The Effect of Hypoxia on Mechanical Stretch-Activated Hypertrophic Gene Expression in Neonatal Mouse Ventricular Cardiomyocytes

Abstract

At birth, the heart is challenged with a shift from relative hypoxia to normoxia and from lower hemodynamic load on the ventricles to higher load, due to differences between in utero and ex utero oxygen tension and fetal and newborn blood circulation. Evidence suggests that postnatal cardiac hypertrophy may be regulated in part by these changes in hemodynamic loading and oxygen supply to the newborn heart. Here we test the hypothesis that mechanical overload and the shift from hypoxic to normoxic conditions may be signals that regulate neonatal myocyte maturation and growth. NMVMs were cultured on PDMS membranes subjected to 5% equibiaxial stretch for 15 minutes to 4 hours, under both normoxic (5% CO2 in air) and hypoxic (6.8 % CO2 with 5 % O2) conditions, after which we measured protein expression by Western blotting. Under normoxic conditions, whole cell lysates from control membranes (without stretch) were negative for atrial natriuretic factor (ANF) protein expression as evidenced by immunoblotting. In contrast, by one hour of mechanical overload, lysates immunoblotted for ANF showed a nearly 2 -fold increase in ANF protein expression which returned to near baseline levels within 4 hours of mechanical stretch. Conversely, myosin heavy chain (MHC) protein expression was detected in lysates from cardiomyocytes subjected to 15 mins mechanical stretch and increased monotonically as evidenced by a 4-fold increase in protein expression by 4 hours of stretch. Furthermore, connexin-43 protein expression increased almost 3-fold over the unstretched controls after 15 mins of stretch. Preliminary data indicates that compared to normoxic conditions, expression of these signaling proteins decreases under hypoxic conditions. An understanding of the role of mechanical stretch in cardiomyocytes cultured under hypoxic conditions holds promise for advancing cardiac regenerative medicine.