[Can the new technologies of telemedicine applied to health help the caregiver?]. / Le nuove tecnologie telematiche applicate in sanità possono aiutare il caregiver?

During the last few years about the chronic patient assistance the tendency is to privilege the home care model, favouring the permanence of the patient in the familiar nucleus. This determines an always greater involvement in term of time and responsibility of the caregiver that is of the person who takes cure of the patient one worrying itself to answer to its physical needs, psychical and social. The burden of the family caregiver is in the consisting majority of the cases rather. The caregiver is therefore, with full rights, the other protagonist of the disease and it must be necessarily integrated in the assistance plan. The increase of the age associated to an increase of the prevalence of chronic pathologies, determines the necessity to plan new interventions on the territory. In chronic patients alternative assistance models, using telemedicine, seem to be effectives improving both clinical aspects and quality of the life. A new area of interest is delineated therefore that, through the new technologies of the ICT must define been involved the single roles of the operating ones in the participation program. The telemedicine seems to be a useful instrument in order to support patient and caregiver in facing the disease and reducing stress. In our model of domiciliary telesurveillance the patient, the caregiver, the family and all the sanitary figures are been involved. This model integrating the service dedicated to chronic pathology with telepsychology at home seems to give good result even if ulterior studies, above all in the long term, are need.

Beta-adrenergic receptors and intracellular signalling pathway in stunned and non-ischemic regions of pig myocardium.

The beta-adrenergic pathway may have a role in the pathophysiology of ischemic syndromes characterised by reversible left ventricular dysfunction, such as myocardial stunning and other clinical conditions of unstable angina or coronary spasms, or chronic reversible left ventricular dysfunction, which might be a consequence of repeated events of short-term ischemia ("repetitive stunning"). A partial-to-total occlusion of the left anterior descending coronary artery in pigs was used to induce short periods of ischemia (total ischemic time 12 +/- 2 min). Hypokinesis and dyskinesis of the myocardium were considered signs of myocardial dysfunction. We found a maintained function of the beta-adrenergic signalling system. Density and affinity of beta-adrenergic receptors were not different in stunned and non-ischemic regions, nor were cyclic AMP and cyclic GMP intracellular contents and ratio, nor well as the ratio of stimulatory/inhibitory G protein a subunits. Our findings are in agreement with a maintained beta-adrenergic signalling system in the pathophysiology of chronic reversible left ventricular dysfunction.

Role of bradykinin and eNOS in the anti-ischaemic effect of trandolapril.

1. Angiotensin converting enzyme (ACE) inhibitors are under study in ischaemic heart diseases, their mechanism of action being still unknown. 2. The anti-ischaemic effect of trandolapril and the possible involvement of a bradykinin-modulation on endothelial constitutive nitric oxide synthase (eNOS) in exerting this effect, were investigated. 3. Three doses of trandolapril, chronically administered in vivo, were studied in isolated perfused rat hearts subjected to global ischaemia followed by reperfusion. 4. Trandolapril has an anti-ischaemic effect. The dose of 0.3 mg kg(-1) exerted the best effect reducing diastolic pressure increase during ischaemia (from 33.0+/-4.5 to 14.0+/-5.2 mmHg; P<0.05 vs control) and reperfusion (from 86.1+/-9.4 to 22.2+/-4.1 mmHg; P<0.01 vs control), improving functional recovery, counteracting creatine phosphokinase release and ameliorating energy metabolism after reperfusion. 5. Trandolapril down-regulated the baseline developed pressure. 6. Trandolapril increased myocardial bradykinin content (from 31.8+/-6.1 to 54.8+/-7.5 fmol/gww; P<0.05, at baseline) and eNOS expression and activity in aortic endothelium (both P<0.01 vs control) and in cardiac myocytes (from 11.3+/-1.5 to 17.0+/-2.0 mUOD microg protein(-1) and from 0.62+/-0.05 to 0.80+/-0.06 pmol mg prot(-1) min(-1); both P<0.05 vs control). 7. HOE 140 (a bradykinin B(2) receptor antagonist) and NOS inhibitors counteracted the above-reported effects. 8. There was a negative correlation between myocyte's eNOS up-regulation and myocardial contraction down-regulation. 9. Our findings suggest that the down-regulation exerted by trandolapril on baseline cardiac contractility, through a bradykinin-mediated increase in NO production, plays a crucial role in the anti-ischaemic effect of trandolapril by reducing energy breakdown during ischaemia.

Enhanced expression and activity of xanthine oxidoreductase in the failing heart.

The molecular basis for heart failure is unknown, but oxidative stress is associated with the pathogenesis of the disease. We tested the hypothesis that the activity of xanthine oxidoreductase (XOR), a free-radical generating enzyme, increases in hypertrophied and failing heart. We studied XOR in two rat models: (1) The monocrotaline-induced right ventricular hypertrophy and failure model; (2) coronary artery ligation induced heart failure, with left ventricular failure and compensatory right ventricular hypertrophy at different stages at 3 and 8 weeks post-infarction, respectively. XOR activity was measured at 30 degrees C and the reaction products were analysed by HPLC. In both models XOR activity in hypertrophic and control ventricles was similar. In the monocrotaline model, the hearts showed enhanced XOR activity in the failing right ventricle (65+/-5 mU/g w/w), as compared to that in the unaffected left ventricle (47+/-3 mU/g P<0.05, n=6-7). In the coronary ligation model, XOR activities did not differ at 3 and 8 weeks. In the infarcted left ventricle, XOR activity increased from 29.4+/-1.4 mU/g (n=6) in sham-operated rats, to 48+/-3 and 80+/-6 mU/g (n=8 P<0.05 v sham) in the viable and infarcted parts of failing rat hearts, respectively. With affinity-purified polyclonal antibody, XOR was localized in CD68+ inflammatory cells of which the number increased more in the failing than in sham-operated hearts. Our results show that the expression of functional XOR is elevated in failing but not in hypertrophic ventricles, suggesting its potential role in the transition from cardiac hypertrophy into failure.

Reduction of oxidative stress by carvedilol: role in maintenance of ischaemic myocardium viability.

OBJECTIVES: To differentiate the impact of the beta-blocking and the anti-oxidant activity of carvedilol in maintaining myocardium viability. METHODS: Isolated rabbit hearts, subjected to aerobic perfusion, or low-flow ischaemia followed by reperfusion, were treated with two doses of carvedilol, one dose (2.0 microM) with marked negative inotropic effect due to beta-blockage and the other (0.1 microM) with no beta-blockage nor negative inotropism. Carvedilol was compared with two doses of propranolol, 1.0 - without - and 5.0 microM - with negative inotropic effect. Anti-oxidant activity was measured as the capacity to counteract the occurrence of oxidative stress and myocardium viability as recovery of left ventricular function on reperfusion, membrane damage and energetic status. RESULTS: Carvedilol counteracted the ischemia and reperfusion induced oxidative stress: myocardial content of reduced glutathione, protein and non-protein sulfhydryl groups after ischaemia and particularly after reperfusion, was higher in hearts treated with carvedilol, while the myocardial content of oxidised glutathione was significantly reduced (0.30+/-0.03 and 0.21+/-0.02 vs. 0.39+/-0.03 nmol/mg prot, both P<0.01, in 0.1 and 2.0 microM). At the same time, carvedilol improved myocardium viability independently from its beta-blocking effect. On the contrary, propranolol maintained viability only at the higher dose, although to a lesser extent than carvedilol. This suggests that the effects of propranolol are dependent on energy saving due to negative inotropism. The extra-protection observed with carvedilol at both doses is likely due to its anti-oxidant effect. CONCLUSIONS: Our data show that the anti-oxidant activity of carvedilol is relevant for the maintenance of myocardium viability.

New insights on myocardial pyridine nucleotides and thiol redox state in ischemia and reperfusion damage.

OBJECTIVE: to investigate the changes of pyridine nucleotides and thiol redox state in cardiac tissue following ischemia and reperfusion. NADH/NAD and NADPH/NADP redox couples were specifically studied and the influence of NADPH availability on cellular thiol redox was also investigated. METHODS: isolated rabbit hearts were Langendorff perfused and subjected to a protocol of ischemia and reperfusion. An improved technique for extraction and selective quantitation of pyridine nucleotides was applied. RESULTS: ischemia and reperfusion induced an increase in diastolic pressure, limited recovery in developed pressure and loss of creatine phosphokinase. Creatine phosphate and ATP were decreased by ischemia and only partially recovered during reperfusion. NADH was increased (from 0. 36+/-0.04 to 1.96+/-0.15 micromol/g dry wt. in ischemia, P<0.001), whereas NADPH decreased during ischemia (from 0.78+/-0.04 to 0. 50+/-0.06 micromol/g dry wt., P<0.01) and reperfusion (0.45+/-0.03 micromol/g dry wt.). Furthermore, we observed: (a) release of reduced (GSH) and oxidised glutathione (GSSG) during reperfusion; (b) decreased content of reduced sulfhydryl groups during ischemia and reperfusion (GSH: from 10.02+/-0.76 to 7.11+/-0.81 nmol/mg protein, P<0.05, and to 5.48+/-0.57 nmol/mg protein; protein-SH: from 280.42+/-12.16 to 135.11+/-17.00 nmol/mg protein, P<0.001, and to 190.21+/-11.98 nmol/mg protein); (c) increased content in GSSG during reperfusion (from 0.17+/-0.02 to 0.36+/-0.02 nmol/mg protein, P<0.001); (d) increased content in mixed disulphides during ischemia (from 6.14+/-0.13 to 8.31+/-0.44 nmol/mg protein, P<0.01) and reperfusion (to 9.87+/-0.82 nmol/mg protein, P<0.01). CONCLUSIONS: under severe low-flow ischemia, myocardial NADPH levels can decrease despite the accumulation of NADH. The reduced myocardial capacity to maintain NADPH/NADP redox potential can result in thiol redox state changes. These abnormalities may have important consequences on cellular function and viability.

The blood perfused isolated heart: characterization of the model.

We have characterized the aerobic blood-perfused isolated heart model evaluating the hemodynamics and metabolism of both the blood donor animal and the isolated organ. Anaesthesia of the blood donor with sodium pentobarbital (30 mg/kg) increases arterial concentration of non esterified fatty acids (NEFA) from 80 +/- 6 to 452 +/- 70 microM; p < 0.01. Injection of 1,000 U/kg heparin causes a second significant increase from 452 +/- 70 to 1012 +/- 104 microM; p < 0.01. Insertion of the perfusion circuit, without the isolated heart, causes a reduction in blood pressure of the blood donor and a significant increase in norepinephrine from 277 +/- 44 to 634 +/- 130 pg/ml; p < 0.05. Two hours of aerobic perfusion of the isolated heart inserted in the perfusion circuit, decreases arterial pressure of the blood donor with a concomitant increase of plasma norepinephrine from 475 +/- 150 to 841 +/- 159 pg/ml; p < 0.05. Developed pressure, oxygen consumption, glucose and NEFA uptake of the isolated heart remain constant during two hours of aerobic perfusion, NEFA being the preferred substrate. Tissue content of high energy phosphates at the end of the perfusion is high and similar to that observed "in vivo". Despite this, there is a release of lactate and CPK from the isolated heart. We conclude that: 1) the model allows accurate measurement of hemodynamics and metabolism of both the isolated heart and the blood donor animal; 2) the perfusion procedure modifies the substrates concentration of the blood donor animal which, in turn, results in the preferential NEFA utilization of the isolated heart. These changes do not affect the functional parameters of the perfused heart.

Role of A2A receptor in the modulation of myocardial reperfusion damage.

Adenosine protects myocardium from ischemia and reperfusion damage; however, the mechanism of action is still under discussion. We investigated whether (a) adenosine protects isolated crystalloid-perfused rabbit heart from ischemia/ reperfusion injury; (b) this action is receptor mediated and what receptor subtypes are involved, and (c) this action is dependent on an enhanced nitric oxide production. Our results showed a cardioprotective effect of adenosine (10(-4) M), of nonselective adenosine-receptor agonist 5'-N-ethyl-carboxamidoadenosine (NECA; 5 x 10(-6) M), and of A2A agonists CGS 21680 (10(-8) and 10(-6) M), 2-hexynylNECA (10(-7) M). On the contrary, A1 agonist CCPA (10(-8) and 10(-6) M) does not provide any protection. The effect has been achieved in terms of significant reduction in contracture development during reperfusion [diastolic pressure was 46.8 +/- 7.1 mm Hg (p < 0.01); 46.1 +/- 7.8 mm Hg (p < 0.01); 46.9 +/- 5.5 mm Hg (p < 0.01); and 59.3 +/- 6.7 mm Hg (p < 0.05) with 10(-4) M adenosine, 5 x 10(-6) M NECA, 10(-6) M CGS 21680, and 10(-7) M 2-hexynylNECA, respectively, versus 77.6 +/- 5.0 mm Hg in control]; reduced creatine phosphokinase release (13.5 +/- 1.6, 22.2 +/- 7.9, 14.2 +/- 3.3, and 14.1 +/- 4.5 U/gww in treated hearts vs. 34.6 +/- 7.2 U/gww in controls; p < 0.05); improved energy metabolism [adenosine triphosphate (ATP) content is 9.9 +/- 0.5, 10.4 +/- 0.6, 9.8 +/- 0.5, and 10.5 +/- 0.5 micromol/gdw in treated hearts vs. 7.6 +/- 0.2 micromol/gdw; p < 0.05]. Moreover, our data indirectly show a functional presence of A2A receptors on cardiomyocytes as the protection is A2A mediated and exerted only during reperfusion, although in the absence of blood and coronary flow changes. These activities appear independent of nitric oxide pathways, as adenosine and 2-hexynylNECA effects are not affected by the presence of a nitric oxide-synthase inhibitor (10(-4) M L-NNA).

Changes in oxidative stress and cellular redox potential during myocardial storage for transplantation: experimental studies.

BACKGROUND: Cardioplegic solutions assure only a sub-optimal myocardial protection during prolonged storage for transplantation. The ultimate cause of myocardial damage during storage is unknown, but oxygen free radicals might be involved. We evaluated the occurrence of oxidative stress and changes in cellular redox potential after different periods of hypothermic storage. METHODS: Langendorff-perfused rabbit hearts were subjected to a protocol mimicking each stage of a cardiac transplantation procedure: explantation, storage and reperfusion. Three periods of storage were considered: Group A = 5 hours, Group B = 15 hours, and Group C = 24 hours. Oxidative stress was determined in terms of myocardial content and release of reduced (GSH) and oxidized (GSSG) glutathione, and cellular redox potential as oxidized and reduced pyridine nucleotides ratio (NAD/NADH). Data on mechanical function, cellular integrity and myocardial energetic status were collected. RESULTS: At the end of reperfusion, despite the different timings of storage, recovery of left ventricular developed pressure (46.1+/-7.0, 54.7+/-6.7, and 45.7+/-7.4% of the baseline pre-ischaemic value), energy charge (0.81+/-0.02, 0.81+/-0.02, and 0.77+/-0.01) and NAD/NADH ratio (8.87+/-1.08, 9.39+/-1.72, and 10.26+/-1.98) were similar in all groups (A, B and C). On the contrary, the rise in left ventricular resting pressure (LVRP) and GSH/GSSG ratio were significantly different between Group C, and Groups A and B (p<0.0001, analyzed by Generalized Estimating Equations model for repeated measures, and p<0.05, respectively). CONCLUSIONS: The pathophysiology of myocardial damage during hypothermic storage cannot be considered as a normothermic ischaemic injury and parameters other than energetic metabolism, such as thiolic redox state, are more predictive of functional recovery upon reperfusion.

Skeletal muscle myosin heavy chain expression in rats with monocrotaline-induced cardiac hypertrophy and failure. Relation to blood flow and degree of muscle atrophy.

BACKGROUND: In congestive heart failure (CHF) the skeletal muscle of the lower limbs develops a myopathy characterised by atrophy and shift from the slow to the fast type fibres. The mechanisms responsible for these changes are not clear yet. OBJECTIVES: We investigated the influence of blood flow and degree of muscle atrophy on the myosin heavy chains (MHC) composition of the soleus and extensor digitorum longus (EDL) of rats with right ventricle hypertrophy and failure. METHODS: CHF was induced in 16 rats by injecting 30 mg/kg monocrotaline. Eight animals had the same dose of monocrotaline but resulting in compensated right ventricle hypertrophy. Two age- and diet-matched groups of control animals (nine and five respectively) were also studied. The relative percentage of MHC1 (slow isoform), MHC2a (fast oxidative) and MHC2b (fast glycolytic) was determined by densitometric scan after electrophoretic separation. The relative weights of soleus and EDL (muscle weight/body weight) were taken as an index of muscle atrophy. Skeletal muscle blood flow was measured by injecting fluorescent micropheres. RESULTS: CHF and Control (Con) rats showed similar degree of atrophy both in soleus (0.40 +/- 0.06 vs. 0.44 +/- 0.06 p = NS), and EDL (0.47 +/- 0.04 vs. 0.45 +/- 0.02, p = 0.09). In CHF rats these two muscles showed a statistically significant MHCs redistribution toward the fast type isozymes. In fact in EDL of CHF rats MHC2a was 30.5 +/- 6.1% vs. 35.8 +/- 8.6% of the Con (p < 0.05). MHC2b was however higher (68.5 +/- 6.6% vs. 61.0 +/- 9.6%, p = 0.017). In the soleus of CHF rats MHC1 was decreased (87.6 +/- 3.4% vs. 91.9 +/- 5.2%, p = 0.02), while MHC2a was increased (12.04 +/- 3.5% vs. 7.9 +/- 5.2%; p = 0.028). Similar changes were not found in the muscles of the compensated hypertrophy animals. No correlation was found between MHC pattern and the relative muscle weight in the CHF animals. Soleus blood flow in CHF rats was significantly lower than that of Con (0.11 +/- 0.03 ml/min/g vs. 0.22 +/- 0.03 p < 0.05), while no differences were found in EDL (0.06 +/- 0.02 ml/min/g vs. 0.08 +/- 0.02, p = NS). CONCLUSIONS: In rats with CHF a skeletal muscle myopathy characterised by a shift of the MHCs toward the fast type isoforms occurs. The magnitude of the shift correlates neither with the degree of atrophy, nor with the skeletal muscle blood flow, suggesting that these two factors do not play a pivotal role in the pathogenesis of the myopathy.

Is stunning an important component of preconditioning?

We tested the hypothesis that stunning following a brief period of ischaemia is a component of cardioprotection afforded by preconditioning in an in vitro model of global normothermic ischaemia. Isolated Langendorff-perfused rat hearts, after 120-150 min of aerobic perfusion, were divided into four groups. Groups 1 and 2 constituted the aerobic and ischaemic controls. The other hearts were preconditioned by two 2-min ischaemia/reperfusion cycles. Two ischaemic preconditioning protocols were used, the only difference being prolongation of the reperfusion cycle from 5 (group 3) to 20 min (group 4) before the onset of severe ischaemic insult. Mechanical function, energetic metabolism and the rate of enzyme release were followed throughout. In group 3, myocardial function remained significantly downregulated before the onset of severe ischaemia. This resulted in cardiac protection as evidenced by enhanced recovery of systolic pressure (37.7 +/- 3.6 v 61.9 +/- 5.7 mmHg for groups 2 and 3, respectively; P < 0.02), reduced rise in diastolic pressure (55.8 +/- 5.9 v 34.3 +/- 5.2 mmHg; P < 0.02), reduced creatine kinase (CK) release (957.3 +/- 175.7 v 541.5 +/- 85.9 mU/min/gww; P < 0.05) and higher contents of high-energy phosphate at the end of ischaemia [3.6 +/- 0.3 v 25.3 +/- 2.9 mumol/gdw for creatine phosphate (CP), P < 0.001] as well as after reperfusion (16.8 +/- 2.4 v 31.4 +/- 1.8 for CP, P < 0.01, and 3.9 +/- 0.5 v 6.2 +/- 0.8 mumol/gdw for ATP, P < 0.05). When severe ischaemia was started only after complete recovery of mechanical function (group 4), no protection was observed. Our data suggest that a decrease in mechanical function or stunning occurring after the short period of ischaemia causes ATP sparing and constitutes an additional mechanism of preconditioning cardioprotection in vitro.

Skeletal muscle metabolism in experimental heart failure.

We studied peripheral skeletal muscle metabolism in monocrotaline-treated rats. Two distinct groups emerged: a percentage of the animals developed ventricular hypertrophy, with no signs of heart failure (compensated group), whilst others, besides ventricular hypertrophy, developed the syndrome of congestive heart failure (CFH group). Oxidative metabolism and redox cellular state were expressed in terms of creatine phosphate, purine (ATP, ADP and AMP) and pyridine (NAD and NADH) nucleotides tissue content. Skeletal muscles with different metabolism were studied: (a) Soleus (oxidative), (b) extensor digitorium longus (glycolytic) and tibialis anterior (oxidative and glycolytic). The results showed that in CFH animals a decreased high-energy phosphates content occurs in the soleus and extensor digitorum longus, but not in the tibialis anterior. In the soleus. ATP declined from 20.31 +/- 2.5 of control group to 9.55 +/- 0.61 mumol/g dry wt. while in the extensor digitorum longus ATP declined from 30.92 +/- 2.68 to 22.7 +/- 1.54 mumol/g dry wt. In both these muscles, a shift of NAD/NADH couple towards oxidation was also observed (from 26.58 +/- 3.34 to 6.95 +/- 0.97 and from 18.88 +/- 3.43 to 10.57 +/- 1.61, respectively). These alterations were more evident in the aerobic soleus muscle. On the contrary, no major changes occurred in skeletal muscle metabolism of compensated animals. The results show that: (1) a decrease in muscle high-energy phosphates occurs in CFH; (2) this is accompanied by a decrease of NAD/NADH couple suggesting an impairment in oxygen utilization or availability.

Metabolic adaptation during a sequence of no-flow and low-flow ischemia. A possible trigger for hibernation.

BACKGROUND: Myocardial hibernation is an adaptive phenomenon occurring in patients with a history of acute ischemia followed by prolonged hypoperfusion. METHODS AND RESULTS: We investigated, in isolated rabbit heart, whether a brief episode of global ischemia followed by hypoperfusion maintains viability. Four groups were studied; group 1,300 minutes of aerobia; group 2,240 minutes of total ischemia and 60 minutes of reperfusion; group 3, 10 minutes of total ischemia, 230 minutes of hypoperfusion (90% coronary flow reduction), and 60 minutes of reperfusion; and group 4, 240 minutes of hypoperfusion followed by reperfusion. In group 3, viability was maintained. Ten minutes of ischemia caused quiescence, a fall in interstitial pH (from 7.2 +/- 0.01 to 6.1 +/- 0.8), creatine phosphate (CP), and ATP (from 54.5 +/- 5.0 and 25.0 +/- 1.9 to 5.0 +/- 1.1 and 15.3 +/- 2.5 mumol/g dry wt, P < .01). Subsequent hypoperfusion failed to restore contraction and pH but improved CP (from 5.0 +/- 1.1 to 20.1 +/- 3.4, P < .01). Reperfusion restored pH, developed pressure (to 92.3%), and NAD/NADH and caused a washout of lactate and creatine phosphokinase with no alterations of mitochondrial function or oxidative stress. In group 4, hypoperfusion resulted in progressive damage. pH fell to 6.2 +/- 0.7, diastolic pressure increased to 34 +/- 5.6 mm Hg, CP and ATP became depressed, and oxidative stress occurred. Reperfusion partially restored cardiac metabolism and function (47%). CONCLUSIONS: A brief episode of total ischemia without intermittent reperfusion maintains viability despite prolonged hypoperfusion. This could be mediated by metabolic adaptation, preconditioning, or both.

Effects of felodipine on the ischemic heart: insight into the mechanism of cytoprotection.

To assess whether the administration of felodipine protects the myocardium in a dose-dependent manner against ischemia and reperfusion, isolated rabbit hearts were infused with three different concentrations of felodipine: 10(-10), 10(-9), and 10(-8) M. Diastolic and developed pressures were monitored; coronary effluent was collected and assayed for CPK activity and for noradrenaline concentration; mitochondria were harvested and assayed for respiratory activity; and ATP production and calcium content and tissue concentration of ATP, creatine phosphate (CP), and calcium were determined. The occurrence of oxidative stress during ischemia and reperfusion was also monitored in terms of tissue content and release of reduced (GSH) and oxidized (GSSG) glutathione. Treatment with felodipine at 10(-10) and 10(-9) M had no effect on the hearts when perfused under aerobic conditions, whilst the higher dose reduced developed pressure from 57.7 +/- 2.6 to 30.0 +/- 2.6 mmHg (p < 0.01). On reperfusion treated hearts recovered better than the untreated hearts with respect to left ventricular performance, replenishment of ATP and CP stores, and mitochondrial function. Recovery of developed pressure was 100% at 10(-8) M, 55% at 10(-9) M, and 46% at 10(-10) M. The reperfusion-induced tissue and mitochondrial calcium overload, release of CPK and noradrenaline, and oxidative stress were also significantly reduced. The effects of felodipine were dose dependent. Felodipine inhibited the initial rate of ATP-driven calcium uptake but failed to affect the initial rate of mitochondrial calcium transport. It is concluded that felodipine infusion provides dose-dependent protection of the heart against ischemia and reperfusion. Because this protection also occurred at 10(-9) M and 10(-10) M in the absence of a negative inotropic effect during normoxia and of a coronary dilatory effect during ischaemia, it cannot be attributed to an energy-sparing effect or to improvement in oxygen delivery. From our data we can envisage two other major mechanisms-(1) membrane protection and (2) reduction in oxygen toxicity. The ATP-sparing effect occurring at 10(-8) M is likely to be responsible for the further protection.

Dichotomy in the post-ischemic metabolic and functional recovery profiles of isolated blood-versus buffer-perfused heart.

There is evidence that buffer- and blood-perfused hearts differ in their postischemic functional recoveries. The present study was designed to: (i) compare ischemia-induced contracture and post-ischemic functional recovery, and (ii) investigate whether the recovery profiles were related to either the release of purines and norepinephrine or high-energy phosphate content. Rat hearts (n = 8/group) were perfused at 37 degrees C with buffer (60 mmHg) or blood (60 mmHg from a support rat), made globally ischemic (15 min) and reperfused (15 min). The onset and severity of ischemic contracture were identical in both models [left ventricular end-diastolic pressure (LVEDP) at the end of 15 min ischemia was 30 +/- 5 and 27 +/- 4 mmHg respectively; P = N.S.]. However, the rate and extent of post-ischemic left ventricular developed pressure (LVDP) differed considerably. Blood-perfused hearts exhibited an initial rapid and complete recovery of LVDP followed by a steady decline to approximately 60% of pre-ischemic values. Buffer-perfused hearts recovered to only 80% after 5 min reperfusion and remained at this level for the duration of reperfusion LVEDP was higher in buffer-perfused than in blood-perfused hearts during the first 5 min of reperfusion; thereafter, LVEDP fell in buffer-perfused hearts to a level than was not significantly different from the observed in blood-perfused hearts. In buffer-perfused hearts, coronary flow recovered to 90% within 5 min and then remained constant; in blood-perfused hearts flow recovered to 100% by 1 min and continued to rise to a maximum by 7 min (201 +/- 15%). This increase appeared to mirror the secondary decline in LVDP. During the first 4 min of reperfusion, in both preparations, venous norepinephrine increased to six- to nine-fold of pre-ischemic values and then fell rapidly to near control levels by 6-9 min. Total purine release was high in early reperfusion in both groups. At the end of 15 min reperfusion, the tissue adenylate pool was similar in both groups. This study demonstrates that the nature of the perfusate used for an isolated rat heart preparation: (i) does not appear to influence the severity of ischemic injury as assessed by ischemic contracture, but (ii) does influence the qualitative and quantitative characteristics of the temporal profile that describes the recovery of systolic and diastolic function during the first 15 min of reperfusion: and (iii) it has no effect upon the changes seen in a number of metabolic indices that are often used for the assessment of injury and protection.

In vitro administration of ergothioneine failed to protect isolated ischaemic and reperfused rabbit heart.

Ergothioneine, a natural thiol-containing molecule, has recently been proposed to protect the heart against damage caused by ischaemia and reperfusion. We investigated the possibility that ergothioneine can have a role in maintaining the myocardial thiol/disulfide balance and consequently also a protective effect against ischaemic and reperfusion injury. We used isolated Langendorff-perfused rabbit hearts subjected to 45 min global and total ischaemia followed by 30 min reperfusion at baseline coronary flow (22 ml/min). Ergothioneine was delivered at 10(-5) M and 10(-4) M 60 min before ischaemia and during reperfusion. Myocardial damage was determined in terms of mechanical function, creatine kinase (CK) and lactate release, energy phosphate stores and the occurrence of oxidative stress. In our experimental conditions the treatment was unable to prevent myocardial damage. Ergothioneine, independently from the dosage used, failed to: (i) increase recovery of developed pressure upon reperfusion (14.4 +/- 2.3 mmHg in control hearts vs. 10.3 +/- 2.9 and 12.5 +/- 2.3 mmHg in 10(-5) M and 10(-4) M ergothioneine treated hearts, respectively); (ii) decrease the rise in diastolic pressure (44.3 +/- 4.4 mmHg in control hearts vs. 49.8 +/- 5.8 and 48.0 +/- 7.7 mmHg in treated hearts); (iii) decrease the release of CK and lactate; (iv) increase the levels of adenosine triphosphate (ATP) and creatine phosphate (CP) in tissue upon reperfusion; (v) maintain ratio between oxidized and reduced forms of adenine nucleotide coenzyme, as index of aerobic metabolism; (vi) prevent the decline of reduced glutathione (GSH), or the accumulation of oxidized glutathione (GSSG) as an index of oxidative stress.

Biochemical and morphological changes in isolated rabbit hearts after prolonged hypothermic ischaemia: comparison of two cardioplegic solutions.

This work evaluates the myocardial protective potential of potassium cardioplegia on ischaemically arrested and reperfused hearts by two cardioplegic solutions: the University of Wisconsin solution (UW) and the standard crystalloid solution of St. Thomas' Hospital (ST). Evaluation of myocardial preservation was based on creatine kinase and lactate releases and on high-energy phosphate preservation of isolated rabbit hearts after 4 hours' hypothermic ischaemia. A morphometric ultrastructural evaluation of mitochondria in cardiomyocytes was also performed. The hearts of 24 rabbits were normothermally perfused with oxygenated Krebs-Henseleit solution for 30 min (Langendroff preparation), and the baseline contractile performance and biochemical parameters were evaluated. The hearts were then arrested and stored in the cardioplegic solutions (12 UW and 12 ST) at 4 degrees C for 4 hours. The hearts were then rewarmed and reperfused with oxygenated Krebs-Henseleit solution for further 30 min. At the end of reperfusion, creatine phosphate and high energy phosphates were higher with UW (p < 0.05); creatine kinase release during reperfusion was significantly lower with UW both at 15 min (p < 0.01) and at 30 min (p < 0.05). Lactate release during the first 15 min of reperfusion was about doubled (p < 0.05) with respect to controls in both groups; at 30 min this increase had almost vanished (+8%) with UW but not with ST (+30%). Ultrastructural morphometry did not show any significant difference at the level of mitochondria between the two treatments. The results indicate, for UW, an improved myocardial preservation associated with relative retention of high-energy phosphates and higher recovery of mechanical function, accelerated metabolic recovery and reduced stress of cell membranes.

Extraction and assay of creatine phosphate, purine, and pyridine nucleotides in cardiac tissue by reversed-phase high-performance liquid chromatography.

The levels of creatine phosphate, purine, and pyridine nucleotides in tissues provide important information on energetic and oxidative cellular states. Nevertheless, technical, theoretical, and methodological difficulties in extraction and quantification procedures have so far limited our understanding of the exact role that these substances play in metabolic processes which take place in cells. The objective of our study was to find an easy and rapid method for extracting, separating, and quantifying creatine phosphate, purine, and pyridine nucleotides in solid tissues. We adapted the classic acid-extraction procedure with HClO4 for purine and oxidized pyridine nucleotides and then developed a new alkaline extraction with phenol in a phosphate buffer solution (pH 7.8) for reduced pyridine nucleotides. Biopsies of myocardial tissue were frozen and ground at -180 degrees C using the appropriate extraction procedure. The separation and quantification of the metabolites were performed using a reversed-phase 3-microns Supelchem C18 column, with the addition of tetrabutylammonium as an ion-pair agent to the buffer solution, by ultraviolet detection. The recovery of the external and internal standards always exceeded 90%. The autooxidation or interconversion processes were almost insignificant for each reduced form. This technique allowed us to avoid complex enzymatic procedures and difficulties in the selective assay of pyridine nucleotides with chemiluminescence and surface spectroscopy.

How do calcium antagonists differ in clinical practice?

The majority of calcium antagonists used clinically belong to three distinct chemical classes: the phenylalkylamines, the dihydropyridines, and the benzothiazepines. In recent years their mode of action has been unravelled, their limitations recognized, and their efficacy and use in the management of patients with a broad spectrum of cardiovascular and other disorders defined. It is clear, however, that these drugs are not all alike, providing an explanation for their differing effects. The final therapeutic effect in humans depends on the mechanisms of action at the molecular level, the tissue selectivity, and the hemodynamic changes of each agent. All these aspects are examined in detail in this article. Concepts that are highlighted are as follows: (a) Molecular biology has allowed recognition of the polypeptide components of the alpha 1 subunit of the L-type Ca2+ channel and the finding of peptide segments covalently labelled by all three classes of drugs. (b) The location of these segments within the peptides is different: Binding sites for dihydropyridines are located externally, whereas those for verapamil and diltiazem are located internally, in the cytosolic part of the membrane. (c) Dihydropyridine binding is voltage dependent. This explains the selectivity of this class of drugs for vascular smooth muscle, which is more depolarized than cardiac muscle. (d) Phenylalkylamines and benzothiazepines reach their receptors at the internal surface of the sarcolemma through the channel lumen. Their binding is facilitated by the repetitive depolarization of atrioventricular and cardiac tissue, a phenomenon described as use dependence. This explains why these drugs are not highly selective, but equipotent for the myocardium, the atrioventricular conducting tissue, and the vasculature. (e) Dihydropyridines act through selective vasodilatation and may increase heart rate and contractility via a reflex mechanism. On the contrary, phenylalkylamines and diltiazem act through a combination of effects, including reduction of afterload, heart rate, and contractility. When taken together, all these differences distinguish the preferential clinical utilization of one of these compounds for a given cardiovascular pathology.