Protective Role of Klotho in Cardiomyocyte Injury Caused by Hypoxia-Reoxygenation via Regulation of the NF-κb Signaling Cascade

Summary

This protocol evaluates the protective role of klotho overexpression in H9C2 cardiomyocytes under hypoxia-reoxygenation using molecular and functional assays.

Abstract

This study aimed to investigate the protective role of klotho in cardiomyocytes subjected to hypoxia/reoxygenation (H/R) injury. An in vitro H/R model was established using H9C2 rat cardiomyoblasts, which were transfected with klotho-overexpressing lentiviral vectors. Cells were divided into six experimental groups: control, H/R, negative control (NC), klotho-overexpression, PDTC (an NF-κB inhibitor), and a combination group treated with klotho and lipopolysaccharide (LPS). Klotho expression levels were determined by quantitative real-time PCR and western blotting. Cell viability was measured via the MTT assay, while apoptosis was assessed using flow cytometry. The expression of proteins involved in the NF-κB signaling pathway was analyzed by western blotting, and the cellular expression of pro-inflammatory cytokines was evaluated through immunofluorescence staining. Klotho overexpression significantly elevated klotho levels in H9C2 cells and led to improved cell viability, reduced apoptosis, and decreased cellular expression of pro-inflammatory cytokines compared to the H/R group. Western blot analysis revealed that klotho overexpression, as well as PDTC treatment, suppressed NF-κB pathway activation, as indicated by a reduced p-P65/P65 ratio and increased p-IκBα/IκBα levels. These effects were partially reversed upon co-treatment with LPS. These results suggest that klotho confers cardioprotection against H/R-induced injury by modulating NF-κB signaling, reducing inflammation, and promoting cell survival. This protocol provides a reproducible method for studying the molecular mechanisms underlying myocardial ischemia-reperfusion injury and the therapeutic potential of klotho in cardiac inflammation.

Introduction

Acute myocardial infarction (AMI) is primarily caused by ischemia followed by reperfusion, which has prompted extensive research into the mechanisms of myocardial hypoxia/reoxygenation (H/R) injury^1,2,3. A pivotal aspect of myocardial H/R injury is cell apoptosis, which contributes significantly to the pathological progression of AMI^4,5.

Recent studies have implicated the nuclear factor kappa B (NF-κB) signaling pathway in myocardial H/R injury through its modulation of inflammatory responses to stimuli such as lipopolysaccharide (LPS)^6,7,8. Activation of NF-κB occurs via two distinct mechanisms: the canonical and non-canonical pathways⁹. In the canonical pathway, activation relies on the phosphorylation-induced degradation of inhibitor of kappa B alpha (IκBα)¹⁰. However, it has also been reported that phosphorylation of the NF-κB subunit p65 can drive apoptosis, potentially independently of IκBα degradation¹¹. Klotho, a single-pass transmembrane protein, regulates phosphate and calcium metabolism and inhibits insulin signaling^12,13,14. Emerging evidence indicates that Klotho exerts inhibitory effects on NF-κB activation in diverse tissues, including the kidney, hippocampus, and aging cells, thereby mitigating inflammation and tissue damage^15,16,17. Despite these advances, the role of Klotho in AMI and its potential interaction with the NF-κB pathway during myocardial H/R injury remain largely unexplored.

From a methodological perspective, the in vitro H/R model using H9C2 rat cardiomyoblasts provides several advantages over animal models, including controlled oxygen tension, reproducibility, and reduced variability. Moreover, lentiviral-mediated Klotho overexpression enables stable and efficient modulation of gene expression, allowing direct investigation of its functional impact. These approaches make the protocol practical, cost-effective, and accessible for mechanistic studies, while also avoiding the ethical and logistical challenges of in vivo experimentation.

In terms of practical considerations, H9C2 cells are widely used in cardiovascular research as they retain many cardiomyocyte-like properties, although they do not fully mimic the phenotype of mature cardiomyocytes. The selected H/R condition (5 h hypoxia followed by 1 h reoxygenation) falls within a range commonly employed to model ischemia-reperfusion injury in vitro. Similarly, lentiviral transduction with multiplicities of infection (MOI) around 100 is a standard approach to achieve efficient overexpression with manageable cytotoxicity. While these features enhance the applicability of the protocol, limitations such as incomplete recapitulation of the in vivo environment should be acknowledged. Therefore, the present study aims to explore how Klotho protects myocardial cells from H/R injury and to establish a reproducible protocol for assessing its interaction with NF-κB signaling.

Protocol

All procedures involving the H9C2 rat cardiomyoblast cell line (ATCC, CRL-1446) were performed in compliance with institutional biosafety regulations. No animal or human subjects were directly involved in this study. Figure 1 illustrates the graphical workflow of the experimental design and analysis pipeline. The reagents and the equipment used are listed in the Table of Materials.

1. Cell culture

The H9C2 rat cardiomyoblast cell line (ATCC, CRL-1446) was cryopreserved in liquid nitrogen using a freezing medium composed of Dulbecco's Modified Eagle Medium (DMEM, high glucose, 4.5 g/L) supplemented with 10% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO). For recovery, frozen vials were rapidly thawed in a 37 °C water bath for 2-3 min and immediately transferred into pre-warmed DMEM. The culture medium was prepared with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 3.7 g/L sodium bicarbonate, and 1 mM sodium pyruvate. Cells were maintained at 37 °C in a humidified incubator with 5% CO₂. H9C2 cells between passages 6 and 20 were used for all experiments. Early passages (<P6) and higher passages (>P20) were not used to avoid passage-related phenotypic drift.

2. Lentiviral overexpression of Klotho in H9C2 cells

A third-generation, self-inactivating lentiviral vector encoding the full-length rat Klotho (Kl) gene under the control of the EF1α promoter was applied to establish stable overexpression in H9C2 cells. The construct contained a P2A-EGFP reporter for visualization, woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) elements for transcript stability, and a puromycin-resistance cassette driven by the PGK promoter for selection. Lentiviral particles were produced in HEK293T cells by co-transfecting the transfer plasmid with psPAX2 (gag/pol/rev) and pMD2.G (VSV-G) plasmids at a ratio of 4:3:1 using polyethyleneimine (PEI; DNA: PEI = 1:3, w/w). Supernatants were harvested at 48 h and 72 h, clarified by centrifugation at 3,000 × g for 10 min, filtered through a 0.45 µm PES membrane, and concentrated by ultracentrifugation at 25,000 × g for 2 h at 4 °C. Viral pellets were resuspended in PBS containing 1% bovine serum albumin (BSA), and functional titers (TU/mL) were determined by limiting-dilution infection of HEK293T cells with EGFP readout at 72 h. H9C2 cells were seeded in 6-well plates at 2 × 10⁵ cells per well, resulting in approximately 60% confluence at the time of transduction. Lentiviral particles were added at a multiplicity of infection (MOI) of 100 in 1 mL of fresh culture medium supplemented with polybrene (8 µg/mL) to enhance infection efficiency. The inoculum was removed after 12 h and replaced with 2 mL of complete medium. At 72 h post-infection, transduction efficiency was verified by EGFP fluorescence under a fluorescence microscope. For the establishment of stable cell lines, puromycin (2 µg/mL, pre-titrated) was applied for 3-5 days starting 48 h after infection, with medium changes every 48 h. Overexpression of Klotho was confirmed by RT-qPCR and immunoblotting. All lentiviral procedures were performed under biosafety level 2 (BSL-2) containment, and all viral waste was inactivated with 10% bleach for at least 30 min prior to disposal.

3. Experiment groups

H9C2 cells were seeded in 6-well plates (growth area 9.6 cm²) at 3 × 10⁵ cells per well, which yielded approximately 70%-80% confluence after 24 h, the required density for hypoxia/reoxygenation (H/R) processing and subsequent assays. After overnight attachment, cells were allocated into six experimental groups: (1) Control group, which received no additional treatment; (2) H/R group, which underwent hypoxia/reoxygenation (H/R) in a sealed modular hypoxia chamber, followed by exposure to 5 h of hypoxia (1% O₂, 94% N₂, 5% CO₂) and 1 h of reperfusion (5% CO₂), as previously described¹⁸; (3) Negative Control (NC) group, in which cellswere transfected with an empty vector 24 h before H/R treatment; (4) Klotho group, in which cells were transfected with lentiviral vectors encoding Klotho 24 h before H/R treatment; (5) Pyrrolidine dithiocarbamate treatment group (PDTC), which was treated with 200 mg/L NF-κB pathway inhibitor PDTC for 24 h before H/R treatment¹⁹; (6) Klotho + LPS group, in which cells were transfected with lentiviral vectors encoding Klotho and subsequently treated with lipopolysaccharide at 1 mg/L for 24 h before H/R treatment²⁰.

4. qRT-PCR

H9C2 cells were seeded in 6-well plates at a density of 1 × 10⁶ cells per well. After treatment, total RNA was isolated using a phenol-chloroform extraction protocol. Briefly, 1 mL of reagent was added per well, followed by chloroform extraction, isopropanol precipitation, and washing with 75% ethanol. The RNA pellet was air-dried and dissolved in RNase-free water. RNA concentration and purity were determined spectrophotometrically. For cDNA synthesis, 1 µg of total RNA was reverse-transcribed using a commercial reverse transcription kit according to the manufacturer's protocol, including genomic DNA removal and random hexamer priming. Quantitative real-time PCR was performed in a real-time PCR system with SYBR Green chemistry under the following conditions: initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. Primer sequences were designed using Primer-BLAST (NCBI) and synthesized by a commercial supplier: (1) Klotho: F 5′-TAAGGTTCAAGTATGGAGAC-3′, R 5′-GGGCGTTCACACTTATTTAT-3′; (2) β-actin (internal control): F 5′-TGTCACCAACTGGGACGATA-3′, R 5′-GGGGTGTTGAAGGTCTCAAA-3′. Relative expression levels were calculated using the 2^-ΔΔCT method, and amplification specificity was confirmed by a single melting-curve peak.

5. Western blot

Proteins were extracted from H9C2 cells using radioimmunoprecipitation assay (RIPA) buffer (200 µL per well of a 6-well plate) supplemented with a protease inhibitor cocktail on ice. Protein concentrations were measured by bicinchoninic acid (BCA) assay, and equal amounts of protein (20-30 µg) were loaded per lane onto a 10% SDS-PAGE gel for electrophoretic separation. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes using a wet transfer system at 100 V for 90 min at 4 °C. Membranes were blocked with 5% non-fat milk prepared in TBST for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against Klotho (1:500), phospho-p65 (1:500), p65 (1:500), phospho-IκBα (1:500), IκBα (1:500), and β-actin (1:1000). After three washes with TBST, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000) for 1 h at room temperature. Protein bands were visualized using an enhanced chemiluminescence detection system, and signal intensities were quantified with ImageJ software using identical threshold settings.

6. MTT assay

H9C2 cells were seeded into 96-well plates at a density of 2 × 10³ cells per well in 100 µL of complete medium and incubated overnight to allow adherence. A 10 µL volume of MTT solution (5 mg/mL in PBS) was added to each well and incubated for 4 h at 37 °C under humidified conditions with 5% CO₂. After incubation, the supernatant was carefully removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals, followed by incubation for 10 min at room temperature with gentle shaking. The absorbance was then measured at 490 nm using a microplate reader. The expected visual outcome was the appearance of purple formazan crystals in viable cells before DMSO addition, and a homogenous purple solution after dissolution.

7. Flow cytometry

Cells were harvested 24 h post-treatment by trypsinization and collected by centrifugation at 1,500 × g for 5 min at 4 °C. The cell pellet was resuspended in 100 µL of 1× binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4). Annexin V-APC and propidium iodide (PI) were mixed at a 1:2 ratio to prepare a 15 µL staining solution, which was added to the cell suspension. The mixture was incubated for 10 min at room temperature in the dark. After incubation, 400 µL of the same binding buffer was added, and the samples were analyzed immediately using a flow cytometer. A total of 10,000 events per sample were collected. Data were analyzed using FlowJo software. Cells were first gated based on forward scatter (FSC) and side scatter (SSC) to exclude debris, followed by quadrant gating of Annexin V vs. PI to distinguish viable cells (Annexin V-/PI-), early apoptotic cells (Annexin V+/PI-), and late apoptotic/necrotic cells (Annexin V+/PI+). Annexin V-positive/PI-negative staining indicated early apoptosis, while Annexin V/PI double-positive staining indicated late apoptosis or necrosis.

8. Immunofluorescence

H9C2 cells were seeded on sterile glass coverslips in 6-well plates at a density of 1 × 10⁵ cells per well and subjected to treatments. Cells were fixed with 4% paraformaldehyde for 15 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and blocked with 5% bovine serum albumin (BSA) in PBS for 30 min at room temperature. Coverslips were incubated overnight at 4 °C with primary antibodies against TNF-α (1:200), IL-1β (1:200), and IL-6 (1:100). After three washes in PBS, cells were incubated with phycoerythrin (PE)-labeled secondary antibodies (1:500) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI for 5 min. Coverslips were mounted on glass slides with antifade mounting medium, and fluorescence signals were visualized using a fluorescence microscope. Images were analyzed with ImageJ (version 1.54). Red fluorescence indicated cytokine expression, while blue fluorescence marked nuclei.

9. Statistical analysis

All data were analyzed using SPSS software. Results are expressed as mean ± standard deviation (SD) from at least three independent experiments. For multiple group comparisons, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was applied. A P-value < 0.05 was considered statistically significant.

Results

Klotho overexpression improves cell survival and attenuates NF-κB-mediated inflammatory injury

To confirm the efficiency of gene delivery, Klotho expression was quantified at both the mRNA and protein levels. Klotho-overexpressing cells showed a robust increase compared with both hypoxia/reoxygenation (H/R) and negative control groups (p < 0.05; Figure 2A,B). Functionally, Klot...

Discussion

In this study, the protective role of Klotho in cardiomyocytes exposed to hypoxia/reoxygenation (H/R) injury was investigated, and Klotho overexpression was found to significantly improve cell viability, reduce apoptosis, and suppress pro-inflammatory cytokine expression. These effects were accompanied by reduced phosphorylation of p65 and enhanced phosphorylation of IκBα, suggesting that inhibition of NF-κB signaling contributes to the protective phenotype. Pharmacological inhibition of NF-κB by PDTC...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
4% Paraformaldehyde	Biosharp	XG1050
Annexin V APC/PI Staining Solution	Elabscience	E-CK-A217
cDNA Synthesis Kit	Qiagen	205410
DAPI Solution	BioFroxx	1155MG010
Dulbecco’s Modified Eagle Medium (DMEM)	HyClone	SH30243.01B
ECL Detection Reagent Kit	Meilunbio	MA0186-1
Electrophoresis Power Supply (POWER PAC 200)	Bio-Rad	POWER PAC 200
Fetal Bovine Serum (FBS)	VivaCell	C04001-500, C04002-500
Flow Cytometer (Gallios)	Beckman Coulter	Gallios
Fluorescence Microscope (BK6000)	Chongqing Optec	BK6000
Formazan Solvent	Solarbio	M1020
HRP-Conjugated Goat Anti-Rabbit IgG Antibody	Bethyl	A120-101P
LPS (NF-κB Pathway Agonist)	Sigma-Aldrich	L2630
Microplate Reader (Model K3)	Thermo	K3
Modular Incubator Chamber (Hypoxia Chamber)	Billups-Rothenberg	MIC-101
MTT Solution	Cayman	21795
PDTC (NF-κB Pathway Inhibitor)	Sigma-Aldrich	P8765
PE-Conjugated Goat Anti-Rabbit IgG Antibody	Bioss	bs-0295G-PE
Polybrene (Hexadimethrine Bromide)	Invitrogen	X2351
PVDF Membrane	Merck	ISEQ00010
Rabbit Anti-Rat IL-1β Antibody	Biorbyt	orb378927
Rabbit Anti-Rat IL-6 Antibody	ZEN BIO	384702
Rabbit Anti-Rat IκBα Antibody	Biorbyt	orb223182
Rabbit Anti-Rat Klotho Antibody	Biorbyt	orb333711
Rabbit Anti-Rat p65 Antibody	Biorbyt	orb229138
Rabbit Anti-Rat Phospho-IκBα Antibody	Biorbyt	orb223035
Rabbit Anti-Rat Phospho-NF-κB p65 Antibody	Biorbyt	orb304662
Rabbit Anti-Rat TNF-α Antibody	Biorbyt	orb106548
Rabbit Anti-Rat β-actin Antibody	Huabio	ET1702
Real-Time Quantitative PCR System (LightCycler 96)	Roche	Light Cycler96
RIPA Lysis Buffer	Mreda	MO52447
SDS-PAGE Gel Preparation Kit	Elabscience	E-IR-R305
TRIzol Reagent Kit	Life Technologies	15596-018