Determining the Association Between Density of Muscarinic Acetylcholine Receptor M3 in Myocardium and Tunica Media of Coronary Vasculature and Self‐Reported Disease States

Abstract

Myocardial infarction causes parasympathetic dysfunction in cardiovascular tissue, where central parasympathetic drive decreases but local acetylcholine levels are unchanged. However, muscarinic acetylcholine receptor M3 (AChM3R) is shown to have mediating effects in cardiac tissue and vasculature, such as regulation of heart rate and vasodilation of coronary arteries. The objective of this study is to determine the association between AChM3R levels in the heart wall and in the coronary vasculature with self‐reported disease states from cadaver donor records.Biopsies of the left anterior descending (LAD) artery and the underlying anterior interventricular septum (AIVS) were taken from 14 cadavers as opportunity samples. A 5 mm biopsy of the LAD and its underlying AIVS were harvested 2.54‐3.81cm from the bifurcation point of the left coronary artery, depending on the tissue integrity of the cadaver specimen. The tissues were immunostained for AChM3R (CHRM3 NB100‐58975 at 1:2000) and visualized using a DAB chromogen. Slides were digitally scanned into a virtual image at 20x using the Motic Easyscan (Motic, Inc.). Images were segmented for tunica media and myocardium using Adobe Photoshop CS (Adobe, Inc.). We developed code in Python to calculate the strong positive and positive staining of the tissues. The number of pixels stained was normalized to the tissue area. The donors from which tissue was biopsied were classified into cardiovascular, cerebrovascular, Alzheimer’s disease dementia (AD), or both cerebrovascular and AD disease categories based on self‐reported donor declarations. We used SPSS (v27, IBM, Inc.) to run a Pearson correlation to determine the association of staining positivity in the anatomical regions and a MANOVA to determine significant differences in the amount of pixel positivity in the tunica media and myocardium as a function of disease classification.Strong positive and positive staining of the myocardium were not correlated with strong positive in the tunica media. There was a significant, strong, and positive relationship between strong myocardium staining and positive tunica media staining (r =.727, n=14, p=.003) as well as positive myocardium and positive tunica media staining (r=.674, n=14, p=.008). There is no significant difference between the amount of AChM3R staining and disease classification.The lack of correlation between strong positive in the tunica media and positive and strong positive in the myocardium suggests that changes in the AChM3R of coronary vascular smooth muscle are independent of the AChM3R state in the heart wall. However, significant correlation between strong positive in the myocardium and positive in the tunica media indicates that changes in AChM3R in the myocardium could be dependent on the presence of a baseline amount of AChM3R in the coronary vascular smooth muscle. Categorization of donor disease states may be too broad to provide statistical significance. Many donors reported co‐morbid diseases, which could have affected the influence of cardiovascular disease and dementia on AChM3R modulation. These findings highlight that interventions targeting parasympathetic dysfunction of coronary vasculature may leave the underlying heart wall unaffected. Future studies should consider cardiovascular disease diagnoses to assess AChM3R distribution in the heart wall and coronary vasculature.