Protocatechuic aldehyde protects cardiomycoytes against ischemic injury via regulation of nuclear pyruvate kinase M2

Abstract

Rescuing cells from stress damage emerges a potential therapeutic strategy to combat myocardial infarction. Protocatechuic aldehyde (PCA) is a major phenolic acid in Chinese herb Danshen (Salvia miltiorrhiza root). This study investigated whether PCA regulated nuclear pyruvate kinase isoform M2 (PKM2) function to protect cardiomyocytes. In rats subjected to isoprenaline, PCA attenuated heart injury and protected cardiomyocytes from apoptosis. Through DARTS and CETSA assays, we identified that PCA bound and promoted PKM2 nuclear translocation in cardiomyocytes exposed to oxygen/glucose deprivation (OGD). In the nucleus, PCA increased the binding of PKM2 to β-catenin via preserving PKM2 acetylation, and the complex, in cooperation with T-cell factor 4 (TCF4), was required for transcriptional induction of genes encoding anti-apoptotic proteins, contributing to rescuing cardiomyocyte survival. In addition, PCA ameliorated mitochondrial dysfunction and prevented mitochondrial apoptosis dependent on PKM2. Consistently, PCA increased the binding of PKM2 to β-catenin, improved heart contractive function, normalized heart structure and attenuated oxidative damage in mice subjected to artery ligation, but the protective effects were lost in Pkm2-deficient heart. Together, we showed that PCA regulated nuclear PKM2 function to rescue cardiomyocyte survival via β-catenin/TCF4 signaling cascade, suggesting the potential of pharmacological intervention of PKM2 shuttle to protect the heart.

Keywords: Apoptosis; CETSA, cellular thermal shift assay; CK-MB, creatine kinase isoenzyme-MB; DARTS, drug affinity responsive target stability; Heart ischemia; ISO, isoprenaline; LDH, lactate dehydrogenase; Mitochondrial damage; Myocardial infarction; NRVMs, neonatal rat ventricular myocytes; Nuclear translocation; OGD, oxygen and glucose deprivation; PCA, protocatechuic aldehyde; PKM2; PKM2, pyruvate kinase isoform M2; Protocatechuic aldehyde; ROS, reactive oxygen species; TCF4; TCF4, T-cell factor 4; TUNEL, deoxynucleotidyl transferase-mediated dUTP nick end-labeling; shRNA, short hairpin RNA; β-Catenin.

Graphical abstract

Figure 1

PCA protects the heart from ISO challenge in rats. The levels of circulating (A) CK-MB and (B) LDH in the plasma (n = 6). (C) The ratio of heart weight to tibia length (HW/TL) (n = 6). (D) One of representative H&E stained heart sections and cardiomyocyte cross-sectional area (n = 6, scale bar = 50 μm). 4-HNE (E) and 8-OHDG (F) content in the heart (n = 6). (G) Immunocytochemical staining of BAX, BCL-2 and c-caspase 3 protein expression (one of 6 independent experiments; scale bar = 50 μm). (H) Representative TUNEL staining images and quantification of TUNEL positive cells (n = 6). Cell survival of primary neonatal rat ventricular myocytes (NRVMs) when exposed to 1% O₂ in glucose-free DMEM (OGD) for 6 h (I) or H₂O₂ for 8 h (J) (n = 6). (K) Intracellular ROS production in NRVMs exposed to OGD for 4 h (n = 6). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 2

Identification of PKM2 as a direct binding target for PCA. (A) The employed drug affinity responsive target stabilization (DARTS) assay detects a marked increase in ∼60 kD band upon PCA incubation in pronase digested H9C2 cell lysates. (B) Immunoblot analysis of PKM2 in pronase-digested cell lysate (n = 3). (C) Recombinant PKM2 in pronase-digested H9C2 cell lysates. PKM2 degradation in H9C2 cell lysates (D) and in intact cells (E) (n = 3). (F) Surface plasmon resonance (SPR) binding curve fit to a K_d = 5.76 nmol/L for PCA and PKM2 (n = 3). Results are shown as mean ± SD; ∗P < 0.05, ∗∗∗P < 0.001.

Figure 3

PCA PKM2-dependently protects cardiomyocytes. Primary neonatal rat ventricular myocytes (NRVMs) were cultured in glucose-free DMEM under 1% O₂ (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). (A) Cell survival in NRVMs exposed to OGD for 6 h (n = 6). (B) Intracellular ROS production (n = 6). (C) Representative images of mitochondrial permeability transition pore (mPTP) and quantification of relative fluorescence intensity (n = 6, one of three independent experiments). Scale bar: 10 μm. (D) Mitochondrial fission was detected by Mito-Tracker Red (one of 3 independent experiments) and quantification analysis. Scale bar: 10 μm. (E) Caspase 3 activity in NRVMs (n = 6). (F) Representative Western blots of BAX, BCL-2, caspase 3, cleaved caspase 3 (c-caspase 3) and the ratio of BAX/BCL-2 (n = 3). (G) Representative images of TUNEL staining and quantification analysis the percentage of apoptosis cells. Scale bars, 100 μm (n = 3). (H) Data of Annexin V/PI double staining flow cytometry; the percentage for each panel indicates the percentage of apoptotic cells and quantification of the percentage of apoptotic cells (n = 3). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 4

PCA promotes the nuclear localization of PKM2. Primary neonatal rat ventricular myocytes (NRVMs) were exposed to 1% O₂ in glucose-free DMEM (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). (A) PKM2 activity in NRVMs (n = 6). (B) Immunofluorescence image of endogenous PKM2 (one of three independent experiments. Scale bar: 10 μm. (C) Nuclear PKM2 protein expression (n = 3). (D) Cytoplasmic PKM2 protein expression (n = 3). (E) Total PKM2 protein expression (n = 3). (F) Docking analysis illustrates the interaction between PCA and PKM2. The residues that are likely to participate in the interactions with PCA are labeled. (G) Representative co-immunoprecipitation (co-IP) analysis of PKM2 and SIRT6 in the NRVMs (n = 3). (H) Representative co-IP of PKM2 and β-catenin in the NRVMs (n = 3). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 5

PCA regulates β-catenin/TCF4 cascades. Primary neonatal rat ventricular myocytes (NRVMs) were exposed to 1% O₂ in glucose-free DMEM (OGD) for 4 h in the presence of protocatechuic aldehyde (PCA). Representative co-IP analysis to of TCF4 and β-catenin in the NRVMs exposed to OGD for 4 h (A) or H₂O₂ for 8 h (B) (n = 3). (C) TCF4 luciferase reporter activity in the presence or absence of PNU-74654 (PUN, 10 μmol/L) (n = 3). The mRNA levels of Myc (D), Ccnd1 (E) and Sgk1 (F) (n = 4). (G) Cell survival of NRVMs (n = 6). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Figure 6

The myocardial protection effect of PCA is dependent on PKM2. Mice were orally administrated with protocatechuic aldehyde (PCA) for 3 weeks after coronary artery ligation. (A) Representative photomicrographs for HE staining of cardiac tissue sections (n = 6), Scale bar, 50 μm. (B) Representative photomicrographs for masson staining of cardiac tissue sections (n = 6). Scale bar, 50 μm. (C) 4-HNE contents in the heart (n = 6). (D) Representative M mode images of echocardiography from the assayed groups under treatments as indicated (n = 6). (E) Ejection fractions (EF) and shortening fraction (FS) in mice (n = 6). (F) Representative co-IP analysis of PKM2 and β-catenin in the heart (n = 3). The mRNA levels of Myc (G), Ccnd1 (H) and Sgk1 (I) in the heart (n = 6). (J) Immunocytochemical staining of BAX, BCL-2 and c-caspase 3 protein expression (n = 6), scale bar = 50 μm. (K) Representative TUNEL staining images and quantification of TUNEL positive cells (n = 6). Results are shown as mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.