrophy in vivo, associated with normalization of myocardial structure and function. The effects of 8MI were reversible, and did not prevent training effects of swimming. Mechanistically, 8MI blocked stress-induced MEF2 acetylation, nuclear export of class II histone deacetylases HDAC4 and -5, and p300 induction, without impeding HDAC4 phosphorylation. Correspondingly, 8MI transformed the transcriptional response to pressure overload, normalizing almost all 232 genes dysregulated by hemodynamic stress. We conclude that MEF2 acetylation is required for development and maintenance of pathological cardiac hypertrophy, and that blocking MEF2 acetylation can permit recovery from hypertrophy without impairing physiologic adaptation.

A cardiac myocyte-restricted Lin28/let-7 regulatory axis promotes hypoxia-mediated apoptosis by inducing the AKT signaling suppressor PIK3IP1.

RATIONALE: The let-7 family of microRNAs (miRs) regulates critical cell functions, including survival signaling, differentiation, metabolic control and glucose utilization. These functions may be important during myocardial ischemia. MiR-let-7 expression is under tight temporal and spatial control through multiple redundant mechanisms that may be stage-, isoform- and tissue-specific. OBJECTIVE: To determine the mechanisms and functional consequences of miR-let-7 regulation by hypoxia in the heart. METHODS AND RESULTS: MiR-let-7a, -7c and -7g were downregulated in the adult mouse heart early after coronary occlusion, and in neonatal rat ventricular myocytes subjected to hypoxia. Let-7 repression did not require glucose depletion, and occurred at a post-transcriptional level. Hypoxia also induced the RNA binding protein Lin28, a negative regulator of let-7. Hypoxia ineither induced Lin28 nor repressed miR-let-7 in cardiac fibroblasts. Both changes were abrogated by treatment with the histone deacetylase inhibitor trichostatin A. Restoration of let-7g to hypoxic myocytes and to ischemia-reperfused mouse hearts in vivo via lentiviral transduction potentiated the hypoxia-induced phosphorylation and activation of Akt, and prevented hypoxia-dependent caspase activation and death. Mechanistically, phosphatidyl inositol 3-kinase interacting protein 1 (Pik3ip1), a negative regulator of PI3K, was identified as a novel target of miR-let-7 by a crosslinking technique showing that miR-let-7g specifically targets Pik3ip1 to the cardiac myocyte Argonaute complex RISC. Finally, in non-failing and failing human myocardium, we found specific inverse relationships between Lin28 and miR-let-7g, and between miR-let-7g and PIK3IP1. CONCLUSION: A conserved hypoxia-responsive Lin28-miR-let-7-Pik3ip1 regulatory axis is specific to cardiac myocytes and promotes apoptosis during myocardial ischemic injury.