beta-adrenergic blockade in developing heart failure: effects on myocardial inflammatory cytokines, nitric oxide, and remodeling.

BACKGROUND: Whether beta-adrenergic blockade modulates myocardial expression of inflammatory cytokines and nitric oxide (NO) in heart failure is unclear. METHODS AND RESULTS: We administered oral metoprolol or no therapy to rats for 12 weeks after large myocardial infarction and subsequently examined left ventricular (LV) remodeling; myocardial tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6 expression; and NO. In untreated rats, echocardiography revealed significant (P<0.001) LV dilatation and systolic dysfunction compared with sham. Papillary muscle studies revealed isoproterenol hyporesponsiveness to be unaltered by NO synthase (NOS) inhibition. Circulating NO metabolites were undetectable. In noninfarcted myocardium, although inducible NOS (iNOS) mRNA was absent, TNF-alpha, IL-1beta, and IL-6 mRNA and protein were markedly elevated compared with sham (P<0.001), with 2-fold higher expression (P<0.025) of IL-6 compared with TNF-alpha or IL-1beta. Metoprolol administration starting 48 hours after infarction (1) attenuated (P<0.02) LV dilatation and systolic dysfunction, (2) preserved isoproterenol responsiveness (P<0.025) via NO-independent mechanisms, and (3) reduced myocardial gene expression and protein production of TNF-alpha and IL-1beta (P<0. 025) but not IL-6, which remained high. CONCLUSIONS: During heart failure development, adrenergic activation contributes to increased myocardial expression of TNF-alpha and IL-1beta but not IL-6, and one mechanism underlying the beneficial effects of beta-adrenergic blockade may involve attenuation of TNF-alpha and IL-1beta expression independent of iNOS and NO.

Chronic beta-adrenergic stimulation induces myocardial proinflammatory cytokine expression.

BACKGROUND: The sympathetic nervous system and proinflammatory cytokines are believed to play key roles in the pathophysiology of congestive heart failure. To evaluate a possible relationship between these neurohormonal systems, we studied the effects of chronic beta-adrenergic stimulation on the myocardial and systemic elaboration of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6. METHODS AND RESULTS: Male rats received either L-isoproterenol (2.4 mg. kg(-1). d(-1), n=8) or saline (n=7) via miniosmotic pumps for 7 days. Myocardial cytokine expression was analyzed by both Northern and Western blotting and localized in the tissue using immunohistochemistry. ELISA was performed to measure circulating levels of cytokines. In myocardium from control animals, neither TNF-alpha nor IL-1beta were detected, whereas IL-6 was present at very low levels. Isoproterenol led to a significant (P<0.01) increase in mRNA and protein expression of all 3 cytokines. Immunohistochemistry did not detect immunoreactivity for either cytokine in myocardium from controls; however, all 3 cytokines were readily detected (P<0.05) throughout the myocardium, localized to resident cells and vessels, in animals treated with isoproterenol. Neither treatment group had detectable levels of cytokines in the serum. CONCLUSIONS: Chronic beta-adrenergic stimulation induces myocardial, but not systemic, elaboration of TNF-alpha, IL-1beta, and IL-6.

Altered LV inotropic reserve and mechanoenergetics early in the development of heart failure.

To test the hypothesis that alterations in left ventricular (LV) mechanoenergetics and the LV inotropic response to afterload manifest early in the evolution of heart failure, we examined six anesthetized dogs instrumented with LV micromanometers, piezoelectric crystals, and coronary sinus catheters before and after 24 h of rapid ventricular pacing (RVP). After autonomic blockade, the end-systolic pressure-volume relation (ESPVR), myocardial O(2) consumption (MVO(2)), and LV pressure-volume area (PVA) were defined at several different afterloads produced by graded infusions of phenylephrine. Short-term RVP resulted in reduced preload with proportionate reductions in stroke work and the maximum first derivative of LV pressure but with no significant reduction in baseline LV contractile state. In response to increased afterload, the baseline ESPVR shifted to the left with maintained end-systolic elastance (E(es)). In contrast, after short-term RVP, in response to comparable increases in afterload, the ESPVR displayed reduced E(es) (P < 0.05) and significantly less leftward shift compared with control (P < 0.05). Compared with the control MVO(2)-PVA relation, short-term RVP significantly increased the MVO(2) intercept (P < 0.05) with no change in slope. These results indicate that short-term RVP produces attenuation of afterload-induced enhancement of LV performance and increases energy consumption for nonmechanical processes with maintenance of contractile efficiency, suggesting that early in the development of tachycardia heart failure, there is blunting of length-dependent activation and increased O(2) requirements for excitation-contraction coupling, basal metabolism, or both. Rather than being adaptive mechanisms, these abnormalities may be primary defects involved in the progression of the heart failure phenotype.

Load sensitivity of left ventricular relaxation in normal and failing hearts: evidence of a nonlinear biphasic response.

OBJECTIVE: This investigation sought to define the effect of heart failure (HF) on the load sensitivity of left ventricular (LV) relaxation and to correlate alterations in load sensitivity with the variables of ejection timing, systolic load profile and elastic recoil. METHODS: Nine dogs instrumented with LV micromanometers and piezoelectric crystals were studied before and after HF produced by prolonged rapid LV pacing. After pharmacologic autonomic blockade and atrial pacing at 160 bpm, hemodynamic measurements were recorded at steady-state and during vena caval occlusion. LV relaxation for individual beats during caval occlusion was assessed using tau, the monoexponential time constant, and systolic load was estimated using end-systolic circumferential force (ESF). RESULTS: The tau-ESF relation was nonlinear and biphasic, with an initial decrease in tau followed by a delayed increase, and was described by a parabolic equation with a curvilinearity coefficient a. Examination of ejection variables and systolic load profile indicated that the initial acceleration of relaxation reflected the influence of increased elastic recoil, whereas the late slowing reflected the influence of earlier end-ejection and delayed systolic loading. HF produced significant baseline prolongations of tau (P < 0.005), time to relaxation onset (P < 0.001) and time to peak force (P < 0.015) compared to control. The curvilinearity coefficient of the tau-ESF relation was significantly increased (18.1 +/- 20.1.10(-5) vs. 3.99 +/- 2.89.10(-5) g-2, P = 0.048), indicating increased load sensitivity of relaxation. This increased load sensitivity correlated with delayed onset but increased overall magnitude of the effects of earlier end-ejection and late systolic loading on relaxation. CONCLUSIONS: The tau-ESF relationship during transient load reduction is nonlinear and biphasic with an initial acceleration of relaxation, reflecting the impact of elastic recoil, and delayed slowing, reflecting changes in ejection timing and systolic loading sequence. This relation is more curvilinear in the failing heart, indicating increased load sensitivity of LV relaxation. These changes primarily occur due to alterations in the impact of ejection timing and systolic load profile rather than increased elastic recoil.

Nitric oxide effects on myocardial function and force-interval relations: regulation of twitch duration.

As the precise role of nitric oxide (NO) as a modulator of myocardial contraction and the force-interval relationship remains unclear, the objective of this study was to examine the effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) on baseline myocardial contraction, and the impact of both SNAP and the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on the force interval relation. Studies were performed using isolated rat papillary muscles. In the presence of baseline NOS blockade, nanomolar to micromolar concentrations of SNAP exerted a modest positive inotropic effect with a small but significant increase in twitch isometric tension (P<0.007). Nanomolar concentrations of SNAP also reduced overall twitch duration (P<0.007). These effects were not seen in control experiments using N-acetyl-penicillamine instead of SNAP. The force-frequency response (FFR) and post-rest contractile potentiation, mechanical correlates of sarcoplasmic reticulum (SR) Ca(2+)handling, were also examined. Neither L-NAME nor SNAP had any effect on post-rest potentiation following rest intervals as long as 6 min, or on the negative FFR at stimulation frequencies between 0.3 to 1.7 Hz. However, L-NAME significantly blunted the net reduction in twitch duration between 0.3 Hz and 1.7 Hz compared to control (P=0.006), an effect reversed by 100 n m SNAP. These results indicate that low concentrations of NO can modulate myocardial function by influencing myocardial inotropy and the time course of myofilament interaction, but do not impact significantly on the force-interval relation and, by inference, SR Ca(2+)handling. Moreover, modulation of twitch duration occurs over a range of stimulation frequencies, suggesting a mechanistic role for NO in the changes in contraction and relaxation time intervals seen during changes in heart rate.

Hemodynamic effects of nitric oxide synthase inhibition at steady state and following tumor necrosis factor-alpha-induced myodepression.

OBJECTIVE: Nitric oxide (NO) has been proposed as a common mediator of tumor necrosis factor-alpha (TNF alpha)-induced vasodilation and myocardial dysfunction. Accordingly, we performed an extensive assessment of the influence of NO synthase inhibition on left ventricle (LV) and circulatory performance in conscious dogs at steady state and after establishment of TNF alpha mediated myodepression. METHODS: Autonomically blocked, chronically instrumented dogs were studied at steady state and 6 h after initiation of a 1-h rhTNF alpha infusion (40 micrograms/kg). Ventricular performance was evaluated using the pressure-volume framework. Dogs were then treated with either NG-nitro-L-arginine methylester (L-NAME, 40 mg/kg bolus) or angiotensin II (250-500 ng/kg). RESULTS: L-NAME, under control conditions or following recombinant human (rh) TNF alpha-induced ventricular dysfunction, produced marked increases in afterload with attendant increases in LV pressure, volume, and prolonged isovolumic relaxation without adversely influencing coronary blood flow. regardless of whether the dogs received rhTNF alpha, L-NAME did not affect the slopes of the end-systolic pressure-volume and stroke-work (SW)-end-diastolic volume (EDV) relations (force-based measure of contractility), whereas the slope of the dP/dtmax-EDV relation, a velocity dependent parameter of LV systolic function, declined. Overall ventricular performance, as seen by the circulation, was reduced by L-NAME in control as well as rhTNF alpha-treated dogs, evidenced by rightward shifts of the SW-EDV and dP/dtmax-EDV relations. Similar findings were observed in the separate cohorts of dogs, at steady state and 6 h after rhTNF alpha, following angiotensin II at matched systolic pressure. CONCLUSIONS: Systemic NO synthase inhibition with L-NAME does not acutely reverse rhTNF alpha-induced myocardial dysfunction. The detrimental influence of L-NAME on LV size, relaxation, and velocity-based measures of contractility is likely attributable to its effects on increasing afterload.

Ryanodine and the left ventricular force-interval and relaxation-interval relations in closed-chest dogs: insights on calcium handling.

OBJECTIVE: Although the myocardial force-interval and relaxation-interval relations are considered to be mechanical expressions of myocardial Ca2+ handling, correlation of these phenomena with altered Ca2+ kinetics in the intact state is limited. Thus, I sought to determine the impact of selective impairment of physiologic sarcoplasmic reticulum Ca2+ release, achieved by the use of the drug ryanodine, on these relations in the intact animal. METHODS: Twelve dogs instrumented with left ventricular manometers and piezoelectric dimension crystals were studied before and after ryanodine (4 micrograms/kg intravenously). End-systolic elastance was measured at paced heart rates of 120-180 bpm to determine the force-frequency response. Mechanical restitution and relaxation restitution were determined by measuring contractile (single beat elastance) and relaxation (peak negative dP/dt) responses for beats delivered at graded extrasystolic intervals, with normalized responses expressed as a function of extrasystolic interval. RESULTS: Ryanodine accelerated mechanical restitution (time constant 60.3 +/- 3.9 versus 81.7 +/- 10.1 ms, p < 0.05) and reduced maximal contractile response (107.5 +/- 2.1 versus 122.1 +/- 5.7%, p < 0.05), slowed early relaxation restitution (time constant 65.5 +/- 13.8 versus 36.8 +/- 3.8 ms, p < 0.05) without changing late relaxation restitution kinetics, and amplified the force-frequency response (end-systolic elastance, 180 bpm, 19.4 +/- 4.3 versus 11.4 +/- 1.2 mm Hg/ml, p < 0.05). CONCLUSIONS: These findings suggest that in the intact animal, Ca2+ handling by the sarcoplasmic reticulum is a primary determinant of mechanical restitution and early relaxation restitution, but not late relaxation restitution. Conversely, ryanodine induced augmentation of the force-frequency response indicates a central role for sarcolemmal Ca2+ influx in producing frequency potentiation.

Ryanodine and left ventricular function in intact dogs: dissociation of force-based and velocity-based indexes.

After anesthesia and autonomic blockade, nine dogs chronically instrumented with left ventricular (LV) micromanometers and piezoelectric dimension crystals were studied before and after the intravenous administration of 4 micrograms/kg ryanodine, a specific inhibitor of the sarcoplasmic reticulum Ca2+ release channel. Ryanodine prolonged LV contraction and relaxation (P < 0.001) without changing heart rate, end-diastolic volume (EDV), or end-systolic pressure. Velocity-dependent mechanical parameters were significantly depressed, including the maximal rate of LV pressure rise (dP/dtmax; P < 0.002), the mean velocity of circumferential fiber shortening (P < 0.002), the slope of the dP/dtmax-EDV relation (P < 0.05), and the time constant of LV relaxation (P < 0.01). In contrast, the slopes of the end-systolic pressure-volume (PES-VES) and stroke work (SW)-EDV relations, both force-based parameters, were increased (P < 0.05) or maintained, respectively. Ryanodine reduced overall LV contractile performance, evidenced by significant rightward shifts of the PES-VES, dP/dtmax-EDV, and SW-EDV relations and reduced SW at constant preload (P < 0.02). Thus, in the closed-chest dog, low-dose ryanodine resulted in 1) generalized slowing of LV mechanical events without changes in heart rate or load, 2) dissociation of velocity-based and force-based measures of LV function, with depression of the former but enhancement or maintenance of the latter, and 3) reduced overall LV inotropic performance. These effects are consistent with ryanodine-induced alterations of the Ca2+ transient and altered sarcoplasmic reticulum Ca2+ availability.

Syncope: current diagnosis and treatment.

The patient with syncope often poses a formidable diagnostic challenge. A large number of underlying causes must be considered, ranging in severity from benign to life-threatening. A careful, systematic clinical evaluation beginning with a history, physical examination, and ECG will establish the diagnosis in most patients, and the judicious use of specialized testing will confirm or uncover the cause in many of the remaining cases. Further basic and clinical research into the pathogenesis and treatment of neurocardiogenic syncope, the role of HUT testing in neurally mediated syncope, and the optimal use of EPS in patients with cardiac disease will markedly improve our management of these patients in the future.

Postextrasystolic mechanical restitution in closed-chest dogs. Effect of heart failure.

BACKGROUND: Postextrasystolic mechanical restitution (MRPES) is thought to be an expression of intracellular Ca2+ handling by cardiac sarcoplasmic reticulum (SR). Since congestive heart failure is characterized by abnormal intracellular Ca2+ homeostasis, we sought to delineate MRPES behavior before and after the production of heart failure to obtain insights into the relation between altered mechanical performance and Ca2+ handling. METHODS AND RESULTS: Ten dogs instrumented with left ventricular (LV) micromanometers and piezoelectric dimension crystals were studied under control conditions; 6 dogs also were studied after tachycardia heart failure (THF) produced by rapid LV pacing for 4 weeks. After priming at a basic cycle length of 375 ms, test pulses were delivered at fixed extrasystolic intervals (ESIs; 300, 375, or 450 ms) and graded postextrasystolic intervals (PESIs). Postextrasystolic mechanical response was assessed using single-beat elastance. MRPES curves were constructed by expressing normalized mechanical response as a function of the PESI. Control MRPES was a monoexponential function whose time constant (TC) and PESI-axis intercept (PESI0) increased significantly (P < .01) with increases in the antecedent ESI. THF significantly slowed MRPES kinetics at each antecedent ESI (P < .025), increased normalized maximal contractile response (CRmax, P < .01), and shortened PESI0 (P < .025). Increases in the TC and CRmax were most pronounced with the smallest antecedent ESI (percent control postextrasystolic TC 363.7 +/- 60.5%, ESI of 300 ms versus 139.0 +/- 15.1%, ESI of 450 ms, P < .005; percent control CRmax 128.6 +/- 4.9%, ESI of 300 ms versus 104.9 +/- 1.0%, ESI of 450 ms; P < .005). CONCLUSIONS: MRPES is much less dynamic in THF: The failing heart operates at lower levels of contractile performance after higher stimulation frequencies and cannot increase its speed of contractile recovery to compensate for higher heart rate. Prolongation of MRPES kinetics is consistent with depression of SR Ca2+ release mechanisms in THF and implicates this site in the loss of the capacity of the failing heart to maintain mechanical performance with tachycardia.

Effect of tachycardia heart failure on the restitution of left ventricular function in closed-chest dogs.

BACKGROUND: Cardiac mechanical restitution and relaxation restitution are thought to be physiological correlates of the recovery kinetics of Ca2+ release mechanisms and sequestration capacity of the sarcoplasmic reticulum (SR). Since congestive heart failure is characterized by abnormal intracellular Ca2+ handling, we sought to delineate changes in mechanical and relaxation restitution produced by heart failure. METHODS AND RESULTS: Six dogs instrumented with left ventricular (LV) micromanometers and piezoelectric dimension crystals were studied under control conditions and after tachycardia heart failure (THF) produced by rapid LV pacing for 3 to 4 weeks. After priming at a basic cycle length of 375 ms, test pulses were delivered at graded extrasystolic intervals (ESIs). Mechanical response was assessed from single-beat elastance. Relaxation was assessed from the time constant of isovolumic relaxation (tau), the average rate of pressure fall during isovolumic relaxation (Ravg), and peak negative dP/dt, the first derivative of LV pressure. Normalized mechanical and relaxation responses plotted against ESI produced monoexponential curves of mechanical and relaxation restitution. THF depressed baseline contractile and relaxation parameters compared with control (end-systolic elastance, 4.7 +/- 0.4 versus 7.1 +/- 0.5 mm Hg/mL, P < .005; tau, 34.8 +/- 2.2 versus 26.7 +/- 1.2 ms, P < .05; all values mean +/- SEM). THF slowed mechanical restitution and delayed development of peak contractile response, with the time constant of mechanical restitution increasing from 61.8 +/- 6.9 to 100.2 +/- 9.6 ms, P < .01. THF abolished the biphasic behavior of relaxation restitution, and this relation was approximated by a single monoexponential function. There was no difference in the time constants of the first phase of relaxation restitution at control and after THF (TCR1, normalized 1/Ravg, 44.3 +/- 5.6 versus 42.0 +/- 8.5 ms, P = NS; TCR1, normalized (dP/dtmin)-1, 42.2 +/- 6.3 versus 36.7 +/- 4.3 ms, P = NS). CONCLUSIONS: These results indicate that THF alters the recovery kinetics of SR Ca2+ release to a significantly greater extent than those of SR Ca2+ sequestration and that the abnormal time course of Ca2+ availability to the myofilaments is the rate-limiting step in the recovery of cardiac function after a depolarization.

Left ventricular energetics in closed-chest dogs.

Studies of ventricular energetics using the relation between myocardial O2 consumption (MVO2) and pressure-volume area (PVA) have been performed extensively in the isolated heart, but not in the intact animal. We characterized the MVO2-PVA relation and its response to heart rate (HR) in eight closed-chest dogs instrumented with high-fidelity micromanometers, piezoelectric crystals, coronary flow probes, and coronary sinus oximetric catheters. The effect of dobutamine was studied in five dogs. MVO2 is linearly related to PVA with lower MVO2 required for the generation of smaller PVAs. Baseline contractile efficiency (EFF) was 25.6 +/- 2.8%. High pacing rates reduced EFF (25.7 +/- 3.2% at a HR of 107 +/- 3 beats/min vs. 16.3 +/- 2.4% at a HR of 194 +/- 5 beats/min, P < 0.0167) and load-independent MVO2 per beat (0.562 +/- 0.119 vs. 0.377 +/- 0.074 J.beat-1 x 100 g LV-1, P < 0.0167) while increasing end-systolic elastance (Ees) (9.4 +/- 1.3 vs. 18.6 +/- 3.1 mmHg/ml, P < 0.0167). Dobutamine administration increased load-independent MVO2 per beat (0.392 +/- 0.108 vs. 0.607 +/- 0.083 J.beat-1 x 100 g LV-1, P < 0.05) and contractility (Ees 10.1 +/- 1.5 vs. 32.0 +/- 7.6 mmHg/ml, P < 0.05) without changing EFF (28.8 +/- 3.8 vs. 30.3 +/- 3.8%, P = NS). Thus the intact animal displays loss of EFF at high heart rates but maintains EFF during dobutamine stimulation. Both interventions increased load-independent MVO2 per minute, indicating increased O2 requirements for excitation-contraction coupling.

An analysis of variability of left ventricular pressure decay.

This study evaluated whether the time course of left ventricular (LV) pressure decay is consistent from beat to beat in the normal heart under tightly controlled experimental conditions. We determined the variability of LV isovolumic relaxation and compared it with that of other hemodynamic parameters. Pressure decay was evaluated using a monoexponential time constant (T), a half-time (T1/2), and an average rate (Ravg) in nine chronically instrumented dogs. To eliminate physical factors that could lead to variability, the dogs were studied at paced heart rates after autonomic blockade and during apnea. At a heart rate of 160 beats/min the coefficient of variation (SD/mean, expressed as a percent) was higher for T (4.7%, P < 0.005), T1/2 (5.0%, P < 0.005), and Ravg (3.2%, P < 0.005) than for dP/dtmax (1.9%), as well as for end-diastolic volume (1.2%), end-systolic volume (1.2%), or end-systolic pressure (1.8%). Similar differences were present at 200 beats/min. Pressure decay was also assessed during major loading shifts induced by rapid caval occlusion. Surprisingly, comparison of first and last beats did not show significant differences for T or T1/2 but did for all standard hemodynamic parameters and for Ravg. While the best correlation with a relaxation parameter and hemodynamic parameters during changing loading conditions was for Ravg, the correlations were not consistent in every case. We conclude that LV pressure decay shows marked variability, unrelated to the algorithm used to assess it. Ravg, a model independent parameter, may be a useful way to quantify LV pressure fall.

Kinetics of restitution of left ventricular relaxation.

Although the kinetics of cardiac systolic force restitution have been well described, the restitution kinetics of left ventricular relaxation have not been examined. To define relaxation restitution behavior, we studied seven dogs chronically instrumented with left ventricular high-fidelity micromanometers and piezoelectric dimension crystals. After a priming period at a basic cycle length of 375 msec, test extrastimuli were introduced after a range of extrasystolic intervals (ESIs). Relaxation behavior of control and extrasystolic beats was characterized by the time constant of isovolumic relaxation, tau. Relaxation restitution can be described by two concatenated monoexponential curves, an early phase described by a rapid time constant and a late phase described by a slower time constant (TC1, 36.21 +/- 7.90 msec; TC2, 75.94 +/- 10.65 msec; p less than 0.05). The first phase of relaxation restitution parallels systolic force restitution over the same range and displays faster recovery (TCs, 58.93 +/- 10.01 msec, p less than 0.05). Postextrasystolic restitution of test pulses after beats at fixed ESIs depends on the initial ESI. Relaxation recovery of postextrasystolic beats proceeds faster with smaller initial ESIs (TC1 for ESI of 300 msec, 13.27 +/- 4.05 msec; TC1 for ESI of 450 msec, 72.85 +/- 21.72 msec; p less than 0.0001). The monoexponential pattern of restitution was seen with model-independent descriptors of relaxation as well as with tau.

Reactive disulfide compounds induce Ca2+ release from cardiac sarcoplasmic reticulum.

Reactive disulfide compounds (RDSs) with a pyridyl ring adjacent to a disulfide bond, 2,2'dithiodipyridine (2,2' DTDP) and 4,4' dithiodipyridine (4,4' DTDP), induce Ca2+ release from isolated canine cardiac sarcoplasmic reticulum (SR) vesicles. RDSs are absolutely specific to free sulfhydryl (SH) groups and oxidize SH sites of low pKa via a thiol-disulfide exchange reaction, with the stoichiometric production of thiopyridone in the medium. As in skeletal SR, this reaction caused large increases in the Ca2+ permeability of cardiac SR and the number of SH sites oxidized by RDSs was kinetically and quantitatively measured through the absorption of thiopyridone. RDS-induced Ca2+ release from cardiac SR was characterized and compared to the action of RDSs on skeletal SR and to Ca2(+)-induced Ca2+ release. (i) RDS-induced Ca2+ release from cardiac SR was dependent on ionized Mg2+, with maximum rates of release occurring at 0.5 and 1 mM Mg2+free for 2,2' DTDP and 4,4' DTDP, respectively. (ii) In the presence of adenine nucleotides (0.1-1 mM), the oxidation of SH sites in cardiac SR by exogenously added RDS was inhibited, which, in turn, inhibited Ca2+ release induced by RDSs. (iii) Conversely, when the oxidation reaction between RDSs and cardiac SR was completed and Ca2+ release pathways were opened, subsequent additions of adenine nucleotides stimulated Ca2+ efflux induced by RDSs. (iv) Sulfhydryl reducing agents (e.g., dithiothreitol, DTT, 1-5 mM) inhibited RDS-induced Ca2+ efflux in a concentration-dependent manner. (v) RDSs elicited Ca2+ efflux from passively loaded cardiac SR vesicles (i.e., with nonfunctional Ca2+ pumps in the absence of Mg-ATP) and stimulated Ca2(+)-dependent ATPase activity, which indicated that RDS uncoupled Ca2+ uptake and did not act at the Ca2+, Mg2(+)-ATPase. These results indicate that RDSs selectively oxidize critical sulfhydryl site(s) on or adjacent to a Ca2+ release channel protein channel and thereby trigger Ca2+ release. Conversely, reduction of these sites reverses the effects of RDSs by closing Ca2+ release channels, which results in active Ca2+ reuptake by Ca2+, Mg2(+)-ATPase. These compounds can thus provide a method to covalently label and identify the protein involved in Ca2+ release from cardiac SR.

The heavy metal ions Ag+ and Hg2+ trigger calcium release from cardiac sarcoplasmic reticulum.

Heavy metal ions have been shown to induce Ca2+ release from skeletal sarcoplasmic reticulum (SR) by binding to free sulfhydryl groups on a Ca2+ channel protein and are now examined in cardiac SR. Ag+ and Hg2+ (at 10-25 microM) induced Ca2+ release from isolated canine cardiac SR vesicles whereas Ni2+, Cd2+, and Cu2+ had no effect at up to 200 microM. Ag(+)-induced Ca2+ release was measured in the presence of modulators of SR Ca2+ release was compared to Ca2(+)-induced Ca2+ release and was found to have the following characteristics. (i) Ag(+)-induced Ca2+ release was dependent on free [Mg2+], such that rates of efflux from actively loaded SR vesicles increased by 40% in 0.2 to 1.0 mM Mg2+ and decreased by 50% from 1.0 to 10.0 mM Mg2+. (ii) Ruthenium red (2-20 microM) and tetracaine (0.2-1.0 mM), known inhibitors of SR Ca2+ release, inhibited Ag(+)-induced Ca2+ release. (iii) Adenine nucleotides such as cAMP (0.25-2.0 mM) enhanced Ca2(+)-induced Ca2+ release, and stimulated Ag(+)-induced Ca2+ release. (iv) Low Ag+ to SR protein ratios (5-50 nmol Ag+/mg protein) stimulated Ca2(+)-dependent ATPase activity in Triton X-100-uncoupled SR vesicles. (v) At higher ratios of Ag+ to SR proteins (50-250 nmol Ag+/mg protein), the rate of Ca2+ efflux declined and Ca2(+)-dependent ATPase activity decreased gradually, up to a maximum of 50% inhibition. (vi) Ag+ stimulated Ca2+ efflux from passively loaded SR vesicles (i.e., in the absence of ATP and functional Ca2+ pumps), indicating a site of action distinct from the SR Ca2+ pump. Thus, at low Ag+ to SR protein ratios, Ag+ is very selective for the Ca2+ release channel. At higher ratios, this selectivity declines as Ag+ also inhibits the activity of Ca2+,Mg2(+)-ATPase pumps. Ag+ most likely binds to one or more sulfhydryl sites "on" or "adjacent" to the physiological Ca2+ release channel in cardiac SR to induce Ca2+ release.