Effect of Yixinshu Capsules on calcium regulatory protein in cardiac myocytes of rats with heart failure
(2.Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China 100700)
【Abstract】To explore the molecular mechanism of Yixinshu Capsules (YXS) in restoring cardiac function in rats with heart failure (HF) from the perspective of calmodulin in cardiac myocytes in the context of the therapeutic effect of YXS on HF. The SD rats were subjected to the ligation of the anterior descending branch of the left coronary artery for four weeks to establish myocardial ischemia-induced HF animal model. The rats were randomly assigned into a sham operation group (Sham, saline), a model group (HF, saline), a high dose YXS group (HF + YXS-H, 1 600 mg·kg−1·d−1), a low dose YXS group (HF + YXS-L, 800 mg·kg−1·d−1), and positive drug valsartan group (HF + VST, 8 mg·kg−1·d−1). After continuous intragastric administration for six weeks, the rats were sacrificed and myocardial tissues were collected. Real time quantitative polymerase chain reaction (RT-PCR) and Western blot were used to detect the expression of genes and proteins related to calcium regulation in cardiac myocytes. Results showed that compared with the model group, YXS increased the transcription levels of Atp2a2, Ryr2, CACNA1C, and PRKACA, and increased the expression levels of p-Ryr2, CACNA1C, and SERCA2a, while decreased the level of NCX1. On the other hand, YXS medication significantly decreased the RIP3 level and the phosphorylation of its substrate CaMKⅡ protein, and enhanced the phosphorylation expression of PLB. In summary, YXS medication could regulate the expression of genes and proteins related to calcium regulation in cardiac myocytes, inhibit RIP3 and the phosphorylation of CaMKⅡ protein, promote the phosphorylation of PLB at Ser16, and increase the expression of SERCA2a protein, suggesting that YXS may regulate myocardial calcium homeostasis through CaMKⅡ/PLB/SERCA2a pathway to improve the ability of calcium uptake in sarcoplasmic reticulum and stabilize intracellular free Ca2+, so as to improve the cardiac function in rats with HF. This study revealed the possible mechanism of YXS in the treatment of HF, especially from the intervention of calmodulin, promoting the clinical application of YXS.
【Keywords】 heart failure; Yixinshu Capsules; calcium/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ); sarcoplasmic reticulum calcium adenodine triphosphatase 2a (SERCA2a);
 LONG W J, YANG Z Q, YANG Y. 肌浆网钙离子泵治疗心衰的研究进展 [J]. Chinese Journal of Heart and Heart Rhythm, 2016, 4 (1): 50 (in Chinese).
 MATHERS C D, LONCAR D. Projections of global mortality and burden of disease from 2002 to 2030 [J]. PLoS Med, 2006, 3 (11): e442.
 WU R G M L, BAO B R B Y L, TONG Y Q. 蒙医药防治心肌缺血损伤机制研究进展 [J]. World Latest Medicine Information, 2018, 18 (4): 39 (in Chinese).
 MA R Q, YANG Z Q, KOU J P, et al. Analysis on new research and development ideas and technical points of traditional Chinese medicine for prevention and treatment of chronic heart failure [J]. China Journal of Chinese Materia Medica, 2020, 45 (11): 2720 (in Chinese).
 LI H Y, ZHAO S H, WANG S H, et al. Research progress of SERCA2a in heart failure [J]. Chinese Pharmacological Bulletin, 2020, 36 (2): 171 (in Chinese).
 LV X, LIU Y, WANG L, et al. Effect of overexpression of β2 receptor on SERCA2a/PLB and its molecular mechanism in cardiac muscle cells of rats with heart failure [J]. Jiangsu Science & Technology Information, 2016 (30): 42 (in Chinese).
 LIU Y M, JIANG M X. The role of phospholamban in heart failure [J]. Chinese Journal of Clinical Medicine, 2010, 17 (2): 164 (in Chinese).
 MARKS A R. Calcium cycling proteins and heart failure: mechanisms and therapeutics [J]. J Clin Invest, 2013, 123 (1): 46.
 MA Y Y, ZHANG M, MA S H, et al. New idea of treatment of heart failure with traditional Chinese medical [J]. China Journal of Chinese Materia Medica, 2011, 36 (22): 3210 (in Chinese).
 SHI M S, QU F Z. Sarcoplasmic reticulum calcium adenodine triphosphatase 2a and heart failure [J]. Medical Recapitulate, 2011, 17 (11): 1632 (in Chinese).
 SANDE J B, Sjaastad I, HOEN I B, et al. Reduced level of se-rine 16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity [J]. Cardiovasc Res, 2002, 53 (2): 382.
 QU F Z. 辛伐他汀对心力衰竭家兔肌浆网钙调控相关蛋白基因的影响 [J]. Chinese Journal of Multiple Organ Diseases in the Elderly, 2009, 8 (5): 458 (in Chinese).
 MCKINSEY T A. Derepression of pathological cardiac genes by members of the CaM kinase superfamily [J]. Cardiovasc Res, 2007, 73 (4): 667.
 ZHANG J J, GUO F F, WU H W, et al. Yixin-Shu facilitated cardiac-like differentiation of mesenchymal stem cells in vitro [J]. RSC Adv, 2018, 8 (18): 10032.
 Chinese Pharmacopoeia Commission. Chinese Pharmacopoeia. Vol. One [S]. Beijing: China Medical Science Press, 2015: 1397 (in Chinese).
 TANG H X, DONG L L, WANG X T, et al. Analysis of clinical efficacy of Yixinshu Capsule in the Treatment of Diabetes Ⅱ Mellitus Combined with Coronary Heart Disease Angina Pectoris [J]. World Chinese Medicine, 2017, 12 (8): 1804 (in Chinese).
 LIU J W, LIU X Y, LI J H, et al. Protective effect of Yixinshu capsule on myocardial ischemia reperfusion injury in rats [J]. China Journal of Chinese Materia Medica, 2013, 38 (12): 2005 (in Chinese).
 ZHANG J J, GENG Y, GUO F F, et al. Screening and identification of critical transcription factors involved in the protection of cardiomyocytes against hydrogen peroxide-induced damage by Yixin-shu [J]. Sci Rep, 2017, 7 (1): 13867.
 ZHANG M Y, WU H W, GUO F F, et al. Identification of active components in Yixinshu Capsule with protective effects against myocardial dysfunction on human induced pluripotent stem cell-derived cardiomyocytes by an integrative approach [J]. Mol Biosyst, 2017, 13 (8): 1469.
 WEI J Y, GUO F F, ZHANG M Y. Signature-oriented investigation of the efficacy of multicomponent drugs against heart failure [J]. FASEB J, 2019, 33 (2): 2187.
 LIU X G, YAO X, LI M, et al. CaMKⅡ promotes TLR-triggered proinflammatory cytokine and type Ⅰ interferon production by directly binding and activating TAK1 and IRF3 in macrophages [J]. Blood, 2008, 112 (13): 4961.
 NOMURA N, SATOH H, TERADA H, et al. CaMKⅡ-dependent reactivation of SRCA uptake and contractile recovery during intracellular acidosis [J]. J Mol Cell Cardiol, 2001, 33 (6).
 WEI Y J, SHEN Y, JIN C L. 心力衰竭时心肌收缩功能的变化、调节及干预作用 [J]. Jilin Journal of Traditional Chinese Medicine, 2013, 33 (4): 430 (in Chinese).
 LEE Y, GUSTAFSSON S B. Role of apoptosis in cardiovascular disease [J]. Apoptosis, 2009, 14 (4): 536.
 ZHAO Y, XU L, QIAO Z, et al. YiXin-Shu, a Shengmai-San-based traditional Chinese medicine formula, attenuates myocar-dial ischemia/reperfusion injury by suppressing mitochondrial mediated apoptosis and upregulating liver-X-receptor α [J]. Sci Rep, 2016, 6: 23025.
 XIAN M H, WANG T T, ZHANG W, et al. Yixinshu ameliorates hippocampus abnormality induced by heart failure via the PPARγ signaling pathway [J]. RSC Adv, 2017, 7 (84): 53415.
 ZHONG S G. Effect of rstrogen on calcium concentration in myocardial cells suffering from hypoxia/reoxygenation [J]. Journal of Northeast Normal University (Natural Science Edition), 2011, 43 (4): 118 (in Chinese).
 LI J P, GUO L L, CHEN Z, et al. Relationship between calcium overload and myocardial ischemia reperfnsion injury and intervention strategy of Chinese herbal medicine [J]. China Journal of Chinese Materia Medica, 2016, 41 (11): 2168 (in Chinese).
 MATTILA M, KOSKENVUO J, SÖDERSTRÖM M, et al. Intramyocardial injection of SERCA2a-expressing lentivirus improves myocardial function in doxorubicin-induced heart failure [J]. J Gene Med, 2016, 18 (7): 124.
 GUO D D, YU S M, ZHANG H T, et al. Effect of Suixin Capsule on cardiac function and gene expression of calcium pump in myocardial sarcoplasmic reticulum in rats with heart failure after myocardial infarction [J]. Journal of Emergency in Traditional Chinese Medicine 2019, 28 (9): 1594 (in Chinese).
 CHANG R, GE R L. Role of CaMKⅡ in modulating Ca2+homeostasis and cardioprotection [J]. Progress in Physiological Sciences, 2011, 42 (1): 55 (in Chinese).
 CHEN H H, ZHANG C, LV R. The role of RyR2 phosphorylation in heart failure [J]. Fudan University Journal of Medical Sciences, 2017, 44 (3): 359 (in Chinese).
 JIA H, LIU Y P, QU X Y, et al. SERCA2a在糖尿病心肌病中表达与调控研究进展 [J]. Journal of Cardiovascular and Pulmonary Diseases, 2018, 37 (2): 165 (in Chinese).
 REBROVA T Y, MUSLIMOVA E F, KONDRATIEVA D S, et al. The role of Ca2+-ATPase 2a (ATP2A2), ryanodine receptors (RYR2), and calsequestrin (CASQ2) gene polymorphisms in the development of heart failure [J]. Russ J Genet, 2018, 54 (6): 604.
 FABIATO A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac Purkinje cell [J]. J Gen Physiol, 1985, 85 (2): 247.
 KIRCHHOF P, FABRITZ L, KILI A, et al. Ventricular arrhythmias, increased cardiac calmodulin kinase Ⅱ expression, and altered repolarization kinetics in ANP receptor deficient mice [J]. J Mol Cell Cardiol, 2004, 36 (5): 691.
 MAIER L, BERS D.Role of Ca2+/calmodulin-dependent protein kinase (CaMK) in excitation-contraction coupling in the heart [J]. Cardiovasc Res, 2007, 73 (4): 631.
 TERRACCIANO C M N, MACLEOD K T. Overexpression of the Na/Ca exchanger and reduced SERCa function [J]. Cardiovasc Res, 2001, 50 (1): 167.
 FENG L, ANDERSON M E. CaMKⅡ is a nodal signal for multiple programmed cell death pathways in heart [J]. J Mol Cell Cardiol, 2017, 103: 102.
 GRUETER C E, ABIRIA S A, DZHURA I, et al. L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKⅡ [J]. Mol Cell, 2006, 23 (5): 641.
 TOMBES R M, FAISON M O, TURBEVILLE J M. Organization and evolution of multifunctional Ca2+/CaM-dependent protein kinase genes [J]. Gene, 2003, 322: 17.