Altered cardiac excitation-contraction coupling in mutant mice with familial hypertrophic cardiomyopathy.

Excitation-contraction coupling in cardiac muscle of familial hypertrophic cardiomyopathy (FHC) remains poorly understood, despite the fact that the genetic alterations are well defined. We characterized calcium cycling and contractile activation in trabeculae from a mutant mouse model of FHC (Arg403Gln knockin, alpha-myosin heavy chain). Wild-type mice of the same strain and age ( approximately 20 weeks old) served as controls. During twitch contractions, peak intracellular Ca2+ ([Ca2+]i) was higher in mutant muscles than in the wild-type (P < 0.05), but force development was equivalent in the two groups. Ca2+ transient amplitude increased dramatically in both groups as stimulation rate increased from 0.2 to 4 Hz. Nevertheless, developed force fell at the higher stimulation rates in the mutants but not in controls (P < 0.05). The steady-state force-[Ca2+]i relationship was less steep in mutants (Hill coefficient, 2.94 +/- 0.27 vs. 5.28 +/- 0.64; P > 0.003), with no changes in the [Ca2+]i required for 50% activation or maximal Ca2+-activated force. Thus, calcium cycling and myofilament properties are both altered in FHC mutant mice: more Ca2+ is mobilized to generate force, but this does not suffice to maintain contractility at high stimulation rates.

The relationship between contractile force and intracellular [Ca2+] in intact rat cardiac trabeculae.

The control of force by [Ca2+] was investigated in rat cardiac trabeculae loaded with fura-2 salt. At sarcomere lengths of 2.1-2.3 microns, the steady state force-[Ca2+]i relationship during tetanization in the presence of ryanodine was half maximally activated at a [Ca2+]i of 0.65 +/- 0.19 microM with a Hill coefficient of 5.2 +/- 1.2 (mean +/- SD, n = 9), and the maximal stress produced at saturating [Ca2+]i equalled 121 +/- 35 mN/mm2 (n = 9). The dependence of steady state force on [Ca2+]i was identical in muscles tetanized in the presence of the Ca(2+)-ATPase inhibitor cyclopiazonic acid (CPA). The force-[Ca2+]i relationship during the relaxation of twitches in the presence of CPA coincided exactly to that measured at steady state during tetani, suggesting that CPA slows the decay rate of [Ca2+]i sufficiently to allow the force to come into a steady state with the [Ca2+]i. In contrast, the relationship of force to [Ca2+]i during the relaxation phase of control twitches was shifted leftward relative to the steady state relationship, establishing that relaxation is limited by the contractile system itself, not by Ca2+ removal from the cytosol. Under control conditions the force-[Ca2+]i relationship, quantified at the time of peak twitch force (i.e., dF/dt = 0), coincided fairly well with steady state measurements in some trabeculae (i.e., three of seven). However, the force-[Ca2+]i relationship at peak force did not correspond to the steady state measurements after the application of 5 mM 2,3-butanedione monoxime (BDM) (to accelerate cross-bridge kinetics) or 100 microM CPA (to slow the relaxation of the [Ca2+]i transient). Therefore, we conclude that the relationship of force to [Ca2+]i during physiological twitch contractions cannot be used to predict the steady state relationship.

Characterisation of decay of frequency induced potentiation and post-extrasystolic potentiation.

STUDY OBJECTIVE: The aim was to elucidate the processes underlying the beat by beat decay of frequency induced and post-extrasystolic potentiation. DESIGN: The ventricular pacing protocol consisted of a "priming period" followed by a "decay" period of pacing at 1 s intervals, characterised by a decaying potentiation of left ventricular (LV) dP/dtmax; these were identified as test beats 1,2,3,4,5. The magnitude of potentiation of test beat 1 (P1) was increased both by increased priming frequency (frequency potentiation) and by alternately shorter priming intervals (paired pulse stimulation) at a given average frequency (post-extrasystolic potentiation). The exponential decay constant (P2) and the asymptotic value (P3) were determined and compared with the measured values and with the slope of the linear relationship between the contractility of one beat and that of the preceding beat. The lowest values after decay were related to the magnitude of preceding potentiation. EXPERIMENTAL MATERIAL: Six anaesthetised dogs with induced heart block and beta adrenergic blockade were used. Beat to beat interval was controlled by ventricular pacing from a programmable stimulator. MEASUREMENTS AND MAIN RESULTS: Contractility of each beat was assessed from maximum rate of rise of LV pressure (LVdP/dtmax) obtained from an intraventricular micromanometer. The asymptotic value of the exponential fit to the decay of potentiation (P3) was found to be below the measured nadir value, which was followed by an increase in LVdP/dtmax to the final steady state value P4. The decay constant (P2) was found to be equivalent to the natural logarithm of the slope of the linear relationship between the contractility of one beat and that of the preceding beat; it was unaffected by priming frequency or interval at a given average priming frequency. The asymptote P3 was inversely related to P1. CONCLUSIONS: P1 was interpreted as the expression of accumulation of activator in an internal release store; P3 was interpreted as a manifestation of negative feedback control of activator entry by the released activator itself, and the slow recovery to P4 as due to the slow lengthening of action potential duration and/or recovery from accumulation of an intracellular metabolite or ion.

Is endoscopic sphincterotomy plus large-balloon dilation a better option than endoscopic large-balloon dilation alone in removal of large bile duct stones? A retrospective comparison study.

BACKGROUND: Several comparison studies have demonstrated that endoscopic sphincterotomy (EST) combined with large-balloon dilation (LBD) may be a better option than EST alone to manage large bile duct stones. However, limited data were available to compare this combination method with LBD alone in removal of large bile duct stones. OBJECTIVE: To compare EST plus LBD and LBD alone for the management of large bile duct stones, and analyze the outcomes of each method. PATIENTS AND METHODS: Sixty-one patients were included in the EST plus LBD group, and 48 patients were included in the LBD alone group retrospectively. The therapeutic success, clinical characteristics, procedure-related parameters and adverse events were compared. RESULTS: Compared with EST plus LBD, LBD alone was more frequently performed in patients with potential bleeding diathesis or anatomical changes (P = 0.021). The procedure time from successful cannulating to complete stone removal was shorter in the LBD alone group significantly (21.5 vs. 17.3 min, P = 0.041). The EST plus LBD group and the LBD alone group had similar outcomes in terms of overall complete stone removal (90.2% vs. 91.7%, P = 1.000) and complete stone removal without the need for mechanical lithotripsy (78.7% vs. 83.3%, P = 0.542). Massive bleeding occurred in one patient of the EST plus LBD group, and successfully coagulated. Postoperative pancreatitis did not differ significantly between the EST plus LBD group and the LBD alone group (4.9% vs. 6.3%; P = 1.000). CONCLUSION: Endoscopic sphincterotomy combined with LBD offers no significant advantage over LBD alone for the removal of large bile duct stones. LBD can simplify the procedure compared with EST plus LBD in terms of shorten the procedure time.

Regulation of intracellular calcium in cardiac muscle.

We have investigated the regulation of intracellular free calcium in heart muscle using the free acid form of the Ca2+ indicator fura-2 iontophoretically microinjected into rat cardiac trabeculae or ferret papillary muscles. This method shows great promise in elucidating a number of crucial questions in cardiac excitation-contraction coupling.

Intrinsic myofilament alterations underlying the decreased contractility of stunned myocardium. A consequence of Ca2+-dependent proteolysis?

We investigated the mechanism of the decreased myofilament Ca2+ responsiveness in stunned myocardium. The steady state force-[Ca2+] relationship was measured before and after skinning in thin ventricular trabeculae from control or stunned (20 minutes of ischemia, 20 minutes of reperfusion) rat hearts.[Ca2+]i was determined using microinjected fura 2 salt in intact muscles, whereas the myofilaments of chemically skinned trabeculae were activated directly with solutions of varied [Ca2+]. Maximal Ca2+- activated force (F max) before and after skinning was identical within either the control or stunned groups but was markedly depressed in both groups of stunned trabeculae (P < .001)). After ischemia and reperfusion, the [Ca2+] required for 50% of maximal activation (Ca50) was increased in both intact (control, 0.60 +/- 0.09 micromol/L; stunned, 0.85 +/- 0.09 micromol/L;P < .001) and skinned (control, 1.13 +/- 0.24 micromol/L; stunned 1.39 +/- 0.21 micromol/L; P = .0025) trabeculae. These data indicate that the decreased Ca2+ responsiveness of stunned myocardium is due to intrinsic alterations of the myofilaments. Therefore, we tested the hypothesis that activation of proteases by reperfusion-induced Ca2+ overload decreases the Ca2+ responsiveness of the cardiac myofilaments. Force-[Ca2+] relations were compared before and 5 to 30 minutes after direct exposure of skinned trabeculae to calpain I (18 microgram/mL, 20 minutes at [Ca2+]=10.8 micromol/L), a Ca2+-activated protease that is present in myocardium. Calpain I reduced F max from 94.3 +/- 8.3 to 56 +/- 8.5 mN/mm2 while increasing Ca50 from 0.94 +/- 0.11 to 1.36 +/- 0.21 micromol/L (P < .01). Calpastatin, a specific calpain inhibitor prevented the effects of calpain I on skinned trabeculae. The results show that the reduced Ca2+ responsiveness of stunned myocardium reflects alteration of the myofilaments themselves, not of soluble cytosolic factors, which can be faithfully reproduced by exposure to Ca2+-dependent protease.

Novel myofilament Ca2+-sensitizing property of xanthine oxidase inhibitors.

Antioxidants are known to mitigate the cardiac contractile dysfunction that follows brief periods of ischemia ("myocardial stunning"). Stunning decreases contractility at the level of the contractile proteins; therefore, we asked whether antioxidant treatment preserves myofilament Ca2+ responsiveness after global ischemia and reflow. Right ventricular trabeculae were dissected from rat hearts subjected either to 20 minutes ischemia and reperfusion in the absence of drugs (stunned group) or to the same protocol in the presence of allopurinol, an inhibitor of xanthine oxidase (XO), and mercaptopropionylglycine (MPG), a hydroxyl radical scavenger (antioxidant group). At 20 minutes of reflow, isovolumic developed pressure recovered completely in the antioxidant group, but in the stunned group it recovered by only 57%. [Ca2+]i and contractile force measurements in trabeculae revealed the expected depression of myofilament function in the stunned group, with no change in Ca2+ transients relative to nonischemic controls. In contrast, Ca2+ transients were smaller, but force was greater, in the antioxidant group relative to both the stunned group and to nonischemic controls. Steady-state [Ca2+]i-force relationships revealed a striking increase of maximal force and a modest shift of activation to a lower range of [Ca2+]i. The increase in maximal force was reproduced by allopurinol+MPG or by allopurinol alone under nonischemic conditions and also by oxypurinol (100 micromol/L), a potent inhibitor of XO. We conclude that allopurinol and oxypurinol sensitize the cardiac myofilaments to Ca2+. This Ca2+-sensitizing effect underlies the preservation of contractility observed with an allopurinol+MPG antioxidant cocktail in a model of stunned myocardium. These serendipitous findings identify allopurinol and oxypurinol as the lead compounds of a novel class of inotropic agents.

Alterations of excitation-contraction coupling in stunned myocardium and in failing myocardium.

Although both myocardial stunning and chronic heart failure are characterized by contractile dysfunction, there are profound differences in their underlying mechanisms. Changes in cardiac contractile force can be effected by modulation of intracellular [Ca2+] or by alteration of the contractile protein response to intracellular Ca2+. New evidence suggests that the principal lesion in the stunned myocardium resides at the level of the contractile proteins, which may be injured by proteases activated early during reperfusion. In contrast, failing myocardium is known to display abnormal intracellular Ca2+ handling, indicative of dysfunction of the sarcoplasmic reticulum. Alterations of gene expression and isoform switching of the myofilaments also occur in failing myocardium, consistent with an observed shift of the kinetics of crossbridge cycling. In conclusion, changes in both intracellular Ca2+ homeostasis and myofilament function occur in failing myocardium, while stunned myocardium primarily reflects an uncoupling between Ca2+ and contractile force.

Selective effects of oxygen free radicals on excitation-contraction coupling in ventricular muscle. Implications for the mechanism of stunned myocardium.

BACKGROUND: Oxygen free radicals (OFRs) have been implicated in the pathogenesis of myocardial stunning, but the precise mechanism by which OFRs foster stunning remains unclear. We investigated the pathophysiology of the contractile dysfunction that occurs after direct exposure of OFRs to cardiac muscle and compared the results with the pathophysiology of stunned myocardium. METHODS AND RESULTS: Trabeculae from the right ventricles of rat hearts were loaded iontophoretically with fura-2 to determine [Ca2+]i. Steady-state force-[Ca2+]i relations were obtained by rapid electrical stimulation in the presence of ryanodine. Two exogenous OFR-generating systems were used: H2O2 + Fe(3+)-nitrilotriacetic acid (H2O2 + Fe3+) to produce hydroxyl radical, and xanthine oxidase+purine (XO + P) to produce superoxide. In muscles exposed to H2O2 + Fe3+ for 10 minutes, both twitch force and Ca2+ transients were decreased (eg, in 1.5 mmol/L external [Ca2+], force decreased from 41 +/- 7 to 23 +/- 4 mN/mm2, P < .05, and Ca2+ transient amplitude from 0.96 +/- 0.09 to 0.70 +/- 0.05 mumol/L, P < .05). Maximal Ca(2+)-activated force (Fmax) decreased slightly, from 103 +/- 5 to 80 +/- 12 mN/mm2 (P = NS). Neither the [Ca2+]i required to achieve 50% of Fmax (Ca50) nor the Hill coefficient was changed. In muscles exposed to XO + P for 20 minutes, twitch force was reduced (in 1.5 mmol/L external [Ca2+]) from 50 +/- 9 to 39 +/- 8 mN/mm2 (P < .05). Ca2+ transients, on the other hand, were not affected. Fmax decreased insignificantly from 100 +/- 16 to 81 +/- 14 mN/mm2. Ca50 increased from 0.71 +/- 0.06 to 1.07 +/- 0.07 mumol/L (P < .05), with no change in the Hill coefficient. CONCLUSIONS: These results indicate that exposure to the H2O2 + Fe3+ free radical-generating system reduces activator Ca2+ availability, whereas XO + P decreases the Ca2+ sensitivity of the myofilaments. Exogenously generated OFRs, particularly those produced by XO + P, mimic the effects of myocardial stunning on cardiac excitation-contraction coupling.

Calcium cycling and contractile activation in intact mouse cardiac muscle.

1. Excitation-contraction coupling in mouse cardiac muscle remains poorly characterized, despite the fact that the mouse is the mammalian species of choice for genetic manipulation. In this study, we characterized the relationship between internal calcium concentration ([Ca2+]i) and contraction in intact mouse ventricular muscle loaded with fura-2 salt at 20-22 degrees C. 2. Both Ca2+ transient amplitude and twitch force increased monotonically as external Ca2+ concentration ([Ca2+]o) was increased up to 8.0 mM, with no changes in diastolic levels or in the times to peak of either Ca2+ transients or force. The decay of Ca2+ transients was accelerated as [Ca2+]o increased, while relaxation was prolonged. Both Ca2+ transient amplitude and twitch force increased as stimulation rate increased from 0.2 to 4 Hz, but the increase in force was much greater than the underlying increase in [Ca2+]i. 3. The steady-state force-[Ca2+]i relationship revealed an [Ca2+]i required for 50 % of maximal activation (Ca50) of 0.95 +/- 0.08 microM, a Hill coefficient of 9.9 +/- 2.6, and a maximal Ca2+-activated force (Fmax) of 60 +/- 5 mN mm-2. 4. Unlike rat ventricular myocardium, mouse cardiac muscle resists supraphysiological [Ca2+]o. The strong positive force-frequency relationship in mouse cardiac muscle, with increases of force disproportionate to the increases in Ca2+ transients, suggests frequency-dependent 'sensitization' of the myofilaments. During steady-state activation, mouse muscle exhibits decreased Ca2+ responsiveness relative to other species, but high co-operativity. 5. These physiological features of mouse cardiac muscle merit consideration when interpreting the phenotypic consequences of genetic manipulations

Force-interval relations of twitches and cold contractures in rat cardiac trabeculae. Effect of ryanodine.

The twitch force (Ft)-interval relation of cardiac muscle reflects recovery of calcium release from the sarcoplasmic reticulum (SR). The calcium content of the SR is thought to be reflected by force developed during a contracture (Fc), induced by rapid cooling to near 0 degrees C. In right ventricular trabeculae of rat, under control conditions, the Ft-interval relation consisted of recovery of Ft to steady state (early recovery), followed by a secondary increase of Ft up to a maximum at an interval of approximately 100 seconds (rest potentiation) and a decline of Ft at intervals greater than 100 seconds (rest depression). The mechanisms that may underlie recovery of force after the last twitch at short intervals are 1) time-dependent transport of Ca2+ from the uptake compartment of the SR to the release compartment, 2) recovery of slow inward Ca2+ current during the action potential, and 3) recovery of the Ca2+ release channels in the SR. The Fc-interval relation was similar to the Ft-interval relation in that both a rest potentiation and a rest depression phase were present. However, at short interstimulus intervals (less than 1 second), Fc was independent of time, suggesting that the mechanism underlying early recovery was bypassed. Ryanodine (0.1-10 nM) reduced rest potentiation in a dose-dependent manner and accelerated rest depression of both Ft and Fc. At high ryanodine concentration, a significant Fc could only be induced after short intervals. Significant acceleration of rest depression was observed at low ryanodine concentrations, when Ft at intervals of 5 seconds was kept constant by increasing the stimulus frequency of [Ca2+]o, suggesting that the ryanodine effect was enhanced by increased [Ca2+]i. Ryanodine also increased the rate of decay of postextrasystolic potentiation in a dose-dependent manner. A significant effect was observed in 10 nM ryanodine. The twitch was not prolonged by ryanodine at these concentrations. These results suggest that the small magnitude of the twitch at short intervals is due to the finite time required by SR Ca2+ release channels to fully recover after a twitch. Furthermore, the results offer support for the hypothesis that ryanodine (in the nanomolar range) promotes Ca2+ leak from the SR in a dose-dependent manner and thereby limits Ca2+ accumulation during the interstimulus interval. Therefore, it may be expected that the negative inotropic effect of ryanodine is due to the SR Ca2+ depletion, and it is not necessary to postulate that ryanodine "blocks" the Ca2+ release channels in the SR.

Relationship between intracellular calcium and contractile force in stunned myocardium. Direct evidence for decreased myofilament Ca2+ responsiveness and altered diastolic function in intact ventricular muscle.

To elucidate the abnormalities of excitation-contraction coupling in stunned myocardium, we measured [Ca2+]i and force in thin fura 2-loaded ventricular trabeculae from control or stunned (20 minutes ischemia followed by 20 minutes reflow at 37 degrees C) rat hearts. At any given [Ca2+]o, force development was significantly lower in the stunned trabeculae than in control trabeculae. In contrast, there was no difference in the amplitude of Ca2+ transients between the two groups. The steady state force-[Ca2+]i relationship, assessed by tetanization in the presence of ryanodine, revealed both a decrease in maximal Ca(2+)-activated force and an increase in the [Ca2+]i required for 50% activation in stunned trabeculae. Postischemic myocardium also exhibited an accelerated rate of diastolic relaxation that was not due to changes in the rate of Ca2+ transient decay. Destabilization of attached cross-bridges in a quantitative model of cardiac myofibrils accurately reproduced the salient systolic and diastolic features of the stunned phenotype, suggesting an abnormality of the thin filaments. In response to supraphysiological increases in [Ca2+]o, diastolic [Ca2+]i and diastolic tone increased much more in stunned trabeculae than in controls, with the frequent occurrence of aftercontractions. This novel experimental model lends further support to the hypothesis that the primary lesion of excitation-contraction coupling resides at the level of the contractile proteins. The finding of enhanced susceptibility to calcium overload helps to rationalize the functional deterioration of stunned myocardium during intense inotropic stimulation and additionally suggests that stunned myocardium may represent a favorable substrate for triggered arrhythmias.

Synergistic modulation of ATP-sensitive K+ currents by protein kinase C and adenosine. Implications for ischemic preconditioning.

Ischemic preconditioning has been shown to involve the activation of adenosine receptors, protein kinase C (PKC), and ATP-sensitive K+ (K ATP) channels. We investigated the effects of PKC activation and adenosine on K(ATP) current (I KATP) and action potentials in isolated rabbit ventricular myocytes. Responses to pinacidil (100 to 400 micromol/L), an opener of K(ATP) channels, were markedly increased by preexposure to the PKC activator phorbol 12-myristate 13-acetate (PMA, 100 nmol/L). I(KATP) measured at 0 mV was increased by PMA pretreatment from 0.55 +/- 0.32 to 3.25 +/- 0.47 nA (n=6, P < .01). We next determined whether PKC activation abbreviates the time required to turn on I(KATP) developed after an average of 15.1 +/- 2.4 minutes (n=8). Ten-minute pretreatment with PMA alone (PMA+MI) did not significantly alter this latency (11.9 +/- 2.0 minutes, n=8). Since adenosine receptor activation has been shown to play an important role in the preconditioning response, two groups of myocytes were studied with adenosine (10 micromol/L) included during MI. Without PMA, adenosine alone (MI+Ado) did not affect the latency to develop I(KATP) (12.3 +/- 1.5 minutes, n=8). However, if cells were pretreated with PMA and then subjected to MI in the presence of adenosine (PMA+MI+Ado), the latency was greatly shortened to 5.5 +/- 1.6 minutes (n=8;P < .02 versus MI, PMA+MI, and MI+Ado groups). This effect could not be reproduced by an inactive phorbol but was completely abolished by the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline. The opening of K(ATP) channels may be cardioprotective because of the abbreviation of action potential duration (APD) during ischemia. Therefore, we tested whether PKC activation could modify the time course of APD shortening during MI. Consistent with the ionic current measurements, PMA pretreatment significantly accelerated APD shortening, but only when adenosine (10 micromol/L) was included during MI. The effects were not attributable to accelerated ATP consumption: PMA pretreatment did not alter the time required to induce rigor during MI, whether or not adenosine was included. Our results indicate that PKC activation increases the I(KATP) Induced by pinacidil or by MI. The latter effect requires concomitant adenosine receptor activation. The synergistic modulation of I(KATP) by PKC and adenosine provides an explicit basis for current paradigms of ischemic preconditioning.

Myofilament Ca2+ sensitivity in intact versus skinned rat ventricular muscle.

The Ca2+ sensitivity of myofilaments was compared before and after skinning in the same rat trabeculae at a diastolic sarcomere length of 2.2 to 2.3 microns. Trabeculae from rat right ventricle were loaded with fura-2 salt by iontophoretic microinjection, and [Ca2+]i was determined from the epifluorescence at 510 nm when excited at 340 and 380 nm. Steady-state activation was achieved by stimulating the muscle at 10 Hz after 10 to 20 minutes of application of ryanodine (5 mumol/L). The muscles were then skinned with Triton X-100 (1%) for 15 to 25 minutes and subsequently activated with solutions containing varied [Ca2+]. The intact force-[Ca2+] relation was highly cooperative (Hill coefficient, 4.87 +/- 0.35; n = 10), with a low [Ca2+]i required for half-maximal activation (K1/2) (0.62 +/- 0.03 mumol/L). After skinning, the Hill coefficient fell to 2.72 and the K 1/2 shifted rightward to 2.2 mumol/L in the presence of 1.2 mmol/L free Mg2+. Because of uncertainty regarding the appropriate [Mg2+], we measured [Mg2+]i at 0.72 +/- 0.06 mmol/L (n = 11) with Mg-fura-2 salt. When activating solutions were modified to contain [Mg2+] = 0.5 mmol/L, the force-[Ca2+] relation was shifted to the left (K 1/2 = 0.93 +/- 0.1, n = 10) with a Hill coefficient of 3.75 +/- 0.37, but the changes were not sufficient to superimpose with the intact force-[Ca2+] relation (P < .05 versus intact). These results suggest that, despite the significant effect of Mg2+ on the force-[Ca2+] relation in skinned muscles, the Ca2+ responsiveness of the myofilaments is still altered by skinning.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca2+]i and cross-bridge kinetics.

1. We investigated the mechanism of force inhibition by 2,3-butanedione monoxime (BDM) on rat cardiac trabeculae. [Ca2+]i was measured by iontophoretic injection of fura-2 salt. Isometric force was recorded at an end-systolic sarcomere length of 2.1-2.2 microns. 2. With an external [Ca2+] of 1 mM, peak twitch force was monotonically reduced with increasing [BMD]; at 5 and 20 mM [BDM], force was 35 and 1% of the control force. In contrast, the mean peak [Ca2+]i during transients was only reduced at [BDM] > or = 10 mM. 3. The duration of the twitch was dramatically reduced by BDM in a dose-dependent fashion with no significant change in the time course of the underlying Ca2+ transients. The abbreviation of twitch force duration was much greater than expected for the observed reduction in peak force by this agent. 4. The mechanism of the inhibition of force by BDM was explored by examining the relationship between twitch force and Ca2+ transients at various values of external [Ca2+]. In the presence of BDM, the steepness of the relationship between peak force and peak [Ca2+]i was reduced compared to control conditions. As a result, significant elevation in the [Ca2+]i transient was unable to reverse the reduction in force observed in the presence of BDM. 5. The direct inhibitory effects of BDM on the contractile system were examined using ryanodine tetani in intact trabeculae to measure the steady-state force-[Ca2+]i relationship. In contrast to the effects on twitch force at 5 mM BDM, maximal force was only reduced to 71% of control. Furthermore, the [Ca2+]i required for half-maximal activation (Ca50) was increased while the Hill coefficient was reduced slightly by BDM. 6. BDM dramatically slowed the rate of rise of tetanic force. At maximal activation, the time required to reach 90% maximal force was prolonged by a factor of 3-8 in the presence of 5 mM BDM. This suggests that the observed reduction in twitch force and steady-state force may result from slowed kinetics of cross-bridge attachment, consistent with recent biochemical studies. 7. The contribution of altered cross-bridge kinetics to the effects of BDM was investigated using a co-operative cross-bridge model of the contractile system. Changing the rate constants for cross-bridge attachment in the model to mimic the reported biochemical effects of BDM reproduced the observed effects of BDM.(ABSTRACT TRUNCATED AT 400 WORDS)

Excitation-transcription coupling mediated by zinc influx through voltage-dependent calcium channels.

Electrical activity initiates a program of selective gene expression in excitable cells. Although such transcriptional activation is commonly attributed to depolarization-induced changes in intracellular Ca2+, zinc represents a viable alternative given its prominent role as a cofactor in DNA-binding proteins coupled with evidence that Zn2+ can enter excitable cells in a voltage-dependent manner. Here it is shown that Zn2+ entry into heart cells depends upon electrical stimulation and occurs via dihydropyridine-sensitive Ca2+ channels. The addition of extracellular Zn2+ to spontaneously depolarizing GH3 pituitary tumor cells induced the expression of a reporter gene driven by the metallothionein promoter, an effect that was prevented by exposure to dihydropyridine Ca2+ channel blockers. Thus, Zn2+ influx through L-type Ca2+ channels can mediate voltage-dependent gene expression.

Cell-type specificity of preconditioning in an in vitro model.

We investigated whether preconditioning could protect several cultured cell lines, to determine whether the protection is specific for cells derived from different myogenic and non-myogenic sources. Ischemia was simulated by centrifugation of cells into a pellet, and cell viability was determined by hypotonic trypan blue solution. Preconditioning was produced by brief exposures to either glucose-free solution or metabolic inhibition. Freshly isolated rabbit ventricular myocytes were studied to confirm that preconditioning occurs in this model. We then compared these results to those in several cultured cell lines, including HEK 293 cells derived from human embryonic kidney, HIT-T15 cells from Syrian hamster pancreatic islets, and C2C12 cells from mouse skeletal muscle. In the latter cell line, we also determined whether differentiation alters preconditioning. Preconditioning protected rabbit ventricular myocytes: the percentage of dead cells was decreased from 36.8 +/- 4.7% in the control group to 23.0 +/- 5.2% in the preconditioned group after 60 min and from 50.7 +/- 2.1% in the control group to 25.5 +/- 4.5% in the preconditioned group after 120 min ischemia (p < 0.02). In contrast, there was no protection from preconditioning in HEK 293 cells or HIT-T15 cells. Preconditioning did not protect C2C12 myoblasts either. Interestingly, after C2C12 myoblasts had differentiated into myotubes (induced by exposing the cells to low-serum medium), they could then be protected by preconditioning (46.3 +/- 3.6% in the control group vs 26.0 +/- 2.7% in the preconditioned group after 60 min and 67.4 +/- 3.6% in the control group vs 46.0 +/- 4.6% in the preconditioned group after 120 min ischemia; p < 0.05). In conclusion, protection from preconditioning is cell-type specific. The presence of endogenous KATP channels (which are plentiful in HIT-T15 cells) is insufficient to enable preconditioning of the cell. Among the various cell types studied, only differentiated muscle cells (rabbit ventricular myocytes and C2C12 myotubes) exhibited preconditioning.

Priming effect of adenosine on K(ATP) currents in intact ventricular myocytes: implications for preconditioning.

Activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA) has been shown to shorten the time to turn on ATP-sensitive potassium currents (I(K,ATP)) during metabolic inhibition (MI) but only when adenosine (Ado) is included. In the present study we tested whether pretreatment with Ado could mimic the effect of PMA in isolated rabbit ventricular myocytes. When I(K,ATP) was measured by conventional whole cell clamp, pretreatment with 100 microM Ado did not alter the time to I(K,ATP) activation: 13.5 +/- 2.1 vs. 12.4 +/- 1.9 min with Ado during MI. We repeated the experiment using the perforated patch technique. Consistent with the previous results in conventional whole cell patch recordings, the time to turn on I(K,ATP) during MI (with Ado included) was shortened from 27.1 +/- 2.2 to 12.6 +/- 2.4 min (P < 0.01) when cells were pretreated with PMA and Ado was included during MI. In contrast to conventional whole cell recordings, Ado pretreatment also abbreviated the time for I(K,ATP) activation during MI (with Ado included) to 16.4 +/- 1.8 min. This effect was partially eliminated by simultaneous administration of an Ado receptor blocker or a PKC inhibitor during Ado pretreatment, suggesting that pretreatment with Ado stimulates PKC by activating Ado receptors. Our results demonstrate that Ado can prime I(K,ATP) during subsequent MI in the presence of Ado. This priming effect appears to be mediated by PKC upregulation of the channel. These results support the notion that Ado plays a dual role to initiate and to mediate ischemic preconditioning and links the roles of Ado receptors, PKC, and I(K,ATP) in ischemic preconditioning.

Unstimulated force during hypoxia of rat cardiac muscle: stiffness and calcium dependence.

The stiffness of rat cardiac trabeculae was measured in vitro to distinguish between an increase in unstimulated force (Fu) caused by rapid cycling of cross bridges or caused by rigor bridges during hypoxia. The force was measured with a strain gauge, the sarcomere length was determined by laser diffraction techniques, and muscle length was controlled by means of a motor. Stiffness was analyzed by using small (less than 1% of muscle length) sinusoidal length perturbations of 1 and 100 Hz. The stiffness at 100 Hz increased linearly with force during tetani at a varied [Sr2+] (0.25-10 mM) in the Krebs-Henseleit (K-H) buffer, but remained virtually unchanged at 1 Hz. In contrast, the stiffness of both the passive muscle and the muscle exposed to either CN- or to PO2 less than 1.5 mmHg up to development of maximal Fu (Fumax) was similar at 1- and 100-Hz perturbations. Less profound hypoxia (PO2 6-10 mmHg) resulted in spontaneous sarcomere activity during the rise in Fu, and an increase in the ratio of stiffness at 100 Hz to stiffness at 1 Hz was detected. When oxidative phosphorylation was inhibited by CN- (2 mM) while the muscle was stimulated in the absence of both Ca2+ and Na+ (choline+substituted), the addition of Na+ at the time at which Fu had reached 30-40% of Fumax did not affect the rate of rise of Fu. These results show that the development of Fu during more complete anoxia in rat trabeculae is completely due to the formation of rigor links and that Ca2(+)-dependent cross-bridge activation can contribute to the rise in Fu during less severe hypoxia.