CaM promotes cardiomyocyte mitophagy in myocardial ischemia-reperfusion injury involving in the regulation of the IP3R3-GRP75-VDAC1 complex

Abstract

The pathogenesis of myocardial ischemia-reperfusion injury (MIRI) is not fully clear. This study aims to investigate the role of mitochondrial-associated endoplasmic reticulum membrane (MAM)-related calcium overload in mitophagy. In vitro and in vivo models were established to simulate MIRI. Cellular injury, apoptosis and mitophagy were measured and gene expression was analysized. The expression levels of glucose-regulated protein 75 (GRP75), receptor for inositol 1,4,5-trisphosphate (IP3R3), voltage-dependent anion-selective channel 1 (VDAC1), and calmodulin (CaM) and the mitochondrial calcium content, mitophagy and apoptosis were significantly increased in MIRI or hypoxia/reoxygenation (H/R) cells when compared to controls, but the mitochondrial membrane potential and ATP significantly decreased. GRP75 knockdown significantly inhibited CaM expression, mitochondrial calcium overload and mitophagy of H9C2 cells, whereas had no significant effect on IP3R3 and VDAC1 expression. CaM knockdown had no significant effect on the expression of GRP75, IP3R3 and VDAC1, and on mitochondrial calcium concentration, ATP levels and mitochondrial membrane potential of H9C2 cells, but significantly inhibited mitophagy and apoptosis. Collectively, these data suggest that the IP3R3-GRP75-VDAC1/CaM axis plays an important role in mitochondrial autophagy injury during myocardial ischemia-reperfusion and that it is a potential target for MIRI treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-07977-5.

Introduction

Cardiovascular diseases have become one of the major causes of disease-related deaths worldwide, and myocardial infarction is one of the most common cardiovascular diseases noted in clinical practice¹. It is well known that the pathogenesis of myocardial infarction is mainly caused by cardiac insufficiency of blood supply, which leads to abnormal cardiomyocyte function, such as apoptosis and necrosis². Timely recovery of cardiomyocyte blood supply is a common treatment of myocardial infarction. However, in addition to the benefits, the restoration of the blood supply can also exert adverse effects on cardiomyocytes, known as reperfusion injury³. The molecular mechanism of cardiomyocyte reperfusion injury is very complex, including oxidative stress injury, calcium overload, and autophagy^3,4. Recent studies have shown that the aforementioned mechanisms are related to mitochondrial damage; however, the exact mechanism remains to be elucidated⁵. Mitophagy is very common in mammalian cells, such as cardiomyocytes and nerve cells, and has both protective and damaging effects⁶. It has been shown that mitophagy is associated with ischemia-reperfusion injury in cardiomyocytes and that this process may involve mitochondrial calcium overload⁷. For example, in a previous study by Thai et al.⁸, it was shown that when cardiomyocytes underwent calcium overload mitophagy was significantly increased⁸. Although both calcium overload and mitophagy were involved in cardiomyocyte injury, the exact mechanism of action remained unclear.

As an important structure connecting the mitochondria with the endoplasmic reticulum, the mitochondrial-associated endoplasmic reticulum membrane (MAM) is involved in a variety of biological processes in mammalian cells, such as ER stress and calcium overload⁹. The receptor for inositol 1,4,5-trisphosphate (IP3R3)- glucose-regulated protein 75(GRP75)-voltage-dependent anion-selective channel 1 (VDAC1) complex is one of the most widely studied MAMs and its main function is associated with the transfer of calcium ions from the endoplasmic reticulum to the mitochondria. Therefore, the IP3R1-GRP75-VDAC1 axis affects the majority of the cell functions¹⁰. A number of studies have shown that the high expression of the IP3R1-GRP75-VDAC1 complex is closely related to mitochondrial calcium overload¹¹. By contrast, knockdown of GRP75 expression or inhibition of the IP3R1-GRP75-VDAC1 complex inhibits mitochondrial calcium overload¹². This fully indicates that the IP3R1-GRP75-VDAC1 complex plays an important role in mitochondrial calcium overload. The involvement of MAMs in autophagy of mammalian cells has been widely recognized. However, whether MAMs are involved in the process of mitophagy in cardiomyocytes and the exact mechanism of action remains unclear.

Calmodulin (CaM) plays a key role in calcium signal regulation, and its abnormal expression may lead to the occurrence and development of various diseases¹³. Previous studies have shown that CaM is involved in the development of autophagy during myocardial ischemia-reperfusion injury (MIRI)¹⁴. Considering the role of CaM in the calcium signaling cascade and the role of MAMs in mitochondrial calcium overload, it was hypothesized that the IP3R1-GRP75-VDAC1 complex is involved in cardiomyocyte mitophagy associated with the regulation of CaM expression.

In the present study, a series of experiments were conducted to demonstrate the possible mechanism of the IP3R1-GRP75-VDAC1 complex in mitophagy. This will provide a new theoretical basis and experimental evidence for the treatment of MIRI.

Results

MIRI leads to myocardial cells injury

The TTC staining shown that IR lead to myocardial infarction( figure S1 A and B). As shown in figure S1 C, the relatively ROS production levels significantly increased in IR when compared to sham. These data shown that myocardial cell injury has occurred.

MIRI induces upregulation of the IP3R3-GRP75-VDAC1 complex and mitochondrial Ca²⁺ overload in cardiomyocytes

The data indicated that the expression levels of IP3R3, GRP75, and VDAC1 in MIRI were apparently increased compared with those of the sham group (Fig. 1A, B, C, and D). This suggested that the expression levels of the IP3R3-GRP75-VDAC1 complex proteins were apparently increased in MIRI. Considering that the IP3R3-GRP75-VDAC1 complex is mainly related to Ca²⁺ transfer between the ER and the mitochondria, the mitochondrial Ca²⁺ levels were assessed in cardiomyocytes. As shown in Fig. 1E F, the mitochondrial Ca²⁺ levels were significantly increased in the MIRI group compared with those of the control group. Inversely, the ATP content and the mitochondrial membrane potential were significantly decreased in the MIRI group compared with those of the sham group (Fig. 1G,H, and I). These data suggested that MIRI contributed to mitochondrial Ca²⁺ overload in cardiomyocytes.

Fig. 1.

MIRI induces upregulation of the IP3R3-GRP75-VDAC1 complex proteins and mitochondrial Ca²⁺ overload in cardiomyocytes. (A). IP3R3, GRP75 and VDAC1 protein expression; (B, C and D). The relative ratio; (E).Rhod-2 fluorescence; (F).Mitochondrial calcium content; (G) ATP level; (H). Mitochondrial membrane potential; (I) Fold-change. A post-hoc test was used following the one-way ANOVA.^*P < 0.05 vs. sham; ^**P < 0.01 vs. sham. IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75,, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

MIRI induces mitophagy and cell injury in cardiomyocytes

The expression levels of CaM, a key protein in calcium signal transduction, were measured. The results indicated that the expression levels of CaM (both mRNA and protein) in MIRI were significantly increased compared with those of the sham group (Fig. 2A,B and C). Subsequently, the expression levels of genes related to mitophagy were investigated. As shown in Fig. 2B,D, and E, the expression levels of ATG4 and LC3 were significantly increased in MIRI compared with those of the control group. Additionally, the levels of specific indicators that are related to cardiomyocyte injury were investigated. As shown in Fig. 2F,G, the CK activity and the TnT levels in MIRI were significantly increased compared with those of the control group. These results indicated that myocardial injury occurred in MIRI rats. Subsequently, the cytotoxicity caused on cardiomyocytes and their induction of apoptosis, were investigated. The data indicated that compared with the sham group, the LDH release and the induction of apoptosis in cardiomyocytes were apparently increased in MIRI (Fig. 2H, I, and J). The co-staining of LC3 and the mitochondria indicated an apparent increase in the co-localization of the mitochondria and LC3 in MIRI compared with the control group (Fig. 2K). These results indicated that MIRI led to mitophagy and apoptosis in cardiomyocytes.

Fig. 2.

MIRI induces mitophagy and cell injury in cardiomyocytes. (A). CaM mRNA expression levels; (B). CaM, ATG4, LC3-2, and LC3-1 protein expression; (C, D, and E). The corresponding quantification analysis of protein expression; (F). CK activity; (G) TnT levels; (H). LDH levels; (I). TUNEL/Hoechst double staining. (J). TUNEL positive cell counting. (K). Co-localization of mitochondria and LC3. A post-hoc test was used following the one-way ANOVA analysis. ^*P < 0.05 vs. sham;^**P < 0.01 vs. sham;^***P < 0.001 vs. sham. MIRI myocardial ischemia-reperfusion injury, CaM calmodulin, ATG4 autophagy-regulating protease 4, LC3 microtubule-associated protein 1 A/1B-light chain 3, CK creatine kinase, TnT troponin-T, LDH lactate dehydrogenase, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA analysis of variance.

Knockdown of GRP75 expression attenuates H/R-induced mitochondrial calcium overload in H9C2 cells

To further confirm the important role of the IP3R3-GRP75-VDAC1 complex in mitochondrial calcium overload in MIRI cardiomyocytes, a hypoxic reoxygenation injury model was established in H9C2 cells to simulate MIRI injury. As shown in Fig. 3A, B, C and D, the expression levels of GRP75 (mRNA) were significantly increased in H/R-treated H9C2 cells. However, this effect was significantly inhibited by transfection of the cells with GRP75 siRNA, but not by transfection with NC siRNA. This indicated that the GRP75 siRNA was functioning appropriately. Subsequently, the expression levels of GRP75, IP3R3, and VDAC1 (protein) were detected. The expression levels of GRP75, IP3R3, and VDAC1 were significantly increased in H/R-treated H9C2 cells compared with those of the control group (Fig. 3C, D and E, F). It was expected that the GRP75 siRNA sequence could cause a significant downregulation in the expression levels of GRP75, whereas it had no apparent effect on IP3R3 and VDAC1, indicating that the GRP75 siRNA had a specific function. Subsequently, the mitochondrial calcium content of the cells was measured. The mitochondrial calcium levels were significantly increased in H/R-treated H9C2 cells compared with those in the control cells, whereas the ATP content and the mitochondrial membrane potential were decreased (Fig. 3G, H, I, J and K). By contrast, knockdown of GRP75 expression could significantly reduce mitochondrial calcium levels and increase the ATP content and the mitochondrial membrane potential in H/R-treated cells (Fig. 3G, H, I, J and K).

Fig. 3.

GRP75 knockdown attenuates H/R-induced mitochondrial calcium overload in H9C2 cells. (A) and (B). GRP75 mRNA expression levels; (C) IP3R3, GRP75, and VDAC1 protein expression; (D, E, and F). The corresponding quantification analysis of protein expression; (G). Rhod-2 fluoresence; (H).Mitochondrial calcium content; (I). ATP levels; (J). Mitochondrial membrane potential; (K). Fold-change. A post-hoc test was used following the one-way ANOVA analysis. ^**P < 0.01 vs. control; ^***P < 0.01 vs. NC siRNA;^##P < 0.01 vs. H/R; ^###P < 0.001 vs. H/R. GRP75 glucose-regulated protein 75, H/R hypoxia/reoxygenation, IP3R3 receptor for inositol 1,4,5-trisphosphate, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

Knockdown of GRP75 expression attenuates H/R-induced mitophagy in H9C2 cells

Since calcium signaling plays an important role in mitophagy, the effects of knockdown of GRP75 expression were investigated on CaM. As shown in Fig. 4A, B, the expression levels of CaM were significantly increased in H/R-treated H9C2 cells compared with those of the control cells, which was consistent with the results of the animal experiments; this effect was apparently inhibited by knockdown of GRP75 expression. Subsequently, the effects of knockdown of GRP75 expression were investigated on mitophagy. The expression levels of ATG4 and LC3 (two autophagy-related genes) were significantly increased in H/R-treated H9C2 cells (Fig. 4A, C, D, E, and F). As expected, knockdown of GRP75 expression significantly inhibited the expression levels of ATG4 and LC3 in H/R-treated H9C2 cells. This indicated that knockdown of GRP75 expression could inhibit mitophagy. Subsequently, the damage caused to the cells was examined. As shown in Fig. 4G,  H, and I, the release of LDH and the induction of apoptosis were significantly increased in H/R-treated H9C2 cells, while this phenomenon was apparently inhibited following knockdown of GRP75 expression. Co-staining of LC3 and the mitochondria indicated an apparent decrease in the number of mitochondria and an increase in the co-localization of mitochondria and LC3 in H/R-treated H9C2 cells compared with the corresponding indices detected in the control cells. By contrast, knockdown of GRP75 expression partly reversed these effects (Fig. 4J). These results suggested that the GRP75 complex was involved in mitophagy.

Fig. 4.

GRP75 knockdown attenuates H/R-induced mitophagy in H9C2 cells. (A). CaM, ATG4, LC3-2, and LC3-1 protein expression; (B). The corresponding quantification analysis of CaM protein expression; (C) ATG4 mRNA expression; (D). The corresponding quantification analysis of ATG4 protein expression; (E) ATG4 mRNA expression; (F). The corresponding quantification analysis of the ratio of LC3-2 to LC3-1; (G). LDH levels; (H). TUNEL/Hoechst double staining. (I). TUNEL positive cell counting. (J). Co-localization of mitochondria and LC3. Post-hoc test was used following the one-way ANOVA analysis. ^*P < 0.05 vs. control;^**P < 0.01 vs. control; ^#P < 0.05 vs. H/R. GRP75 glucose-regulated protein 75, H/R hypoxia/reoxygenation, CaM calmodulin, ATG4 autophagy-regulating protease 4, LC3 microtubule-associated protein 1 A/1B-light chain 3, LDH lactate dehydrogenase, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA analysis of variance.

Knockdown of CaM expression exhibits no apparent effects on H/R-induced mitochondrial calcium overload in H9C2 cells.

Considering the critical role of CaM in calcium signaling, the effects of knockdown of its expression were determined in the cells. In Fig. 5A, B, C and D, the expression levels of CaM (mRNA and protein) were significantly inhibited by CaM siRNA, but not by NC siRNA. This indicated that the CaM siRNA sequence achieved efficient knockdown. Furthermore, the effects of knockdown of CaM expression were determined on the IP3R3-GRP75-VDAC1 complex proteins. The data indicated that knockdown of CaM expression exhibited no apparent effect on the expression levels of GRP75, IP3R3, and VDAC1 (Fig. 5C, E, F, G). Moreover, the effects of knockdown of CaM expression on mitochondrial calcium levels, ATP levels, and mitochondrial membrane potential were investigated in H/R-treated H9C2 cells. The present study indicated that knockdown of CaM expression exhibited no apparent effects on mitochondrial calcium levels, ATP content, and mitochondrial membrane potential in H/R-treated H9C2 cells (Fig. 5H, I, J, K and L). These results suggested that CaM may play a role by affecting calcium signal transduction.

Fig. 5.

CaM knockdown exhibits no apparent effect on H/R-induced mitochondrial calcium overload in H9C2 cells. (A) and (B). CaM mRNA expression levels; (C). CaM, IP3R3, GRP75, and VDAC1 protein expression levels; (D, E, F, and G). The corresponding quantification analysis of protein expression; (H). Rhod-2 fluoresence; (I). Mitochondrial calcium content; (J). ATP levels; (K): mitochondrial membrane potential; (L). fold-change. A post-hoc test was used following the one-way ANOVA analysis.^*P < 0.05 vs. control; ^**P < 0.01 vs. control; ^***P < 0.01 vs. NC siRNA;^##P < 0.01 vs. H/R; ^###P < 0.001 vs. H/R. CaM calmodulin,H/R hypoxia/reoxygenation, IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75, VDAC1 voltage-dependent anion-selective channel 1, ANOVA analysis of variance.

Effects of knockdown of Cam expression on H/R-induced mitophagy in H9C2 cells

The effects of knockdown of CaM expression on mitophagy were investigated in H/R-induced H9C2 cells. As shown in Fig. 6A, B and C, the expression levels of ATG4 and LC3 in H/R-induced H9C2 cells were significantly inhibited by knockdown of CaM expression. The effects of knockdown of CaM expression were investigated on LDH release and apoptosis in H/R-induced H9C2 cells. The data indicated that the LDH release and cell apoptosis in H/R-induced H9C2 cells were significantly inhibited following knockdown of CaM expression (Fig. 6D, E, and F). Co-staining of LC3 and mitochondria indicated an apparent decrease in the number of mitochondria and an increase in the co-localization of mitochondria and LC3 in H/R-treated cells compared with the control cells, while this effect could be partly reversed by CaM knockdown (Fig. 6G). These results demonstrated that CaM was involved in the process of mitophagy induced by mitochondrial calcium overload in cardiomyocytes.

Fig. 6.

Effects of CaM knockdown on H/R-induced mitophagy in H9C2 cells. (A). ATG4, LC3-2, and LC3-1 protein expression; (B and C). The corresponding quantification analysis of protein expression; (D). LDH levels; (E). TUNEL/Hoechst double staining; (F). TUNEL positive cell counting. (G). Co-localization of mitochondria and LC3. A post-hoc test was used following the one-way ANOVA test.^*P < 0.05 vs. control; ^**P < 0.01 vs. control; ^#P < 0.05 vs. H/R. ^##P < 0.01 vs. H/R. CaM, calmodulin,H/R, hypoxia/reoxygenation, ATG4, autophagy-regulating protease 4,LC3, microtubule-associated protein 1 A/1B-light chain 3, LDH, lactate dehydrogenase, TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling, ANOVA, analysis of variance.

Discussion

Mitochondrial autophagy and calcium overload are important causes of cardiomyocyte injury and MAMs play an important role in mitochondrial function. The present study found that high expressed IP3R3-GRP75-VDAC1 complex and CaM contributed to mitochondrial calcium overload and mitophagy in cardiomyocytes. Knockdown of GRP75 significantly inhibited both the occurrence of mitochondrial calcium overload and mitophagy in cardiomyocytes, while knockdown of CaM only reduced the mitophagy and without significant effect on mitochondrial calcium overload. This suggested that the IP3R3-GRP75-VDAC1 complex promoted mitochondrial calcium overload in cardiomyocytes, which promoted mitophagy and cell injury through the mediation of CaM (Fig. 7).

Fig. 7.

Schematic diagram of MIRI. MIRI led to an upregulated expression of the IP3R3-GRP75-VDAC1 complex proteins in cardiomyocytes, which resulted in mitochondrial calcium overload. In addition, MIRI led to an upregulated in the expression levels of CaM. The Ca²⁺ bind to CaM forming the Ca²⁺-CaM complex, which induces mitophagy-related gene (LC3 and ATG4) expression. This in turn leads to induction of mitophagy. Excessive mitophagy results in cell damage. MIRI myocardial ischemia-reperfusion injury, IP3R3 receptor for inositol 1,4,5-trisphosphate, GRP75 glucose-regulated protein 75, VDAC1 voltage-dependent anion-selective channel 1, CaM calmodulin; Ca²⁺, calcium ions, LC3 microtubule-associated protein 1 A/1B-light chain 3, ATG4 autophagy-regulating protease 4.

It well demonstrated that mitochondria are the energy factory of mammalian cell and play an important role in cell survival and apoptosis and calcium ions, the secondary messengers, are considered to be key to normal mitochondrial function¹⁵. Previous studies have shown that calcium has a dual role in mitochondrial function. Normal Ca²⁺ levels are necessary for the production of ATP by the mitochondrial oxidative phosphorylation. However, excessive calcium levels can cause mitochondrial membrane permeability changes, which will inhibit ATP production, increase cytochrome C release, and induce apoptosis, resulting in cell damage¹⁶. This suggested that mitochondrial Ca²⁺ overload is an important cause for cardiomyocytes injury. Thus, elucidating the regulation mechanism of mitochondrial Ca²⁺ level is very important and necessary to understanding cardiomyocytes injury. In this study, we found that the mitochondrial function was regulated, at least partly, by the IP3R3-GRP75-VDAC1 complex, which in accordance with previous studies that the IP3R3-GRP75-VDAC1 complex plays a key role in regulating calcium transfer between the endoplasmic reticulum and the mitochondria¹⁷. Previous studies shown that IP3R1 is located on the endoplasmic reticulum membrane and related to the production of high Ca²⁺ levels, and VDAC1 is located on the outer mitochondrial membrane and is an important calcium uptake channel, and GRP75 is mainly a structure connecting IP3R3 and VDAC1 channels⁹. This suggests that IP3R3-GRP75-VDAC1 complex play important roles in mitochondrial Ca²⁺ overload. Thus the dysfunction of IP3R3-GRP75-VDAC1 complex will lead to cardiomyocytes injury and this was demonstrated by many studies, for example, Luan Y et al. have found that the expression levels of IP3R3 significantly increased in cardiac hypertrophy, heart failure, and ischemic cardiomyopathy, which are important causes of heart failure and result in the development of other diseases¹⁸ and Paillard M have found that inhibition of the CypD/VDAC1/Grp75/IP3R1 complex reduces mitochondrial Ca²⁺ overload to protect cardiomyocytes(H9C2 cells) from injury¹⁹. Thus, knocking down any member of the IP3R3-GRP75-VDAC1 complex will cause its dysfunction theoretically. Therefore, inhibition of GRP75 will inhibit the IP3R3-GRP75-VDAC1 complex formation and resulting in inhibition of mitochondrial calcium overload. This was confirmed by the present study that during MIRI or hypoxic reoxygenation injury, the expression levels of the IP3R3-GRP75-VDAC1 complex proteins in cardiomyocytes were increased, concomitantly with calcium overload and cardiomyocytes apoptosis. Additionally, knockdown of GRP75 expression or interference of the formation of the IP3R3-GRP75-VDAC1 complex can significantly reduce mitochondrial Ca²⁺ levels and cell apoptosis. The results above suggest that the IP3R3-GRP75-VDAC1 complex is a potential target for cardiomyocyte injury.

Mitophagy is an important biological process in mammalian cells. The normal function of mitophagy is to remove damaged mitochondria, thus achieving a protective effect; however, excessive mitophagy will reduce ATP generation and cause cell death²⁰. As the main organelle of cardiomyocytes, mitochondria are important determinants of cardiomyocyte survival or death. Previous studies have shown that mitochondrial damage is an important manifestation of cardiomyocyte injury or apoptosis²¹. It has been shown that cardiomyocytes exhibit detailed mitophagy during MIRI. Ji et al. summarized the molecular mechanism of mitophagy in cardiomyocytes and demonstrated that mitophagy was mainly mediated via the PTEN-induced kinase 1/parkin RBR E3 ubiquitin protein ligase (Parkin) pathway, and FUN14 domain containing 1 and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 proteins²¹. In-depth studies of the role of mitophagy in MIRI have demonstrated its application as an important target for the research and development of drugs related to cardiovascular diseases. For example, Yang et al. demonstrated that the mechanism of traditional Chinese medicine Tongxinluo in reducing MIRI involved Parkin-mediated mitophagy²². Consistently, the present study indicated that the expression levels of ATG4 and LC3 were increased in cardiomyocytes following myocardial I/R, indicating that detailed mitophagy occurred in these cells. Considering that mitochondrial calcium overload is closely related to mitochondrial damage, it was hypothesized that this process may be an important cause of mitophagy. In fact, certain studies have shown that mitochondrial calcium overload can promote the occurrence of mitophagy and damage the recycling of mitochondria in nerve cells, thus leading to the occurrence and development of Alzheimer’s disease²³. In addition, many studies have found that mitochondrial calcium overload can cause changes in mitochondrial permeability, resulting in mitochondrial damage and ultimately heart failure²⁴. In the present study, we found that inhibition of GRP75 expression could significantly inhibit the occurrence of mitochondrial calcium overload and reduce the occurrence of mitophagy in H9C2 cells, thereby reducing cell apoptosis. Furthermore, the present study indicated an apparent decrease in the number of mitochondria and an increase in the co-localization of mitochondria and LC3 in MIRI rats or H/R-treated H9C2 cells compared with the corresponding control groups; these effects were partly reversed by knockdown of GRP75 expression. This demonstrated that mitophagy occurred in MIRI cardiomyocytes and mitochondrial calcium overload was an important factor causing mitophagy in cardiomyocyte injury.

Ca²⁺ is an important intracellular secondary messenger and its signaling plays an important role in cardiomyocyte stress. Therefore, it was speculated that the regulation of calcium signaling-related proteins was involved in the occurrence and development of mitophagy which caused by mitochondrial calcium overload. Among the various regulatory proteins, the present study focused on CaM for the following reason:¹ previous studies have proved that CaM play important roles in Ca²⁺ signaling¹⁹;² in our previous experiment we found that high expression level of CaM was associated with myocardial ischemia reperfusion injury. Thus, we designed experiments to observe what is mainly affected by CaM, is the downstream or upstream of mitochondrial Ca²⁺ overload. As we known, CaM is a key protein involved in the regulation of calcium signaling, which is required for ubiquitination and degradation. For example, in the study of Mukherjee et al., it was found that CaM can regulate the interaction of Mahogunin ring finger 1-gp78 and mediate the ubiquitin-protease degradation system²⁵. In addition, Zhou et al. indicated that doxorubicin could induce excessive autophagy of myocardial cells, whereas sphingosylphosphorylcholine could inhibit myocardial cell mitochondrial autophagy²⁶. The associated mechanisms of action involved the inhibition of CaM expression²⁶. The present study indicated that the expression levels of CaM were significantly increased in MIRI rats or H/R-induced H9C2 cells, which was accompanied with an apparent increase in mitophagy. This suggested a potential correlation between CaM and mitophagy. The present result shown that knockdown of CaM expression could significantly reduce the mitophagy in H9C2 cells, but had no significant effect on mitochondrial calcium ion levels and the ATP content. In addition, we also found there is an apparently increased co-localization of mitochondria and LC3 was noted in MIRI rats or H/R-treated H9C2 cells compared with the control groups, but it can be partly reversed by CaM knockdown. This suggested that mitophagy induced by mitochondrial calcium overload in MIRI was mediated, at least in part, by CaM.

Although the present study provides in vivo and in vitro experimental evidence, it contains certain limitations as follows:¹ The GRP75 and CaM knockdown experiments were carried out in vitro and they were not applied to animals;² only male rats were used in the animal experiment and no female rats were included;³ the mechanism of CaM effect on mitophagy was not investigated;⁴ for insufficient of research funding, partial data only has in vitro experiments. The potential differences arising due to the different genders were not fully considered. Therefore, additional experiments should be performed in future studies.

Taken together, these results indicated that the IP3R3-GRP75-VDAC1/CaM axis plays an important role in mitochondrial autophagy injury during myocardial ischemia-reperfusion and that it is a potential target for MIRI treatment.

Materials and methods

Animal experiments

Sprague Dawley male rats (8-week old) weighing 250 g were purchased from Hunan SJA Laboratory Animal Co., Ltd. The animals underwent a week of acclimatization prior to the experiments. The environmental conditions were the following: 25 °C, 65% humidity and natural light. During the whole process, the rats had free access to water and food. The rats were grouped into the following two groups: sham and ischemia reperfusion (I/R)(n = 8 rats for each group). The surgical MIRI model was established following a previously published method²⁷. Briefly, the rats were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg) and subsequently surgery was conducted to isolate the heart. During the surgical process, the vital signs of each rat were monitored, including blood pressure, heart rate, body temperature, and electrocardiogram (ECG) analysis. Subsequently, the left anterior descending coronary artery was occluded using a vein clamp. If the coronary artery was occluded successfully, the ECG presented an apparent change as determined by the ST segment upgrade. Subsequently, the vein clamp was removed to restore the blood supply of the coronary artery. The rats were subjected to a 0.5 h time period of ischemia and a 2 h time period of reperfusion. The study protocol was approved by the ethics committee of the affiliated Changsha hospital of Hunan normal university (approval number: CSSDSYY-LLSC-XYXM-2021-8-8) and all methods were carried out in accordance with the National Institute of Health guidelines.

At the end of the experiments, the animals were anesthetized by intraperitoneal injection of 3% pentobarbital sodium (40 mg/kg) and immediately sacrificed by removal of the aortic blood. The heart tissues were collected for subsequent experiments.

Isolation of rat cardiomyocytes by enzymatic digestion

At the end of ischemia reperfusion, the heart was isolated and washed twice with PBS to remove blood. The heart tissues were cut into pieces and homogenate in digestion buffer containing: Collagenase II (0.8 mg/mL), Trypsin (0.025%), and 10 µM CaCl₂ for 5 min. The homogenation was centrifuged at 1000r/min for 10 min to collect the cell pellet and then suspended with PBS. Repeat it twice. Then the cells were used for subsequent experiments, such as hoechrst staining, mitochondrial membrane potential determination.

TTC staining

The ventricles were cut into 5 slices along the longitudinal axis of the heart, each with a thickness of about 2 mm. The heart sections were placed in TTC solution and incubated for 40 min at 37 °C in an incubator. Yellow and white areas are myocardial infarction areas and dark red areas are non-infarcted areas. The Image analysis software Image-Pro Plus was used to analyze and calculate the area of each color.

Determination of creatine kinase isoenzymes (CK-MB) activity

The CK-MB activity measurements were performed using the heart homogenates and a CK-MB test kit (Solarbio). Following the instructions provided by the manufacturer, a total of 1,000 µl reaction solutions was prepared and the absorption value was measured at 340 nm using a spectrophotometer (colorimetric method). At the end of the experiment, the CK-MBactivity was calculated.

Determination of troponin T (TnT) levels

For determining the TnT levels, a commercial ELISA kit was used. The detailed process was performed according to the instructions provided by the manufacturer as follows: Formation of the solid phase antigen was achieved by linking specific antibodies to a solid phase carrier and formation of the antigen-antibody complex was performed by addition of serum and enzyme-labeled antibody. In the final step, a substrate was used for development and the absorbance value was measured at 450 nm corresponding to the TnT content, which was calculated according to the kit instructions. The TnT concentration was calculated according to a formula provided by the kit supplier: TnT concentration = Absorbance of treated sample/ Absorbance of control*concentration of standard control.

Lactate dehydrogensae (LDH) release

To determine the cytotoxicity of myocardial cells, an LDH assay kit was used. Briefly, an LDH measurement work solution was prepared according to the manufacturer’s instructions; this work solution was mixed with serum and incubated for 30 min in the dark. At the end of the experiment, the absorbance value was measured at 490 nm and the LDH release was calculated according to the kit instructions. The LDH concentration was measured according to a formula provided by the kit supplier: LDH concentration=(Absorbance of treated sample)/(Absorbance of control)/* concentration of standard control.

ATP content measurement

The ATP content in the cells was measured by an ATP assay kit (Beyotime). The ATP lysis solution was used for tissues or cells lysis. The supernatant of the cells or the tissue lysates were used for ATP detection according to the kit instruction. The ATP content was calculated according to a formula provided by the kit supplier:

ATP concentration=(RLU value of treated sample)/ RLU value of control)*ATP concentration of standard control.

Determination of mitochondrial membrane potential

The mitochondrial membrane potential was determined using a commercial kit (JC-1, Beyotime). Briefly, the JC-1 work solution was prepared by mixing the work solution with the cells. The resulting mixture was incubated at 37℃ for 20 min. The cells were imaged with a fluorescence microscope and the mitochondrial membrane potential was measured.

Determination of ROS level

The relative content of ROS in cardiomyocytes was detected by reactive oxygen species detection kit (DCFH-DA). DCFH-DA was mixed with the cell suspension to a final concentration of 10 μm and incubated for 30 min at 37 ° C in an incubator according to the manufacturer’s instructions. The fluorescence intensity of each group was measured by a multifunctional microplate reader (excitation wavelength 488 nm, emission wavelength 525 nm).

Cell culture

H9C2 cells were purchased from CTCC (Chinese Academy of Sciences; Shanghai). A compound media that consisted of DMEM, 10% FBS, and antibiotics was utilized for cell culture experiments. The cells were placed in an incubator at 37˚C in the presence of 5% CO₂ and 95% air.

Cell model of hypoxia/reoxygenation (H/R)

To mimic the MIRI, an in vitro H9C2 cell H/R model was established. Briefly, the cells were subjected to a 5 h time period of hypoxia and a 20 h time period of reoxygenation. During the hypoxic conditions, the cells were incubated with DPBS, whereas during the reoxygenation, the cells were incubated with regular compound media.

Cell transfection

GRP75 small interfering RNA (siRNA) (sense: 5’-UGAAACAAAUCUAAAAGCCUC-3’; antisense: 5’-GGCUUUUAGAUUUGUUUCAAG-3’), CaM siRNA (sense:5’-UGAAUUCAGCAAUCUGUUCUU-3’; antisense: 5’-GAACAGAUUGCUGAAUUCAAG-3’), and negative control (NC) siRNA(.

sense: 5′-GAA UUA AUU AAA GAU GGC CCG UUG UAC U-3′; anti-.

sense: 5′-UCA UCG AAG UUA UAG GGA UAC AUU ACG UGA UC-3′) were utilized for functional analysis of GRP75 and CaM in cardiomyocytes. Initially, a transfection solution that consisted of GRP75, CaM, or NC siRNAs, and Lipofectamine 2000 (Ruibo, China) was prepared. Subsequently, H9C2 cells were treated with the transfection solution so as to achieve a final concentration of 100 nM GRP75 or CaM siRNA and further incubated for an additional 24 h. Finally, the cells were collected for subsequent analysis, such as apoptosis.

Measurement of calcium ion (Ca²⁺) levels

The Rhod-2 kit was used for myocardial cell mitochondrial Ca²⁺ level measurement. Briefly, a Rhod-2 working solution was initially prepared according to the manufacture’s instruction and the cells were incubated with this working solution for 1 h at 37˚C. Finally, the absorbance value of was measured at 549/578 nm (excitation/emission) and the Ca²⁺ content was calculated.

Terminal Deoxynucleotidyl transferase dUTP Nick end labeling (TUNEL)/Hoechst double-labeling

TUNEL/Hoechst double-labeling was used for myocardial cell apoptosis measurements according to a previously published method by Mao et al.²⁸. Briefly, the heart was initially cut into sections of 5 μm thickness and the heart section was fixed with 4% w/v formaldehyde and washed with PBS. Subsequently, the sections were immersed into buffer in the presence of deoxynucleotide transferase, rinsed to remove the residual reaction solution, and incubated with Hoechst 33,342 for 5 min. Finally, the sections were captured at x200 magnification utilizing an epifluorescence microscope (Nikon). The cells were prepared in a similar way as the tissues. The number of TUNEL-positive cells was calculated by the ratio of TUNEL positive cell to total cells (nuclei number plus Tunnel positive cells).

Immunofluorescence staining

The co-staining of microtubule-associated protein 1 A/1B-light chain 3 (LC3) and the mitochondria was conducted using an immunofluorescence assay as reported Niu et al.²⁹. Briefly, the cells were subjected to the following steps: Fixation with 4% paraformaldehyde for 30 min, permeabilization with 0.1% Triton X-100 for 10 min, washing with PBS thrice for 10 min, incubation with a blocking buffer at 37 °C for 1 h, incubation with an LC3B antibody at 4 °C for 16 h, washing with PBS, incubation with a secondary antibody at 4 °C for 1 h in the dark, washing with PBS, and a final incubation with MitoTracker Green (a mitochondria fluorescence probe) at 37 °C in the dark for 30 min. Finally, the co-localization of LC3B and mitochondria was observed using fluorescence microscopy.

Reverse transcription real-time PCR

TRIzol™ reagent (Takara) was utilized for total RNA extaction. A NanoDrop One spectrophotometer (Thermo Fisher) was used for the measurement of RNA purity and concentration. A reverse transcription assay was established according the manufacture’s instruction to obtain the cDNA. The amplification reaction was established according to a SYBR kit (Takara) and the amplification was carried out using the ABI 7500 plus system (Applied Biosystems). The 2-ΔΔCq method was used for data analysis³⁰. The following primers were used: GRP75 forward (F): 5’-UGAAACAAAUCUAAAAGCCUC-3’, reverse (R): 5’-GGCUUUUAGAUUUGUUUCAAG-3’;CaM F: 5’-GCTCGCACCATGGCTGAT-3’, R: 5’-CCTTTGTTGTGATGGTGCCG-3’; autophagy-regulating protease 4 (ATG4) F: 5’-AAGGCTCATGGGAGTGTTCTC-3’, R: 5’-CTCCATGGATAGTGCAGCGG-3’;LC3F: 5’-GCGTGTGTAACATATTTAGAGGGG-3’, R: 5’-AGTTGCTCAGGTATTCGCCG-3’; β-actin F, 5’CCCATCTATGAGGGTTACGC-3’ and R, 5’-TTTAATGTCACGCACGATTTC-3’.

Western blotting

The total protein was extracted using a cell lysis buffer. The bicinchoninic acid protein concentration measurement kit was used for measurement of the total protein concentration. The protein was diluted with 5x loading buffer and denatured at 99℃. For western blot analysis, the protein was separated with SDS-PAGE and the target protein was isolated and blotted to a polyvinylidene fluoride (PVDF) membrane. Subsequently, these PVDF membranes containing proteins were incubated with primary and secondary antibodies successively. The primary antibodies of GRP75, IP3R3, VDAC1, ATG4, LC3, CaM, and β-actin were purchased from Santa Cruz Biotechnology, Inc. For primary antibody incubation, the membrane was maintained at 4℃ for 16 h in the dark. For secondary antibody incubation, the membranes were maintained at 25℃ for 2 h. All antibodies used in this study were diluted at a 1:1,000. Finally, the band signals were detected using a chemiluminescence imaging system (ChemiDox XRS). The results were normalized using β-actin as the loading control.

Statistical analysis

The data are expressed as mean ± SD and analyzed utilizing SPSS version 22. Three independent experiments were carried out. The Student’s t-test and analysis of variance tests were used to assess statistical differences between the two and more than two groups, respectively. One-way ANOVA was performed for comparisons between multiple groups, followed by a post-hoc test (Tukey’s). P < 0.05 was considered to indicate a statistically significant difference.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Bi-Ying Liu, Li Mao and Zhong-Bao Yang worte the main manuscript text and Bi-Ying Liu, Li Mao, Zhao-Hui Dai, and Ling-Zhi Guo prepared figures1-figure6. All authors reviewed the manuscript.

Funding

This work was supported by Hunan Provincial Natural Science Foundation of China (No. 2023JJ30431 to Zhong-BaoYang; No.2022JJ70044 to Zhao-Hui Dai).

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was approved by the Ethics Committee of the Affiliated Changsha Hospital of Hunan Normal University.