Melatonin protected cardiac microvascular endothelial cells against oxidative stress injury via suppression of IP3R-[Ca2+]c/VDAC-[Ca2+]m axis by activation of MAPK/ERK signaling pathway

Abstract

The cardiac microvascular reperfusion injury is characterized by the microvascular endothelial cells (CMECs) oxidative damage which is responsible for the progression of cardiac dysfunction. However, few strategies are available to reverse such pathologies. This study aimed to explore the mechanism by which oxidative stress induced CMECs death and the beneficial actions of melatonin on CMECs survival, with a special focused on IP3R-[Ca²⁺]c/VDAC-[Ca²⁺]m damage axis and the MAPK/ERK survival signaling. We found that oxidative stress induced by H₂O₂ significantly activated cAMP response element binding protein (CREB) that enhanced IP3R and VDAC transcription and expression, leading to [Ca²⁺]c and [Ca²⁺]m overload. High concentration of [Ca²⁺]m suppressed ΔΨm, opened mPTP, and released cyt-c into cytoplasm where it activated mitochondria-dependent death pathway. However, melatonin could protect CMECs against oxidative stress injury via stimulation of MAPK/ERK that inactivated CREB and therefore blocked IP3R/VDAC upregulation and [Ca²⁺]c/[Ca²⁺]m overload, sustaining mitochondrial structural and function integrity and ultimately blockading mitochondrial-mediated cellular death. In summary, these findings confirmed the mechanisms by which oxidative injury induced CMECs mitochondrial-involved death and provided an attractive and effective way to enhance CMECs survival.

Keywords: Apoptosis; Calcium overload; Endothelial; IP3R; Melatonin; Reperfusion injury; VDAC.

Fig. 1

Characterization of CMECs. a Isolated ADMSCs displayed f a spindle shape and cobblestone-like morphology. b, c Immunofluorescence results showed that CMECs were uniformly positive for CD31 and could absorb DiL-Ac-LDL. Bar = 50 μm

Fig. 2

Effect of melatonin on cell viability and proliferation. a Melatonin had no cytotoxic effect on CMECs. b The growth curve of CMECs under different concentrations of melatonin. *P < 0.05 vs. control group

Fig. 3

Melatonin protected CMECs against oxidative stress injury. a, b Cell death was determined by TUNEL assay. a Annexin V/PI assay. b The change of caspase activity under different doses of melatonin. c The LDH release assay indicated that melatonin could reduce CMECs oxidative stress damage. Blue are cell nuclei; *P < 0.05 vs. control group, #P < 0.05 vs. H₂O₂ group

Fig. 4

Melatonin reduce CMECs death through alleviation of IP3R-dependent [Ca²⁺]c overload. a The co-immunofluorescence of [Ca²⁺]c and PI. The higher [Ca²⁺]c was indicative of more PI⁺ death cell. BAPTA, a calcium chelator that reduce the levels of intracellular Ca²⁺. Ion, ionomycin, a calcium agonist that induced [Ca²⁺]c overload. b, c The expression of IP3R and RyR. Oxidative mainly increased IP3R expression that was blocked by melatonin. d Flow cytometry was used to quantitatively detect the change of [Ca²⁺]c under melatonin treatment. Meanwhile, siRNA knockdown of IP3R could alleviate the contents of [Ca²⁺]c in response to oxidative stress. *P < 0.05 vs. control group, #P < 0.05 vs. H₂O₂ group, and P < 0.05 vs. H₂O₂ + melatonin group

Fig. 5

IP3R-dependent [Ca²⁺]c overload activated VDAC-mediated [Ca²⁺]m overload. a The co-immunofluorescence of [Ca²⁺]c and [Ca²⁺]m. Higher [Ca²⁺]c was associated with [Ca²⁺] through VDAC because siRNA knockdown of VDAC could significantly abate the increase in [Ca²⁺]m under oxidative stress. b, c The [Ca²⁺]m map via confocal microscopy by Rhod-2. Fluorescence intensity of Rhod-2 was measured by excitation wavelengths of 550 nm and emission wavelengths of 570 nm, respectively. Data (F/F0) were obtained by dividing fluorescence intensity (F) by (F0) at resting level (t = 0) which was normalized by control groups. *P < 0.05 vs. control group, #P < 0.05 vs. H₂O₂ group

Fig. 6

[Ca²⁺]m overload aggregated mitochondrial damage and triggered the mitochondria-related cell death pathways. a The re-location of cyt-c from mitochondria into cytoplasm suggesting the activation of mitochondria-related cell death pathways. b, c The change of mitochondrial membrane potential (ΔΨm). d Melatonin could reduce the mPTP opening. P < 0.05 vs. control group, #P < 0.05 vs. H₂O₂ group

Fig. 7

Melatonin signaled MAPK/ERK pathways to inactivate CREB transcription promoter which reduces the expression of IP3R and VDAC under H₂O₂. a The activation of melatonin on MAPK/ERK. b MAPK/ERK was responsible for the inhibitory role of melatonin on IP3R and VDAC. c Melatonin could reduce the expression of CREB and its activation. SGC-CBP30, the specific inhibitor of CREB pathways. d, e The transcription promoter CREB was the downstream of ERK, which was activated by oxidative stress and contributed to the elevation of IP3R and VDAC. Melatonin impaired the activation of CREB and therefore reduced the IP3R and VDAC. *P < 0.05 vs. control group, #P < 0.05 vs. H₂O₂ group