Defective sarcoplasmic reticulum-mitochondria calcium exchange in aged mouse myocardium.

Mitochondrial alterations are critically involved in increased vulnerability to disease during aging. We investigated the contribution of mitochondria-sarcoplasmic reticulum (SR) communication in cardiomyocyte functional alterations during aging. Heart function (echocardiography) and ATP/phosphocreatine (NMR spectroscopy) were preserved in hearts from old mice (>20 months) with respect to young mice (5-6 months). Mitochondrial membrane potential and resting O2 consumption were similar in mitochondria from young and old hearts. However, maximal ADP-stimulated O2 consumption was specifically reduced in interfibrillar mitochondria from aged hearts. Second generation proteomics disclosed an increased mitochondrial protein oxidation in advanced age. Because energy production and oxidative status are regulated by mitochondrial Ca2+, we investigated the effect of age on mitochondrial Ca2+ uptake. Although no age-dependent differences were found in Ca2+ uptake kinetics in isolated mitochondria, mitochondrial Ca2+ uptake secondary to SR Ca2+ release was significantly reduced in cardiomyocytes from old hearts, and this effect was associated with decreased NAD(P)H regeneration and increased mitochondrial ROS upon increased contractile activity. Immunofluorescence and proximity ligation assay identified the defective communication between mitochondrial voltage-dependent anion channel and SR ryanodine receptor (RyR) in cardiomyocytes from aged hearts associated with altered Ca2+ handling. Age-dependent alterations in SR Ca2+ transfer to mitochondria and in Ca2+ handling could be reproduced in cardiomyoctes from young hearts after interorganelle disruption with colchicine, at concentrations that had no effect in aged cardiomyocytes or isolated mitochondria. Thus, defective SR-mitochondria communication underlies inefficient interorganelle Ca2+ exchange that contributes to energy demand/supply mistmach and oxidative stress in the aged heart.

Percutaneous electrocatheter technique for on-line detection of healed transmural myocardial infarction.

Healed myocardial infarction has been recognized by its particular tissue electrical impedance spectrum measured with intramural needle electrodes in animal models. The aim of this study was to develop a percutaneous approach for in vivo recognition of areas of healed myocardial infarction by measuring myocardial electrical impedance with an intracavitary contact electrocatheter. Electrical impedance (resistance and phase angle) of normal myocardium and of a 2-month-old anterior transmural infarction were measured in nine chloralose anesthetized pigs by applying alternating currents from 1 kHz to 1 MHZ between a bipolar intracavitary catheter and a reference electrode placed on the epicardium (group I, n = 4) or on the precordium (group II, n = 5). Resistance of the infarcted myocardium was lower than that of healthy tissue at all current frequencies (ANOVA, P < 0.001) (i.e., at 1 kHz: 15 +/- 4 omega vs 50 +/- 19 omega in group I, and 64 +/- 13 omega vs 76 +/- 13 omega in group II). Phase angle at 316 kHz best differentiated transmural infarction from normal tissue (group I: -2.5 +/- 1.9 degrees vs -14.8 +/- 4.6 degrees, P < 0.001; group II: +0.7 +/- 1.0 degrees vs -2.7 +/- 1.4 degrees, P < 0.001). This study shows that analysis of myocardial impedance spectrum using a percutaneous intracavitary contact catheter approach permits on-line recognition of areas of healed transmural myocardial infarction. This technique may be useful to optimize clinical application of energy sources (i.e., radiofrequency ablation, laser myocardial revascularization).

Neurally mediated depressor hemodynamic response induced by intracoronary catheter balloon inflation in pigs.

OBJECTIVES: To assess whether intracoronary catheter balloon inflation triggers a neurally mediated hemodynamic response that interacts with the ischemia-induced myocardial dysfunction. METHODS: Forty-eight chloralose anesthetized pigs underwent a 60 s intraluminal catheter balloon inflation of the proximal left anterior descending (LAD) coronary artery before and after one of these treatments: disruption of LAD pericoronary nerves with phenol (n=6), bilateral stellectomy (n=8), bilateral cervical vagotomy (n=6), atropine (n=5), and ganglionic blockade with hexamethonium (n=10). In 13 other pigs, we assessed the reproducibility of two balloon inflations spaced 15 min (n=6) or 60 min (n=7). The ECG, left ventricular (LV) pressure, and LV dP/dt were recorded during each intervention. Right ventricular (RV) pressure, RV dP/dt, and aortic blood flow were also measured in a subset of pigs. RESULTS: Balloon inflation induced an early (10 s) and reproducible (ANOVA, P<0.001) drop in systolic pressure and peak dP/dt; a decrease in aortic blood flow; a rise in end-diastolic pressure; and elevation of the ST segment. Pericoronary denervation, stellectomy and ganglionic blockade attenuated (P<0.001) the drop in LV parameters during coronary inflation, but atropine and vagotomy did not. CONCLUSIONS: A depressor hemodynamic response subserved by pericoronary nerves worsens the LV dysfunction induced by brief coronary catheter balloon inflation in anesthetized pigs. Cholinergic fibers do not appear to play a major role.

Cardiovascular reflex responses induced by epicardial chemoreceptor stimulation.

The cardiac mechano- and chemoreceptors are broadly distributed in the myocardium and coronary vessels. A portion of these receptors extends over the epicardium and pericardium and therefore can be excited by mechanical or chemical stimuli directly applied to the surface of the heart. Excitation of epicardial receptors by topical application of chemical compounds elicits a variety of reflex cardiovascular responses, without the vascular or systemic effects of the drug administered systemically. A considerable number of studies has used the epicardial sensory field as a tool to delineate the functional characteristics of the cardiac afferent neurones in normal as well as in pathological conditions. In this review we analyze the cardiovascular reflex responses induced by epicardial application of a variety of substances like bradykinin, nicotine, muscarine, isoprenaline, adenosine, potassium chloride, capsaicin, prostaglandins or substance P in physiological models and also in models with acute myocardial ischemia or heart failure. The data highlight the contribution of the epicardial sensory neurites to the overall control of the cardiovascular system and, on the other hand, strengthen the need for further investigations directed to better elucidate the reflex cardiovascular responses that may develop in patients with pericardial abnormalities.

[Sudden death (II). Myocardial ischemia and ventricular arrhythmias in experimental models: triggering mechanisms]. / Muerte súbita (II). Isquemia miocárdica y arritmias ventriculares en modelos experimentales: mecanismos desencadenantes.

Metabolic and electrolytic alterations generated in the acute ischemic myocardium, such as an increase in extracellular potassium or acidosis, are responsible for the occurrence of ventricular arrhythmias. In the first 5-10 minutes following coronary occlusion, reentry seems to have an important role, although not in the next 15 minutes. If the patient survives, a subacute arrhythmia period appears, 6 to 72 hours after the onset of ischemia, probably due to abnormal automaticity in the surviving Purkinje fibers. Finally, reentry in the epicardial border zone is the most likely mechanism for chronic arrhythmias. In this review we focus on the studies dealing with the mechanisms of ischemia-induced arrhythmias, with special reference to those conducted in experimental models.

In vivo and in situ ischemic tissue characterization using electrical impedance spectroscopy.

The investigation of processes of ischemia in different organ tissues is very important for the development of methods of protection and preservation during surgical procedures. Electrical impedance spectroscopy was used to distinguish between different tissues and their degree of ischemia. We describe mathematical methods used to adjust experimental data to Cole-Cole models for one-circle and two-circle impedance loci and a study of the main parameters for representing the behavior of ischemia in time. In vivo and in situ postmortem measurements of different tissues from pigs are shown in the 100 Hz to 1 MHz range. The Cole parameters that best characterize the ischemia are R0 and fc.

Passive transmission of ischemic ST segment changes in low electrical resistance myocardial infarct scar in the pig.

OBJECTIVES: To analyze the passive electrical properties of a healed infarction and assess their role on transmission of contiguous ischemic ST segment potential changes. METHODS: We measured tissue resistivity (omega cm) at 1 kHz and the epicardial ST segment during 1 h of proximal reocclusion of the left anterior descending (LAD) coronary artery in 12 anesthetized pigs with one-month-old transmural infarction elicited by LAD ligature below the first branch. The impedance spectrum (1 to 1000 kHz) of normal and infarcted myocardium was measured in seven other pigs with similar infarctions. Electrical transmission of current pulses (30 microA) in infarcted tissue and in test solutions was also investigated. RESULTS: The infarct scar has a lower than normal resistivity (110 +/- 30 omega cm vs. 235 +/- 60 omega cm, p < 0.0001) and, unlike the normal myocardium, resistivity and phase angle of the scar did not change at increasing current frequencies, reflecting no capacitative response. LAD reocclusion induced a resistivity rise (510 +/- 135 omega cm, p < 0.01) and a ST segment elevation (0.6 +/- 0.7 to 9.5 +/- 5.1 mV, p = 0.002) in the ischemic peri-infarction zone, whereas the infarcted area showed ST segment elevation (0.5 +/- 0.5 to 3.8 +/- 2.6 mV, p = 0.03) with no resistivity changes. Potential decay of both ST segment and current pulses in the scar and in 0.9% NaCl solution was less than 1 mV/mm. Transmural deposition of connective tissue was seen in the center of the infarction. CONCLUSIONS: A one-month-old transmural infarction is a low resistance, noncapacitative medium that allows a good transmission of current pulses and of ST segment potential changes generated by contiguous peri-infarction ischemia.

Rhythmic oscillating complexes in gastrointestinal tract of chickens: a role for motilin.

Rhythmic oscillating complex (ROC) is a highly organized gastrointestinal motility pattern recently described in fasted avian species. ROCs show several high-speed aborad-propagated contractions that progressively change into others of orad direction. In addition, chickens show migrating motor complexes (MMC) in both fed and fasting states. Recently, motilin was isolated and characterized from chicken small intestine. Accordingly, the aim of this study was to learn whether chicken motilin might be involved in either ROC or MMC induction. Electromyographic recordings were obtained from different areas of the gastrointestinal tract of chickens while motilin was infused. The response to chicken motilin was dose dependent in both fed and fasted animals; a bolus of 4 x 10(-11) mol/kg (n = 5) did not modify the intestinal motor pattern, whereas 4 x 10(-10) and 4 x 10(-9) mol/kg (n = 5 each) induced a complete ROC pattern of 5.2 +/- 0.6 and 10.8 +/- 0.9 min, respectively. ROCs induced by chicken motilin presented exactly the same pattern as that described during a spontaneous ROC. Furthermore, motilin concentration in plasma, measured by radioimmunoassay, increased during a spontaneous ROC. This study suggests that chicken motilin triggers an ROC in chickens. The fact that plasma motilin levels increased during spontaneous ROC strongly suggests that motilin is involved in the induction of the ROC pattern. Motilin seems to play a different role in avian and mammalian species, because a phase III of the MMC was never induced by motilin infusion.

Lack of evidence of M-cells in porcine left ventricular myocardium.

OBJECTIVE: The aim of this study was to analyze whether cells with long action potential duration, fast Vmax, and spike-and-dome configuration (M-cells) are present in porcine left ventricular myocardium. METHODS: Transmembrane action potentials (n = 505) of the left ventricle were recorded with conventional glass microelectrodes in an epicardial-endocardial direction at 2000 ms basic cycle length in 14 pigs. In 3 pigs, potentials were obtained at 1000, 2000, and 5000 ms cycle length before and after superfusion with quinidine HCl 1 microgram/ml. In addition, transmembrane potentials (n = 52) were recorded in 4 dogs at 2000 ms cycle length to verify the ability of our protocol to detect M-cells. RESULTS: In pigs, action potential duration at 90% repolarization was shorter (ANOVA, P < 0.001) and Vmax slower (P < 0.001) in the epicardium than in the other transmural sites, but there were no regional differences in resting membrane potential or in action potential amplitude. Potentials with particularly long phase 3 or with spike-and-dome configuration were not observed. All myocardial sites displayed rate dependence of action potential duration (P = 0.02) which was transmurally homogeneous and persisted after quinidine exposure. The drug did not induce afterdepolarizations. In dogs, potentials with spike-and-dome configuration, long duration, and fast Vmax, like those described in M-cells, were detected in deep epicardial and midmyocardial areas. CONCLUSION: The porcine left ventricular myocardium shows transmural differences in action potential duration and Vmax, but, unlike dogs, it lacks M-cells.

CCK is involved in both peripheral and central mechanisms controlling food intake in chickens.

The aim of this work was to study the involvement of cholecystokinin (CCK) in the control of food intake in chickens. The following aspects were studied: 1) the effects of intravenous and intracerebroventricular sulfated octapeptide of CCK (CCK-8s) on voluntary food intake; 2) the effects of two CCK-receptor antagonists. L-365,260 and L-364,718, on food intake; and 3) the ability of such drugs to block the effects of CCK-8s on food intake in the chicken. Intravenous and intracerebroventricular CCK-8s caused a decrease in food intake. Intraperitoneal L-365,260, a CCK-receptor antagonist with low affinity for the two CCK receptors described in the chicken, increases food intake. Intracerebroventricular L-364,718, a drug that has high affinity for the chicken central CCK-receptor type, increased food intake. The effect of intravenous CCK-8s on food intake was not blocked by L-364,718 or L-365,260, whereas that of intracerebroventricular CCK-8s was blocked by intracerebroventricular L-364,718. It is concluded that central endogenous CCK plays a role in the control of food intake, which is dependent on central CCK-receptor type; nevertheless, peripheral CCK also decreases food intake acting on the peripheral CCK-receptor type. The fact that intracerebroventricular L-364,718 is able to increase food intake is related to its high affinity for the central CCK-receptor type of this species. Finally, three different speculations that might explain the fact that intraperitoneal L-365,260 increases food intake are discussed.

L-364,718 and L-365,260, two CCK antagonists, have no affinity for central benzodiazepine binding sites in chickens.

It has recently been demonstrated that L-365,260, a CCK-B antagonist in mammals, causes an increase in food intake in chickens. In contrast, L-364, 718, a CCK-A antagonist in mammals, shows this effect only at very high dose levels. It has been shown that L-365,260 has very low affinity for chicken CCK receptors. Thus, the mechanism of action of L-365,260 remains unknown. As L-365,260 is a benzodiazepine derivative, one may hypothesize that it would be acting on benzodiazepine binding sites. The aims of this work were to establish the existence of benzodiazepine binding sites in the chicken brain, and to check the possibility that L-365,260 was acting on these receptors, determining the affinity of L-364,718 and L-365,260 for them. We have found specific binding for tritiated flunitrazepam (a benzodiazepine agonist) ([3H]-flunitrazepam) in chicken brain membranes. A single binding site was detected with a Kd of 3.58 +/- 0.97 nM and a Bmax of 451.6 +/- 23.3 fmol/mg protein L-365,260 and L-364,718 exhibited very low affinity for these binding sites (Ki = 1.17 x 10(-6) +/- 0.16 x 10(-6) M and Ki > 10(-5) M, respectively). Thus, these results demonstrate that the increase in food intake caused by L-365,260 in the chicken is not due to a direct action on benzodiazepine receptors. Other possible explanations for its effect are discussed.

Central and NO mediated mechanisms are involved in the inhibitory effects of CCK on the chicken cecorectal area.

In chickens CCK-8s induces defecation and causes an inhibition of rectal electrical activity (EA) and an increase in cecal motility. In contrast, CCK-4 inhibits the motility of both rectum and ceca. The cecorectal responses to CCK-8s and CCK-4, given intravenously (i.v.), were studied in conscious chickens prepared with electrodes for electromyography; the influence of atropine, phentolamine plus propranolol, hexamethonium and L-NAME on such responses was determined. Atropine and phentolamine plus propranolol did not cause any change in the response to CCK-8s or CCK-4 in the cecorectal area. Hexamethonium only induced a significant decrease in the number of defecations (ND) induced by CCK-8s. L-NAME slightly modified the decrease in rectal EA due to CCK-8s. The effects of intracerebroventricular (i.c.v.) administration of CCK-8s and CCK-4 were also studied. CCK-8s and CCK-4, given i.c.v., caused, in conscious chickens, a slight decrease in cecal EA, in the 15 minutes following administration. This effect was similar to that seen after i.v. administration of CCK-4. In conclusion, our results suggest that the inhibitory action of CCK on chicken rectum is mediated, at least in part, through nitric oxide release. In addition, nicotinic receptors mediate the increase in the ND caused by CCK-8s. Ganglionic, muscarinic, adrenergic and nitrergic blockade were not able to modify the excitatory cecal response to CCK-8s, which may indicate that the receptor mediating this effect is located on the cecal smooth muscle. Finally, the inhibitory action of i.v. CCK-4 on chicken cecum seems to be centrally mediated, as suggested by the fact that i.c.v. administration of either CCK-8s or CCK-4 induce a similar effect.

Central and peripheral cholecystokinin receptors in chickens differ from those in mammals.

Specific binding for the radioligand [3H]CCK-8s has been identified in chicken brain, hypothalamus, pancreas, gallbladder and caecum membranes. This binding was found to be of high affinity, low capacity and saturable, suggesting the presence of specific CCK receptors in these tissues. Scatchard analysis indicated the existence of a single binding site for each tissue. Dissociation constant (kd) values were 0.63 +/- 0.18, 0.73 +/- 0.13, 0.85 +/- 0.12, 1.47 +/- 0.21 and 0.96 nM for brain, hypothalamus, pancreas, caecum and gallbladder, respectively. Binding densities (Bmax) were higher for brain, pancreas and caecum (32.60 +/- 10.70, 30.33 +/- 2.40 and 35.83 +/- 5.10 fmol/mg protein, respectively) than for the other two tissues (9.75 +/- 1.90 and 6.31 fmol/mg protein for hypothalamus and gallbladder, respectively). As in mammals, CCK-4 shows high affinity for CCK receptors located in chicken brain and hypothalamus, and very low affinity for those located in peripheral structures. L-364,718 (a CCK-A antagonist) showed a relative selectivity and a high affinity for those receptors located in central tissues, whereas L-365,260 (a CCK-B antagonist) is almost inactive in all studied tissues. These results give support for the existence of at least two distinct CCK receptors in birds and that these receptors are relatively different from those described in mammals.

Effects of cholecystokinin on chicken cecal motility: mechanisms involved.

The aims of this work are to characterize the effects of cholecystokinin (CCK) on chicken ceca and to study in vitro the mechanisms through which such actions are mediated. Longitudinal and circular cecal strips kept in vitro in organ baths were responsive to CCK sulphated octapeptide (CCK-8s). On longitudinal strips the response consisted of a fast phasic contraction followed by a sustained increase in tone which was dose dependent and decreased markedly in the presence of tetrodotoxin (TTX). Ketanserin (10(-5) M) also caused a decrease in the CCK-8s response. CCK tetrapeptide (CCK-4) and CCK unsulphated octapeptide (CCK-8ns) induced slightly less contractile effects at concentrations of 2 x 10(-6) M only. L365,260 and L364,718 decreased the response of longitudinal strips to CCK-8s with similar efficacy. On circular strips CCK-8s caused rhythmic phasic contractions of dose dependent amplitude and frequency, and both effects were resistant to TTX. The EC50 for the amplitude was about 4 times higher than that for the frequency. CCK-8ns (2x 10(-6) M) also caused phasic contractions, whereas the same concentrations of CCK-4 did not elicit any motor effects. L365,260 and L364,718 showed different efficacy in decreasing amplitude or frequency of contraction. These results suggest that 1) Both muscularly and neurally located CCK receptors are present on the longitudinal layer of chicken ceca whereas only muscular receptors are present on the circular muscle. 2) 5HT2 receptors seem to be involved in the neurally mediated CCK-8s response observed in the longitudinal layer. 3) The different potency of CCK-8s, CCK-8ns and CCK4 to induce contractile effects and of the CCK-A and CCK-B antagonists to block such effects suggests the existence of two different CCK receptors on the circular layer.

Rhythmic oscillating complex: characterization, induction, and relationship to MMC in chickens.

Migrating myoelectrical complexes (MMCs) and rhythmic oscillating complexes (ROCs) have been investigated in chickens prepared for electromyography. Animals were chronically implanted with electrodes in stomach, duodenum, jejunum, ileum, ceca, and rectum. MMCs showing phases I-III were found in the jejunum and ileum both in fed and fasted states. Repetitive spike bursts were recorded in the duodenum (0.5-1/h), disrupting the gastroduodenal coordination and preceding a phase III in the jejunum. ROCs appeared spontaneously in fasted animals and in 75% of the recordings during the dark period. Four consecutive intestinal myoelectrical patterns have been described during a ROC. Briefly, they consisted in series of high-speed propagated abroad contractions of great amplitude that progressively changed into others of orad direction. In relation to the MMC, the ROC pattern appeared just after a phase III reached the distal ileum, and a pattern of duodenal repetitive spike bursts, followed by a migrating phase III in the jejunum, started at the duodenum after a ROC. No myoelectrical changes were recorded in cecorectal activity during ROC. Vagotomized animals showed the ROC pattern. Neither apomorphine (5-100 micrograms/kg iv) nor cholecystokinin (10(-9) mol/kg iv) induced ROCs. Naloxone (5 x 10(-7) mol/kg iv) and atropine (0.1 mg/kg iv) induced isolated orad contractions. Myoelectrical and functional similarities can be found between retrograde giant contractions, described in mammals, and ROCs. However, they differ in their origin and mechanism of induction.

Cecocolonic motility in the chicken. Effects of cholecystokinin.

The aims of this work were 1) to define electromyographically the motility pattern of chicken ceca and colon; 2) to study the changes induced by photoperiod and food intake on the motility of this area and 3) to characterize the motor effects of intravenous (i.v.) cholecystokinin in vivo, measuring changes in electrical activity and intracecal pressure. Electromyographical studies show that in ceca, the spike burst frequency is higher during the day than during the night and in the fed than in the fasted state; about 90% of the bursts propagate towards the apex and corresponde to filling movements. In the colon the spike burst frequency during the day is 2.9 bursts/min in animals fed ad libitum. Nocturnal recordings in animals fed ad libitum and diurnal recordings in fasted animals show a significantly decreased electrical activity. Both CCK-8s and CCK-4 induce a dose-dependent decrease of colonic electrical activity and a dose-dependent increase in the number of colonic defecations. CCK-4 also causes a slight inhibition in the cecum, whereas CCK-8s induces an increase in cecal electrical activity. Intracecal pressure recordings performed in anaesthetized animals provide similar results. In conclusion, the cecocolonic motility of the chicken displays a circadian pattern and undergoes substantial modifications in the fed compared to the fasted state. CCK-8s is not mediating the increased colonic activity that follows food intake, as its effects on colonic motility are inhibitory. In contrast, i.v. CCK-8s induces defecation and a dose dependent increase in cecal electrical activity, intraluminal pressure and colonic defecation. Intravenous CCK-4 induces inhibitory effects both on ceca and colon.