Freezing preservation of the mammalian cardiac explant. V. Cryoprotection by ethanol.

We studied the colligative cryoprotective effect of ethanol (EtOH) in preserving the isolated rat heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C. Addition of 4.7% (v/v) EtOH to a cardioplegic solution, CP-14, raised the osmolality from 280 to 1100 mOsm/kg H2O and lowered the melting point from -0.52 to -2.1 degrees C. Freezing of the cardiac explant at -3.4 degrees C for 6 h resulted in 34.3 +/- 1.9% of the tissue water as ice; recovery of cardiac output (CO) was 50%. Polyethylene glycol, which at 5% (w/v) has been shown to cryoprotect the hearts during freezing at -1.4 degrees C, did not improve the protective effect of 4.7% EtOH. CP-14 + 4.7% EtOH did not freeze at -1.4 degrees C. After 6 h storage, CO in hearts flushed with CP-14 + 4.7% EtOH oxygenated with 95% O2/5%CO2 returned to almost control level and was much higher than that in hearts flushed with 100% O2 saturated-CP-14 + 4.7% EtOH. Storage of 8 and 12 h reduced CO to 87 +/- 9 and 60 +/- 5% of control. By employing EtOH as a colligative cryoprotectant, we preserved the adult mammalian heart frozen at -3.4 degrees C or unfrozen at -1.4 degrees C, suggesting that this small molecular weight, penetrating substance may be a suitable cryoprotectant for long-term storage of the cardiac explant at high subzero temperatures.

Freezing preservation of the mammalian heart explant. III. Tissue dehydration and cryoprotection by polyethylene glycol.

Isolated rat hearts perfused with hyperosmotic Krebs-Henseleit buffer containing 60 mmol/L NaCl lose 10% of their tissue water. Perfusion of the rat hearts with Krebs-Henseleit buffer containing polyethylene glycol 8000 caused a concentration-dependent reduction in tissue water. In a study of the effect of different cryoprotectants on cardiac preservation, isolated rat hearts were flushed with a cardioplegic solution (CP-14), or CP-14 with either 50 mmol/L glycerol (CP-15), or 5% polyethylene glycol (CP-16) and frozen at -1.4 degrees C for 5 hours. Thawed hearts were reperfused in working mode to assess function. There was no recovery in CP-14 hearts. Hearts treated with CP-15 recovered 39.3% +/- 2.9% (mean +/- SEM) of control cardiac output. CP-16 boosted the recovery of cardiac output to 54.4% +/- 5.7% (p less than 0.05 vs CP-15). Glycerol significantly reduced tissue ice content; PEG further decreased the ice content to 31.7% +/- 0.6%, which was distinctively lower than that in CP-14 (44.7% +/- 1.1%) and in CP-15 hearts (34.6% +/- 1.1%). Tissue water content of CP-14 and CP-15 hearts was similar (3.83 and 3.87 gm H2O/gm dry weight). Polyethylene glycol reduced the tissue water content to 3.24 +/- 0.04 gm H2O/gm dry (p less than 0.01 vs CP-14 and CP-15 by ANOVA). Thus both glycerol and polyethylene glycol offered cryoprotection to the heart explant by reducing tissue ice formation. Polyethylene glycol was superior to glycerol by dehydrating myocardial tissue and further minimizing freezing damage.

Freezing preservation of the mammalian cardiac explant. II. Comparing the protective effect of glycerol and polyethylene glycol.

We compared the cryoprotective ability of glycerol and polyethylene glycol (PEG) during freezing. Isolated rat hearts were flushed with one of three cardioplegic solutions (CP-14, CP-15, and CP-16), frozen at -1.4 degrees C, and reperfused after thawing to assess function. After 3 h freezing, cardiac output (CO) in CP-14-flushed hearts recovered to 58.1% of control. CP-16 (CP-14 with 5% PEG) improved CO to 77.5%. Five hours of freezing abolished recovery in CP-14 hearts, but CP-15 (CP-14 with 50 mM glycerol) and CP-16 hearts produced 40.0 and 49.0% CO, respectively. With 6 h freezing, CP-15 hearts did not recover, whereas CP-16 hearts recovered 37.5% CO. In CP-14 hearts frozen for 3 h, 37.4% of the tissue water was ice that increased to 44.7% with 5 h freezing. CP-15 and CP-16 hearts had 34.4 and 30.9% tissue ice, respectively, after 5 h freezing. Tissue water contents in CP-14 and CP-15 hearts (3.83 to 3.96 g H2O/g dry) were 14 to 24% higher than that in CP-16 hearts. Six hours of freezing elevated AMP and ADP contents and reduced ATP levels in CP-15 and CP-16 hearts. Total adenine nucleotide (TAN) content of CP-15 hearts was 72% of control, while that of CP-16 hearts was normal. In conclusion, both glycerol and PEG offered cryoprotection by reducing tissue ice formation. PEG was superior by reducing tissue ice content further via dehydration and by better preserving TAN content.

Metabolic and functional cardiac impairment after reperfusion with persantine.

The effect of dipyridamole (DYP) on postischemic myocardial function and metabolism was studied using the isolated rabbit heart model. Twenty-one isolated rabbit heart preparations were divided into two groups: KH (control N = 10) were reperfused after 24 min normothermic hyperkalemic arrest with modified Krebs-Henseleit buffer (KH) while DYP (N = 11) were reperfused with KH and 5 X 10(-6) M DYP. Hearts were analyzed for myocardial function (DP, developed pressure, +dp/dt, -dp/dt) and metabolic function (ATP, CrP, ADP, AMP, purines, and lactate levels). Data analysis revealed significant reperfusion depression in DYP myocardial function compared with KH (P less than 0.05): DP (42 +/- 6 vs 89 +/- 7 mm Hg), +dp/dt (390 +/- 21.6 vs 1227 +/- 48.4), and -dp/dt (280 +/- 20.1 vs 677 +/- 19.8). Comparison of DYP to KH metabolic parameters was also significantly different (P less than 0.05): ATP (5.8 +/- 0.7 vs 9.5 +/- 1.4), ADP (2.1 +/- 0.2 vs 3.2 +/- 0.6), CrP (9.6 +/- 0.3 vs 17.2 +/- 1.3). Tissue purines (adenosine and inosine) were significantly elevated (P less than 0.01) in the DYP group, while coronary sinus purines and lactate loss were similar. Thus, the data suggest that DYP, present during postischemic reperfusion, depresses myocardial function by inhibiting adenosine phosphorylation, thereby decreasing the generation of high-energy phosphates without increased substrate loss or ischemia.