Impact of current cryopreservation procedures on mechanical and functional properties of human aortic homografts.

We evaluated the impact f standard cryopreservation on mechanical and functional properties of human aortic homografts. From 14 human heart-valve donors, the thoracic descending aorta was obtained. Effects of cryopreservation on mechanical (elastic properties and breaking stress) and smooth muscle cell (SMC) and endothelium function were tested. Cryopreservation (cryo) did not significantly affect Young's modulus of elastin (fresh: 3.1 +/- 1.0, cryo: 2.7 +/- 0.9 x 10(5) Nm(-2)), collagen recruitment pressure (fresh: 1.1 +/- 0.3, cryo: 1.1 +/- 0.4 x 10(4) Nm(-2)), distensibility (fresh: 3.8 +/- 1.8, cryo: 3.6 +/- 1.6 x 10(5) N(-1)m2), or breaking stress (fresh: 2.4 +/- 1.0, cryo: 2.2 +/- 1.0 x 10(6) Nm(-2)). Following explantation, no endothelium-dependent relaxation was found. SMC function and endothelium-independent relaxation were mainly intact after explantation but significantly decreased after cryopreservation. Aortic mechanical properties are not influenced by cryopreservation. Following explantation, almost no endothelial cell function is present, and SMC contractility is strongly affected after cryopreservation.

ECG of the "newborn" mouse (Mus domesticus) with specific reference to comparative AV transmission.

INTRODUCTION: The objective of this study was to record the ECG of the smallest living mammal to extend the domain of data for comparative AV nodal electrophysiologic purposes. These data are needed to establish the relationship between the PR interval and heart size in mammalian species of all sizes. METHODS AND RESULTS: In recently born mice (age 1.5 to 8 weeks) weighing between 2.5 and 10 g and with estimated heart weights between 15 and 60 mg, ECGs, using bipolar limb leads, were recorded during general anesthesia. The PR interval, representing AV transmission time was about 40 msec, which is quite long for hearts of this size. On the basis of detailed analysis of the data, we postulate the presence of a fixed delay or discontinuous propagation in the AV node not only in newborn mice, but in mammals of all sizes. CONCLUSION: AV transmission times obtained in mammals (including humans) cannot be explained on the basis of generally accepted, classic AV conduction theories. The acceptance of the presence of a fixed delay in the AV node may ultimately be of value to better understand AV node function during sinus rhythm and supraventricular arrhythmias.

Immunogenic human leukocyte antigen class II antigens on human cardiac valves induce specific alloantibodies.

BACKGROUND: The kinetics of panel reactive antibodies (PRA) and incidence of antibodies directed against human leukocyte antigen (HLA) class II were studied in patients who received a cryopreserved cardiac valve allograft. METHODS: A complement-dependent microlymphocytotoxicity test was used to determine the percentage of panel reactive antibodies. Anti-HLA class II antibodies were measured by two-color fluorescence assays. RESULTS: The panel reactive antibodies became positive in 25 (78%) of 32 recipients between 1 and 16 months after implantation. Antibodies against HLA class II antigens were detected in 11 (37%) of 30 patients. In 9 (82%) of 11 cases these antibodies were donor specific. The induction of antibodies against donor HLA class II antigens suggests that intact HLA class II antigens are expressed by viable cells within the graft. Dithiothreitol analysis showed that the antibodies were of the immunoglobulin G type. Apparently, the HLA class II antigens are expressed in an immunogenic way, as activation of specific T-helper cells is essential for the switch from immunoglobulin M to immunoglobulin G antibodies. CONCLUSIONS: Allogeneic valve transplantation is associated with the production of donor-specific anti-HLA class I and II antibodies that could contribute to graft failure. This possibly detrimental effect might be prevented by cross matching in sensitized patients.

Effects of cryopreservation on contractile properties of porcine isolated aortic valve leaflets and aortic wall.

Human semilunar donor heart valves can be stored in banks, awaiting transplantation. To evaluate the result of the preservation protocols, a quantitative description of the tissue is necessary. In this study we investigated in a quantitative way the contractile properties of fresh and cryopreserved porcine isolated aortic heart valve leaflets in response to a number of endogenous vasoactive compounds. The responses of strips of the aortic wall were included for comparison. Contraction was measured isometrically in response to potassium (K+; 100 mmol/L), 5-hydroxytryptamine (1 nmol/L to 100 micromol/L), noradrenaline (1 nmol/L to 100 micromol/L), endothelin-1 (0.01 nmol/L to 0.3 micromol/L), and prostaglandin F(2alpha) (0.1 nmol/L to 10 micromol/L). The pharmacologic parameters E(MAX) (the maximal response expressed as a percentage of contraction to a 100 mmol/L dose of K+) and EC50 (the concentration that produces 50% of the maximal effect) were calculated for every compound (n = 6 to 7 each). We observed that all specimens contracted in response to potassium. Its magnitude in fresh leaflets equaled 1.6 +/- 0.14 mN compared with 26.6 +/- 2.6 mN in fresh aortic wall. Noradrenaline, endothelin-1, and prostaglandin F(2alpha) all caused contraction in valvular leaflets and aortic wall, whereas 5-hydroxytryptamine caused contraction in the valvular leaflets but relaxation in aortic wall. After cryopreservation, the response to K+ amounted to 24% of the response of the fresh specimens in valvular leaflets (n = 25) and 14% in aortic wall (n = 26). The values of E(MAX) and EC50 of the responses to noradrenaline, endothelin-1, and prostaglandin F(2alpha) remained unchanged. Although the physiologic relevance of contraction of valvular leaflets needs further study, its measurement may provide an additional model to verify the consequences of alternative methods of preservation.

Donor-specific cellular immune response against human cardiac valve allografts.

We studied the presence of donor-specific T lymphocytes in explanted human cardiac valve allografts in vivo. From five of seven explants we propagated lymphocyte cultures in an interleukin-2 conditioned medium. Phenotyping revealed the presence of T-cell receptors in more than 95% of the lymphocytes obtained in each culture. Donor-specific cytotoxicity was demonstrated in three patients with known HLA status of the donor. Cytotoxicity was directed against only HLA class I in one patient, and against class I and/or class II in the others. These results indicate that donor-specific cellular reactivity can be induced by transplantation of human cardiac valve allografts.

Cracks in cryopreserved aortic allografts and rapid thawing.

Cryopreserved aortic allografts are shipped in a frozen state. Cracks in the graft, appearing after thawing, can pose serious problems in the planned operative procedures. Cracks were encountered in approximately 3% of all our shipped cryopreserved human material and were strongly associated with transportation in a "dry shipper." We practice two other modes of transport: on dry ice and directly to our operating room. We studied the temperature behavior during thawing of 17 porcine aortic valves in three groups. The valves were frozen and stored like our human valves. Before thawing, they were subjected to stimulated transport in either a dry shipper, on dry ice, or to the operating room. Differences between the groups were noted in the maximal rate of thawing of the inner as well as the outer wall. The highest maximal thawing rates were observed in the dry shipper group, in which 66% of the valves cracked. We therefore suspended transportation of cryopreserved human valves by means of a dry shipper.

Different endothelin receptors involved in endothelin-1- and sarafotoxin S6B-induced contractions of the human isolated coronary artery.

1. Endothelin receptors, that mediate contraction of the human isolated coronary artery, were characterized by use of a number of agonists and antagonists. Contraction induced by the non-selective agonists, endothelin (ET)-1 and sarafotoxin S6b, was compared in endothelium-intact and endothelium-denuded ring segments. The effects of ET-1 and BQ-123 (an ETA receptor antagonist) were investigated both in ring segments and in spirally cut strips. Lastly, the effect of phosphoramidon was studied on contraction induced by big-ET-1. 2. The order of agonist potency (pD2) in endothelium-intact coronary artery ring segments was: ET-1 (8.27) approximately sarafotoxin S6b (8.16) > big-ET-1 (< 7.1) approximately ET-3 (< 6.9). [Ala1,3,11,15]ET-1 (ETB receptor agonist) caused significant contraction only at 1 microM, whereas 0.3 microM big-ET-3 had no effect. Removal of the endothelium in ring segments did not affect the contractile response to ET-1 or to sarafotoxin S6b. 3. After a full concentration-response curve had been obtained to ET-1 or sarafotoxin S6b, further contractions of the endothelium-intact coronary artery segments could only be achieved by applying ET-1 in segments exposed to sarafotoxin S6b, and not the reverse. 4. BQ-123 (0.1 microM) antagonized contractions of endothelium-intact ring segments induced by sarafotoxin S6b (pKB 7.86). Only 10 microM BQ-123 antagonized contractions induced by ET-1 (pKB 5.75). FR139317 was also more potent against sarafotoxin S6b (pKB 8.24-8.47) than against ET-1 (pKB 6.11). [Ala1,3,11,15]ET-1 (1 microM) had no effect on the contractile response to ET-1 or to sarafotoxin S6b. 5. In strip preparations with intact endothelium, the pD2 of ET-l increased to 9.04 =/- 0.16 (vs.8.50 +/- 0.07 in rings), and BQ-123 (1 microM) caused a rightward shift of the ET-l induced concentration response curve (pKB 6.62 vs. 5.75 in rings).6. Contractile responses to big-ET-1 of endothelium-intact coronary artery segments were attenuated in the presence of phosphoramidon (100 microM), indicating conversion of big-ET-1 to ET-1 within the coronary artery segment.7. The present study indicates that ET-1 and sarafotoxin S6b contract the human isolated coronary artery via different receptors, which can probably be best characterized as subtypes of the ETA receptor.Furthermore, it is demonstrated that the type of preparation (ring or strip) may affect the potency of ET-1 as an agonist and of BQ-123 as an antagonist.

Effect of cryopreservation and HLA-DR matching on the cellular immunogenicity of human cardiac valve allografts.

The cellular immunogenicity of fresh and cryopreserved human cardiac valve leaflets was measured in a lymphocyte proliferation assay. One fresh leaflet and a cryopreserved leaflet derived from the same valve were cut into 2 mm diameter pieces and incubated with responder peripheral blood mononuclear cells, matched or mismatched for HLA-DR. The tritiated thymidine incorporation into the lymphocytes measured after 7 days, was expressed as stimulation index. Fresh, HLA-mismatched valve pieces induced high stimulation index in all cases (median 9, range 4 to 117). The cryopreservation procedure resulted in a significantly lower stimulation index (p = 0.002, Wilcoxon), with a median stimulation index of 2 (range 0 to 9). In the instances where HLA-DR matched combinations were studied, cryopreservation was also associated with lower stimulation index. HLA matching itself was able to reduce the stimulation index both with cryopreserved and fresh valve pieces as stimulator, resulting in a median stimulation index of 4 (range 2 to 117) for the HLA-DR-mismatched and 1 (range 0 to 5) for the matched lymphocytes (p = 0.006, Wilcoxon). In conclusion, human cardiac valves are able to stimulate immune competent cells in vitro, even after cryopreservation. The cellular allogeneic response in vitro could be an explanation for valve allograft degeneration observed in the clinic. Matching for HLA-DR may reduce these effects.

Electrocardiogram of the humpback whale (Megaptera novaeangliae), with specific reference to atrioventricular transmission and ventricular excitation.

OBJECTIVES: The objective of the study was to record the electrocardiogram (ECG) of a large whale to obtain crucial data for comparative electrophysiologic analysis. BACKGROUND: The data were needed to establish the mismatch between heart size and PR interval and QRS duration in mammals. METHODS: In the waters off the coast of Newfoundland, in two humpback whales (Megaptera novaeangliae) with an estimated weight of 30,000 kg a 1-lead ECG was recorded, enabling reliable assessment of P waves and QRS complexes. RESULTS: It was found that both the PR interval (atrioventricular [AV] transmission time) and QRS duration (ventricular excitation) are extremely short for animals of this size. These findings are difficult, if not impossible, to explain on the basis of currently accepted electrophysiologic theories. However, the narrow QRS complex may be due to a very dense His-Purkinje network in the ventricular wall of whales. Alternative mechanisms that can explain the function of the mammalian AV node need to be considered and explored. CONCLUSIONS: The results of the study may be of value for the understanding of the ECG in humans.