Rack1 Contributes to the Upregulation of Embryonic Genes in a Model of Cardiac Hypertrophy

doi:10.21203/rs.3.rs-4826518/v1

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Rack1 Contributes to the Upregulation of Embryonic Genes in a Model of Cardiac Hypertrophy

https://doi.org/10.21203/rs.3.rs-4826518/v1

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published 28 Oct, 2024

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Receptors for activated C kinase (RACKs) have been shown to coordinate PKC-mediated hypertrophic signaling in the mouse. However, rare information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart’s culture model by using Phenylephrine (PE) or a growth factors cocktail (GFs) as pro-hypertrophic/regeneration stimuli. Blebbistatin (BL) inhibitory action treatment was also studied for its involvement in blocking some of PE’s signal transduction actions. qRT-PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1’s expression. Immunohistochemical analyses of the heart showed the co-localization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation treatment, whereas resulted less expressed in the BL treatment. Cultured ZF heart cells activated via GFs treatment the enhanced expression of RACK1. Overexpression of RACK1 induced by transfection of recombinant RACK1 cDNA in ZF heart cells, enhances the expression of embryonic genes, especially after a week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as a mammal's alternative study model.

Biological sciences/Biochemistry

Biological sciences/Cell biology

Biological sciences/Developmental biology

Cardiac Hypertrophy

RACK1

zebrafish

GATA4

WT1

NFAT2

Heart

Phenylephrine

Growth Factors

Blebbistatin

The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins with homology to the β subunit of G-proteins [1, 2]. Although WD-repeat proteins lack enzymatic activity, they are subject to post-translational modifications, suggesting that their cellular location and function can be reversibly modulated [3]. RACK1, constituted by WD folders appear to have multiple roles in the cell and serve as a hub for diverse signal transduction pathways [4]. RACK1 phosphorylates eIF6 and also acts as a component of the alternative eIF3d-dependent translation [5-7]. Additionally, RACK1 represses translation by binding to the Argonauta/miRNA (miRISC) complex in both invertebrates and vertebrates [8]. Altering RACK1 expression impacts on miRISC functions and impairs the association of the complex with the translating ribosomes [9]. RACK1 also plays an important role in the nucleus by activating the P300 protein-dependent acetylation of nucleosomes [10]. Coherently with its multiple functions, RACK1 expression is essential, and RACK1 knock-out mice die during embryonal development [11]. In the mouse heart, PKC-RACK interactions and RACK expression modulate PKC-mediated manifestation of cardiac phenotype and in particular, cardiac-speciﬁc transgenic overexpression of a PKB-subunit (PKCbII) cDNA provoke cardiac hypertrophy [12].

RACK1 signal transduction cascades, associated with cardiac hypertrophy, are still to be refined [10]. RACK1 could be implied in the activation of mammalian cardiac fibroblasts and their transformation into active myofibroblasts, as responsible for excessive extracellular matrix production and deposition, typical of cardiac fibrosis [13, 14]. This process is due to RACK1 activity and the downregulation of key miRNAs, such as miR133a and miR1 [15]. Recently, research on mice established a correlation between the Speckle-type pox virus-zinc finger (POZ) protein and RACK1 in heart fibrosis [16].

Some differences in activating fibroblasts have been found in zebrafish (ZF) compared to mammals. In ZF, cardiac hypertrophy is characterized by hyperplasia and a growing number of myofibrils in myocytes [17]. Resident ZF fibroblasts deposit fewer connective fibrils than mammalian fibroblasts and are eventually pushed to transdifferentiate into myocytes and components of the vascular system [18, 19]. This process occurs in both regeneration and hypertrophy-induced processes through the downregulation of miR1, miR133a, and miR133b, which directly or indirectly control the expression of embryonal genes such as Gata Binding Protein 4 (GATA4), nuclear factor of activated T cells-2 (NFAT2), and Wilms tumor-suppressor gene-1 (WT1) [17, 18]. In the hypertrophy process induced by Phenylephrine (PE), cells undergo to a tonic contraction through the involvement of various calcium channels in the inner and plasma membrane. The consequent calcium waves can activate PKC phosphorylation processes, miR-downregulation, RACK1 activation and re-expression of embryonic genes [10, 17, 20].

We previously demonstrated that microRNAs (miR1, miR133a, and miR133b) are essential for controlling the expression of key embryonic genes involved in differentiating cardiac tissue components (epicardium, myocardium, and endocardium) during ZF heart regeneration and in PE-inducing hypertrophy [17, 18, 21, 22]. Moreover, Blebbistatin (BL) was shown to counteract the activity of PE, probably by blocking the calcium waves [22]. Herein, by using the same ex vivo culture model of the ZF heart, we aimed to determine the role of RACK1 in embryonic gene re-expression during the cardiac hypertrophy process.

The study was conducted in conformity to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes, and approved by the protocol code N. 10-87/2010 (and the up-grades until 2020) authorization/s from the Italian Health Ministry.

2.1. Ex-vivo experiments

Adult ZF (n = 339) with weigh of 0.40 ± 0.05 g and lengths of 3.4 ± 0.07 cm were used for the experiments. The ZF were sacrificed with 4 mg/ml of ethyl 3-aminobenzoate methanesulfonate salt (MS222, Sigma-Aldrich). Heart was extracted in a flow laminar hood (PBI, International) was washed in L15c media [(L15, Liebovitz, Sigma-Aldrich) supplemented with 80 units penicillin/streptomycin + 0.5 mg/ml of L-glutamine + 5% fetal calf serum (GIBCO) and 0.8 mM CaCl₂ (Sigma-Aldrich)] and transferred to 12-well Petri dishes and divided into four groups: 1) the control (CTR) cultured with L15c medium; 2) the batch treated with Phenylephrine 500µM (PE) for 96h; 3) The batch treated with a cocktail of Growth Factors (GFs); 4) the batch treated simultaneously with PE and Blebbistatin (BL) 10 µM for 96h (PE/BL). This latter group was considered to confirm the inhibitory action of BL on treatment with PE. To evaluate the inhibitory entity of BL on treatment with PE, the other two experimental groups: 5) the batch treated with PE for 72h, and then treated with (BL) for 24h (called with the acronym PE + BL), and 6) the batch treated with BL for 24h and then treated with PE for 72h (called by the acronym, BL + PE). The cocktail of GFs was developed inour laboratory and already published [21]. Briefly it consisted of L15c spiked with a mixture enriched in growth factors constituted by 5 ng/ml of Recombinant Human Cardiothropin-1 (CT, Peprotech Inc.), 2 nM Thrombin (TR, Sigma-Aldrich), 40 ng/ml of Recombinant Human, Fibroblast Growth Factor-basic (FGFb, FGF2, Invitrogen, Biosource),10 ng/ml ﬁbroblast growth factor-4 (FGF-4, Sigma-Aldrich) and 10 ng/ml of Platelet-Derived Growth Factor- BB (PDGF-BB, Sigma-Aldrich).

2.2. RNA extraction and qRT PCR analysis

The collected hearts (N= 6 done in triplicate experiments) were placed in 0.5 mL in RNALater (stabilization Solution, Invitrogen, US ) and stored at -80°C. The extraction and purification of total RNA were done using the previously published procedure [22]. Briefly, after the isolation and purification of total RNA by using the ThermoFisher Kits (US), the quantity and purity of the extracted RNA (ratio A 260/280) were tested by Molecular Device SpectraMax QuickDrop SPM. The Agilent Bioanalyzer 2100 with RNA 6000 Pico Kit has been used to verify the integrity of RNA. The TaqMan GEX Assay (Thermo Fisher/Applied Biosystem) was used in combination with DNase directly in the sample already extracted. For these cDNAs, the amplification in Real-Time PCR was performed using the PowerUp SYBR Green Master Mix and appropriate primers: Receptor for Activated C Kinase 1 (RACK1, Dr03118827_m1), Myosin Heavy Chain 7 (MYH7, Dr- 03431146_m1), Brain Natriuretic Peptide (BNP, specific for ZF ventriculus, ID ARAADCC) and S18 (HS99999901_S1) were purchase as primers by ThermoFisher Scientific (US). The relative expression of the genes was calculated by comparing the cycle times for each target PCR. The Ct values of each PCR target were normalized by subtracting the cycle threshold (Ct) value of S18, which was represented by the value ΔCt. The level of relative expression was calculated using the following equation:

Relative gene expression = 2 ^{– (ΔCt_sample – ΔCt_control)}

Significant differences (probability values, P) between the experimental groups and control/untreated animals were calculated based on the mean from three parallel experiments ±standard deviation (SD) and analyzed by a one-tailed ANOVA-Bonferroni test.

2.3. Immunoblot analysis

The hearts (n= 10 for each group done in triplicate experiments) were homogenized manually by pestle in a lysis buffer [Tris-HCl (20 mM, pH 7.4) + EGTA (10 mM) + NaCl (150 mM) + Triton X-100 (1%) glycerol (10%) + Sodium Dodecyl Sulfate (SDS, 1%)] supplemented with protease and phosphatase inhibitors (PPI). Protein extract from cells were performed in RIPA buffer [Tris-HCl (10 mM, pH 8.0) + NaCl (140 mM), EDTA (1 mM) + Triton X-100 (1%) + SDS (0.1%)] supplemented with PPI. Following the standard protocol, the proteins were quantiﬁed by BCA protein assay kit (Pierce, Rockford, IL, US). Protein were loaded on 4–20 % polyacrylamide precast gels from 30-50 µg of total (Criterion TGX Stain-free precast gels; Bio-Rad). The blotted proteins were transferred onto a nitrocellulose membrane using a Trans-Blot Turbo SystemTM (7 min at 2.5 A) and Transfer packTM (Bio-Rad). Primary antibodies used: anti-mouse RACK1 IgM (Bioscience, US); and rabbit anti-actin (Invitrogen, US). After the incubation with the appropriate horseradish peroxidase-conjugated secondary antibody, bands were visualized using the Clarity Western ECL substrate with a ChemiDoc MP imaging system (Bio-Rad). Bands were quantiﬁed for densitometry using the BioRad Image Lab software.

2.4. Histology and immunohistochemistry

2.4.1. Sample preparation for histology and immunohistochemistry

The hearts cultured in ex-vivo (n= 7 for each group) were placed in fixed solution: 4% paraformaldehyde in PBS (Santa Cruz Biotechnology, US) + 10% sucrose for 24h at 4°C. The fixed hearts were incubated overnight in PBS containing 10% sucrose. Some heart samples (Three for each group) were embedded with paraffin procedure. The sections obtained by Reichert-OME microtome were mounted on slides APES-treated (Sigma-Aldrich). The dewaxed sections were processed for histochemistry and DAB immnunohistochemistry. The other hearts (4 for each group) were embedded in OCT-compound (Invitrogen) at -40°C and sectioned by cryo-microtome (Leitz) for fluorescence-immunohistochemistry. The slides superfrost Plus Gold (Espredia, NL/USA) were used The sections were observed with an Axioscope (Zeiss) microscope supported with software for image analysis (Axiovision 100 software, Zeiss). The confocal images were acquired by a Zeiss LSM 710 Scanning module with 34 spectral R/FL detection channels, and 6 laser lines (405,458, 488, 514, and 633 nm) by acquiring at least 6-8 analysis plans.

2.4.2. Masson's trichrome staining

The de-paraffinized sections were stained by following the procedure already published in [22] and described in supplementary material.

2.4.3. Immunohistochemistry

The cryosections were obtained from 4 hearts of each group (N=24) were treated with phosphate buffer (PBS) added with 2% Tween + 1 mM EDTA (Sigma-Aldrich), and slides were placed in the citrate buffer to evidence the antigens for 20 min at 40°C. Subsequently, slides were washed in PBSTX (PBS+ 0.8% Triton X100, Sigma-Aldrich). The sections were treated for 12hrs with Sudan Black 0.01% in PBSTX. The sections were then processed in 0.1%Triton + 0.1% DMSO + 2% BSA and incubated at 4°C with the primary antibody. The primary antibodies used: 1) anti-Wilms Tumour 1 (WT1, rabbit recombinant polyclonal protein antibody, Abcam) 1:100 [17] or 2) anti-Gata4 (GATA4 rabbit recombinant polyclonal protein antibody, Abcam) 1:100 [21] or 3) anti-Nfat2 (NFAT2 rabbit recombinant polyclonal protein antibody, Abcam) 1:1500 [17]. These antibodies were used in contemporary with the RACK1 anti-mouse IgM antibody (BD, Bioscience, USA). The antibodies were diluted in PBST + 1% normal goat serum (NGS, Sigma-Aldrich). After 48 hrs of incubation/ 4°C, the slides were washed in PBSTX and incubated with PBSTX + 1% NGS + 2% BSA before the incubation of ﬂuorophore-conjugate secondary antibody: 1) red fluorescent goat anti-mouse IgM (GAM, 1:200, Alexa-conjugates 594 wavelength, Invitrogen) and 2) green florescent goat anti-rabbit (GAR, 1:200, Invitrogen, 488 nm wavelength) for 1 h at room temperature. The nuclei were counterstained with 4',6-diamidin-2-fenilindolo (DAPI, 1:1000, Vectastain). Slides were mounted with Fluoreshield Mounting medium. The confocal analysis was done by using ZEISS LSM 710- Scanning module with 34 spectral R/FL detection channels, 6 laser lines (405, 458, 488, 514, and 633 nm) with at least six analysis plans for each sample. To obtain a correct and standardized fluorescence intensity from the samples, each fluorescence channel was first set on a sample treated only with secondary antibodies/DAPI and then set on the control sample without treatments (CTR group). The analysis of antibodies staining: WT1/RACK1/DAPI; NFAT2/RACK1/DAPI and GATA4/RACK1/DAPI immunostaining was carried out on a single section. Each image acquired from the relative fluorescent canal has been converted to RGB, merged for the three color canals, and normalized to the background by Adobe Photoshop 6 program, (Adobe Systems, Mountain View, CA, USA). The resolution of each image has been selected to be 1024 × 1024 pixels (single stack).

2.5. Cell culture, RACK1/HA transfection and immunocytochemistry

2.5.1. Cell culture

Nine hearts have been cultured in GFs medium: GFs medium has composed of basic cardiac medium (L15-Sigma/Aldrich added with 100 X penicillin/streptomycin, 0.5 mg/ ml of L-glutamine, 5% Fetal Calf Serum (FCS, GIBCO), 0.8 mM CaCl₂) spiked with a mixture enriched in growth factors constituted by 5 ng/ml of Recombinant Human Cardiothropin-1 (CT, Peprotech Inc.), 2 nM Thrombin (TR, Sigma-Aldrich), 40 ng/ ml of Recombinant Human, Fibroblast Growth Factor-basic (FGFb, FGF2, Invitrogen, Biosource),10 ng/ml ﬁbroblast growth factor-4 (FGF-4, Sigma-Aldrich) and 10 ng/ ml of Platelet-Derived Growth Factor- BB (PDGF-BB, Sigma-Aldrich) and maintained for 24h post explantation as described in [21]. After that period the non-adherent cells have been collected and used to prepare primary culture of cardiac-activated cells. The cells were distributed, in triplicate, in wells of polystyrene plate with 0.5 ml each of GFs medium. A part of the cells has been used for immunocytochemistry with double staining RACK1/WT1; RACK1/NFAT2; RACK1/GATA4. The nucleus is marked by DAPI staining (1:1000, Vectastain). The other part has been used to transfect the cells or, as a control maintained with lypofectamine/medium (see section 2.5.3).

2.5.2. Immunocytochemistry from cytospin cells (CSc)

About 100 µl of cultured cells were taken from the wells, ﬁxed with 4% PFA in PBS+ 20% sucrose and centrifuged at 1000 rpm for 10 minutes/9°C on slides (superfrost Plus Gold-Espredia, NL/USA), dried for 15 min at room temperature under UV-steril flow. Once dry, the pellets on the CSc-slide were washed with 500 µl of PBS for 5 min and processed for the immunostaining. The CSc were washed with PBSTX 0.8% 15’ followed by unmaskin solution (20’/37°C) and consequent whashing with PBSTX 0.8% 10’/RT. The slides were lied in Sudan Black solution (0.01 gr/ 70% alcohol, Sigma-Aldrich) then treated with H₂O₂ 5% in PBSTX for 10’ to erase the possible auto-fluorescence due to peroxisomes and washed in PBSTX 0.8%. The slides were treated with unspecific blocking reagent (normal mouse serum 1%, normal goat serum 1%, bovine serum albumin 2% in PBSTX). The slides finally were incubated with a double mixture of primary antibodies: RACK1/WT1, RACK1/GATA4, and RACK1/NFAT2 for 12hrs at 4°C. As a control of the reaction, a slide was treated with the solution used to dilute the antibodies (PBSTX 0.8 + BSA 1%). After 12 hrs the slides were washed in PBSTX 0.8% and treated with PBSTX 0.8%+ BSA 5%) to block the non-specific reaction of secondary antibodies. The slides were treated with a mixture of secondary antibodies: goat-anti-rabbit-green fluoresceinated (GAR-FITC, Alexafluor 488 nm, 1:200, Invitrogen)/ goat-anti-mouse-red fluoresceinated (GAM-TRITC, Alexafluor 586 nm, Invitrogen) in PBS added of 5% BSA (Sigma-Aldrich) and mounted with 25 µl of PBS-glycerol/DAPI (Sigma-Aldrich). The slides were observed with the Axioscope microscope (Zeiss) equipped with image analysis software.

2.5.3. Transfection procedure

The protocol has been adapted to ZF cells from [24]. One day before transfection, 0.5 X 10⁵cells were seed per well in 500 μl of 1:1 Opti-MEM I (Invitrogen /31985) /Basic medium (without antibiotics/fetal calf serum until the time of transfection, L15-OT). For each transfection sample, prepare DNA–Lipofectamine 2000 complexes by a dilution of 0.8 μg DNA (plasmid contains: Rack1 and haemagglutinin; RACK1/HA Invitrogen, V79020) in 50 μl Opti-MEM I. Parallelly, a control of Transfection (CTR) was prepared using 50 μl Opti-MEM without RACK1/HA. Four μl of Lipofectamine 2000 (Thermofisher Scientific US/IT) has been diluted in 100 μl Opti-MEM I and after a gentle mix was divided in two Eppendorf vials. After 5 minutes at room temperature, the liquid Lipofectamine/Opti-MEM I from each vial was added to the Eppendorf without plasmid (CTR) or to the Eppendorf with the plasmid (RACK1/HA) to reach the final amount of 100 μl each. The two vials containing the 100 μl of transfection complex have been added to each well-containing cells and L15-OT. The buffers/cells in the wells have been mixed gently by rocking the plate back and forth under the steril flow. The plate was incubated at 28 °C in a humidified incubator for 24h until they were ready to assay for transgene expression (T0 and T1). Due to the low metabolism of hetherothermic cells an incubation of 24h has been necessary to obtain 80% success of transfection. After this period the cells are washed in osmolar PBS, and centrifuged at 1000rpm/10’. The half amount of the cells was fixed by a mixture of PFA 4% /PBS added to 5% sucrose at 4°C for 24h (called T0 samples). The remaining part was added to CTR or RACK1/HA wells containing L15c/GFs added with 1% Gentamicin (to prevent the expulsion of RACK1/HA plasmids), and the plate was posed in a cell incubator at 28°C. The L15c/GFs (150 µl) were added to the wells two times during a week (samples called T1). After this period the transfected and control cells were washed in osmolar PBS, centrifuged at 1000 rpm/10’, and fixed as described above.

The immunocytochemical assay has been realized from CSc (CTR and RACK/HA from T0 and T1 samples) upon slide Poly-L-Lysine-coated (Bright-GB). Double immunostaining as performed as described in the 2.5.2 section. The primary antibodies used: 1) rabbit-anti haemagglutinin (batch C29F4/ ThermoFisher Scientific (US), 1:100; HA-TAG) in combination with 2) mouse anti-RACK1 (see above) to ascertain the presence of both in the cells. To study the presence of RACK1+ in the transfected cells in combination with eventually enhanced expression of embryonic genes, it has been used 3) mouse HA-TAG monoclonal antibody (Ab1424, Abcam (UK) 1:200) in combination with 4) rabbit-NFAT2 or rabbit-WT1 or rabbit-GATA4 (see above).

The secondary antibodies (IIAb) used were green-fluorescent Alexa Fluor® 488 goat anti-mouse IgG antibody (1:200, Invitrogen, US) and Alexa Fluor® 596 goat anti-rabbit (1:200, Invitrogen,US). IIAb were incubated at Room temperature for 30’ and then washed in PBSTX 0.8% and then coated by PBS/Glycerol/DAPI mounting media (Sigma-Aldrich, US) and the cover glass. As control of immunohistochemical reaction were used cytoslides marked with the secondary antibodies (i.e. GAM and GAR) in combination with DAPI, to avoid the false positive. The confocal analysis of immuno-marked slides was done using ZEISS LSM 710 with a merge of four to six plans of laser scanning. To obtain a correct and standardized fluorescence intensity from the samples, each fluorescence channel was first set on a sample treated only with secondary antibodies/DAPI and then set on the control sample without treatments (CTR group).

3.1. RACK1 is co-espressed with hypertrophy markers in the ZF heart

Morphological analysis of cardiac tissue treated with GFs or with PE by Masson trichomic staining confirmed the hypertrophy induced by PE and the counteracting effects of BL [22]. GFs treatment, caused a sparse enhancement in myofibril quantity in the explanted ZF heart, accompanied by moderate hyperplasia of epicardial cells (Supplementary Materials). Thus, GFs treatment not determined hypertrophy like PE treatment but only a proliferation stimuli. To test whether hypertrophy and/or hyperplasia affects the expression of RACK1, cardiac Myosin/Heavy chain 7 (MYH7) and Brain Natriuretic Peptide (BNP), a hypertrophy marker specific to the ventricular portion of the heart, we perfomed quantitative Real-Time PCR (qRT-PCR) analyses of RNA isolated from ZF heart subjected or not to ex vivo treatments. These analyses revealed that PE and GFs treatment increased the expression of RACK1 (Fig. 1; PE and GFs vs CTR or other groups; ANOVA Bonferroni test p<0.001). Samples treated simultaneously with PE and BL (PE / BL) and PE+BL showed non-significative modifications. In contrast, the group post-treated with PE evidenced a significative reduction in RACK1 expression compared to the control group (BL + PE vs CTR, p< 0,05). MYH7 expression showed a significant upregulation in PE and PE + BL. The highest expression was observed in PE treatment with statistical significance in all other groups (p<0.001 or p<0.005), indicating a higher expression of this sarcomeric myosin in PE-treated hearts. The PE + Bl group evidenced an increase of MYH7 (vs CTR, p<0,01). The GFs group showed a slight increase compared to the CTR group (GFs vs. CTR, p<0.05). BNP has upregulated in the PE group (PE vs. CTR; p<0.001; PE vs. all groups p<0.05) and PE+BL group (PE+BL vs CTR p<0.01). Conversely, this upregulation was partially repressed after pre- and co-treatment with BL group. The GFs group also showed a higher mean than the control, but variability among samples did not yield statistical significance.

Figure 1. Quantitative RT-PCR of RACK1 and hypertrophy markers. The graph shows the relative expression of Rack1 (RACK1), myosin heavy chain 7 (MYH7), and brain natriuretic peptide (BNP). In the PE treated samples, a significant increase in markers was seen compared to the control (*, PE vs CTR, PE vs all groups, p<0.001). The GFs group, despite the variability of the samples, showed a significant increase compared to the CTR and the other groups, except PE (GFs vs CTR, PE / BL, PE + BL BL + PE, p<0.01). PE / BL and PE + BL showed non-significative modifications, whereas BL + PE group evidenced a reduction (BL + PE vs CTR, p< 0,05). MYH7 expression showed a significant upregulation in PE and PE + BL (PE vs all groups p<0.001; PE + BL vs all groups lacks PE, p<0.005). The GFs group showed a slight increase compared to the CTR group (GFs vs. CTR, p<0.05). BNP has upregulated in the PE group (PE vs. CTR; p<0.001; PE vs. all groups p<0.05) and PE + BL group (PE+BL vs CTR p<0.01). Conversely, this upregulation was partially repressed after pre- and co-treatment with BL. The GFs group also showed a higher mean than the control, but variability among samples did not yield statistical significance. Hearts from fish used N= 6 in each group done in triplicate experiments—the statistical analysis: ANOVA- Bonferroni Test.

3.3. Immunoblot analysis of RACK1 expression

Western blot analyses were performed to evaluate the Rack1 protein (RACK1) expression level in treated heart samples (PE, PE / BL, PE+BL, BL+PE, and GFs). The normalization of actin levels indicated that the PE-treated group displayed a fourfold upregulation of RACK1 compared to the CTR group (Figure 2, p<0.001). The other treated groups also showed slight increase in the presence of the protein. In particular, the GFs and PE / BL groups significantly increased (p<0.05). The PE + BL group expressed less protein than the CTR (**; p<0.05) in contrast with the mRNA quantified in qRTPCR.

Figure 2. Immunoblot analysis of RACK1 protein expression in experimental groups vs CTR. Immunoblot images of Rack1 protein (RACK1) expression (upper part) and non-sarcomere actin (below) were used as housekeeping protein expressions. The graph reports the means of at least three independent experiments of the density ratio between RACK1 and actin bands (mean ± S.D.). RACK1 expression is strongly marked in the PE group compared to CTR (n=18), but appreciable positivity is also observed in the GFs group to CTR. The RACK1 is comparable to CTR in other treated groups. The PE+BL group expressed less protein than CTR (**; p<0.05). Hearts from fish used N= 10 in each group done in triplicate experiments—the statistical analysis: ANOVA- Bonferroni Test.

3.4. Localization of RACK1 on tissue

Immunohistochemistry analysis via nickel enhancement/DAB on whole hearts showed RACK1 positivity at the level of the epicardium, endocardium, and myocardium. Particularly, hearts treated with PE or GFs showed a detectable staining to RACK1 at the epicardium and endocardium levels compared to untreated controls (CTR vs. PE vs. GFs Figure S2, Supplementary Data). Hearts treated simultaneously with PE and BL showed positivity in the epicardium, which was higher than the untreated control, but less intense than PE and GFs. By contrast, hearts pre-treated with PE showed an intense reaction at the level of muscle fibers and epicardium compared to the untreated control, but less intense than PE and GFs. Particularly the endocardium, resulted low marked as compare PE and GFs-treated group. The hearts post-treated with PE showed a similar pattern to GFs samples.

3.5. Immunohistochemistry and cytochemistry of RACK1/embryonal markers support their co-localization in tissue

To verify whether RACK1 co-localized with embryonic gene expression, a double labeling confocal analysis was conducted with RACK1 different embryonic markers (GATA4, WT1, NFAT2; Figures 3–5, merge of staining data: S3—Supplementary Data). All the sections showed RACK1 labeling similar to that observed with DAB/nickel-enhancement immunohistochemistry while highlighting its co-expression with different embryonic genes (GATA4, WT1, and NFAT2 proteins). In particular, GATA4 is expressed in the epicardium, endocardium, and myocardium, resulting in complete overlap with RACK1 expression (Fig. 3). The co-localization of WT1 with RACK1 showed exclusive positivity in the epicardium (Fig. 4). The NFAT2 marker showed clear positivity in the endocardium lining cardiomyocytes, and in co-localization with RACK1 (Fig. 5). In heart specimens treated with GFs, these proteins had similar staining to the PE group, but embryonic markers displayed lower intensity. In the PE/BL group (treated simultaneously with PE and BL) RACK1 positivity was greater than in the control. This positivity was mainly localized around the fibers (in cardiomyocytes), where it co-localized with GATA4 and NFAT2. Furthermore, WT1’s positivity in the epicardium was less marked. By contrast, in hearts pre-treated with PE, GATA4 staining was intense and greater than RACK1 positivity. WT1 and NFAT2 showed less intense reactions in the epicardium and endocardium, respectively, than PE and GFs. Post-PE-treated hearts showed a less intense reaction to RACK1 and a lower signal for cardiac markers. The figure relative to the merge staining of RACK1/GATA4/DAPI, RACK1/WT1/DAPI, and RACK1/NFAT2/DAPI is shown in the Supplementary Materials (Figure S3).

Figure 3. Double staining with RACK1 and GATA4 analyzed via confocal microscopy. RACK1 (red fluorescence) is fundamentally expressed in the CTR myocardium. In all experimental groups, RACK1 is enhanced and co-localizes with the GATA4 antibody (green fluorescence). GATA4 is strongly expressed in PE and GFs and moderately expressed in PE+BL, PE/BL. Moreover, it is less expressed in BL+PE. The hearts utilised for experiments were N= 4 /5 sections in each group in triplicate experiments. Blue fluorescence: DAPI (nuclear marker); Bar: 500µm.

Figure 4. Double staining with RACK1 and WT1 analyzed via confocal microscopy. RACK1 (red fluorescence) is less expressed in the epicardium than in the myocardium in CTR. In all experimental groups, RACK1 is enhanced and particularly co-localized with the WT1-antibody marking the epicardium (green fluorescence). WT1 is strongly expressed in PE and GFs and moderately expressed in PE+BL, PE/BL, and BL+PE. The hearts utilised for experiments were N= 4 /5 sections in each group in triplicate experiments. Blue fluorescence: DAPI (nuclear marker); Bar: 500µm.

Figure 5. Double staining with RACK1 and NFAT2 analyzed via confocal microscopy. RACK1 (red fluorescence) is fundamentally expressed in the myocardium of CTR. In all experimental groups, RACK1 is enhanced and co-localized with the NFAT2 antibody marking the endocardium (green fluorescence). NFAT2 is strongly expressed in PE and GFs and moderately expressed in PE+BL, PE/BL, and BL+PE. The hearts utilised for experiments were N= 4 /5 sections in each group in triplicate experiments. Blue fluorescence: DAPI (nuclear marker); Bar: 500µm.

3.6. Rack1+_transfected epicardial/endocardial cells showed higher expression of embryonal markers

Cardiac-activated cells by GFs in ex vivo cultured hearts leave the organ as transdifferentiate cells and start to proliferate in the culture buffer. Previously we have identified two types: epicardium-activate and endocardium-activate cells that express embryonal genes. In particular Epicardium-activated express WT1/GATA4 and endocardium-activated express NFAT2/GATA4 [21]. To indagate if RACK1 is linked with the expression of these embryonal genes, the GFs-activated cells were transfected with the plasmid containing rack1/hemagglutinin-Tag (RACK1/HA) and analyzed 24 hours after the transfection event (T0, Fig. 6) or for one week (T1, Fig. 7). T1 cells were also cultured in culture media containing GFs to still give both proliferative and expression stimulus to embryonic genes. Transfected cells at T0 and T1 were compared to control cells (CTR) that did not contain the plasmid. Immunocytochemistry was conducted with double labeling and an anti-HA antibody to detect RACK1 expression due to the plasmid (green fluorescence) and endogenous embryonic genes GATA4, WT1, and NFAT2 (red fluorescence). The labeling analysis was conducted with a confocal microscope to have a clear signal of the labels and evaluate their possible relationship with RACK1 upregulation.

CTR cells did not show any positivity to HA and, therefore, to plasmid-encoded RACK1. They also supported the normal expression of endogenous RACK1 at T0 (Fig. 4) and T1 (Fig. 7) due to stimulation by GFs. At T0, the transfected cells all expressed RACK1/HA (Fig. 6), whose presence largely co-localized with GATA4, WT1, and NFAT2 expression.

In particular, GATA4 was more expressed in RACK1/HA-positive cells than in CTR cells. Moreover, GATA4 in RACK1/HA cells was locally diffused at the cytoplasmic level but was poorly detectable or comparable to CTR at the nuclear level. At T1, RACK1/HA-positive cells displayed a marked expression of GATA4 at both the nuclear and cytoplasmic levels. Figure 7 shows CTR cell labeling at both the nuclear and cytoplasmic levels.

Regarding WT1 expression in the CTR at T0 (Fig. 6), the positivity was mild and mainly localized to the nucleus. At T1 (Fig. 7), the positivity was mainly cytoplasmic. In RACK1/HA-positive cells, WT1 positivity was mainly nuclear and co-localized in some parts with RACK1/HA positivity.

At T0, NFAT2 (Fig. 6) showed positive immunostaining with a few spots at the nuclear level in both RACK1/HA cells and CTRs. At T1 (Fig. 7), the control displayed slightly higher positivity for NFAT2 than at T0, also showing positivity at the cytoplasmic level. Interestingly, at T1 in RACK1/HA-transfected cells (Fig. 7), NFAT2-immunostaining became more intense in the cytoplasmic than at the nuclear levels. NFAT2 positivity also appeared to co-localize with RACK1/HA.

Figure 6. Confocal analysis at 24 hours post-transfection (T0). The plasmid containing rack1⁺ and the reporter hemagglutinin (RACK1⁺/HA) showed green fluorescence. The cells were double-labeled with embryonal markers (GATA4, WT1, and NFAT2; red fluorescence) and compared to non-transfected cells (CTR). GATA4 is enhanced in transfected cells and mainly localized in the cytoplasm. WT1 is comparable to the CTR. NFAT2 is sparsely expressed in the CTR and, in both cases, is localized in the nucleus. The nucleus is marked with DAPI (blue fluorescence).DAPI/Ab: DAPI labeling+ antibody (GATA4 or WT1 or NFAT2). TRANSF/MERGE: tranfected cells labeled with all antibodies (against HA and one embryonal marker). CTR/MERGE: non-transfected cells labeled with all antibodies. The hearts utilised for experiments were N= 4 /5 sections in each group in triplicate experiments. The hearts utilised for experiments were N= 3 and analysis was done from 10⁴ cells in each group in triplicate experiments. Bar: 20 µm.

Figure 7. Confocal analysis after one week of post-transfection cultured in a medium supplemented with GFs (T1). The plasmid containing RACK1/HA showed green fluorescence. The cells are double-labeled with embryonal markers (GATA4, WT1, and NFAT2; red fluorescence) and compared to non-transfected cells (CTR). GATA4 is strongly enhanced in transfected cells and mainly localized in the nucleus. WT1 is highly expressed in the nucleus and cytoplasm compared to CTR. Compared to the nucleus, NFAT2 is marked in the cytoplasm and is more expressed than CTR, which is localized in the nucleus. Nucleus marked with DAPI (blue fluorescence); DAPI/Ab: DAPI labeling+ antibody (GATA4 or WT1 or NFAT2). TRANSF/MERGE: transfected cells labeled with all antibodies (against HA and one embryonal marker). CTR/MERGE: non-transfected cells labeled with all antibodies. The hearts utilised for experiments were N= 3 and analysis was done from 10⁴ cells in each group in triplicate experiments. Bar: 20 µm.

4.1 RACK1 is expressed in cardiac tissue and extensively in PE- and GFs-treated hearts

In ZF, RACK1 has been detected in some organs/tissues, such as the epidermis, intestinal tract, liver [25], and also in the cardiovascular system [26]; however, a possible correlation between RACK1 and embryonal gene expression is sparse. At the moment, what is known about the RACK1/embryonal genes-correlation is molecule-necessity from gastrula to adult, because any knockout-RACK1 model results in not life-compatible [27].

In ZF, as well as in mammals, the epithelial cells of the heart undergo proliferation and transdifferentiation by re-expressing embryonic genes when subjected to hypertrophy regeneration [21]. The expression of GATA4, NFAT2, and WT1 embryonal markers appears dependent on miR expression [18], possibly involving RACK1 in our previous analysis of their translational control.

In the present research, in a cardiac control group, RACK1 had low expression and was widespread in the epicardium, myocardium, and endocardium. This observation is consistent with others in vertebrate or invertebrate tissues due to the constitutive expression of differentiated cells or in a pre-differentiating process [28]. For example, RACK1 is expressed transiently in the skeletal muscle of post-natal mice, being abundant in the early phase of muscle growth and almost disappearing in mature adult fibers [23]. During oogenesis in Drosophila, somatic RACK1 plays a non-cell-autonomous role in maintenance and function [29].

Contrary to the CTR or BL-treated groups, RACK1 was markedly enhanced in PE samples by about six times in qRT-PCR and double in immuno-blot. This observation suggests a possible RACK1’s involvement in the hypertrophy process. This can be supported by evidence of high expression in hypertrophy markers, such as MYH7 and BNP in line with RACK1 expression in different samples. Similarly, the heart samples cultured with the buffer supplemented with the GFs, already standardized in our laboratory [21], showed double the amount of RACK1 expression than in the CTR.

Previously, in both PE and GFs chemo-stimulations are related to the downregulation of key microRNAs (i.e., miR1, miR133a, and miR133b) [17, 18]. These miRs lead to different pathways that cause cells to differentiate in various cardiac typologies [epicardial, endocardial, and myocytes] by repressing embryonic gene expression directly or indirectly [17, 18]. In this view, the observation of high expression of RACK1 and, contemporarily a notable embryonic gene expression, may indicate direct or indirect relationships between these molecules. An interesting detail observed during the comparison of the analysis of RACK1 mRNA and protein in the groups treated with BL. Intersting observation is that the pre-treatment with BL and also in the contemporary treatment with PE and BL the expression of RACK1 is comparable on average to the control. Instead, in the group post-treated with BL (PE+BL) mRNA is significantly higher than the control, while the protein appears to have a significant quantitative reduction. An explanation could be given by the knowledge in mammals cardiomyocytes that the eIF6/p27BBP-his-myc protein complex is also localized in the Z discs directly activated by cytoskeletal disruption generated by the BL, and is able to decrease protein synthesis by attaching to the 60S subunit [30]. Furthermore, eIF6 directly interacts in the cytoplasm with RACK1 limiting its presence [31].

4.2 RACK1 co-localized with embryonal markers in activated cells

A zinc finger protein, GATA4, is a hypertrophy-responsive transcription factor in mammals that forms a complex with an intrinsic histone acetyltransferase, p300. Previous studies indicated that Cdk9 forms a functional complex with p300/GATA4 and is required for the p300/GATA4-transcriptional pathway during cardiomyocyte hypertrophy. RACK1 also plays an important role in this phase [10,32]. Moreover, in rats, cardiomyocytes cultured with PE have shown a block of hypertrophy when RACK1 phosphorylation was inhibited [10]. In our model of cardiac hypertrophy in ZF, GATA4 was highly expressed in the PE and GFs groups, and partially expressed in the PE and BL groups compared to CTR. These findings, together with high RACK1 expression in PE/ samples, contrasted those of Suzuki et al. [10]. However, considering RACK1’s multiple functions in the cytoplasm, we can hypothesize that its major function in PE and GFs is to favor GATA4 translation instead of phosphorylating the p300/GATA4 complex [33]. Further transfection experiments have confirmed this hypothesis (see below).

The Wilms’ tumor suppressor gene wt1 encodes a C protein with four C-terminal Kruppel-type zinc fingers transcription factors, which play an important role in developing the mammalian genitourinary system and heart [34,35]. This transcription factor’s expression has been exclusive to activated epicardium in mammals as well as in ZF [18,36]. In ZF, wt1a- and wt1b genes expressing cardiomyocytes changed their cell adhesion properties, delaminated from the myocardium, and upregulated epicardial gene expression, which led to their transdifferentiation into epicardial-like cells [37]. In the present research, WT1 demonstrated high expression in PE- and GFs-treated groups compared to other groups, confirming previous findings. Interestingly, RACK1 co-localized with this expression. WT1⁺ cells showed mutually exclusive binding of either protein phosphatase 2A or integrin to RACK1, which is controlled by an agonist-dependent interaction between RACK1 and the insulin-like growth factor I receptor [38]. Insulin-dependent regulation of this RACK1 protein complex’s composition impacts cell migration [39]. The integrins are likely related to hyaluronic acid in the extracellular matrix, which causes the cells to migrate after the epithelial-mesenchymal transdifferentiation [20]. In the hypertrophy or regeneration cardiac ex vivo model, we identified WT1’s high expression in activated epicardial cells [18,21].

NFAT is a family of transcription factors (NFAT 1–5, [40-42]) found in the cytosol in its phosphorylated, inactive form [43]. NFAT2 is mediated by activation and controlled by calcineurin, a Ca2⁺-dependent phosphatase and RACK1. At sustained elevations of cytoplasmic calcium, calcineurin dephosphorylates NFATC1–C4, allowing NFAT to translocate to the nucleus and activate gene transcription [43-45]. NFAT2 is found in muscles, including the heart in differentiation, maturation, and hypertrophy [43]. Transgenic mice with cardiomyocyte-specific G protein-coupled receptor kinase overexpression activate NFAT-reporters in mice basally and after hypertrophic stimulation, including transverse aortic constriction and phenylephrine treatment [45]. In ZF, NFAT2 enhancement expression was demonstrated during PE treatment in ex vivo cultured hearts [22]. Due to PE treatment, the calcium wave could activate calmodulin which, in a cascade, activates serine/threonine phosphatase and calcineurin. The latter can bind to NFAT’s N-terminal regulatory domain, inducing dephosphorylation and conformation changes. In the present research, NFAT2 was enhanced by GFs treatment and partially by PE. Interestingly, by nickel-enhancement/DAB immunostaining, RACK1 revealed a localization in the endocardium after PE treatments (PE, PE+BL, PE/BL, and BL+PE groups), demonstrating a role of endocardium activation in hypertrophy. BL and PE treatments, as indicated by the lower expression, align with the previously described Ca+-wave blocking capacity of BL.

4.3 The transfection of activated cells with a plasmid containing RACK1/hemagglutinin showed involvement in embryonic gene expression

In our ZF model, the activated epicardial and endocardial cells showed RACK1 and basic embryonic gene expression levels of GATA4, WT1, and NFAT2 under L15c without GFs. The myocytes remain in the ex vivo organ, thus it was used only for histochemical analyses. However, embryonic genes and RACK1 were enhanced by the culture with a GFs-supplemented medium. This observation aligns with our previous observations, indicating enhanced embryonic gene expression [21]. In the activated/RACK1+-transfected cells, embryonic gene expression was enhanced compared to the non-transfected control. Moreover, after one week of culture in a GFs-supplemented medium, all the genes were strongly expressed in transfected cells, suggesting some RACK1’s involvement in their translation. In particular, GATA4 is strongly expressed at the nucleus level in transfected/GFs-cultivated cells. Thus, RACK1 overexpression enhances the presence of GATA4 protein (detected by specific antibodies) and increases GATA4 activity in the nucleus. Unfortunately, there are no previous reports about the possible implication of RACK1 in GATA4 translation,

Epicardium-derived cells, which become activated upon injury and migrate to the injured area, can differentiate into various types of myocardial cells. These cells were WT1-positive in both mammals and fish [21,46]. WT1, the Wilms’ tumor suppressor protein, is a zinc finger-containing transcription factor that activates or represses transcription depending on cell type and promoter context and to adpter protein with WD40 motif [47]. After activation, WT1⁺ cells undergo mesenchymal transition and can activate angiogenesis and a new extracellular matrix by stimulating resident fibroblasts [48]. In this research, WT1 expression was enhanced in RACK1⁺/HA-transfected cells after 1 week of culturing in a GFs-supplemented medium and 24 h after transfection. WT1 localization was mainly nuclear. Comparably, in non-transfected cells, WT1 expression was also enhanced after 1 week but less marked as compare the transfected cells. Altogether, these observations suggest a possible RACK1’s role in WT1 protein translation

NFAT2 is a marker of endocardial-activated cells and is expressed early in ZF development. NFAT can change dynamically in phosphorylation status by the action of additional molecules such as nuclear p300, GATA, and RACK1 [49,50]. Transfected RACK1⁺/HA cells after 1 week of culture in GFs-supplemented medium showed high NFAT2 gene expression. The localization was principally cytoplasmatic instead of nuclear. Interestingly, after 1 week of culture in a GFs-supplemented medium, NFAT2 was significantly enhanced in RACK1+/HA cells compared to non-transfected or T0-transfected cells. In previous observations, the overexpression of RACK1 was hypothesized to play a role in NFAT-binding and block its nucleus translocation [45]. However, in our case, the high presence in the cytoplasm and also on the nucleus of NFAT2 in T1 RACK1/HA transfected cells could not simply be explained as a block of translocation because it seems to link to increasing the protein's translation. Interestingly, when RACK1 expression was decreased by myocardial infarction, the cardiomyocyte went into apoptosis [50]. This observation was confirmed by apoptosis blocking in RACK1⁺/HA -transfected cardiomyocytes. In this view, it can also be involved in NFAT2 protein because it regulates PD-1 (programmed death-1) expression [51].

5. Conclusions and mechanistic hypothesis of RACK1 interactions

RACK1, as an adapter protein, interacting with a variety of signaling molecules to act phosphorilation in combination with PKC [52-54]; it is also a component of the 40S subunit of the ribosome by interacting with Scp160 [5] and necessary for the 60s subunit assembly [30, 55]. In particular, RACK1 may be involved in the alternative-translation eIF-3d-dependent by attaching the 5'UTR loop of mRNA [54,30]. RACK1 is also involved in other alternative translation machinery by functioning as an adapter favoring the ribosome 40S/60S junction in the complex ITAF-Internal Ribosome Entry Site (IRES) [56]. The IRES sequence has a longer loop than the eIF3d-dependent, highly structured 5'UTR, which lacks a methylated cap structure at the 5' end and represents the most ancient translation system (conserved from viruses, [57]). Since the translated eIF4E-dependent cap represents a recent machinery type in evolution, it is possible that ancient embryonic genes (common in vertebrates) could be translated by the eIF-3d-dependent or IRES-ITAF typology [57] (Fig.8).

The present research demonstrates the involvement of RACK1 in heart hypertrophy or GFs activation in ex vivo conditions. In samples treated with PE or GFs, RACK1 was enhanced, and we observed significant expressions of GATA4, WT1, and NFAT2. Several indications in mammals confirm that PE stimulation activates signaling pathways for cardiomyocyte hypertrophy, including ERK/JNK/p38 (MAPK), calcineurin-NFAT, JAK/STAT, and p300/GATA4 [33]. In chicken, fibroblast growth factors induced RACK1 and such induction of RACK1 expression was accompanied by a significant augmentation in the number of active PKC molecules/PKC enzymatic activity [58]. Thus, it is not surprising that some key embryonic genes are re-expressed by PE in GFs treatments; however, RACK1 may play a role in this process. By overexpressing a plasmid containing rack1, we confirmed molecule’s involvement in activating cardiac cells and enhancing embryonic gene expression. Although some remarks can be made regarding possible outcomes of RACK1’s loss of function, this was not investigated in this study. Since loss of function or KO models of RACK1 are not viable in mammals [27,59], we decided to limit the experiment to overexpression. Further studies on RACK1 should highlight its direct versus indirect involvement in transcribing and translating embryonic genes since an enhancement in the canonical translation mechanism has also been reported in heart regeneration or hypertrophy [60]. Moreover, indirectly RACK1 could be involved to the translation of specific RBP-bound mRNAs [61].

Ideally, RACK1 may be involved in eIF-3d-dependent translation and enhance GATA4 protein quantity (Fig. 8) or NFAT2. An ongoing bioinformatic analysis found a dimensional loop in the 5’mRNA of GATA4 that attaches to the eIF-3d cap complex and, thus, RACK1, whereas for NFAT2 is still difficult to prove. In the several variants described of WT1 mRNA, there is evidence for a non-AUG (CUG) translation initiation codon upstream of the first AUG. A long 5’UTR sequence, compatible with IRES sequence, could suggests a possible role of RACK1 in the translation [47 51] (Fig. 8). Since NFAT2 translation was enhanced in transfected cells, RACK1 may also be directly involved in the translation of the protein by interacting with alternative translation machinery. This ancient, early-expressing gene may be derived from alternative translation motifs. Further bioinformatic and experimental tests should be conducted to confirm this hypothesis (Fig.8).

Signals identified by comparative studies should be the focus of follow-up candidate approaches to determine the exact roles key signals or genes play in regeneration/disease. They should also investigate how this signal can be manipulated to facilitate reparative process control. In this view, this research represents RACK1’s potential as a candidate marker of cardiac cell activation.

Figure 8. RACK1 activation (A) and hypothesis about RACK1’s possible direct interaction with embryonal marker expression (B). A) From previous research, RACK1 can be activated by PKC that, in turn, is activated by calcium waves induced by PE or a cocktail of FGF (GFs). BL treatment depletes the calcium wave. B) The translation of mRNA of GATA4 could be by IRES sequence because a our bioinformatic analysis find a long sequence in the 5’ UTR. RACK1 interact with ITAF/IRES complex to permit the ribosome assembly. The acetylation of GATA4 by the transcriptional co-activator p300 induces its multimerization and activates its DNA binding activity. GATA4 permits the transcription of cardiac hypertrophic response genes, including atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), and myosin heavy chain 7 (MYH7). WT1 has shown, by preliminary bioinformatic analysis, a long 5’UTR compatible with the IRES sequence. RACK1 can interact with the ITAF/IRES complex for the ribosome assembly during the translation. WT1 as a transcriptional factor can induce the epicardial cells to proliferation and transdifferentiation. NFAT2 can interact with RACK1 in two ways: in the translation by eIF-3d 5’UTR and, by cooperating in transporting the phosphorylated protein to the nucleus. NFAT2 is a powerful transcription factor for proliferation and transdifferentiation of the endocardium and endothelial cells

Author Contributions: Conceptualization, N.R.; methodology, M.C., C.S., D.B.; software, E.C.; validation, F.P, F.S.; formal analysis, D.B.,F.P and E.C.; investigation, N.R.; resources, D.C. and R.G.; data curation, D.B. and F.S.; writing—original draft preparation, N.R.; writing—review and editing, N.R, M.C..; visualization, R.G and C.S; supervision, NR. and C.S.; funding acquisition, D.C. All authors have read and agreed to the published version of the manuscript.”

Funding: This research was funded by Ministry for Universities and Research (MUR): PRIN n.2017FJSM9S_003.

Acknowledgments: The authors are indebted to Dr. Silvia Bongiorni for the help in bioinformatics analysis of Gata4, Wt1 and Nfat2 zebrafish sequences and to Dr. Riccardo Marangon for some help in immunocytochemical experiments.

Disclosers: The authors declare no conflicts of interest. The authors did not use generative AI or AI-assisted technologies in the development of this manuscript

Data availability statement. All authors have seen and approved the submitted manuscript's final version. All data generated or analysed during this study are included in this published article and in its supplementary files. Further data can be available from the corresponding author on reasonable request.

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Rack1 Contributes to the Upregulation of Embryonic Genes in a Model of Cardiac Hypertrophy

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