Phospholamban ablation rescues the enhanced propensity to arrhythmias of mice with CaMKII-constitutive phosphorylation of RyR2 at site S2814.

KEY POINTS: Mice with Ca(2+) -calmodulin-dependent protein kinase (CaMKII) constitutive pseudo-phosphorylation of the ryanodine receptor RyR2 at Ser2814 (S2814D(+/+) mice) exhibit a higher open probability of RyR2, higher sarcoplasmic reticulum (SR) Ca(2+) leak in diastole and increased propensity to arrhythmias under stress conditions. We generated phospholamban (PLN)-deficient S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice, to test the hypothesis that PLN ablation can prevent the propensity to arrhythmias of S2814D(+/+) mice. PLN ablation partially rescues the altered intracellular Ca(2+) dynamics of S2814D(+/+) hearts and myocytes, but enhances SR Ca(2+) sparks and leak on confocal microscopy. PLN ablation diminishes ventricular arrhythmias promoted by CaMKII phosphorylation of S2814 on RyR2. PLN ablation aborts the arrhythmogenic SR Ca(2+) waves of S2814D(+/+) and transforms them into non-propagating events. A mathematical human myocyte model replicates these results and predicts the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a CaMKII-dependent leaky RyR2. ABSTRACT: Mice with constitutive pseudo-phosphorylation at Ser2814-RyR2 (S2814D(+/+) ) have increased propensity to arrhythmias under ß-adrenergic stress conditions. Although abnormal Ca(2+) release from the sarcoplasmic reticulum (SR) has been linked to arrhythmogenesis, the role played by SR Ca(2+) uptake remains controversial. We tested the hypothesis that an increase in SR Ca(2+) uptake is able to rescue the increased arrhythmia propensity of S2814D(+/+) mice. We generated phospholamban (PLN)-deficient/S2814D(+/+) knock-in mice by crossing two colonies, S2814D(+/+) and PLNKO mice (SD(+/+) /KO). SD(+/+) /KO myocytes exhibited both increased SR Ca(2+) uptake seen in PLN knock-out (PLNKO) myocytes and diminished SR Ca(2+) load (relative to PLNKO), a characteristic of S2814D(+/+) myocytes. Ventricular arrhythmias evoked by catecholaminergic challenge (caffeine/adrenaline) in S2814D(+/+) mice in vivo or programmed electric stimulation and high extracellular Ca(2+) in S2814D(+) /(-) hearts ex vivo were significantly diminished by PLN ablation. At the myocyte level, PLN ablation converted the arrhythmogenic Ca(2+) waves evoked by high extracellular Ca(2+) provocation in S2814D(+/+) mice into non-propagated Ca(2+) mini-waves on confocal microscopy. Myocyte Ca(2+) waves, typical of S2814D(+/+) mice, could be evoked in SD(+/+) /KO cells by partially inhibiting SERCA2a. A mathematical human myocyte model replicated these results and allowed for predicting the increase in SR Ca(2+) uptake required to prevent the arrhythmias induced by a Ca(2+) -calmodulin-dependent protein kinase (CaMKII)-dependent leaky RyR2. Our results demonstrate that increasing SR Ca(2+) uptake by PLN ablation can prevent the arrhythmic events triggered by SR Ca(2+) leak due to CaMKII-dependent phosphorylation of the RyR2-S2814 site and underscore the benefits of increasing SERCA2a activity on SR Ca(2+) -triggered arrhythmias.

Glibenclamide action on myocardial function and arrhythmia incidence in the healthy and diabetic heart.

Myocardial sarcolemmal ATP-dependent potassium (KATP) channels, which are normally closed by high ATP concentration, open during ischemia when ATP generation decreases favoring K(+) efflux. This reduces action potential duration (APD) decreasing the time of Ca(2+) influx and Ca(2+) overload. This behavior suggested that they might be involved in the protection against stunning and arrhythmias and in the mechanism of ischemic preconditioning. Sulfonylureas, used as hypoglycemic agents for the treatment of type 2 diabetes also block myocardial KATP channels prolonging APD during ischemia, which by allowing Ca(2+) entry for a longer period of time, is potentially harmful to the heart. Controversial findings have been reported regarding the protective effect of sulfonylureas. Due to their importance in the clinical setting, their action on the heart of large conscious animal models is relevant. The effect of glibenclamide, a representative sulfonylurea, has been studied in a conscious sheep model submitted to regional 12 min ischemia. Glibenclamide (0.4 mg/kg) completely blocked KATP channels, as assessed by monophasic APD, producing a deleterious effect on reperfusion-induced arrhythmias and myocardial recovery from stunning in normal animals. This adverse effect was more noticeable in alloxan-induced diabetic sheep, where a lower dose (0.1 mg/kg) inhibited KATP channel opening worsening mechanical recovery and arrhythmia incidence. However, glibenclamide did not abolish ischemic late preconditioning against stunning and arrhythmias in normal animals. Because diabetic sheep do not develop this cardioprotective phenomenon, probably due to KATP channel dysfunction, it was not possible to assess glibenclamide effect on preconditioning in this pathological condition. In conclusion, in large conscious animals, glibenclamide interferes with the beneficial action of KATP channel opening during acute ischemia-reperfusion events both in normal and diabetic animals. Therefore, despite some studies claiming no added cardiovascular risk due to glibenclamide treatment, this pharmacological agent should be further investigated to ensure its safe administration in patients with concurrent heart disease.

Glibenclamide effects on reperfusion-induced malignant arrhythmias and left ventricular mechanical recovery from stunning in conscious sheep.

INTRODUCTION: Sulfonylureas have been associated with a high incidence of cardiovascular death in diabetic patients treated with these drugs. Although the evidence on the cardiovascular effects of sulfonylureas is contradictory and scarce, many experiments have shown that the second-generation compound glibenclamide has a protective effect on mechanical function and against generation of malignant arrhythmias. OBJECTIVE: The purpose of this study was to assess whether glibenclamide elicits protection on postischemic myocardial functional recovery (stunning) and against reperfusion-induced arrhythmias in a conscious sheep model. METHODS: Sheep were divided into three groups: control, glibenclamide (0.4 mg/kg) and vehicle. After a 12-min ischemic period, the heart was reperfused and recordings for index calculation were acquired during 2 h of reperfusion. Percent systolic wall thickening fraction (%WTH), radial diastolic compliance (CR), arrhythmia incidence and Bernauer's arrhythmia severity index (ASI) were calculated for each group. RESULTS: Glibenclamide infusion had a high proarrhythmic action (ASI: glibenclamide 143, control 54 and vehicle 23; ANOVA P < 0.001 drug vs. control and vehicle) and a detrimental effect on regional systolic (%WTH: glibenclamide 26.9 +/- 6.7, control 65.7 +/- 3.5 and vehicle 68.6 +/- 5.6, ANOVA P < 0.01 drug vs. control and vehicle) and diastolic function (CR: glibenclamide 76.2 +/- 7.8, control 104.7 +/- 4.2 and vehicle 106 +/- 4.9, ANOVA P < 0.05 drug vs. control and vehicle) during reperfusion. CONCLUSIONS: Glibenclamide infusion resulted in adverse cardiovascular effects. The combined deleterious effects on reperfusion-induced arrhythmias and on myocardial recovery from stunning could be the cause of the unexplained high mortality in diabetic patients treated with sulfonylurea derivatives. The mechanism involved seems to be the blockade of the cardiac ATP sensitive potassium (K-ATP) channel.

Concentration and elongation of attached cross-bridges as pressure determinants in a ventricular model.

A ventricular model based on a muscle model relating sarcomere dynamics to Ca(2+)kinetics was used to establish the relative contribution to pressure development of the two components of cross-bridge dynamics: attached cross-bridge concentration and elongation of its elastic structure. The model was tested by reproduction of experiments reflecting myofibrillar behavior at the ventricular level as well as chamber mechanical properties. It was then used to study cross-bridge behavior independently of Ca(2+)activation, by simulation of flow-clamp experiments at constant Ca(2+)concentration. During the volume ramp, both reduced cross-bridge elongation and decreased concentration by cross-bridge detachment caused a fall of pressure; at end-ejection there was a fast partial increase of pressure by recovery of cross-bridge elongation, and during post-ejection there was a slower pressure change towards the value corresponding to end-ejection volume by cross-bridge reattachment according to the rate of constants of Ca(2+)kinetics. Likewise, during a physiological normal ejection, results showed that a maximal decrease in cross-bridge elongation (Deltah) produced a maximal reduction of ejecting pressure with respect to that at constant cross-bridge elongation (DeltaP), both in simulated beats (DeltaP=20%, Deltah=17%), and in experimentally fitted pressure-volume data from open-chest dogs (DeltaP=43.7+/-3.8%, Deltah=30.7+/-8.3%), Deltah being dependent of peak flow (Deltah=0. 1471 peak flow+6.0788, r=0.72). We conclude that normal ejecting pressure depends not only on cross-bridge concentration, but also on the elongation of its elastic structure, which reduces pressure according to flow.

Left ventricular regional systolic and diastolic function in conscious sheep undergoing ischemic preconditioning.

OBJECTIVE: Late preconditioning diastolic protection and cardiac function optimization by the combined effects of late and early preconditioning have not been studied in conscious animals. This study assessed in fully conscious sheep: (1) whether 24 h after a reversible ischemia, a new ischemic episode results in lesser systo-diastolic dysfunction (late preconditioning protection) and (2) whether the addition of early preconditioning (brief episodes of ischemia-reperfusion before the subsequent sustained ischemia) on the second day of late preconditioning optimized the second window of protection. METHODS: Three protocols were assessed: (a) late preconditioning, 9 min ischemia and 2 h reperfusion was done on two consecutive days; (b) early plus late preconditioning, as in protocol (a) except that on day 2 the heart underwent three periods of 3 min ischemia-6 min reperfusion prior to the sustained 9 min ischemia; (c) early preconditioning, the same protocol as in (b) except that day 2 was separated 1 week from day 1. RESULTS: Late preconditioning decreased regional radial diastolic stiffness from 147 +/- 26% (day 1) to 96 +/- 14% (day 2), at 2 h of reperfusion (mean +/- SEM, p < 0.05), but did not protect against systolic stunning (thickening fraction and regional stroke work). Early plus late preconditioning did not improve late preconditioning findings. Early preconditioning alone did not protect either systolic or diastolic functions. CONCLUSION: In conscious sheep, there is diastolic but not systolic mechanical protection with late preconditioning. Diastolic protection is not enhanced by the addition of early preconditioning.

A cardiac muscle model relating sarcomere dynamics to calcium kinetics.

A muscle model establishing the link between cross-bridge dynamics and intracellular Ca2+ kinetics was assessed by simulation of experiments performed in isolated cardiac muscle. The model is composed by the series arrangement of muscle units formed by inextensible thick and thin filaments in parallel with an elastic element. Attached cross-bridges act as independent force generators whose force is linearly related to the elongation of their elastic structure. Ca2+ kinetics is described by a four-state system of sites on the thin filament associated with troponin C: sites with free troponin C (T), sites with Ca2+ bound to troponin C (TCa); sites with Ca2+ bound to troponin C and attached cross-bridges (TCa*); and sites with troponin C not associated with Ca2+ and attached cross-bridges (T*). The intracellular Ca2+ concentration ([Ca2+]) is controlled solely by the sarcoplasmic reticulum through an inflow function and a saturated outflow pump function. All the simulations were performed using the same set of parameters. The model was able to reproduce the following experiments in cardiac muscle: (a) time course of isometric force (peak force: 46.5 mN/mm2), intracellular [Ca2+] (peak [Ca2+]: 1.5 microM); (b) force-length-[Ca2+] relations; (c) transient response of force to step changes in length; (d) force-velocity relation (maximum velocity: 3 microns/s); (e) the force response to length pulses to estimate the time course of [TCa]; (f) force response to quick releases showing the superactivating and deactivating effects of shortening; (g) stiffness response to sinusoidal length changes; and (h) time course of active state. The good accordance of the simulations with experimental results indicates that the model is an adequate representation of the link between cross-bridge dynamic behaviour and Ca2+ kinetics.

The contractile mechanism as an approach to building left ventricular pump models.

OBJECTIVE: The aim was to construct a model linking a simplified interpretation of the contractile process at the myofilament level to the mechanical behaviour of the left ventricle to improve the ability of elastic-resistive models to represent the pumping response of the left ventricle. The mechanical model, consisting of an elastic component connected in series with a contractile component and an elastic component parallel to both the series elastic and contractile components, is able to develop pressure by the binding of a structural substance T to an excitatory substance C, the behaviour of which is a simplification of miofibrillar Ca2+ kinetics. METHODS: Theoretically, the model was validated for its ability to reproduce by computer simulation, experiments that described the pumping properties of the left ventricle--namely, elasticity, resistivity, deactivating and positive effect of ejection, and the behaviour of intracellular Ca2+. Experimentally, the model was tested to fit intraventricular pressure (P(t)) and volume (V(t)) of single ejective beats in nine open chest dogs fitted with a pressure microtransducer to measure intraventricular P(t) and an aortic flowprobe to measure ventricular outflow and calculate V(t). Parameters were estimated up to maximum negative dP/dt adjusting P(t) or V(t) data of the ejective beats, and the goodness of the fit was evaluated through the root mean square error normalised with respect to the corresponding mean P(t) or V(t) in the fitting interval (NE). RESULTS: Descriptive validation of the model showed that the mean NE for the ejective P(t) fit was 0.03(SD 0.005) and for the V(t) fit 0.014(0.003). Predictive validation of P(t) and V(t) data of beats with partial occlusion of the aorta was performed up to end ejection, with parameters estimated from the P(t) or V(t) fit of the preceding ejective beat. Results gave a mean NE equal to 0.05(0.02) for predicted P(t) and 0.02(0.007) for predicted V(t), from either source of estimated parameters. Explanative validation showed that all the estimated parameters were in the same range used in simulation and that derived indexes [isovolumic maximum pressure (Pmax) = 166(13) mm Hg, time to maximum pressure (TPmax) = 0.186(0.012) s and the slope of the end systolic pressure volume relation (Emax) = 5.45(1.5) mm Hg.ml-1] were within reported experimental values. Finally, the model responded to increased inotropic state [dobutamine (5-35 micrograms.kg-1.min-1)] causing the estimated Pmax and Emax to increase by 33% and 25%, respectively, and TPmax to decrease by 10%. CONCLUSION: This model represented an improvement over previous pump models because (1) the model was able to represent behaviours other than purely elastic-resistive ones, such as the deactivation and positive effect of ejection; (2) left ventricular properties were the response of model behaviour and not constitutive elements of its structure; and (3) it adequately fulfilled model validation procedures.

Single-beat evaluation of left ventricular inotropic state in conscious dogs.

Two competing left ventricular elastic-resistive (ER) models were used to predict parameter values from pressure, volume, and time data of a single ejective beat in conscious dogs during control, enhanced (dobutamine), and decreased (propranolol) inotropic states. The animals were instrumented with three pairs of microcrystals and a transducer to measure intraventricular volume and pressure. Results showed that with the ER nonlinear model (ERNL), parameter values in all animals lay within the physiological range. These were the slope (Emax) and the intercept (V0) of the isovolumic end-systolic pressure-volume relationship (ESPVR), the slope of the end-diastolic pressure-volume relationship (Ed), the time to Emax (Tmax), the normalized time to end of activation (A), and the resistive constant (K). In the two models, the normalized SE of the estimate of data fitting was below 0.2 Emax, as estimated from a single beat, responded to changes in contractility in a significantly more consistent fashion than the slope of ESPVRs (Ees) generated by preload maneuvers in conscious dogs. Single-beat estimated Tmax and K with the ERNL model did also respond consistently to contractility changes, whereas with the elastic resistive linear (ERL) model, K did not reproduce the experimental findings with decreased inotropic state. We conclude that 1) the ERNL model can be employed to assess contractility changes in conscious dogs from data of a single ejective beat, and 2) these changes are better indicated by single-beat estimated Emax than by Ees calculated from conventional ESPVRs.

A computer study of the relation between chamber mechanical properties and mean pressure-mean flow of the left ventricle.

Computer simulation of left ventricular contraction was used to analyze the mean left ventricular pressure-mean flow relation with changes of parameter values: end-diastolic volume, contractile state, internal resistance, characteristic resistance, capacitance, end-diastolic stiffness, and heart rate and with changes of experimental conditions: filling kinetics (constant atrial pressure as opposed to constant end-diastolic volume) and coronary perfusion pressure (constant or varying with atrial pressure, i.e., self-perfused). The chamber mechanical properties used in the simulation were defined in terms of a modified purely elastic behavior model with a flow-dependent resistive component. Computed results showed that at constant end-diastolic volume and constant ventricular perfusion pressure the mean pressure-mean flow relation was linear, except for changes in internal resistance where a cubic fit of points was more appropriate. In these conditions, parameter variations in the accepted linear relation produced changes in the slope and mean pressure axis intercept. Imposition of changes in experimental conditions gave rise to nonlinear mean pressure-mean flow relations. The results indicate that with elastic-resistive chamber mechanical properties as a starting point, the experimental conditions would be responsible for the different shapes of the mean pressure-mean flow relation obtained in isolated heart experiments. However, a more complete description of chamber properties (such as the addition of a deactivation component) could also give rise to nonlinear pump function graphs.

Relationship between changes of chamber mechanical parameters and mean pressure-mean flow diagrams of the left ventricle.

A theoretical relationship between mean ventricular pressure (P) and mean ventricular outflow (Q) was developed based on a model of the left ventricle with elastic-resistive properties. Using a polynomial interpolation method, a fifth-order polynomial equation for the P-Q relationship was obtained. Its coefficients are functions of end-diastolic volume (VD), heart rate (HR), contractile state (CS), diastolic elastance (ED), asymmetry (S) of the elastance function E(t), and ventricular internal resistance factor (K). Effect of changes of these parameters indicated that normal and enhanced CS relations diverge toward the P axis but have a common intercept toward the Q axis. A similar effect was obtained with increased asymmetry of E(t). Changes in VD, HR and ED produced a parallel shift of the P-Q relation. The effect of K was negligible, however, which would reduce the description of the P-Q relationship to a third-order polynomial equation. A flow-dependent deactivation component was introduced, altering the asymmetry factor S, which decreases in a linear proportion to Q. This factor shifted the pump function graph downwards. We conclude that the theoretical description of the P-Q relation we present reproduces the experimental behavior of pump function diagrams reported in the literature (changes in VD, HR, and CS) and predicts the possible behavior due to other parameter changes.

Relationship between chamber mechanical properties and mean pressure-mean flow diagram of the left ventricle.

We undertook a theoretical analysis of the source resistance of the left ventricle represented in a mean pressure-mean flow (P-Q) diagram, using the chamber properties established in terms of the pressure-volume relationship. This analysis showed that P-Q pairs of points should lie above the linear function proposed by Elzinga and Westerhof. A third-order polynomial function would theoretically explain better than a linear relation or a parabolic fit the curved shape of experimentally obtained P-Q relationships. The analysis resolves the discrepancy between Elzinga and Westerhof's theoretical concept of linear source resistance and the actual nonlinear P-Q relationship.

Characterization of ventricular dynamics in dogs using computerized variables and their statistical treatment.

To improve the analysis of mechanical events of the cardiac cycle and the selection of indexes of cardiac function, it is necessary to obtain the most probable time course of a given variable or of the shape of a loop. With this objective, a special program has been devised to obtain mean time tracings of left ventricular pressure (LVP) and posterior wall thickness (PWTH). The time scale can be normalized or beats with small variation in duration can be chosen. Mean loops are obtained in dogs by relating mean LVP and PWTH values. The distribution of pairs of individual values is assumed as normal bivariate with respect to its corresponding mean value, yielding confidence and tolerance ellipses. Results show that for a 95% confidence limit the loop is open in normal conditions. We postulate that this statistical treatment, by giving a more precise course of a mechanical signal allows a more accurate comparison between different experimental or clinical situations in the whole cardiac cycle, as well as the development of cardiac indexes.

Sensitivity of enzymatic release due to ischemia in the isolated rat heart.

The sensitivity of cardiac enzymes (GOT, a-HBDH, LDH, LDH-1, CPK and CPK-MB) was studied in the isolated rat heart subjected to 0 (non-ischemic), 20, 30 and 40 min of total global ischemia (0 ml/min) followed by reperfusion in which three consecutive perfusate samples were collected for enzymatic measurements. Cardiac function, estimated by peak tension (Tp), maximal rate of tension development (Tc) and relaxation (Tr) decreased to 0 with ischemia and recovered with reperfusion to 100% in the 20 min, 50% in the 30 min and 10% in the 40 min ischemic groups. In these three groups, enzymatic release, measured as the relative increment (RI) with respect to the control samples, gave significantly higher values than those of non-ischemic hearts (P less than 0.001). LDH-1 was the most sensitive enzyme (P less than 0.0005) for the detection of ischemia in these experimental conditions.

Sensitivity of enzymatic release due to ischemia in the isolated rat heart.

The sensitivity of cardiac enzymes (GOT, a-HBDH, LDH, LDH-1, CPK and CPK-MB) was studied in the isolated rat heart subjected to 0 (non-ischemic), 20, 30 and 40 min of total global ischemia (0 ml/min) followed by reperfusion in which three consecutive perfusate samples were collected for enzymatic measurements. Cardiac function, estimated by peak tension (Tp), maximal rate of tension development (Tc) and relaxation (Tr) decreased to 0 with ischemia and recovered with reperfusion to 100

in the 20 min, 50

in the 30 min and 10

in the 40 min ischemic groups. In these three groups, enzymatic release, measured as the relative increment (RI) with respect to the control samples, gave significantly higher values than those of non-ischemic hearts (P less than 0.001). LDH-1 was the most sensitive enzyme (P less than 0.0005) for the detection of ischemia in these experimental conditions.

Myocardial triglycerides increased by fasting. Effects of hypoxia on contractility and enzymatic release.

The effect of fasting on the mechanics of contraction was studied in the isolated perfused rat heart, in oxygenated and hypoxic conditions. Animals were subjected to 48 h of fasting, a condition which produces augmented endogenous triglycerides (TG). Normal and fasted rats were submitted to a 10-min period of hypoxia, which depressed peak tension (Tp), maximal rate of contraction (Tc), and relaxation (Tr); all three parameters recovered with reoxygenation. However, Tp and Tc of hearts of fasted animals were less affected by hypoxia, and Tp, Tc, and Tr attained higher levels during reoxygenation compared with hearts of normally fed animals. These results suggest that as triglycerides are augmented, they might have a beneficial action on the hypoxic heart, though other unknown effects of fasting cannot be discarded. LDH was the only cardiac enzyme whose release in the perfusion medium significantly increased in hypoxic hearts, irrespective of the fasting state of the animal. The positive correlation between LDH and an index of relative recovery (IH) for Tp, Tc, and Tr, indicate that not only hypoxia but a good recovery are necessary to yield high LDH values.

One trial passive avoidance learning in neonatally hormone treated rats. Its relation with activity.

The performance in one trial passive avoidance learning and the degree of activity were determined in adulthood, from neonatal hormone treated and intact rats. Activity was measured in the same environmental conditions in which the learning test takes place, i.e., shuttle box unequally illuminated. The treated groups were androgenized females and castrated males. Intact groups were composed by normal males and females. Intact females showed more activity than intact males. Castrated males and androgenized females displayed a degree of activity similar to the normal females. This score was negatively correlated with the total time that each animal remained in the dark compartment of the box. In this respect the normal males showed greater dark preference than the other three groups, which had no differences among them. Similar results to that of dark preference were obtained in performance on one trial passive avoidance learning, that is, androgenization in females did not produce a significant effect in this test and castration in males decreased their performance. A linear correlation between activity and passive avoidance score could be established.