# IGF1 Knockdown Hinders Myocardial Development through Energy Metabolism Dysfunction Caused by ROS-Dependent FOXO Activation in the Chicken Heart

Authors: Yafan Gong; Jie Yang; Qi Liu; Jingzeng Cai; Yingying Zheng; Yuan Zhang; Dahai Yu; Honggui Liu; Ziwei Zhang
Journal: Oxidative Medicine and Cellular Longevity
 (2019)

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## Abstract

Insulin-like growth factor 1 (IGF1) is a multifunctional cellular regulatory factor that can regulate cell growth and development by mediating growth hormone stimulation. However, the mechanism of IGF1 dysfunction in cardiomyocyte development is seldom reported. To study this, we employed the models of IGF1 knockdown in chicken embryo in vivo and in cardiomyocytes in vitro. We detected the antioxidant capacity, PI3K/Akt pathway, energy metabolism-related genes, and myocardial development-related genes. Our results revealed that the low expression of IGF1 can significantly suppress the antioxidant capacity and increase the ROS (P<0.05) levels, activating the AMPK and PI3K pathway by inhibiting the expression of IRS1. We also found that myocardial energy metabolism is blocked through IGF1, GLUT, and IGFBP inhibition, further inducing myocardial developmental disorder by inhibiting Mesp1, GATA, Nkx2.5, and MyoD expression. Altogether, we conclude that low IGF1 expression can hinder myocardial development through the dysfunction of energy metabolism caused by ROS-dependent FOXO activation.


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## Body

## 1. Introduction

Insulin-like growth factors (IGFs) are a group of polypeptides with growth-promoting function. The secretory cells are widely distributed in tissues such as the liver, kidney, lung, heart, brain, and intestine [1]. IGFs play an important role in cell proliferation, differentiation, individual growth, and development [2]. The IGF family has two subtypes: insulin-like growth factor 1 (IGF1) and insulin-like growth factor 2 (IGF2). The production of IGF1 is dependent on the growth hormone (GH), which is an important growth factor in life processes. Myocardial development is a complex process that is regulated by complex molecular networks composed of many development-related factors. Many studies have shown that various signal pathways are involved in the development of vertebrate hearts, including the bone morphogenetic protein (BMP), Wnt, Notch, and fibroblast growth factor 4 (FGF 4) signal transduction pathways. The BMP and Wnt signaling pathways play an important role in the development of early mesoderm cells into cardiomyocytes; they act on the cardiac-specific transcription factor GATA4 and Nkx2.5 through a signal cascade process, promoting the differentiation of cardiac precursor cells into cardiomyocytes [3, 4]. Musarò et al. demonstrated that localized synthesis of IGF1 is closely related to skeletal muscle hypertrophy, the molecular pathways of which are similar to those responsible for cardiac hypertrophy [5].Insulin is a hormone secreted by isletβ cells, and it is the only hormone that reduces blood sugar and promotes the synthesis of glycogen, fat, and protein in animals [6]. Insulin has been proven to regulate metabolism and growth in the body [7]. The insulin receptor (IR) is a tetramer formed by two alpha subunits and two beta subunits linked by disulfide bonds. The two alpha subunits are located on the outer side of the plasma membrane and have a binding site for insulin; the two beta subunits are transmembrane proteins that play a role in signal transduction. The IR family contains IR, insulin-like growth factor receptor (IGFR), and insulin receptor-related receptor (IRR). Intracellular signaling is initiated by activating intracellular tyrosine kinases through a series of structural conformational changes after IR binding to ligands, which exerts important physiological functions in the body [8]. The cardiac cell membrane is rich in IR, making cardiomyocytes a very important target organ for insulin action. Insulin plays a key role in the regulation of various aspects of cardiovascular metabolism through glucose metabolism, protein synthesis, and vascular tone. The IGF family can regulate cardiac lineage induction by expanding the mesodermal cell population [9]. Bisping et al. demonstrated that although IGF1 is unnecessary for cardiac structure and function, GATA4 must be activated by the IGF1 pathway to exert its function [10].Conformational changes occur in the beta receptor subunit when insulin binds to IR to form a complex, and this leads to autophosphorylation and activation of tyrosine kinase (TK). The complex phosphorylates insulin receptor substrate (IRS) and activates the phosphatidylinositol 3-kinase (PI3K) pathway and mitogen-activated protein kinase (MAPK) pathway. Insulin augments cardiomyocyte contraction, increases ribosomal biogenesis and protein synthesis, stimulates vascular endothelial growth factor (VEGF), and thereby suppresses apoptosis, promoting cell survival and increasing blood perfusion of the myocardium principally through the PKB/Akt signaling pathway [11]. IGF1 can regulate the process of membrane assembly at the axonal growth cone by activating the PI3K pathway [12]. Zhu et al. found that IGF1 can upregulate VEGF-C in breast cancer by mediating the PI3K/Akt and MAPK/ERK1/2 signaling pathways [13]. Treating the smooth muscle cells of the saphenous vein with IGF1 can induce phosphorylation of PI3K-Akt/PKB and promote proliferation of saphenous vein smooth muscle cells [14].Organisms can produce free radicals during normal metabolism, and excessive oxygen free radicals can cause damage to human tissues and cellular structures [15, 16]. The free radical balances can be maintained depending on the antioxidant system. The body can mediate the accumulation of excess reactive oxygen species (ROS) through some cell signal transduction, which enhances the expression of many protective proteins in the cell. IGF can sense the changes in ROS levels and thus affect the insulin pathway [17]. Papaiahgari et al. demonstrated that ROS can mediate the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) through the PI3K/Akt pathway [18].In our previous study, we demonstrated that selenium deficiency disrupted insulin responsiveness through inhibition of the PI3K/Akt pathway by producing excessive oxygen free radicals [19, 20]; meanwhile, selenium deficiency can downregulate the expression of IGF1. However, the role of IGF1 in myocardial development is still less reported; in our present study, we developed models for IGF1 knockdown in cardiomyocyte cultures (siRNA) in vitro and IGF1 knockdown in a chicken embryo model in vivo to detect the effect of IGF1 suppression on energy metabolism, insulin pathways, and myocardial development.


## 2. Materials and Methods

All procedures used in this study were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University (SRM-11).


### 2.1. Primary Cardiomyocyte Culture

Twelve-day-old chicken embryos were used to obtain primary cardiomyocytes for culture. Subsequent to surface disinfection (using 75% alcohol), the chest was dissected to collect the apical portion of the pericardium (approximately 1/3 of the heart), which was immediately transferred to phosphate-buffered solution (PBS) (4°C) and washed to remove fat, connective tissue, and blood clots. Subsequently, the myocardial tissue was cut into small pieces and washed 3 times with PBS. After enzymatic digestion with collagenase-II (0.1%) for 15 minutes on a constant temperature magnetic stirrer (37°C, 100 r/min) for acclimatization and centrifugation, an equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM)/F12w containing 10% fetal bovine serum and 1x mycillin was added to terminate digestion. The pellet was digested until the small tissue fragments were completely digested. All supernatants were collected with 300 mesh and 500 mesh filters. The cell suspension was centrifuged at 600 rpm for 5 minutes and resuspended in DMEM/F12w twice in disposable Petri dishes for differential adhesion (the first was 1 h; the second was 1.5 h). Nonadherent cells (cardiomyocytes) were collected, centrifuged at 600 rpm, counted, plated in 6-well plates at2×105, and incubated at 37°C, 5% CO2, in an adherent culture incubator for 48 h [21].


### 2.2. Establishment of the IGF1 Knockdown Model In Vitro

Cardiomyocytes attain 80% confluence after approximately 48 h of incubation. In chicken cardiomyocyte primary cultures, which are the same as described in our previous experimental method, IGF1 was knocked down using siRNA (sense 5′-GTTCGTATGTGGAGACAGA-3′, anti-sense 5′-TCTGTCTCCACATACGAAC-3′) subsequent to two washes with Opti-MEM (prewarmed). All cells were randomly divided into two groups: C (control group) and KD (knockdown group). For each group, 3 replicates were prepared with 6×105 cardiomyocytes per replicate (n=3). Cells in the knockdown group were transfected with 3 μL of 20 μM siRNA and 3 μL of Lipofectamine RNAi MAX Reagent (Invitrogen) in 2 mL of Opti-MEM. Cells in the control group were treated with 2 mL of Opti-MEM (Invitrogen), which only contain the same volume of Lipofectamine RNAi MAX Reagent. Approximately 48 h posttransfection, the cells were harvested for analysis.


### 2.3. Establishment of the IGF1 Silence Model In Vivo

First, 50μg of nucleic acid was diluted with pure water without endotoxin to a concentration of 1 μg/μL; the final concentration of glucose was 5%, and the final volume was 100 μL. Then, 25 μL of Entranster™ in vivo reagent was diluted with 50 μL of 10% glucose solution and supplemented with pure water. The final concentration of running glucose was 5%, and the final volume was 100 μL of liquid.The diluted transfection reagent was added to the diluted nucleic acid solution to form a transfection complex, which was then left at room temperature for 15 min. Ninety hatching eggs were randomly divided into two groups (45 per group), viz., the normal group (N) and siRNA group (Si). The subgerminal cavity of each egg was injected with 1μg siRNA and sealed with a sealing film. All of the eggs were incubated in a constant temperature incubator. The hearts were taken at 6, 8, and 10 days for subsequent experiments.


### 2.4. Detection of Intracellular ROS Accumulation

Posttransfection, ROS activities were measured using an ROS assay kit (Nanjing Jiancheng Bioengineering Institute, China). First, 10μM DCFH-DA (2,7-dichlorofurescin diacetate) was added to the culture medium, containing the cell samples to be tested, which was then incubated at a constant temperature (37°C) for 45 min. Then, the medium was discarded, and PBS (37°C preheat) was used to wash the cells three times. Finally, the cells were collected for detecting the activities of ROS at the excitation wavelength of 500±15nm and emission wavelength of 530±20nm. The cardiomyocytes were visualized using fluorescence microscopy.


### 2.5. Determination of Oxidative Stress Markers

Cells were grown on 6-well plates at a density of3×105mL−1, collected with jets of saline, and centrifuged at 700 × g; then, the supernatant was collected. The hearts of chicken embryos were taken for homogenization in saline solution and centrifuged at 700 g for 20 min, and the supernatants were collected. The hydrogen peroxide (H2O2), glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), induced nitric oxide synthase (iNOS), superoxide dismutase (SOD), and total antioxidant capability (T-AOC) contents were measured by detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s protocols. The SOD activity was measured at 25°C using autooxidation of pyrogallol in 50 Mm Tris/HCl, pH 8, with 100 mM pyrogallol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).


### 2.6. Determination of the mRNA Expression of the Genes Related to IGF1, the PI3K/Akt Pathway, Insulin, and Cardiac Differentiation

Total RNA was isolated from heart tissues of three points in time and cardiomyocytes by using the TRIzol reagent according to the manufacturer’s instructions (Roche, Basel, Switzerland). The dried RNA pellets were resuspended in 50μL of diethyl pyrocarbonate-treated water. The concentration and purity of the total RNA were determined using a spectrophotometer. cDNA was synthesized from 5 μg of the total RNA using oligo-dT primers and Superscript II reverse transcriptase according to the manufacturer’s instructions (Promega, Beijing, China). cDNA was diluted at a ratio of 1 : 5 with sterile water and stored at −80°C.Primer Premier Software (PREMIER Biosoft International, USA) was used to design specific primers for IGF1 and AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), c-Jun N-terminal kinase (JNK), threonine-protein kinase (Akt), forkhead box protein (FOXO), insulin-like growth factor 1 receptor (IGF1R), glucose transporter-1 (GLUT1), glucose transporter-3 (GLUT3), glucose transporter-8 (GLUT8), insulin-like growth factor-binding protein-1 (IGFBP1), insulin-like growth factor-binding protein-2 (IGFBP2), insulin-like growth factor-binding protein-3 (IGFBP3), insulin-like growth factor-binding protein-4 (IGFBP4), insulin-like growth factor-binding protein-5 (IGFBP5), insulin-like growth factor-binding protein-7 (IGFBP7), insulin receptor (IR), insulin receptor substrate-1 (IRS1), MyoD, myogenin (MyoG), cardiac transcription factor mesoderm posterior 1 (Mesp1), myogenic factor-5 (MYF5), myogenic factor-6 (MYF6), GATA-binding protein 4 (GATA4), GATA-binding protein 6 (GATA6), NK2 homeobox 5 (Nkx2.5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) based on known chicken sequences (Table1). First, general PCR was performed to confirm the specificity of the primers. Quantitative real-time PCR (qPCR) was then performed with a Roche detection system (Applied Biosystems, Foster City, CA). The reactions were conducted in a 20 μL reaction mixture containing 10 μL of 2x SYBR Green I PCR Master Mix (Roche, Basel, Switzerland), 2 μL of cDNA, 0.4 μL of each primer (10 μM), 0.4 μL of 50x ROX reference Dye II, and 6.8 μL of PCR-grade water. The PCR procedure for IGF1 and AMPK, PI3K, JNK, Akt, FOXO, IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, IRS1, MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, Nkx2.5, and GAPDH consisted of 95°C for 30 s followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 60°C for 30 s. For each PCR, Dissociation Curve 1.0 Software (Applied Biosystems) was used to analyze the dissociation curves in order to detect and eliminate possible primer dimers and nonspecific amplification.Table 1
The primers used in the present study.
Target gene
Primer sequence (5′-3′)
IGF1
Forward 5′-GCTTTTGTGATTTCTTGAAGGTGAA-3′
Reverse 5′-CATACCCTGTAGGCTTACTGAAGTA-3′
AMPK
Forward 5′-CCATCTGTCATTAGTCTTCTG-3′
Reverse 5′-AGGCTTCGTCATCAATCAT-3′
PI3K
Forward 5′-GTCCTTGAGCCACTGATG-3′
Reverse 5′-TGTTGCCTTACGGTTGTT-3′
Akt
Forward 5′-AGGAGGAAGAGATGATGGAT-3′
Reverse 5′-GAATGGATGCCGTGAGTT-3′
JNK
Forward 5′-CAGATAAGCAGTTAGATGAGAG-3′
Reverse 5′-GACAGATGACGACGAAGAT-3′
FOXO
Forward 5′-CAGCAATGTCAAGGAGAGCA-3′
Reverse 5′-TGAAGAGGTTGTCCGAGTCC-3′
IGF1R
Forward 5′-GCGTGAGAGGATAGAGTTC-3′
Reverse 5′-TGTTGGCGTTGAGGTATG-3′
GLUT1
Forward 5′-TAGTACTGGAGCAGGTGGCAGA-3′
Reverse 5′-CGGCACAAGAATGGATGAAA-3′
GLUT3
Forward 5′-TCCCCAGAGCTTCTTACCTCAC-3′
Reverse 5′-CAGCAAAAGCCAAGACATTCAC-3′
GLUT8
Forward 5′-CCAAATGGGAACAACTCATCAA-3′
Reverse 5′-GGGCAAAACCAGCAACAAA-3′
IGFBP1
Forward 5′-TGGCTCGGGCTAGCTGGATG-3′
Reverse 5′-ACCAGCACCCAGCGGAATCT-3′
IGFBP2
Forward 5′-TGTGACAAGCATGGCTTGTACA-3′
Reverse 5′-TCTCCACGCTGCCCATTC-3′
IGFBP3
Forward 5′-ATGGTCCCTGTCGTAGAG-3′
Reverse 5′-ATCCAGGAAGCGGTTGT-3′
IGFBP4
Forward 5′-TGGTGCGTGGACCGCAAGAC′-3′
Reverse 5′-AGCGATGGGGGCGTCCCATA-3′
IGFBP5
Forward 5′-TGTGCCTCTGGCAGGGGGTA-3′
Reverse 5′-CAACACAGCCCACGCTTCCG-3′
IGFBP7
Forward 5′-TGTGAAGTCATTGGCATCC-3′
Reverse 5′-CCTCTCCTTTGGCATTTGA-3′
IR
Forward 5′-CAAACGGTGACCAAGCCTCA-3′
Reverse 5′-CATCCTGCCCATCAAACTCC-3′
IRS1
Forward 5′-TCCACCACCACCACCATCAC-3′
Reverse 5′-ACAGCAGCCGCATCCGAAT-3′
MyoD
Forward 5′-CCGCCGATGACTTCTATG-3′
Reverse 5′-GTTGGTGGTCTTCCTCTTG-3′
MyoG
Forward 5′-AGGCTGAAGAAGGTGAAC-3′
Reverse 5′-GCTCGATGTACTGGATGG-3′
Mesp1
Forward 5′-GGTCATCACCCTCCTACA-3′
Reverse 5′-CCATCTCTGCATCCACAA-3′
MYF5
Forward 5′-GAGGAGGAGGCTGAAGAA-3′
Reverse 5′-CGGCAGGTGATAGTAGTTC-3′
MYF6
Forward 5′-GGAGGAGGCTGAAGAAGA-3′
Reverse 5′-CTCTCGATGTAGCTGATGG-3′
GATA4
Forward 5′-TCAGACAAGGAAGCGTAAG-3′
Reverse 5′-ATGGCAGAGACCGAGAAT-3′
GATA6
Forward 5′-CCGACCACTTGCTATGAA-3′
Reverse 5′-TTGCTACAGTCATCTGAGTT-3′
Nkx2.5
Forward 5′-GACAGAGGAAGAGGAGGAA-3′
Reverse 5′-CGTTCGCTAGATGGTCTC-3′
GAPDH
Forward 5′-AGAACATCATCCCAGCGT-3′
Reverse 5′-AGCCTTCACTACCCTCTTG-3′


### 2.7. Determination of the Protein Expression of the Proteins Related to IGF1, the PI3K/Akt Pathway, Insulin, and Cardiac Differentiation

For total protein extraction, protein lysates were subjected to 15% SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were then transferred to a nitrocellulose membrane for 2 h at 100 mA in a transfer apparatus containing Tris-glycine buffer and 20% methanol. The membrane was blocked with 5% skim milk for 24 h and incubated overnight with diluted primary antibodies against IGF1 (1 : 500, Proteintech, China), PI3K (1 : 1000, Santa Cruz Biotechnology, USA), Akt (1 : 500, Santa Cruz Biotechnology, USA), P-Akt (1 : 500, Proteintech, China), FOXO (1 : 1000, Santa Cruz Biotechnology, USA), P-FOXO (1 : 1000, Santa Cruz Biotechnology, USA), JNK (1 : 1000, Santa Cruz Biotechnology, USA), P-JNK (1 : 1000, Santa Cruz Biotechnology, USA), 14-3-3 (1 : 1000, Abcam, Cambridge, UK), P-14-3-3 (1 : 1000, Santa Cruz Biotechnology, USA), GLUT3 (1 : 300, Santa Cruz Biotechnology, USA), IGF1Rβ (1 : 500, Proteintech, China), IGFBP2 (1 : 500, Proteintech, China), MyoG (1 : 1000, Abcam, Cambridge, UK), and MyoD (1 : 1000, Abcam, Cambridge, UK) followed by a horseradish peroxidase- (HRP-) conjugated secondary antibody against rabbit (IGF1, PI3K, Akt, P-Akt, FOXO, P-FOXO, JNK, P-JNK, 14-3-3, P-14-3-3, GLUT, IGF1Rβ, IGFBP2, MyoG, and MyoD) IgG (1 : 5000, Santa Cruz Biotechnology, USA). To verify equal loading of the samples, the membrane was incubated with a monoclonal GAPDH antibody (1 : 1500, Santa Cruz Biotechnology, USA), followed by an HRP-conjugated goat anti-mouse IgG (1 : 3000) secondary antibody. The signal was detected with X-ray films (TransGen Biotech Co., Beijing, China). The optical density (OD) of each band was determined using an Image VCD gel imaging system, and the relative abundance of IGF1, PI3K, Akt, P-Akt, FOXO, P-FOXO, JNK, P-JNK, 14-3-3, P-14-3-3, GLUT3, IGF1Rβ, IGFBP2, MyoG, and MyoD proteins was calculated and presented as the ratios of OD of each of these proteins to that of GAPDH.


### 2.8. Measurement of ATP

Cardiomyocytes were grown on 6-well plates at a density of2×105cells/mL, gathered with the lysis solution, and centrifuged at 700 × g. The supernatant was collected, resuspended by salt water and incubated for 35 min at 25°C. The level of adenosine triphosphate (ATP) in the cardiomyocytes was measured by using an ATP detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The detection was carried out using an ultraviolet spectrophotometer (Synergy NEO, BioTek Instruments) with a detection wavelength of 636 nm.


### 2.9. Histopathological Examination

Cardiomyocytes were grown at a density of2×105cells/mL and then washed with PBS three times; 4% paraformaldehyde solution was added in a 24-well plate for cell fixing. After 12 h, the 4% paraformaldehyde solution in the 24-well plate was removed and 0.01 M PBS was added; then, the wells were soaked for 5min×3times. Hematoxylin staining solution was added to the wells and immersed for 1 min. The staining solution was removed, and distilled water was added to soak for 5 min. The distilled water was then removed and placed in 1% hydrochloric acid alcohol. After 1-3 s, it was aspirated. Tap water was added to soak for 5 min to return the cells to blue. Then, the tap water was removed, and Yihong dye solution was added to soak for 1 min. The eosin staining solution was aspirated and soaked in distilled water for 1 min. The climbing pieces were removed, and glycerol ethanol was added dropwise. The staining effect was observed under a microscope and photographed under a 200x microscope [22].The myocardial tissues were rapidly fixed in 10% formaldehyde for at least 24 h and embedded in paraffin for microscopic examination. From the prepared paraffin blocks, sections (5μm thick) were cut, obtained, and stained with hematoxylin and eosin (H.E.) for light microscopic observation.


### 2.10. High-Resolution Respirometry of Mitochondrial Function

Cardiomyocytes were grown on 6-well plates at a density of2×105cells/mL. All cells were randomly divided into two groups: C (control group) and KD (knockdown group). The cells in the knockdown group were transfected with 3 μL of 20 μM siRNA and 3 μL of Lipofectamine RNAi MAX Reagent (Invitrogen) in 2 mL of Opti-MEM. The cells in the control group were treated with 2 mL of Opti-MEM (Invitrogen) containing the same volume of Lipofectamine RNAi MAX Reagent. Approximately 48 h posttransfection, the cells were harvested for analysis, gathered with the lysis solution, and centrifuged at 700 × g. The supernatant was collected and resuspended by the medium for high-resolution respirometry. The mitochondrial respiratory function was analyzed in a two-channel titration injection respirometer (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). The cell suspension was transferred separately to oxygraph chambers at a final density of approximately 2×105cells/mL. After a short stabilization period, the chambers were closed and data were recorded using DatLab software 5.2 (Oroboros Instruments, Innsbruck, Austria).


### 2.11. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 5.0 software, and all data was assessed using the unpairedt-test, where P<0.05 was considered a statistically significant difference.


## 2.1. Primary Cardiomyocyte Culture

Twelve-day-old chicken embryos were used to obtain primary cardiomyocytes for culture. Subsequent to surface disinfection (using 75% alcohol), the chest was dissected to collect the apical portion of the pericardium (approximately 1/3 of the heart), which was immediately transferred to phosphate-buffered solution (PBS) (4°C) and washed to remove fat, connective tissue, and blood clots. Subsequently, the myocardial tissue was cut into small pieces and washed 3 times with PBS. After enzymatic digestion with collagenase-II (0.1%) for 15 minutes on a constant temperature magnetic stirrer (37°C, 100 r/min) for acclimatization and centrifugation, an equal volume of Dulbecco’s Modified Eagle’s Medium (DMEM)/F12w containing 10% fetal bovine serum and 1x mycillin was added to terminate digestion. The pellet was digested until the small tissue fragments were completely digested. All supernatants were collected with 300 mesh and 500 mesh filters. The cell suspension was centrifuged at 600 rpm for 5 minutes and resuspended in DMEM/F12w twice in disposable Petri dishes for differential adhesion (the first was 1 h; the second was 1.5 h). Nonadherent cells (cardiomyocytes) were collected, centrifuged at 600 rpm, counted, plated in 6-well plates at2×105, and incubated at 37°C, 5% CO2, in an adherent culture incubator for 48 h [21].


## 2.2. Establishment of the IGF1 Knockdown Model In Vitro

Cardiomyocytes attain 80% confluence after approximately 48 h of incubation. In chicken cardiomyocyte primary cultures, which are the same as described in our previous experimental method, IGF1 was knocked down using siRNA (sense 5′-GTTCGTATGTGGAGACAGA-3′, anti-sense 5′-TCTGTCTCCACATACGAAC-3′) subsequent to two washes with Opti-MEM (prewarmed). All cells were randomly divided into two groups: C (control group) and KD (knockdown group). For each group, 3 replicates were prepared with 6×105 cardiomyocytes per replicate (n=3). Cells in the knockdown group were transfected with 3 μL of 20 μM siRNA and 3 μL of Lipofectamine RNAi MAX Reagent (Invitrogen) in 2 mL of Opti-MEM. Cells in the control group were treated with 2 mL of Opti-MEM (Invitrogen), which only contain the same volume of Lipofectamine RNAi MAX Reagent. Approximately 48 h posttransfection, the cells were harvested for analysis.


## 2.3. Establishment of the IGF1 Silence Model In Vivo

First, 50μg of nucleic acid was diluted with pure water without endotoxin to a concentration of 1 μg/μL; the final concentration of glucose was 5%, and the final volume was 100 μL. Then, 25 μL of Entranster™ in vivo reagent was diluted with 50 μL of 10% glucose solution and supplemented with pure water. The final concentration of running glucose was 5%, and the final volume was 100 μL of liquid.The diluted transfection reagent was added to the diluted nucleic acid solution to form a transfection complex, which was then left at room temperature for 15 min. Ninety hatching eggs were randomly divided into two groups (45 per group), viz., the normal group (N) and siRNA group (Si). The subgerminal cavity of each egg was injected with 1μg siRNA and sealed with a sealing film. All of the eggs were incubated in a constant temperature incubator. The hearts were taken at 6, 8, and 10 days for subsequent experiments.


## 2.4. Detection of Intracellular ROS Accumulation

Posttransfection, ROS activities were measured using an ROS assay kit (Nanjing Jiancheng Bioengineering Institute, China). First, 10μM DCFH-DA (2,7-dichlorofurescin diacetate) was added to the culture medium, containing the cell samples to be tested, which was then incubated at a constant temperature (37°C) for 45 min. Then, the medium was discarded, and PBS (37°C preheat) was used to wash the cells three times. Finally, the cells were collected for detecting the activities of ROS at the excitation wavelength of 500±15nm and emission wavelength of 530±20nm. The cardiomyocytes were visualized using fluorescence microscopy.


## 2.5. Determination of Oxidative Stress Markers

Cells were grown on 6-well plates at a density of3×105mL−1, collected with jets of saline, and centrifuged at 700 × g; then, the supernatant was collected. The hearts of chicken embryos were taken for homogenization in saline solution and centrifuged at 700 g for 20 min, and the supernatants were collected. The hydrogen peroxide (H2O2), glutathione (GSH), glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), induced nitric oxide synthase (iNOS), superoxide dismutase (SOD), and total antioxidant capability (T-AOC) contents were measured by detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer’s protocols. The SOD activity was measured at 25°C using autooxidation of pyrogallol in 50 Mm Tris/HCl, pH 8, with 100 mM pyrogallol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).


## 2.6. Determination of the mRNA Expression of the Genes Related to IGF1, the PI3K/Akt Pathway, Insulin, and Cardiac Differentiation

Total RNA was isolated from heart tissues of three points in time and cardiomyocytes by using the TRIzol reagent according to the manufacturer’s instructions (Roche, Basel, Switzerland). The dried RNA pellets were resuspended in 50μL of diethyl pyrocarbonate-treated water. The concentration and purity of the total RNA were determined using a spectrophotometer. cDNA was synthesized from 5 μg of the total RNA using oligo-dT primers and Superscript II reverse transcriptase according to the manufacturer’s instructions (Promega, Beijing, China). cDNA was diluted at a ratio of 1 : 5 with sterile water and stored at −80°C.Primer Premier Software (PREMIER Biosoft International, USA) was used to design specific primers for IGF1 and AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), c-Jun N-terminal kinase (JNK), threonine-protein kinase (Akt), forkhead box protein (FOXO), insulin-like growth factor 1 receptor (IGF1R), glucose transporter-1 (GLUT1), glucose transporter-3 (GLUT3), glucose transporter-8 (GLUT8), insulin-like growth factor-binding protein-1 (IGFBP1), insulin-like growth factor-binding protein-2 (IGFBP2), insulin-like growth factor-binding protein-3 (IGFBP3), insulin-like growth factor-binding protein-4 (IGFBP4), insulin-like growth factor-binding protein-5 (IGFBP5), insulin-like growth factor-binding protein-7 (IGFBP7), insulin receptor (IR), insulin receptor substrate-1 (IRS1), MyoD, myogenin (MyoG), cardiac transcription factor mesoderm posterior 1 (Mesp1), myogenic factor-5 (MYF5), myogenic factor-6 (MYF6), GATA-binding protein 4 (GATA4), GATA-binding protein 6 (GATA6), NK2 homeobox 5 (Nkx2.5), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) based on known chicken sequences (Table1). First, general PCR was performed to confirm the specificity of the primers. Quantitative real-time PCR (qPCR) was then performed with a Roche detection system (Applied Biosystems, Foster City, CA). The reactions were conducted in a 20 μL reaction mixture containing 10 μL of 2x SYBR Green I PCR Master Mix (Roche, Basel, Switzerland), 2 μL of cDNA, 0.4 μL of each primer (10 μM), 0.4 μL of 50x ROX reference Dye II, and 6.8 μL of PCR-grade water. The PCR procedure for IGF1 and AMPK, PI3K, JNK, Akt, FOXO, IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, IRS1, MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, Nkx2.5, and GAPDH consisted of 95°C for 30 s followed by 40 cycles of 95°C for 15 s, 60°C for 30 s, and 60°C for 30 s. For each PCR, Dissociation Curve 1.0 Software (Applied Biosystems) was used to analyze the dissociation curves in order to detect and eliminate possible primer dimers and nonspecific amplification.Table 1
The primers used in the present study.
Target gene
Primer sequence (5′-3′)
IGF1
Forward 5′-GCTTTTGTGATTTCTTGAAGGTGAA-3′
Reverse 5′-CATACCCTGTAGGCTTACTGAAGTA-3′
AMPK
Forward 5′-CCATCTGTCATTAGTCTTCTG-3′
Reverse 5′-AGGCTTCGTCATCAATCAT-3′
PI3K
Forward 5′-GTCCTTGAGCCACTGATG-3′
Reverse 5′-TGTTGCCTTACGGTTGTT-3′
Akt
Forward 5′-AGGAGGAAGAGATGATGGAT-3′
Reverse 5′-GAATGGATGCCGTGAGTT-3′
JNK
Forward 5′-CAGATAAGCAGTTAGATGAGAG-3′
Reverse 5′-GACAGATGACGACGAAGAT-3′
FOXO
Forward 5′-CAGCAATGTCAAGGAGAGCA-3′
Reverse 5′-TGAAGAGGTTGTCCGAGTCC-3′
IGF1R
Forward 5′-GCGTGAGAGGATAGAGTTC-3′
Reverse 5′-TGTTGGCGTTGAGGTATG-3′
GLUT1
Forward 5′-TAGTACTGGAGCAGGTGGCAGA-3′
Reverse 5′-CGGCACAAGAATGGATGAAA-3′
GLUT3
Forward 5′-TCCCCAGAGCTTCTTACCTCAC-3′
Reverse 5′-CAGCAAAAGCCAAGACATTCAC-3′
GLUT8
Forward 5′-CCAAATGGGAACAACTCATCAA-3′
Reverse 5′-GGGCAAAACCAGCAACAAA-3′
IGFBP1
Forward 5′-TGGCTCGGGCTAGCTGGATG-3′
Reverse 5′-ACCAGCACCCAGCGGAATCT-3′
IGFBP2
Forward 5′-TGTGACAAGCATGGCTTGTACA-3′
Reverse 5′-TCTCCACGCTGCCCATTC-3′
IGFBP3
Forward 5′-ATGGTCCCTGTCGTAGAG-3′
Reverse 5′-ATCCAGGAAGCGGTTGT-3′
IGFBP4
Forward 5′-TGGTGCGTGGACCGCAAGAC′-3′
Reverse 5′-AGCGATGGGGGCGTCCCATA-3′
IGFBP5
Forward 5′-TGTGCCTCTGGCAGGGGGTA-3′
Reverse 5′-CAACACAGCCCACGCTTCCG-3′
IGFBP7
Forward 5′-TGTGAAGTCATTGGCATCC-3′
Reverse 5′-CCTCTCCTTTGGCATTTGA-3′
IR
Forward 5′-CAAACGGTGACCAAGCCTCA-3′
Reverse 5′-CATCCTGCCCATCAAACTCC-3′
IRS1
Forward 5′-TCCACCACCACCACCATCAC-3′
Reverse 5′-ACAGCAGCCGCATCCGAAT-3′
MyoD
Forward 5′-CCGCCGATGACTTCTATG-3′
Reverse 5′-GTTGGTGGTCTTCCTCTTG-3′
MyoG
Forward 5′-AGGCTGAAGAAGGTGAAC-3′
Reverse 5′-GCTCGATGTACTGGATGG-3′
Mesp1
Forward 5′-GGTCATCACCCTCCTACA-3′
Reverse 5′-CCATCTCTGCATCCACAA-3′
MYF5
Forward 5′-GAGGAGGAGGCTGAAGAA-3′
Reverse 5′-CGGCAGGTGATAGTAGTTC-3′
MYF6
Forward 5′-GGAGGAGGCTGAAGAAGA-3′
Reverse 5′-CTCTCGATGTAGCTGATGG-3′
GATA4
Forward 5′-TCAGACAAGGAAGCGTAAG-3′
Reverse 5′-ATGGCAGAGACCGAGAAT-3′
GATA6
Forward 5′-CCGACCACTTGCTATGAA-3′
Reverse 5′-TTGCTACAGTCATCTGAGTT-3′
Nkx2.5
Forward 5′-GACAGAGGAAGAGGAGGAA-3′
Reverse 5′-CGTTCGCTAGATGGTCTC-3′
GAPDH
Forward 5′-AGAACATCATCCCAGCGT-3′
Reverse 5′-AGCCTTCACTACCCTCTTG-3′


## 2.7. Determination of the Protein Expression of the Proteins Related to IGF1, the PI3K/Akt Pathway, Insulin, and Cardiac Differentiation

For total protein extraction, protein lysates were subjected to 15% SDS-polyacrylamide gel electrophoresis under reducing conditions. The separated proteins were then transferred to a nitrocellulose membrane for 2 h at 100 mA in a transfer apparatus containing Tris-glycine buffer and 20% methanol. The membrane was blocked with 5% skim milk for 24 h and incubated overnight with diluted primary antibodies against IGF1 (1 : 500, Proteintech, China), PI3K (1 : 1000, Santa Cruz Biotechnology, USA), Akt (1 : 500, Santa Cruz Biotechnology, USA), P-Akt (1 : 500, Proteintech, China), FOXO (1 : 1000, Santa Cruz Biotechnology, USA), P-FOXO (1 : 1000, Santa Cruz Biotechnology, USA), JNK (1 : 1000, Santa Cruz Biotechnology, USA), P-JNK (1 : 1000, Santa Cruz Biotechnology, USA), 14-3-3 (1 : 1000, Abcam, Cambridge, UK), P-14-3-3 (1 : 1000, Santa Cruz Biotechnology, USA), GLUT3 (1 : 300, Santa Cruz Biotechnology, USA), IGF1Rβ (1 : 500, Proteintech, China), IGFBP2 (1 : 500, Proteintech, China), MyoG (1 : 1000, Abcam, Cambridge, UK), and MyoD (1 : 1000, Abcam, Cambridge, UK) followed by a horseradish peroxidase- (HRP-) conjugated secondary antibody against rabbit (IGF1, PI3K, Akt, P-Akt, FOXO, P-FOXO, JNK, P-JNK, 14-3-3, P-14-3-3, GLUT, IGF1Rβ, IGFBP2, MyoG, and MyoD) IgG (1 : 5000, Santa Cruz Biotechnology, USA). To verify equal loading of the samples, the membrane was incubated with a monoclonal GAPDH antibody (1 : 1500, Santa Cruz Biotechnology, USA), followed by an HRP-conjugated goat anti-mouse IgG (1 : 3000) secondary antibody. The signal was detected with X-ray films (TransGen Biotech Co., Beijing, China). The optical density (OD) of each band was determined using an Image VCD gel imaging system, and the relative abundance of IGF1, PI3K, Akt, P-Akt, FOXO, P-FOXO, JNK, P-JNK, 14-3-3, P-14-3-3, GLUT3, IGF1Rβ, IGFBP2, MyoG, and MyoD proteins was calculated and presented as the ratios of OD of each of these proteins to that of GAPDH.


## 2.8. Measurement of ATP

Cardiomyocytes were grown on 6-well plates at a density of2×105cells/mL, gathered with the lysis solution, and centrifuged at 700 × g. The supernatant was collected, resuspended by salt water and incubated for 35 min at 25°C. The level of adenosine triphosphate (ATP) in the cardiomyocytes was measured by using an ATP detection kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The detection was carried out using an ultraviolet spectrophotometer (Synergy NEO, BioTek Instruments) with a detection wavelength of 636 nm.


## 2.9. Histopathological Examination

Cardiomyocytes were grown at a density of2×105cells/mL and then washed with PBS three times; 4% paraformaldehyde solution was added in a 24-well plate for cell fixing. After 12 h, the 4% paraformaldehyde solution in the 24-well plate was removed and 0.01 M PBS was added; then, the wells were soaked for 5min×3times. Hematoxylin staining solution was added to the wells and immersed for 1 min. The staining solution was removed, and distilled water was added to soak for 5 min. The distilled water was then removed and placed in 1% hydrochloric acid alcohol. After 1-3 s, it was aspirated. Tap water was added to soak for 5 min to return the cells to blue. Then, the tap water was removed, and Yihong dye solution was added to soak for 1 min. The eosin staining solution was aspirated and soaked in distilled water for 1 min. The climbing pieces were removed, and glycerol ethanol was added dropwise. The staining effect was observed under a microscope and photographed under a 200x microscope [22].The myocardial tissues were rapidly fixed in 10% formaldehyde for at least 24 h and embedded in paraffin for microscopic examination. From the prepared paraffin blocks, sections (5μm thick) were cut, obtained, and stained with hematoxylin and eosin (H.E.) for light microscopic observation.


## 2.10. High-Resolution Respirometry of Mitochondrial Function

Cardiomyocytes were grown on 6-well plates at a density of2×105cells/mL. All cells were randomly divided into two groups: C (control group) and KD (knockdown group). The cells in the knockdown group were transfected with 3 μL of 20 μM siRNA and 3 μL of Lipofectamine RNAi MAX Reagent (Invitrogen) in 2 mL of Opti-MEM. The cells in the control group were treated with 2 mL of Opti-MEM (Invitrogen) containing the same volume of Lipofectamine RNAi MAX Reagent. Approximately 48 h posttransfection, the cells were harvested for analysis, gathered with the lysis solution, and centrifuged at 700 × g. The supernatant was collected and resuspended by the medium for high-resolution respirometry. The mitochondrial respiratory function was analyzed in a two-channel titration injection respirometer (Oxygraph-2k; Oroboros Instruments, Innsbruck, Austria). The cell suspension was transferred separately to oxygraph chambers at a final density of approximately 2×105cells/mL. After a short stabilization period, the chambers were closed and data were recorded using DatLab software 5.2 (Oroboros Instruments, Innsbruck, Austria).


## 2.11. Statistical Analysis

Statistical analyses were performed using GraphPad Prism 5.0 software, and all data was assessed using the unpairedt-test, where P<0.05 was considered a statistically significant difference.


## 3. Results

### 3.1. Development of an IGF1 Knockdown Model in Cells and Chicken Embryos

The mRNA and protein levels of IGF1 were significantly decreased (P<0.05) in the KD group (Figures 1(a) and 1(b)). The results confirmed that we successfully established the model of IGF1 knockdown in vitro.Figure 1
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of the IGF1 gene in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of the IGF1 gene in the myocardium. The results were calculated from at least three independent experiments,n=3. The data are expressed as the means±SD. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. ∗ indicates a significant difference from the corresponding control (P<0.05).
(a)
(b)
(c)
(d)The mRNA levels of IGF1 were detected at 6, 8, and 10 days, and we found that the expression of IGF1 was significantly decreased at 10 days. For further verification, we took chicken embryos at 10 days to detect the protein level of IGF1. Compared with the N group, the mRNA levels in the Si group decreased (P<0.05) at 8 days and 10 days (Figure 1(c)). The Si group exhibited significantly decreased protein levels compared with the N group (Figure 1(d)). The results confirmed that we successfully established the IGF1 silence model in vivo.


### 3.2. Detection of the Antioxidant Capacity in Cells and Chicken Embryos

To assess the relationship between oxidative stress and IGF1, the production of ROS, the levels of H2O2, MDA, and T-AOC and the activities of GSH, GSH-Px, SOD, CAT, and iNOS were measured in cardiomyocytes. As presented in Figure 2, the ROS activities were significantly increased (P<0.05) compared with the C group. The levels of H2O2, MDA, and iNOS were significantly increased in the KD group (P<0.05) (Figures 3(d)–3(f)). The levels of GSH, GSH-Px, CAT, SOD, and T-AOC in the KD group were significantly lower than those in the C group (P<0.05) (Figures 3(a)–3(c), 3(g), and 3(h)).Figure 2
(a) ROS generation was performed by immunofluorescence using DCFH-DA (green fluorescence, 5 mM) in cells. C indicates the control group; KD indicates the knockdown group. Cardiomyocytes were visualized using fluorescence microscopy. (b) The effects of IGF1 knockdown on the ROS levels in cardiomyocytes were detected by using a fluorescence microplate reader. C indicates the control group; KD indicates the knockdown group.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)Figure 3
(a–h) Oxidative stress markers of the GSH, GSH-Px, CAT, H2O2, MDA, iNOS, SOD, and T-AOC contents were measured in cardiomyocytes. (i–p) Oxidative stress markers of GSH, GSH-Px, CAT, H2O2, MDA, iNOS, SOD, and T-AOC contents were measured in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. ∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)To further demonstrate the antioxidant capacity of IGF1 silence in vivo, we also studied the antioxidant capacity of the myocardium. As shown in Figure3, the levels of H2O2, MDA, and iNOS were significantly increased in the Si group (P<0.05) at 6, 8, and 10 days (Figures 3(l)–3(n)). The levels of GSH, GSH-Px, CAT, SOD, and T-AOC in the Si group were significantly lower than those in the N group (P<0.05) at 6, 8, and 10 days (Figures 3(i), 3(j), 3(l), 3(o), and 3(p)).


### 3.3. Protein and mRNA Expression of the PI3K/Akt Pathway-Related Genes in Cells and Chicken Embryos

To examine whether IGF1 knockdown changed the expressions of the PI3K/Akt pathway-related genes, we detected the mRNA expression levels of AMPK, PI3K, JNK, Akt, and FOXO as well as the protein expression levels of FOXO, P-FOXO, JNK, P-JNK, PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3. The effects of IGF1 knockdown on the mRNA abundance of PI3K-related genes in chicken cardiomyocytes are shown in Figure4(a). Compared with the C group, the qPCR results revealed that the mRNA expression of AMPK, JNK, and FOXO was significantly increased (P<0.05) in the KD group. However, the mRNA expression of PI3K and Akt was decreased (P<0.05). The results revealed that compared with the C group, the protein expression of FOXO, P-FOXO, JNK, and P-JNK in the KD group was significantly increased (P<0.05). However, the protein expression of PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3 decreased in the KD group (P<0.05) (Figure 4(b)).Figure 4
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of PI3K-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of PI3K-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)Moreover, we detected the effects of IGF1 silencing on the mRNA abundance of PI3K-related genes (AMPK, PI3K, JNK, Akt, and FOXO) in the myocardium of chicken embryos as shown in Figure4(c). The qPCR results revealed that the mRNA expression of AMPK, JNK, and FOXO was significantly increased (P<0.05) in the Si group at 6, 8, and 10 days. The mRNA expression of PI3K increased at 6 and 8 days but significantly decreased (P<0.05) at 10 days. The mRNA expression of Akt increased at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. A western blot analysis was performed to determine the protein expression of PI3K-related genes. The results revealed that compared with the N group, the protein expression of FOXO, P-FOXO, JNK, and P-JNK in the Si group was significantly increased (P<0.05). However, the protein expression of PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3 decreased in the Si group (P<0.05) (Figure 4(d)).


### 3.4. Protein and mRNA Expression of Insulin-Related Genes in Cells and Chicken Embryos

We also examined the effects of IGF1 knockdown on the mRNA abundance of insulin-related genes (IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1) in chicken cardiomyocytes (Figure5(a)); the qPCR results revealed that the mRNA expression of IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1 was significantly decreased (P<0.05) in the KD group. A western blot analysis was performed to determine the protein expression of insulin-related genes. The results revealed that compared with the C group, the protein expression of GLUT3, IGF1Rβ, and IGFBP2 in the KD group was significantly decreased (P<0.05) (Figure 5(b)).Figure 5
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of insulin-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of insulin-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)Furthermore, we examined the effects of IGF1 knockdown on the mRNA abundance of insulin-related genes (IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1) in the myocardium of chicken embryos as shown in Figure5(c). The qPCR results revealed that the mRNA expression of IGF1R, GLUT1, GLUT8, IGFBP1, IGFBP2, and IGFBP7 increased in the Si group at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. The mRNA expression of GLUT3, IGFBP3, IR, and IRS1 increased at 6 and 8 days, but significantly decreased (P<0.05) at 10 days. The mRNA expression of IGFBP4 increased at 6 days but significantly decreased at 8 and 10 days. The mRNA expression of IGFBP5 significantly decreased (P<0.05) at 8 and 10 days. A western blot analysis was performed to determine the protein expression of insulin-related genes. The results revealed that compared with the N group, the protein expression of GLUT3, IGF1Rβ, and IGFBP2 in the Si group was significantly decreased (P<0.05) (Figure 5(d)).


### 3.5. The Oxygen Consumption Rate and ATP Content in Myocardial Cells

To assess the effect of IGF1 knockdown on energy metabolism, the oxygen consumption rate and ATP content were detected. The results revealed that IGF1 knockdown significantly decreased the ATP content (Figure6(a)). The results of the oxygen consumption rate revealed that the oxygen consumption rate of the IGF1 knockdown group was 15.166 while the oxygen consumption rate of the control group was 24.019, indicating that IGF1 knockdown significantly decreased the myocardial oxygen consumption rate under the same conditions and the same initial oxygen concentration (Figure 6(b)).Figure 6
(a) The level of ATP was determined to investigate the function of energy metabolism. The data are represented as themeans±SD. Samples with an asterisk (∗) represent significant differences (P<0.05), n=3. The myocardial oxygen consumption rate results are shown in (b). The red curve indicates the oxygen consumption, and the blue curve indicates the oxygen concentration. C indicates the control group; KD indicates the knockdown group.
(a)
(b)The instantaneous oxygen consumption rates of the cardiomyocytes for different groups are provided in Table2.Table 2
Oxygen concentration and the oxygen consumption rate.
Time (min)
1A: O2 concentration (nmol/mL)
1A: O2 flux per V (pmol/(s∗mL))
1B: O2 concentration (nmol/mL)
1B: O2 flux per V (pmol/(s∗mL))
0.03
134.4705
222.2083
0.07
134.361
222.1058
0.1
134.3131
222.0585
0.13
134.2618
222.1088
0.17
134.1943
31.2253
222.157
1.3718
0.2
134.1481
25.9247
222.0753
-5.4236
0.23
134.0865
26.9524
221.9699
6.9917
0.27
134.0053
29.6908
221.9236
24.3313
0.3
133.9386
31.3172
221.8753
32.2136
0.33
133.8958
29.6935
221.8517
14.5802
0.37
133.8531
28.1554
221.8585
13.5949
0.4
133.8078
27.8145
221.8034
15.271
0.43
133.7513
27.2173
221.7768
17.5375
0.47
133.694
26.4491
221.7354
17.44
0.5
133.6496
27.3053
221.6911
14.1902
0.53
133.5888
26.5373
221.6527
14.0926
0.57
133.5316
26.3677
221.6044
15.079
0.6
133.4914
26.0266
221.5699
16.3605
0.63
133.4358
25.6004
221.5187
16.5588
0.67
133.3802
26.457
221.499
14.8846
0.7
133.3238
25.9453
221.4684
14.7868
0.73
133.2365
26.033
221.431
15.2803
0.77
133.1827
26.1198
221.3995
15.7737
0.8
133.1467
25.9497
221.365
15.676
0.83
133.1014
26.4639
221.3246
14.7904
0.87
133.0279
26.2092
221.2931
14.6927
0.9
132.9774
26.2105
221.2497
14.9893
0.93
132.9338
26.1261
221.2143
15.2857
0.97
132.8663
26.0422
221.1739
15.2868
1
132.8064
26.4713
221.1473
14.6963
1.03
132.7414
26.3019
221.1256
14.5984
1.07
132.6687
26.3892
221.0951
14.6976
1.1
132.6345
26.3901
221.0586
14.8956
1.13
132.5969
26.3055
221.0182
14.7981
1.17
132.5524
26.4776
220.9788
14.5035
1.2
132.4772
26.3085
220.9493
14.4057
1.23
132.4361
26.3095
220.9168
14.5051
1.27
132.3746
26.3111
220.8714
14.6047
1.3
132.3147
26.2271
220.836
14.6056
1.33
132.2617
26.3994
220.7926
14.4097
1.37
132.189
26.3157
220.7394
14.411
1.4
132.124
26.4028
220.7089
14.5103
1.43
132.089
26.4037
220.6685
14.7083
1.47
132.024
26.3198
220.6439
14.6104
1.5
131.9581
26.407
220.6064
14.5128
1.53
131.9094
26.4082
220.5749
14.3166
1.57
131.8777
26.409
220.5385
14.3175
1.6
131.8495
26.3242
220.4872
14.4173
1.63
131.7649
26.4118
220.4695
14.4177
1.67
131.6828
26.4994
220.4242
14.5174
1.7
131.6366
26.5005
220.3641
14.5189
1.73
131.5896
26.5017
220.3326
14.5197
1.77
131.5374
26.503
220.3099
14.5202
1.8
131.5041
26.5039
220.2744
14.5211
1.83
131.4545
26.4196
220.2311
14.5222
1.87
131.3698
26.4217
220.2134
14.5227
1.9
131.2988
26.4235
220.1651
14.6224
1.93
131.2518
26.4247
220.1099
14.6238
1.97
131.1979
26.426
220.0469
14.8224
2
131.1304
26.4277
220.0232
14.9215
2.03
131.0833
26.4289
219.9888
14.9223
2.07
131.0269
26.4303
219.9523
15.0217
2.1
130.9645
26.5173
219.909
15.1213
2.13
130.9021
26.5189
219.8597
15.2211
2.17
130.8559
26.5201
219.8272
15.3204
2.2
130.802
26.6069
219.7829
15.3215
2.23
130.7396
26.6085
219.7464
15.421
2.27
130.6797
26.61
219.7228
15.5201
2.3
130.6318
26.6112
219.6735
15.6198
2.33
130.5899
26.6122
219.639
15.7192
2.37
130.5532
26.6131
219.71
15.5204
2.4
130.5147
26.6141
219.71
15.3233
2.43
130.4591
26.53
219.6095
15.1288
2.47
130.3608
26.5324
219.5297
15.1308
2.5
130.3129
26.5336
219.4824
15.132
2.53
130.2658
26.4493
219.441
15.133
2.57
130.2085
26.4507
219.3996
15.1341
2.6
130.1632
26.3664
219.3977
15.0356
2.63
130.1119
26.3676
219.3455
15.0369
2.67
130.0546
26.2836
219.2676
15.0389
2.7
129.9785
26.371
219.2115
15.1388
2.73
129.9281
26.3722
219.1632
15.2385
2.77
129.887
26.3733
219.114
15.4368
2.8
129.8391
26.2889
219.0834
15.536
2.83
129.781
26.2904
219.048
15.6354
2.87
129.7194
26.2919
219.0145
15.6363
2.9
129.6715
26.2931
218.979
15.7357
2.93
129.6159
26.209
218.9317
15.7369
2.97
129.5441
26.2108
218.8736
15.8368
3
129.4774
26.2125
218.8726
15.8369
3.03
129.4099
26.2997
218.8539
15.9358
3.07
129.368
26.3007
218.8224
15.9366
3.1
129.3235
26.3018
218.7642
15.9381
3.13
129.2585
26.3035
218.7248
15.9391
3.17
129.2055
26.2193
218.6795
15.9402
3.2
129.1627
26.2203
218.6342
15.9413
3.23
129.1157
26.2215
218.5977
15.9422
3.27
129.0456
26.2233
218.5652
15.943
3.3
128.9874
26.2247
218.5278
16.0425
3.33
128.9421
26.2259
218.4579
16.1428
3.37
128.9062
26.2268
218.4214
16.2422
3.4
128.8455
26.2283
218.3987
16.2428
3.43
128.7796
26.2299
218.3524
16.3424
3.47
128.7463
26.2308
218.316
16.4418
3.5
128.6693
26.2327
218.2786
16.5413
3.53
128.5992
26.3199
218.2559
16.5419
3.57
128.5308
26.4072
218.2234
16.6412
3.6
128.4803
26.4939
218.18
16.6423
3.63
128.4231
26.5809
218.1288
16.7421
3.67
128.3837
26.5819
218.0855
16.8417
3.7
128.3162
26.5836
218.0559
16.6454
3.73
128.2469
26.5853
218.0185
16.3508
3.77
128.1947
26.5866
217.9643
16.2536
3.8
128.1494
26.6732
217.9436
16.2541
3.83
128.1032
26.6744
217.9298
16.0574
3.87
128.0656
26.6753
217.8875
15.96
3.9
128.0075
26.5913
217.8421
15.9611
3.93
127.9288
26.5932
217.7978
15.7652
3.97
127.8724
26.5947
217.7387
15.6682
4
127.845
26.5953
217.6865
15.768
4.03
127.7877
26.5968
217.6372
15.7692
4.07
127.7099
26.5987
217.6225
15.8681
4.1
127.6654
26.5998
217.6008
15.8686
4.13
127.6081
26.6013
217.5614
15.8696
4.17
127.5363
26.6031
217.5259
15.8705
4.2
127.4713
26.6047
217.4767
15.8717
4.23
127.408
26.6063
217.4402
15.8727
4.27
127.3618
26.6074
217.3969
15.8737
4.3
127.3105
26.6942
217.3565
15.9733
4.33
127.2763
26.6951
217.325
15.9741
4.37
127.2233
26.6964
217.2836
15.9751
4.4
127.1626
26.6979
217.2422
15.8776
4.43
127.1216
26.6989
217.1742
15.8793
4.47
127.054
26.7006
217.1378
15.9787
4.5
126.9813
26.7024
217.0875
15.98
4.53
126.942
26.7034
217.0294
16.08
4.57
126.8941
26.7046
217.0038
16.1791
4.6
126.8342
26.6206
216.9585
16.2788
4.63
126.7932
26.6216
216.8984
16.3788
4.67
126.7145
26.6236
216.8846
16.4776
4.7
126.6461
26.6253
216.858
16.4783
4.73
126.5948
26.5411
216.8235
16.5777
4.77
126.5401
26.5424
216.7762
16.5789
4.8
126.4862
26.5438
216.7457
16.6781
4.83
126.4529
26.4591
216.7033
16.6792
4.87
126.3981
26.4605
216.6708
16.68
4.9
126.3494
26.4617
216.6294
16.681
4.93
126.2955
26.3775
216.5881
16.6821
4.97
126.2314
26.3791
216.5703
16.6825
5
126.1527
26.3811
216.5102
16.684
5.03
126.0963
26.468
216.4748
16.6849
5.07
126.0638
26.4688
216.4482
16.6856
5.1
126.0159
26.47
216.4009
16.6868
5.13
125.9492
26.4717
216.3457
16.6881
5.17
125.9039
26.4728
216.3388
16.5898
5.2
125.844
26.3888
216.4088
16.2925
5.23
125.7901
26.3902
216.3132
16.0979
5.27
125.726
26.3918
216.2078
16.1005
5.3
125.6944
26.3071
216.1861
16.0025
5.33
125.6525
26.2226
216.1142
16.1028
5.37
125.5875
26.1387
216.0659
16.2026
5.4
125.5413
26.1399
215.997
16.3028
5.43
125.4806
26.0559
216.0039
16.2041
5.47
125.4224
25.9718
215.9891
16.106
5.5
125.3745
25.973
215.9359
16.1073
5.53
125.3335
25.974
215.8955
16.0098
5.57
125.2753
25.9755
215.8601
16.0107
5.6
125.2163
25.977
215.8039
16.0121
5.63
125.1462
25.9787
215.7743
15.9143
5.67
125.0855
25.9802
215.7409
15.9151
5.7
125.0393
25.8959
215.6946
15.8178
5.73
124.9889
25.8971
215.6443
15.819
5.77
124.9119
25.8991
215.5921
15.9189
5.8
124.8709
25.9001
215.5665
15.9195
5.83
124.8264
25.9012
215.5192
15.9207
5.87
124.7777
25.9024
215.4818
16.0201
5.9
124.7204
25.8183
215.464
16.1191
5.93
124.6631
25.8198
215.4227
16.1201
5.97
124.5827
25.8218
215.3586
16.2202
6
124.5117
25.9091
215.3468
16.3191
6.03
124.4827
25.9953
215.3212
16.3197
6.07
124.4408
25.9963
215.2867
16.3206
6.1
124.4091
25.9971
215.2256
16.3221
6.13
124.3552
25.9985
215.1675
16.422
6.17
124.3014
25.9143
215.1261
16.4231
6.2
124.2501
25.9156
215.0995
16.4237
6.23
124.1782
25.8319
215.0641
16.5231
6.27
124.1115
25.8335
215.001
16.5247
6.3
124.0619
25.8348
214.9695
16.624
6.33
124.014
25.9215
214.9439
16.6247
6.37
123.9456
26.0087
214.9252
16.6251
6.4
123.8875
26.0102
214.8729
16.6264
6.43
123.8302
26.0116
214.8158
16.6279
6.47
123.7609
26.0989
214.7577
16.7278
6.5
123.7301
26.1851
214.7321
16.7285
6.53
123.684
26.1863
214.7045
16.5321
6.57
123.6215
26.1878
214.672
16.3359
6.6
123.5685
26.2747
214.6276
16.337
6.63
123.5138
26.2761
214.5715
16.3384
6.67
123.4497
26.2777
214.535
16.3393
6.7
123.4009
26.2789
214.4897
16.439
6.73
123.3778
26.1939
214.4395
16.5388
6.77
123.3077
26.1957
214.3794
16.6388
6.8
123.2479
26.1972
214.339
16.7383
6.83
123.2119
26.1126
214.3065
16.7391
6.87
123.1401
26.1144
214.2681
16.8386
6.9
123.1025
26.0298
214.2287
16.8396
6.93
123.0332
26.0315
214.1705
16.9395
6.97
122.987
25.9472
214.1252
17.0392
7
122.9255
25.9487
214.0888
17.0401
7.03
122.875
25.95
214.0799
17.0403
7.07
122.8015
25.9518
214.0504
17.0411
7.1
122.7596
26.0384
214.01
17.0421
7.13
122.7339
25.9535
213.9587
17.1419
7.17
122.663
25.9553
213.8967
17.2419
7.2
122.6134
25.9565
213.8632
17.2428
7.23
122.5544
25.8725
213.8346
17.2435
7.27
122.5013
25.8738
213.8248
17.1452
7.3
122.4304
25.9611
213.7834
17.1462
7.33
122.3731
26.048
213.7528
17.0485
7.37
122.3132
26.0495
213.7213
16.9508
7.4
122.279
26.0504
213.6662
16.8536
7.43
122.2243
26.0518
213.6041
16.8552
7.47
122.1619
25.9678
213.5509
16.8565
7.5
122.0986
25.9694
213.4987
16.9563
7.53
122.0456
25.9707
213.4484
16.9576
7.57
122.0139
25.9715
213.4031
16.9587
7.6
121.9806
25.8868
213.3824
17.0578
7.63
121.9173
25.8029
213.3529
17.0585
7.67
121.8626
25.8043
213.3036
17.0597
7.7
121.8078
25.8056
213.2652
16.9622
7.73
121.7326
25.8075
213.2327
16.963
7.77
121.6873
25.8086
213.213
16.865
7.8
121.6248
25.8957
213.1834
16.7672
7.83
121.5855
25.8967
213.1174
16.7688
7.87
121.547
25.8122
213.0642
16.7702
7.9
121.4957
25.8134
213.0258
16.7711
7.93
121.4495
25.8146
212.9894
16.6735
7.97
121.3803
25.7308
212.949
16.6745
8
121.3161
25.8179
212.9175
16.5768
8.03
121.2648
25.8192
212.9027
16.5772
8.07
121.2092
25.8206
212.8455
16.4801
8.1
121.1742
25.736
212.8081
16.481
8.13
121.1349
25.6514
212.7677
16.5806
8.17
121.0835
25.5672
212.7234
16.5817
8.2
121.0117
25.569
212.6643
16.5831
8.23
120.9604
25.4848
212.614
16.6829
8.27
120.8852
25.5722
212.5707
16.7825
8.3
120.8227
25.5737
212.5559
16.7829
8.33
120.7919
25.5745
212.5165
16.8824
8.37
120.7458
25.4901
212.4958
16.7844
8.4
120.6979
25.4913
212.4791
16.6863
8.43
120.6346
25.4929
212.4111
16.688
8.47
120.5679
25.4946
212.4022
16.5897
8.5
120.5183
25.4958
212.4633
16.3911
8.53
120.4773
25.4968
212.3402
16.2957
8.57
120.4277
25.4126
212.2249
16.3971
8.6
120.355
25.4144
212.1825
16.3982
8.63
120.3105
25.4155
212.1116
16.4984
8.67
120.2857
25.4161
212.0653
16.4996
8.7
120.237
25.4173
212.0722
16.4009
8.73
120.166
25.4191
212.0702
16.3025
8.77
120.1292
25.3345
212.018
16.3038
8.8
120.0754
25.3359
211.955
16.3053
8.83
119.9967
25.4233
211.9087
16.405
8.87
119.9497
25.4245
211.8781
16.5043
8.9
119.9009
25.4257
211.8998
16.4052
8.93
119.8265
25.4276
211.8703
16.3075
8.97
119.7752
25.4289
211.7816
16.3097
9
119.7059
25.5161
211.7107
16.41
9.03
119.6717
25.517
211.6584
16.5098
9.07
119.617
25.6039
211.5944
16.6099
9.1
119.5503
25.6055
211.5461
16.7096
9.13
119.4981
25.6923
211.5245
16.8087
9.17
119.434
25.7795
211.4782
16.8098
9.2
119.3853
25.7807
211.4289
17.0081
9.23
119.3348
25.8675
211.3816
17.1078
9.27
119.2698
25.8691
211.3787
17.2064
9.3
119.2356
25.8699
211.3639
17.2067
9.33
119.2006
25.8708
211.3087
17.2081
9.37
119.1458
25.8722
211.354
17.01
9.4
119.1022
25.8733
211.3797
16.8123
9.43
119.0672
25.7886
211.2358
16.7174
9.47
119.0167
25.7044
211.1373
16.7198
9.5
118.9432
25.6207
211.0743
16.8199
9.53
118.8842
25.6222
211.0181
17.0183
9.57
118.8551
25.5374
211.0181
17.1169
9.6
118.8012
25.5387
210.9698
17.2166
9.63
118.726
25.5406
210.9294
17.3161
9.67
118.6618
25.6277
210.8605
17.4163
9.7
118.6148
25.5434
210.8221
17.5158
9.73
118.5567
25.5449
210.7679
17.5172
9.77
118.5036
25.5462
210.7699
17.5171
9.8
118.4224
25.6337
210.8802
17.2188
9.83
118.3933
25.72
210.7452
16.9267
9.87
118.3591
25.6353
210.6369
16.8309
9.9
118.2958
25.6369
210.5807
16.9308
9.93
118.2266
25.7241
210.5226
17.0307
9.97
118.1958
25.7249
210.4714
17.229
10
118.1376
25.6408
210.4832
17.4258
10.03
118.0855
25.6422
210.428
17.4272
10.07
118.0299
25.558
210.3935
17.428
10.1
117.994
25.4734
210.3591
17.4289
10.13
117.9307
25.3895
210.3059
17.4302
10.17
117.8666
25.3911
210.294
17.4305
10.2
117.8016
25.3927
210.3728
17.2315
10.23
117.7708
25.3935
210.2822
16.9382
10.27
117.7272
25.3946
210.166
16.8426
10.3
117.6562
25.3963
210.1246
16.7451
10.33
117.5972
25.3978
210.0576
16.8453
10.37
117.5716
25.3985
210.0162
16.9449
10.4
117.51
25.4
210.0113
16.945
10.43
117.4536
25.4014
209.968
17.0446
10.47
117.4193
25.4023
209.9088
17.1446
10.5
117.3783
25.4033
209.8714
17.244
10.53
117.2954
25.4054
209.8419
17.2448
10.57
117.2355
25.4924
209.7877
17.4432
10.6
117.1962
25.5789
209.7512
17.4441
10.63
117.1551
25.5799
209.7059
17.4452
10.67
117.1004
25.4958
209.6665
17.4462
10.7
117.0474
25.4971
209.6143
17.349
10.73
116.9721
25.499
209.6833
16.9532
10.77
116.9345
25.4999
209.6232
16.6592
10.8
116.9046
25.3296
209.4882
16.7611
10.83
116.8396
25.3313
209.4616
16.8602
10.87
116.7891
25.247
209.3897
16.9605
10.9
116.7464
25.1626
209.3463
17.0601
10.93
116.6891
25.0785
209.3286
17.1591
10.97
116.6395
24.9942
209.3444
17.0602
11
116.5856
24.9956
209.2793
17.1603
11.03
116.5377
24.9967
209.2153
17.2604
11.07
116.4676
24.9985
209.1789
17.3599
11.1
116.4163
24.9998
209.1345
17.361
11.13
116.3522
25.0869
209.0971
17.1649
11.17
116.3248
25.0021
209.1651
16.8676
11.2
116.2735
25.0034
209.0853
16.7711
11.23
116.2059
25.005
208.969
16.8725
11.27
116.1444
25.0921
208.9582
16.8728
11.3
116.0871
25.0935
208.9119
17.071
11.33
116.0229
25.1806
208.8528
17.0725
11.37
115.9836
25.1816
208.837
17.0729
11.4
115.946
25.1826
208.7878
17.1726
11.43
115.8827
25.1841
208.7444
17.1737
11.47
115.8254
25.1856
208.6873
17.2736
11.5
115.7809
25.2722
208.6548
17.2745
11.53
115.7219
25.3592
208.6193
17.0783
11.57
115.6792
25.3603
208.5819
16.9807
11.6
115.6125
25.3619
208.6223
16.7827
11.63
115.5646
25.4486
208.637
16.5853
11.67
115.5295
25.4495
208.5366
16.4893
11.7
115.4825
25.4507
208.4577
16.4913
11.73
115.4235
25.4522
208.442
16.3932
11.77
115.3713
25.4535
208.3848
16.2961
11.8
115.3294
25.369
208.311
16.3964
11.83
115.2867
25.2846
208.3159
16.2978
11.87
115.2268
25.2861
208.2676
16.299
11.9
115.1704
25.2875
208.2243
16.3001
11.93
115.1088
25.3745
208.1819
16.3011
11.97
115.0558
25.2903
208.1504
16.3019
12
114.9959
25.2918
208.0982
16.3032
12.03
114.9549
25.2929
208.0578
16.3042
12.07
114.8967
25.3798
208.0282
16.108
12.1
114.8258
25.3816
208.1248
15.7115
12.13
114.7727
25.3829
208.0134
15.7143
12.17
114.7326
25.4694
207.8992
15.7171
12.2
114.6787
25.4708
207.8736
15.8163
12.23
114.6334
25.3864
207.8223
15.9161
12.27
114.6043
25.3016
207.7583
16.0162
12.3
114.553
25.3029
207.7741
16.0158
12.33
114.4846
25.2191
207.7534
15.9178
12.37
114.4127
25.2209
207.6982
15.9192
12.4
114.3537
25.2224
207.6588
15.9202
12.43
114.3093
25.309
207.6095
16.0199
12.47
114.2426
25.3107
207.5268
16.1205
12.5
114.2015
25.3117
207.5051
16.0225
12.53
114.1733
25.3124
207.5603
15.8241
12.57
114.1314
25.2279
207.5573
15.7257
12.6
114.0604
25.2297
207.4293
15.7289
12.63
113.9963
25.2313
207.3593
15.7306
12.67
113.9535
25.3179
207.2973
15.9292
12.7
113.9056
25.3191
207.247
16.029
12.73
113.8509
25.3205
207.2224
16.1281
12.77
113.8184
25.3213
207.246
16.226
12.8
113.7663
25.2371
207.1948
16.2273
12.83
113.7141
25.2384
207.1377
16.3273
12.87
113.6551
25.2398
207.1002
16.4267
12.9
113.5833
25.2416
207.0451
16.6251
12.93
113.5285
25.3285
206.9948
16.6264
12.97
113.4841
25.3296
206.9603
16.4302
13
113.4413
25.3307
206.9298
16.431
13.03
113.3883
25.332
206.8579
16.5313
13.07
113.3327
25.3334
206.8756
16.5308
13.1
113.2848
25.3346
206.9396
16.3322
13.13
113.2258
25.2506
206.8372
16.3348
13.17
113.1839
25.2516
206.7466
16.337
13.2
113.1506
25.1669
206.716
16.3378
13.23
113.0719
25.0834
206.651
16.3394
13.27
113.0155
25.0848
206.5899
16.538
13.3
112.9573
25.0863
206.5939
16.5379
13.33
112.918
25.0872
206.5535
16.5389
13.37
112.8684
25.0885
206.517
16.6383
13.4
112.8025
25.0901
206.4658
16.6396
13.43
112.7624
25.1766
206.4106
16.4439
13.47
112.7145
25.1778
206.3643
16.4451
13.5
112.6572
25.1793
206.3121
16.5449
13.53
112.5965
25.1808
206.2688
16.6445
13.57
112.5306
25.268
206.2363
16.7438
13.6
112.493
25.1834
206.2116
16.843
13.63
112.4554
25.1843
206.1821
16.8437
13.67
112.3998
25.1002
206.1545
16.8444
13.7
112.3425
25.1871
206.122
16.7467
13.73
112.2963
25.1883
206.0875
16.7476
13.77
112.2279
25.2755
206.053
16.7484
13.8
112.1638
25.3626
206.0176
16.7493
13.83
112.1219
25.3637
205.988
16.75
13.87
112.0791
25.3648
205.925
16.6531
13.9
112.0124
25.3664
205.9171
16.3578
13.93
111.962
25.4532
205.986
16.0605
13.97
111.9286
25.454
205.8836
15.866
14
111.8696
25.4555
205.7969
15.9667
14.03
111.826
25.4566
205.7732
15.9673
14.07
111.7644
25.4581
205.7102
15.9689
14.1
111.7251
25.3736
205.659
15.9702
14.13
111.6738
25.3749
205.6639
15.8715
14.17
111.6105
25.291
205.5861
15.8735
14.2
111.5566
25.2923
205.522
15.8751
14.23
111.4865
25.3796
205.4826
15.9746
14.27
111.4455
25.3806
205.4521
16.0739
14.3
111.4164
25.3813
205.4186
16.0747
14.33
111.36
25.3827
205.4018
16.0751
14.37
111.3009
25.3842
205.3506
16.1749
14.4
111.2325
25.3859
205.3201
16.1757
14.43
111.1906
25.387
205.2836
15.9795
14.47
111.1479
25.388
205.3152
15.6832
14.5
111.0923
25.3039
205.324
15.486
14.53
111.0324
25.3054
205.2265
15.3899
14.57
110.9657
25.3071
205.1408
15.392
14.6
110.9332
25.3079
205.0945
15.4917
14.63
110.876
25.3093
205.0373
15.4931
14.67
110.811
25.3965
204.992
15.4943
14.7
110.7622
25.3977
204.9782
15.4946
14.73
110.7118
25.3989
204.9142
15.5947
14.77
110.6485
25.4005
204.8728
15.6943
14.8
110.6066
25.4016
204.8482
15.7934
14.83
110.5844
25.4021
204.8029
15.8931
14.87
110.5262
25.3181
204.7418
16.0916
14.9
110.4715
25.3194
204.7162
16.2893
14.93
110.4108
25.3209
204.6926
16.3884
14.97
110.3586
25.3223
204.6591
16.4877
15
110.297
25.3238
204.6118
16.5874
15.03
110.2474
25.325
204.5763
16.5883
15.07
110.203
25.3261
204.5605
16.5887
15.1
110.1371
25.3278
204.5162
16.5898
15.13
110.079
25.4148
204.4719
16.6894
15.17
110.0328
25.4159
204.4098
16.691
15.2
109.9926
25.4169
204.3803
16.7902
15.23
109.9302
25.4185
204.3744
16.6919
15.27
109.8883
25.505
204.4561
16.2958
15.3
109.8609
25.4202
204.3576
16.0027
15.33
109.8122
25.3359
204.2778
15.9062
15.37
109.7386
25.3378
204.2266
15.9075
15.4
109.6762
25.3393
204.1566
15.9092
15.43
109.63
25.255
204.1133
16.0088
15.47
109.5796
25.2562
204.1172
15.8117
15.5
109.5377
25.2573
204.0798
15.8126
15.53
109.4975
25.1728
204.0148
15.9128
15.57
109.4633
25.1736
203.9606
16.0126
15.6
109.3966
25.0898
203.933
16.1118
15.63
109.3188
25.0917
203.8966
16.1127
15.67
109.264
25.0931
203.867
16.1135
15.7
109.2221
25.0086
203.8246
16.2131
15.73
109.1512
25.0959
203.7872
16.214
15.77
109.1204
25.0967
203.7557
16.2148
15.8
109.0853
25.012
203.7251
16.117
15.83
109.0297
24.9279
203.6769
15.8227
15.87
108.9853
24.929
203.6355
15.7252
15.9
108.9297
24.9304
203.5941
15.7262
15.93
108.8775
24.8462
203.5567
15.7272
15.97
108.8151
24.8478
203.5193
15.7281
16
108.7647
24.849
203.4917
15.8273
16.03
108.7245
24.85
203.4651
15.7295
16.07
108.6757
24.8512
203.4207
15.7306
16.1
108.6364
24.8522
203.3498
15.8309
16.13
108.5765
24.8537
203.2897
15.9309
16.17
108.5167
24.7697
203.272
16.0298
16.2
108.4594
24.7711
203.2375
16.1292
16.23
108.3978
24.7727
203.1922
16.2289
16.27
108.3499
24.7739
203.1518
16.3284
16.3
108.3037
24.775
203.137
16.3288
16.33
108.2576
24.7762
203.1055
16.4281
16.37
108.1986
24.7777
203.0612
16.4292
16.4
108.1558
24.7787
203.0011
16.4307
16.43
108.1131
24.7798
202.9705
16.5299
16.47
108.0446
24.867
202.9508
16.5304
16.5
107.9856
24.8685
202.9173
16.5313
16.53
107.9352
24.9553
202.8809
16.6307
16.57
107.8805
25.0422
202.8375
16.5333
16.6
107.8497
25.0429
202.7804
16.3377
16.63
107.8086
24.9584
202.7489
16.2399
16.67
107.7633
24.8741
202.7193
16.1422
16.7
107.7094
24.8754
202.6908
16.0444
16.73
107.6436
24.8771
202.6484
16.1439
16.77
107.6111
24.8779
202.6307
16.0459
16.8
107.5435
24.8796
202.6031
15.948
16.83
107.4939
24.8808
202.5489
15.8509
16.87
107.4486
24.8819
202.5046
15.852
16.9
107.3905
24.7979
202.4721
15.8528
16.93
107.3152
24.7998
202.409
15.8544
16.97
107.2827
24.8861
202.3755
15.8552
17
107.2391
24.8872
202.3469
15.8559
17.03
107.1724
24.8888
202.3184
15.8567
17.07
107.1126
24.9758
202.275
15.7592
17.1
107.0561
25.0628
202.2445
15.76
17.13
106.9997
25.0642
202.1962
15.7612
17.17
106.9544
25.1508
202.1489
15.7624
17.2
106.9202
25.1517
202.1135
15.6647
17.23
106.8783
25.0672
202.1834
15.466
17.27
106.8158
25.0688
202.1263
15.3689
17.3
106.7577
25.1557
202.0248
15.3714
17.33
106.7184
25.1567
201.9992
15.2736
17.37
106.6611
25.1582
201.946
15.1764
17.4
106.5995
25.1597
201.8967
15.1776
17.43
106.5499
25.0754
201.8958
15.1776
17.47
106.5011
25.0766
201.8524
15.1787
17.5
106.4687
25.0775
201.8022
15.18
17.53
106.4208
25.0786
201.7657
15.1809
17.57
106.3541
25.0803
201.7174
15.2806
17.6
106.3173
25.0812
201.6593
15.3806
17.63
106.2609
25.0826
201.617
15.4801
17.67
106.1907
25.0844
201.5825
15.481
17.7
106.1454
25.0855
201.547
15.5804
17.73
106.0924
25.0869
201.5155
15.5812
17.77
106.0445
25.0881
201.4692
15.6809
17.8
105.9975
25.0892
201.4396
15.7801
17.83
105.9376
25.1762
201.3845
15.7815
17.87
105.8923
25.1774
201.4436
15.6815
17.9
105.8376
25.1787
201.4495
15.4843
17.93
105.788
25.18
201.3254
15.4874
17.97
105.7521
25.1809
201.2899
15.4883
18
105.6973
25.0967
201.219
15.5886
18.03
105.6221
25.0986
201.151
15.6888
18.07
105.5785
25.0997
201.148
15.7874
18.1
105.5417
25.0151
201.1106
15.7883
18.13
105.475
25.0168
201.0584
15.7896
18.17
105.4186
25.0182
200.9953
15.8897
18.2
105.3724
25.0193
200.9648
15.8905
18.23
105.3262
25.0205
200.9313
15.9898
18.27
105.2826
25.0216
200.9047
16.089
18.3
105.2287
24.9374
200.8781
16.0897
18.33
105.168
24.9389
200.9707
15.8903
18.37
105.1201
24.9401
200.8722
15.8928
18.4
105.074
24.9413
200.7747
15.8952
18.43
105.0363
24.9422
200.7491
15.8959
18.47
104.973
25.0293
200.6919
15.8973
18.5
104.9235
25.0306
200.6338
16.0958
18.53
104.8781
25.0317
200.6141
16.0963
18.57
104.8123
24.9478
200.5757
15.9987
18.6
104.7413
25.0351
200.5382
15.9012
18.63
104.708
25.036
200.5028
15.902
18.67
104.6703
25.0369
200.4446
15.9035
18.7
104.631
24.9524
200.4141
16.0028
18.73
104.5805
24.9536
200.3727
16.0038
18.77
104.5181
24.9552
200.3323
16.0048
18.8
104.4651
24.9565
200.3195
16.0051
18.83
104.4044
24.958
200.3796
15.8066
18.87
104.3625
24.9591
200.2663
15.7109
18.9
104.3197
24.8746
200.1777
15.8117
18.93
104.2607
24.8761
200.1461
15.8124
18.97
104.2154
24.8772
200.1077
15.9119
19
104.1701
24.7929
200.0664
16.0115
19.03
104.1111
24.7943
200.0752
16.0112
19.07
104.0623
24.7956
200.0181
16.1112
19.1
104.0222
24.7966
199.9629
16.1126
19.13
103.9862
24.712
199.9215
16.2121
19.17
103.9221
24.7136
199.8821
16.3116
19.2
103.8853
24.629
199.8792
16.2132
19.23
103.8263
24.6304
199.823
16.0175
19.27
103.763
24.632
199.7767
15.9202
19.3
103.7194
24.5476
199.8575
15.7211
19.33
103.6767
24.4632
199.7807
15.526
19.37
103.6245
24.4645
199.6605
15.6276
19.4
103.5476
24.5519
199.6437
15.628
19.43
103.5022
24.553
199.5817
15.728
19.47
103.4595
24.5541
199.5324
15.8278
19.5
103.4107
24.5553
199.5166
15.9267
19.53
103.3654
24.5564
199.4851
16.026
19.57
103.3218
24.5575
199.4585
16.0267
19.6
103.2722
24.4733
199.4103
16.1264
19.63
103.2064
24.4749
199.3679
16.1274
19.67
103.1653
24.4759
199.3068
15.9319
19.7
103.12
24.4771
199.2645
15.933
19.73
103.0618
24.4785
199.2507
15.9333
19.77
103.0063
24.4799
199.3393
15.6356
19.8
102.9524
24.4813
199.2241
15.6385
19.83
102.9028
24.4825
199.1285
15.6409
19.87
102.8412
24.5696
199.0999
15.7401
19.9
102.8045
24.5705
199.0379
15.8402
19.93
102.7514
24.6573
198.9975
15.8412
19.97
102.6916
24.6588
198.9935
15.9398
20
102.6463
24.7455
198.9512
15.9408
20.03
102.5941
24.7468
198.8931
16.0408
20.07
102.5342
24.7483
198.8428
16.1406
20.1
102.4821
24.7496
198.8133
16.2398
20.13
102.4573
24.7502
198.7857
16.339
20.17
102.4111
24.7513
198.7561
16.1427
20.2
102.3624
24.7525
198.697
16.1442
20.23
102.2982
24.7542
198.6813
16.1446
20.27
102.2452
24.841
198.7532
16.0443
20.3
102.2127
24.8418
198.6172
16.0477
20.33
102.1597
24.7576
198.5364
16.1482
20.37
102.087
24.8449
198.5
16.2477
20.4
102.0477
24.8459
198.4488
16.3474
20.43
101.9964
24.8472
198.4094
16.4469
20.47
101.9553
24.7627
198.4143
16.4468
20.5
101.9134
24.7638
198.3493
16.547
20.53
101.8647
24.6795
198.3089
16.548
20.57
101.798
24.6811
198.2586
16.6477
20.6
101.7338
24.6827
198.2271
16.747
20.63
101.6817
24.6841
198.1926
16.6494
20.67
101.6372
24.6852
198.1808
16.4527
20.7
101.5928
24.6863
198.1335
16.4538
20.73
101.5508
24.6873
198.1197
16.4542
20.77
101.4953
24.6887
198.1936
16.2553
20.8
101.4431
24.69
198.0685
16.1599
20.83
101.3952
24.6912
197.9907
16.1619
20.87
101.3499
24.6923
197.968
16.1624
20.9
101.296
24.6937
197.901
16.1641
20.93
101.2285
24.6954
197.8577
16.1652
20.97
101.1797
24.6966
197.8616
16.1651
21
101.131
24.6978
197.8104
16.1664
21.03
101.0771
24.6992
197.7444
16.2665
21.07
101.0275
24.7004
197.6823
16.3666
21.1
100.9796
24.7016
197.6518
16.1703
21.13
100.9437
24.7025
197.64
16.0721
21.17
100.9027
24.618
197.6114
16.0728
21.2
100.8556
24.6192
197.571
16.0739
21.23
100.8095
24.6203
197.5286
16.0749
21.27
100.759
24.5361
197.5996
15.9746
21.3
100.7017
24.5375
197.505
15.78
21.33
100.6427
24.539
197.4085
15.8809
21.37
100.5948
24.5402
197.373
15.8818
21.4
100.5392
24.6271
197.3257
15.9815
21.43
100.4896
24.6283
197.2981
15.9822
21.47
100.4452
24.6295
197.2804
15.9826
21.5
100.3879
24.6309
197.2311
15.9838
21.53
100.3468
24.5464
197.1917
15.9848
21.57
100.2964
24.5477
197.1474
16.0844
21.6
100.2357
24.6347
197.1178
15.7896
21.63
100.1818
24.5505
197.0804
15.7906
21.67
100.1219
24.6375
197.0607
15.7911
21.7
100.0886
24.6384
197.1238
15.5925
21.73
100.039
24.6396
197.0646
15.4954
21.77
99.9937
24.6407
196.9642
15.4979
21.8
99.9338
24.6422
196.9326
15.4987
21.83
99.8817
24.558
196.8568
15.5006
21.87
99.8415
24.559
196.8016
15.6005
21.9
99.7902
24.5603
196.8026
15.699
21.93
99.7397
24.5616
196.7612
15.7986
21.97
99.6961
24.5627
196.7208
15.7996
22
99.6294
24.5643
196.6834
15.8005
22.03
99.5918
24.5653
196.6351
15.9002
22.07
99.5405
24.5666
196.5928
15.9013
22.1
99.4909
24.5678
196.5691
15.7048
22.13
99.4396
24.5691
196.5238
15.6075
22.17
99.3865
24.5704
196.5021
15.608


### 3.6. Protein and mRNA Expression of Cardiac Differentiation-Related Genes in Cells and Chicken Embryos

The effects of IGF1 knockdown on the mRNA abundance of cardiac differentiation-related genes (MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5) in chicken cardiomyocytes are shown (Figure7(a)), and the qPCR results revealed that the mRNA expression of MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5 was significantly decreased (P<0.05) in the KD group. A western blot analysis was performed to determine the protein expression of cardiac differentiation-related genes. The results revealed that compared with the C group, the protein expression of MyoG and MyoD in the KD group was significantly decreased (P<0.05) (Figure 7(b)).Figure 7
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of cardiac differentiation-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of cardiac differentiation-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)The effects of IGF1 knockdown on the mRNA abundance of cardiac differentiation-related genes (MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5) in the myocardium of chicken embryos are shown in Figure7(c). The qPCR results revealed that the mRNA expression of MyoD, Mesp1, and MYF5 increased in the Si group at 6 and 8 days, but significantly decreased (P<0.05) at 10 days. The mRNA expression of MyoG, GATA4, and GATA6 increased at 6 days, but significantly decreased at 8 and 10 days. The mRNA expression of MYF6 increased at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. The mRNA expression of Nkx2.5 decreased (P<0.05) at 6, 8, and 10 days. A western blot analysis was performed to determine the protein expression of cardiac differentiation-related genes. The results revealed that compared with the N group, the protein expression of MyoG and MyoD in the Si group was significantly decreased (P<0.05) (Figure 7(d)).


### 3.7. Intracellular Morphological Observation and H.E. Stain in Cells and Chicken Embryos’ Myocardium

As observed under a microscope, normal cardiomyocytes were fusiform and tightly connected and the whole looked similar to paving stones accompanied by protruding pseudopodia that stretched out between cells, interweaving into a mesh in the control group (Figure8(a)). In the KD group, as the density of cell growth decreased, the volume of the cardiomyocytes and intercellular junctions was evidently reduced. We observed that myocardial fibers and muscle fiber bundles were disintegrated, and the pseudopodia between cells did not interweave into a mesh in the KD group (Figure 8(b)). We observed myocardial cells stained by hematoxylin and eosin (H.E.). The cardiomyocytes in the control group displayed normal morphologies (Figure 8(c)). However, many slender cardiomyocytes appeared in the IGF1 knockdown group (Figure 8(d)), which indicated that cardiomyocyte development was blocked.Figure 8
Morphological observation was performed in cardiomyocytes transfected with Opti-MEM (C group, a) and siRNA (KD group, b) for 24 h. Cardiomyocytes were stained by H.E., and the results are shown in (c) and (d). H.E. staining for myocardial tissues in the N group (e) and the Si group (f). C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo.
(a)
(b)
(c)
(d)
(e)
(f)Myocardial injury and ultrastructural damage are shown in Figure8(f). Blood vessel rupturing and increased tissue gaps were observed in the IGF1-deficient chicken heart group more than in the normal group (Figure 8(e)).


## 3.1. Development of an IGF1 Knockdown Model in Cells and Chicken Embryos

The mRNA and protein levels of IGF1 were significantly decreased (P<0.05) in the KD group (Figures 1(a) and 1(b)). The results confirmed that we successfully established the model of IGF1 knockdown in vitro.Figure 1
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of the IGF1 gene in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of the IGF1 gene in the myocardium. The results were calculated from at least three independent experiments,n=3. The data are expressed as the means±SD. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. ∗ indicates a significant difference from the corresponding control (P<0.05).
(a)
(b)
(c)
(d)The mRNA levels of IGF1 were detected at 6, 8, and 10 days, and we found that the expression of IGF1 was significantly decreased at 10 days. For further verification, we took chicken embryos at 10 days to detect the protein level of IGF1. Compared with the N group, the mRNA levels in the Si group decreased (P<0.05) at 8 days and 10 days (Figure 1(c)). The Si group exhibited significantly decreased protein levels compared with the N group (Figure 1(d)). The results confirmed that we successfully established the IGF1 silence model in vivo.


## 3.2. Detection of the Antioxidant Capacity in Cells and Chicken Embryos

To assess the relationship between oxidative stress and IGF1, the production of ROS, the levels of H2O2, MDA, and T-AOC and the activities of GSH, GSH-Px, SOD, CAT, and iNOS were measured in cardiomyocytes. As presented in Figure 2, the ROS activities were significantly increased (P<0.05) compared with the C group. The levels of H2O2, MDA, and iNOS were significantly increased in the KD group (P<0.05) (Figures 3(d)–3(f)). The levels of GSH, GSH-Px, CAT, SOD, and T-AOC in the KD group were significantly lower than those in the C group (P<0.05) (Figures 3(a)–3(c), 3(g), and 3(h)).Figure 2
(a) ROS generation was performed by immunofluorescence using DCFH-DA (green fluorescence, 5 mM) in cells. C indicates the control group; KD indicates the knockdown group. Cardiomyocytes were visualized using fluorescence microscopy. (b) The effects of IGF1 knockdown on the ROS levels in cardiomyocytes were detected by using a fluorescence microplate reader. C indicates the control group; KD indicates the knockdown group.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)Figure 3
(a–h) Oxidative stress markers of the GSH, GSH-Px, CAT, H2O2, MDA, iNOS, SOD, and T-AOC contents were measured in cardiomyocytes. (i–p) Oxidative stress markers of GSH, GSH-Px, CAT, H2O2, MDA, iNOS, SOD, and T-AOC contents were measured in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. ∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
(i)
(j)
(k)
(l)
(m)
(n)
(o)
(p)To further demonstrate the antioxidant capacity of IGF1 silence in vivo, we also studied the antioxidant capacity of the myocardium. As shown in Figure3, the levels of H2O2, MDA, and iNOS were significantly increased in the Si group (P<0.05) at 6, 8, and 10 days (Figures 3(l)–3(n)). The levels of GSH, GSH-Px, CAT, SOD, and T-AOC in the Si group were significantly lower than those in the N group (P<0.05) at 6, 8, and 10 days (Figures 3(i), 3(j), 3(l), 3(o), and 3(p)).


## 3.3. Protein and mRNA Expression of the PI3K/Akt Pathway-Related Genes in Cells and Chicken Embryos

To examine whether IGF1 knockdown changed the expressions of the PI3K/Akt pathway-related genes, we detected the mRNA expression levels of AMPK, PI3K, JNK, Akt, and FOXO as well as the protein expression levels of FOXO, P-FOXO, JNK, P-JNK, PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3. The effects of IGF1 knockdown on the mRNA abundance of PI3K-related genes in chicken cardiomyocytes are shown in Figure4(a). Compared with the C group, the qPCR results revealed that the mRNA expression of AMPK, JNK, and FOXO was significantly increased (P<0.05) in the KD group. However, the mRNA expression of PI3K and Akt was decreased (P<0.05). The results revealed that compared with the C group, the protein expression of FOXO, P-FOXO, JNK, and P-JNK in the KD group was significantly increased (P<0.05). However, the protein expression of PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3 decreased in the KD group (P<0.05) (Figure 4(b)).Figure 4
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of PI3K-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of PI3K-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)Moreover, we detected the effects of IGF1 silencing on the mRNA abundance of PI3K-related genes (AMPK, PI3K, JNK, Akt, and FOXO) in the myocardium of chicken embryos as shown in Figure4(c). The qPCR results revealed that the mRNA expression of AMPK, JNK, and FOXO was significantly increased (P<0.05) in the Si group at 6, 8, and 10 days. The mRNA expression of PI3K increased at 6 and 8 days but significantly decreased (P<0.05) at 10 days. The mRNA expression of Akt increased at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. A western blot analysis was performed to determine the protein expression of PI3K-related genes. The results revealed that compared with the N group, the protein expression of FOXO, P-FOXO, JNK, and P-JNK in the Si group was significantly increased (P<0.05). However, the protein expression of PI3K, Akt, P-Akt, 14-3-3, and P-14-3-3 decreased in the Si group (P<0.05) (Figure 4(d)).


## 3.4. Protein and mRNA Expression of Insulin-Related Genes in Cells and Chicken Embryos

We also examined the effects of IGF1 knockdown on the mRNA abundance of insulin-related genes (IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1) in chicken cardiomyocytes (Figure5(a)); the qPCR results revealed that the mRNA expression of IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1 was significantly decreased (P<0.05) in the KD group. A western blot analysis was performed to determine the protein expression of insulin-related genes. The results revealed that compared with the C group, the protein expression of GLUT3, IGF1Rβ, and IGFBP2 in the KD group was significantly decreased (P<0.05) (Figure 5(b)).Figure 5
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of insulin-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of insulin-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)Furthermore, we examined the effects of IGF1 knockdown on the mRNA abundance of insulin-related genes (IGF1R, GLUT1, GLUT3, GLUT8, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP7, IR, and IRS1) in the myocardium of chicken embryos as shown in Figure5(c). The qPCR results revealed that the mRNA expression of IGF1R, GLUT1, GLUT8, IGFBP1, IGFBP2, and IGFBP7 increased in the Si group at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. The mRNA expression of GLUT3, IGFBP3, IR, and IRS1 increased at 6 and 8 days, but significantly decreased (P<0.05) at 10 days. The mRNA expression of IGFBP4 increased at 6 days but significantly decreased at 8 and 10 days. The mRNA expression of IGFBP5 significantly decreased (P<0.05) at 8 and 10 days. A western blot analysis was performed to determine the protein expression of insulin-related genes. The results revealed that compared with the N group, the protein expression of GLUT3, IGF1Rβ, and IGFBP2 in the Si group was significantly decreased (P<0.05) (Figure 5(d)).


## 3.5. The Oxygen Consumption Rate and ATP Content in Myocardial Cells

To assess the effect of IGF1 knockdown on energy metabolism, the oxygen consumption rate and ATP content were detected. The results revealed that IGF1 knockdown significantly decreased the ATP content (Figure6(a)). The results of the oxygen consumption rate revealed that the oxygen consumption rate of the IGF1 knockdown group was 15.166 while the oxygen consumption rate of the control group was 24.019, indicating that IGF1 knockdown significantly decreased the myocardial oxygen consumption rate under the same conditions and the same initial oxygen concentration (Figure 6(b)).Figure 6
(a) The level of ATP was determined to investigate the function of energy metabolism. The data are represented as themeans±SD. Samples with an asterisk (∗) represent significant differences (P<0.05), n=3. The myocardial oxygen consumption rate results are shown in (b). The red curve indicates the oxygen consumption, and the blue curve indicates the oxygen concentration. C indicates the control group; KD indicates the knockdown group.
(a)
(b)The instantaneous oxygen consumption rates of the cardiomyocytes for different groups are provided in Table2.Table 2
Oxygen concentration and the oxygen consumption rate.
Time (min)
1A: O2 concentration (nmol/mL)
1A: O2 flux per V (pmol/(s∗mL))
1B: O2 concentration (nmol/mL)
1B: O2 flux per V (pmol/(s∗mL))
0.03
134.4705
222.2083
0.07
134.361
222.1058
0.1
134.3131
222.0585
0.13
134.2618
222.1088
0.17
134.1943
31.2253
222.157
1.3718
0.2
134.1481
25.9247
222.0753
-5.4236
0.23
134.0865
26.9524
221.9699
6.9917
0.27
134.0053
29.6908
221.9236
24.3313
0.3
133.9386
31.3172
221.8753
32.2136
0.33
133.8958
29.6935
221.8517
14.5802
0.37
133.8531
28.1554
221.8585
13.5949
0.4
133.8078
27.8145
221.8034
15.271
0.43
133.7513
27.2173
221.7768
17.5375
0.47
133.694
26.4491
221.7354
17.44
0.5
133.6496
27.3053
221.6911
14.1902
0.53
133.5888
26.5373
221.6527
14.0926
0.57
133.5316
26.3677
221.6044
15.079
0.6
133.4914
26.0266
221.5699
16.3605
0.63
133.4358
25.6004
221.5187
16.5588
0.67
133.3802
26.457
221.499
14.8846
0.7
133.3238
25.9453
221.4684
14.7868
0.73
133.2365
26.033
221.431
15.2803
0.77
133.1827
26.1198
221.3995
15.7737
0.8
133.1467
25.9497
221.365
15.676
0.83
133.1014
26.4639
221.3246
14.7904
0.87
133.0279
26.2092
221.2931
14.6927
0.9
132.9774
26.2105
221.2497
14.9893
0.93
132.9338
26.1261
221.2143
15.2857
0.97
132.8663
26.0422
221.1739
15.2868
1
132.8064
26.4713
221.1473
14.6963
1.03
132.7414
26.3019
221.1256
14.5984
1.07
132.6687
26.3892
221.0951
14.6976
1.1
132.6345
26.3901
221.0586
14.8956
1.13
132.5969
26.3055
221.0182
14.7981
1.17
132.5524
26.4776
220.9788
14.5035
1.2
132.4772
26.3085
220.9493
14.4057
1.23
132.4361
26.3095
220.9168
14.5051
1.27
132.3746
26.3111
220.8714
14.6047
1.3
132.3147
26.2271
220.836
14.6056
1.33
132.2617
26.3994
220.7926
14.4097
1.37
132.189
26.3157
220.7394
14.411
1.4
132.124
26.4028
220.7089
14.5103
1.43
132.089
26.4037
220.6685
14.7083
1.47
132.024
26.3198
220.6439
14.6104
1.5
131.9581
26.407
220.6064
14.5128
1.53
131.9094
26.4082
220.5749
14.3166
1.57
131.8777
26.409
220.5385
14.3175
1.6
131.8495
26.3242
220.4872
14.4173
1.63
131.7649
26.4118
220.4695
14.4177
1.67
131.6828
26.4994
220.4242
14.5174
1.7
131.6366
26.5005
220.3641
14.5189
1.73
131.5896
26.5017
220.3326
14.5197
1.77
131.5374
26.503
220.3099
14.5202
1.8
131.5041
26.5039
220.2744
14.5211
1.83
131.4545
26.4196
220.2311
14.5222
1.87
131.3698
26.4217
220.2134
14.5227
1.9
131.2988
26.4235
220.1651
14.6224
1.93
131.2518
26.4247
220.1099
14.6238
1.97
131.1979
26.426
220.0469
14.8224
2
131.1304
26.4277
220.0232
14.9215
2.03
131.0833
26.4289
219.9888
14.9223
2.07
131.0269
26.4303
219.9523
15.0217
2.1
130.9645
26.5173
219.909
15.1213
2.13
130.9021
26.5189
219.8597
15.2211
2.17
130.8559
26.5201
219.8272
15.3204
2.2
130.802
26.6069
219.7829
15.3215
2.23
130.7396
26.6085
219.7464
15.421
2.27
130.6797
26.61
219.7228
15.5201
2.3
130.6318
26.6112
219.6735
15.6198
2.33
130.5899
26.6122
219.639
15.7192
2.37
130.5532
26.6131
219.71
15.5204
2.4
130.5147
26.6141
219.71
15.3233
2.43
130.4591
26.53
219.6095
15.1288
2.47
130.3608
26.5324
219.5297
15.1308
2.5
130.3129
26.5336
219.4824
15.132
2.53
130.2658
26.4493
219.441
15.133
2.57
130.2085
26.4507
219.3996
15.1341
2.6
130.1632
26.3664
219.3977
15.0356
2.63
130.1119
26.3676
219.3455
15.0369
2.67
130.0546
26.2836
219.2676
15.0389
2.7
129.9785
26.371
219.2115
15.1388
2.73
129.9281
26.3722
219.1632
15.2385
2.77
129.887
26.3733
219.114
15.4368
2.8
129.8391
26.2889
219.0834
15.536
2.83
129.781
26.2904
219.048
15.6354
2.87
129.7194
26.2919
219.0145
15.6363
2.9
129.6715
26.2931
218.979
15.7357
2.93
129.6159
26.209
218.9317
15.7369
2.97
129.5441
26.2108
218.8736
15.8368
3
129.4774
26.2125
218.8726
15.8369
3.03
129.4099
26.2997
218.8539
15.9358
3.07
129.368
26.3007
218.8224
15.9366
3.1
129.3235
26.3018
218.7642
15.9381
3.13
129.2585
26.3035
218.7248
15.9391
3.17
129.2055
26.2193
218.6795
15.9402
3.2
129.1627
26.2203
218.6342
15.9413
3.23
129.1157
26.2215
218.5977
15.9422
3.27
129.0456
26.2233
218.5652
15.943
3.3
128.9874
26.2247
218.5278
16.0425
3.33
128.9421
26.2259
218.4579
16.1428
3.37
128.9062
26.2268
218.4214
16.2422
3.4
128.8455
26.2283
218.3987
16.2428
3.43
128.7796
26.2299
218.3524
16.3424
3.47
128.7463
26.2308
218.316
16.4418
3.5
128.6693
26.2327
218.2786
16.5413
3.53
128.5992
26.3199
218.2559
16.5419
3.57
128.5308
26.4072
218.2234
16.6412
3.6
128.4803
26.4939
218.18
16.6423
3.63
128.4231
26.5809
218.1288
16.7421
3.67
128.3837
26.5819
218.0855
16.8417
3.7
128.3162
26.5836
218.0559
16.6454
3.73
128.2469
26.5853
218.0185
16.3508
3.77
128.1947
26.5866
217.9643
16.2536
3.8
128.1494
26.6732
217.9436
16.2541
3.83
128.1032
26.6744
217.9298
16.0574
3.87
128.0656
26.6753
217.8875
15.96
3.9
128.0075
26.5913
217.8421
15.9611
3.93
127.9288
26.5932
217.7978
15.7652
3.97
127.8724
26.5947
217.7387
15.6682
4
127.845
26.5953
217.6865
15.768
4.03
127.7877
26.5968
217.6372
15.7692
4.07
127.7099
26.5987
217.6225
15.8681
4.1
127.6654
26.5998
217.6008
15.8686
4.13
127.6081
26.6013
217.5614
15.8696
4.17
127.5363
26.6031
217.5259
15.8705
4.2
127.4713
26.6047
217.4767
15.8717
4.23
127.408
26.6063
217.4402
15.8727
4.27
127.3618
26.6074
217.3969
15.8737
4.3
127.3105
26.6942
217.3565
15.9733
4.33
127.2763
26.6951
217.325
15.9741
4.37
127.2233
26.6964
217.2836
15.9751
4.4
127.1626
26.6979
217.2422
15.8776
4.43
127.1216
26.6989
217.1742
15.8793
4.47
127.054
26.7006
217.1378
15.9787
4.5
126.9813
26.7024
217.0875
15.98
4.53
126.942
26.7034
217.0294
16.08
4.57
126.8941
26.7046
217.0038
16.1791
4.6
126.8342
26.6206
216.9585
16.2788
4.63
126.7932
26.6216
216.8984
16.3788
4.67
126.7145
26.6236
216.8846
16.4776
4.7
126.6461
26.6253
216.858
16.4783
4.73
126.5948
26.5411
216.8235
16.5777
4.77
126.5401
26.5424
216.7762
16.5789
4.8
126.4862
26.5438
216.7457
16.6781
4.83
126.4529
26.4591
216.7033
16.6792
4.87
126.3981
26.4605
216.6708
16.68
4.9
126.3494
26.4617
216.6294
16.681
4.93
126.2955
26.3775
216.5881
16.6821
4.97
126.2314
26.3791
216.5703
16.6825
5
126.1527
26.3811
216.5102
16.684
5.03
126.0963
26.468
216.4748
16.6849
5.07
126.0638
26.4688
216.4482
16.6856
5.1
126.0159
26.47
216.4009
16.6868
5.13
125.9492
26.4717
216.3457
16.6881
5.17
125.9039
26.4728
216.3388
16.5898
5.2
125.844
26.3888
216.4088
16.2925
5.23
125.7901
26.3902
216.3132
16.0979
5.27
125.726
26.3918
216.2078
16.1005
5.3
125.6944
26.3071
216.1861
16.0025
5.33
125.6525
26.2226
216.1142
16.1028
5.37
125.5875
26.1387
216.0659
16.2026
5.4
125.5413
26.1399
215.997
16.3028
5.43
125.4806
26.0559
216.0039
16.2041
5.47
125.4224
25.9718
215.9891
16.106
5.5
125.3745
25.973
215.9359
16.1073
5.53
125.3335
25.974
215.8955
16.0098
5.57
125.2753
25.9755
215.8601
16.0107
5.6
125.2163
25.977
215.8039
16.0121
5.63
125.1462
25.9787
215.7743
15.9143
5.67
125.0855
25.9802
215.7409
15.9151
5.7
125.0393
25.8959
215.6946
15.8178
5.73
124.9889
25.8971
215.6443
15.819
5.77
124.9119
25.8991
215.5921
15.9189
5.8
124.8709
25.9001
215.5665
15.9195
5.83
124.8264
25.9012
215.5192
15.9207
5.87
124.7777
25.9024
215.4818
16.0201
5.9
124.7204
25.8183
215.464
16.1191
5.93
124.6631
25.8198
215.4227
16.1201
5.97
124.5827
25.8218
215.3586
16.2202
6
124.5117
25.9091
215.3468
16.3191
6.03
124.4827
25.9953
215.3212
16.3197
6.07
124.4408
25.9963
215.2867
16.3206
6.1
124.4091
25.9971
215.2256
16.3221
6.13
124.3552
25.9985
215.1675
16.422
6.17
124.3014
25.9143
215.1261
16.4231
6.2
124.2501
25.9156
215.0995
16.4237
6.23
124.1782
25.8319
215.0641
16.5231
6.27
124.1115
25.8335
215.001
16.5247
6.3
124.0619
25.8348
214.9695
16.624
6.33
124.014
25.9215
214.9439
16.6247
6.37
123.9456
26.0087
214.9252
16.6251
6.4
123.8875
26.0102
214.8729
16.6264
6.43
123.8302
26.0116
214.8158
16.6279
6.47
123.7609
26.0989
214.7577
16.7278
6.5
123.7301
26.1851
214.7321
16.7285
6.53
123.684
26.1863
214.7045
16.5321
6.57
123.6215
26.1878
214.672
16.3359
6.6
123.5685
26.2747
214.6276
16.337
6.63
123.5138
26.2761
214.5715
16.3384
6.67
123.4497
26.2777
214.535
16.3393
6.7
123.4009
26.2789
214.4897
16.439
6.73
123.3778
26.1939
214.4395
16.5388
6.77
123.3077
26.1957
214.3794
16.6388
6.8
123.2479
26.1972
214.339
16.7383
6.83
123.2119
26.1126
214.3065
16.7391
6.87
123.1401
26.1144
214.2681
16.8386
6.9
123.1025
26.0298
214.2287
16.8396
6.93
123.0332
26.0315
214.1705
16.9395
6.97
122.987
25.9472
214.1252
17.0392
7
122.9255
25.9487
214.0888
17.0401
7.03
122.875
25.95
214.0799
17.0403
7.07
122.8015
25.9518
214.0504
17.0411
7.1
122.7596
26.0384
214.01
17.0421
7.13
122.7339
25.9535
213.9587
17.1419
7.17
122.663
25.9553
213.8967
17.2419
7.2
122.6134
25.9565
213.8632
17.2428
7.23
122.5544
25.8725
213.8346
17.2435
7.27
122.5013
25.8738
213.8248
17.1452
7.3
122.4304
25.9611
213.7834
17.1462
7.33
122.3731
26.048
213.7528
17.0485
7.37
122.3132
26.0495
213.7213
16.9508
7.4
122.279
26.0504
213.6662
16.8536
7.43
122.2243
26.0518
213.6041
16.8552
7.47
122.1619
25.9678
213.5509
16.8565
7.5
122.0986
25.9694
213.4987
16.9563
7.53
122.0456
25.9707
213.4484
16.9576
7.57
122.0139
25.9715
213.4031
16.9587
7.6
121.9806
25.8868
213.3824
17.0578
7.63
121.9173
25.8029
213.3529
17.0585
7.67
121.8626
25.8043
213.3036
17.0597
7.7
121.8078
25.8056
213.2652
16.9622
7.73
121.7326
25.8075
213.2327
16.963
7.77
121.6873
25.8086
213.213
16.865
7.8
121.6248
25.8957
213.1834
16.7672
7.83
121.5855
25.8967
213.1174
16.7688
7.87
121.547
25.8122
213.0642
16.7702
7.9
121.4957
25.8134
213.0258
16.7711
7.93
121.4495
25.8146
212.9894
16.6735
7.97
121.3803
25.7308
212.949
16.6745
8
121.3161
25.8179
212.9175
16.5768
8.03
121.2648
25.8192
212.9027
16.5772
8.07
121.2092
25.8206
212.8455
16.4801
8.1
121.1742
25.736
212.8081
16.481
8.13
121.1349
25.6514
212.7677
16.5806
8.17
121.0835
25.5672
212.7234
16.5817
8.2
121.0117
25.569
212.6643
16.5831
8.23
120.9604
25.4848
212.614
16.6829
8.27
120.8852
25.5722
212.5707
16.7825
8.3
120.8227
25.5737
212.5559
16.7829
8.33
120.7919
25.5745
212.5165
16.8824
8.37
120.7458
25.4901
212.4958
16.7844
8.4
120.6979
25.4913
212.4791
16.6863
8.43
120.6346
25.4929
212.4111
16.688
8.47
120.5679
25.4946
212.4022
16.5897
8.5
120.5183
25.4958
212.4633
16.3911
8.53
120.4773
25.4968
212.3402
16.2957
8.57
120.4277
25.4126
212.2249
16.3971
8.6
120.355
25.4144
212.1825
16.3982
8.63
120.3105
25.4155
212.1116
16.4984
8.67
120.2857
25.4161
212.0653
16.4996
8.7
120.237
25.4173
212.0722
16.4009
8.73
120.166
25.4191
212.0702
16.3025
8.77
120.1292
25.3345
212.018
16.3038
8.8
120.0754
25.3359
211.955
16.3053
8.83
119.9967
25.4233
211.9087
16.405
8.87
119.9497
25.4245
211.8781
16.5043
8.9
119.9009
25.4257
211.8998
16.4052
8.93
119.8265
25.4276
211.8703
16.3075
8.97
119.7752
25.4289
211.7816
16.3097
9
119.7059
25.5161
211.7107
16.41
9.03
119.6717
25.517
211.6584
16.5098
9.07
119.617
25.6039
211.5944
16.6099
9.1
119.5503
25.6055
211.5461
16.7096
9.13
119.4981
25.6923
211.5245
16.8087
9.17
119.434
25.7795
211.4782
16.8098
9.2
119.3853
25.7807
211.4289
17.0081
9.23
119.3348
25.8675
211.3816
17.1078
9.27
119.2698
25.8691
211.3787
17.2064
9.3
119.2356
25.8699
211.3639
17.2067
9.33
119.2006
25.8708
211.3087
17.2081
9.37
119.1458
25.8722
211.354
17.01
9.4
119.1022
25.8733
211.3797
16.8123
9.43
119.0672
25.7886
211.2358
16.7174
9.47
119.0167
25.7044
211.1373
16.7198
9.5
118.9432
25.6207
211.0743
16.8199
9.53
118.8842
25.6222
211.0181
17.0183
9.57
118.8551
25.5374
211.0181
17.1169
9.6
118.8012
25.5387
210.9698
17.2166
9.63
118.726
25.5406
210.9294
17.3161
9.67
118.6618
25.6277
210.8605
17.4163
9.7
118.6148
25.5434
210.8221
17.5158
9.73
118.5567
25.5449
210.7679
17.5172
9.77
118.5036
25.5462
210.7699
17.5171
9.8
118.4224
25.6337
210.8802
17.2188
9.83
118.3933
25.72
210.7452
16.9267
9.87
118.3591
25.6353
210.6369
16.8309
9.9
118.2958
25.6369
210.5807
16.9308
9.93
118.2266
25.7241
210.5226
17.0307
9.97
118.1958
25.7249
210.4714
17.229
10
118.1376
25.6408
210.4832
17.4258
10.03
118.0855
25.6422
210.428
17.4272
10.07
118.0299
25.558
210.3935
17.428
10.1
117.994
25.4734
210.3591
17.4289
10.13
117.9307
25.3895
210.3059
17.4302
10.17
117.8666
25.3911
210.294
17.4305
10.2
117.8016
25.3927
210.3728
17.2315
10.23
117.7708
25.3935
210.2822
16.9382
10.27
117.7272
25.3946
210.166
16.8426
10.3
117.6562
25.3963
210.1246
16.7451
10.33
117.5972
25.3978
210.0576
16.8453
10.37
117.5716
25.3985
210.0162
16.9449
10.4
117.51
25.4
210.0113
16.945
10.43
117.4536
25.4014
209.968
17.0446
10.47
117.4193
25.4023
209.9088
17.1446
10.5
117.3783
25.4033
209.8714
17.244
10.53
117.2954
25.4054
209.8419
17.2448
10.57
117.2355
25.4924
209.7877
17.4432
10.6
117.1962
25.5789
209.7512
17.4441
10.63
117.1551
25.5799
209.7059
17.4452
10.67
117.1004
25.4958
209.6665
17.4462
10.7
117.0474
25.4971
209.6143
17.349
10.73
116.9721
25.499
209.6833
16.9532
10.77
116.9345
25.4999
209.6232
16.6592
10.8
116.9046
25.3296
209.4882
16.7611
10.83
116.8396
25.3313
209.4616
16.8602
10.87
116.7891
25.247
209.3897
16.9605
10.9
116.7464
25.1626
209.3463
17.0601
10.93
116.6891
25.0785
209.3286
17.1591
10.97
116.6395
24.9942
209.3444
17.0602
11
116.5856
24.9956
209.2793
17.1603
11.03
116.5377
24.9967
209.2153
17.2604
11.07
116.4676
24.9985
209.1789
17.3599
11.1
116.4163
24.9998
209.1345
17.361
11.13
116.3522
25.0869
209.0971
17.1649
11.17
116.3248
25.0021
209.1651
16.8676
11.2
116.2735
25.0034
209.0853
16.7711
11.23
116.2059
25.005
208.969
16.8725
11.27
116.1444
25.0921
208.9582
16.8728
11.3
116.0871
25.0935
208.9119
17.071
11.33
116.0229
25.1806
208.8528
17.0725
11.37
115.9836
25.1816
208.837
17.0729
11.4
115.946
25.1826
208.7878
17.1726
11.43
115.8827
25.1841
208.7444
17.1737
11.47
115.8254
25.1856
208.6873
17.2736
11.5
115.7809
25.2722
208.6548
17.2745
11.53
115.7219
25.3592
208.6193
17.0783
11.57
115.6792
25.3603
208.5819
16.9807
11.6
115.6125
25.3619
208.6223
16.7827
11.63
115.5646
25.4486
208.637
16.5853
11.67
115.5295
25.4495
208.5366
16.4893
11.7
115.4825
25.4507
208.4577
16.4913
11.73
115.4235
25.4522
208.442
16.3932
11.77
115.3713
25.4535
208.3848
16.2961
11.8
115.3294
25.369
208.311
16.3964
11.83
115.2867
25.2846
208.3159
16.2978
11.87
115.2268
25.2861
208.2676
16.299
11.9
115.1704
25.2875
208.2243
16.3001
11.93
115.1088
25.3745
208.1819
16.3011
11.97
115.0558
25.2903
208.1504
16.3019
12
114.9959
25.2918
208.0982
16.3032
12.03
114.9549
25.2929
208.0578
16.3042
12.07
114.8967
25.3798
208.0282
16.108
12.1
114.8258
25.3816
208.1248
15.7115
12.13
114.7727
25.3829
208.0134
15.7143
12.17
114.7326
25.4694
207.8992
15.7171
12.2
114.6787
25.4708
207.8736
15.8163
12.23
114.6334
25.3864
207.8223
15.9161
12.27
114.6043
25.3016
207.7583
16.0162
12.3
114.553
25.3029
207.7741
16.0158
12.33
114.4846
25.2191
207.7534
15.9178
12.37
114.4127
25.2209
207.6982
15.9192
12.4
114.3537
25.2224
207.6588
15.9202
12.43
114.3093
25.309
207.6095
16.0199
12.47
114.2426
25.3107
207.5268
16.1205
12.5
114.2015
25.3117
207.5051
16.0225
12.53
114.1733
25.3124
207.5603
15.8241
12.57
114.1314
25.2279
207.5573
15.7257
12.6
114.0604
25.2297
207.4293
15.7289
12.63
113.9963
25.2313
207.3593
15.7306
12.67
113.9535
25.3179
207.2973
15.9292
12.7
113.9056
25.3191
207.247
16.029
12.73
113.8509
25.3205
207.2224
16.1281
12.77
113.8184
25.3213
207.246
16.226
12.8
113.7663
25.2371
207.1948
16.2273
12.83
113.7141
25.2384
207.1377
16.3273
12.87
113.6551
25.2398
207.1002
16.4267
12.9
113.5833
25.2416
207.0451
16.6251
12.93
113.5285
25.3285
206.9948
16.6264
12.97
113.4841
25.3296
206.9603
16.4302
13
113.4413
25.3307
206.9298
16.431
13.03
113.3883
25.332
206.8579
16.5313
13.07
113.3327
25.3334
206.8756
16.5308
13.1
113.2848
25.3346
206.9396
16.3322
13.13
113.2258
25.2506
206.8372
16.3348
13.17
113.1839
25.2516
206.7466
16.337
13.2
113.1506
25.1669
206.716
16.3378
13.23
113.0719
25.0834
206.651
16.3394
13.27
113.0155
25.0848
206.5899
16.538
13.3
112.9573
25.0863
206.5939
16.5379
13.33
112.918
25.0872
206.5535
16.5389
13.37
112.8684
25.0885
206.517
16.6383
13.4
112.8025
25.0901
206.4658
16.6396
13.43
112.7624
25.1766
206.4106
16.4439
13.47
112.7145
25.1778
206.3643
16.4451
13.5
112.6572
25.1793
206.3121
16.5449
13.53
112.5965
25.1808
206.2688
16.6445
13.57
112.5306
25.268
206.2363
16.7438
13.6
112.493
25.1834
206.2116
16.843
13.63
112.4554
25.1843
206.1821
16.8437
13.67
112.3998
25.1002
206.1545
16.8444
13.7
112.3425
25.1871
206.122
16.7467
13.73
112.2963
25.1883
206.0875
16.7476
13.77
112.2279
25.2755
206.053
16.7484
13.8
112.1638
25.3626
206.0176
16.7493
13.83
112.1219
25.3637
205.988
16.75
13.87
112.0791
25.3648
205.925
16.6531
13.9
112.0124
25.3664
205.9171
16.3578
13.93
111.962
25.4532
205.986
16.0605
13.97
111.9286
25.454
205.8836
15.866
14
111.8696
25.4555
205.7969
15.9667
14.03
111.826
25.4566
205.7732
15.9673
14.07
111.7644
25.4581
205.7102
15.9689
14.1
111.7251
25.3736
205.659
15.9702
14.13
111.6738
25.3749
205.6639
15.8715
14.17
111.6105
25.291
205.5861
15.8735
14.2
111.5566
25.2923
205.522
15.8751
14.23
111.4865
25.3796
205.4826
15.9746
14.27
111.4455
25.3806
205.4521
16.0739
14.3
111.4164
25.3813
205.4186
16.0747
14.33
111.36
25.3827
205.4018
16.0751
14.37
111.3009
25.3842
205.3506
16.1749
14.4
111.2325
25.3859
205.3201
16.1757
14.43
111.1906
25.387
205.2836
15.9795
14.47
111.1479
25.388
205.3152
15.6832
14.5
111.0923
25.3039
205.324
15.486
14.53
111.0324
25.3054
205.2265
15.3899
14.57
110.9657
25.3071
205.1408
15.392
14.6
110.9332
25.3079
205.0945
15.4917
14.63
110.876
25.3093
205.0373
15.4931
14.67
110.811
25.3965
204.992
15.4943
14.7
110.7622
25.3977
204.9782
15.4946
14.73
110.7118
25.3989
204.9142
15.5947
14.77
110.6485
25.4005
204.8728
15.6943
14.8
110.6066
25.4016
204.8482
15.7934
14.83
110.5844
25.4021
204.8029
15.8931
14.87
110.5262
25.3181
204.7418
16.0916
14.9
110.4715
25.3194
204.7162
16.2893
14.93
110.4108
25.3209
204.6926
16.3884
14.97
110.3586
25.3223
204.6591
16.4877
15
110.297
25.3238
204.6118
16.5874
15.03
110.2474
25.325
204.5763
16.5883
15.07
110.203
25.3261
204.5605
16.5887
15.1
110.1371
25.3278
204.5162
16.5898
15.13
110.079
25.4148
204.4719
16.6894
15.17
110.0328
25.4159
204.4098
16.691
15.2
109.9926
25.4169
204.3803
16.7902
15.23
109.9302
25.4185
204.3744
16.6919
15.27
109.8883
25.505
204.4561
16.2958
15.3
109.8609
25.4202
204.3576
16.0027
15.33
109.8122
25.3359
204.2778
15.9062
15.37
109.7386
25.3378
204.2266
15.9075
15.4
109.6762
25.3393
204.1566
15.9092
15.43
109.63
25.255
204.1133
16.0088
15.47
109.5796
25.2562
204.1172
15.8117
15.5
109.5377
25.2573
204.0798
15.8126
15.53
109.4975
25.1728
204.0148
15.9128
15.57
109.4633
25.1736
203.9606
16.0126
15.6
109.3966
25.0898
203.933
16.1118
15.63
109.3188
25.0917
203.8966
16.1127
15.67
109.264
25.0931
203.867
16.1135
15.7
109.2221
25.0086
203.8246
16.2131
15.73
109.1512
25.0959
203.7872
16.214
15.77
109.1204
25.0967
203.7557
16.2148
15.8
109.0853
25.012
203.7251
16.117
15.83
109.0297
24.9279
203.6769
15.8227
15.87
108.9853
24.929
203.6355
15.7252
15.9
108.9297
24.9304
203.5941
15.7262
15.93
108.8775
24.8462
203.5567
15.7272
15.97
108.8151
24.8478
203.5193
15.7281
16
108.7647
24.849
203.4917
15.8273
16.03
108.7245
24.85
203.4651
15.7295
16.07
108.6757
24.8512
203.4207
15.7306
16.1
108.6364
24.8522
203.3498
15.8309
16.13
108.5765
24.8537
203.2897
15.9309
16.17
108.5167
24.7697
203.272
16.0298
16.2
108.4594
24.7711
203.2375
16.1292
16.23
108.3978
24.7727
203.1922
16.2289
16.27
108.3499
24.7739
203.1518
16.3284
16.3
108.3037
24.775
203.137
16.3288
16.33
108.2576
24.7762
203.1055
16.4281
16.37
108.1986
24.7777
203.0612
16.4292
16.4
108.1558
24.7787
203.0011
16.4307
16.43
108.1131
24.7798
202.9705
16.5299
16.47
108.0446
24.867
202.9508
16.5304
16.5
107.9856
24.8685
202.9173
16.5313
16.53
107.9352
24.9553
202.8809
16.6307
16.57
107.8805
25.0422
202.8375
16.5333
16.6
107.8497
25.0429
202.7804
16.3377
16.63
107.8086
24.9584
202.7489
16.2399
16.67
107.7633
24.8741
202.7193
16.1422
16.7
107.7094
24.8754
202.6908
16.0444
16.73
107.6436
24.8771
202.6484
16.1439
16.77
107.6111
24.8779
202.6307
16.0459
16.8
107.5435
24.8796
202.6031
15.948
16.83
107.4939
24.8808
202.5489
15.8509
16.87
107.4486
24.8819
202.5046
15.852
16.9
107.3905
24.7979
202.4721
15.8528
16.93
107.3152
24.7998
202.409
15.8544
16.97
107.2827
24.8861
202.3755
15.8552
17
107.2391
24.8872
202.3469
15.8559
17.03
107.1724
24.8888
202.3184
15.8567
17.07
107.1126
24.9758
202.275
15.7592
17.1
107.0561
25.0628
202.2445
15.76
17.13
106.9997
25.0642
202.1962
15.7612
17.17
106.9544
25.1508
202.1489
15.7624
17.2
106.9202
25.1517
202.1135
15.6647
17.23
106.8783
25.0672
202.1834
15.466
17.27
106.8158
25.0688
202.1263
15.3689
17.3
106.7577
25.1557
202.0248
15.3714
17.33
106.7184
25.1567
201.9992
15.2736
17.37
106.6611
25.1582
201.946
15.1764
17.4
106.5995
25.1597
201.8967
15.1776
17.43
106.5499
25.0754
201.8958
15.1776
17.47
106.5011
25.0766
201.8524
15.1787
17.5
106.4687
25.0775
201.8022
15.18
17.53
106.4208
25.0786
201.7657
15.1809
17.57
106.3541
25.0803
201.7174
15.2806
17.6
106.3173
25.0812
201.6593
15.3806
17.63
106.2609
25.0826
201.617
15.4801
17.67
106.1907
25.0844
201.5825
15.481
17.7
106.1454
25.0855
201.547
15.5804
17.73
106.0924
25.0869
201.5155
15.5812
17.77
106.0445
25.0881
201.4692
15.6809
17.8
105.9975
25.0892
201.4396
15.7801
17.83
105.9376
25.1762
201.3845
15.7815
17.87
105.8923
25.1774
201.4436
15.6815
17.9
105.8376
25.1787
201.4495
15.4843
17.93
105.788
25.18
201.3254
15.4874
17.97
105.7521
25.1809
201.2899
15.4883
18
105.6973
25.0967
201.219
15.5886
18.03
105.6221
25.0986
201.151
15.6888
18.07
105.5785
25.0997
201.148
15.7874
18.1
105.5417
25.0151
201.1106
15.7883
18.13
105.475
25.0168
201.0584
15.7896
18.17
105.4186
25.0182
200.9953
15.8897
18.2
105.3724
25.0193
200.9648
15.8905
18.23
105.3262
25.0205
200.9313
15.9898
18.27
105.2826
25.0216
200.9047
16.089
18.3
105.2287
24.9374
200.8781
16.0897
18.33
105.168
24.9389
200.9707
15.8903
18.37
105.1201
24.9401
200.8722
15.8928
18.4
105.074
24.9413
200.7747
15.8952
18.43
105.0363
24.9422
200.7491
15.8959
18.47
104.973
25.0293
200.6919
15.8973
18.5
104.9235
25.0306
200.6338
16.0958
18.53
104.8781
25.0317
200.6141
16.0963
18.57
104.8123
24.9478
200.5757
15.9987
18.6
104.7413
25.0351
200.5382
15.9012
18.63
104.708
25.036
200.5028
15.902
18.67
104.6703
25.0369
200.4446
15.9035
18.7
104.631
24.9524
200.4141
16.0028
18.73
104.5805
24.9536
200.3727
16.0038
18.77
104.5181
24.9552
200.3323
16.0048
18.8
104.4651
24.9565
200.3195
16.0051
18.83
104.4044
24.958
200.3796
15.8066
18.87
104.3625
24.9591
200.2663
15.7109
18.9
104.3197
24.8746
200.1777
15.8117
18.93
104.2607
24.8761
200.1461
15.8124
18.97
104.2154
24.8772
200.1077
15.9119
19
104.1701
24.7929
200.0664
16.0115
19.03
104.1111
24.7943
200.0752
16.0112
19.07
104.0623
24.7956
200.0181
16.1112
19.1
104.0222
24.7966
199.9629
16.1126
19.13
103.9862
24.712
199.9215
16.2121
19.17
103.9221
24.7136
199.8821
16.3116
19.2
103.8853
24.629
199.8792
16.2132
19.23
103.8263
24.6304
199.823
16.0175
19.27
103.763
24.632
199.7767
15.9202
19.3
103.7194
24.5476
199.8575
15.7211
19.33
103.6767
24.4632
199.7807
15.526
19.37
103.6245
24.4645
199.6605
15.6276
19.4
103.5476
24.5519
199.6437
15.628
19.43
103.5022
24.553
199.5817
15.728
19.47
103.4595
24.5541
199.5324
15.8278
19.5
103.4107
24.5553
199.5166
15.9267
19.53
103.3654
24.5564
199.4851
16.026
19.57
103.3218
24.5575
199.4585
16.0267
19.6
103.2722
24.4733
199.4103
16.1264
19.63
103.2064
24.4749
199.3679
16.1274
19.67
103.1653
24.4759
199.3068
15.9319
19.7
103.12
24.4771
199.2645
15.933
19.73
103.0618
24.4785
199.2507
15.9333
19.77
103.0063
24.4799
199.3393
15.6356
19.8
102.9524
24.4813
199.2241
15.6385
19.83
102.9028
24.4825
199.1285
15.6409
19.87
102.8412
24.5696
199.0999
15.7401
19.9
102.8045
24.5705
199.0379
15.8402
19.93
102.7514
24.6573
198.9975
15.8412
19.97
102.6916
24.6588
198.9935
15.9398
20
102.6463
24.7455
198.9512
15.9408
20.03
102.5941
24.7468
198.8931
16.0408
20.07
102.5342
24.7483
198.8428
16.1406
20.1
102.4821
24.7496
198.8133
16.2398
20.13
102.4573
24.7502
198.7857
16.339
20.17
102.4111
24.7513
198.7561
16.1427
20.2
102.3624
24.7525
198.697
16.1442
20.23
102.2982
24.7542
198.6813
16.1446
20.27
102.2452
24.841
198.7532
16.0443
20.3
102.2127
24.8418
198.6172
16.0477
20.33
102.1597
24.7576
198.5364
16.1482
20.37
102.087
24.8449
198.5
16.2477
20.4
102.0477
24.8459
198.4488
16.3474
20.43
101.9964
24.8472
198.4094
16.4469
20.47
101.9553
24.7627
198.4143
16.4468
20.5
101.9134
24.7638
198.3493
16.547
20.53
101.8647
24.6795
198.3089
16.548
20.57
101.798
24.6811
198.2586
16.6477
20.6
101.7338
24.6827
198.2271
16.747
20.63
101.6817
24.6841
198.1926
16.6494
20.67
101.6372
24.6852
198.1808
16.4527
20.7
101.5928
24.6863
198.1335
16.4538
20.73
101.5508
24.6873
198.1197
16.4542
20.77
101.4953
24.6887
198.1936
16.2553
20.8
101.4431
24.69
198.0685
16.1599
20.83
101.3952
24.6912
197.9907
16.1619
20.87
101.3499
24.6923
197.968
16.1624
20.9
101.296
24.6937
197.901
16.1641
20.93
101.2285
24.6954
197.8577
16.1652
20.97
101.1797
24.6966
197.8616
16.1651
21
101.131
24.6978
197.8104
16.1664
21.03
101.0771
24.6992
197.7444
16.2665
21.07
101.0275
24.7004
197.6823
16.3666
21.1
100.9796
24.7016
197.6518
16.1703
21.13
100.9437
24.7025
197.64
16.0721
21.17
100.9027
24.618
197.6114
16.0728
21.2
100.8556
24.6192
197.571
16.0739
21.23
100.8095
24.6203
197.5286
16.0749
21.27
100.759
24.5361
197.5996
15.9746
21.3
100.7017
24.5375
197.505
15.78
21.33
100.6427
24.539
197.4085
15.8809
21.37
100.5948
24.5402
197.373
15.8818
21.4
100.5392
24.6271
197.3257
15.9815
21.43
100.4896
24.6283
197.2981
15.9822
21.47
100.4452
24.6295
197.2804
15.9826
21.5
100.3879
24.6309
197.2311
15.9838
21.53
100.3468
24.5464
197.1917
15.9848
21.57
100.2964
24.5477
197.1474
16.0844
21.6
100.2357
24.6347
197.1178
15.7896
21.63
100.1818
24.5505
197.0804
15.7906
21.67
100.1219
24.6375
197.0607
15.7911
21.7
100.0886
24.6384
197.1238
15.5925
21.73
100.039
24.6396
197.0646
15.4954
21.77
99.9937
24.6407
196.9642
15.4979
21.8
99.9338
24.6422
196.9326
15.4987
21.83
99.8817
24.558
196.8568
15.5006
21.87
99.8415
24.559
196.8016
15.6005
21.9
99.7902
24.5603
196.8026
15.699
21.93
99.7397
24.5616
196.7612
15.7986
21.97
99.6961
24.5627
196.7208
15.7996
22
99.6294
24.5643
196.6834
15.8005
22.03
99.5918
24.5653
196.6351
15.9002
22.07
99.5405
24.5666
196.5928
15.9013
22.1
99.4909
24.5678
196.5691
15.7048
22.13
99.4396
24.5691
196.5238
15.6075
22.17
99.3865
24.5704
196.5021
15.608


## 3.6. Protein and mRNA Expression of Cardiac Differentiation-Related Genes in Cells and Chicken Embryos

The effects of IGF1 knockdown on the mRNA abundance of cardiac differentiation-related genes (MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5) in chicken cardiomyocytes are shown (Figure7(a)), and the qPCR results revealed that the mRNA expression of MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5 was significantly decreased (P<0.05) in the KD group. A western blot analysis was performed to determine the protein expression of cardiac differentiation-related genes. The results revealed that compared with the C group, the protein expression of MyoG and MyoD in the KD group was significantly decreased (P<0.05) (Figure 7(b)).Figure 7
The effects of IGF1 knockdown on the mRNA levels (a) and protein levels (b) of cardiac differentiation-related genes in cardiomyocytes and the effects of IGF1 silencing on the mRNA levels at 6, 8, and 10 days (c) and on the protein levels (d) of cardiac differentiation-related genes in the myocardium. C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo. GAPDH was selected as the reference.∗ shows a significant difference from the corresponding control (P<0.05). n=3.
(a)
(b)
(c)
(d)The effects of IGF1 knockdown on the mRNA abundance of cardiac differentiation-related genes (MyoD, MyoG, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5) in the myocardium of chicken embryos are shown in Figure7(c). The qPCR results revealed that the mRNA expression of MyoD, Mesp1, and MYF5 increased in the Si group at 6 and 8 days, but significantly decreased (P<0.05) at 10 days. The mRNA expression of MyoG, GATA4, and GATA6 increased at 6 days, but significantly decreased at 8 and 10 days. The mRNA expression of MYF6 increased at 6 days, showed no significant change at 8 days, and significantly decreased (P<0.05) at 10 days. The mRNA expression of Nkx2.5 decreased (P<0.05) at 6, 8, and 10 days. A western blot analysis was performed to determine the protein expression of cardiac differentiation-related genes. The results revealed that compared with the N group, the protein expression of MyoG and MyoD in the Si group was significantly decreased (P<0.05) (Figure 7(d)).


## 3.7. Intracellular Morphological Observation and H.E. Stain in Cells and Chicken Embryos’ Myocardium

As observed under a microscope, normal cardiomyocytes were fusiform and tightly connected and the whole looked similar to paving stones accompanied by protruding pseudopodia that stretched out between cells, interweaving into a mesh in the control group (Figure8(a)). In the KD group, as the density of cell growth decreased, the volume of the cardiomyocytes and intercellular junctions was evidently reduced. We observed that myocardial fibers and muscle fiber bundles were disintegrated, and the pseudopodia between cells did not interweave into a mesh in the KD group (Figure 8(b)). We observed myocardial cells stained by hematoxylin and eosin (H.E.). The cardiomyocytes in the control group displayed normal morphologies (Figure 8(c)). However, many slender cardiomyocytes appeared in the IGF1 knockdown group (Figure 8(d)), which indicated that cardiomyocyte development was blocked.Figure 8
Morphological observation was performed in cardiomyocytes transfected with Opti-MEM (C group, a) and siRNA (KD group, b) for 24 h. Cardiomyocytes were stained by H.E., and the results are shown in (c) and (d). H.E. staining for myocardial tissues in the N group (e) and the Si group (f). C indicates the control group; KD indicates the knockdown group in vitro; N indicates the normal group; Si indicates the knockdown group in vivo.
(a)
(b)
(c)
(d)
(e)
(f)Myocardial injury and ultrastructural damage are shown in Figure8(f). Blood vessel rupturing and increased tissue gaps were observed in the IGF1-deficient chicken heart group more than in the normal group (Figure 8(e)).


## 4. Discussion

IGF1 is a polypeptide neurotropic factor with a structure and function similar to insulin. IGF1 is a single-chain protein that promotes cell differentiation and proliferation, and it has a wide range of biological functions and participates in the regulation of various organs. IGF1 plays an important role in the development of human and vertebrate embryos [23]. In the present study, we established an IGF1 knockdown model in vivo and in vitro through transfection of small interfering RNA (siRNA) and the mRNA and protein levels of IGF1 were detected to support this point. Significant damage to myocardial tissue, rupturing of blood vessels, and increased tissue gaps were observed in IGF1-deficient chicken heart through histopathological observation, demonstrating that IGF1 suppression leads to dysplasia of cardiomyocytes and myocardial tissue.The concentration of free radicals is low under physiological conditions, which is important to cell signal regulation, metabolism, survival, and apoptosis [24, 25]. However, a large number of free radicals are induced when organisms are stimulated by physical factors or exogenous chemical substances; free radicals can covalently bind to biomacromolecules and peroxidize biofilm lipids to produce various toxic effects [26], which can lead to the occurrence of many diseases such as myocardial infarction [27, 28] and various cancers [29]. Oxygen free radicals can make lipid fatty acids into lipid peroxides and further decompose into a series of complex compounds, including MDA; therefore, the level of lipid oxidation can be detected by the level of MDA [30]. iNOS is an oxidative stress (free radical) that utilizes nitric oxide, which can be produced when cells are stimulated and activated. The cells can form a complex antioxidant enzyme defense system, which mainly includes SOD, CAT, GSH, and so on, to protect the body from peroxidative damage [26, 31]. SOD can eliminate free radicals in the body and protect cells from free radical damage. The level of SOD activity reflects the ability of antifree radicals. SOD can convert superoxide anion (O2-) into H2O2, and CAT converts H2O2 into water such that toxic O2- and H2O2 are converted into harmless water molecules [32, 33]. GSH is catalyzed to convert to oxidized glutathione (GSSH) with the assistance of GSH-Px, which can reduce oxidized substances and relieve their toxicity [34, 35]. In recent years, the antioxidant functions of IGF1 have been gradually discovered; Tumati et al. found that low IGF1 can induced excessive ROS, which will be further moderated by JNK-induced epithelial cytoprotection [36]. ROS, as an important endogenous stimulator in the body, can stimulate multiple pathways including myocardial development. Huk et al. found that ROS serves as secondary messengers to influence cardiac valve development [37]. ROS may be involved in adverse cardiac remodeling [38]. In our present study, we found that decreasing expression of IGF1 results in increasing ROS generation, suggesting that IGF1 may be involved in the regulation of ROS and the occurrence of oxidative stress. IGF1 significantly decreased the CAT, SOD, GSH, and GSH-Px activities in vivo and in vitro. These changes were accompanied by reduced T-AOC, which is an important indicator for determining the body’s antioxidant capacity; meanwhile, the expression of iNOS was significantly increased. All of these results demonstrate that IGF1 suppression can significantly reduce the body’s antioxidant capacity and enrich many oxygen free radicals in the body; this may be one of the important causes of myocardial damage and dysplasia.FOXO, a highly conserved transcriptional regulatory protein, is a downstream protein of the PI3K/Akt and AMPK/14-3-3 signaling pathways [39, 40]. The PI3K/Akt/FOXO signaling pathway is involved in the regulation of various physiological processes such as proliferation, apoptosis, and insulin resistance. The PI3K/Akt pathway can be activated by several of these stimuli, and ROS is one of the most important factors. Adipogenic differentiation can be mediated through activation of the PI3K/Akt pathway by oxidative stress in primary rat osteoblasts [41]. Stitt et al. found that the IGF1/PI3K/Akt pathway plays an important role in preventing the expression of ubiquitin ligases by inhibiting FOXO transcription factors [42]. AMPK is an important protein kinase in eukaryotic cells. The energy regulation of AMPK can maintain normal ATP levels in cardiomyocytes [43]. AMPK regulates the utilization of energy throughout the body, and it is considered the energy regulator [44]. FOXO is an important downstream molecule of AMPK involved in the signal transduction and regulation of various cellular biological processes such as inhibiting cell proliferation and promoting apoptosis [45, 46]. IGF1 stimulation can decrease the level of AMPK phosphorylation in F1 and F3/4 granulosa cells [47]. Hinchy proved that the ROS released from mitochondria can activate AMPK indirectly [48]. Furthermore, FOXO has been proven to be required in endothelial but not myocardial cell lineages during cardiovascular development [49]. Evans-Anderson et al. demonstrated that a FOXO transcription factor is a negative regulator of cardiomyocyte proliferation during heart development [50]. In our present study, we found that IGF1 depletion inhibited the expression of IRS1, PI3K, and Akt and upregulated both the mRNA and protein expression of AMPK and FOXO; meanwhile, the expression of JNK significantly increased in the IGF1 suppression group, which is important upstream of IRS1. All of these results suggest that IGF1 knockdown can activate FOXO through inhibiting the expression of IRS1. Combined with the results of ROS, we concluded that IGF1 suppression triggers ROS release to activate FOXO, which further inhibits myocardial development.Mitochondria, as an energy converter in animal cells, are a main site for intracellular oxidative phosphorylation and ATP formation. Pawlikowska et al. showed that mitochondria are essential organelles for insulin-mediated muscle formation and that insulin stimulates mitochondria and promotes mitochondrial function [51]. Insulin-like growth factor-binding proteins (IGFBPs) are important parts of the IGF family; IGFBPs cannot bind with insulin but rather form a complex with IGFs. They can regulate the normal growth of embryonic and postnatal organs, which is also a very important tool for IGF1 transport and storage. The IGF1-IGFBP complex will decompose and release IGF1, which will further combine with IGF1R in the cell membrane under the catalysis of IR [52]. IGF1 is protected from decomposition by its high affinity with IGFBP, prolonging the half-life of IGF1 in the body’s circulation cells. IGFBPs avoid the body’s negative effects for insulin overdose by reducing the concentration of free IGF1 in the blood. In addition, studies have shown that IGFBP can help IGF1 recognize target cell and regulate the activity of IGF1 [53]. The IGF1-IGFBP complex can cause a cascade of various phosphorylation processes in the cell, ultimately leading to the entry of the glucose transporter molecules (GLUT1, 3, and 8) into the cell membrane and increasing the rate of glucose transport in the cell. Glucose transport is mainly dependent on GLUT, and it plays an important role in regulating glucose transport and maintaining cardiac energy balance [54]. IGF1RS/IRS signaling regulates cellular energy metabolism through the PI3K/Akt signaling pathway, and its downstream genes have been found [55]. FOXO plays an important role in mediating the effects of insulin and growth factors on diverse physiological functions [56]. In the present study, we demonstrated that IGF1 knockdown significantly decreased the expression of GLUT3 and IGFBP, indicating that IGF1 suppression blocks energy metabolism. To further verify these results, we also detected the ATP content and oxygen consumption rate in two different groups; the results revealed that IGF1 knockdown can significantly impede the ability of using oxygen for cardiomyocytes, thereby reducing the production of ATP. Considering our results showing FOXO activation by IGF1 knockdown, we conclude that FOXO can inhibit myocardial development by interfering with myocardial energy metabolism.Myogenic regulatory factors (MRFs) are a class of muscle-specific regulators that determine the function of cell-directed differentiation. MRFs are essential for muscle development in vertebrates [56]. MRFs include MYF5, MyoD, MyoG, and MRF4; the function of MRFs is transforming mesenchymal stem cells into myoblasts, which will further activate and maintain the differentiation state of myocytes. Each member of the MRF family plays different roles at different states. MyoD and MYF5 play an important role in the early proliferative phase of myocytes [57]. MYF5 is responsible for myoblast proliferation, and MyoD regulates the differentiation process for myoblasts. MyoG and MRF4 can drive terminal differentiation. The proliferation process of myoblasts is normal after knocking out MyoG, but the subsequent differentiation process is significantly inhibited [58]. Mesp1 and Mesp2 are key genes in regulating cardiac differentiation. Mesp1 can directly activate other genes in the cardiac core by binding to other gene promoters in cardiomyocytes. Mesp1 and Mesp2 reduction can impede heart development [59]. GATA4 and Nkx2.5 are transcription factors involved in heart development, and overexpression of GATA4 can accelerate myocardial differentiation [60]. Early embryo development requires the production and expenditure of large amounts of cellular energy for cell growth. Insulin can regulate skeletal muscle cell differentiation by mediating the activation of MAPK and PKB phosphorylation [61]. Montarras et al. demonstrated that insulin or IGF is necessary for cell differentiation [62]. Cellular energy metabolism that contains fatty acid is involved in fetal heart development [63]. In the present study, we found that IGF1 knockdown can significantly decrease the expression of MyoD, Mesp1, MYF5, MYF6, GATA4, GATA6, and Nkx2.5, indicating that IGF1 suppression can block myocardial development. We suggest that reducing energy metabolism inhibits the expression of myocardial development to factors through comprehensive results on energy metabolism.In summary, we conclude that IGF1 knockdown hinders myocardial development through the energy metabolism dysfunction caused by ROS-dependent FOXO activation in the chicken heart. Our results indicate a novel hypothesis for IGF1 in the development of cardiomyocytes.



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