Several biological mechanisms have been proposed to account for age-related cognitive decline including cerebrovascular disease, mitochondrial dysfunction, inflammation, impaired neuronal signaling, and reduced antioxidant capacity. Oxidative stress has been well studied although more intervention trials are needed. A limited number of clinical trials using nutraceuticals to target oxidative stress and improve cognitive function have been undertaken. Of these, the most promising interventions are Coenzyme Q10 (CoQ10), pycnogenol, and the nonflavonoid polyphenols, curcumin, and resveratrol. This chapter discusses some of the mechanisms underlying cognitive aging, with a particular focus on oxidative stress and reviews the efficacy and mechanisms by which nutraceuticals improve cognitive function. We conclude that while there is some evidence that nutraceuticals with antioxidant activity may reduce cognitive decline, and that there are plausible biological mechanisms by which this may occur, large and long-term clinical trials are lacking and results are somewhat inconsistent. A recently completed trial completed by our group, the Australian Research Council Longevity Intervention, may shed some light on this topic.

Background. Use of donation after circulatory death (DCD) hearts is becoming more prevalent in cardiac transplantation. However, there is no standardized approach to myocardial preservation, and little data exists on ultrastructural changes in DCD hearts. We have previously identified increased proapoptotic and proinflammatory activity in brain dead donor (BDD) hearts that subsequently exhibit primary graft failure and lower levels in DCD left atrial tissue. This study further investigates these markers and correlates them with cardiac function in DCD hearts. Methods. This prospective study used donor hearts deemed unsuitable for transplant after gaining institutional ethics approval; 11 human hearts were obtained from 5 DCD donors and 6 BDDs. All hearts were preserved by continuous microperfusion for 4 hours with a cold crystalloid solution and then were evaluated on a blood perfusion bench rig. After 4 hours perfusion and working assessment, tissues from all cardiac chambers were stored for later messenger RNA (mRNA) analysis for proapoptotic and proinflammatory markers. Results. Significantly raised levels of caspase-1, BNIP3, and NADPH oxidase mRNA expression were identified in cardiac chambers from BDD hearts compared to DCD hearts, and these differences were exaggerated in older donors. In the pooled analysis, lower expression of caspase-1, NF-KB1, and BNIP3 mRNA correlated with developed pressure at 1 hour after reperfusion in the right ventricle, but not the left. Conclusion. Compared to BDD hearts, DCD hearts exhibit less stimulation of proapoptotic cascades and reactive oxygen species, potentially reducing their susceptibility to ischemic reperfusion injury.

Background. Donation after circulatory death (DCD) represents an increasing source of organs. However, evaluating the suitability of DCD hearts for transplantation represents a challenge. Contractile function is the ultimate determinant of recovery. We developed a novel technique in an ex vivo rig for the measurement of contractility using intraventricular balloons. We compared this technique with the measurement of lactate metabolism, the current gold standard. Methods. Human DCD (n = 6) and donation after brain death (n = 6) hearts were preserved by perfusion with a cold oxygenated crystalloid solution for 4 h, transferred to a blood perfusion rig at 37 °C where balloons were inserted into the left (LV) and right (RV) ventricles to measure developed pressure (DP = systolic minus diastolic). Perfusate lactate levels were measured for metabolic assessment. Concordance between LVDP and lactate was assessed during 4 h using cutoffs for LVDP of 70 mm Hg and for lactate of 10 mmol/L. Results. Measurements of contractile function (LVDP) and metabolism (lactate levels) were deemed concordant in 7 hearts with either a high LVDP (mean 100 mm Hg) with low lactate (mean 6.7 mmol/L)) or a low LVDP (15 mm Hg) with high lactate (mean 17.3 mmol/). In the remaining 5 hearts, measurements were deemed discordant: 4 hearts had high LVDP (mean 124 mm Hg), despite high lactate levels 17.3 mmol/L) and 1 had low LVDP (54 mm Hg) but low lactate (6.9 mmol/L). Conclusions. The intraventricular balloon technique provides useful information regarding contractile recovery of donor hearts that if combined with lactate metabolism has potential application for the evaluation of DCD and marginal donation after brain death hearts before transplant.

Heart failure (HF) is one of the most common causes of death in Western society. Recent results underscore the utility of coenzyme Q(10) (CoQ(10)) addition to standard medications in order to reduce mortality and to improve quality of life and functional capacity in chronic heart failure (CHF). The rationale for CoQ(10) supplementation in CHF is two-fold. One is the well-known role of CoQ(10) in myocardial bioenergetics, and the second is its antioxidant property. Redox balance is also improved by oral supplementation of CoQ(10), and this effect contributes to enhanced endothelium-dependent relaxation. Previous reports have shown that CoQ(10) concentration is decreased in myocardial tissue in CHF and by statin therapy, and the greater the CoQ(10) deficiency the more severe is the cardiocirculatory impairment. In patients with CHF and hypercholesterolaemia being treated with statins, the combination of CoQ(10) with a statin may be useful for two reasons: decreasing skeletal muscle injury and improving myocardial function. Ubiquinol, the active reduced form of CoQ(10), presents higher bioavailability than the oxidised form ubiquinone, and should be the preferred form to be added to a statin. The combination ezetimibe/simvastatin may have advantages over single statins. Since ezetimibe reduces absorption of cholesterol and does not affect CoQ(10) synthesis in the liver, the impact of this combination on CoQ(10) tissue levels will be much less than that of high dose statin monotherapy at any target low density lipoprotein-cholesterol (LDL-C) level to be reached. This consideration makes the ezetimibe/statin combination the ideal LDL-lowering agent to be combined with ubiquinol in CHF patients. However, particular caution is advisable with the use of strategies of extreme lowering of cholesterol that may negatively impact on myocardial function. All in all there is a strong case for considering co-administration of ubiquinol with statin therapy in patients with depressed or borderline myocardial function.

Introduction: Statin drugs have become the most highly prescribed drugs for cardiovascular disease. However, there is disagreement as to the existence of adverse effects of statin administration on cognitive function. Therefore, it is important to better understand the effects of statins on cognition and possible mechanisms of these effects. Areas covered: We analyzed relevant studies of the relationship between cognitive performance and statin and usage. We included articles published between 2018 and 1992. We identified three randomized trials, one observational study and 66 case reports that provided credible evidence of statin-induced cognitive impairment. We also identified seven randomized trials and two observational studies reporting no significant evidence of statin-induced cognitive impairment. Expert opinion: We found methodological differences that may have contributed to the divergence of these results. Evaluation of all these studies indicated that statin-associated cognitive decline is a real entity. Likely mechanisms to explain the adverse effects include 1. Reduction of synthesis of coenzyme Q(10) with consequent increasing oxidative stress and reduction of cerebral energy production; 2. Depletion of central nervous system myelin by inhibition of cholesterol synthesis. We conclude that statin-induced cognitive decline does exist, needs to be better recognized and requires more studies of prevention and treatment.

Introduction: With an aging population there is an important need for the development of effective treatments for the amelioration of cognitive decline. Multiple mechanisms underlie age-related cognitive decline including cerebrovascular disease, oxidative stress, reduced antioxidant capacity and mitochondrial dysfunction. CoQ10 is a novel treatment which has the potential to improve brain function in healthy elderly populations due to established beneficial effects on mitochondrial function, vascular function and oxidative stress. Methods and Analysis: We describe the protocol for a 90-day randomized controlled trial which examines the efficacy of Ubiquinol (200 mg/day) vs. placebo for the amelioration of cognitive decline in a healthy (non-demented) elderly sample, aged 60 years and over. The primary outcome is the effect of Ubiquinol at 90 days compared to baseline on CogTrack composite measures of cognition. Additional cognitive measures, as well as measures of cardiovascular function, oxidative stress, liver function and mood will also be monitored across 30-, 60- and 90- day time points. Data analyses will involve repeated measures analysis of variance (ANOVA). Discussion: This study will be the first of its kind to provide important clinical and mechanistic data regarding the efficacy of Ubiquinol as a treatment for age-related cognitive decline in the healthy elderly with important implications for productivity and quality of life within this age group. Clinical Trial Registration: The trial has been registered with the Australian and New Zealand Clinical Trials Registry (ANZCTRN12618001841268).

Background The aim of this study was to define the status of preoperative zinc levels in patients with heart disease presenting for cardiac surgery and to identify any predictors for and any clinical consequences of low zinc levels. Methods Adult patients presenting for elective surgery, either coronary artery bypass graft surgery and/or valve replacement, provided a fasting blood sample on the day of admission for surgery. Plasma and erythrocyte zinc levels were analysed and the levels correlated with the patient's characteristics and clinical outcomes. Results Of 56 patients 53% (n=30) had abnormally low plasma zinc levels (<12μmol/L) and 5.5% (n=3) had abnormally low erythrocyte zinc levels (<160μmol/L), indicative of deficiency. There were significant associations between lower plasma zinc levels and the presence of hypertension (p=0.02), hypercholesteraemia (p=0.02) and higher body mass index (BMI) (p=0.034) but no effect on major postoperative clinical outcomes. Conclusions This small study shows that zinc deficiency is common in cardiac surgery patients, especially in the presence of hypertension, hypercholesterolaemia or obesity. The effects of zinc deficiency in cardiac surgery need to be further investigated.

Background We previously showed that donation after circulatory death (DCD) canine hearts can be resuscitated if perfused with warm blood. However, clinical application of this technique is complex and difficult. We have developed a simplified system of cold crystalloid perfusion and compared it with standard cold storage for DCD heart preservation. Methods Anesthetized greyhounds underwent 30 minutes DCD by withdrawal of ventilation followed by assignment to either 4 hours of perfusion (n = 6) or cold storage (n = 7). Nonpreserved hearts (n = 5) served as a normal reference group. Perfusion hearts were reperfused with a protective solution then perfused for 4 hours with a novel oxygenated, nutrient-containing solution at 20 mL/min at 4°C to 10°C. Cold storage hearts were flushed with St Thomas' cardioplegic solution and stored in ice. After preservation, the recovery of the hearts was assessed on a blood-perfused working heart rig. Results During preservation, perfusion hearts consumed oxygen (0.09 ± 0.01 mL/100 g per minute) and showed decreasing lactate production in the perfusate (initial: 0.031 ± 0.004 vs final: 0.007 ± 0.002 mmol/min; P = 0.001). After preservation, compared to cold storage hearts, perfusion hearts had higher cardiac output (P = 0.004), LV dP/dt max (P = 0.003) and myocardial oxygen efficiency (P = 0.01), with lower blood perfusate lactate (P = 0.007). Hemodynamic values of perfused hearts reached 60% or more those in the normal reference group. Conclusions Continuous cold crystalloid perfusion in a canine model of DCD: (1) facilitates aerobic metabolism and resuscitates the DCD heart, (2) provides functional and metabolic recovery superior to cold storage, (3) shows promise for improved clinical preservation of DCD and marginal donor hearts.

BACKGROUND: Donation after circulatory death (DCD) represents a potential new source of hearts to increase the donor pool. We showed previously that DCD hearts in Greyhound dogs could be resuscitated and preserved by continuous cold crystalloid perfusion but not by cold static storage and could demonstrate excellent contractile and metabolic function on an in vitro system. In the current study, we demonstrate that resuscitated DCD hearts are transplantable.METHODS: Donor Greyhound dogs (n = 12) were divided into perfusion (n = 8) and cold static storage (n = 4) groups. General anesthesia was induced and ventilation ceased for 30 minutes to achieve circulatory death. Donor cardiectomy was performed, and for 4 hours the heart was preserved by controlled reperfusion, followed by continuous cold perfusion with an oxygenated crystalloid perfusate or by static cold storage, after which orthotopic heart transplantation was performed. Recovery was assessed over 4 hours by hemodynamic monitoring.RESULTS: During cold perfusion, hearts showed continuous oxygen consumption and low lactate levels, indicating aerobic metabolism. The 8 dogs in the perfusion group were weaned off bypass, and 4 hours after bypass produced cardiac output of 4.73 +/- 0.51 liters/min, left ventricular power of 7.63 +/- 1.32 J/s, right ventricular power of 1.40 +/- 0.43 J/s, and left ventricular fractional area shortening of 39.1% +/- 5.2%, all comparable to pre-transplant values. In the cold storage group, 3 of 4 animals could not be weaned from cardiopulmonary bypass, and the fourth exhibited low-level function.CONCLUSIONS: Cold crystalloid perfusion, but not cold static storage, can resuscitate and preserve the DCD donor heart in a canine model of heart transplantation, thus rendering it transplantable. Controlled reperfusion and cold crystalloid perfusion have potential for clinical application in DCD transplantation. (C) 2016 Published by Elsevier Inc. on behalf of International Society for Heart and Lung Transplantation. All rights reserved.

Reactive oxygen species (ROS) play an important role in the regulation of normal cellular function. When ROS are produced in excess they can have detrimental effects, a state known as oxidative stress. Thus ROS play both physiological and pathophysiological roles in the body. In clinical practice oxidative stress and its counterpart, antioxidant capacity can be measured and can guide remedial therapy. Oxidative stress can have a negative impact in all forms of major surgery including cardiac surgery, general surgery, trauma surgery, orthopedic surgery and plastic surgery; this is particularly marked in an ageing population. Many different therapies to reduce oxidative stress in surgery have been tried with variable results. We conclude that in surgical patients the assessment of oxidative stress, improvement of the understanding of its role, both positive and negative, and devising appropriate therapies represent fruitful fields for future research.

Irreversible electroporation (IRE) is a non-thermal focal ablation technique that uses needle electrodes to deliver a series of brief (100μs duration) electric pulses into the targeted region. These alter cellular transmembrane potentials, destabilizing the membranes in a manner that kills the cells while sparing major vasculature and other sensitive structures. IRE can therefore be used in regions ineligible for surgical resection or thermal ablation. Treatments result in rapid lesion creation and resolution [1], are unaffected by the blood perfusion “heat sink”, can be planned with numerical modeling [2], and its effects can be readily monitored with various imaging modalities [3]. Therapeutic ire has proven effective in the treatment of experimental [4] and clinical tumors. A human safety study attained complete regression in 46 of 69 tumors ineligible or unresponsive to conventional treatment [5], and veterinary case studies convey its utility in large difficult tumors [6, 7].

PURPOSE:Electroporation-based therapies deliver brief electric pulses into a targeted volume to destabilize cellular membranes. Nonthermal irreversible electroporation (IRE) provides focal ablation with effects dependent on the electric field distribution, which changes in heterogeneous environments. It should be determined if highly conductive metallic implants in targeted regions, such as radiotherapy brachytherapy seeds in prostate tissue, will alter treatment outcomes. Theoretical and experimental models determine the impact of prostate brachytherapy seeds on IRE treatments.MATERIALS AND METHODS:This study delivered IRE pulses in nonanimal, as well as in ex vivo and in vivo tissue, with and in the absence of expired radiotherapy seeds. Electrical current was measured and lesion dimensions were examined macroscopically and with magnetic resonance imaging. Finite-element treatment simulations predicted the effects of brachytherapy seeds in the targeted region on electrical current, electric field, and temperature distributions.RESULTS:There was no significant difference in electrical behavior in tissue containing a grid of expired radiotherapy seeds relative to those without seeds for nonanimal, ex vivo, and in vivo experiments (all p > 0.1). Numerical simulations predict no significant alteration of electric field or thermal effects (all p > 0.1). Histology showed cellular necrosis in the region near the electrodes and seeds within the ablation region; however, there were no seeds beyond the ablation margins.CONCLUSION:This study suggests that electroporation therapies can be implemented in regions containing small metallic implants without significant changes to electrical and thermal effects relative to use in tissue without the implants. This supports the ability to use IRE as a salvage therapy option for brachytherapy.

Despite increasing use of donation after cardiac death (DCD) and encouraging results for non-cardiac transplants, DCD cardiac transplantation has not been widely adopted because, (1) the DCD heart sustains warm ischaemic injury during the death process and (2) conventional static cold storage significantly adds to the ischaemic injury. We have developed a simple system for perfusion of the DCD heart with cold crystalloid solution using gravity-feed that can reduce ischaemic injury and potentially render the heart suitable for transplantation. This report describes the first application of this technique to a human DCD heart with good functional metabolic recovery over 12h on an ex vivo rig.

younger muscle. 4)Oral CoQ10 therapy before cardiac surgery improves efficiency of mitochondrial energy production, improves post-operative heart function, reduces intra-operative myocardial damage and shortens hospital stay.

Background-Ca2+ over-load plays an important role in the pathogenesis of cardioplegic ischemia-reperfusion injury. The standard technique to control Ca2+ overload has been to reduce Ca2+ in the cardioplegic solution (CP). Recent reports suggest that Na+/H+ exchange inhibitors can also prevent Ca2+ overload. We compared 4 crystalloid CPs that might minimize Ca2+ overload in comparison with standard Mg2+-containing CP: (1) low Ca2+ CP (0.25 mmol/L), (2) citrate CP/normal Mg2+ (1 mmol/L Mg2+), (3) citrate CP/high Mg2+ (9 mmol/L Mg2+), and (4) the addition of the Na+/H+ exchange inhibitor HOE-642 (Cariporide). We also tested the effect of citrate titration in vitro on the level of Free Ca2+ and Mg2+ in CPs.Methods and Results-Isolated working rat heart preparations were perfused with oxygenated Krebs-Henseleit buffer and subjected to 60 minutes of 37 degreesC arrest and reperfusion with CPs with different Ca2+ concentrations. Cardiac performance, including aortic flow (AF), was measured before and after ischemia. Myocardial high-energy phosphates were measured after reperfusion. The in vitro addition of citrate to CP (2%, 21 mmol/L) produced parallel reductions in Mg2+ anti Ca2+. Because only Ca2+ was required to be low, the further addition of Mg2+ increased free Mg2+, but the highest level achieved was 9 mmol/L. Citrate Cf significantly impaired postischemic function (AF 58.3+/-2.5% without citrate versus 41.6+/-3% for citrate with normal Mg2+, P<0.05, versus 22.4+/-6.2% for citrate with high Mg2+, P<0.05). Low-Ca2+ CP (0.25 mmol/L Ca2+) significantly improved the recovery of postischemic function in comparison with standard CP (1.0 mmol/L Ca2+) (AF 47.6+/-1.7% versus 58.3+/-2.5%, P<0.05). The addition of HOE-642 (1 mol/L) to CP significantly improved postischemia function (47.6+/-1.7% without HOE-642 versus 62.4+/-1.7% with HOE-642, P<0.05). Postischemia cardiac high-energy phosphate levels were unaffected by Ca2+ manipulation.Conclusions-(1) A lowered Ca2+ concentration in CP is beneficial in Mg2+-containing cardioplegia. (2) The use of citrate to chelate Ca2+ is detrimental in the crystalloid-perfused isolated working rat heart, especially with high Mg2+. (3) The mechanism of citrate action is complex, and its use limits precise simultaneous control of Ca2+ and Mg2+. (4) HOE-642 in CP is as efficacious in preservation of the ischemic myocardium as is the direct reduction in Ca2+.

Background —Ca 2+ overload plays an important role in the pathogenesis of cardioplegic ischemia-reperfusion injury. The standard technique to control Ca 2+ overload has been to reduce Ca 2+ in the cardioplegic solution (CP). Recent reports suggest that Na + /H + exchange inhibitors can also prevent Ca 2+ overload. We compared 4 crystalloid CPs that might minimize Ca 2+ overload in comparison with standard Mg 2+ -containing CP: (1) low Ca 2+ CP (0.25 mmol/L), (2) citrate CP/normal Mg 2+ (1 mmol/L Mg 2+ ), (3) citrate CP/high Mg 2+ (9 mmol/L Mg 2+ ), and (4) the addition of the Na + /H + exchange inhibitor HOE-642 (Cariporide). We also tested the effect of citrate titration in vitro on the level of free Ca 2+ and Mg 2+ in CPs. Methods and Results —Isolated working rat heart preparations were perfused with oxygenated Krebs-Henseleit buffer and subjected to 60 minutes of 37°C arrest and reperfusion with CPs with different Ca 2+ concentrations. Cardiac performance, including aortic flow (AF), was measured before and after ischemia. Myocardial high-energy phosphates were measured after reperfusion. The in vitro addition of citrate to CP (2%, 21 mmol/L) produced parallel reductions in Mg 2+ and Ca 2+ . Because only Ca 2+ was required to be low, the further addition of Mg 2+ increased free Mg 2+ , but the highest level achieved was 9 mmol/L. Citrate CP significantly impaired postischemic function (AF 58.3±2.5% without citrate versus 41.6±3% for citrate with normal Mg 2+ , P <0.05, versus 22.4±6.2% for citrate with high Mg 2+ , P <0.05). Low-Ca 2+ CP (0.25 mmol/L Ca 2+ ) significantly improved the recovery of postischemic function in comparison with standard CP (1.0 mmol/L Ca 2+ ) (AF 47.6±1.7% versus 58.3±2.5%, P <0.05). The addition of HOE-642 (1 μmol/L) to CP significantly improved postischemia function (47.6±1.7% without HOE-642 versus 62.4±1.7% with HOE-642, P <0.05). Postischemia cardiac high-energy phosphate levels were unaffected by Ca 2+ manipulation. Conclusions —(1) A lowered Ca 2+ concentration in CP is beneficial in Mg 2+ -containing cardioplegia. (2) The use of citrate to chelate Ca 2+ is detrimental in the crystalloid-perfused isolated working rat heart, especially with high Mg 2+ . (3) The mechanism of citrate action is complex, and its use limits precise simultaneous control of Ca 2+ and Mg 2+ . (4) HOE-642 in CP is as efficacious in preservation of the ischemic myocardium as is the direct reduction in Ca 2+ .

Following pericardiotomy in rats, subsequent fibrosis of the visceral pericardium becomes a site of high-density angiotensin-converting enzyme (ACE) binding. This study was undertaken to determine whether this exteriorized site of ACE activity is associated with angiotensin II (AngII) production. Four weeks after pericardiotomy, hearts were isolated and maintained by Krebs–Henseleit perfusion; coronary venous and Thebesian drainage were removed by cannulae. Following a 30-min period of stabilization, a balloon containing superfusate was placed around the heart. Superfusate composition was controlled and included either lisinopril (10−7mol/l), angiotensin I (AngI, 10−7mol/l), or angiotensinogen (10−6mol/l). Sixty min later, superfusate AngII concentration was determined (high-performance liquid chromatography followed by radioimmunoassay). Pericardial fibrosis was confirmed by picrosirius red staining and its high-density ACE binding by quantitativein vitroautoradiography (125I-351A). ACE activity was measured by hippuryl-histidyl-leucine degradation. In coronary effluent, AngII concentration and ACE activity were not different between controls and hearts with pericardial fibrosis. Compared to unoperated, age/sex-matched control hearts, however, we found those with pericardial fibrosis to have: (a) significantly (P<0.05) greater tissue ACE activity (118.42±6.66v89.45±7.70 nmol/min/g); (b) significantly (P<0.01) greater superfusate AngII concentration (4.98±0.94v1.43±0.28 pg/ml); (c) lisinopril markedly attenuated superfusate AngII concentration to that seen in controls; (d) exogenous AngI markedly increased AngII production (13.76±1.65v4.98±0.94 pg/ml); and (e) exogenous angiotensinogen did not alter superfusate AngII. Thus, high-density ACE binding and ACE activity of fibrosed pericardium is responsible for AngII production in thisin vitromodel. Cells involved in generating AngI at this site are uncertain and may involve fibroblast-like cells that express ACE and have ACE activity. The role of local AngII production is unknown, but its autocrine/paracrine properties may regulate collagen turnover of these cells

The binding characteristics of [125I]endothelin-1 (ET-1) to cardiac membranes isolated from rat hearts subjected to Ca(2+)-free perfusion or the Ca2+ paradox were examined. The effect of treatment with 2, 3 butanedione monoxime (BDM), which inhibits the tissue damage associated with the calcium paradox, was also investigated. Membranes from rat hearts perfused under control conditions bound [125I]ET-1 to a single population of sites with a Bmax of 107.7 +/- 3.7 fmol/mg protein and an affinity (KD) of 153 +/- 12 pM. Ten minutes of Ca(2+)-free perfusion resulted in a significant (P < 0.01) increase in Bmax to 167.5 +/- 8.3 fmol/mg protein without change in KD. Ca2+ repletion following Ca(2+)-free perfusion tended to increase further the Bmax (180.6 +/- 10.4 fmol/mg protein) without change in KD. Treatment with BDM attenuated but did not prevent the rise in Bmax following Ca(2+)-free perfusion. Following Ca2+ repletion, however, Bmax returned to control levels in the BDM treated group. These changes were not associated with changes in the ability of ET-1 and ET-3 to inhibit [125I]ET-1 binding. The results demonstrate that Ca(2+)-free perfusion is associated with an increase in the binding site density of [125I]ET-1 which is maintained or further increased upon Ca2+ repletion. If, however, the tissue damage associated with the Ca2+ paradox is prevented with BDM, Ca2+ repletion is associated with a reversal of the increase due to Ca(2+)-free perfusion.