Acellular hemoglobin impairs calcium release and contractility in cardiomyocytes from healthy animals

Hemoglobin (Hb)-based oxygen (O 2 ) carriers (HBOC) have been proposed as an alternative for blood transfusion. Phase III clinical trials of previous HBOC generations presented issues, such as vasoconstriction and myocardial infarction, that were not detected in pre-clinical trials. These problems have been attributed to extravasation of the low molecular weight (LMW) Hb species, nitric oxide (NO) scavenging and O 2 oversupply to the vasculature. However, the mechanisms responsible for the cardiac toxicity of HBOCs remain unclear. To explore the effects of acellular (acell) Hb and HBOC in cardiac tissues, we exposed cardiomyocytes (CMCs) to highly purified stroma-free acell bovine Hb (bHb) or human Hb (hHb). CMCs were loaded with Ca2+ fluorescent indicator (fluo-3) and put on laminin-coated coverslips. Cells were superfused with standard Tyrode solution with and without acell Hb. CMCs were stimulated with current pulses given at 1 Hz, and free cytosolic Ca2+ concentration was measured. To eliminate the effects of NO scavenging on CMCs, L-Name was added to the superfused with bHb and hHb. To study the effect of acell Hb MW on CMCs, LMW polymerized hHb (PolyhHb) was used to challenge CMCs. To explore the effect of Hb oxidation on CMCs, LMW MetHb was used to challenge the CMCs and compared to LMW PolyhMetHb. To explore the effects of antioxidants and heme toxicity, the studies with hHb and MethHb were repeated in the presence of the antioxidant N-acetylcysteine (NAC) or purified human Haptoglobin (Hp). Mitochondrial reactive oxygen species (ROS) production was measured using the fluorescent indicator MitoSOX Red after removing the Hb by washing the cells. After ROS determination, the maximal ROS production by CMCs was measured by challenging the cells with a high concentration of H 2 O 2 . Acell Hb increased the Ca 2+ transient decay time constant (tau) relative to control. Removing the effects of NO production with L-Name, increased the tau of the Ca 2+ transient in control and acell Hb challenge cells. Challenging CMCs with PolyhHb reduced the effects observed with acell Hb in Ca 2+ transients. MetHb appears to be worse than reduced Hb in terms of the changes induced to CMCs Ca 2+ transients. The antioxidant NAC mitigated the effects of acell Hb and MetHb but did not eliminate the changes on increased Ca2+ transients. The Hb scavenger, Hp, mitigated the effects of acell hHb and MethHb on Ca 2+ transients. The MitoSOX Red fluorescence was higher for CMCs exposed to acell Hb and MetHb compared to the control. PolyhHb and PolyMetHb challenge decreased the MitoSOX compared to acell hHb and MethHb. Cells with higher levels of ROS showed lower changes in fluorescence when exposed to H 2 O 2 . Here, we show that the toxicity of acell Hb is impart determined by facilitated diffusion, NO scavenging, heme release, and ROS, potentially affecting the energy supply and metabolism of CMCs. This study was supported by National Institutes of Health grants R01HL162120, R01HL159862 and the Department of Defense under grants W81XWH1810059. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.