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Decreased mRNA levels of cardiac Cx43 and ZO1 in sudden cardiac death related to coronary atherosclerosis: a pilot study

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Abstract

Sudden cardiac death (SCD) is the most frequent cause of sudden unexplained death in forensic practice. The most common cause of SCD is coronary artery disease related to coronary atherosclerosis. Previous study suggested the possible application of connexin 43 (Cx43) and zonula occludens-1 (ZO1) immunostaining in the early diagnosis of myocardial ischemia. However, there appears to be insufficient data with regard to their mRNA levels. The present study investigated the cardiac mRNA levels of Cx43 and ZO1, using forensic autopsy materials consisting of 41 control cases without any disease or structural abnormality of the heart (group 1), 32 deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis (group 2), and 29 traumatic deaths with coronary atherosclerosis (group 3). Ten candidate reference genes were evaluated in the left ventricles of 10 forensic autopsy cases. EEF1A1, PPIA, TPT1, and RPL13A were identified as the most stable reference genes. Using these validated reference genes, mRNA levels of Cx43 and ZO1 were examined in the bilateral ventricles and atria of the heart. Relative mRNA quantification demonstrated decreased calibrated normalized relative quantity (CNRQ) values of Cx43 and ZO1 in bilateral ventricles of group 2. When using one conventional reference gene (GAPDH or ACTB) for normalization, nearly no difference was detected among the three groups. These findings indicate that ventricular gap junction remodeling may be a key contributor to rhythm disturbances. Analysis of cardiac Cx43 and ZO1 using real-time PCR is useful in diagnosis of SCD, and validation of reference genes is crucial.

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Acknowledgments

This research was supported by the National Natural Science Foundation of China (No. 81401556), Guangdong Natural Science Foundation (No. 2014A030310504 and 2014A030310293), and the Special Foundation of President of School of Public Health and Tropical Medicine of Southern Medical University (No. GW201402).

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    Authors and Affiliations

    1. Department of Forensic Medicine, School of Basic Medical Science, Southern Medical University, No. 1838, Guangzhou, 510515, Guangdong Province, China

      Ye Xue, Si-Hao Du, Dong-Ri Li, Jing-Tao Xu & Qi Wang

    2. Department of Forensic Pathology, China Medical University School of Forensic Medicine, Shenyang, China

      Rui Zhao

    3. Collaborative Innovation Center of Judicial Civilization, Beijing, China

      Dong Zhao

    4. Ministry of Education, Key Laboratory of Evidence Science (China University of Political Science and Law), Beijing, China

      Dong Zhao

    5. Department of Toxicology, School of Public Health and Tropical Medicine, Southern Medical University, No. 1838, Guangzhou, 510515, Guangdong Province, China

      Xiao-Li Xie

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    1. Ye Xue
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    Correspondence to Xiao-Li Xie or Qi Wang.

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    Ye Xue, Rui Zhao and Si-Hao Du contributed equally to this work.

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    Supplementary Material 1

    Cx43 mRNA expression levels after normalization against GAPDH. No difference was detected among three groups. CNRQ, calibrated normalized relative quantity; LV, left ventricle; RV, right ventricle; Group 1, control cases without any disease or structural abnormality of the heart; Group 2, deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis; Group 3, traumatic deaths with coronary atherosclerosis. (PPT 534 kb)

    Supplementary Material 2

    ZO1 mRNA expression levels after normalization against GAPDH. No difference was detected among three groups. CNRQ, calibrated normalized relative quantity; LV, left ventricle; RV, right ventricle; Group 1, control cases without any disease or structural abnormality of the heart; Group 2, deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis; Group 3, traumatic deaths with coronary atherosclerosis. (PPT 519 kb)

    Supplementary Material 3

    Cx43 mRNA expression levels after normalization against ACTB. No difference was detected among three groups, except for an increased Cx43 mRNA level in the left atrial wall of Group 1 than in Group 2. CNRQ, calibrated normalized relative quantity; LV, left ventricle; RV, right ventricle; Group 1, control cases without any disease or structural abnormality of the heart; Group 2, deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis; Group 3, traumatic deaths with coronary atherosclerosis. (PPT 492 kb)

    Supplementary Material 4

    ZO1 mRNA expression levels after normalization against ACTB. No difference was detected among three groups. CNRQ, calibrated normalized relative quantity; LV, left ventricle; RV, right ventricle; Group 1, control cases without any disease or structural abnormality of the heart; Group 2, deaths due to acute ischemic heart disease related to coronary atherosclerosis without apparent myocardial necrosis; Group 3, traumatic deaths with coronary atherosclerosis. (PPT 551 kb)

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    Xue, Y., Zhao, R., Du, SH. et al. Decreased mRNA levels of cardiac Cx43 and ZO1 in sudden cardiac death related to coronary atherosclerosis: a pilot study. Int J Legal Med 130, 915–922 (2016). https://doi.org/10.1007/s00414-016-1353-0

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