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. 2015 Dec 1;142(23):4026-37.
doi: 10.1242/dev.126649.

Dynamic microRNA-101a and Fosab Expression Controls Zebrafish Heart Regeneration

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Free PMC article

Dynamic microRNA-101a and Fosab Expression Controls Zebrafish Heart Regeneration

Megan Beauchemin et al. Development. .
Free PMC article

Abstract

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world owing to the limited regenerative capacity of the mammalian cardiovascular system. In lieu of new muscle synthesis, the human heart replaces necrotic tissue with deposition of a noncontractile scar. By contrast, the adult zebrafish is endowed with a remarkable regenerative capacity, capable of de novo cardiomyocyte (CM) creation and scar tissue removal when challenged with an acute injury. In these studies, we examined the contributions of the dynamically regulated microRNA miR-101a during adult zebrafish heart regeneration. We demonstrate that miR-101a expression is rapidly depleted within 3 days post-amputation (dpa) but is highly upregulated by 7-14 dpa, before returning to uninjured levels at the completion of the regenerative process. Employing heat-inducible transgenic strains and antisense oligonucleotides, we demonstrate that decreases in miR-101a levels at the onset of cardiac injury enhanced CM proliferation. Interestingly, prolonged suppression of miR-101a activity stimulates new muscle synthesis but with defects in scar tissue clearance. Upregulation of miR-101a expression between 7 and 14 dpa is essential to stimulate removal of the scar. Through a series of studies, we identified the proto-oncogene fosab (cfos) as a potent miR-101a target gene, stimulator of CM proliferation, and inhibitor of scar tissue removal. Importantly, combinatorial depletion of fosab and miR-101a activity rescued defects in scar tissue clearance mediated by miR-101a inhibition alone. In summation, our studies indicate that the precise temporal modulation of the miR-101a/fosab genetic axis is crucial for coordinating CM proliferation and scar tissue removal during zebrafish heart regeneration.

Keywords: Cardiomyocyte proliferation; Fosab; Heart regeneration; MicroRNA-101a; Scarring; Zebrafish.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
miRNAs are dynamically regulated in response to heart resection injury. Total RNA was extracted from uninjured and 6 h post-amputation (hpa) ventricles. cDNAs were synthesized from small RNAs and hybridized onto microarray chips in triplicate, to detect changes in miRNA expression. (A) Heat-map of the microarray data shows dynamic upregulation and downregulation of miRNAs in response to cardiac injury (green indicates lower expression; red indicates increased expression). (B) Real-time qRT-PCR validation of a subset of the most highly differentially expressed miRNAs from uninjured and 6 hpa wild-type hearts. miRNA levels were normalized to U6 and represented as fold-change relative to expression levels in uninjured hearts. (C) Time course of miR-101a expression levels during heart regeneration. qPCR studies reveal that relative to uninjured hearts (red dashed line) miR-101a expression (blue line) is at its lowest levels at 3 days post-amputation (dpa), increases to 1.5-fold at 14 dpa and is restored to pre-injury levels by 30 dpa. *P<0.05, **P<0.001 (Student's t-test); error bars represent s.e.m.
Fig. 2.
Fig. 2.
Depletion of miR-101a expression promotes cardiomyocyte proliferation in the injured apex. (A-D) Control or Tg(hs:miR-101a-sp) animals were injured and subjected to daily heat treatment. Hearts were collected at 3 dpa for histology, cryosectioned at 10 µm and stained with antibodies to detect Mef2 (green) and Pcna (red) to identify proliferating cardiomyocytes (CMs) (arrowheads) at the injured apex (A,C) and lateral wall (remote zone) (B,D). Insets in A,C are higher magnifications of the areas within dashed boxes. (E) CM proliferation indices were determined by representing Mef+Pcna+ cells as a percentage of total Mef2+ cells. Depletion of miR-101a expression doubled CM proliferation indices at 3 dpa in the injured apex but not in the remote zone. (F) qPCR studies show ∼80% reduction of miR-101a expression levels in Tg(hs:miR-101a-sp) ventricles  (blue line) compared with the control group (red dashed line) under conditions of heat treatment. n=5-7; *P<0.05 (Student's t-test); error bars represent s.e.m. HT, heat treatment.
Fig. 3.
Fig. 3.
Sustained suppression of miR-101a leads to increased scar tissue. (A-F) Wild-type and Tg(hs:miR-101a-sp) hearts were resectioned, heat treated daily and collected at 7, 14 and 30 dpa for histology. Hearts were sectioned at 10 µm and stained with Acid Fucshin Orange G (AFOG) to detect muscle (brown), collagen (blue) and fibrin (red). Arrowheads in F mark newly regenerated muscle; dashed lines indicate approximate resection injury plane. The penetrance of each phenotype is indicated (number showing the illustrated phenotype/total number of samples). (G) Scarring indices were established by quantifying the percentage of collagen and fibrin within the total injury area at the indicated time points using CellProfiler. Prolonged depletion of miR-101a led to greater scar tissue at 14 and 30 dpa compared with control. (H) Injury size was comparable between the groups as shown by quantification of total injury area. n=5-14; *P<0.001 (Student's t-test); error bars represent s.e.m.
Fig. 4.
Fig. 4.
Upregulation of miR-101a between 7 and 14 dpa is essential for scar tissue removal. (A-I) Control and Tg(hs:miR-101a-sp) animals were subjected to ventricular resection and heat treated at defined intervals. Hearts were collected at 30 dpa and processed for AFOG staining to identify scar tissue. (A-C) Heat-treated induced depletion of miR-101a from 0 to 7 dpa did not alter scar tissue removal between control and Tg(hs:miR-101a-sp) hearts. (D-F) Suppression of miR-101a expression between 7 and 30 dpa resulted in increased scarring in Tg(hs:miR-101a-sp) hearts, compared with controls. (G-I) miR-101a suppression from 14 to 30 dpa, however, led to normal scar tissue clearance in control and Tg(hs:miR-101a-sp) hearts. Dashed lines indicate approximate resection injury plane. (J) Quantification of scarring indices demonstrates that increased miR-101a expression at 7-14 dpa is required for scar tissue clearance. ct, control. n=4-8; *P<0.01 (Student's t-test); NS, not significant; error bars represent s.e.m.
Fig. 5.
Fig. 5.
Long-term miR-101a depletion results in muscle regeneration and defects in scar tissue clearance. (A-F) Control, Tg(hs:miR-101a-sp) and Tg(hs:miR-133a1-pre) hearts were resectioned, heat treated daily for 30 dpa and extracted for histology. Hearts were cryosectioned at 10 µm and stained with either AFOG (A-C) or antibodies directed against Tropomyosin (D-F). Compared with heat-treated control animals, Tg(hs:miR-101a-sp) hearts reveal more scar tissue and less new muscle regeneration in the wounded apex. Tg(hs:miR-133a1-pre) hearts, which overexpress miR-133a1, show defects in both new muscle regeneration and scar tissue removal. Brackets represent approximate injury area; arrowheads in F mark gaps in myocardium and the lack of muscle regeneration. (G-I) Quantification of scarring indices, Tropomyosin expression and injury area were performed with CellProfiler. n=6-7; *P<0.05 (Student's t-test); error bars represent s.e.m.
Fig. 6.
Fig. 6.
miR-101a controls fosab activity through interactions at the 3′-UTR. (A) qRT-PCR studies show dynamic fosab expression during a time course of normal heart regeneration. (B) Western blots show increased Fosab protein expression in Tg(hs:miR-101a-sp) ventricles at 3 and 14 dpa relative to controls. Graph shows quantification of western blots using ImageJ software and normalized to β-actin. (C-E′) RNA in situ hybridization studies to detect miR-101a (blue) and fosab (red) in uninjured and 3 dpa regenerating hearts. miR-101a expression is restricted to CMs in uninjured hearts, whereas fosab expression is enriched in CMs at the border zone and non-muscle cells of the wound environment at 3 dpa. (F-H′) Antibody staining to detect Fosab protein expression confirms RNA in situ hybridization expression patterns. E′ and H′ are magnified images of boxed areas in E and H; arrowheads mark miR-101a (E′) and Fosab (H′) expression in CMs; brackets in H′ indicate Fosab in the microenvironment. (I,J) mRNA EGFP-fosab-3′-UTR sensor assays reveal ∼30% decrease in fosab expression when the sensor is co-injected with miR-101a RNA duplex, relative to EGFP-fosab-3′-UTR sensor injection alone. This miR-101a regulation of fosab is abolished in response to a three-nucleotide mutation of the predicted miR-101a binding site (fosabm sensor). n=6-8 embryos per treatment; *P<0.05, **P<0.001 (Student's t-test); NS, not significant; error bars represent s.e.m.
Fig. 7.
Fig. 7.
Knockdown of fosab activity rescues defects mediated by miR-101a depletion. (A,B) Experimental design for fosab vivo MO and combinatorial injections with anti-miR-101a antisense oligonucleotides. (C) Wild-type animals were injured and treated with vehicle, anti-miR-101a oligonucleotides or anti-miR-101a and fosab vivo MO for three days. Hearts were extracted and western blot hybridizations were performed to detect changes to Fosab protein. Fosab levels increase with anti-miR-101a treatment, but expression was restored to near control levels when anti-miR-101a and fosab vivo MO were co-injected. (D) Combinatorial knockdown of fosab and miR-101a activity is sufficient to rescue elevated CM proliferation indices mediated by miR-101a suppression alone. Treatment of wild-type animals with fosab vivo MO alone significantly reduced CM proliferation indices at 3 dpa compared with vehicle treatment. (E) Quantification of scar tissue was assessed at 30 dpa after vehicle, anti-miR-101a, or anti-miR-101a and fosab-vivo-MO treatment. Suppression of fosab expression between 7 and 30 dpa is sufficient to significantly decrease the scarring effect seen with suppression of miR-101a between 7 and 30 dpa. n=6-8; *P<0.05, **P<0.001 (Student's t-test); NS, not significant; error bars represent s.e.m.
Fig. 8.
Fig. 8.
Temporal modulation of miR-101a/fosab genetic circuit controls heart regeneration. In response to injury, miR-101a levels are reduced, enabling fosab expression to increase. This enhanced fosab expression promotes CM proliferation. However, between 7 and 14 dpa, miR-101a levels are significantly elevated, leading to repression of fosab activity. This decline in Fosab protein attenuates scar tissue deposition. miR-101a regulation of fosab during these defined regeneration intervals is crucial for normal heart regeneration.

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