Fig. 1

Experimental protocol and assessment of effect of isoproterenol. A Study design. B Heart rates recorded 2 min after injection of isoproterenol showed a comparable increase due to β-adrenergic activation in both males and females. C Increments in heart weight normalized to tibia length indicated a buildup of edema in both sexes. D The percentage of the area of intact myofibers on histological slides of the myocardium declined due to isoproterenol therapy in both sexes compared to controls. E The percentage of picrosirius red stained area was not significantly changed in response to ischemia at the end of the experimental period. F Representative photomicrographs of hematoxylin & eosin stained slides demonstrate diffuse necrotic areas (arrows) embedded within healthy areas of the myocardium after isoproterenol-induced ischemia. Further images (second row) demonstrate how after H&E color deconvolution the eosin color component can be used to quantify the relative area of intact myofibers in the myocardium. First, the overall area of the myocardium is detected. Areas devoid of any myocardial tissue are outlined with yellow and are excluded from the calculation of the overall myocardial area. Then careful thresholding on the eosin color component can identify viable cardiomyocytes with intact myofibrillar structures. These areas (colored red in the software) are then used for the calculation of intact myofibrillar areas which are inversely proportional to and thus are an indirect measure of the extent of myocardial damage, myocardial necrosis. G: representative photomicrographs of picrosirius stained slides. Statistical significance of post-hoc test compared to same sex control is highlighted as follows: *P < 0.05, **P < 0.01, ***P < 0.001. M-Co: male control; M-Isch: male ischemic; F-Co: female control; F-Isch: female ischemic; HR: heart rate; HW/TL: heart weight normalized to tibia length