Abstract 443: Overexpression of Myoglobin Modulates Nitric Oxide/cGMP Signaling and Reduces Oxidative Stress in the Ischemic Heart
Myoglobin (Mb) is a cytoplasmic hemoprotein that is expressed in cardiomyocytes and has multiple cellular functions including facilitated oxygen delivery and scavenging nitric oxide (NO)/reactive oxygen species. To define the cardioprotective role of Mb during ischemia, we generated Mb overexpressing mice (TGMb) and previously observed in a Langendorff preparation that the TGMb hearts were more resistant to an ischemia-reperfusion injury compared to wild-type hearts (+dP/dtmax 1750±115 vs. 1475±68mmHg/sec, respectively; p<0.05; n=6). Mechanistic in vitro studies were undertaken to investigate the cytoprotective role of Mb within the myocyte. An adenovirus overexpressing Mb was generated and infection assays revealed increased cellular viability in anoxic Mb overexpressing myotubes (MbMyt) vs. control anoxic myotubes (Myt) (<0.1% O2 for 12 hours) (87±1.6% vs. 33±3.0%, respectively; p<0.05; n=3). Measurement of oxidative stress by H2DCFDA fluorescence revealed a qualitative reduction in free radical production in anoxic MbMyt vs. control anoxic Myt. Biochemical assays revealed a 22% decrease in total NO production and a reduction in cGMP concentrations in anoxic MbMyt vs. control anoxic Myt (0.1±0.05 pmol/mg vs. 0.49±0.04 pmol/mg, respectively; p<0.05, n=3). Since many of the cellular effects of inducible nitric oxide synthase (iNOS) generated NO are transduced via the cGMP pathway, we evaluated the role of Mb in scavenging iNOS-generated NO in the ischemic heart. We undertook a loss of function approach with the use of previously engineered Mb null (Mb−/−) and Mb/iNOS null (DKO) mice. We utilized the Langendorff preparation to induce an ischemia-reperfusion injury in hearts from Mb−/− and DKO mice. DKO hearts were significantly more resistant to 15 minutes of ischemia compared to control Mb−/− hearts (+dP/dtmax 1900±115 vs. 1027±188mmHg/ sec, respectively; p<0.05; n=3). These data highlight the importance of the interaction between myoglobin and NO under ischemic conditions and establish that myoglobin is an essential cardiac hemoprotein that functions as a key regulator of O2, NO, and free radical homeostasis.