Human Atrial Trabeculae Research Articles

Coenzyme Q10 improves the tolerance of the senescent myocardium to aerobic and ischemic stress: studies in rats and in human atrial tissue.

The inferior recovery of cardiac function after interventional cardiac procedures in elderly patients compared to younger patients suggests that the aged myocardium is more sensitive to stress. We report two studies that demonstrate an age-related deficit in myocardial performance after aerobic and ischemic stress and the capacity of CoQ10 treatment to correct age-specific diminished recovery of function. In Study 1 the functional recovery of young (4 mo) and senescent (35 mo) isolated working rat hearts after aerobic stress produced by rapid electrical pacing was examined. After pacing, the senescent hearts, compared to young, showed reduced recovery of pre-stress work performance. CoQ10 pretreatment (daily intraperitoneal injections of 4 mg/kg CoQ10 for 6 weeks) in senescent hearts improved their recovery to match that of young hearts. Study 2 tested whether the capacity of human atrial trabeculae (obtained during surgery) to recover contractile function, following ischemic stress in vitro (60 min), is decreased with age and whether this decrease can be reversed by CoQ10. Trabeculae from older individuals (> or = 70 yr) showed reduced recovery of developed force after simulated ischemia compared to younger counterparts (< 70 yr). Notably, this age-associated effect was prevented in trabeculae pretreated in vitro (30 min at 24 degrees C) with CoQ10 (400 MicroM). We measured significantly lower CoQ10 content in trabeculae from > or = 70 yr patients. In vitro pretreatment raised trabecular CoQ10 content to similar levels in all groups. We conclude that, compared to younger counterparts, the senescent myocardium of rats and humans has a reduced capacity to tolerate ischemic or aerobic stress and recover pre-stress contractile performance, however, this reduction is attenuated by CoQ10 pretreatment.

Human SERCA2a levels correlate inversely with age in senescent human myocardium

Objectives. This study sought to characterize functional impairment after simulated ischemia-reperfusion (I/R) or Ca2+bolus in senescent human myocardium and to determine if age-related alterations in myocardial concentrations of SERCA2a, phospholamban, or calsequestrin participate in senescent myocardial dysfunction.Background. Candidates for elective cardiac interventions are aging, and an association between age and impairment of relaxation has been reported in experimental animals. Function of the sarcoplasmic reticulum resulting in diastolic dysfunction could be dysregulated at the level of cytosolic Ca2+uptake by SERCA2a, its inhibitory subunit (phospholamban), or at the level of Ca2+binding by calsequestrin.Methods. Human atrial trabeculae from 17 patients (45–75 years old) were suspended in organ baths, field simulated at 1 Hz, and force development was recorded during I/R (45/120 min). Trabeculae from an additional 12 patients (53–73 years old) were exposed to Ca2+bolus (2–3 mmol/L bath concentration). Maximum ± dF/dt and the time constant of force decay (tau) were measured before and after I/R or Ca2+bolus and related to age. SERCA2a, phospholamban, and calsequestrin from 12 patients (39–77 years old) were assessed by immunoblot.Results. Functional results indicated that maximum ±dF/dt and tau were prolonged in senescent (>60 years) human myocardium after I/R (p < 0.05). Calcium bolus increased the maximum ±dF/dt and decreased tauin younger, but not older patients (p < 0.05). SERCA2a and the ratio of SERCA2a to either phospholamban or calsequestrin were decreased in senescent human myocardium (p < 0.05).Conclusions. Senescent human myocardium exhibits decreased myocardial SERCA2a content with age, which may, in part, explain impaired myocardial function after either I/R or Ca2+exposure.

Therapeutic Antidysrhythmic and Functional Protection in Human Atria

Approximately 30% of patients suffer supraventricular dysrhythmias after cardiac bypass. While the heart can be constructively preconditioned to maintain function against subsequent ischemic insult using a variety of stimuli across many species, preconditioning in experimental animals is associated with decreased postischemic reperfusion cardiac dysrhythmias. This mode of therapeutic preconditioning has not been previously examined in human atrial myocardium. We therefore hypothesized that preconditioning provides both antidysrhythmic and functional protection to human atria. To study this, human atrial trabeculae were suspended in organ baths, paced at 1 Hz, while force development and ectopy were recorded before and after simulated ischemia. The study consisted of five groups: (1) control trabeculae (n= 12), (2) trabeculae exposed to dysrhythmogenic stimuli (phenylephrine 50 μM and isoproterenol 25 μM (n= 8)), (3) trabeculae exposed to ischemia-reperfusion (I/R) injury and then drug stimulated (n= 10), (4) trabeculae preconditioned with adenosine (ADO 125 μM) then drug stimulated (n= 10), and (5) trabeculae preconditioned with ischemic preconditioning (IPC) then drug stimulated (n= 6) each at end reoxygenation. Differences between groups were assessed using χ2analysis and ANOVA (Bonferroni/Dunn). Results demonstrated that human atrial trabeculae did not exhibit dysrhythmia at baseline or when stimulated with α and β agonists. After I/R, control trabeculae exhibited stimulated reperfusion dysrhythmia, while trabeculae preconditioned with either ADO or transient ischemia exhibited decreased stimulated dysrhythmia (eachP< 0.05 vs. I/R). Functionally, I/R decreased developed force (DF) to 16 ± 2% of baseline (%BDF) while ADO pretreatment increased postischemic DF to 41 ± 3% BDF (P< 0.05 vs. I/R) while IPC increased DF to 49 ± 3% BDF (P< 0.05 vs. I/R). We conclude that (1) human atrial trabeculae can be functionally preconditioned with either ADO or IPC, and (2) protective preconditioning/cardioprotection does extend to dysrhythmia control and is therapeutically accessible in human atrial myocardium.

Adenosine Reduces Cardiac TNF-α Production and Human Myocardial Injury Following Ischemia-Reperfusion

Myocardial tumor necrosis factor-α (TNF-α) is an autocrine contributor to myocardial dysfunction and cardiomyocyte death in ischemia-reperfusion injury (I/R), sepsis, chronic heart failure, and cardiac allograft rejection. Cardiac resident macrophages and cardiomyocytes themselves produce TNF-α. In this regard, adenosine (ADO) has been reported to reduce macrophage TNF-α production. Our purposes were to determine whether (1) I/R induces rat myocardial TNF-α production; (2) ADO decreases ischemia-induced rat myocardial TNF-α production; (3) ADO functionally protects human myocardium against I/R; and (4) TNF-α-binding protein (TNFBP; p55) confers similar protection when substituted for ADO pretreatment. To study this, human atrial trabeculae were obtained during cardiac surgery and suspended in organ baths, paced at 1 Hz, and force development was recorded during I/R (45/120 min) with or without ADO pretreatment (125 μM × 10 min), or TNFBP (1 μg/ml) during I/R. Isolated rat hearts were perfused using the Langendorff method undergoing I/R (20/40 min) with or without ADO pretreatment (125 μM × 2 min) and rat myocardial expression of TNF-α was assessed by ELISA. Results demonstrated that I/R increased rat myocardial TNF-α levels from 324 ± 36 to 902 ± 77 pg/g (P< 0.05; ANOVA and Bonferroni/Dunn) and decreased human myocardial developed force (DF) to 18 ± 2% of baseline (%BDF;P< 0.05). ADO pretreatment decreased ischemia-induced rat myocardial TNF-α production (356 ± 107 pg/g;P< 0.05) and increased postischemic DF of human myocardium to 39 ± 3% BDF (P< 0.05). Further substantiating the link between ischemia-induced TNF-α production and injury, TNFBP administration similarly improved post-I/R function of human myocardium (55 ± 5% BDF;P< 0.05 vs. I/R alone). We conclude that (1) I/R induces rat myocardial TNF-α production; (2) ADO pretreatment decreases I/R-induced rat myocardial TNF-α production; (3) ADO improves human myocardial function; (4) TNFBP confers similar protection; and (5) inhibition/ neutralization of TNF-α represents a novel strategy for protecting human myocardium against ischemia and reperfusion injury.

Calcium Preconditioning in Human Myocardium

Background. Ischemic stress and other protein kinase C (PKC)–linked receptor stimuli can induce rapid cardiac protection against ischemia-reperfusion injury. We and others have demonstrated that exogenous calcium (Ca 2+) pretreatment confers PKC-mediated cardiac functional and infarct protection in animal models, but it remains unknown whether Ca 2+ preconditioning confers similar postischemic functional protection in human myocardium, and, if so, whether the mechanism is mediated by PKC. We postulated that Ca 2+ preconditioning confers ischemic tolerance to human myocardium by a PKC-dependent mechanism. Methods. Human atrial trabeculae were suspended in organ baths and paced at 1 Hz, and force development was recorded. After 90 minutes of equilibration, all trabeculae were subjected to ischemia (45 minutes) and reperfusion (120 minutes). Exogenous CaCl 2 (3.0 mmol/L for 5 minutes) or vehicle (saline solution) was administered before simulated ischemia, with or without concurrent PKC inhibition (bisindolylmaleimide I, 150 nmol/L). Results. Ischemia-reperfusion resulted in decreased postischemic developed force, Ca 2+ preconditioning protected human myocardium against ischemia-reperfusion injury ( p < 0.05 versus control ischemia-reperfusion), and concurrent PKC inhibition abolished the salutary effect of Ca 2+ preconditioning in human myocardium ( p < 0.05 versus Ca 2+ preconditioning). Conclusions. Preconditioning with Ca 2+ represents a potent means of accessing PKC-mediated protection of the human myocardium against ischemia-reperfusion injury.

The Effect of Oxytocin on the Contractile Force of Human Atrial Trabeculae

We performed an in vitro examination of the inotropic effect of oxytocin, chlorobutanol, and their combination to assess the effect of these drugs on the contractile force of human atrial trabeculae.Right atrial tissue samples were obtained during cardiac surgery with cardiopulmonary bypass. Trabeculae of the atrial appendage were dissected and mounted on muscle stands in a modified Krebs-Henseleit buffer bath. This isometric atrial trabecula preparation was subjected to a cumulative pharmacological protocol of either pure oxytocin, pure chlorobutanol, or a combination of the two drugs until no further change occurred in either developed force or resting force of the atrial trabeculae. A "no drug" buffer solution was used to assess the effect of time on the natural decay of the atrial preparation. The relative developed force of oxytocin plus chlorobutanol solution and pure chlorobutanol were similar in magnitude and lower than that in control experiments (P < 0.001) Pure oxytocin did not change the contractile force of atrial tissue. We conclude that pure oxytocin does not have a cardiodepressive effect in this human atrial preparation. Chlorobutanol has a negative inotropic effect, which is of a magnitude similar to a combined solution of chlorobutanol and oxytocin. Therefore, chlorobutanol added as a preservative to the commercial synthetic oxytocin solution may contribute to hypotension observed in patients after an intravenous bolus injection. Implications: We obtained specimens of heart tissue from patients undergoing cardiac surgery and conducted a laboratory study of the effects of oxytocin and its preservative (chlorobutanol) on these tissue samples. Chlorobutanol decreased the ability of the heart to contract, while as pure oxytocin had no effect. This explains why maternal blood pressure may decrease and provides impetus to produce oxytocin with another, safer preservative. (Anesth Analg 1988;86:40-4)

The effect of oxytocin on the contractile force of human atrial trabeculae.

We performed an in vitro examination of the inotropic effect of oxytocin, chlorobutanol, and their combination to assess the effect of these drugs on the contractile force of human atrial trabeculae. Right atrial tissue samples were obtained during cardiac surgery with cardiopulmonary bypass. Trabeculae of the atrial appendage were dissected and mounted on muscle stands in a modified Krebs-Henseleit buffer bath. This isometric atrial trabecula preparation was subjected to a cumulative pharmacological protocol of either pure oxytocin, pure chlorobutanol, or a combination of the two drugs until no further change occurred in either developed force or resting force of the atrial trabeculae. A "no drug" buffer solution was used to assess the effect of time on the natural decay of the atrial preparation. The relative developed force of oxytocin plus chlorobutanol solution and pure chlorobutanol were similar in magnitude and lower than that in control experiments (P < 0.001) Pure oxytocin did not change the contractile force of atrial tissue. We conclude that pure oxytocin does not have a cardiodepressive effect in this human atrial preparation. Chlorobutanol has a negative inotropic effect, which is of a magnitude similar to a combined solution of chlorobutanol and oxytocin. Therefore, chlorobutanol added as a preservative to the commercial synthetic oxytocin solution may contribute to hypotension observed in patients after an intravenous bolus injection. We obtained specimens of heart tissue from patients undergoing cardiac surgery and conducted a laboratory study of the effects of oxytocin and its preservative (chlorobutanol) on these tissue samples. Chlorobutanol decreased the ability of the heart to contract, while as pure oxytocin had no effect. This explains why maternal blood pressure may decrease and provides impetus to produce oxytocin with another, safer preservative.

Felodipine protects human atrial muscle from hypoxia–reoxygenation dysfunction: a force–frequency relationship study in an in vitro model of stunning

Aims: We aimed at investigating contractile changes after hypoxia–reoxygenation and dobutamine challenge in superfused human atrial pectinate muscle to see whether high versus low stimulation rate during hypoxia might account for outcome differences compatible with the definition of an in vitro model of myocardial stunning and whether pretreatment with the dihydropyridine Ca2+ entry blocker felodipine might afford protection. Methods: Human right atrial trabeculae obtained from adult patients were superfused in an organ bath with oxygenated (O2 content 16 ml/l) and modified (NaHCO3 25.7 mmol/l) Tyrode's solution at 37°C. Dobutamine (1 nmol/l to 10 μmol/l) was superfused in 10 oxygenated preparations to select the optimal drug concentration to be used in another 22 which were randomized. Group (A) consisted of time-related controls (Tyrodes's solution for 225 min at cycle length (CL) 1600 ms and no dobutamine). There were two test groups, respectively: (B) low (1600 ms CL) and (C) high (400 ms CL) stimulation rate. After 60 min of stabilization, in groups B and C, hypoxic superfusion (O2 content 5 ml/l) lasted 60 min, then reoxygenation (60 min) and dobutamine challenge (1 μmol/l, 15 min) were performed. Analysis of variance for repeated measures with the Greenhouse–Geisser correction, and a repeated measures model with structured covariance (preparation mass, length, width and time-varying time to peak tension) matrices were used whereby grouping (G), time (T) and G·T interaction were weighted. Force–frequency relationship and post-pausal potentiation were studied after each phase. Electrophysiology, histomorphometry and electron microscopy were carried out (n=6). Felodipine (0.1 μmol/l, n=5) pretreatment (15 min before hypoxia) was given in parallel experiments. Results: Time-related controls showed =̃ 10% per hour decrease of developed tension and the Paradise test provided ≅80% of control values. In test groups (as compared to baseline values) contractility was decreased ≅65% after hypoxia–reoxygenation and it increased ≅25% after dobutamine (G, 0.0155<P<0.0065; T, P=0.00005; G·T, P=0.00005). High stimulation rate during hypoxia worsened hypoxia–reoxygenation contractile changes, whereas reversibility after dobutamine was less. In both B and C groups during hypoxia, contractility decreased quite rapidly, although by 10 min or so a plateau (≅50%) was reached in group B, whereas in group C contractility decreased to <20%. None of the covariates contributed significantly to predict the dependent variables investigated. Force–frequency relationship and post-pausal potentiation were repeatable, paralleled overall changes due to hypoxia, reoxygenation and dobutamine challenge and were useful to discriminate Ca2+-related diastolic processes thus helping index myocardial contractile reserve. Force–frequency relationship was negative at high stimulation rates, concomitant to an abrupt change of shape and duration of action potential with little time for Ca2+-related Ca2+ release and ensuing systolic processes. Felodipine pretreatment enabled an unblunted response to dobutamine. Histomorphometry showed an unexpected `fibrotic core'. At electron microscopy, subendocardial and deep part of the same pectinate muscles showed identical degrees of degenerative lesions. Superfused samples showed, unexpectedly, less anoxic lesions than preparations fixed within 15 min from surgical explant, although lesions were higher than in samples fixed immediately after explant. Conclusions: This might be a relevant model, whereby pharmacological or physical interventions are tested. Native human atrial trabeculae might be used without dissection and/or preservatives. If high stimulation rate during hypoxia is used the power of hypothesis testing is maximized. Future studies with this material will be easier and comparatively smaller series might be investigated. Felodipine at low concentration was protective in this model.

Ischemic preconditioning of human myocardium: protein kinase C mediates a permissive role for alpha 1-adrenoceptors.

The purposes of this study were to determine whether ischemic preconditioning (IPC) in human atrial trabeculae is mediated by alpha 1-adrenoceptors and protein kinase C (PKC) and whether the protection of IPC is replicated with alpha 1-adrenoceptor stimulation [alpha 1-adrenoceptor preconditioning (alpha 1-PC)]. Atrial trabeculae were obtained during coronary bypass surgery. The trabeculae were suspended in organ baths containing Tyrode solution and field stimulated at 1 Hz, and developed force was recorded. The trabeculae underwent 45 min of simulated ischemia (SI) and 120 min of reperfusion (I/R injury). IPC trabeculae received transient SI before I/R injury, alpha 1-Adrenoceptor blockade with BE-2254 and PKC inhibition with chelerythrine were independently combined with IPC before I/R injury. alpha 1-PC before I/R was examined with alpha 1-adrenergic agonist (phenylephrine) pre-treatment. Improved recovery of developed force and higher tissue creatine kinase activity were present in IPC trabeculae, and the protective effect of IPC was eliminated with either alpha 1-adrenoceptor blockade or PKC inhibition. alpha 1-PC trabeculae also exhibited enhanced functional recovery after I/R injury but lacked preservation of tissue creatine kinase activity. PKC inhibition eliminated the functional protection of alpha 1-PC. These results suggest that, in human atrial trabeculae, alpha 1-adrenoceptors and PKC mediate, in part, the functional and tissue CK preservation conferred by IPC, but alpha 1-PC does not replicate the protection of IPC.

Oral sulfonylurea hypoglycemic agents prevent ischemic preconditioning in human myocardium. Two paradoxes revisited.

Patients receiving oral hypoglycemic agents for diabetes mellitus are at increased risk of cardiovascular mortality. Oral hypoglycemic agents are inhibitors of the ATP-sensitive potassium (KATP) channel. Ischemic preconditioning is mediated by KATP channel activation. We therefore hypothesized that myocardium from patients taking long-term oral hypoglycemic agents would be resistant to the protection by ischemic preconditioning. Isolated human right atrial trabeculae were suspended in an organ bath at 37 degrees C, with field stimulation at 1 Hz. Control trabeculae were then subjected to 45 minutes of simulated ischemia (hypoxic, glucose-free buffer with pacing at 3 Hz) and 120 minutes of reperfusion. Ischemic preconditioned (IPC) trabeculae from patients without oral hypoglycemic therapy and from patients taking insulin (Ins+IPC) were given 5 minutes of simulated ischemia before this injury. Trabeculae (Oral Hypo+IPC) were obtained from patients taking long-term oral hypoglycemic agents and were also exposed to 5 minutes of simulated ischemia before this injury. Developed force (DF) was recorded. Recovery of DF relative to preischemic values was 28 +/- 4% in control trabeculae, whereas IPC trabeculae showed 52 +/- 5% recovery (P < .05 versus control). In patients receiving long-term oral hypoglycemic agents (Oral Hypo+IPC), recovery of DF was 27 +/- 3%, but in trabeculae from insulin-treated patients (Ins+IPC), it was 45 +/- 6%. Human myocardium from patients without long-term exposure to oral hypoglycemic agents is functionally protected by preconditioning. Long-term oral hypoglycemic intake blocks the protection by preconditioning. These data suggest that ischemic preconditioning in human myocardium relies on KATP channels, and long-term inhibition of KATP channels with oral hypoglycemic agents may explain the excess cardiovascular mortality in these patients.

Angiotensin-Converting Enzyme Inhibitors Potentiate Preconditioning Through Bradykinin B2Receptor Activation in Human Heart

Objectives. This study was designed to determine whether angiotensin-converting enzyme (ACE) inhibitors play a role in cardioprotection in a human model of preconditioning.Background. Recent studies have suggested that bradykinin may contribute to the protective effects of preconditioning in animal models. ACE inhibitors are known to inhibit the degradation of bradykinin and hence may be able to potentiate the effect of preconditioning.Methods. We examined the effects of the ACE inhibitors captopril and lisinopril in combination with a subthreshold preconditioning stimulus (i.e., insufficient to have any protective effects alone). Human atrial trabeculae were superfused with Krebs buffer and paced at 1 Hz. They were subjected to a full or subthreshold preconditioning stimulus consisting of either 3 or 1.5 min of simulated ischemia and 7 min of reoxygenation. In each instance, this stimulus was followed by 90 min of simulated ischemia and 2 h of reoxygenation. In addition, the subthreshold preconditioned group had 20 min of previous ACE inhibitor treatment.Results. Recovery of contractile function (percent of baseline) was 22 ± 1% (mean ± SEM) in the control group versus 61 ± 1% in the preconditioned group. The subthreshold preconditioned group and the ACE inhibitor-alone groups did not exhibit any protection; however, in combination, the protection was significant (71 ± 4% in the captopril group, 58 ± 8% in the lisinopril group, p < 0.005) compared with the control group. There was no significant difference between these values and recovery after the full preconditioning stimulus. Furthermore, Hoe 140, a specific bradykinin B2receptor antagonist, abolished the protection.Conclusions. To our knowledge, these are the first results in human muscle to suggest that ACE inhibitors may augment ischemic preconditioning, possibly through B2receptor activation.(J Am Coll Cardiol 1997;29:1599–606)

Stereoselectivity of actions of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264.

The thiadiazinone derivative [+]-EMD 60263 ((+)-5-(l-(alpha-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4-tetrah ydroquinoline -6-yl)-6-methyl-3,6-dihydro-2H-1,3,4 -thiadiazine-2-on) is a Ca(2+)-sensitizing agent with only minor phosphodiesterase inhibitory activity. Our aim was to characterize the inotropic and electrophysiological effects of [+]-EMD 60263 and its enantiomer [-]-EMD 60264 in several cardiac muscle preparations. The Ca(2+)-sensitizing activity resided in the [+]-enantiomer only. [+]-EMD 60263 (3 microM) shifted the EC50 of Ca2+ for contractile activation of skinned fibers of pig heart from 2.41 microM to 0.73 microM, whereas [-]-EMD 60264 (30 microM) was ineffective. In Langendorff-perfused guinea pig hearts, [+]-EMD 60263 and [-]-EMD 60264 induced concentration-dependent positive and negative inotropic effects, respectively; both enantiomers reduced spontaneous heart rate but did not influence perfusion pressure. The maximum increase in force of human atrial trabeculae was 35% of pre-drug control with [+]-EMD 60263 in comparison to 113% with forskolin. In guinea-pig papillary muscles, [+]-EMD 60263 and [-]-EMD 60264 had opposite inotropic responses, however, both agents similarly prolonged action potential duration. Both enantiomers concentration-dependently blocked the rapidly activating component IKr of the delayed rectifier in guinea-pig myocytes. The block saturated at potentials positive to +30 mV, closely resembling the effects of the antiarrhythmic agent E-4031 which had been originally used to define IKr.

Adenosine Preconditioning of Human Myocardium is Dependent upon the ATP-sensitive K+Channel

Evidence supports the involvement of adenosine receptor stimulation and activation of KATPchannels in ischemic preconditioning of human myocardium. It is unknown, however, whether protection mediated by adenosine receptors is dependent upon the KATPchannel in the human heart. The purpose of this study was to determine whether adenosine-mediated protection against a simulated ischemia–reperfusion injury in human myocardium is dependent upon KATPchannels. Isolated human right atrial trabeculae were placed in tissue baths at 37°C, oxygenated with a modified Tyrode solution, and field stimulated at 1 Hz. Trabeculae were subjected to 45 min of normothermic simulated ischemia (hypoxic, substrate-free buffer with pacing at 3 Hz.) and 60 min of reperfusion (I/R trabeculae). Trabeculae were preconditioned with simulated ischemia (IPC trabeculae) or adenosine receptor stimulation (adenosine, 125 μmol/l) for 5 min (ADO trabeculae) prior to simulated ischemic–reperfusion injury. Inhibition of the KATPchannel with glibenclamide (10 μmol/l) was combined with adenosine pretreatment (ADO+GLI trabeculae) or alone (GLI trabeculae) prior to simulated ischemic–reperfusion injury. Developed force (DF) at end reperfusion (mean±s.e.) was compared to baseline developed force, and tissue creatine kinase (CK) activity at end reperfusion was measured. I/R trabeculae showed 27±2% of baseline DF, whereas IPC trabeculae or ADO trabeculae showed 50±4% and 43±3% of baseline DF, respectively. ADO+GLI trabeculae showed 25±2% and GLI trabeculae showed 23±4% of baseline DF. Tissue CK activity was enhanced in the IPC and ADO trabeculae (433±63 U/g wet myocardium, and 415±28 U/g wet myocardium, respectively). I/R trabeculae had 196±26 U/g wet myocardium and ADO+GLI trabeculae had 277±38 U/g wet myocardium at end reperfusion. The results suggest that ischemic preconditioning and adenosine receptor stimulation confer functional protection against simulated ischemic-reperfusion, and adenosine mediated protection is eliminated by KATPchannel inhibition in human myocardium.

Preconditioning and Hypothermic Cardioplegia Protect Human Heart Equally Against Ischemia

Background. The purpose of this study was to determine whether transient ischemic preconditioning protects human myocardium against normothermic ischemic injury. Methods. Isolated human right atrial trabeculae were suspended in an organ bath with oxygenated Tyrode’s solution at 37°C and field stimulated at 1 Hz. Developed force was recorded. Trabeculae (Warm I/R) received normoxic perfusion before 45 minutes of normothermic simulated ischemia (hypoxic, substrate-free buffer with pacing at 3 Hz) and 120 minutes of reperfusion. Preconditioned trabeculae (Warm IPC) were subjected to 5 minutes of normothermic simulated ischemia and 10 minutes of perfusion before normothermic simulated ischemia-reperfusion injury. Trabeculae (Cold I/R) were subjected to hypothermic (4°C) ischemia (hypoxic buffer) for 4 hours and 60 minutes of reperfusion (37°C). Preconditioned trabeculae (Cold IPC) were pretreated with 5 minutes of normothermic simulated ischemia before hypothermic ischemia and 60 minutes of reperfusion. At the end of reperfusion, trabeculae were frozen at −70°C and assayed for tissue creatine kinase activity. Results. At the end of reperfusion, warm preconditioned trabeculae (Warm IPC) recovered 51% ± 5% of baseline developed force, whereas warm I/R trabeculae recovered 24% ± 3% ( p < 0.05). Tissue creatine kinase levels reflecting preserved tissue viability were sustained in Warm IPC trabeculae (1,183 ± 204 U/g), whereas nonpreconditioned control trabeculae (Warm I/R) exhibited lower levels of enzymatic activity (403 ± 32 U/g) ( p < 0.05). In contrast, Cold IPC trabeculae recovered 47% ± 5% and Cold I/R, 56% ± 8% of baseline developed force at the end of reperfusion ( p > 0.05). Conclusions. We conclude that transient ischemic preconditioning protects human myocardium against normothermic ischemic injury. (Ann Thorac Surg 1997;63:147–52)

The shape of human atrial action potential accounts for different frequency-related changes in vitro

We aimed at investigating frequency-related changes of human atrial action potential (AP) in vitro to see whether baseline AP shape might account for different responses to increasing stimulation rates. Human right atrial trabeculae ( n = 48) obtained from adult ( n = 38, mean age 59 ± 8, range 45–72 years) consecutive patients (≅30% of those operated upon by a single surgeon; 1.26 preparations per patient, range 1–2) were superfused in an organ bath with oxygenated (O 2 content 16 ml/l) and modified (NaHCO 3 25.7 mmol/l) Tyrode's solution at 31 °C. Baseline electrophysiology (pacing: 1 ms duration, 2–4 mA current intensity) at cycle length (CL) of 1000 ms was recorded in 90% (43 out of 48) of the preparations. The frequency-related protocol (CL from 1600 to 300 ms) was, however, undertaken in 23 (48%) preparations because 20 (42%) became pacing unresponsive immediately after baseline recordings. No statistical differences were seen when baseline electrophysiological parameters (mean ± SD) were grouped according to late pacing responsiveness ( n = 43 vs. n = 23): respectively, resting membrane potential (RMP) was −74 ± 6 vs. −75 ± 4 mV, maximal upstroke velocity (Vmax) 172 ± 60 vs. 173 ± 39 V/s, AP amplitude (APA) 89 ± 11 vs. 91 ± 8 mV and AP durations were at 30% (APD30%) 10 ± 13 vs. 13 ± 18 ms, 50% (APD50%) 45 ± 79 vs. 62 ± 91 ms and 90% (APD90%) 383 ± 103 vs. 407 ± 108 ms. To classify baseline AP shape, two criteria were adopted: criterion 1 (“objective”), based on APA (cut-off 90 mV) and APD90% (cut-off 500 ms) computed values and criterion 2 (“visual”) derived from the literature. These criteria enabled us to differentiate three AP shape types: type 1 (spike and dome), type 3 (no dome) and type 4 (extremely prolonged). At baseline, the two criteria diagnosed different proportions of AP shape types. There were, however, no intra-type statistical differences among electrophysiological parameters. By criterion 1, analysis of variance (ANOVA) showed significant inter-type differences of RMP, Vmax, APA, APD50 and 90% and by criterion 2 of APA, APD30, 50 and 90%, respectively. To facilitate comparisons with previous published data, criterion 2 was selected to analyse frequency-related changes of AP shape types. At low stimulation rate, ANOVA for repeated measures (with Greenhouse-Geisser \\ ̂ g3 correction) showed inter-type differences for APD30, 50 and 90% ( P = 0.00005). RMP, Vmax, APA and APD90% were overall frequency-related ( P = 0.00005). Inter-type frequency-related differences were however seen only for APD90%. Human atrial AP durations (30, 50 and 90%) enable differentiation among AP shape types (1, 3 and 4). By a standardized use-dependent protocol overall RMP, Vmax, APA and APD90% are frequency-related. AP shape accounts for frequency-related changes of APD90% only. A type 4 AP shape with much prolonged AP duration had a flat frequency dependence. At high stimulation rates, adult type 1 and 3 AP shapes are indistinguishable. Use-dependent and pharmacological investigations in human atrial myocytes need to take AP shape into account.

Effects of hypoxia, simulated ischemia and reoxygenation on the contractile function of human atrial trabeculae.

Hypoxia, ischemia and reoxygenation cause contractile dysfunction which will be characterized by the time course of isometric contraction of human atrial trabeculae. Post-rest potentiation (PRP) and postextrastimulatory potentiation (PEP) were elicited to obtain indirect information about the the role of the sarcoplasmic reticulum (SR) in excitation-contraction coupling. As lipid peroxidation could cause SR dysfunction, thiobarbituric acid reactive substances (TBARS) were measured. After 30 min of hypoxia (H) or simulated ischemia (H combined with acidosis-SI), contractile force decreased to 15% and 6%, respectively, of control (p < or = 0.05), whereas the normalized rate of both contraction and relaxation increased. In group H, rapid reoxygenation produced a recovery of contractile force to about 60%. After post-hypoxic reoxygenation the TBARS concentration was increased. In group SI, rapid reoxygenation and a rather gradual correction of acidosis produced complete recovery of contractile force. PRP and PEP were maintained during H and SI. Particularly post-ischemic reoxygenation caused a marked depression of PRP and partly of PEP. Thus, alteration of SR Ca2+ handling occurs predominantly during reoxygenation rather than during H or SI, probably associated with the damaging effect of increased oxygen radicals. The depression of potentiation occurred along with delayed relaxation, temporary increased resting force, mechanical alternans, and spontaneous activity which are further characteristics for SR dysfunction. Thus, for a possibly beneficial effect of low pH during SI combined with its gradual correction during reoxygenation on the recovery of contractile function, developed force should not be the only index.

Preconditioning in isolated superfused human muscle

Studies in isolated superfused rabbit papillary muscles indicate that preconditioning (PC) is not confined to arterially perfused myocardium. In the present study PC of isolated human right atrial trabeculae was investigated avoiding the problems of invasive experimentation in patients. Atrial trabeculae were suspended in an organ bath, superfused with Tyrode's solution and field stimulated at 1 Hz. After stabilization, muscles were randomly allocated to five groups (n = 8 per group). Control (C) muscles had no additional treatment. PC was induced by 3 min rapid pacing at 3 Hz with hypoxic substrate-free buffer, followed by reoxygenation with substrate for 12 min. In two additional groups 8-p-sulfophenyltheophylline (SPT) was added to the superfusate either during stabilization in controls (C+SPT) or during preconditioning (PC+SPT). In the final group, R-phenyl-isopropyl adenosine (R-PIA) was added to the superfusate for 5 min to see whether or not this could substitute for preconditioning. All muscles were then exposed to 90 min hypoxia with no substrate and pacing at 3 Hz, followed by 120 min reoxygenation at 1 Hz. Recovery of developed tension was significantly improved by PC 46.5 +/- 2.4% v 24.6 +/- 2.3% in controls) and this protective effect was blocked by the addition of SPT without adversely affecting controls (recovery in PC+SPT, 25.8 +/- 4.1% and C+SPT, 22.7 +/- 2.9%). R-PIA protected the muscles to a similar extent as PC (43.8 +/- 1.9%). These data provide evidence for the involvement of adenosine in preconditioning in human myocardium.

Effects of the new phosphodiesterase-III inhibitor R80122 on contractility and calcium current in human cardiac tissue.

The selective phosphodiesterase III (PDE-III) inhibitor R80122 ((E)-N-cyclohexal-N-methyl-2-[[[phenyl-(1,2,3,5- tetrahydro-2-oxoimidazo-[2,1b]-quinazolin-7-yl)-methylene]-a mino]-oxy]-acetamide) has been reported to possess greater cardiotonic potency and less side effects than the standard compounds milrinone or enoximone. To characterize this new compound further, we investigated the effects of R80122 on force of contraction (Fc) and calcium current (ICa) in human right atrium (HRA) and human left ventricle (HLV) with reference to the nonselective PDE-inhibitor IBMX (3-isobutyl-1-methylxanthine). With "late" exposure (300- to 330-min equilibration) of human atrial trabeculae, R80122 (3 microM) increased Fc by 60 +/- 11%; log EC50 was -6.2 +/- 0.1. R80122 (3 microM) induced a relative leftward shift of forskolin concentration-response curves by 0.34 log units; the respective value for IBMX (20 microM) was 0.46. A positive inotropic effect of R80122 was also shown in guinea pig papillary muscles. ICa was measured in voltage-clamped isolated myocytes of human right atrial and left ventricular (LV) tissue, and, for comparison, guinea pig ventricle. With clamp steps from -40 to +5 mV, R80122 (3 microM) increased peak ICa from 3.1 +/- 0.2 to 5.4 +/- 0.3 pA/pF in HRA cells, from 2.9 +/- 0.4 to 5.1 +/- 0.6 pA/pF in HLV cells, and from 4.4 +/- 0.3 to 6.6 +/- 0.5 pA/pF in guinea pig myocytes. IBMX 20 microM increased ICa to a greater extent. Washout or addition of carbachol 10 microM partially reversed the effect of R80122. Voltage dependence, inactivation time course, and steady-state inactivation of ICa were little changed by either compound. Stimulation of Ca2+ influx by L-type Ca2+ channels contributes to the positive inotropic effect of the selective PDE-III inhibitor R80122.

Studies of the nucleoside transporter inhibitor, draflazine, in the human myocardium.

1. The aim of the present study was to determine the effect of the nucleoside transporter inhibitor, draflazine, on the force of contraction in human myocardium and the affinity of the compound for the nucleoside transporter. Nucleoside transport inhibitors, like draflazine, are of potential importance for cardiopreservation of donor hearts for heart transplantation. 2. Functional experiments were performed in isolated electrically driven (1 Hz, 1.8 mmol l-1 Ca2+) human atrial trabeculae and ventricular papillary muscle strips. The affinity of draflazine for the myocardial nucleoside transporter was studied in isolated membranes from human ventricular myocardium and human erythrocytes in radioligand binding experiments using [3H]-nitrobenzylthioinosine ([3H]-NBTI). Dipyridamole was studied for comparison. 3. In membranes from human myocardium and erythrocytes, [3H]-NTBI labelled 1.18 pmol mg-1 protein and 23.0 pmol mg-1 protein, respectively, nucleoside transporter molecules with a KD value of 0.8 nmol l-1. Draflazine concentration-dependently inhibited binding of [3H]-NBTI to myocardial and erythrocyte membranes with a K(i)-value of 4.5 nmol l-1. The potency as judged from the K(i) values was ten times greater than that of dipyridamole in both myocardial and erythrocyte membranes. 4. Draflazine, at concentrations up to 100 mumol l-1, did not produce negative inotropic effects in atrial and ventricular myocardium. (-)-N6-phenylisopropyladenosine (R-PIA) and carbachol did not reduce force of contraction in ventricular myocardium, but exerted concentration-dependent direct negative inotropic effects in atrial myocardium. 5. The data provide evidence that draflazine specifically binds to the nucleoside transporter of the human heart and erythrocytes with high affinity. The compound does not produce negative inotropic effects at concentrations as high as 100 micromol 1-1.6. Draflazine could be a useful agent for cardio preservation because it does not produce cardio depressant effects. Thus, it may be possible to perfuse explanted hearts directly with this agent without the hazard of cardiodepression.

Triggered propagated contractions in human atrial trabeculae

Mechanical stretch and rapid release of locally damaged regions of rat cardiac trabeculae due to contraction of undamaged cells during the regular twitch have been shown to trigger local aftercontractions in the damaged region. These aftercontractions appear to propagate along the length of the trabeculae and to precede triggered arrhythmias. The aim of this study was to determine whether triggered propagated contractions can also be elicited in human cardiac tissue. Trabeculae were dissected from a biopsy of the right atrial appendage which was obtained during cardiac surgery. They were mounted horizontally, superfused with a modified Krebs-Henseleit solution, and monitored with a video system. Force was measured with a silicon strain gauge; sarcomere length was measured with laser diffraction techniques. Triggered contractions were elicited in 10 trabeculae following trains of 15 stimuli at a rate of 2 Hz, separated by 15 s rest intervals, at 21 degrees C and a [Ca2+]o above 5.0 mM. Both video analysis and sarcomere length recordings showed that the contractions started at one end of the trabeculae and travelled from there as a localised contraction along the muscle. Increasing [Ca2+]o or the number of conditioning stimuli increased force of the stimulated twitches up to a maximum; further increase of [Ca2+]o or the number of stimuli was accompanied by a decrease of force. Force of the triggered propagated contractions increased monotonically with increasing [Ca2+]o and number of stimuli, while the interval between last stimulus and the peak of the force transient (or latency) decreased progressively, and propagation velocity increased monotonically. Propagation velocity was calculated from the interval between peak sarcomere shortening at two sites and the distance between these sites, as well as from the ratio between the length of the muscle and the duration of the force transient in the remainder of the trabeculae. With increasing temperature at constant [Ca2+]o twitch force increased while latency, force, and duration of the force transient decreased. At 37 degrees C, localised contractions travelling along the muscle could still be induced at sufficiently increased [Ca2+]o. For all interventions, propagation velocity varied from 0.8 to 3.9 mm.s-1. Contractions can be elicited in human atrial cardiac trabeculae. These contractions have the same basic characteristics as the triggered propagated contractions that have been described previously in rat ventricular trabeculae.