Modulation of TNFα Activity by the microRNA Let-7 Coordinates Zebrafish Heart Regeneration

Abstract

The adult zebrafish is capable of regenerating heart muscle, resolving collagen tissue, and fully restoring heart function throughout its life. In this study, we show that the highly upregulated, epicardium-enriched microRNA let-7i functions in wound closure and cardiomyocyte proliferation. RNA sequencing experiments identified upregulated expression of members of the tumor necrosis factor (TNF) signaling pathway in the absence of let-7. Importantly, co-suppression of TNF and let-7 activity rescued epicardium migration and cardiomyocyte proliferation defects induced by depletion of let-7 alone. Sensitizing animals to low levels of TNF activity before injury culminated in repressed cardiomyocyte proliferation and wound closure defects, suggesting that levels of inflammation at the onset of injury are critical for heart regeneration. Our studies indicate that injury-induced reduction in TNF signaling by let-7 in the epicardium creates a pro-regenerative environment for cardiomyocyte proliferation during adult heart regeneration.

Keywords: Cell Biology; Developmental Biology; Genetics; Molecular Biology; Physiology.

Graphical abstract

Figure 1

Members of the Let-7 microRNA Family Are Upregulated in Response to Heart Resection (A) Real-time qPCR analysis reveal let-7i as the most highly upregulated family member throughout regeneration. (B–D) Representative fluorescence-activated cell sorting plots of Tg(cmcl2:GFP), Tg(tcf21:Dsred);Tg(fli1a:GFP), and Tg(mpeg1:YFP) transgenic hearts from uninjured ventricles. (E and F) qPCR analysis of let-7i expression in cmlc2, tcf21, fli1a, and mpeg1 sorted cells in either uninjured (E) or 3-dpa hearts (F). Let-7i is confined to tcf21 cells in the uninjured state and enriched in tcf21, fli1a, and to a lesser extent, mpeg1-positive cells at 3 dpa. (G and H) Real-time qPCR studies show elevated levels of cmlc2, tcf21, fli1a, and mpeg1 expression in the appropriate fluorescent cells isolated from uninjured hearts. Values are means ± SE. *p < 0.05 compared with uninjured ventricles (A), relative to cmlc2-positive cells (E and F) or fluorescence-negative cells (G and H); dpa = days post-amputation.

Figure 2

Let-7 Depletion Results in Defects in Wound Closure (A) Schematic of locked nucleic acid (LNA) microinjection paradigm to deplete let-7 expression. (B and C) (B) Real-time qPCR analyses show that LNA treatment results in significant knockdown of mature let-7 expression at 3 dpa, when compared with scrambled control LNA oligonucleotides. Values are means ± SE. *p < 0.05, (n = 4). (C) Representative images of tcf21:Dsred expression in scrambled LNA control and LNA-let-7-treated hearts at 3, 14, and 21 dpa showing defects in wound closure. Scale bar, 50 μm. Brackets indicate approximate resection plane. (D) Quantification of tcf21:Dsred expression levels within the resection zone. Values are means ± SE. *p < 0.05, (n = 8–10). i.p., intraperitoneal; dpa, days post-amputation; NS, not significant.

Figure 3

Let-7 Activity Is Required for Heart Regeneration (A) Representative images showing proliferating CMs in scrambled LNA control and LNA-let-7-treated fish. Arrowheads indicate Mef2+ PCNA+ cells at the injury zone (n = 10–12). Scale bars, 50 μm in top three panels and 25 μm in magnified images (bottom panel). (B) CM proliferation indices are suppressed by 72% and 48% in LNA-let-7 hearts at 3 and 7 dpa, respectively, when compared with scrambled LNA control treatment. Values are means ± SE; *p < 0.05. (C) Representative images of hematoxylin and eosin staining of 14 dpa control and LNA-let-7-treated hearts reveal a spectrum of heart regeneration defects, categorized as normal, mild, and severe. (N = 21). Dashed lines mark approximate resection plane; dpa = days post-amputation. (D) Representative images of Acid Fushin Orange G (AFOG) stains of control and LNA-let-7-administered hearts reveal more collagen and fibrin tissue within the injury zone (N = 10). Brackets mark the injury zone. Brown, muscle; red, fibrin; blue, collagen.

Figure 4

CM Dedifferentiation Is Normal under Conditions of Decreased Let-7 Activity (A) Representative transmission electron microscopic images of remote and injury zones of resected 7-dpa hearts from scrambled control and LNA-let-7-treated hearts. Brackets mark intact CM sarcomeres in a remote zone. By contrast, asterisks highlight disorganized and dedifferentiated myosin bundles at the injury zone, (n = 4). Scale bar, 500 nm. (B) Representative images showing activation of Tg(gata4:GFP) expression in the primordial muscle layer in scrambled control and LNA-let-7-treated hearts at 7 dpa. Brackets in upper panels show approximate resection injury plane. Arrowheads in magnified bottom panels show gata4:GFP expression in both scrambled LNA control and LNA-let-7-treated hearts. (n = 6). (C) Quantification of gata4:GFP expression levels at the injury border and remote zones show no differences between control and LNA-let-7-treated hearts. Values are means ± SE. (N = 6).

Figure 5

RNA Sequencing Analysis Reveals Enrichment of Tumor Necrosis Factor Signaling in LNA-let-7-Treated Hearts (A) A gene network that depicts genes associated with TNFα function and were upregulated (red) and downregulated (green) in LNA-let-7 samples, compared with control treatment. Predicted direct target genes of let-7 are highlighted within the blue rectangle. (B) Nine TNFα-associated mRNAs with predicted binding sites for let-7 are significantly upregulated in the absence of let-7 expression, as confirmed by qPCR studies. (C) qPCR studies reveal the inverse expression relationship between let-7i and TNFα transcripts in the early stages following heart injury. (D) qPCR studies show the relative TNFα expression levels in cmlc2, tcf21, fli1a, and mpeg1 3-dpa sorted cells. TNFα expression is elevated in mpeg1 cells and downregulated in tcf21 cells. (E) Representative images of Tg(BAC:TNFα:GFP) and (tcf21:dsRed) expression in 1- and 3-dpa double transgenic hearts show TNFα-GFP activation in the epicardium and resection zone. (n = 4–6). Arrowheads indicate co-labelled cells, brackets mark approximate injury zone. Scale bar, 50 μm. Values are means ± SE. *p < 0.05 compared with control heart ventricles (B), uninjured heart ventricles (C), or CM expression levels (D).

Figure 6

Co-suppression of Let-7 and TNF-α Activity Restores Defective Regeneration Processes Induced By Let-7 Depletion Alone (A–G) (A–C) Representative tcf21:Dsred expression at 14 dpa and (E–G) CM proliferation at 3 dpa, in vehicle, LNA-let-7, and LNA-let-7 with CAY10500 treatment. Brackets indicate approximate amputation planes. Arrowheads mark Mef2+ PCNA+ cells. (n = 9–14). Scale bar, 50 μm. (D) Quantification of tcf21:Dsred expression within the injury zone reveals migration of epicardial cells across the resected zone under conditions of CAY10500 and LNA-let-7 co-treatment. (n = 10). (H) Quantification of CM proliferation indices reveals LNA-let-7 treatment alone suppressed proliferation indices by 48%, whereas co-treatment with LNA-let-7 and CAY10500 restores CM proliferation indices to levels comparable to vehicle treatment. Values are means ± SE. *p < 0.05 compared with scrambled heart ventricles.

Figure 7

Preconditioning Hearts with CAY10500 Culminates in Pleiotropic Defects in Regeneration (A) An outline of the treatment paradigm for preconditioning animals with CAY10500. (B) Representative images of 3-dpa hearts stained with antibodies to detect Mef2 and PCNA. Arrowheads indicate Mef2+PCNA+ cells. (C) Quantification of cardiomyocyte proliferation indices was determined by calculating the percent of Mef+ PCNA+ cells over total Mef2+ cells. Preconditioning animals with CAY10500 suppressed CM proliferation by 49% (n = 12). (D) Representative hematoxylin and eosin images of 3-dpa hearts show normal wound closure in vehicle (control) and enlarged clots in CAY10500 hearts. (n = 10). Values are means ± SE. *p < 0.05 compared with control heart ventricles. Brackets represent approximate resection plane in (B and D).

Figure 8

A Model of Let-7/TNF-α Control of Heart Regeneration Heart resection stimulates upregulation of let-7i levels prominently in the epicardium, and to a lesser extent, in macrophages. Let-7i represses TNFα expression to a proliferation instructive level by direct binding and degrading transcripts. Optimized levels of TNFα create a regeneration-instructive microenvironment resulting in CM proliferation and epicardial cell migration during heart regeneration.