The PKC/CPI-17/MLC2 Signaling Pathway Is Involved In Molecular Mechanism Studies Of Coronary Artery Spasm

Objective: In forensic cases,we often encounter cases of rapid death after stress factors such as quarrel,cold,and minor trauma.Some cases have no obvious fatal disease or injury after systematic autopsy,and the cause of death is often identified as unexplained cause or sudden cardiac death.Making the case protracted,resulting in adverse social effects.Coronary artery spasm(CAS)refers to a reversible hypercontractile response of local or diffuse coronary arteries to various stimuli,resulting in partial or total occlusion of normal or atherosclerotic coronary arteries,which can lead to myocardial ischemia,myocardial infarction and even sudden death.It is suggested that it may be one of the important mechanisms leading to cardiac death without obvious cardiac diseases.CAS can also cause acute arrhythmia in patients with coronary heart disease and lead to death.Whether stress factors such as minor trauma lead to rapid death by causing CAS remains to be confirmed.Therefore,revealing the pathological molecular mechanism of CAS death is an urgent scientific and technical problem in forensic pathology.Studies have confirmed that the contractile hyperresponsiveness of vascular smooth muscle cells is the main mechanism of vascular hyperconstriction caused by CAS.The phosphorylation of Myosin II regulatory light chain(MLC2)is critical for the contractile response of local vascular smooth muscle.The efficiency of smooth muscle contraction depends on the sensitivity of contractile proteins to calcium.Protein Kinase C-Potentiated Phosphatase Inhibitor 17(CPI-17)is an important molecule that regulates calcium sensitivity.CPI-17 was initially identified as a PKC phosphorylation substrate,and then it was found that Rho Kinase,Proteinkinase N(PKN),Integrin-Linked kinase(ILK),etc.,could also phosphorylate CPI-17.It is suggested that CPI-17 may be a common key hub for multiple intracellular kinases to regulate Myosin light chain phosphatase(MLCP)-induced CAS.PKC plays a crucial role in vasoconstriction by regulating downstream signals,such as Endothelin-1(ET-1),mitogen-activated protein kinase(MAPK),CPI-17 and so on.However,it is unclear whether PKC regulates CPI-17/MLC2 signaling to play a role in CAS.Therefore,this project intends to establish a spasm injury model from the overall animal level and cell level.To investigate the mechanism of CAS induced by PKC/CPI-17/MLC2 pathway,and to explore the changes of key factors in the pathway leading to cardiac death.Screening reliable markers of CAS may provide new ideas for clinical treatment and reduction of mortality.Methods:1.At the global animal level:(1)Establishment of coronary artery spasm rat model;(2)The experiments were divided into Control group and CAS group.(3)HE staining was used to observe the folding of the coronary elastic membrane and the contractile band of coronary smooth muscle.(4)The ultrastructure of myocardium was observed by transmission electron microscope.(5)The levels of myocardial enzymes were detected by mechanical biochemistry.(6)The expression of PKC/CPI-17/MLC2 pathway-related proteins p-CPI-17(Thr38),CPI-17,p-MLC2(Ser19)and MLC was detected by Western blot,immunohistochemical staining and immunohistochemical fluorescence staining.2.At the cellular level:(1)Human coronary artery smooth muscle cells(h CASMCs)were cultured in vitro,and the vasoconstrictor(angiotensin,Ang II)was added to the medium at different doses(0,0.05,0.1,0.5,0.1 u M/m L)or at different times(0,5,15,30,60 min).The expression of p-MLC2(Ser19)was observed to select the optimal Ang II concentration and time for cell contraction.(2)The cells were divided into Veh group,Calphostin C group,GF-109203 X group,Ang II group,Ang II+GF-109203 X group and Ang II+ Calphostin C group.(3)The expression of PKCα,PKCδ,PKCε,p-CPI-17(Thr38),CPI-17,p-MLC2(Ser19),and MLC in PKC/CPI-17 pathway was detected by immunofluorescence staining.(4)PKC inhibitor(GF-109203 X,5 μmol/L;Calphostin C,3 μmol/L)and h CASMCs in the control group were cultured under the same conditions for the same time.Laser confocal immunofluorescence was used to observe the changes in the arrangement of myofilaments in each group.The expressions of PKCα,PKCδ,PKCε,p-CPI-17(Thr38),CPI-17,p-MLC2(Ser19)and MLC2 were detected by immunocytofluorescence staining.Results:1.Findings at the global animal level(1)The results of HE staining showed that the coronary smooth muscle cells of CAS group were vacuolated,and the coronary intimal folding was increased compared with the control group.(2)The results of transmission electron microscopy showed that compared with the control group,the CAS group had swollen mitochondria,damaged membrane,shallow and dissolved matrix,broken and shortened cristae.The sarcoplasmic reticulum was markedly dilated and vacuolated.(3)There was no significant difference in serum creatine kinase(CK),creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH)and lactate dehydrogenase isoenzyme(LDH1)between the CAS rats treated with pituitrin for 2 min and the control rats;The levels of CK,CK-MB,LDH and LDH1 in the serum of CAS rats were increased in the 10 min and 30 min groups injected with pituitrin through the tail vein.(4)Immunohistochemical staining,immunohistochemical fluorescence staining and Western blot showed that the phosphorylation levels of p-CPI-17(Thr38)and p-MLC2(Ser19)in CAS group were significantly higher than those in control group.2.Findings at the cellular level(1)h CASMCs were cultured in vitro,and the expression of smooth muscle cell marker protein(a-SMA)was detected by cell immunofluorescence staining.This cell line basically has the morphological,phenotypic and functional characteristics of primary cultured coronary smooth muscle cells.(2)Immunocytochemical staining results showed that: During Ang II treatment,h CASMCs showed an increase in the phosphorylation level of MLC2,which reached the highest level at a dose of 0.1 μM/ml for 30 min.Therefore,Ang II at 0.1 μM/ml for 30 min was selected for subsequent experiments.(3)Immunocytochemical staining showed that PKCα translocated from the cytoplasm to the cell membrane in the Ang II group compared with the control group.However,PKCδ and PKCε were also expressed in the cytoplasm of h CASMCs with no significant difference compared with the control group.(4)Immunocytochemical staining results showed that: PKCα was mainly located in the cytoplasm in the Veh group,Calphostin C group,GF-109203 X group and Ang II+ Calphostin C group,while PKCα was translocated from the cytoplasm to the cell membrane in the Ang II group and Ang II+GF-109203 X group.(5)Immunocytochemical staining showed that compared with the Veh group,the expression of p-CPI-17(Thr38)and p-MLC2(Ser19)protein in the Calphostin C and GF-109203 X groups had no significant difference.Compared with the Veh group,the positive expression of p-CPI-17(Thr38)and p-MLC2(Ser19)protein in the Ang II group was significantly increased.Compared with the Ang II group,the positive expression of p-CPI-17(Thr38)and p-MLC2(Ser19)protein was decreased in the Ang II+GF-109203 X and Ang II+Calphostin C groups.Conclusions:At the global animal level,the expression of p-CPI-17(Thr38)and pMLC2(Ser19)in coronary arteries was increased in the coronary artery spasm group.At the cellular level,PKCα was displaced,and p-CPI-17(Thr38)and pMLC2(Ser19)were increased in h CASMCs of Ang II group,suggesting that PKC/CPI-17 signaling pathway is involved in the occurrence of coronary artery spasm.p-CPI-17(Thr38)and p-MLC2(Ser19)may be used as molecular markers for the diagnosis of CAS before death.