EXPLORING THE BENEFICIAL EFFECTS OF S100A6 OVEREXPRESSION IN ACUTE INFARCTION-REPERFUSION: PREVENTION OF FIBROSIS, ENHANCED ANGIOGENESIS AND ATTENUATION OF MYOCARDIAL HYPERTROPHY

BackgroundWe have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses.MethodsUsing UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson’s Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation.ResultsS100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours.ConclusionS100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion. BackgroundWe have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses. We have previously shown that pre-delivery of S100A6 gene, an EF-hand Ca2+-binding protein, by ultrasound-targeted microbubble destruction (UTMD) results in lower mortality and improved LV systolic function after cardiac infarction-reperfusion (I/R). The S100A6 therapeutic effect on myocardial I/R was due in part to its anti-apoptotic effects and regulatory function on intracellular calcium cycling. Here we hypothesize that S100A6 therapeutic effects may also involve other pathways including angiogenesis and hypertrophic responses. MethodsUsing UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson’s Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation. Using UTMD, we pre-delivered GFP-tagged human-S100A6 plasmid and empty plasmid to the LV in 10-12 weeks old Fischer- 344 male rats, 2 days prior to LAD-ligation (30 min) and reperfusion, with control animals receiving no UTMD. We measured the ratio of heart weight to tibia length at day 28 post I/R. Cardiomyocyte size was measured off haemotoxylin-eosin stained sections. Relative expression of βMHC and ANF were studied in the infarct and non-infarct regions of myocardium 28 days post I/R. Masson’s Trichrome staining was performed to detect collagen fibers in the infarct zones. Myocardial contrast echo (MCE) was performed at day 28 to measure myocardial blood flow in the infarct and peri-infarct regions. For in vitro studies, human umbulical vein endothelial cells (HUVECs) were transduced with either adenoviral packaged GFP-tagged human-S100A6 plasmid, empty plasmid or rat S100A6-shRNA. Non-transduced cells served as additional controls. Matrigel tube formation assay was performed to observe the effects of S100A6 on capillary-like network formation. ResultsS100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours. S100A6-treated hearts after cardiac I/R had significantly lower heart weight to tibia length (HW/TL) ratio, lower expression of β MHC and ANF in the infarct and non-infarct regions of myocardium and smaller cardiomyocyte size compared to controls at day 28. Extent of fibrosis/infarct size at day 28 was significantly lower in S100A6 pre-treated animals compared to empty plasmid and non-treated controls. MCE-derived myocardial blood flow at day 28 post ischemia/reperfusion in the risk area (infarct and peri-infarct regions) normalized to the remote non-infarct regions was significantly higher in the S100A6 treated group confirmed with IHC staining showing increased vessel density there. S100A6 over-expressing HUVECs formed significantly greater number of nodes and tubes compared to null-transduced and non-transduced controls after 3 hours. ConclusionS100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion. S100A6 overexpression results in reduced infarct size, not only by prevention of cardiomyocyte apoptosis (based on our previous data), but also via prevention of late fibrosis, enhancement of angiogenesis within the infarct and peri-infarct regions and attenuation of myocyte hypertrophy after acute infarction-reperfusion.