This document summarizes research on the regulation of zebrafish myocardial regeneration through microRNA-133. Zebrafish have the ability to regenerate up to 20% of their heart tissue after injury. Experiments showed that the regenerated cardiomyocytes come from existing cardiomyocytes that dedifferentiate, proliferate, and redifferentiate. MicroRNAs play a key role in regulating gene expression and zebrafish myocardial regeneration. Specifically, experiments identified that microRNA-133 regulates cardiomyocyte proliferation during regeneration by inhibiting it, as levels of miR-133 decrease during regeneration and return to normal levels once regeneration is complete. Understanding which microRNAs regulate regeneration in zebrafish can help researchers develop cellular engineering techniques to potentially induce cardiomyocyte differentiation for
2. ZEBRAFISH
• Zebrafish, along with many fish, planarians, and amphibians,
possess the extraordinary capacity of regeneration.
Dresden Technical University:
Biolab
4. • Zebrafish regenerate cardiomyocytes for up to 20% of ventricular injury
(Poss et al., 2002)
• Seconds after mutation: erythrocytes clotted the wound site.
• 4 dpa: fibrin proteins replaced erythrocytes.
• 9 dpa: fibrin surrounded and penetrated by myofibers, forming a myocardium.
• 30 dpa: fibrin proteins began to disappear.
• 60 dpa: heart completely regenerated.
MYOCARDIA
L
REGENERATI
ON
DISCOVERY
5. SOURCE OF
REGENERATED
CARDIOMYOCYTES
• The regenerated cardiomyocytes come from
dedifferentiating cardiomyocytes who
proliferate.
• Experiments used GFP-staining and
Cre/Lox lineage tracing, where all
cardiomyocytes are stained with GFP, which
binds on a genetical level.
• Regenerating hearts have disorganized
mitochondrion and sarcomeres (muscle
tissue) as opposed to healthy hearts,
allowing cardiomyocytes to reenter cell
cycle and proliferate.
7. MICRORNAS
• MicroRNAs are short strands of
noncoding and endogenous (from
within) that are 22 base pairs long.
• They regulate gene expression
through binding to the 3’-UTR (3’-
untranslated region), which targets
the mRNA and directs it to
degradation.
• Crucial factor in apoptosis, cell
differentiation, cell movement, and
cell growth.
• Source: Amber et al., 2004
8. REGENERATION
SIGNALING PATHWAY
• Regeneration occurs when the
Fibroblast Growth Factor (FGF)
signaling pathway activates the
Mps1 protein kinase in response to
injury (Yin et al., 2008).
• The Mps1 protein kinase is an
enzyme that regulates cell mitosis,
is a part of cellular development,
and is involved in cytokinesis
through phosphorylation (Liu et al.,
2012).
• Picture from Lan et al., 2010.
9. MICRORNA-133 REGULATION
• MicroRNA-133, or miR-133, is the
microRNA that regulates myocardial
proliferation through inhibition (Yin et
al., 2012).
• Yin et al. experimented by using a
microarray to detect gene expression
levels in cardiomyocytes.
• miR-133 levels decreased during
cardiomyocyte regeneration, and
returned to normal after regeneration
was complete.
10. FURTHER EXPERIMENTS
• The knowledge of which
microRNA regulates
myocardial regeneration in
zebrafish is extremely useful,
because it can help
researchers with cellular
engineering. This could
potentially allow us to “force”
cells to differentiate into
cardiomyocytes, and this
could help with treatment for
-Regenerates through formation of blastema and proliferation of cells.
-Regenerates brain, retina, heart, liver, spinal cord, and appendages.
-
-Up until 2002, there were still debates over whether zebrafishes could regenerate their hearts, as opposed to simply creating scar tissue.
-In 2002, Poss et al. published an article which showed that zebrafish could, in fact, regenerate their cardiomyocytes, if there was only a 20% injury.
-After amputating about 20% of heart, they observed the heart at set time periods (days post-amputation).
Jopling et al. stained the existing cardiomyocytes in a zebrafish heart before ventricular resection with GFP (Green Fluorescent Protein) in a type of experiment called Cre/Lox lineage tracing, which traces where a cell comes from. After resection, they found that all the regenerated cardiomyocytes also glowed green, meaning that they came from the original differentiated cardiomyocytes. This is because GFP binds on a genetical level, and is thus passed down through proliferation of pre-existing cardiomyocytes.
The original cardiomyocytes reenter the cell cycle through disruption of z-lines, which are units of sarcomeres and mitochondrion arranged in an organized manner. The disruption of the z-lines creates intercellular spaces which allows for reentry into cell cycle. Moreover, this was also supported through the constant activity that occurred with GFP-positive cardiomyocytes.
