Overexpression of SERCA2a Alleviates Cardiac Microvascular Ischemic Injury by Suppressing Mfn2-Mediated ER/Mitochondrial Calcium Tethering

Abstract

Our previous research has shown that type-2a Sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) undergoes posttranscriptional oxidative modifications in cardiac microvascular endothelial cells (CMECs) in the context of excessive cardiac oxidative injury. However, whether SERCA2a inactivity induces cytosolic Ca²⁺ imbalance in mitochondrial homeostasis is far from clear. Mitofusin2 (Mfn2) is well known as an important protein involved in endoplasmic reticulum (ER)/mitochondrial Ca²⁺ tethering and the regulation of mitochondrial quality. Therefore, the aim of our study was to elucidate the specific mechanism of SERCA2a-mediated Ca²⁺ overload in the mitochondria via Mfn2 tethering and the survival rate of the heart under conditions of cardiac microvascular ischemic injury. In vitro, CMECs extracted from mice were subjected to 6 h of hypoxic injury to mimic ischemic heart injury. C57-WT and Mfn2^KO mice were subjected to a 1 h ischemia procedure via ligation of the left anterior descending branch to establish an in vivo cardiac ischemic injury model. TTC staining, immunohistochemistry and echocardiography were used to assess the myocardial infarct size, microvascular damage, and heart function. In vitro, ischemic injury induced irreversible oxidative modification of SERCA2a, including sulfonylation at cysteine 674 and nitration at tyrosine 294/295, and inactivation of SERCA2a, which initiated calcium overload. In addition, ischemic injury-triggered [Ca²⁺]c overload and subsequent [Ca²⁺]m overload led to mPTP opening and ΔΨm dissipation compared with the control. Furthermore, ablation of Mfn2 alleviated SERCA2a-induced mitochondrial calcium overload and subsequent mito-apoptosis in the context of CMEC hypoxic injury. In vivo, compared with that in wild-type mice, the myocardial infarct size in Mfn2^KO mice was significantly decreased. In addition, the findings revealed that Mfn2^KO mice had better heart contractile function, decreased myocardial infarction indicators, and improved mitochondrial morphology. Taken together, the results of our study suggested that SERCA2a-dependent [Ca²⁺]c overload led to mitochondrial dysfunction and activation of Mfn2-mediated [Ca²⁺]m overload. Overexpression of SERCA2a or ablation of Mfn2 expression mitigated mitochondrial morphological and functional damage by modifying the SERCA2a/Ca²⁺-Mfn2 pathway. Overall, these pathways are promising therapeutic targets for acute cardiac microvascular ischemic injury.

Keywords: CMEC; Mfn2; SERCA2a; hypoxia; ischemia injury; mitochondria.

FIGURE 1

Hypoxic injury-induced oxidative modification of sarco/endoplasmic reticulum CSERCA2a and SERCA2a-induced Ca²⁺ imbalance (n = 3/group). (A,B) A calcium map was used to quantitatively analyze the change in [Ca²⁺]c (cytoplasmic calcium) in the different groups. (C–E) TUNEL assays and DCFH-DA fluorescence were used to assess cell survival. (F–I) ELISA results showing the changes in MDA, GPX, and SOD levels and the activity of SERCA2a after hypoxic injury in CMECs. (J–M) The relative expression levels of SERCA2a with nitrotyrosine and SERCA2a with sulfonylated cysteine were assessed by coimmunoprecipitation assays. *p < 0.05 compared with the Ad-Ctrl; ^#p < 0.05 compared with the Hypo(Ad-Ctrl).

FIGURE 2

Excessive [Ca²⁺]m was followed by SERCA2a-dependent [Ca²⁺]c overload and mitochondrial dysfunction (n = 3/group). (A–D) The [Ca²⁺]m probe Fluo-3AM and the [Ca²⁺]c probe Rhod2 were detected by flow cytometry to assess changes in Ca²⁺ concentration in the different groups. (E–H) The change in membrane potential (ΔΨm) was detected by JC-1 staining. The arbitrary mitochondrial permeability transition pore (mPTP) opening time was determined as the time when the TMRE fluorescence intensity decreased by half between the initial and final fluorescence intensities. *p < 0.05 compared with the Ad-Ctrl; ^#p < 0.05 compared with the Hypo(Ad-Ctrl).

FIGURE 3

SERCA2a-dependent [Ca²⁺]c overload activated mitofusin 2 (Mfn2)-mediated [Ca²⁺]m overload and was accompanied by increased Parkin/PINK-dependent mitophagy (n = 3/group). (A,B) Western blot analysis was performed to detect the expression of Mfn2. Hypoxia and pretreatment with tBHQ increased the expression of Mfn2, while overexpression of SERCA2a, pretreatment with BAPTA and knockdown of the Mfn2 gene reversed the Mfn2 expression level. (C,D) Coimmunofluorescence of SERCA2a and Mfn2 in cardiac microvascular endothelial cells (CMECs). The overlap of fluorescence intensity changed in the different groups and was consistent with the changes observed in WB. (E,F) The [Ca²⁺]c probe (Fluo-3) and [Ca²⁺]m probe (Rhod2) were used to observe changes in Ca²⁺ concentration via fluorescence. (G,H) Western blot analysis was performed to detect Parkin/PINK-dependent mitophagy- and mito-apoptosis-related protein expression in the different groups. (I) Electron microscopy was used to observe autophagosome formation. *p < 0.05 compared with the Ad-Ctrl; ^#p < 0.05 compared with the Hypo(Ad-Ctrl).

FIGURE 4

SERCA2a-dependent [Ca²⁺]c overload triggered Mfn2-mediated [Ca²⁺]m overload and was accompanied by increased Drp-1-dependent mitochondrial fission (n = 3/group). (A,B) A Mito-ROS probe (MitoSOX) was used to compare the mito-oxidative stress levels between the different groups. (C–E) ELISA and tests on extracted mitochondria were performed to detect the function of mitochondria in the different groups. (F,G) Coimmunofluorescence of mitochondria and lysosomes in CMECs. The overlap of fluorescence intensity changed in the different groups to reveal the morphology of mitochondria and the function of lysosomes. (H,I) Western blot analysis was performed to detect Drp-1- and Fis-1-dependent mito-fission expression in different groups. *p < 0.05 compared with the Ad-Ctrl; ^#p < 0.05 compared with the Hypo(Ad-Ctrl); ^&p < 0.05 compared with the Hypo(Ad-SERCA).

FIGURE 5

Knockdown of Mfn2 decreased heart infarction under cardiac ischemic injury conditions (n = 3/group). (A,B) Representative images of heart sections with TTC and Evans Blue staining of the infarcted area. The bar graph indicates the infarct size (ischemia group 42.3 ± 4.5% vs. Mfn2^KO group 21.9 ± 2.4%, p < 0.05). (C). qPCR was used to test the efficiency of Mfn2 knockout in the Mfn2^KO mice. (D,E) The ischemic injury group presented more TUNEL-positive cells than the sham group. (F,J) Western blot analysis was performed to detect apoptosis-related protein expression in different groups. (G–I) ELISA analysis found significantly higher levels of LDH, troponin T, and CK-MB after ischemic injury, while the Mfn2^KO group showed some reversal of these effects. (K–N) Representative M-mode echocardiograms were performed after 1 h of heart ischemic injury in each group, and quantitative analysis of the cardiac data derived from echocardiograms was performed. *p < 0.05 compared with the Sham(WT); ^#p < 0.05 compared with the Ische(WT).

FIGURE 6

Knockdown of Mfn2 attenuated cardiac microcirculatory ischemic injury (n = 3/group). (A) Electron microscopy revealed fragmented interfibrillar mitochondria. (B) HE staining for red blood cell morphology and arrangement among heart microcirculation in the different groups. (C–E) Immunohistochemistry of CD68 and eNOS expression in endothelial cells was performed to assess the integrity of microvessels in the different groups. (F–H) Blood was collected after cardiac ischemic injury, and the concentrations of inflammatory factors in the different groups were analyzed using MCP-1, TNF, and IL-6 ELISAs. *p < 0.05 compared with the Sham(WT); ^#p < 0.05 compared with the Ische(WT).

FIGURE 7

Schematic of the overall mechanism of heart ischemic injury in CMECs.