Application Of Smooth Muscle Contraction-related Protein As A Diagnostic Marker For Coronary Spasm

Objective: In the forensic cases,we often encounter cases of rapid death after emotional stress or mild trauma during disputes.Some of them have no clear fatal diseases or injuries through system autopsy.The cause of death is often identified as unknown cause of death or sudden cardiac death.In such cases,the lack of specific pathomorphological diagnostic indicators has led to disputes in the appraisal opinions.Many re-appraisals are often initiated,resulting in cases placed for a long time and no results,producing adverse social impacts.Coronary artery spasm(CAS)is a local persistent excessive contraction of the coronary arteries.In mild cases,only angina pectoris and arrhythmia are the main manifestations;in severe cases,persistent and irreversible spasms involving multiple coronary arteries can cause myocardial infarction or sudden death.At present,it is believed that CAS may be one of the important mechanisms of sudden cardiac death(SCD)after minor trauma.It is extremely important to screen the exact and reliable forensic pathology diagnostic markers related to CAS.It is currently believed that CAS is an acute coronary hyperconstriction syndrome induced by multiple factors,and the abnormally increased excitability of vascular smooth muscle and the imbalance of the release of vasomotor factors caused by endothelial dysfunction are the reasons for the hyperresponsiveness of vasoconstriction,especially the former is more important.In recent years,studies have found that the phenotype of smooth muscle cells can affect the ability of cells to contract.Therefore,the study detects changes of proteins related with coronary artery smooth muscle cell contraction hyperresponsiveness pathway and smooth muscle cell contraction phenotype in autopsy cases with different causes of death,and explores their roles in coronary artery spasm-induced sudden cardiac death.To explore their feasibility as diagnostic markers for coronary artery spasm.Methods: In this study,54 cases from the Forensic Medical Identification Center of Hebei Medical University from 2016 to 2020 were selected and divided into three groups according to the cause of death.The inclusion criteria was that all cases were dissected within 6 days from the time of death.Among them,30 cases of coronary luminal stenosis < 50%,clear cause of death,and non-cardiac sudden death were the control group.9 cases of coronary luminal stenosis < 50%,minor trauma or strong emotional stress,exclusion of other cardiomyopathy,and death due to sudden cardiac death were the SCD group with mild coronary atherosclerosis.15 cases of coronary luminal stenosis >50%,minor trauma or strong emotional stress,and death due to the acute attack of coronary heart disease were the SCD group with severe coronary atherosclerosis.HE staining,special staining techniques Masson staining and EVG staining were used to observe the morphology of coronary arteries.At the same time,immunohistochemical staining technique was used to observe the smooth muscle cell contraction hyperresponsiveness pathway proteins p CPI-17、MLC and p MLC(S20),contractile smooth muscle cell phenotype protein α-SMA.Results processing and statistical analysis: Statistical analysis was performed using SPSS21.0 statistical software.All data are measurement data,expressed as mean ± standard deviation(Mean ± SD).For the comparison of the mean of each group,when the variances are uniform,one-way ANOVA(one-way ANOVA)is used,and the Tukey post hoc test is used to compare any two groups;when the variances are not uniform,the non-parametric Kruskal-Wallis test,and use Dunn’s multiple comparison test method to compare any two groups.All statistical results are statistically different with P<0.05.Results:1.The results of HE staining showed that: In the control group,the vascular tissue structure is complete,the endothelium is smooth、intact and continuous,the intimal thickening is not obvious,the media structure is normal,the lumen stenosis is less than 50%,and there is no sclerosis or mild sclerosis.In the SCD group with mild coronary atherosclerosis,the vascular tissue structure is relatively complete,the endothelium is smooth and still maintains its integrity and continuity,the intimal thickening degree is mild,the media structure is normal,there is no serious extrusion and thinning phenomenon,and the lumen stenosis is less than 50%,mild hardening.In the SCD group with severe coronary atherosclerosis,the vascular tissue structure is disordered,the endothelium is not smooth,its integrity and continuity are damaged,the intima is thickened with obvious atheromatous plaques,a large number of foam cells infiltration and cholesterol crystals can be seen.The media is squeezed by the thickened intimal structure,with varying thickness,localized thinning and deep staining,lumen stenosis more than 50%,and severe hardening.2.Masson staining results showed that: Masson staining can dye collagen fibers blue,muscle fibers,cellulose and red blood cells red.In the control group,collagen fibers are abundant and smooth muscle cells are intertwined with each other,and the coloring is uniform.In the SCD group with mild coronary atherosclerosis,the content of collagen fibers in the intimal plaque is more and the range is wider.In the SCD group with severe coronary atherosclerosis,the content of collagen fibers in the intimal plaque is significantly reduced,or even almost absent.The local smooth muscles in the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis showed dense and deep red staining,and the local thickness was reduced,especially in the SCD group with severe coronary atherosclerosis.3.EVG staining results showed that: EVG dyeing can dye elastic fibers into purple-black,collagen fibers into red,and the background is yellow.In the control group,the internal and external elastic membrane structure is continuous,and the dyeing is dense,the internal elastic membrane is wavy,the middle membrane is scattered with elastic fibers,and the arrangement is orderly and compact;the internal elastic membrane is broken and the outer elastic membrane is intact in the SCD group with mild coronary atherosclerosis.In the SCD group with severe coronary atherosclerosis,the internal and external elastic membrane were significantly reduced,the continuity was lost,and the dyeing became shallow,and the elastic fibers in the media layer were sparse or even missing.4.The results of immunohistochemical staining showed that:4.1The result of p CPI-17 immunohistochemistry staining: p CPI-17 is a protein related to the contraction hyperresponsiveness pathway of smooth muscle cells,which is mainly expressed in the cytoplasm of smooth muscle cells.There was no difference between the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis respectively compared with the control group.There was no difference between the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis.4.2The result of MLC immunohistochemistry staining: MLC is a smooth muscle cell contraction-related protein,which is mainly expressed in the cytoplasm of smooth muscle cells.There was no difference in the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis respectively compared with the control group.There was a significant difference between the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis(P<0.05),the average positive intensity is higher in the SCD group with mild coronary atherosclerosis.4.3The result of p MLC(S20)immunohistochemistry staining: p MLC(S20)is a smooth muscle cell contraction-related protein,mainly expressed in the cytoplasm of smooth muscle cells,and its expression intensity is positively correlated with the contraction intensity.The p MLC(S20)of the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis was higher than that of the control group,and there were significant differences compared with the control group(P<0.05);while there was no difference between the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis.4.4The result of α-SMA immunohistochemistry staining: α-SMA is smooth muscle actin,a characteristic marker protein of the contractile phenotype of smooth muscle cells,and is mainly expressed in the cytoplasm of smooth muscle cells.There was a significant difference in the SCD group with mild coronary atherosclerosis compared with the control group(P<0.05),the average positive intensity is higher in the SCD group with mild coronary atherosclerosis,while there was no significant difference in the SCD group with severe coronary atherosclerosis compared with the control group;And there was no significant difference between between the SCD group with mild coronary atherosclerosis and the SCD group with severe coronary atherosclerosis.Conclusions:1.Smooth muscle cells have a high contraction reserve capacity in mild coronary atherosclerosis,which may be the mechanism of coronary artery spasm and even cardiac death after emotional stress or minor trauma.2.Smooth muscle contraction-related proteins such as p MLC(S20)andα-SMA are expected to be used as diagnostic markers for sudden cardiac death caused by coronary spasm.