Antifreeze glycoproteins promote intracellular freezing of rat cardiomyocytes at high subzero temperatures

Despite recent reports that antifreeze glycoproteins (AFGPs) protect mammalian cells during low-temperature preservation, T. Wang, Q. Zhu, X. Yang, J. R. Layne, and A. L. DeVries (Cryobiology 31: 185-192, 1994) reported that AFGPs failed to protect rat hearts during freezing. Rather, the presence of AFGPs exacerbated cardiac damage after freezing. This study examined the effects of freezing (-4 degrees C) in the presence of AFGPs at the cellular level with the use of cryomicroscopy. Large, blunt ice crystals formed in the solutions without AFGPs and excluded most cardiomyocytes from the plane of ice formation. After thawing, cells appeared similar in morphology to unfrozen cells. Ice in 0.5 mg/ml AFGP solution was more dendritic and prismatic than ice formed in the absence of AFGPs. On thawing, many cells exhibited spontaneous contraction, resulting in cell death. Spicular ice formed rapidly in the 10 mg/ml AFGP solution. These needlelike ice crystals appeared to penetrate the cardiomyocytes, resulting in intracellular freezing followed by cell lysis. These AFGP-induced changes in ice crystal structure may account for the injury observed in whole heart and cardiomyocyte experiments.