Panax Notoginseng Saponins Protect Cardiac Myocytes Against Endoplasmic Reticulum Stress and Associated Apoptosis Through Mediation of Intracellular Calcium Homeostasis.

Jun Chen,Xueyang Bai,Gangqiong Liu,Wenjing Zhang,Li Li,Rui Xue,Li Li Xiao,Ling Li,Jiahong Shangguan

doi:10.3389/fphar.2019.01013

Jun Chen, Xueyang Bai + Show 7 more

Open Access

https://doi.org/10.3389/fphar.2019.01013

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Abstract

Endoplasmic reticulum (ER) stress has been demonstrated to play important roles in the pathogenesis of various cardiovascular diseases. The ER stress pathway is therefore a promising therapeutic target in cardiovascular disease. Although Panax notoginseng saponins (PNS) are one of the patent medicines that are traditionally used to treat cardiovascular disorders, their effects on ER stress in cardiac myocytes remain unexploited so far. This study investigates the effects of PNS on ER stress and its associated cell apoptosis along with the related mechanism in cardiac myocytes. PNS compounds were identified via high-performance liquid chromatograph (HPLC) assay. PNS-pretreated H9c2 cells, HL-1 cells, and primary cultured neonatal rat cardiomyocytes were stimulated with thapsigargin (TG) to induce ER stress response and apoptosis. ER stress response was tested by immunofluorescence or immunoblot of the ER protein chaperones—calnexin, binding immunoglobulin protein (BiP) and the C/EBP homologous protein (CHOP). Cell viability was tested by methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis was detected by immunoblot of Cleaved caspase-3 and flow cytometry analysis of Annexin V/propidium iodide (PI) staining. Cytosolic, mitochondrial, and ER calcium dynamics were investigated by calcium imaging. Moreover, a ryanodine receptor type-2 (RyR2) overexpression stable cell line was generated to verify the mechanism of RyR2 involved in PNS in the inhibition of ER stress and cell apoptosis. We demonstrate here that PNS protected cardiac myocytes from ER stress response and associated cell death in a concentration-dependent manner. Importantly, PNS reduced the elevation of cytosolic calcium, mitochondria calcium, as well as ER calcium in response to either TG or histamine treatment. PNS protection in ER stress was regulated by RyR2 expression. In summary, PNS protection against TG-induced ER stress response and its associated cell apoptosis in cardiac myocytes is calcium dependent. Through the regulation of ER calcium release mediated by RyR2, a novel mechanism for PNS in the prevention of cardiovascular diseases is thereby identified.

Highlights

The endoplasmic reticulum (ER) is a multifunctional organelle essential for the synthesis, folding, and processing of secretory and transmembrane proteins
We mainly examined the effects of Panax notoginseng saponins (PNS) on TG-induced alternations of Endoplasmic reticulum (ER) network morphology, expression of unfolded protein response (UPR)-involved proteins chaperone binding immunoglobulin protein (BiP, known as the glucose-regulated protein 78/ Grp78) and the C/EBP homologous protein (CHOP, known as growth arrest- and DNA damage-inducible gene 153/GADD153), cell viability, expression of apoptotic gene caspase-3, as well as the intracellular calcium homeostasis and associated calcium handling proteins
Annexin V/propidium iodide (PI) doublestaining analyses by flow cytometry showed that PNS pretreatment significantly reduced the number of Annexin V/PI double-positive cells induced by TG stimulation in non-targeted control (NTC) HL-1 cells, while having no protection effects on ryanodine receptor type-2 (RyR2)-multifunctional GFP (mfGFP) stably transfected HL-1 cells (Figure 6D). These results suggest that PNS protection against ER stress and its associated apoptosis is through RyR2-mediated ER Ca2+ release

Summary

Introduction

The endoplasmic reticulum (ER) is a multifunctional organelle essential for the synthesis, folding, and processing of secretory and transmembrane proteins. Pathological stimuli that disrupt the ER homeostasis resulting in an accumulation of misfolded and unfolded proteins are known as ER stress. ER stress evokes a protective and compensatory mechanism referred to as the unfolded protein response (UPR), which serves multiple functions, including the assistance of protein folding via the upregulated ER protein chaperones and the enhanced degradation of misfolded proteins via the upregulation of molecules involved in the ER-associated protein degradation (ERAD) pathway (Brewer et al, 1997; Friedlander et al, 2000; Hampton, 2000). TG disturbs the calcium homeostasis and leads to protein misfolding, causing the accumulated misfolded/ unfolded proteins to induce ER stress. Prolonged TG treatment initiates the intrinsic apoptotic pathway by permeabilizing the mitochondrial membrane, releasing cytochrome c and apoptosis inducing factor (AIF) to cytosol, resulting in apoptosome formation, and leading to the activation of caspase-3 (Rao et al, 2002)

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