Comparison of University of Wisconsin and St Thomas' Hospital solutions on endothelium-derived hyperpolarizing factor-mediated function in coronary micro-arteries

BACKGROUND: It is controversial whether coronary endothelial function is impaired after cold exposure to University of Wisconsin (UW) or St. Thomas' Hospital (ST) solution during heart transplantation. We therefore examined the effects of cold storage of coronary micro-arteries with UW or ST solution on endothelium-derived hyperpolarizing factor (EDHF)-mediated function. METHODS: Porcine and human coronary arteries were immersed in either UW or ST solution at 4 degrees C for 4 hr and then normalized in a wire myograph. RESULTS: In the rings (normalized diameter: 200-500 microM) precontracted by U46619, EDHF-mediated relaxation and hyperpolarization were initiated by bradykinin (BK) or A23187 in the presence of indomethacin and NG-nitro-L-arginine. In the human coronary arteries, the EDHF-mediated relaxation to BK was reduced by UW solution from 53.2+/-5.6% to 24.0+/-2.7% (P=0.006). The reduced EDHF-mediated relaxation occurred concurrently with the decreased hyperpolarization to BK (17.0+/-1.5 vs. 10.5+/-1.1 mV, n=10, P=0.004) or A23187 in porcine coronary arteries. In the control arteries, K+ channel blockers, either glybenclamide or tetraethylammonium reduced the EDHF-mediated relaxation. After exposure to UW solution, the EDHF-mediated relaxation was further significantly inhibited. In contrast, ST solution did not affect these responses. CONCLUSIONS: These results show that in coronary micro-arteries, UW, but not ST, solution impairs the EDHF-mediated function and inhibits the Ca2+-activated and ATP-sensitive K+ channels. Our comparative study suggests that ST solution may be superior to UW solution in preserving the EDHF-related endothelial function of coronary micro-arteries.     

Histidine-tryptophan-ketoglutarate solution maximally preserves endothelium-derived hyperpolarizing factor-mediated function during heart preservation: comparison with University of Wisconsin solution.

BACKGROUND: The University of Wisconsin (UW) solution is used widely in heart preservation but has been demonstrated to be detrimental to the endothelial function. The present study compares the effect of histidine-tryptophan-ketoglutarate (HTK) and UW solutions on endothelium-derived hyperpolarizing factor (EDHF)-mediated function in porcine small coronary arteries. METHODS: An isometric force study was performed in a myograph and the membrane potential of a single smooth muscle cell was measured electrophysiologically. Small coronary arteries (diameter 457 +/- 15 microm) were incubated with UW (n = 8), HTK (n = 7) or Krebs solution (n = 15) at 4 degrees C for 4 hours. After washout, in the presence of indomethacin (Indo; 7 micromol/liter), N(G)-nitro-l-arginine (l-NNA; 300 micromol/liter) and oxyhemoglobin (HbO; 20 micromol/liter), bradykinin (BK; -10 to -6.5 log M)-induced relaxation was compared in U46619 (-8 log M) pre-contraction. EDHF-mediated hyperpolarization was elicited by BK (-6.5 log M) in the presence of Indo, l-NNA and HbO. RESULTS: BK-induced, EDHF-mediated relaxation was reduced from 93.6 +/- 2.8% to 79.7 +/- 4.6% after UW preservation (p = 0.01 by unpaired t-test and p = 0.005 by 2-way analysis of variance [ANOVA]), whereas HTK incubation did not decrease EDHF-mediated relaxation (87.0 +/- 6.5%, p = 0.3 by unpaired t-test and p = 0.6 by 2-way ANOVA, compared with control, and p = 0.001 by 2-way ANOVA, compared with UW). EDHF-mediated hyperpolarization (10.3 +/- 1.6 mV) was attenuated by UW exposure (3.4 +/- 0.6 mV, [p = 0.002] vs control), but not by HTK exposure (8.3 +/- 1.1 mV, [p = 0.3] vs control). CONCLUSIONS: HTK is superior to UW solution in protecting EDHF-mediated endothelial function in porcine small coronary arteries. The present findings supports the use of HTK solution in heart preservation.     

Epoxyeicosatrienoic acids (EET(11,12)) may partially restore endothelium-derived hyperpolarizing factor-mediated function in coronary microarteries.

Background. Endothelial cells derive nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The cytochrome P-450–monooxygenase metabolites of arachidonic acid (epoxyeicosatrienoic acids [EETs]) have been suggested to be EDHF. This study was designed to examine the effect of EET_11,12 with regard to the possibility of restoring EDHF function when added into hyperkalemic cardioplegic solution.

Methods. Porcine coronary microartery rings were studied in a myograph. In groups 1 and 2, paired arteries were incubated in either hyperkalemic solution (K⁺ 20 mmol/L) or Krebs’ solution (control). In group 3, the paired arteries were incubated in hyperkalemia plus EET_11,12 (1 × 10^−6.5 mol/L) or hyperkalemia alone (control) at 37°C for 1 hour, followed by Krebs’ washout and then precontracted with 1 × 10^−8.5 mol/L U46619. The EDHF-mediated relaxation to EET_11,12 (group 1) or bradykinin (groups 2 and 3) was studied in the presence of N^G-nitro-l-arginine, indomethacin, and oxyhemoglobin.

Results. After exposure to hyperkalemia, the EDHF-mediated maximal relaxation by bradykinin (72.5% ± 7.8% versus 41.6% ± 10.6%; p < 0.05), but not by EET_11,12 (18.4% ± 3.3% versus 25.1% ± 4.9%; p > 0.05) was significantly reduced. Incubation with EET_11,12 partially restored EDHF function (33.3% ± 9.5% versus 62.0% ± 8.5%; p < 0.05).

Conclusions. In coronary microarteries, hyperkalemia impairs EDHF-mediated relaxation, and EET_11,12 may partially mimic the EDHF function. Addition of EET_11,12 into cardioplegic solution may partially restore EDHF-mediated function reduced by exposure to hyperkalemia.     

Procaine in cardioplegia: the effect on EDHF-mediated function in porcine coronary arteries

OBJECTIVES: Hyperkalemia in cardioplegia impairs the endothelium-derived hyperpolarizing factor (EDHF)-mediated function. This study examined the effect of procaine in cardioplegia on the EDHF-mediated response in porcine coronary arteries. METHODS: An isometric force study was performed in a myograph. Two rings taken from the same artery (diameter 200-450 microm) were incubated with Krebs solution (group I) or 20 mM K+ (group II) with/without procaine (1 mM) at 37 degrees C for 1 hour. The EDHF-mediated relaxation was induced by bradykinin (BK, -10 approximately -6.5 log M) after U46619 (-8 log M, in group I) or K+-precontraction (in group II) in the presence of indomethacin (7 microM), NG-nitro-L-arginine (300 microM), and hemoglobin (20 microM). The membrane potential of a single smooth muscle cell was measured by a microelectrode after superfusion with Krebs solution with/without procaine for 1 hour. RESULTS: The EDHF-mediated relaxation was increased by the treatment with procaine with the EC50 shifted leftward (97.3 +/- 0.6% vs. 83.0 +/- 5.1% at -7 log M and 99.4 +/- 0.6% vs. 96.7 +/- 1.6% at -6.5 log M, p < 0.05; EC50: -8.57 +/- 0.24 vs. -7.92 +/- 0.23 log M, p < 0.05). Procaine decreased the BK-induced hyperpolarization from -72.3 +/- 0.7 mV to -68.8 +/- 0.8 mV (-6.5 log M, p < 0.01). The EDHF-mediated relaxation in arteries exposed to 20 mM K+ was not altered by procaine (49.9 +/- 7.4% vs. 55.8 +/- 7.6%, p > 0.05). CONCLUSIONS: In the coronary arteries, procaine has a depolarizing effect but it enhances EDHF-mediated relaxation. Addition of procaine in cardioplegia did not change the EDHF-mediated endothelial function.     

Cellular electrophysiological and mechanical effects of celsior solution on endothelial function in resistance coronary arteries

BACKGROUND: We investigated a relatively new organ preservation (Celsior) solution regarding its effect on the endothelium-derived hyperpolarizing factor (EDHF)-mediated function with comparison to St. Thomas Hospital (ST) solution. METHODS: The EDHF-mediated relaxation was induced by bradykinin (BK, -10 to -6.5 logM) in the presence of inhibitors of nitric oxide and prostacyclin in porcine small resistance coronary arteries, before and after incubation in ST (Group Ia, n=11), Celsior (Group Ib, n=13), or Krebs (Group Ic, control, n=12) at 4 degrees C for 4 hr. The EDHF-mediated hyperpolarization of the membrane potential of smooth muscle cells was measured by microelectrode with simultaneous relaxation after cold storage in ST (IIa, n=7), Celsior (IIb, n=6), or Krebs (IIc, control, n=6), or followed by washout with Krebs (ST: IIIa, n=6, Celsior: IIIb, n=6). RESULTS: The EDHF-mediated relaxation was significantly decreased in Group Ia (56.4+/-7.2% vs. 71.2+/-5.3%, P<0.05) and Ib (44.8+/-4.9% vs. 74.7+/-3.3%, P<0.05) but not in Ic. The sensitivity to BK was also significantly decreased (Ia: -7.51+/-0.14 vs. -7.76+/-0.12 log M, P<0.05; Ib: -7.36+/-0.09 vs. -7.60+/-0.09 logM, P<0.05). The resting membrane potential was depolarized in IIa (-44.3+/-1.9 mV, n=7, P<0.05) and IIb (-33.0+/-2.2 mV, n=6, P<0.05) compared with IIc (-57.1+/-1.5 mV, n=6). The EDHF-mediated hyperpolarization decreased significantly in IIa and IIb (3.4+/-0.3 and 3.0+/-0.2 vs. 6.3+/-0.5 mV, P<0.05) and partially restored in IIIa (5.0+/-0.2 vs. 3.4+/-0.3 mV, P<0.05) and IIIb (4.1+/-0.3 vs. 3.0+/-0.2 mV, P<0.05). CONCLUSIONS: Storage with Celsior and ST solutions reduces the EDHF-mediated endothelial function (hyperpolarization and associated relaxation) in porcine small resistance coronary arteries.     

Epoxyeicosatrienoic acids (EET11,12) may partially restore endothelium-derived hyperpolarizing factor–mediated function in coronary microarteries

Background. Endothelial cells derive nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). The cytochrome P-450–monooxygenase metabolites of arachidonic acid (epoxyeicosatrienoic acids [EETs]) have been suggested to be EDHF. This study was designed to examine the effect of EET_11,12 with regard to the possibility of restoring EDHF function when added into hyperkalemic cardioplegic solution.Methods. Porcine coronary microartery rings were studied in a myograph. In groups 1 and 2, paired arteries were incubated in either hyperkalemic solution (K⁺ 20 mmol/L) or Krebs’ solution (control). In group 3, the paired arteries were incubated in hyperkalemia plus EET_11,12 (1 × 10^−6.5 mol/L) or hyperkalemia alone (control) at 37°C for 1 hour, followed by Krebs’ washout and then precontracted with 1 × 10^−8.5 mol/L U46619. The EDHF-mediated relaxation to EET_11,12 (group 1) or bradykinin (groups 2 and 3) was studied in the presence of N^G-nitro-l-arginine, indomethacin, and oxyhemoglobin.Results. After exposure to hyperkalemia, the EDHF-mediated maximal relaxation by bradykinin (72.5% ± 7.8% versus 41.6% ± 10.6%; p < 0.05), but not by EET_11,12 (18.4% ± 3.3% versus 25.1% ± 4.9%; p > 0.05) was significantly reduced. Incubation with EET_11,12 partially restored EDHF function (33.3% ± 9.5% versus 62.0% ± 8.5%; p < 0.05).Conclusions. In coronary microarteries, hyperkalemia impairs EDHF-mediated relaxation, and EET_11,12 may partially mimic the EDHF function. Addition of EET_11,12 into cardioplegic solution may partially restore EDHF-mediated function reduced by exposure to hyperkalemia.     

Impaired endothelium-derived hyperpolarizing factor-mediated relaxation in porcine pulmonary microarteries after cold storage with Euro-Collins and University of Wisconsin solutions

BACKGROUND: Endothelium plays an important role in mediating the function of transplanted organs. The widely used University of Wisconsin solution impairs the endothelium-derived hyperpolarizing factor-mediated relaxation in coronary arteries, but little is known about effects of lung preservation on endothelium-derived hyperpolarizing factor-mediated endothelial function. This study examined the effect of organ preservation solutions on the endothelium-derived hyperpolarizing factor-mediated relaxation in the pulmonary microarteries (diameter 200 to 450 microm). METHODS: Two segments (1 as control) from the same microartery were allocated in 2 chambers of a myograph. After incubation with hyperkalemia (potassium 115 mmol/L), University of Wisconsin, or Euro-Collins solution (at 4 degrees C for 4 hours), the endothelium-derived hyperpolarizing factor-mediated relaxation was induced by bradykinin (-10 to -6.5 log M, n = 8) or calcium ionophore (A(23187), -9 to -5.5 log M, n = 7) in U(46619) (-7.5 log M) precontracted rings in the presence of indomethacin (7 micromol/L), N(G)-nitro-L-arginine (300 micromol/L), and oxyhemoglobin (20 micromol/L). RESULTS: Exposure to hyperkalemia and storage with Euro-Collins or University of Wisconsin solution significantly decreased the relaxation to bradykinin (51.9 +/- 8.4% vs 60.3 +/- 6.1%, P =.02 or 49.3 +/- 7.3% vs 65.2 +/- 3.5%, P =.04) or A(23187) (12.5 +/- 0.02% vs 33.8 +/- 0.07%, P =.02 or 13.2 +/- 0.03% vs 31.0 +/- 0.05%, P =.03%). CONCLUSIONS: Endothelium-derived hyperpolarizing factor plays an important role in porcine pulmonary microarteries, and the endothelium-derived hyperpolarizing factor-mediated relaxation is impaired when the lung is preserved with University of Wisconsin or Euro-Collins solution. This impairment may affect the lung function during the reperfusion period after lung transplantation.     

Altered endothelium-derived hyperpolarizing factor-mediated endothelial function in coronary microarteries by St Thomas' Hospital solution.

OBJECTIVES: We examined the effect of St Thomas' Hospital solution on endothelium-derived hyperpolarizing factor-mediated function in the porcine coronary microarteries with emphasis on the effect of temperature and washout time. METHODS: Microartery rings (diameter, 200-450 micrometers) were studied in myograph. The arteries were incubated in St Thomas' Hospital or Krebs solution (control) at 4 degrees C for 4 hours followed by 45 minutes (group Ia) or 90 minutes washout (group Ib) or at 22 degrees C for 1 hour followed by 45 minutes (group IIa) or 90 minutes washout (group IIb) and precontracted with -8.5 log M U 46619. The endothelium-derived hyperpolarizing factor-mediated relaxation to bradykinin was studied when endothelium-derived nitric oxide and prostaglandin I2 were inhibited with the presence of 7 micromol/L indomethacin and 300 micromol/L NG-nitro-L -arginine. RESULTS: After exposure to St Thomas' Hospital solution, the maximal endothelium-derived hyperpolarizing factor-mediated relaxation (percentage of the precontraction) was significantly reduced at either temperature after washout for 45 minutes (group Ia, 42.7% +/- 3.5% vs 69.0% +/- 5.3%; n = 9; P =.000; and group IIa, 12.3% +/- 1.6% vs 56.1% +/- 4. 4%; n = 8; P =.000) but fully recovered after washout for 90 minutes. The U46619-induced contraction force was also significantly reduced after washout for 45 minutes (P <.001) but fully recovered at 90 minutes. CONCLUSIONS: Under profound and moderate hypothermia, St Thomas' Hospital solution impairs endothelium-derived hyperpolarizing factor-mediated relaxation and smooth muscle contraction in the coronary microarteries. These effects exist during the reperfusion period for at least 45 minutes after exposure to St Thomas' Hospital solution and may account for the possible myocardial dysfunction during reperfusion.     

University of Wisconsin solution increases hyperpolarizing mechanisms in response to bradykinin

BACKGROUND: The aim of this study was to determine the effect of University of Wisconsin solution (UWS) incubation on bradykinin-induced vasodilation. METHODS: Porcine coronary arteries were incubated in Krebs-Henseleit solution (KHS) or UWS at 4 degrees C for 20 hours. Endothelium-dependent relaxation to bradykinin and endothelium-independent relaxation to nitric oxide were tested after U46619 or KCl pre-contraction. Nitric oxide synthase activity and protein expression was determined by [3H]-L-citrulline formation and western blot analysis, respectively. RESULTS: The relaxation to bradykinin (0.1 to 300 nmol/liter) after U46619 (30 to 300 nmol/liter) pre-contraction was similar with both KHS and UWS pre-incubation; however, it was reduced after KCl pre-contraction (15 to 20 mmol/liter), this reduction being greater after UWS incubation. The inhibitory effect of N(G)-nitro-L-arginine methylester (0.1 mmol/liter) on bradykinin-induced relaxation was lower in UWS- than KHS-incubated segments after U46619 pre-contraction, but similar after KCl pre-contraction; however, the inhibitory effect of 0.5 mmol/liter ouabain was unaffected. Tetraethylammonium (5 mmol/liter) reduced the response to bradykinin more strongly after UWS pre-incubation. UWS did not modify relaxation to nitric oxide (0.1 to 30 micromol/liter) in pre-incubated UWS or KHS segments. UWS failed to modify both total nitric oxide synthase activity and endothelial nitric oxide synthase expression. CONCLUSIONS: UWS incubation decreased nitric oxide participation and increased the hyperpolarizing mechanisms produced by bradykinin.     

Effect of potassium-channel openers on the release of endothelium-derived hyperpolarizing factor in porcine coronary arteries stored in cold hyperkalemic solution

Hyperkalemic solution is widely used to protect the myocardium during open-heart surgery or to preserve donor hearts during heart or heart/lung transplants. The inhibitory effects of hvperkalemic solution on the release of endothelium-derived hyperpolarizing factor (EDHF) of coronary arteries following deep hypothermic storage (4 degrees C) has been well studied. However, it has not been established whether potassium channel openers have protective effects on the coronary endothelial function after cold storage. This study was designed to examine this. Porcine coronary artery rings were studied in organ baths. Relaxation in response to the EDHF stimulus A23187 (nonreceptor-mediated stimulus calcium ionophore) in thromboxane A2 mimetic U46619 (30 nmol/L)-induced precontraction after incubation with hyperkalemic solution (20 mmol/L) with nicorandil (10 micromol/L) (either at 37 degrees C in the oxygenated organ chamber or at 4 degrees C in a refrigerator for 6 h) was compared with the control. There was significant difference between hyperkalemia group and hyperkalemia with nicorandil group under normothermia (p = .04). The difference was significant in the same solution between normothermia and hypothermia. After incubation in hyperkalemic solution without or with nicorandil, the A23187-induced relaxation was 32.8% +/- 9.1% and 72.6% +/- 16.9%, respectively (N = 8, p < .01). Potassium channel opener can attenuate the inhibitory effect of hyperkalemic solution on the release of EDHF after cold storage.     

Alteration of cellular electrophysiologic properties in porcine pulmonary microcirculation after preservation with University of Wisconsin and Euro-Collins solutions

BACKGROUND: The effect of cold storage of porcine pulmonary microvessels in University of Wisconsin (UW) and Euro-Collins (EC) solutions on the cellular electrophysiologic properties remains unknown. METHODS: The pulmonary microarteries (PA, 381.6 +/- 62.8 microm; n = 60) and microveins (PV, 360.8 +/- 54.5 microm; n = 60) were incubated with Krebs (control), UW, or EC solution at 4 degrees C for 4 hours in a myograph. The resting membrane potential and the endothelium-derived hyperpolarizing factor-mediated hyperpolarization to bradykinin (0.1 micromol/L) in the presence of inhibitors of nitric oxide and prostacyclin, N(omega)-nitro-l-arginine, hemoglobin, and indomethacin, in a single smooth muscle cell were directly measured. RESULTS: The resting membrane potential (-60.8 +/- 1.3 mV in PA and -48.1 +/- 0.7 mV in PV, n = 6) was depolarized after exposure to UW solution (to -18.4 +/- 0.7 mV in PA and -13.6 +/- 0.8 mV in PV; n = 8; p < 0.001). The amplitude of endothelium-derived hyperpolarizing factor-mediated hyperpolarization to bradykinin was also decreased (from 7.4 +/- 0.7 mV to 2.6 +/- 0.7 mV in PA and from 4.6 +/- 0.5 mV to 0.9 +/- 0.4 mV in PV; p < 0.001). In comparison, EC depolarized the membrane potential to a lesser extent (to -28.3 +/- 0.9 mV in PA and to -21.3 +/- 0.8 mV in PV; n = 8; p < 0.001) and almost abolished the hyperpolarization to bradykinin. After washout, hyperpolarization was partially restored (UW, 4.9 +/- 0.7 mV in PA and 2.0 +/- 0.3 mV in PV. p < 0.01; EC, 2.3 +/- 0.5 mV in PA and 1.0 +/- 0.3 mV in PV. p < 0.01). CONCLUSIONS: Cold storage of porcine PA and PV with UW or EC solution impairs the electrophysiologic properties (hyperpolarization) related to endothelium-smooth muscle interaction. The alteration is more profound with EC than UW solution and in veins than in arteries. The findings urge further studies on lung preservation solutions.     

Preservation of steatotic livers in IGL-1 solution.

A new Institut Georges Lopez (IGL-1) solution was used to preserve steatotic livers. Steatotic (obese [Ob]) and nonsteatotic (lean [Ln]) livers from Zücker rats (n = 16, 8 Ln and 8 Ob) were preserved for 24 hours at 4 degrees C in University of Wisconsin (UW) or IGL-1 solution, respectively, and then perfused ex vivo for 2 hours at 37 degrees C. Additionally, Ob and Ln livers (n = 16, 8 Ln and 8 Ob) were preserved in IGL-1 plus Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME). Hepatic injury and function (aminotransferases, bile production, bromosulfophthalein clearance), and factors potentially involved in the susceptibility of steatotic livers to ischemia-reperfusion injury, such as oxidative stress, mitochondrial damage, and vascular resistance, were studied. Nitric oxide (NO) production and constitutive and inducible NO synthase were also measured. Steatotic and nonsteatotic livers preserved in IGL-1 solution showed lower transaminases, malondialdehyde, glutamate dehydrogenase levels, and higher bile production than UW-solution-preserved livers. IGL-1 solution protected against oxidative stress, mitochondrial damage and the alterations in vascular resistance associated with cold ischemia-reperfusion. Thus, at the end of reperfusion period, aspartate aminotransferase levels in steatotic livers were 281 +/- 6 U/L in UW vs. 202 +/- 10 U/L in IGL-1 solution. Glutamate dehydrogenase was 463 +/- 75 U/L in UW vs. 111 +/- 4 U/L in IGL-1 solution, and oxidative stress was 3.0 +/- 0.1 nmol/mg prot in UW vs. 2.0 +/- 0.1 nmol/mg prot in IGL-1 solution. These beneficial effects of IGL-1 solution were abolished by the addition of L-NAME, which implicates NO in the benefits of IGL-1. In conclusion, IGL-1 solution provided steatotic livers with better protection against the deleterious effects of cold ischemia-reperfusion injury than did UW solution.     

Impaired EDHF-Mediated Relaxation in Porcine Pulmonary Micro-Arteries by Cold Storage with University of Wisconsin and Euro-Collins Solutions

Background: Vascular endothelium plays a key role in regulation of vascular tone. Hyperkalemia has been demonstrated to impair the EDHF‐mediated endothelial function in coronary circulation. University of Wisconsin (UW) and Eruo‐collins (EC) solutions are used for organ preservation in transplantation surgery. The potassium concentration in UW or EC solutions is as high as 125 mmol/L or 115 mmol/L, respectively. This study was designed to examine whether hyperkalemia or storage with UW and EC solutions affects the relaxation mediated by EDHF in the porcine pulmonary micro‐arteries. Methods: Porcine pulmonary micro‐artery rings (diameter 200–450 μm) were studied in myograph (n = 8 in each group). After incubation with hyperkalemia (K⁺ 125 mmol/L, at 37° C), UW or EC solutions (at 4° C for 4 hours), EDHF‐mediated relaxation induced by bradykinin (BK, ?10 to ?6.5 log M) in the presence of inhibitors for cyclooxygenase (Indomethacin, 7 μM), nitric oxide synthase (N^G‐nitro‐_L‐arginine, 300 μM), and oxyhemoglobin (20 μM) was compared with control (Krebs' solution) in precontraction with U₄₆₆₁₉ (?7.5 log M). Results: The EDHF‐mediated relaxation to BK was 69.6 ± 6.3% compared with 97.1 ± 1.7% (p= 0.003) in control (no inhibitors). After incubation with hyperkalemia, the relaxation significantly decreased (38.6 ± 3.0% vs. 59.1 ± 7.4%, p= 0.03 ). Storage with UW or EC solutions also significantly decreased the relaxation (49.3 ± 7.3% vs. 65.2 ± 3.5%, p= 0.04 and 51.9 ± 8.4% vs. 60.3 ± 6.1%, p= 0.02 , respectively). Conclusions: In porcine pulmonary micro‐arteries, exposure to hyperkalemia or storage with UW or EC solutions at 4°C for 4 hours impairs the EDHF‐mediated endothelial function. The clinical significance of this effect should be further studied.     

Optimized cardiac graft preservation: a comparative experimental study using P-31 magnetic resonance spectroscopy and biochemical analyses.

BACKGROUND: The University of Wisconsin (UW), St. Thomas (ST) and Broussais (B) solutions were compared to the CRMBM solution, that we developed for long term heart preservation. METHODS: Isolated isovolumic rat hearts were arrested with each cardioplegic solution (n = 5) to 8 hearts in each group), submitted to 12 hours of cold storage (4 degrees C) in the same solution and then reperfused for 60 minutes at 37 degrees C. Function was measured during control and reflow. High energy phosphates and intracellular pH were monitored by P-31 magnetic resonance spectroscopy. Analyses were performed by biochemical assays and HPLC in coronary effluents (CK, Pi, lactate, purines) and in freeze-clamped hearts (amino acids, nucleotides, CK, LDH) at the end of reperfusion. RESULTS: Functional recovery was significantly improved with the new cardioplegic solution (50+/-12% recovery for the rate pressure product at the end of reflow vs 8+/-3% with UW, 0% with B and with ST). This result was correlated with the best metabolic and cellular protection as assessed in particular by higher PCr levels during reflow (30+/-3% vs 10+/-3% with UW, 8+/-4% with B, and 7+/-1% with ST) as well as reduced creatine kinase leakage during reflow (110+/-15 IU/60 minute vs 270 +/- 57 IU/60 minute with UW, 323+/-36 IU/60 minute with Broussais solution and 237+/-18 IU/60 minute with ST). CONCLUSION: This new solution is more effective in prolonged myocardial protection than the three most widely used solutions.     

Improved survival of orthotopic liver allograft in swine by addition of trophic factors to University of Wisconsin solution

Serum-free preservation media such as University of Wisconsin (UW) may cause tissue damage through trophic factor (TF) deprivation. This study evaluated whether the addition of TFs to UW solution improves liver graft quality after extended cold preservation time in pigs. UW solution was supplemented with epidermal growth factor, insulin-like growth factor-1, nerve growth factor-beta, bactenecin, and substance P to create TF-supplemented (TFS) UW. Orthotopic liver transplantation was performed after 18 hr of static cold storage at 4 degrees C in UW (n=7) or TFS-UW (n=7) solution. Recipients of grafts preserved with TFS-UW demonstrated significantly better 5-day survival (57%) than those preserved with UW alone (14%) (P<0.05). Adenosine triphosphate content in grafts preserved in TFS-UW was significantly higher than in grafts preserved in UW (17.4+/-5.0 vs. 4.8+/-1.2 nmol/mg protein, respectively) (P<0.05). This study showed that the addition of TFs to UW solution allowed a significant extension of cold ischemic time in pigs.     

Functional recovery of hearts after cardioplegia and storage in University of Wisconsin and in St. Thomas' Hospital solutions

There are conflicting reports of the beneficial effects of University of Wisconsin (UW) cardioplegic solution used in heart preservation techniques. Therefore we investigated the efficacy of myocardial protection in adult rat hearts subjected to single-dose infusion (3 minutes) of nonoxygenated cardioplegic solutions (UW or St. Thomas' Hospital solution No. 2 [STH]) and stored at 4 degrees C by immersion in the same solution or in saline solution. Isolated working-heart preparations (n = 8 per group) were used to assess the prearrest (20 minutes' normothermic perfusion) and postischemic left ventricular functions. Four groups of hearts underwent 5, 8, 10, and 20 hours of cold ischemia (4 degrees C) in UW solution. Hearts stored for 8 to 20 hours showed no postischemic recovery of cardiac pump function (aortic flow, 0%), had decreased levels of myocardial high-energy phosphates, and were highly edematous (50% to 70% increased). After 5 hours of storage there was also poor recovery of aortic flow, coronary flow, and aortic pressure (55.0% +/- 19.4%, 67.1% +/- 5.1%, and 58.1% +/- 11.7%, respectively) but good recovery of adenosine triphosphate, creatine phosphate, and guanosine triphosphate (18.54 +/- 1.42, 29.99 +/- 2.05, and 1.64 +/- 0.14 mumol/gm dry weight, respectively). In contrast, hearts arrested and stored in STH solution for 5 hours rapidly established normal left ventricular functions (aortic flow, 111.5% +/- 2.5%; cardiac output, 99.1% +/- 1.2%; coronary flow, 85.0% +/- 3.4%; heart rate, 95.8% +/- 2.7%; and aortic pressure, 94.6%). A group of hearts arrested with STH solution but stored in saline solution recovered more slowly, had only partial return of function (aortic flow, 73.6% +/- 14.8%; p less than 0.01 vs STH/STH group), and had significantly greater tissue water content (8.020 +/- 0.080 vs 6.870 +/- 0.126 ml/gm dry wt; p less than 0.01). These results demonstrate the superior preservation of explanted hearts at 4 degrees C obtained by STH cardioplegic solution compared with UW solution under conditions used for transplantation.     

Intracellular free calcium accumulation in ferret vascular smooth muscle during crystalloid and blood cardioplegic infusions.

OBJECTIVE: The effects of magnesium- and potassium-based crystalloid and blood-containing cardioplegic solutions on coronary smooth muscle intracellular free calcium ([Ca2+]i) accumulation and microvascular contractile function were examined. METHODS: Isolated ferret hearts were subjected to hyperkalemic (25 mmol/L K+) blood cardioplegic infusion, hypermagnesemic (25 mmol/L Mg2+, K+-free) crystalloid cardioplegic infusion, or hyperkalemic crystalloid cardioplegic infusion for 1 hour. Coronary arterioles were isolated, cannulated, and loaded with fura 2. Reactivity and [Ca2+]i were assessed with videomicroscopy. [Ca2+]i was measured at baseline and after application of 50 mmol/L KCl. In addition, [Ca2+]i and vascular contraction were measured during exposure to Mg2+ and K+ cardioplegic solution at both 4 degrees C and 37 degrees C. RESULTS: From a baseline [Ca2+]i of 177 +/- 52 nmol/L, K+ cardioplegic infusion (302 +/- 80 nmol/L potassium) markedly increased [Ca2+]i, whereas blood cardioplegic infusion (214 +/- 53 nmol/L) and Mg2+ cardioplegic infusion (180 +/- 42 nmol/L) did not alter [Ca2+]i. Although a difference between groups in percentage contraction after application of 50 mmol/L KCl was not observed, [Ca2+]i increased significantly more in vessels in the control group (764 +/- 327 nmol/L) and the K+ crystalloid cardioplegic infusion group (698 +/- 215 nmol/L) than in vessels in the blood cardioplegic infusion group (402 +/- 45 nmol/L) and the Mg2+ cardioplegic infusion group (389 +/- 80 nmol/L). Mg2+ cardioplegic solution induced no microvascular contraction at either 4 degrees C or 37 degrees C, nor was an increase in [Ca2+]i observed. K+ cardioplegic solution induced microvascular contraction at 37 degrees C but not at 4 degrees C; it increased [Ca2+]i at both 4 degrees C and 37 degrees C. CONCLUSION: An Mg2+-based cardioplegic solution, or appropriate Mg2+ or blood supplementation of a K+ crystalloid cardioplegic solution, may decrease the accumulation of [Ca2+]i in the vascular smooth muscle during ischemic arrest.     

Protective effect of magnesium on the endothelial function mediated by endothelium-derived hyperpolarizing factor in coronary arteries during cardioplegic arrest in a porcine model

OBJECTIVES: Hyperkalemia in cardioplegia impairs the function mediated by endothelium-derived hyperpolarizing factor. This study examined the effect and mechanism of magnesium ion on the relaxation mediated by endothelium-derived hyperpolarizing factor. METHODS: In the isometric force study, porcine coronary microarteries in a myograph (diameter 200-450 microm) were incubated in Krebs solution (subgroups Ia, IIa, and IIIa), potassium ion (20 mmol/L, subgroups Ib, IIb, and IIIb), magnesium ion (16 mmol/L, subgroups Ic, IIc, and IIIc), or potassium ion plus magnesium ion (subgroups Id, IId, and IIId) for 1 hour at 37 degrees C in group I or II, followed by washout for 45 minutes in group III (n = 8). Relaxation to bradykinin (groups I and III) or sodium nitroprusside (group II) in U(46619)-stimulated contraction was established. In the electrophysiologic study, the membrane potentials of single smooth muscle cells of arteries were measured by microelectrode after superfusion with the previously described solutions (subgroups IVa-IVc). RESULTS: In group I, 20-mmol/L potassium ion greatly reduced the bradykinin-induced relaxation (35.0% +/- 4.9% vs 86.0% +/- 5.3%, P <.001), which was significantly restored by magnesium ion (51.9% +/- 4.0%, P =.017). In groups II and III, the bradykinin- or nitroprusside-induced relaxation had no significant differences. In group IV, potassium ion depolarized the smooth muscle and decreased the bradykinin-induced hyperpolarization (-72.0 +/- 1.5 vs -61.7 +/- 0.7 mV, n = 7, P <.001), which was significantly restored by magnesium ion (-68.0 +/- 2.5 mV vs -72.5 +/- 1.5 mV, n = 6, P =.029). CONCLUSIONS: Magnesium ion, either alone or added to hyperkalemic solutions, preserves or helps to restore the endothelial function mediated by endothelium-derived hyperpolarizing factor. The mechanism is related to preservation of the membrane hyperpolarization and reversal of the potassium-induced membrane depolarization of the smooth muscle cell.     

Hypoxic preconditioning in coronary microarteries: role of EDHF and K+ channel openers

BACKGROUND: Hypoxic preconditioning may provide a useful method of myocardial protection in cardiac operations. The present study was designed to investigate the possible mechanisms of preconditioning regarding endothelium-derived hyperpolarizing factor (EDHF) and the effect of a potassium channel opener KRN4884 on the porcine coronary microartery in mimicking hypoxic preconditioning. METHODS: Porcine coronary microartery rings (diameter 200 to 500 microm) studied in a myograph were divided into seven groups: (1) control group; (2) hypoxia-reoxygenation group (hypoxia for 60 minutes followed by reoxygenation for 30 minutes); (3) preconditioning group (hypoxia for 5 minutes followed by reoxygenation for 10 minutes before hypoxia reoxygenation); (4) KRN4884 pretreatment group (KRN4884 was added into the myograph chamber 20 minutes before hypoxia reoxygenation); (5) 5-hydroxydecanoate + KRN group (5-hydroxydecanoate was given 20 minutes before KRN4884 pretreatetment); (6) glibenclamide (GBC) + KRN group (GBC was added 20 minutes before KRN4884 pretreatment); and (7) endothelium denuded group (the endothelium was removed). The endothelium-derived hyperpolarizing factor-mediated relaxation to bradykinin was studied in the rings precontracted with U46619 in the presence of N(omega)-nitro-L-arginine and indomethacin. RESULTS: The maximal relaxation induced by bradykinin was reduced in hypoxia reoxygenation (40.7% +/- 2.8% vs 66.9% +/- 2.5% in control, p = 0.000). This reduced relaxation was recovered in either preconditioning (64.6% +/- 4.6%, p = 0.002), or KRN4884 pretreatment (67.1% +/- 3.6%, p = 0.000). The 5-hydroxydecanoate, but not GBC pretreatment abolished the effect of KRN44884 pretreatment (67.1% +/- 3.6% vs 42.9% +/- 3%, p = 0.001). CONCLUSIONS: Hypoxia reoxygenation reduces the relaxation mediated by endothelium-derived hyperpolarizing factor in the coronary microartery. This function can be restored by either hypoxic preconditioning or the K(ATP) channel opener KRN4884, and therefore K(ATP) channel openers may provide similar effect as preconditioning. The mechanism is mainly related to the mitochondrial ATP-sensitive K+ channels.     

Prolonged 24-hour subzero preservation of heterotopically transplanted rat hearts using antifreeze proteins derived from arctic fish

BACKGROUND: Arctic fish survive subzero temperatures by producing a family of antifreeze proteins (AFPs) that noncolligatively lower the freezing temperature of their body fluids. We report 24-hour storage of mammalian hearts for transplantation at subzero temperatures using AFPs derived from arctic fish. METHODS: Forty-two heterotopic transplantations were performed in isoimmune Sprague-Dawley rats. Harvested hearts were retrogradely infused with cold 4 degrees C University of Wisconsin (UW) solution and were preserved in a specialized cooling bath at two target temperatures, 4 degrees C and -1.3 degrees C for 12,18, and 24 hours (6 experiments/group). Preservation solutions were UW alone for the 4 degrees C group, and UW with 15 mg/mL AFP III for the -1.3 degrees C group. After hypothermic storage the hearts were heterotopically transplanted into isoimmune rats. Viability was assessed and graded on a scale of 0 to 6 (0 = no contractions to 6 = excellent contractions). Transplanted hearts were then fixed in vivo and were subject to electron microscopy and histopathologic examination. RESULTS: None of the hearts preserved at -1.3 degrees C in UW/AFP III solution froze. All control hearts preserved at -1.3 degrees C without AFP protection froze and died at reperfusion. Viability of hearts preserved at -1.3 degrees C in UW/AFP III solution was significantly better after 18 hours of preservation, 30 and 60 minutes after reperfusion (median, 5 versus 3 and 6 versus 3, respectively; p < 0.05) and after 24 hours of preservation 30 and 60 minutes after reperfusion (median, 4.5 versus 1.5 and 5 versus 2, respectively; p < 0.05). Histologic and electron microscopy studies demonstrated better myocyte structure and mitochondrial integrity preservation with UW/AFP III solution. CONCLUSIONS: Antifreeze proteins prevent freezing in subzero cryopreservation of mammalian hearts for transplantation. Subzero preservation prolongs ischemic times and improves posttransplant viability.