Fluorescent-based methods for gene knockdown and functional cardiac imaging in zebrafish.

Area of Science:

• Zebrafish as a model organism
• Molecular biology and genetics
• Cardiovascular research

Background:

• Zebrafish morpholino antisense oligonucleotide (MO) injection is a common gene knockdown method.
• Inconsistent phenotypes in MO-injected zebrafish morphants complicate research.
• Accurate quantification of MO dosage per embryo is crucial for reproducible results.

Purpose of the Study:

• To develop a method for estimating the quantity of MO injected into individual zebrafish embryos.
• To apply this method to study the role of cardiac troponin T (tnnt2a) in zebrafish cardiac function.
• To enable precise selection of partially depleted tnnt2a morphants for detailed cardiac analysis.

Main Methods:

• Co-injection of a lissamine-labeled control MO with the target MO to estimate injected quantity.
• Knockdown of cardiac troponin T (tnnt2a) in zebrafish embryos using the developed method.
• Fluorescent cardiac imaging with Bodipy-ceramide for detailed cardiac function analysis.

Main Results:

• The developed method allowed efficient selection of zebrafish with partial tnnt2a depletion.
• tnnt2a-depleted zebrafish exhibited decreased heart rate and impaired cardiac contraction.
• Cardiac image analysis revealed impaired diastolic distensibility and reduced contraction/relaxation velocities.

Conclusions:

• This study presents a novel method for quantifying MO injection in living zebrafish morphants.
• Cardiac troponin T (tnnt2a) plays a significant role in zebrafish cardiac function, affecting contractility and relaxation.
• The combinatorial approach is applicable for studying the function of various proteins in cardiac disease models.