Effect Of Cardiac Muscle Cells Secretome, With And Without Loading Preconditioning, On Hypoxia/Reoxygenation Injury

Abstract

Reperfusion after myocardial infarction (MI) can worsen cardiac tissue damage and in vitro models of hypoxia/reoxygenation (H/R) have been developed to simulate the in vivo ischemia/reperfusion injury. Cardiac muscle stem cells have been used for regeneration after MI due to their paracrine actions in improving myocardial cell survival and function, while interestingly, mechanical loading of cardiac muscle cells may modulate their secretome. PURPOSE: The present study investigated the cardiac muscle cells’ paracrine effects in H/R, by treating them with the secretome of mechanically loaded or unloaded cells, in vitro. METHODS: H9C2 cardiomyoblasts were cultured on elastic membranes and underwent a cyclic stretching (12% elongation at 0.25 Hz for 12h) and then their secretome was collected (stretch media, SM). Secretome of unstretched cardiomyoblasts were also collected (non-stretch media, NSM). Cardiomyoblasts were subjected to 6 h of hypoxia followed by 8 or 4 h of reoxygenation (H/R) while during reoxygenation, they were treated either with SM or NSM. Cell apoptosis was subsequently assessed by ΜΤΤ assay and flow cytometry. RESULTS: After the hypoxia period, cell viability rate was 98±9%, without differing from the normoxia group (p>0.05). However, after 8 or 4 h of reoxygenation, the viability rate was reduced to 51±10% (p<0.01) and 74±10% (p<0.001), respectively. In addition, the percentage of early apoptotic cells was 36% (p<0.001) after 6 h of hypoxia/4 h of reoxygenation (H/R) as assessed by MTT assay. The viability rates in the same H/R protocol increased from 74±10% to 92±10% and 80±2% in SM- and NSM-treated cells, respectively, without exhibiting differences with the normoxia group (p>0.05). Interestingly, a significantly higher viability was observed only in the cells treated with the SM compared to the non-treated cells after H/R injury (p<0.05). CONCLUSIONS: Our findings suggest that cardiomyocytes are susceptible to H/R-induced injury, while the cell death rate depends on the duration of reoxygenation. Moreover, cardiomyoblasts’ secretome inhibits their apoptosis after H/R injury while their mechanical load “preconditioning” appears to boost the anti-apoptotic effects of their secretome, implying the beneficial paracrine action of cardiac muscle cells due to mechanical loading.