Ultrastructure-function correlative studies for cardiac cryopreservation. III. Hearts frozen to −10 ° and −17 °C with and without dimethyl sulfoxide (DMSO)

Abstract

Isolated rat hearts were perfused with balanced salt solution (BSS), then with BSS containing DMSO in one of several concentrations (0.14, 0.70, 1.41, 2.11, or 2.82 m) at +30 °C, sealed in a metal cannister, and cooled slowly (1 °C/min) to a core temperature of −17 °C (total cooling time (TCT) = 37 min), thawed rapidly and reperfused with BSS. Groups of protected (0.70 m DMSO) or nonprotected hearts were cooled to −10 °C; of these, some were thawed immediately upon reaching −10 °C (TCT = 30 min), others were maintained at −10 °C for an additional 7 min and thawed (TCT = 37 min). Contractile activity was recorded during prefreeze and postthaw perfusion periods. Hearts which exhibited spontaneous A-V function after thawing were considered to be survivors. No hearts treated with 0.14 or 2.82 m DMSO survived thawing from −17 °C, but approximately half of all hearts treated with 0.70, 1.41 or 2.11 m DMSO and thawed from −17 °C survived. Survivors had diminished contractile strengths and heart rates, and gave no or negative inotropic responses to ouabain, a cardiotonic drug. Electron microscope study of all hearts revealed subcellular damage which differed little between surviving and nonsurviving hearts in each of these three groups. Cellular and subcellular membranes as well as nuclear fine structure were damaged, but the severity and extent of such injury was markedly less than that seen in thawed hearts treated with 0.14 or 2.82 m DMSO or no DMSO at all. DMSO largely prevented rupture of outer nuclear and mitochondrial membranes, but mitochondrial swelling was evident. Sarcomere alteration was minimal and usually limited to lateral compression by adjacent swollen mitochondria. Some regions of the myocardium contained generally disrupted cells, but many cells showed remarkable ultrastructural preservation. The relative ratio of “damaged” to “undamaged” cells was higher in surviving hearts than in nonsurvivors. All hearts treated with 0.70 m DMSO and thawed immediately from −10 °C survived; only half survived when DMSO was omitted. Seventythree percent of DMSO-treated hearts held at −10 °C for 7 min survived; none survived this brief storage interval when DMSO was not used. Protected hearts thawed immediately from −10 °C showed minor ultrastructural changes, whereas prolonging duration of cooling or omitting DMSO caused increased ultrastructural alteration qualitatively similar to those seen in hearts cooled to −17 °C. These results suggest that physical parameters such as water flux, crystal growth and cell volume are predominately responsible for freeze damage rather than chemical injury, and that percent cell water converted to ice and solute concentration per se are not directly related to ultimate cardiac cryoinjury.