Ischemic preconditioning modulates the DNA methylation process of the rat heart to provide tolerance to withstand ischemia reperfusion injury and its associated mitochondrial dysfunction

Abstract

DNA methylation plays a crucial role in the pathogenesis of myocardial ischemia reperfusion injury(I/R) and the I/R injury can be combated effectively by ischemia preconditioning (IPC), but the role is DNA methylation in this process is unknown. In this study, we uncovered the role of ischemic preconditioning (IPC)- mediated cardioprotection of rat myocardium by using a Langendorff rat heart model with 30 min of ischemia followed by 60 min of reperfusion. Heart conditioned with short cycles of ischemia and reperfusion (IPC procedure) prior to I/R protocol significantly reduced the I/R-induced global DNA hypermethylation level by 32% and the DNMT activity by 33% while rendering cardioprotection. Blocking the PI3K pathway via wortmannin not only negates the cardio-protection by IPC, but also increases the methylation of DNA by 75%. Besides, the correlation analysis showed a negative relationship between PI3K gene expression and the global DNA methylation level (r = − 0.8690, p = 0.0419) in IPC-treated rat hearts. Moreover, the global level DNA hypomethylation induced by IPC exhibited a regulatory effect on the genes involved in I/R pathology mediators like apoptosis (Caspase3), mitochondrial function (PGC 1α, TFAM, ND1) and oxidative stress (CuZnSOD, SOD2), and their corresponding function. The present study results provide novel evidence for the involvement of DNA methylation in the IPC procedure, and suggest DNA methylation as one of the potential therapeutic targets regulated by ischemic preconditioning in rat hearts subjected to ischemia reperfusion.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-024-03965-0.

Introduction

Myocardial ischemia reperfusion injury is one of the key determinants of the degree of infarct size in the heart subjected to revascularization procedures that include percutaneous or surgical interventions adopted in patients (Wang and Peng 2023; Frank et al. 2012). Although a lot of advancements are reported that underpin the pathophysiological mechanisms of reperfusion injury, translation of the understandings from the preclinical studies to clinical benefits in patients has produced mixed responses (Davidson et al. 2019; Frank et al. 2012). Many studies have demonstrated that repeated brief episodes of coronary occlusion and release before or after an ischemic insult to the myocardium increase the resistance towards prolonged ischemia and subsequent reperfusion (Kalogeris et al. 2016). Studies also supported the beneficial effect of preconditioning against post-ischemic dysfunction and ventricular arrhythmias (Spannbauer et al. 2019).

Previous studies have shown that ischemia preconditioning can reduce the infarct size in the heart by 15–25% and works via activating cardioprotective signalling pathways (Granfeldt et al. 2009). The reperfusion injury salvage kinase (RISK) pathway and the survival activating factor enhancement (SAFE) pathway are the two key pro- survival kinase pathways by which the cardiac conditioning techniques like ischemia preconditioning and ischemia post-conditioning works (Hausenloy et al. 2011). Repeated episodes of ischemic incidence before prolonged ischemia can induce an influx of adenosine from the ATP catabolism leading to an activation of the A1 adenosine receptor (Lasley 2018). This activation of adenosine receptor triggers phosphatidylinisitol 3-phosphate (PI3K) signaling pathway and subsequent downstream mediators like Akt/PKB, that in turn modulate GSK3β activity (Gao et al. 2001). GSK3β preserves mitochondria functional activity by selectively opening the K_ATP channel and thereby preventing mPTP transition (Juhaszova et al. 2009). It also activates mTOR and autophagy pathways, promoting cell survival, and thereby providing protection in cardiac I/R (Lal et al. 2015). In addition, nitric oxide synthase (NOS) produces nitric oxide (NO) during I/R, which in turn activates signalling pathway that regulates Ca²⁺-handling proteins, thereby preventing Ca²⁺ overload-induced cell death initiated in the mitochondria during myocardial I/R injury (Schulz et al. 2004).

Recent studies have shown that epigenetic regulations via acetylation and methylation are closely related to the pathogenesis of myocardial ischemia reperfusion injury (Wang et al. 2021). Many investigators have shown that HDAC inhibitors can attenuate myocardial I/R (Pickell et al. 2020), while higher DNA methylation during revascularization procedures was reported in both clinical and preclinical studies (Kim et al. 2020; Ke et al. 2017). However, very limited studies have used epigenetic modifiers as a novel therapeutic target to ameliorate or prevent I/R. Considering the importance of mitochondrial function during I/R by IPC and the absence of histones in the mitochondrial genome, exploring the role of DNA methylation in IPC-mediated cardioprotection, especially its effect on mitochondrial function and quality control genes is relevant. However, the IPC-mediated epigenetic modifications especially DNA methylation is not well understood in I/R rat hearts and is the major objective of the study.

Materials and methods

Animals

All procedures involving the animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), SASTRA University, Thanjavur, India (CPCSEA Approval No. /SASTRA/IAEC/RPP/547) and was conducted in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines. Animals were housed in polycarbonate cages and maintained at 25 ± 2 °C with 12 h of light/dark cycle and relative humidity of 65 ± 2%. Feed and water were provided ad libitum.

Experimental groups

Male Wistar rats (200–250 g) were divided randomly into 5 groups (n = 6 per group). (1) Normal (N), (2) Ischemia reperfusion (I/R), (3) Ischemic precondition control (IPC-C), (4) Ischemic preconditioned and ischemia reperfusion (IPC-I/R), 5) PI3K inhibited Ischemic precondition (W_IPC-I/R). The rats were anaesthetized with sodium thiopentone (60 mg/kg1 i.p.) and the hearts were excised and mounted on a Langendorff apparatus and perfused in a constant pressure mode with Krebs–Henseleit (KH) buffer as per the treatment groups. The normal group rat hearts were perfused with KH buffer for 120 min, while the I/R hearts were perfused for 20 min stabilization followed by stopping the buffer flow for 30 min to induce ischemia, and reperfusing the buffer flow for 60 min to induce reperfusion. IPC-C hearts were stabilized for 20 min, followed by 3 cycles of IPC treatment (2 min ischemia and 3 min reperfusion), while IPC-I/R hearts underwent 20 min stabilization, 3 cycles of IPC treatment, followed by I/R. PI3K inhibited Ischemic precondition hearts were pre-administered 10 nM wortmannin intravenously 1 h before the heart excision, followed by IPC-I/R protocol after mounting the heart.

I/R injury assessment by hemodynamics evaluation

Cardiac recovery was assessed by monitoring the hemodynamic changes in the left ventricle (left ventricular pressure) using Labchart Pro 8 and Power Lab Data Acquisition System (AD Instruments). The heart rate, developed pressure (DP), and maximum and minimum pressure derivative (dp/dt) were calculated from the recorded left ventricular pressure. The rate pressure product (RPP), the measure of cardiac stress, was determined by the product of DP and heart rate.

DNA methylation analysis

TRIzol reagent was used to extract total RNA, followed by the cDNA synthesis kit protocol (Thermo Fisher Scientific, MA, USA). The mRNA expression of DNMT1, DNMT3A, DNMT3B, TET1, TET2 and TET3 genes were evaluated using real-time PCR analysis on the system ABI 7500 (Applied Biosystems, CA, USA) with DyNAmo Flash SYBR from Thermo Fisher Scientific. The primer details are provided in supplementary Table 1. The DNMT enzyme activity was then evaluated in the nuclear extract using the EpiQuik^™ DNMT (DNA Methyltransferase) Activity/Inhibition Assay Ultra Kit. DNA was isolated from the rat heart samples using the Phenol/Chloroform/Isoamyl alcohol method (Yeung 2002) and global DNA methylation was evaluated in the heart DNA using MethylFlash^™ Global DNA Methylation (5-mC) ELISA Easy Kit (Epigentek).

DNA fragmentation analysis

Cardiac tissue homogenate was prepared in Tris–Hcl buffer (pH-8) with EDTA (25 mM) and NaCl (400 mM). DNA was isolated using phenol–chloroform-isoamyl alcohol mixture as per standard protocol and the sample (5 μg/well) was run on an agarose gel (1.8%) for 2 h. Laddering/smearing patterns representing DNA breaks due to apoptosis were evaluated along with a standard DNA ladder and images were taken using the Bio-Rad chemi Doc XRS system (Yeung 2002).

mRNA gene expression analysis

The mRNA expression of the genes responsible for mitochondrial replication, transcription (PGC-1α, POLG, TFAM), mitochondrial fission ((MFN1, MFN2), fusion (MFF, FIS, DNM1) and autophagy (PINK1, PARKIN, OPTN), mitochondrial electron transport chain (ETC) function (Complex 1 (ND1, ND2, ND3, ND4, ND4L, ND5, ND6); Complex III (Cyt B); Complex IV (COX1, COX2, COX3); Complex V (ATP6, ATP8)), apoptosis (Casp 9, casp 3, casp 7, PARP), DNA methylation (DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3), RSK signalling (PI3K, AKT), Oxidative stress (CuZnSOD, Catalase, NFKB, Gpx), mitochondrial oxidative stress (Gltrdxn 1, Prxrdxn 6, Prxrdxn 3, SOD2) were assessed using the real-time PCR analysis with the isolated mRNA samples. The primer sequence of the genes is given in supplementary Table 1. The expression was normalized with GAPDH as control and the relative gene expression was calculated by the method of Livak (Livak and Schmittgen 2001).

MtDNA copy number estimation

Mitochondrial DNA copy number was calculated as the ratio of relative gene expression of ND1 gene and beta-actin gene expression using DNA as a source sample.

Mitochondrial isolation and evaluation of mitochondrial functional activities

Mitochondria were isolated by density-gradient separation method from the homogenised heart tissue as per the protocol mentioned in (Palmer et al. 1977). Briefly, tissue homogenate (10%) was prepared in Tris–Cl (pH-7.4) buffer and subjected to centrifugation (4 ºC) at 600 g for 10 min. Later, the supernatant was subjected to centrifugation at 6000 g (4 ºC) for 10 min, to yield the mitochondrial pellet. The pellet obtained was then purified by dissolving in mitochondrial IB-2 and centrifuged for 10 min at 12000 g to yield mitochondrial pellet. Finally, the mitochondrial pellet was dissolved in a storage buffer and protein concentration was determined using Bradford reagent (BioRad) using bovine serum albumin as a standard. Mitochondrial ATP content was determined using ATP lite (Perkin Elmer) according to the manufacturer’s instructions. ETC enzyme activities were measured spectrophotometrically by using specific donor–acceptors as described previously (Barrientos et al. 2009).

Evaluation of oxidative stress

The total reactive oxygen species (ROS) level was analysed by fluorescence method using 2′, 7′-dichlorofluorescein diacetate (DCHFDA, Cat No. D6883, Sigma Aldrich, USA) by measuring the fluorescence at Ex/Em = 485/530 nm. The level of glutathione (GSH) and GSSG in the heart mitochondrial fraction was estimated by the method of (Shaik and Mehvar 2006). Glutathione peroxidase (Gpx), Catalase and superoxide dismutase (SOD) activity was estimated using the standard method described elsewhere (Nandi and Chatterjee 1988; Goldblith and Proctor 1950).

Statistical analysis

All data were represented as the mean ± SD. The significance level between the groups was assessed with a one-way ANOVA test followed by Dunnett's test, and post hoc analysis using Graph Pad Prism 7.0 software. Correlation analysis was performed using the Pearson coefficient method.

Results

IPC-mediated cardioprotection against I/R in rat hearts

Initially, we reconfirmed the cardioprotective effect of IPC by measuring hemodynamic changes in rats conditioned with IPC prior to I/R and the results are given in Table 1.

Table 1.

Effect of IPC on cardiac hemodynamics during I/R

Groups	LVDP(× 10 mmHg)	RPP(mmHg*beats/min*10^4)	(− dp/dt) (× 10 mmHg)	(+ dp/dt) (× 10 mmHg)
N	11.1 ± 2.0*	3.3 ± 0.19*	82.2 ± 10.1*	109.9 ± 17.2*
I/R	3.4 ± 0.6	1.72 ± 0.01	27 ± 2.6	23.6 ± 1.9
IPC-C	10.7 ± 1.2*	2.94 ± 0.21*	77.3 ± 2.9*	97.1 ± 1.9*
IPC-I/R	8.8 ± 1.1*	2.78 ± 0.19*	70.6 ± 5.1*	84.6 ± 3.1*
W_IPC-I/R	2.3 ± 0.8	0.98 ± 0.1	21 ± 2.5	20.8 ± 2.9

The effect of IPC on cardiac I/R in isolated rat hearts were evaluated by the hemodynamic changes LVDP (left ventricular diastolic pressure); RPP (Rate Pressure Product = heart rate × LVDP) and ± dp/dt maximum/minimum force of contraction. The values are represented as mean ± SEM of 6 independent experiments. *p < 0.05 vs. I/R. N = 6

Rat hearts subjected to ischemia reperfusion injury showed a significant decline in the cardiac hemodynamic indices (69% in LVDP, 48% in RPP) from the normal. However, no significant difference in the hemodynamic index was observed between the normal and IPC control rat hearts. But IPC treated I/R challenged rat heart enhanced the cardiac performance by improving the LVDP, rate of contraction and relaxation by 61, 61 and 72% respectively and the overall RPP by 40% from I/R heart (Table 1), which was negated in the presence of wortmannin (W_IPC-I/R group rat hearts).

DNA methylation and expression of methylation genes in IPC rat heart

In order to assess the role of DNA methylation in I/R protocol, we measured global DNA methylation levels and the mRNA expression of DNMTs and TET genes. I/R rat hearts exhibited a significant increase in DNMT1 and DNMT3A gene expression by 3.4 and 4.7 folds respectively and TET1 expression by 6.3 folds without much significant difference in the DNMT 3B and TET 2, TET 3 from the normal group (Fig. 1a). Further, we evaluated if the changes in gene expression of DNMTs altered its enzyme activity. I/R increased the DNMT activity by 43% (Fig. 1b), which is reflected in the global DNA methylation level as well (52%) from the normal heart (Fig. 1c).

Fig. 1.

Impact of IPC on DNA methylation in the myocardium (a) The fold changes in the gene expression of DNA methylation enzymes (DNMT1, DNMT3A, DNMT3B, TET1, TET2, TET3) of different groups from normal are presented, (b) IPC induced changes in the cardiac DNMT activity are depicted; (c) represent the global DNA methylation level in the myocardium. The graphs represent mean ± SD values. *p < 0.05 vs. I/R. N = 6

Rat hearts from the IPC group exhibited strikingly low expression of the DNMT gene and significantly upregulated TET genes. However, rat hearts conditioned with IPC prior to I/R downregulated the DNMT1 and 3A gene expression to 1.2 and 2.3 folds respectively (Fig. 1a) from normal and induced a decline in DNMT activity by 36% when compared to I/R hearts (Fig. 1b). The demethylation enzymes TET2 and TET3 gene expression were upregulated by 3.4 and 2.6 folds respectively in IPC-I/R hearts from the normal heart (Fig. 1a). Moreover, the I/R induced DNA hypermethylation was abolished by 32% when compared with the I/R rat heart (Fig. 1c). Interestingly, in the presence of wortmannin, IPC-I/R upregulated DNMT gene expression and downregulated TETs that favours increased DNA methylation.

In order to check the impact of the DNA methylation changes on cardiac performance, we performed a correlation analysis. Pearsons coefficient analysis of DNA methylation with rate pressure product (index that measures the cardiac function in isolated rat heart), showed a significant correlation between global DNA methylation and RPP values (r value = − 0.8231, p value = 0.0456) (Table 2).

Table 2.

Correlation data of global DNA methylation with the injury and functional genes during cardiac I/R in IPC treated rats

Parameter	Correlation coefficient r-value	p-value
RPP	− 0.8231	0.0456
Gene expression
Gene
PGC 1α	− 0.9892	0.0319
Dnm1	− 0.6892	0.2843
Parkin	− 0.4723	0.1212
MFN1	0.5934	0.2783
MFN2	0.3498	0.4390
DRP1	0.4937	0.0689
MFF	− 0.6923	0.0384
FIS	− 0.3841	0.1949
PINK1	− 0.3494	0.1809
TFAM	− 0.9521	0.0373
POLG1	− 0.8901	0.0442
ND1	− 0.8132	0.0461
CYTB	0.4888	0.2242
ND6	− 0.5940	0.4938
ND5	− 0.6988	0.4322
ND4L	− 0.8943	0.2947
ND3	− 0.5783	0.0734
COX3	− 0.6880	0.0941
ATP6	− 0.5122	0.0789
ATP8	− 0.7349	0.0611
COX2	− 0.4398	0.1589
COX1	− 0.7412	0.1093
ND2	− 0.5905	0.1892
ND4	− 0.5894	0.1485
Casp 9	0.2392	0.3849
Casp 3	0.9192	0.0490
Casp 7	0.6891	0.1374
PARP	0.4812	0.2945
Gltrdxn 1	− 0.4777	0.1033
Prxrdxn 6	− 0.5496	0.1384
Prxrdxn 3	− 0.7898	0.0588
CuZnSOD	− 0.9238	0.0381
SOD2	− 0.8835	0.0428
Catalase	− 0.6783	0.0589
Gpx1	− 0.3894	0.0832
NFKB	− 0.7644	0.0711
PI3K	− 0.8690	0.0419
AKT	− 0.7998	0.0676

Pearson correlation of global DNA methylation with the cardiac hemodynamic parameters and the listed functional genes has been calculated and the corresponding p-value and r-value are presented. p < 0.05 was considered statistically significant

Effect of IPC on apoptosis

Apoptosis is the key event of cell death in I/R and is known to be reversed upon IPC. Accordingly, I/R induction resulted in a significant upregulation in the apoptotic genes caspase 3,7,9 and PARP (Fig. 2a), producing high DNA fragmentation (Fig. 2b) However, IPC prior to the I/R challenge reversed the changes in the mRNA expression of apoptotic genes to near-normal, resulting in decreased DNA fragmentation (Fig. 2b). Wortmannin presence during IPC increased the apoptosis level, proven via the expression changes in caspase system and DNA fragmentation level.

Fig. 2.

IPC induced changes in the expression of apoptotic genes and RISK signalling pathway genes: (a) The fold changes in the mRNA expression of the apoptotic genes Casp 9, Casp7, Casp 3, PARP in I/R and IPC groups (Fold changes are presented from normal group); (b) Representative gel showing DNA fragmentation in myocardial cell nuclei isolated from different experimental groups (Lane 1- N, 2- I/R, 3- IPC-C, 4- IPC-I/R, 5- W_IPC-I/R). c) The fold changes in the mRNA expression of the genes (from the normal group) like PI3K and AKT in I/R and IPC groups are given. Bars represent the mean ± SD. *p < 0.05 Vs I/R. N = 6

Since the apoptosis and survival of the cell are majorly regulated by the PI3K/AKT signalling pathway, which is also a cardioprotective signalling cascade, we checked for the expression of PI3K/ Akt genes. While I/R significantly downregulated the expression of PI3K and AKT genes, IPC control itself could impart a significant twofold upregulation of these genes from normal. The IPC-I/R and W_IPC-I/R groups PI3K/ AKT expression data were also in line with the apoptotic data.

Correlation analysis showed a significant negative correlation of PI3K with DNA methylation during IPC (r = − 0.8690, p = 0.0419) and a positive correlation of caspase 3 with DNA methylation (r = 0.9192, p = 0.0490) (Table 2). Considering the significant impact of PI3K on mitochondria and our data showing a negative correlation of DNA methylation with PI3K, we further evaluated the mitochondrial changes (Table 2).

Impact of IPC on mitochondrial copy number and mitochondrial dynamics

Figure 3 shows differential mitochondrial quality control gene expression patterns of I/R and IPC hearts in comparison with the normal rat heart. The master regulator of mitochondrial biogenesis, PGC-1α, mitochondrial transcription factor TFAM and mitochondrial polymerase POLG were downregulated significantly by 70, 50 and 48% respectively in I/R rat hearts (Fig. 3a).

Fig. 3.

mRNA expression changes of Mitochondrial quantity, quality genes and mtDNA copy number during I/R and IPC: The influence of IPC protocol on the mitochondrial quantity was assessed by measuring (a) the fold changes in the mRNA expression of genes (PGC-1α, TFAM, POLG) involved in replication control; (b) The mitochondrial DNA copy number in the myocardium tissues of different experimental groups (N, I/R, IPC-C, IPC-I/R, W_IPC-I/R); (c) mRNA levels of mitophagy genes (PINK, PARKIN, OPTN) from different experimental groups (N, I/R, IPC-C, IPC-I/R, W_IPC-I/R) normalized to normal control heart; (d) mRNA levels of mitofission genes (MFF, DNM, FIS1) from different experimental groups (N, I/R, IPC-C, IPC-I/R, W_IPC-I/R) normalized to normal control heart; (e) mRNA levels of mitofusion genes (MFN1, MFN2) from different experimental groups (N, I/R, IPC-C, IPC-I/R, W_IPC-I/R) normalized to normal control heart. Bars represent the mean ± SD of 6 animals in each group. *p < 0.05 vs I/R. N = 6

IPC control heart induced a striking elevation in the expression of PGC-1α and TFAM without significant change in mitochondrial copy number from the normal. IPC prior to I/R reversed the I/R-induced downregulation of TFAM, POLG and PGC-1α in the rat hearts to above-normal levels. In fact, PGC 1α was upregulated 7.9 folds upon IPC, which completely declined in the presence of wortmannin in the IPC group to 0.26 folds, signifying the importance of PGC-1α in IPC-mediated protection.

The mitochondrial DNA copy number, (the end effector of the alterations in these gene expression), which declined by 51% during I/R improved by 69% upon IPC treatment prior to I/R (Fig. 3b) and declined drastically in wortmannin-treated IPC-I/R group rat hearts.

Mitophagy and mitochondrial dynamics (mitofission and mitofusion) are the other key events that determine the quality of mitochondria in the cell (Fig. 3c–e) While both the Mitofusion genes MFN1 and MFN2 were significantly downregulated by 57 and 81% respectively (Fig. 3e), mitofission genes did not show a significant variation upon I/R except Fis1 which was downregulated by 80% (Fig. 3d). However the mRNA expression of the genes responsible for mitophagy declined significantly upon I/R, evident by significant downregulation in PARKIN and OPTN gene in I/R samples (Fig. 3c).

Rat hearts from IPC alone exhibited upregulation of only MFN2 and FIS1 genes. Despite the improvement in mitochondrial copy number by IPC (Fig. 3b), we could not find significant improvement in mitochondrial fission and fusion genes except MFN2 and FIS1 by IPC prior to I/R induction in rat hearts.

Correlation study between the DNA methylation, and these genes (PGC-1α, TFAM, POLG) showed a negative correlation between DNA methylation with PGC 1α and TFAM (Pearson correlation coefficient r value = − 0.9892 and − 0.9521 respectively) in IPC treated I/R rat tissues. Also, the global DNA methylation level showed a significant negative correlation with POLG gene (Pearson correlation coefficient r value = − 0.8901) (Table 2).

Effect of IPC on I/R induced mitochondrial encoded gene expression changes

Mitochondria encodes 13 ETC and the mRNA expression analysis of these 13 genes showed that I/R induction significantly downregulated ND1 and ND4L (C I), Cyt B (CIII) and COX 1 (C IV) genes by 36, 49, 89 and 48% respectively, while ND4 (C I) and ATP8 (C V) genes were significantly upregulated (Fig. 4). There were not much significant changes in the expression of other mitochondrial encoded genes upon I/R.

Fig. 4.

mRNA expression changes of mitochondrial encoded proteinogenic genes during IPC: The impact of I/R and IPC on the mitochondrial bioenergetics were assessed from the fold changes in the expression of the mitochondrial encoded genes that produce proteins for the ETC complexes (a) Complex I encoded genes are ND1, ND2, ND3, ND4, ND4L, ND6; (b) Complex III encoded gene Cyt B c) Complex IV encoded genes are COX1, COX2, COX3; d) Complex V encoded gens are ATP6, ATP8. *p < 0.05 vs. I/R. N = 6. p < 0.05 was considered statistically significant. N = 6

Ischemic precondition hearts were able to upregulate all 13 genes significantly from normal hearts. Meanwhile, I/R induction post-IPC procedure could induce a significant elevation in the expression pattern of 13 genes and the fold changes were comparatively low except for CytB, ND1 and ATP8 genes, when compared to IPC-C. The wortmannin presence prevented the IPC-induced changes in I/R hearts.

Correlation analysis results showed no significant relationship between DNMT and the mitochondrial encoded 13 genes, while the global DNA methylation level negatively correlated with ND1 expression (r = − 0.8132, p = 0.0461) (Table 2).

Influence of IPC on mitochondrial bioenergetics function

Once the DNA methylation’s role in the modification of I/R-induced mitochondrial encoded gene expression alterations was identified, we further checked if this affected the overall mitochondrial ETC function. I/R significantly suppressed the mitochondrial electron transport chain complex activities (I, II, III and IV) by 51, 33, 77 and 64% respectively when compared with normal hearts (Fig. 5a–d). However, IPC preserved the complex I, II, III & IV activities by 48, 24, 62 and 57% respectively on the I/R challenge when compared with the I/R group. Moreover, the ATP production in total mitochondria, which dramatically declined by 40% in the I/R group, was also improved with IPC prior to I/R by 35% (Fig. 5e). The ROS, a product produced due to the dysfunctional ETC, also was reduced upon preconditioning in I/R rat hearts (Fig. 5f).

Fig. 5.

IPC improved the mitochondrial function in I/R induced cardiac tissues: The effect of IPC on mitochondrial function was assessed by evaluating the electron transport chain enzyme activities of complexes (a) NQR (Complex I); (b) SQR (Complex II); (c) QCR (Complex III); (d) COX (Complex IV). The oxidative phosphorylation potential was assessed by measuring the ATP content, given in (e) and the ROS level was depicted in (f). The graphs represent mean ± SD values. *p < 0.05 vs. I/R. N = 6

IPC’s effect on the I/R-induced gene expression changes in oxidative stress and the antioxidant system

I/R imparted a significant downregulation in the mRNA expression of the NFkB and antioxidant enzymes CuZnSOD, glutathione peroxidase and catalase (Fig. 6). The changes in the gene expression were reflected in their activities as well, where the I/R significantly decreased the SOD, catalase and GPx enzyme activity by 54, 55 and 25% respectively (Fig. 6) and the GSH/GSSG ratio by 69% (Fig. 6e). The IPC control group could significantly upregulate all these genes even in the absence of I/R IPC prior to I/R improved all the antioxidant enzymes and GSH: GSSG ratio effectively when compared to I/R. This was in coherence with the IPC-induced upregulation of CuZnSOD, glutathione peroxidase, catalase and NFkB mRNA by 1.1, 1.1, 1.5 and 1.8 folds respectively from the normal group, where I/R imparted a significant downregulation (Fig. 6a). In fact, the wortmannin treated IPC groups abrogated the antioxidant system by its subsequent downregulation.

Fig. 6.

Effect of IPC on I/R-induced Oxidative stress: the effect of IPC on the mRNA expression of antioxidant genes CuZnSOD, GPx, catalase and NfkB and the corresponding enzyme activity of IPC-mediated changes in the oxidative stress was assessed by measuring (a) the fold changes in the mRNA expression of the oxidative stress response genes in the myocardium of different experimental groups (N, I/R, IPC-C, IPC-I/R, W_IPC-I/R) from the normal group. b, c, e represents the enzyme activities of CuZnSOD, GPx and catalase respectively in different experimental groups. d depicts the level of GSG/GSSG. *p < 0.05 vs. I/R. N = 6

Since the majority of the oxidative stress-associated ROS has its source from mitochondria, we analysed the IPC’s effect on mitochondrial antioxidant system gene expression during I/R injury (Fig. 7a). Our results showed that I/R injury induced a significant downregulation in the SOD2, Prxrdxn3 and Prxrdxn6 genes by 0.4, 0.07 and 0.3 folds respectively, and IPC could significantly upregulate the mRNA expression of these genes in both the presence and absence of I/R injury (Fig. 7a). The antioxidant enzyme activities (Fig. 7b–d) and GSH: GSSG ratio (Fig. 7e) in the mitochondrial fraction are supportive of the observed gene expression changes. Wortmannin presence completely abolished the IPC-induced upregulation in these genes and the corresponding antioxidant enzyme activities in mitochondria.

Fig. 7.

Effect of IPC on mitochondrial oxidative stress: IPC-mediated changes in the mitochondrial oxidative stress was assessed by measuring (a) the fold changes in the gene expression of the mitochondrial oxidative stress response genes like SOD2, Gltrdxn1, Prxrdxn3, Prxrdxn6) in I/R and IPC groups (from the normal group); Enzyme activities of mitochondrial antioxidant enzymes are given in (b) MnSOD; (c) Glutathione peroxidase; (d) GSH:GSSG ratio; and (e) Catalase. *p < 0.05 vs. I/R. N = 6

Correlation analysis showed a significant negative correlation of global DNA methylation with CuZnSOD (r = − 0.9238, p = 0.0381) and SOD2 (r = − 0.8835, p = 0.0428) (Table 2).

Discussion

Ischemic preconditioning (IPC) is a powerful protective mechanism in the heart that protects the myocardium against ischemia reperfusion injury (Bousselmi et al. 2014; Zhang and Zhang 2021). IPC is associated with immediate gene responses that are linked with reduced Infarct size (Xu et al. 2017). In the present study, we explored the status of DNA methylation and its enzymes, that may regulate the genes associated with I/R pathology. Accordingly, we found that, unlike the I/R rat heart, the IPC procedure reduced the DNA methylation level, decreased the expression of DNMTs and increased the expression of TET enzymes along with low DNMT1 activity in the myocardium. The consequential impact of these changes in DNA methylation was reflected in the increased gene expression of apoptotic genes like caspase 3, 7, 9 and downregulation of the PARP gene in IPC rat hearts. Besides, IPC induced upregulation of PI3K and AKT gene, thereby modulating the pro-survival pathways for I/R resistance. Another significant finding from the study suggests that the short duration of IPC protocol without I/R was sufficient to upregulate the mitochondrial regulatory genes like TFAM, POLG and PGC 1α and the 13 mitochondrial encoded bioenergetics genes, which remained upregulated in I/R rat heart conditioned with IPC protocol. The change in the gene expression was supported by high ETC enzyme activity and ATP levels in IPC rat hearts, which indicate well-preserved mitochondrial functional activity. In addition, the IPC procedure also upregulated the gene expression of antioxidant genes that include CuZnSOD, SOD2, Catalase, GPx, Gltrdxn 1, Prxrdxn 6, Prxrdxn 3 in IPC conditioned I/R rat heart, which indicate the possible abundance of antioxidant enzymes that may be involved in the reduction of oxidative stress not only in the mitochondria but also in the cytosol.

Epigenetic reprogramming especially DNA methylation is reported to be involved in the pathogenesis of many non-communicable diseases, but not well explored in the field of myocardial ischemia reperfusion injury. In our previous study, we showed that I/R is characterized by global DNA hypermethylation in normal and diseased conditions (Boovarahan and Kurian 2022; Boovarahan et al. 2022a, 2022b, 2022c), and targeting global DNA methylation can effectively reduce I/R associated cardiac injury and physiological dysfunction (Boovarahan and Kurian 2021; Boovarahan et al. 2022a). In the present study, we found a significant decline in DNA methylation level and a corresponding reduction in the enzyme activity and expression. Interestingly, the short duration of IPC (IPC-C) itself could induce a DNA hypomethylation effect without I/R by downregulating DNMTs and upregulating TETs. Moreover, the correlation analysis, suggested a positive correlation with caspase 3 gene expression (index for cardiac injury) and a negative correlation with rate pressure product measured from the cardiac hemodynamics, indicating the possible link between DNA methylation and cardiac recovery mediators. However, this relationship was drawn from the global DNA aspect rather than specific genes. This made us explore the group of genes involved in I/R-associated pathology that converged in mitochondria.

In general, DNA methylation is under the control of DNMTs and TET, where the former acts as writers and the latter as erasers (Moore et al. 2013). In the present study, the IPC-induced lower DNA methylation was due to the significant elevation in the TET2 and TET3 gene expression apart from reduction in DNMT expression, in both the presence and absence of I/R, indicating possible role played by these genes in IPC-mediated cardio-protection. In fact, the involvement of TET family proteins in the I/R pathophysiology has already been established in the kidney, where promoter regions of Cxcl10 and Ifngr2 genes were altered by TET (Huang et al. 2012). Therefore the improvement in TET2 and TET3 in the present study via IPC protocol provides a beneficial effect in IPC-treated I/R rat heart.

Early reports have shown that DNA methylation alterations can occur in response to the activation of pro-survival Kinase pathways like RISK (PI3K-AKT) (Spangle et al. 2017). IPC is known to activate PI3K signalling while rendering cardio-protection in I/R rat heart (Rossello et al. 2018). Hence, we checked whether DNA methylation level has any significant relation with the upregulation of PI3K and AKT genes associated with IPC. Our data demonstrated that IPC-conditioned hearts prior to I/R reduced the DNA methylation level and reduced the expression of DNMTs with increased TETs. However, with wortmannin, the inhibitor of PI3K signalling, DNA methylation was high with increased DNMT expression, where the IPC-mediated cardio-protection was absent. Popkie and his group demonstrated that the PI3K signalling pathway is capable of altering the DNA methylation of imprinting loci (Popkie et al. 2010). In addition, the significant elevation of TET gene expression of IPC and IPC-I/R rat hearts was significantly low with the wortmannin treatment prior to IPC, which indicates the possible involvement of TET enzymes in IPC-mediated cardioprotection. Thus, according to the present study results, we assumed that the IPC protocol triggers PI3K signal that in turn activates the downstream mediators along with the activation of TET enzymes, which resulted in a low DNA methylation level.

In order to answer whether epigenetic activation of IPC has any role in the regulation of genes involved in the mediators, we assessed the expression of genes involved in apoptosis and mitochondrial function. Apoptotic genes showed upregulation of caspase genes like Casp3, Casp7, Casp9 and PARP in I/R rat hearts, which were downregulated in IPC-I/R rat hearts, showing that decreased gene expression of caspase genes during IPC has reduced the apoptosis (measured via DNA fragmentation) and contributed to the survival of the cell. The Poisson correlation analysis suggested a positive correlation between the caspase 3 gene and global DNA methylation level in IPC-mediated I/R protection. An early study by Duan and his coworkers (2021) showed that increased caspase-3 expression correlates with DNA hypomethylation of caspase-3 enhancer regions (Duan et al. 2021). In agreement with the previous study by Hossini (2016) in iPSC cells (Hossini et al. 2016), Wortmannin resulted in the activation of caspase-3 gene expression, indicating the importance of IPC-mediated hypomethylation in the cardioprotection.

Previous studies have emphasized the involvement of mitochondrial functional preservation in the mechanism of IPC-mediated cardio-protection in I/R rat hearts (Rossello et al. 2018). Mitochondria in the heart exhibit different functions and the mitochondrial proteome is estimated to have more than 1500 genes and many are under the control of epigenetic regulations as well. Importantly, mitochondria is the key player that controls some of the substrates involved in nuclear methylation such as one-carbon metabolism and the methionine pathway (A 2020). Hence mitochondrial dysfunction mediated by ischemia reperfusion can sensitize the myocardium and may trigger the methylation of DNA. Accordingly, we analyzed the expression of different specific mitochondrial genes, reported to be involved in I/R condition. An imbalance in energy demand and supply is the crux of I/R pathology and is mainly controlled by mitochondrial bioenergetics. Both nDNA and mtDNA are involved in the assembly of the bioenergetics system, where mtDNA encodes the core genes of oxidative phosphorylation. Unlike the I/R rat heart, IPC treated rat heart exhibited elevated expression of genes like PGC1α, TFAM, and POLG along with mitochondria-encoded ETC genes with a significant increase in the mitochondrial copy number, indicating the improvement in mitochondrial bioenergetics gene expression by IPC. However, in the presence of wortmannin, the gene expression was similar to that in I/R rat hearts suggesting the importance of PI3K signaling in the expression of these genes. As mentioned earlier, PI3K regulate these genes via stimulating DNA hypomethylation. Literature shows that, the chromatin gets modified and decondensed and increased the gene expression when the bioenergetics intermediate increases (Wallace 2010). Thus in the I/R rat heart, most of the bioenergetics gene expressions were elevated as the tissue experienced an adaptive transition from the global ischemia. Interestingly, a few gene alterations were only observed with IPC-treated I/R rat hearts, which were not fully reversed in IPC-treated I/R rat hearts pretreated with wortmannin, indicating the less significant involvement of these pathway genes with PI3K signalling.

Oxidative stress is known to induce hypermethylation and as expected, was increased in I/R rat hearts. IPC induced hypomethylation and subsequently increased the expression of antioxidant enzymes, especially in the mitochondria. Declined expression of antioxidant genes in wortmannin administrated IPC conditioned I/R rat heart, suggested the involvement of methylation in the status of antioxidant enzyme genes especially in the mitochondria.

Conclusion

Based on the results obtained in the present study, we demonstrated that IPC-mediated cardio-protection in I/R rat hearts works via hypomethylation of DNA, in addition to the established mode of action as described by the previous findings. Besides we found that IPC-mediated hypomethylation of DNA required activation of the PI3K signalling pathway, where the administration of wortmannin prior to the IPC procedure not only negated the cardio-protection but also induced hypermethylation of DNA and elevated expression of DNMTs. Furthermore, our results demonstrated that the expression of apoptotic genes was low in the hypomethylated state, but the majority of genes involved in mitochondrial bioenergetics and the antioxidants were high. In fact, these genes showed an opposite trend in the hypermethylated state in I/R or wortmannin-treated rat hearts.

Supplementary Information

Below is the link to the electronic supplementary material.

Data availability

The data will be provided upon reasonable request.

Declarations

Conflict of interest

There exist no potential conflicts of interest including employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding among the authors.

Ethical approval

All procedures involving the animals were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), SASTRA University, Thanjavur, India (CPCSEA Approval No. /SASTRA/IAEC/RPP/547) and was conducted in accordance with the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) guidelines.