Protocol for the Isolation and Super-resolution dSTORM Imaging of RyR2 in Cardiac Myocytes

Abstract

Heart failure is associated with low cardiac output (CO) and low brain perfusion that imposes a significant risk for accelerated brain ageing and Alzheimer's disease (AD) development. Although clinical heart failure can emerge several years following acute myocardial infarction (AMI), the impact of AMI on cerebral blood flow (CBF) at early stages and up to 30 days following MI is unknown. Sixteen months old male mice underwent left anterior descending (LAD) coronary artery ligation. Hemodynamics analyses were performed at baseline and at days 1, 7, and 30 post-MI. Left ventricular (LV) ejection fraction (EF), LV volumes, CO, and right common carotid artery (RCCA) diameter were recorded by echocardiography. RCCA flow (RCCA FL) was measured by Doppler echocardiography. LV volumes consistently increased (P<0.0012) and LV systolic function progressively deteriorated (P<0.0001) post-MI. CO and RCCA FL showed a moderate but significant decrease over the course of MI with similar fluctuation pattern such that both variables were decreased at day 1, increased at day 7, and decreased at 30 days post-MI. Correlation and regression analyses between CO and RCCA FL showed a strong correlation with significance at baseline and day 30 post-MI (R = 0.71, P=0.03, and R = 0.72, P=0.03, respectively). Days 1 and 7 analyses between CO and RCCA FL showed moderate correlation with non-significance post-MI (R = 0.51, P=0.2, and R = 0.56, P=0.12, respectively). In summary, CBF significantly decreased following AMI and remained significantly decreased for up to 30 days, suggesting a potential risk for brain damage that could contribute to cognitive dysfunction later in life.

Live-cell photoactivated localization microscopy correlates nanoscale ryanodine receptor configuration to calcium sparks in cardiomyocytes

“…Single cardiomyocytes were isolated as previously described 39 . Briefly, mice were anesthetized by isoflurane inhalation and euthanized by cervical dislocation.…”

Analysis of RyR2 distribution in HEK293 cells and mouse cardiac myocytes using 3D MINFLUX microscopy

“…Cardiac myocyte isola&on from PA-RFP RyR2 mice PA-RFP RyR2 mice that harbour PA-TagRFP inserted arer T1365, within exon 31 of the mRyR2 gene on chromosome 13 as described 8 , were housed at the University of Oslo and used to provide isolated myocytes. Single cardiomyocytes were isolated as previously described 34 . Briefly, the mice were first anesthe3zed by isoflurane inhala3on before being euthanized by cervical disloca3on.…”