Shao T. Wu

Influence of positive inotropic agents on intracellular calcium transients. Part I. Normal rat heart.

This study, which was designed to evaluate the effects of positive inotropic agents on intracellular calcium transients ([Ca2+]i), is the first to analyze calcium transients in the whole heart. The positive inotropic agents that augment intracellular cyclic adenosine monophosphate (cAMP) (dibutyryl cAMP, amrinone, and isoproterenol) caused an increase in developed pressure and [Ca2+]i transients and a decrease in diastolic [Ca2+]i. On the other hand, the glycoside digoxin and the alpha-adrenoceptor agents, phenylephrine and dobutamine, also caused an increase in [Ca2+]i transients and developed pressure. However, unlike the agents that increase [cAMP]i, they induced an elevation in diastolic [Ca2+]i. With all the positive inotropic agents, developed pressure increased commensurately with the percentage changes in amplitude of the [Ca2+]i transients.

Rapid adaptation of myocardial calcium homeostasis to short episodes of ischemia in isolated rat hearts

Short episodes of ischemia and reperfusion (ischemic preconditioning) have been shown to attenuate the detrimental rise in intracellular free Ca2+ ([Ca2+]i) caused by subsequent sustained ischemia. The purpose of this study was to investigate the detailed time course of [Ca2+]i during such short episodes of ischemia and reperfusion. [Ca2+]i was measured at a high time resolution by surface fluorometry and indo-1 in isolated perfused rat hearts during three episodes of 5 minutes of ischemia followed by 5 minutes of reperfusion. We found that the rise in systolic [Ca2+]i was significantly smaller during the second (191% +/- 67%) and third (194% +/- 75%) episodes of ischemia than during the first episode (289% +/- 75%, P < 0.05 vs both subsequent episodes). This attenuated rise in systolic [Ca2+]i was preserved during perfusion with low extracellular Na+ (105.5 mmol/L [Na+]o). During short episodes of ischemia and reperfusion, [Ca2+]i rises less during the second and third ischemic episode than during the first episode. Thus one transient ischemic stimulus led to a rapid adaptation of [Ca2+]i homeostasis to subsequent ischemic conditions. Such a rapid adaptation might be an important mechanism of the ischemic preconditioning phenomenon in protecting against ischemic injury.

Calcium-dependent fluorescence transients during ventricular fibrillation☆

Using surface fluorometry, calcium-dependent fluorescence transients were recorded during ventricular fibrillation in perfused rat and hamster hearts loaded with INDO 1-AM (fluorimetric reagent for calcium ion). Among a series of 203 consecutive isolated heart studies, 13 instances of ventricular fibrillation occurred. These arrhythmias developed during pretreatment with isoproterenol, dobutamine, norepinephrine, phenylephrine, digoxin, and 4 mmol/L calcium in the perfusate. Alternans behavior of calcium transients occurred in three cases; premature beats preceded ventricular fibrillation in six cases. Premature beats led to a further increase in the free intracellular calcium ([Ca2+]i) concentration, resulting in a stronger contraction with the subsequent beat and/or the initiation of ventricular fibrillation. Two distinct patterns of calcium transients were seen: ventricular fibrillation type 1 showed fast disorganized transients with small amplitude and an irregular, nonuniform tracing; type 2 revealed fast activity and multiform, polymorphous transients with marked changes in amplitude. Independent of the morphologic type of fibrillation, [Ca2+]i remained constant or even increased during an observation time up to 9 minutes. No intracellular hypocalcemia was observed. Isoproterenol pretreatment resulted in [Ca2+]i levels in the range of the end-diastolic calcium level of the last regular contraction. Fibrillating calcium transients after norepinephrine, dobutamine, phenylephrine, digoxin, high calcium, and fast pacing were in the previous end-systolic range. It is suggested that inotropic agents acting without a major elevation of cyclic adenosine monophosphate result in higher [Ca2+]i.

Angiotensin II and phorbol esters depress cardiac performance and decrease diastolic and systolic [Ca2+]i in isolated perfused rat hearts☆

Both angiotensin II and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), significantly depressed developed pressure, oxygen consumption, and coronary flow in isolated perfused rat hearts and caused a decrease in diastolic and systolic [Ca2+]i and [Ca2+]i transients. PMA and angiotensin II did not change the levels of cAMP but moderately decreased PCr/Cr. The decrease in systolic [Ca2+]i and amplitude of [Ca2+]i transients caused by PMA and angiotensin II resulted in depressed cardiac function. Hearts perfused with PMA and angiotensin II had a decreased sensitivity to extracellular calcium. Depressed developed pressure and oxygen consumption in the PMA- and angiotensin II-treated hearts may have been due to a decrease in amplitude of effective [Ca2+]i transients, because the [Ca2+]i threshold for cross-bridge interaction was presumably higher than the diastolic [Ca2+]i in these hearts.

Intracellular Ca2+ Handling and Vulnerability to Ventricular Fibrillation in Spontaneously Hypertensive Rats

Spontaneously hypertensive rats (SHR) with ventricular hypertrophy show an increased vulnerability for the development of potentially lethal ventricular arrhythmias such as ventricular fibrillation (VF). The mechanisms of this increased vulnerability are not fully understood but may be related to abnormal intracellular Ca2+ ([Ca2+]i) handling under stress conditions. We therefore investigated whether [Ca2+]i handling is abnormal in hypertrophied hearts of SHR without heart failure during stimulation stress, and if so whether abnormal [Ca2+]i handling is a determinant of the increased vulnerability to VF in SHR. [Ca2+]i was measured by indo-1 surface fluorescence in perfused hearts of 8- to 10-month-old control Wistar-Kyoto rats (WKY) and age-matched SHR. The state of [Ca2+]i handling was analyzed during three different forms of stimulation stress: rapid pacing, the long rest period after cessation of rapid pacing, and preprogrammed ventricular stimulation that was simultaneously used for the determination of VF threshold. The pulse number VF threshold was used as an index to determine vulnerability to VF and to analyze the relationship of [Ca2+]i handling to vulnerability. Although VF thresholds were lower in SHR than in WKY, we found that both demonstrated similar [Ca2+]i handling during stimulation stress. The extent and rate of [Ca2+]i accumulation during rapid pacing and those of the [Ca2+]i decline after cessation of pacing were similar in SHR and WKY. In addition, the relationship between [Ca2+]i and VF threshold was unaltered in SHR. Thus, we conclude that [Ca2+]i handling is normal in hypertrophied hearts of SHR without heart failure during stimulation stress and that it is not a determinant of the increased vulnerability to VF in SHR.

Intracellular calcium during pacing-induced ventricular fibrillation. Effects of lidocaine.

Hemodynamics and endocardial [Ca2+]i transients were studied during ventricular fibrillation in isolated perfused rat hearts. During 1 minute of pacing-induced ventricular fibrillation, the diastolic fibrillatory [Ca2+]i level increased significantly (p less than 0.001) above the end-systolic [Ca2+]i concentration of the last regular contraction. A bolus of lidocaine led to a decrease in the [Ca2+]i level during fibrillation (p less than 0.007) to the end-diastolic level of baseline conditions and then subsequently converted the heart to sinus rhythm. In 13 (62%) of the 21 studies described, post-lidocaine ventricular fibrillation converted to a brief period of asystole followed by sinus rhythm; in 8 (38%) of the studies, post-lidocaine ventricular fibrillation switched to ventricular tachycardia. Postfibrillatory cardiac dysfunction was related to the duration and degree of elevated diastolic [Ca2+]i. The authors conclude that [Ca2+]i levels are increased during ventricular fibrillation, and that lidocaine treatment leads to a prompt decrease in [Ca2+]i preceding conversion to sinus rhythm.

The effect of dobutamine on myocardial performance and high-energy phosphate metabolism at different stages of heart failure in cardiomyopathic hamsters: A 31P MRS study

Dobutamine has been shown to exert disparate clinical effects in patients with cardiomyopathy and heart failure. This study evaluated the effects of dobutamine on hemodynamics and energetics in isolated, perfused myopathic hamster hearts at a moderate and advanced stage of heart failure. Biochemical changes were correlated with left ventricular developed pressure, coronary flow, and myocardial oxygen consumption. During dobutamine treatment left ventricular developed pressure increased in the control and moderate heart failure group 28.0 +/- 1.0% and 114.2 +/- 11.6%, respectively. Myocardial oxygen consumption increased 50.1 +/- 9.1% and 45.5 +/- 16.0%, respectively. There were no significant changes of left ventricular developed pressure and myocardial oxygen consumption in the advanced heart failure group. Inorganic phosphate (Pi) increased in the control group from 6.8 +/- 0.5 to 11.4 +/- 1.2 mmol (p less than 0.005) and in the advanced heart failure group from 10.4 +/- 1.1 to 15.3 +/- 1.2 mmol (p less than 0.01). Phosphocreatine (PCr) and beta-ATP (adenosine triphosphate) decreased in the control group from 12.2 +/- 0.4 to 8.7 +/- 0.7 mmol (p less than 0.001) and 10.4 +/- 0.8 to 7.7 +/- 0.7 mmol (p less than 0.02), respectively. PCr/Pi ratio, reflecting mitochondrial function, fell in the control and advanced heart failure group from 1.84 +/- 0.14 to 0.84 +/- 0.14 (p less than 0.02) and 0.81 +/- 0.16 to 0.37 +/- 0.08 (p less than 0.03), respectively. Thus in cardiomyopathic hamsters dobutamine improved mechanical performance and thermodynamic efficiency in moderate stages of heart failure by improving mitochondrial activity, but did not improve mechanical performance in an advanced stage of heart failure. These experiments provide into the disparate clinical effects of dobutamine at various stages of heart failure.

Influence of positive inotropic agents on intracellular calcium transients. Part II. Cardiomyopathic hamster hearts

To study the mechanism of dobutamine on end-stage heart failure, we assessed hemodynamic responses, high-energy phosphates (31P-NMR), and free intracellular calcium ([Ca2+]i) transients (surface fluorometry) during perfusion with 10(-6) mol/L dobutamine in Syrian cardiomyopathic hamsters with severe heart failure. These results were compared to perfusion of the heart with 10(-6) mol/L norepinephrine and 10(-6) mol/L isoproterenol. With the positive inotropic agents the rate-pressure product increased immediately (p less than 0.01 with dobutamine, norepinephrine; p less than 0.003 with isoproterenol); after 10 to 15 minutes of perfusion the rate-pressure product remained relatively stable with norepinephrine and isoproterenol but decreased with dobutamine (p = NS vs control values). [Ca2+]i-transients increased significantly in all groups. The end-diastolic [Ca2+]i decreased continuously with norepinephrine and isoproterenol (p less than 0.008; p less than 0.005) but increased during dobutamine by 19%. Alterations in coronary flow, pHi, high-energy phosphates, and the phosphorylation potential were not significantly different among the three catecholamines. In conclusion, in contrast to norepinephrine and isoproterenol, dobutamine depressed myocardial performance and increased end-diastolic [Ca2+]i in late heart failure.

Intracellular calcium transients underlying interval-force relationship in whole rat hearts: effects of calcium antagonists

OBJECTIVES Much of the understanding about the cardiac interval-force relationship of the whole heart, including mechanical restitution and postextrasystolic potentiation (PESP), has been inferred from isolated muscle studies. We tested whether results from isolated muscles about intracellular Ca2+([Ca2+]i) transients underlying the interval-force relationship can be substantiated in whole hearts. Additionally, we investigated whether Ca2+ antagonists could alter [Ca2+]i transients underlying mechanical restitution and postextrasystolic potentiation. METHODS [Ca2+]i transients were studied in isolated perfused rat hearts by surface fluorometry and Indo-1. Using computer-controlled pacing protocols, we performed restitution curves for left ventricular developed pressure and [Ca2+]i (developed pressure and [Ca2+]i plotted as a function of extrasystolic intervals). To quantify restitution curves, we fitted monoexponential functions to plots and analyzed their shift and slope. Then, we used Ca2+ antagonists, low extracellular Ca2+([Ca2+]o) and PESP to modify restitution curves. [Ca2+]i transients in isolated rat hearts were interpreted as Ca2+ released from the sarcoplasmic reticulum. RESULTS Interval-dependent changes in developed pressure were strongly correlated to interval-dependent changes in the amplitude of [Ca2+]i transients in isolated whole rat hearts. Additionally, nifedipine and low [Ca2+]o led to similar downward shifts but not to a changed slope of restitution curves for [Ca2+]i. On the other hand, PESP increased the slope of restitution curves for [Ca2+]i. Furthermore, the effect of PESP on developed pressure was blunted by high concentrations of Ca2+ antagonists. CONCLUSIONS The results from isolated muscles about [Ca2+]i transients underlying the interval-force relationship could be substantiated in whole hearts. Additionally, low [Ca2+]i (induced by nifedipine or low [Ca2+]o) decreased the maximal Ca2+ release of the sarcoplasmic reticulum but did not change the release kinetics. On the other hand, PESP presumably accelerated Ca2+ release kinetics of the sarcoplasmic reticulum.

Relationship between intracellular calcium and oxygen consumption: Effects of perfusion pressure, extracellular calcium, dobutamine, and nifedipine☆

All of the mechanisms that connect the cardiac mechanical work load with energy production have not been clearly defined. The purpose of this study was to evaluate the relationship between intracellular calcium and oxygen consumption in intact hearts, to further understand this relationship. Intracellular calcium was measured in isolated nonworking perfused rat hearts loaded with Indo-1 by means of a surface fluorometry technique. Glucose was used as a substrate. Myocardial contraction and oxygen consumption were modulated by perfusion pressure (80, 110, and 140 cm of water), extracellular calcium (1, 2, 3, and 4 mmol/L), dobutamine (10(-6) mol/L), and nifedipine (10(-6) mol/L). With all of these interventions there was a close correlation between intracellular calcium (systolic, diastolic, and amplitude) and oxygen consumption or left ventricular developed pressure. Observations in this study support the hypothesis that intracellular calcium plays a regulatory role in the link between cardiac mechanics and energy production.