Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C‐Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury

Abstract

Background

The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell‐cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine.

Methods and Results

Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co‐Immunoprecipitation (Co‐IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO‐IP experiments. Results showed that the 1–265 domain of CHK1 directly binds to the 157–400 amino acids of PKM2. Furthermore, hiPSC‐CM (human iPS cell‐derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C‐domain‐mediated cardiac metabolic reprogramming.

Conclusions

This study demonstrates that the 1–265 amino acid domain of CHK1 binds to the 157–400 domain of PKM2 and activates PKM2‐mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Nonstandard Abbreviations and Acronyms

CHK1

checkpoint kinase 1

cTNT

cardiac troponin T

I/R

ischemia/reperfusion

mTOR

mammalian target of rapamycin

PKM2

pyruvate kinase isoform M2

rhCHK1

recombinant human checkpoint kinase 1

Sf9

spodoptera frugiperda 9

Clinical Perspective.

What Is New?

• Intramyocardial administration of rhCHK1 (recombinant human checkpoint kinase 1)‐hydrogel promotes cardiac regeneration and repair by promoting cardiomyocyte proliferation, inhibiting cardiomyocyte apoptosis, and reducing inflammatory responses; in the long term, rhCHK1‐hydrogel effectively reduces scar area, improves cardiac function, and attenuates ventricular remodeling in a porcine ischemia/reperfusion model.

• CHK1 (checkpoint kinase 1) binds to PKM2 (pyruvate kinase isoform M2) C‐domain and activates PKM2‐mediated metabolic reprogramming to promote cardiomyocyte proliferation and cardiac repair.

What Are the Clinical Implications?

• This translational research showed rhCHK1 might be a novel therapeutic concept in patients with myocardial infarction and provided a theoretical basis for further randomized clinical trials to evaluate the safety and efficacy of rhCHK1 in patients with acute myocardial infarction.

Ischemic heart disease is the leading cause of death worldwide. ¹ , ² , ³ Myocardial infarction (MI) is the most severe manifestation of ischemic heart disease. ⁴ Currently, approaches for promoting cardiac repair have been extensively explored, including promoting myocardial regeneration, inhibiting myocardial apoptosis, and alleviating myocardial fibrosis. ⁵ , ⁶ , ⁷ , ⁸ The cardiac regeneration strategy mainly focuses on increasing the number of cardiomyocytes, fundamentally correcting the loss of vital cardiomyocytes after MI, and then reducing scar area. ⁹ , ¹⁰ , ¹¹

Unlike many other tissues, adult mammalian cardiomyocytes generally withdraw from the cell cycle and only remain with limited proliferation capacity, thus it is difficult to stimulate cardiomyocyte regeneration for enhancing cardiac repair after cardiac injury. ¹² Recent studies have shown that neonatal pig hearts can regenerate functional cardiac muscle after cardiac insult. Moreover, there is some evidence that humans also have the same ability to regenerate heart muscle to some extent, suggesting that adult mammalian heart regeneration can be achieved by stimulating adult cardiomyocytes to re‐enter the cell cycle. ¹⁰ , ¹³ , ¹⁴ , ¹⁵ , ¹⁶

Recent studies have found that the function of many genes can affect metabolic processes, and metabolic reprogramming also plays a key role in myocardial regeneration and repair. Promoting glycolysis and inhibiting oxidative phosphorylation of adult cardiomyocytes can stimulate cardiomyocytes to re‐enter the cell cycle. ¹³

Our previous research indicated that overexpression of CHK1 (checkpoint kinase 1) in adult mice could promote myocardial regeneration and improve cardiac function after MI by activating the mechanistic target of rapamycin complex 1 (mTORC1)/70 kDa ribosomal protein S6 kinase (P70S6K) pathway. ¹⁷ However, the relationship between CHK1 and metabolic reprogramming has not been reported.

Protein kinase biotherapy has many advantages such as high activity, low toxicity, and explicit function. Recent studies have found that hydrogel, with good histocompatibility, can deliver appropriate therapeutic agents to the target area. ¹⁸ In this experimental study, we tested the hypothesis that locally applied rhCHK1 (recombinant human checkpoint kinase 1) protein kinase (using hydrogel as the rhCHK1 protein kinase carrier) could stimulate cardiac repair after ischemia/reperfusion (I/R) injury in pigs. This translational research might provide important evidence, bridging the gap between basic research and future clinical application of promoting cardiac repair in patients after ischemic insult with exogenous CHK1.

Methods

The data that support the findings of this study are available from the corresponding author upon request for purposes of reproducing the results or replicating the procedure. Additional methods can be found in Data S1 and Table S1.

Animals and Ethics Statement

Adult Bama pigs (12±2 months old, 25±5 kg) were purchased from Animal Core Facility of Nanjing Medical University. Animals were raised in cages under a controlled temperature of 22 °C to 25 °C and kept on a 12‐hour light–dark cycle. All animal experiments performed for the current study were in strict accordance with the guidelines for the care and use of laboratory animals and were approved by the Committee of Nanjing Medical University (IACUC‐2005033).

Construction and Production of Injectable Thermosensitive Hydrogel

Hydrogel was provided by the State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University. The synthetic procedure was introduced previously. ¹⁹ Briefly, the thermosensitive hydrogel was composed of 3 poly (D,L‐lactic acid‐co‐glycolic acid)‐b‐poly(ethylene glycol)‐b‐poly(D,L‐lactic acid‐co‐gly‐colic acid) (PLGA‐PEG‐PLGA) triblock copolymers with different PLGA/PEG ratios and showed satisfactory sol–gel transition in response to temperature. PLGA‐PEG‐PLGA copolymers were synthesized by ring‐opening copolymerization of lactic acid (LA) and glycolic acid (GA) with PEG as the initiator and stannous octoate as the catalyst. In this study, 2 types of PLGA‐PEG‐PLGA copolymers with different PLGA/PEG ratios were synthesized by initiating PEG1000 or PEG1500. ²⁰ The copolymer self‐assembled into micelles in water, and after heating, the micelles aggregated to form a penetrating micellar network, resulting in sol–gel conversion.

Preparation of rhCHK1

The preparation method of rhCHK1 can be found in a previous report, ²¹ , ²² and rhCHK1 was produced in frugiperda 9 (Sf9 [spodoptera frugiperda 9]) cells by baculovirus expression system. The brief key steps were as follows: To generate the recombinant transfer plasmid pFastBacHTA‐cell penetrating peptide 1 (PEP1)‐CHK1‐porcine teschovirus‐1 2A peptide (P2A)‐enhanced green fluorescent protein (EGFP), the optimized CHK1 genes PEP1‐CHK1‐P2A‐EGFP were cloned into the restriction endonuclease BamHI site and restriction endonuclease HindIII site of the baculovirus transfer vector pFastBacHTA. ²³ Then, the purified plasmid DNA was transformed into DH10Bac (Escherichia coli DH10BAC strain) super competent cells for transposition into the bacmids. Recombinant bacmid DNA, which was appropriate for use in insect cell transfections, was isolated from DH10Bac Escherichia coli (Qiagen) and analyzed by polymerase chain reaction (PCR). After confirming that the recombinant bacmid contains the gene of interest, Sf9 insect cells were transfected with Cellfectin II Reagent (Gibco) to produce recombinant baculovirus according to the guidelines and instructions. After successful cell transfection, the virus‐containing medium was transferred from cell plates to sterile 15‐mL snap‐cap tubes, and the tubes were centrifuged at 500g for 5 minutes. The clarified supernatant was then transferred into fresh 2.0‐mL tubes and defined as P0 virus stock. P0 virus stock was a low‐titer stock, which was amplified by infecting Sf9 cells to generate the higher‐titer baculovirus stock named P1 virus stock. The high titer (1 × 10⁸ pfu/mL) pFastBac baculovirus stock was used for infecting Sf9 insect cells. Next, 125 mL of sf9 cells (1.5 × 10⁶ cells/mL) were cultured in triangle glass bottle, 50 to 100 μL of P1 virus stock was added, and flat‐shake culture was performed in the sf9 incubator at cell culture circumstance (200 rpm, 27 °C) for 72 hours.

Cells were then harvested and lysed with lysis buffer. The lysate supernatant was collected into a 50 mL spin tube after completely pelleting the cell debris. Qiagen's superflow Ni‐NTA resin was used for the purification of the sf9‐expressed protein as follows: after exhausting the resin‐preserving solution and balancing the resin with wash buffer, the balanced resin with the lysate supernatant was transferred into another 50‐mL spin tube and rocked for 4 to 6 hours on a rotary mixer at 4 °C. All of the lysate‐Ni‐NTA mixture was loaded into the 5‐mL plastic column, and the flowthrough was collected. After binding of EGFP‐labeled protein to the resin, the resin (original color is blue) will turn light green. After purification, P2A (2A peptide) underwent a broken process to get rid of the EGFP and reduced the molecular weight, which might facilitate membrane penetration thereafter, and the target rhCHK1 was then collected and stored at −80 °C for the following experiments.

I/R Injury Model Construction and Hydrogel Injection

The preparation method of the adult pig I/R model was based on previous research reports. ²⁴ The brief steps were as follows: Echocardiographic measurement by the Simpson method was first performed to ensure that the animal was healthy before instrumentation and I/R induction. Anesthesia was maintained with a mixture of 1% to 2% isoflurane dissolved in 40% air and 60% oxygen after intramuscular injection of Zoletil50 (4–6 mg/kg) followed by tracheal intubation, intravenous access, and mechanical ventilation with positive pressure. Glucosa‐insulin‐potassium solution was infused intravenously (30 drops per minute), and the vital signs of the pigs (respiration, ECG, heart rate, blood pressure, oxygen saturation) were continuously monitored. The left anterior descending coronary artery was exposed from the median thoracic incision and isolated from surrounding tissue distal to the first diagonal branch with a 5–0 sliding suture; both ends of the suture thread passed through a plastic tube and were tightened to occlude the exposed left anterior descending coronary artery. Ischemic preconditioning was performed by occlusion for 5 minutes before formal occlusion to reduce the occurrence of ventricular arrhythmias. The occlusion was confirmed by the presence of regional myocardial cyanosis, and abnormal echocardiography visualized abnormal segmental movement. After 60 minutes, the suture was released to achieve myocardial reperfusion.

Immediately after reperfusion, we rapidly closed the chest in the pigs. The surviving animals were examined with echocardiography, then the pigs with comparable ejection fraction (ranged from 30% to 45%) were randomly divided into 3 groups and received intramyocardial injections of saline, nonloaded hydrogel, or rhCHK1‐hydrogel equally spaced along the visually identified infarct border zone (8 injection sites per Bama pig heart, 100 μL [rhCHK1: 1 mg/kg] per injection). The sham group underwent thoracotomy to expose the heart, but without the ligation of the left anterior descending coronary artery. Bama pigs were treated with lidocaine (1.5 mg/kg bolus followed by continuous infusion) and amiodarone (5 mg/kg bolus followed by continuous infusion) to prevent and treat ventricular arrhythmias. Electrical defibrillation was used when indicated.

Hearts Sample Collection and Examination

All pigs were subjected to I/R surgery, and 48 of the pigs with left ventricular ejection fraction (LVEF) ranging between 30% and 45% at 1 hour after ligation were selected for subsequent studies. Of the 48 pigs included in the study, 28 were male and 20 were female. Thirty‐six pigs were randomly divided into 3 groups: I/R+Control (n=12), I/R+hydrogel (n=12), and I/R+rhCHK1‐hydrogel (n=12). The pigs in each group were randomly assigned to 3‐, 7‐, and 28‐day postinfarction (DPI) subgroups (n=4 each) for hearts sample collection and related examination per study design (see below). The remaining pigs were used for mechanism study purpose and grouped as follows: I/R+hydrogel group, I/R+rhCHK1‐hydrogel group, I/R+rhCHK1+PKM2 RNAi(PKM2i) group (n=3 each).

On the 3rd, 7th, and 28th day after operation, Bama pigs assigned to the 3‐, 7‐, and 28‐day subgroups were anesthetized and euthanized by injection of 10% KCl. Animals euthanized at 3 days after operation were used to evaluate cardiomyocytes proliferation and apoptosis, cardiac inflammation, and myocardial injury markers. Animals euthanized at 7 days after operation were used to evaluate cardiomyocytes proliferation, apoptosis, and inflammation. Animals euthanized at 28 days after operation were used to evaluate the scar size and degree of interstitial fibrosis. Briefly, the hearts were cross‐sectioned in four 0.5‐cm‐thick slices, starting from the apex toward the base. Then, each slice was divided into 6 regions (indicated by letters). Paraffin sections (5 μm) were used for Masson and hematoxylin and eosin (H&E) staining, and ImageJ (version 1.51) was used on Masson‐stained sections for infarct size calculation of each animal. For mechanistic study, we collected the myocardium from infarct border zone in the I/R+hydrogel and I/R+rhCHK1‐hydrogel groups at 7 days postoperation for RNA sequencing combined with proteomics analysis, respectively, to explore the underlying signaling changes.

Echocardiography Analysis

Cardiac function was evaluated by echocardiography (IE33 digital ultrasonic scanner; Philips Medical Systems) 3 days before I/R surgery, during I/R surgery (1 hour after ligation), and 28 days after surgery. Specifically, after intramuscular anesthesia, the pigs were fixed at a left oblique 30° to 45°. Oxygen saturation and changes on 12‐lead ECGs were monitored. Two‐dimensional echocardiographic images of parasternal long‐axis and apical 4‐chamber view were sequentially collected. LVEF, left ventricular end diastolic volume, and left ventricular end systolic volume were derived from the Simpson method on apical 4‐chamber views. Echocardiography data were obtained and analyzed by experienced cardiologists who were not aware of the study groups.

Cardiac Magnetic Resonance Imaging

All pigs underwent magnetic resonance imaging on the 28th day after the operation. The cardiac function of the pigs was evaluated, and the infarct area was determined. After intramuscular injection of Zoletil50, propofol was used to maintain anesthesia. Pigs were kept in a shallow sleep state with normal breathing. Pigs were fixed in a left lateral position and the ECG, heart rate, and respiration of the pig were continuously monitored during the whole process. In the short‐axis and long‐axis views of the heart, retrospective ECG gating and steady‐state free precession magnetic resonance imaging were used to obtain magnetic resonance cine images.

Cardiac Magnetic Resonance Imaging Data Acquisition

The cardiac magnetic resonance imaging scan was performed on a 3.0 T system (Ingenia; Philips Healthcare, Best, the Netherlands). Cine sequences were performed by balanced steady‐state free pression, breath‐hold technique, and with retrospective ECG triggering in the short‐axis. Overall, 10 to 12 slices of short‐axis, 2‐chamber, 3‐chamber, and 4‐chamber long‐axis (LAX) were performed. T2‐weighted short‐tau inversion recovery (T2W‐STIR) and late gadolinium enhancement (LGE) imaging of short‐axis slices were correlated to cine imaging to ensure they were in the same position. LGE images were scanned 8 to 10 minutes after the contrast medium was injected. The 3 short‐axis slices of T1 and T2 mapping were basal, mid, and apical. The contrast medium was Magnevist (Bayer, German), the dose was 10 mL for each pig, and the injection speed was 4.0 mL/s. Technical parameters are as follows: 1.2 ms echo time, 40 ms repetition time, flip angle 50°, field of view 300 mm, slice thickness 8 mm, and image matrix 256 × 256. Images obtained were analyzed, and the infarct area was measured by experienced radiologists who were not aware of the study groups.

Transcriptomics

Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. Fragmentation was performed using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. The 2 strands of cDNA were then synthesized separately. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina paired‐end (PE) adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 400 to 500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15‐cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent).

The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina). The original data in FASTQ format (raw data) were generated. We use Cutadapt (version 1.15) software to filter the sequencing data to get high‐quality sequencing (clean data) for further analysis. We used HTSeq 0.9.1 statistics to compare the read count values on each gene as the original expression of the gene.

Proteomics

Proteomic digestion was performed as described. Pig hearts were lysed in protein extraction buffer (8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.2, 1% [vol/vol] EDTA‐free protease inhibitor, 1 mM NaF, 1 mM β‐glycerophosphate, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF)). Protein concentration was measured using the Bradford assay. Cysteine residues were reduced with dithiothreitol (DTT) at a 5 mM final concentration for 25 minutes at 56 °C, followed by alkylation in 14 mM iodoacetamide for 30 minutes at room temperature in the dark. Unreacted iodoacetamide was quenched with DTT for 15 minutes. Lysates were then diluted down to 1.6 M urea with 25 mM Tris, pH 8.2. CaCl2 was added to a final concentration of 1 mM. The proteins were then digested overnight at 37 °C with trypsin in a 1:200 enzyme‐to‐protein ratio and subsequently quenched by addition of trifluoroacetic acid. The peptides were desalted using a Sep‐Pak column from Waters (Milford, MA). For tandem mass tag (TMT) labeling, purified peptides were reconstituted in 200 mM triethylammonium bicarbonate. Twenty‐two microliters of TMT reagent (Pierce) was added to 45 μg peptides (measured by A280) and allowed to react for 1 hour at room temperature. MI‐group samples were labeled with 126, 128, and 130 labels and the sham‐group with 127, 129, and 131 labels. The reaction was quenched for 15 minutes by addition of 5% hydroxylamine. After TMT labeling, all 6 samples were combined and purified using an Oasis hydrophilic‐lipophilic balance (HLB) 1‐cc Vac cartridge (Waters) and then lyophilized.

TMT‐labeled peptide mixture was resuspended in 100 μL buffer A (10 mM ammonium acetate, pH 10) and loaded onto a XBridgeTM BEH130 C18 column (300 μm × 150 mm, 1.7 μm; Waters) with the UltiMate 3000 high performance liquid chromatography (HPLC) system at a flow rate of 4 μL per minute. Fractions were collected every 30 seconds in a 73‐minute gradient, and were further pooled to 20 fractions using the nonadjacent pooling scheme. The fractions were then dried under vacuum for proteome quantification.

The tryptic peptides were dissolved in 0.1% formic acid (solvent A), directly loaded onto an inhouse‐packed reversed‐phase analytical column (20 cm, 75 μm ID). The gradient comprised an increase from 3% to 5% solvent B (0.1% formic acid in acetonitrile) over 5 seconds, 5% to 15% in 23.9 minutes, 15% to 28% in 21 minutes, 28% to 38% in 7.5 minutes, and climbing to 100% in 5 seconds and then holding at 100% for the last 12.4 minutes, all at a constant flow rate of 300 nL/min on an EASY‐nLC 1200 ultra‐performance liquid chromatography (UPLC) system.

The peptides were subjected to an nanoelectrospray lonization (NSI) source followed by tandem mass spectrometry in a Thermo Fisher Scientific Orbitrap Fusion Lumos Tribrid coupled online to the UPLC. The electrospray voltage applied was 2.1 kV. The mass/charge (m/z) scan range was 400 to 1600 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 60 000. Peptides were then selected for tandem mass spectrometry using normalized collision energy (NCE) setting as 38, and the fragments were detected in the Orbitrap at a resolution of 15 000. A data‐dependent procedure alternated between 1 mass spectrometry scan followed by 20 tandem mass spectrometry scans. Automatic gain control was set at 1.25E5. The fixed first mass was set as 105 m/z.

Raw files were searched against the pig protein sequences obtained from the Universal Protein Resource database (release 2021_04) using Proteome Discoverer software (version 2.4). False discovery rates (FDRs) were estimated using the target‐decoy strategy, and FDR cutoffs were set to 0.01 for sites, peptides, and proteins. Enzyme specificity was considered to be full cleavage by trypsin, and 2 maximum missed cleavage sites were permitted. The minimum required peptide length was set to 6 residues. Carbamidomethyl was set as fixed modification. Variable modifications included oxidation, and acetylation. Protein quantification was based on the reporter ion intensities of TMT6 reagents at the peptide level.

Gene Enrichment Analysis

We put the data after RNA sequencing and mass spectrometry screening (FDR < 0.05) into the Ingenuity Pathway Analysis software (Qiagen) for analysis. From the canonical pathway library of the Ingenuity Pathway Analysis software, the probability of each biological function assigned to this data set was determined independently using the Fisher exact test based on statistical significance.

Statistical Analysis

All of the statistical data were analyzed using the Prism8 software package. Data are expressed as mean±SD or mean±SEM. Normality distribution of the data was assessed using the Shapiro‐Wilk test. If the data conformed to a normal distribution, the comparisons between 2 groups were performed by the Student t test. For the comparisons among multiple groups, 1‐way ANOVA (with the post hoc Tukey test) or 2‐way ANOVA (with the post hoc Sidak test) were performed to determine the statistical differences. If the data did not conform to the normal distribution, we used a nonparametric test to analyze the data. For all the statistical analyses, significance was accepted at P<0.05.

Results

Construction and Verification of Purified Recombinant Human CHK1

To enhance the purification and transmembrane efficiency of CHK1 protein, the purified rhCHK1 protein was produced by eukaryotic expression systems, which binds the CHK1 protein and pep‐1 (cell‐penetrating peptide). The molecular weight of rhCHK1 was 70 kd. The activated phosphorylation site (Ser317) of rhCHK1 was also confirmed (Figure S1). Importantly, expression of rhCHK1 in primary mouse cardiomyocytes increased dose‐dependently in response to increasing doses of applied rhCHK1 (Figure S1). These results hinted that the purified rhCHK1 was physiologically functional.

Local Injection of rhCHK1‐Hydrogel Upregulated CHK1 Expression in the Infarct Border Zone

The level of CHK1 in the hearts of adult pigs was lower than in the hearts of neonatal and adult mice (Figure S2A). I/R pigs with similarly reduced LVEF (Figure S3A, B) were randomly grouped and received a local injection of saline (I/R+Control), hydrogel (100 μL, I/R+hydrogel), and rhCHK1‐hydrogel (1 mg/kg, I/R+rhCHK1‐hydrogel) in the border zone of infarcted myocardium at 1 hour postreperfusion per study protocol (Figure 1A). To test the delivery efficiency of rhCHK1‐hydrogel in the border zone of the infarcted heart, the hearts at 3 days after I/R were cross‐sectional and sliced into 4 main sections. For further analysis: each segment contained an anterior wall (A region), interventricular septum (S region), posterior wall (P region), and lateral wall (LT region). The effect of rhCHK1 injection was also verified by Western blot (WB) (Figure 1B). Furthermore, the P1 and A3 section in the pig treated with rhCHK1‐hydrogel underwent immunofluorescence staining, and the results indicated that CHK1 was enriched in the cardiomyocytes surrounding the injection sites (Figure 1C and 1D). The above results indicated that intramyocardial injection of exogenous rhCHK1‐hydrogel effectively enhanced CHK1 expression in the border zone of pig hearts with I/R.

Figure 1. Local delivery method of rhCHK1 into the border zone of pig hearts with MI.

A, Study design schematic. B, Cross‐sectional comparison of infarcted myocardium in the I/R+rhCHK1‐hydrogel group. For further analysis, each segment contained anterior wall (A region), interventricular septum (S region), posterior wall (P region), and lateral wall (LT region). C and D, Representative immunofluorescence images of tissue sections of porcine hearts subjected to I/R injury and rhCHK1 injection. The injected rhCHK1 proteins were identified by colocalization of Flag and cTNT. The individual numbers in (C) and (D) represent the I/R+CHK1 group. Scale bar=50 μm. The comparisons were performed by Student t test (D). Data are presented as mean±SD or mean±SEM. ***P<0.001. CHK1 indicates checkpoint kinase 1; I/R, ischemia/reperfusion; MI, myocardial infarction; MRI, magnetic resonance imaging; p‐CHK1(S317), phosphorylated checkpoint kinase 1 at Ser317; and rhCHK1, recombinant human checkpoint kinase 1.

rhCHK1 Promoted Cardiomyocyte Proliferation and Reduced Cardiomyocyte Apoptosis in Pig Hearts After I/R

Three days after surgery, upregulation of CHK1, phosphorylated CHK1 (p‐CHK1), Flag, phosphorylated mTOR (p‐mTOR), phosphorylated P70S6K (p‐P70S6K) proteins was observed and quantified in the infarct border zone of the I/R+rhCHK1‐hydrogel group (Figure S4A, B). Immunofluorescence results showed that the number of Ki‐67 antigen (Ki67)⁺, phospho‐histone H3 (pH3)⁺ and Aurora B⁺ cardiomyocytes was significantly increased in the infarct border zone of the I/R+rhCHK1‐hydrogel group (Figure 2A through 2C, Figure S5A, B). Wheat germ agglutinin staining showed that the cardiomyocyte size was smaller in the rhCHK1‐hydrogel group. The number of total mononucleated cardiomyocytes was significantly higher in rhCHK1‐hydrogel group than in the hydrogel group (Figure 2D through 2G). These results indicated that rhCHK1 could reactivate cell cycle and promote cardiomyocyte proliferation in the infarct border zone of pigs with I/R.

Figure 2. rh‐CHK1 injection promoted cardiomyocyte proliferation and inhibited apoptosis and acute inflammation in 3‐DPI porcine myocardium.

A through C, Porcine tissue sections from normal and infarcted border zone from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups were stained for cTNT to visualize cardiomyocytes, Hoechst to visualize nucleus, proliferation marker Ki67, pH3, and Aurora B to mark endogenous cardiomyocyte proliferation. N=4 for each group. Scale bar=20 μm. D, Representative immunofluorescence images of WGA for CMs size from I/R+CON, I/R+hydrogel, and I/R+rhCHK1 groups. Scale bar=50 μm. E and F, Quantification and comparison of CM size and numbers in the infarcted border zone. N=4. G, Quantification of nucleation (mono‐, bi‐, multi‐) of CMs from I/R+CON, I/R+hydrogel, and I/R+rhCHK1 group. N=4 for each group. H, Representative immunofluorescence images of Tunel⁺ CMs from I/R+CON, I/R+hydrogel, and I/R+rhCHK1 groups. Scale bar=20 μm. I, H&E staining for acute inflammatory infiltration in heart slices from 3 groups at 3 DPI. Scale bar=50 μm. J, mRNA level of acute inflammation markers from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel group was detected by qRT‐PCR 3 days postinjury. In comparisons among multiple groups, the ANOVA was tested to determine the universal difference. If there is a universal difference among groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (A through H, J). Data are presented as mean±SD or mean±SEM. *P<0.05, **P<0.01, ***P<0.001. CMs indicate cardiomyocytes; CON, control; cTNT indicates cardiac troponin T; DPI, days postinfarction; H&E, hematoxylin and eosin; IL‐1β, interleukin 1β; IL‐6, interleukin 6; I/R, ischemia/reperfusion; NS, no significance; and rhCHK1, recombinant human checkpoint kinase 1.

Furthermore, the cardiomyocyte apoptosis was also significantly alleviated in the infarct border zone of I/R+rhCHK1‐hydrogel group (Figure 2H). H&E staining and quantitative reverse transcription polymerase chain reaction (qRT‐PCR) were performed to evaluate the acute reactive inflammatory responses among the different groups at 3 days after I/R. The H&E staining showed that the inflammatory infiltrates were alleviated after rhCHK1 injection (Figure 2I). qRT‐PCR showed that compared with the I/R+Control and I/R+hydrogel group, the mRNA levels of inflammatory cytokines including IL (interleukin)‐6, IL‐1β, tumor necrosis factor alpha (TNF‐α) and NOD‐like receptor family pyrin domain containing 3 (NLRP‐3) were significantly downregulated in the I/R+rhCHK1‐hydrogel group (Figure 2J).

The WB results showed that protein expression of p‐CHK1, p‐mTOR and p‐P70S6K were significantly upregulated in the I/R+rhCHK1‐hydrogel group at 7 DPI (Figure 3A). Compared with the I/R+ Control group and I/R+ hydrogel group, rhCHK1 increased the proliferation rate of cardiomyocytes and reduced the level of apoptosis (Figure 3B through 3E, Figure S6A, B). In addition, qRT‐PCR and the H&E staining results showed that cardiac inflammation was reduced at 7 DPI (Figure 3F and 3G). Levels of cardiac troponin T (cTNT) and cardiac troponin I (cTNI) were similar among the I/R+rhCHK1‐hydrogel, I/R+Control, and I/R+hydrogel groups at 3 DPI, whereas the levels of cTNT and cTNI were lower at 7 DPI in the I/R+rhCHK1‐hydrogel group compared with the I/R+Control and I/R+hydrogel groups. The level of proBNP (pro‐B‐type natriuretic peptide) was also lower at 28 DPI in the I/R+rhCHK1‐hydrogel group (Figure 3H). The WB results showed rhCHK1 (70 kilodalton [Kd]) was not detectable in the liver and kidneys (Figure S7).

Figure 3. The infarcted border zone treated with rhCHK1 after 7 DPI of I/R.

A, The expression levels of downstream proteins were measured by Western blot. B, Porcine tissue sections from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups were stained for cTNT to visualize cardiomyocytes; Hoechst to visualize the nucleus, Ki67, pH3, and Aurora B; and Tunel to mark CM proliferation and apoptosis. Right: Relevant proliferation and apoptosis markers quantification measurements. C, Quantification of nucleation (mono‐, bi‐, multi‐) of cardiomyocytes from I/R+CON, I/R+hydrogel, and I/R+rhCHK1 groups. N=4 for each group. D, mRNA levels of cell cycle‐related markers in myocardium of I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups were quantified by qRT‐PCR 7 days postinjury. E, The expression levels of apoptosis and inflammation‐related proteins were detected by Western blot. F, H&E staining for acute inflammatory infiltrate in heart slices from 3 groups at 7 DPI. G, mRNA level of acute inflammation markers from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups was detected by qRT‐PCR 7 days postinjury. N=4 for each group. H, cTNT, cTNI, and proBNP in the plasma of pigs in the I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups at indicated time points (cTNT, cTNI: intraoperation, 3 and 7 days after I/R; proBNP: intraoperation, 28 days after I/R). N=4 for each group. Comparing multiple groups, the ANOVA was tested to determine the universal difference. If there was a universal difference between groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (A through D, G, and H). Data are presented as mean±SD or mean±SEM. *P<0.05, **P<0.01, ***P<0.001. Bax indicates Bcl2‐associated X protein; Bcl2, B‐cell lymphoma‐2; CM, cardiomyocyte; CON, control; cTNI: cardiac troponin I; cTNT: cardiac troponin T; DPI, days postinfarction; H&E, hematoxylin and eosin; IL‐1β, interleukin 1β; IL‐6, interleukin 6; IL‐18, interleukin 18; I/R, ischemia/reperfusion; mTOR, mammalian target of rapamycin; NS, no significance; p‐CHK1, phosphorylated checkpoint kinase 1; p‐mTOR, phosphorylated mammalian target of rapamycin; proBNP, pro‐B‐type natriuretic peptide; and rhCHK1, recombinant human checkpoint kinase 1.

Collectively, our results indicated that injection of rhCHK1 in the infarct border zone promoted cardiomyocyte proliferation and attenuated I/R‐induced apoptosis and inflammation in pigs.

rhCHK1 Improved Cardiac Function, Reduced Cardiac Scar Area, and Attenuated Ventricular Remodeling in the Long Term of I/R Pigs

Echocardiography was performed immediately after reperfusion, and 28 days postsurgery to monitor the cardiac function of pigs in the different groups. After reperfusion, pigs with decreased LVEF were randomly assigned to 3 groups (Figure 4A through 4C).

Figure 4. rhCHK1 improved cardiac function and alleviated scar and fibrosis formation in the long‐term after I/R injury.

A, Representative Simpson 4‐chamber heart image from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups at the indicated time. B, LVEF of pigs in 3 groups at the indicated time. N=4 for each group. C, LVEF change comparison among 0 to 28 DPI from the I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups. N=4 for each group. D, Representative H&E, Masson, and immunohistochemistry for α‐SMA and cTNT staining for heart slices from 3 groups. Scale bar=3 cm. Scale bar=50 μm (H&E, Masson, immunohistochemistry). E, Quantification of fibrosis area from heart slices. N=4. F through H, mRNA levels of fibrosis markers (Collagen I, Collagen III, and α‐SMA) at 28 DPI detected by qRT‐PCR. Data are presented as mean±SD. *P<0.05. **P<0.01. ***P<0.001. I, Representative cardiac magnetic resonance imaging from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel groups. Scale bar=3 cm. J and K, Gross anatomy and fibrous area quantification of cardiac slices from I/R+CON, I/R+hydrogel, and I/R+rhCHK1‐hydrogel heart at 4 weeks after the surgery. N=4. Data are presented as mean±SD or mean±SEM. *P<0.05, **P<0.01, ***P<0.001. #, I/R+hydrogel compared with I/R+CON, P<0.05. ##, I/R+hydrogel compared with I/R+CON, P<0.01. Comparing multiple groups, the ANOVA was tested to determine the universal difference. If there was a universal difference between groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (B, C, E through H). Two‐way ANOVA (with the post hoc Sidak test) was performed to determine the statistical differences (K). CON indicates control; cTNT, cardiac troponin T; DPI, days postinfarction; EF, ejection fraction; H&E, hematoxylin and eosin; I/R, ischemia/reperfusion; LVEF, left ventricular ejection fraction; NS, no significance; and rhCHK1, recombinant human checkpoint kinase 1.

To explore the long‐term therapeutic effects on the ventricular remodeling after I/R, pathological section, H&E, Masson, and immunohistochemistry staining were conducted at 28 DPI. Compared with the I/R+Control and I/R+hydrogel groups, rhCHK1‐hydrogel reduced the fibrosis area (Figure 4D and 4E). Similarly, the levels of fibrosis markers (alpha smooth muscle actin (α‐SMA), Collagen‐1, Collagen‐3) were significantly lower in the rhCHK1‐hydrogel group than in the nonloaded hydrogel group and I/R+Control group (Figure 4F through 4H). Scar mass of infarcted pig hearts at 28 DPI was also detected by cardiac magnetic resonance imaging. Pigs in the I/R+Control group exhibited prominent fibrous scar accounting for nearly 30% of ventricles from the apex to the base, which was significantly reduced in the I/R+rhCHK1‐hydrogel group (Figure 4I through 4K).

Considering the potential side effects created by vascular invasion after local rhCHK1 injection, we next evaluated the morphological and functional characteristics of other organs after myocardial rhCHK1 injection. There was no organ toxicity or neoplasm formation in the lungs, kidneys, and liver as shown by H&E staining (Figure S8). Liver and kidney function was not affected by rhCHK1 injection (Figure S9), which indicated that local injection of rhCHK1 was safe during the 28‐day follow‐up.

These results indicated that rhCHK1‐hydrogel could improve cardiac function, attenuate left ventricular remodeling, and reduce cardiac fibrosis in pigs with I/R injury.

rhCHK1 Promoted hiPSC Cardiomyocytes Proliferation

To find out whether rhCHK1 can also promote human‐derived CMs proliferation in vitro, we applied it in hiPSC (human iPS cell‐derived) cardiomyocytes, and evaluated it by immunofluorescence staining such as Ki67, pH3, and Aurora B. Immunofluorescence results showed that the number of Ki67⁺, pH3⁺, and Aurora B⁺ hiPSC cardiomyocytes was increased in the rhCHK1 group (Figure 5A through 5C). Immunofluorescence staining also showed that the cardiomyocytes’ size was smaller in the rhCHK1 group. The number of total hiPSC cardiomyocytes per millimeter squared was higher in the rhCHK1 group than in the Control group (Figure 5D).

Figure 5. rhCHK1 promoted hiPSC‐CMs proliferation.

A through C, hiPSC‐CMs from the CON and rhCHK1 groups were stained for cTNT to visualize cardiomyocytes; Hoechst to visualize the nucleus, proliferation marker Ki67, and pH3; and Aurora B to mark cardiomyocyte proliferation. N=6 for each group. Scale bar= 50 μm. D, Representative immunofluorescence images for hiPSC‐CMs size from the CON and rhCHK1 groups. Scale bar=100 μm. Quantification and comparison of hiPSC‐CMs size and numbers in the 2 groups. N=6. Data are presented as mean±SD or mean±SEM. ***P<0.001. The comparisons were performed by Student t test (A through D). CON indicates control; cTNT, cardiac troponin T; hiPSC‐CMs, human iPS cell‐derived cardiomyocytes; and rhCHK1, recombinant human checkpoint kinase 1.

These results indicated that rhCHK1 could promote hiPSC cardiomyocytes proliferation in vitro.

Underlying Mechanisms of rhCHK1‐Triggered Cardiac Repair After I/R Injury

We previously demonstrated the CHK1‐mediated cardiomyocyte proliferation via the mTORC1/P70S6K pathway in mice. In the current study, we further performed transcriptomics and proteomics analysis (FDR < 0.05) on the border regions of pig hearts at 7 DPI with nonloaded hydrogel or rhCHK1‐hydrogel. All of the differentially expressed genes were enriched in the regulation of the eukaryotic initiation factor 4 (eIF4) and P70S6K signaling and mTOR pathway, which were consistent with the WB experimental results of 3 DPI. In addition, the glycolysis process was also enriched (Figure 6A through 6E). Furthermore, the glucose‐6‐phosphate dehydrogenase (G6PD) mRNA level was also found to be prominently higher in rhCHK1‐hydrogel group, showing that the pentose phosphate pathway might also strengthened (Figure 6F). To clarify which rate‐limiting enzyme in glycolysis took part in the metabolic reprogramming promoted by rhCHK1 treatment, the WB results indicated that PKM2 (pyruvate kinase isoform M2) and G6PD expression were all significantly activated in I/R+rhCHK1‐hydrogel group 7 days after I/R, whereas hexokinase 2 (HK2) and phosphofructokinase platelet (PFKP) remained unchanged (Figure 6G and 6H).

Figure 6. Multiomics pathway analysis found that rhCHK1 possibly promoted cardiac metabolic reprogramming via activating PKM2.

A and B, RNA‐sequencing data from 7‐DPI samples were analyzed using the IPA database, and the results also showed multiple pathways related to cell cycle, inflammation, and apoptosis. C, The proteomics data of the 7‐DPI samples were also analyzed using the IPA database, and the relevant results are presented. D, Chord diagrams of the selected 7 pathways. E, The heatmap of pathways enriched by transcriptomics and proteomics analysis 7 days postsurgery (P<0.05). F, The mRNA levels of G6PD were verified by qRT‐PCR. G through H, The relative protein levels of HK2, PFKP, PKM2, and G6PD was verified by WB. Comparing multiple groups, the ANOVA was tested to determine the universal difference. If there was a universal difference between groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (F and H). Data are presented as mean±SD or mean±SEM. *P<0.05, **P<0.01, ***P<0.001. CON indicates control; DPI, days postinfarction; G6PD; glucose‐6‐phosphate dehydrogenase; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; HK2, hexokinase 2; MS, mass spectrometry; NS, no significance; PFKP, phosphofructokinase, platelet; PKM2, pyruvate kinase isozyme type M2; and rhCHK1, recombinant human checkpoint kinase 1.

rhCHK1 Improved Glycolysis and Pentose Phosphate Process by Directly Binding With PKM2

Previous studies found that the transition from glycolysis to fatty acid metabolism in postnatal hearts is consistent with the loss of cardiac regenerative capacity. ²⁵ , ²⁶ Furthermore, the activation of PKM2 was found to promote the G6PD‐dependent pentose phosphate pathway and stimulate cardiomyocyte proliferation after MI. ²⁷ Because PKM2 and G6PD were not activated in the I/R+hydrogel group, the mechanism experiments were mainly conducted in the I/R+hydrogel group and I/R+rhCHK1‐hydrogel group, and relevant data were analyzed in these 2 groups. To further clarify the relationship between CHK1 and glycolysis, WB was performed on pig myocardium at 7 DPI with nonloaded hydrogel or rhCHK1‐hydrogel. The protein expression of phosphorylated PKM2 serine 37 (p‐PKM2 S37), phosphorylated PKM2 tyrosine 105 (p‐PKM2 Y105), G6PD, and lactate dehydrogenase A (LDHA) were increased (Figure 7A). qRT‐PCR experiments indicated that G6PD and LDHA mRNA level were also higher in the I/R+rhCHK1‐hydrogel group (Figure 7B). Furthermore, we used protein CO‐IP experiment to explore the detailed relationship between CHK1 and PKM2, and could rhCHK1 directly bind with PKM2 and increase the protein level of PKM2 (Figure 7C). To verify whether PKM2 could promote cardiac metabolic reprogramming and cardiomyocyte proliferation, we transfected neonatal mouse cardiomyocytes with cTNT‐PKM2 plasmid. The results showed that PKM2 activated cardiac glycolysis and promoted cardiomyocyte proliferation with S37 and Y105 phosphorylated (Figure S10). In the pig myocardium, we also found PK (pyruvate kinase) activity, pyruvate content, LA content, and glucose content were prominently higher in rhCHK1‐hydrogel group (Figure 7D through 7G). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) results indicated that rhCHK1 could improve cardiac glycolysis and inhibit oxidative phosphorylation in hiPSC cardiomyocytes (Figure 7H and 7I). Protein docking prediction showed that CHK1 and PKM2 shared structural motif for direct binding. Prediction results from Schrödinger software revealed that CHK1 and PKM2 may form a protein interaction interface (Figure 7J). To further explore specific interaction between CHK1 and PKM2, truncated mutants of CHK1 were constructed with deletion of 1–265 amino acid fragment (Δ 1–265 aa), 270–327 amino acid fragment (Δ 270–327 aa), or 391–476 amino acid fragment (Δ 391–476 aa; Figure 7K). Truncated mutants of PKM2 were constructed with deletion of 24–124 amino acid fragment (Δ 24–124 aa) and 157–400 amino acid fragment (Δ 157–400 aa; Figure 7L). We transfected them into HEK293T cells for CO‐IP experiments. It was found that the 1–265 domain of CHK1 directly binds to the 157–400 amino acids of PKM2 (Figure 7M through 7P). Further 5‐ethynyl‐2ʹ‐deoxyuridine (EDU), Ki67, and pH3 staining in vitro showed that CHK1 stimulated hiPSC cardiomyocyte proliferation by binding with PKM2 C‐domain (Figure 7Q through 7S).

Figure 7. CHK1 directly interacts with PKM2 and promotes cardiac glycolysis at 7 DPI for pigs.

A, Relative protein level of p‐PKM2(S37), p‐PKM2(Y105), PKM2, G6PD, and LDHA. B, The mRNA levels of LDHA and G6PD were verified by qRT‐PCR. C, Representative coimmunoprecipitation analysis of CHK1 and PKM2 in pig myocardium injected with rhCHK1. D through G, From left to right are PK activity, pyruvate content, lactate content, and glucose content in pig myocardium of the I/R+hydrogel and I/R+rhCHK1‐hydrogel groups. N=4. H, ECAR) were detected by seahorse in PBS and rhCHK1‐treated cardiomyocytes. I, Mitochondrial OCRs were detected by seahorse in PBS‐ and rhCHK1‐treated cardiomyocytes. J, Structure‐based protein interaction interface analysis between CHK1 and PKM2. Green: CHK1, Yellow: PKM2. K, Amino acids 1–265, amino acids 270–327, and amino acids 391–476 of CHK1 were the interaction domain binding to PKM2. The deletion of amino acids 1–265 domain (CHK1^Δ1), the deletion of amino acids 270–327 domain (CHK1^Δ2), the deletion of amino acids 391–476 domain (CHK1^Δ3), the deletion of amino acids 1–265, amino acids 270–327, and amino acids 391–476 (CHK1^Δ4) were constructed. L, Amino acids 24–124 and amino acids 157–400 of PKM2 were the interaction domain binding to CHK1. The deletion of amino acids 24–124 domain (PKM2^Δ1), the deletion of amino acids 157–400 domain (PKM2^Δ2), the deletion of amino acids 24–124 and amino acids 157–400 (PKM2^Δ3) were constructed. M and N, Representative coimmunoprecipitation analysis of CHK1 and PKM2 in HEK293T cells cotransfected with FLAG‐CHK1 (WT, Δ1, Δ2, Δ3, Δ4) and HA‐PKM2. O and P, Representative coimmunoprecipitation analysis of CHK1 and PKM2 in HEK293T cells co‐transfected with HA‐PKM2 (WT, Δ1, Δ2, Δ3) and FLAG‐CHK1. Q through S, Representative immunofluorescence images of Edu⁺, Ki67⁺, and pH3⁺ hiPSC‐CMs from CON, CHK1^WT+PKM2^WT, CHK1^Δ1+PKM2^WT, CHK1^WT+PKM2^Δ2, and CHK1^Δ1+PKM2^Δ2 groups. The comparisons were performed by Student t test (B, D through G). Comparing multiple groups, the ANOVA was tested to determine the universal difference. If there was a universal difference between groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (Q through S). The repeated measures ANOVA (based on GLM) was used, then the Sidak multiple comparisons test to compare the difference between 2 groups in different time points (H‐I). Data are presented as mean±SD or mean±SEM. *P<0.05, ***P<0.001. CHK1 indicates checkpoint kinase 1; CON, control; DPI, days postinfarction; ECAR, extracellular acidification rate; G6PD; glucose‐6‐phosphate dehydrogenase; I/R, ischemia/reperfusion; LDHA, lactate dehydrogenase A; L‐LA, L‐lactic Acid; NS, no significance; OCR, O₂ consumption rates; PKM2, pyruvate kinase isozyme type M2; PK, pyruvate kinase; p‐PKM2 S37, phosphorylated pyruvate kinase isozyme type M2 at Ser37; p‐PKM2 Y105, phosphorylated pyruvate kinase isozyme type M2 at tyrosine 105; rhCHK1, recombinant human checkpoint kinase 1; and WT, wild‐type.

rhCHK1 Stimulated Cardiomyocyte Renewal by Activating PKM2‐Mediated Metabolic Reprogramming

To further confirm the mechanistic role of PKM2‐mediated glycolysis in rhCHK1‐mediated beneficial effects in our in vivo model, we constructed adenoviruses of PKM2 RNAi (si‐PKM2). In vivo experiments were divided into 3 groups: the I/R+hydrogel group, I/R+rhCHK1‐hydrogel group, and I/R+rhCHK1+PKM2i group. At 7 DPI, the echocardiography results showed that cardiac function of I/R+rhCHK1‐hydrogel group was higher than the other 2 groups. The improvement of cardiac function was lower in the I/R+rhCHK1+PKM2i group as compared with the I/R+rhCHK1‐hydrogel group (Figure 8A and 8B). Immunofluorescence results showed that the number of Ki67⁺, pH3⁺, and Aurora B⁺ cardiomyocytes was significantly decreased in the infarct border zone of the I/R+rhCHK1+PKM2i group (Figure 8C through 8E). Furthermore, the Tunel⁺ cardiomyocytes were also significantly increased in the infarct border zone of the I/R+rhCHK1+PKM2i group (Figure 8F). In addition, Masson staining showed that the fibrosis area was also increased in the I/R+rhCHK1+PKM2i group (Figure 8G).

Figure 8. rhCHK1 promotes cardiomyocyte proliferation and cardiac repair in pigs through activating PKM2 in 7‐DPI pigs.

A, LVEF of pigs in the I/R+hydrogel, IR+rhCHK1‐hydrogel, and IR+rhCHK1+PKM2i groups at indicated times. N=3 for each group. B, LVEF change of pigs in I/R+hydrogel, IR+rhCHK1‐hydrogel, and IR+rhCHK1+PKM2i groups from 0 DPI to 7 DPI. N=3 for each group. C through E, Porcine tissue sections from the infarcted border zone from the I/R+hydrogel, IR+rhCHK1‐hydrogel, and IR+rhCHK1+PKM2i groups were stained for cTNT to visualize cardiomyocytes; Hoechst to visualize the nucleus; and proliferation marker Ki67, pH3, and Aurora B to mark endogenous cardiomyocyte proliferation. N=3 for each group. F, Representative immunofluorescence images of Tunel⁺ cardiomyocytes from the I/R+hydrogel, IR+rhCHK1‐hydrogel, and IR+rhCHK1+PKM2i groups. N=3 for each group. G, Representative Masson staining and fibrous area quantification for heart slices from 3 groups. Comparing multiple groups, the ANOVA was tested to determine the universal difference. If there was a universal difference between groups, then 1‐way ANOVA (with the post hoc Tukey test) was performed to determine the statistical differences (B through G). Data are presented as mean±SD or mean±SEM. *P<0.05, **P<0.01, ***P<0.001. CON indicates control; DPI, days postinfarction; EF, ejection fraction; IR and I/R, ischemia/reperfusion; LVEF, left ventricular ejection fraction; NS, no significance; PKM2, pyruvate kinase isozyme type M2; and rhCHK1, recombinant human checkpoint kinase 1.

These results indicated rhCHK1 could stimulate cardiomyocyte proliferation and regenerative repair by activating PKM2‐mediated metabolic reprogramming. The therapeutic effects and potential mechanisms of the rhCHK1 remedy are shown in Figure 9.

Figure 9. Intramyocardial injection of rhCHK1 stimulated cardiac regeneration in a pig model via the activation of PKM2‐mediated glycolysis and proliferative gene activation.

PKM2 indicates pyruvate kinase isozyme type M2; and rhCHK1, recombinant human checkpoint kinase 1.

Discussion

One of the largest challenges in the biomedical field, particularly in cardiology, is the gap between the basic and clinical research arenas. Specifically, the translation study of heart regenerative signals elucidated in rodents into preclinical large animal and clinical stages is scarce. This translational study aimed to bridge the gap between small and large animal studies of cardiac regenerative therapy after MI. We reported that intramyocardial administration of rhCHK1‐hydrogel promoted myocardial repair after I/R injury by promoting cardiomyocyte proliferation, inhibiting cardiomyocyte apoptosis, and reducing inflammatory responses thereby effectively reduces infarct size and scar area to improve cardiac function and ventricular remodeling in a preclinical pig model of acute I/R. Future clinical studies are warranted to see if these results could be effectively and safely translated to patient treatment.

We have previously demonstrated that the overexpression of CHK1 in the mouse heart could constitutively activate the cell cycle in cardiomyocytes. Intramyocardially injection of adeno‐associated virus serotype 9 (AAV9)‐CHK1 could promote remuscularization of infarcted immunodeficient mouse hearts, thus reducing left ventricular remodeling and cardiac dysfunction. ¹⁷ However, the clinical relevance of using rodent models was limited by significant physiological differences between rodent and human hearts in regard to size and electric conduction as well as the formation and distribution of collateral coronary arteries. ²⁸ Therefore, large animal research models, especially those based on porcine models, are important for developing and characterizing novel therapeutic approaches, proving their efficacy and safety, as well as optimizing the dose, frequency, and routes of administration. ²⁹ The data presented in this report demonstrated that local injection of rhCHK1 protein could upregulate rhCHK1 expression in cardiomyocytes and induce significant proliferative activity (cell cycle activation) in resident cardiomyocytes in this pig model of acute myocardial infarction (AMI). Our results are of certain clinical relevance on designing future human studies.

Local injection of hydrogel into the myocardium can remove reactive oxygen species (ROS) and improve mitochondrial function and was shown to be a therapeutic option in the reconstruction of cardiac function after myocardial infarction. ¹⁸ A series of studies also aimed to explore how to optimize the preparation of hydrogel patches with good biocompatibility, electrical conductivity, and stable mechanical properties in the complex microenvironment of the heart. ³⁰ Here, we designed an eukaryotic expression method to recombine and purify rhCHK1 protein that could penetrate the myocardial cell membrane. Hydrogel was used as an injection vector to slowly release the rhCHK1 protein in the infarct zone and marginal zone. In our study, the results showed a lower level of scar area and cardiac fibrosis in the I/R+no‐loading hydrogel group than in the I/R group at 28 days after surgery. In addition, if rhCHK1 protein is carried by hydrogel, there will be more benefits for cardiac function and myocardial repair after myocardial infarction. These results demonstrated that rhCHK1 could stimulate cardiomyocyte proliferation and cell cycle re‐entry is of importance on improving the cardiac function in this animal model.

Our previous research proved that the overexpression of CHK1 kinase could promote cardiac repair by activating the mTORC1/P70S6K pathway in adult MI mice. To further exploit the role of signaling pathways such as mTORC1/P70S6K in cardiac repair, we performed RNA‐sequencing (FDR < 0.05) and mass spectrometry (MS) analysis (FDR < 0.05) on pig myocardium 7 days after I/R with nonloaded hydrogel or rhCHK1‐hydrogel. Interestingly, the differentially expressed genes were not only enriched in the regulation of eIF4 and p70S6K signaling and the mTOR pathway, but the glycolysis process was also activated with elevated expression of the glycolysis rate‐limiting enzyme PKM2. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into HEK293T cells for CO‐IP experiments. It was found that the 1–265 domain of CHK1 directly binds to the 157–400 amino acids of PKM2. Evidence showed that the energy metabolism of vertebrate postpartum cardiomyocytes was converted from glycolysis to oxidative phosphorylation. ³¹ This induced mitochondria to produce a large amount of reactive oxygen species, activated oxidative DNA damage, and caused cardiomyocytes to rapidly exit the cell cycle. Metabolic remodeling of cardiomyocytes, especially switching from the fatty acid metabolic pathway to the glycolytic pathway, promoted cardiomyocyte cytokinesis and mitosis. ³² Activation of the Y105 phosphorylation site of PKM2 promoted tetramer‐to‐dimer conversion, catalyzed glycolysis and biosynthetic pathways, and initiated metabolic reprogramming. ³³ Activation of the ser37 phosphorylation site of PKM2 can promote the increase of PKM2 entry into the nucleus, thus upregulating the β‐catenin gene to promote myocardial regeneration. ²⁷ In addition, PKM2 activated metabolic enzyme pathways in the myocardium, initiated adult cardiomyocyte reentry into the cell cycle, and promoted myocardial regeneration after MI. Combined with the previous studies reporting that the enhanced glycolysis could promote cardiomyocyte proliferation and cardiac regenerative capacity, our results show that rhCHK1 could promote cardiomyocyte proliferation and alleviate myocardial inflammation and cardiomyocyte death jointly by activating PKM2 pathways.

Sources of Funding

This work was supported by grants from the Key Clinical Frontier Technology Project of Department of Science and Technology of Jiangsu Provincial (number BE2022806), the National Natural Science Foundation of China Innovative Research Group Project (number 82121001), the National Natural Science Foundation of China (number 82070367), and a Project Funded by the Scientific Research Innovation Projects of Graduate Students in Jiangsu Province (number KYCX22_1844, KYCX22_1838).

Disclosures

None.

Supporting information

Data S1

Table S1

Figures S1–S10