Fig. 4. Aspn protects against cardiomyocytes cell death induced by ischemia-reperfusion injury and improves cardiac function.

(A) Differentiated H9c2 cardiomyocytes were treated with vehicle or rASPN protein (1 nM) for 24 hrs prior to exposing the cells to hypoxia (2 hrs) and reoxygenation (2 hrs) (HR). The media was collected and LDH levels were determined by ELISA. Bar graph representing the levels of LDH in the media from different groups normalized to control normoxia group (n = 4). Data are expressed as mean with SD and one-way ANNOVA with Turkey’s multiple comparison test, ***p<0.001, *p<0.01; (B) TUNEL positive nuclei were counted from many fields, each containing 10–15 cells and expressed as a ratio to DAPI stained cells in the field. Data are expressed as mean with SD. (C) Wild type and Aspn KO mice were subjected to ischemia (30 mins) – reperfusion (24 hrs) injury and left ventricular tissue were stained with TTC. Representative infarct size images, showing viable tissue (in blue), area at risk (in red) and infarct (in white). (D) Infarct size as a ratio to area at risk was calculated and represented as bar graph, with individual points presented (n = 6 for wild type; n = 7 for Aspn KO). (E) Bar graph representing area at risk in 2 groups. (F) Heart weight is represented as ratio to body weight as assessed at Day 28 after I/R (n = 7 for wild type; n = 5 for Aspn KO). Data are expressed as mean with SD and unpaired t-test was employed to test for significance *p<0.05, ****p<0.0001. (G) Line graph represents ejection fraction and (H) fractional shortening as determined by echocardiography at Day 0 (before surgery), Day 3, 7, 14 and 28 of I/R injury model.