Sarcolemmal Calcium Influx Research Articles

Cardiovascular effects of Sp-CTx, a cytolysin from the scorpionfish (Scorpaena plumieri) venom

Fish venom cytolysins are multifunctional proteins that in addition to their cytolytic/hemolytic effects display neurotoxic, cardiotoxic and inflammatory activities, being described as “protein lethal factors”. A pore-forming cytolysin called Sp-CTx (Scorpaena plumieriCytolytic Toxin) has been recently purified from the venom of the scorpionfish Scorpaena plumieri. It is a glycoprotein with dimeric constitution, comprising subunits of approximately 65 kDa. Previous studies have revealed that this toxin has a vasorelaxant activity that appears to involve the L-arginine–nitric oxide synthase pathway; however its cardiovascular effects have not been fully comprehended. The present study examined the cardiovascular effects of Sp-CTx in vivo and in vitro. In anesthetized rats Sp-CTx (70 μg/kg i.v) produced a biphasic response which consisted of an initial systolic and diastolic pressure increase followed by a sustained decrease of these parameters and the heart rate. In isolated rats hearts Sp-CTx (10−9 to 5 × 10−6 M) produced concentration-dependent and transient ventricular positive inotropic effect and vasoconstriction response on coronary bed. In papillary muscle, Sp-CTx (10−7 M) also produced an increase in contractile isometric force, which was attenuated by the catecholamine releasing agent tyramine (100 μM) and the β-adrenergic antagonist propranolol (10 μM). On isolated ventricular cardiomyocytes Sp-CTx (1 nM) increased the L-type Ca2+ current density. The results show that Sp-CTx induces disorders in the cardiovascular system through increase of sarcolemmal calcium influx, which in turn is partially caused by the release of endogenous noradrenaline.

Chronic exposure to low doses of HgCl2 avoids calcium handling impairment in the right ventricle after myocardial infarction in rats.

Right ventricle systolic dysfunction is a major risk factor for death and heart failure after myocardial infarction (MI). Heavy metal exposure has been associated with the development of several cardiovascular diseases, such as MI. The aim of this study was to investigate whether chronic exposure to low doses of mercury chloride (HgCl2) enhances the functional deterioration of right ventricle strips after MI. Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks- 1st dose 4.6 µg/kg, subsequent dose 0.07 µg/kg/day, i.m. to cover daily loss); MI surgery induced and HgCl2-MI groups. One week after MI, the morphological and hemodynamic measurements and isometric tension of right ventricle strips were investigated. The chronic HgCl2 exposure did not worsen the injury compared with MI alone in the morphological or hemodynamic parameters evaluated. At basal conditions, despite similar maximum isometric force at L-max, relaxation time was increased in the MI group but unaffected in the HgCl2-MI compared to the Control group. Impairment of the sarcoplasmic reticulum (SR) function and reduction in the sarcolemmal calcium influx were observed in MI group associated with SERCA2a reduction and increased PLB protein expression. Induction of MI in chronic HgCl2 exposed rats did not cause any alteration in the developed force at L-max, lusitropic function or −dF/dt except for a tendency of a reduction SR function. These findings could be partially explained by the normalization in the sarcolemmal calcium influx and the increase in NCX protein expression observed only in this group. These results suggest that chronic exposure to low doses of HgCl2 prevents the impaired SR function and the reduced sarcolemmal calcium influx observed in MI likely by acting on NCX, PLB and SERCA2a protein expression.

Chronic exposure to low doses of HgCl2 avoids the impaired on calcium handling in the right ventricle after myocardial infarction in rat (547.3)

The aim of this study was to investigate if chronic exposure to low doses of mercury chloride (HgCl2) causes functional alterations in right ventricle (RV) strips after myocardial infarction (MI). Male Wistar rats were divided into four groups: Control (vehicle); HgCl2 (exposure during 4 weeks‐ 1st dose 4.6 μg/kg, subsequent dose 0.07 μg/kg/day, i.m to cover daily loss); MI surgery induced and HgCl2‐MI group. One week after MI RV strips, superfused with Krebs solution, were studied using an isometric tension system. Contractile function of HgCl2 group did not change compared to Control. On basal conditions, despite similar maximum isometric force at L‐max, relaxation time was reduced in the MI group but preserved in the HgCl2‐MI compared to Control group. The MI group presented a depression in the tetanic peak and plateau of developed force. Additionally, impaired sarcoplasmic reticulum (SR) function and a reduced sarcolemmal calcium influx were observed only in MI animals. The SERCA2a protein expression was reduced in both MI groups while PLB protein expression was increased only in MI group. These results suggest that chronic exposure to low doses of HgCl2 avoids the impaired SR function and the reduced sarcolemmal calcium influx observed in MI probably acting on PLB and SERCA2a protein expression.Grant Funding Source: Supported by: PRONEX ‐ FAPES, CNPq, CAPES

Acute exposure to lead increases myocardial contractility independent of hypertension development

We studied the effects of the acute administration of small doses of lead over time on hemodynamic parameters in anesthetized rats to determine if myocardial contractility changes are dependent or not on the development of hypertension. Male Wistar rats received 320 µg/kg lead acetate iv once, and their hemodynamic parameters were measured for 2 h. Cardiac contractility was evaluated in vitro using left ventricular papillary muscles as were Na+,K+-ATPase and myosin Ca2+-ATPase activities. Lead increased left- (control: 112 ± 3.7 vs lead: 129 ± 3.2 mmHg) and right-ventricular systolic pressures (control: 28 ± 1.2 vs lead: 34 ± 1.2 mmHg) significantly without modifying heart rate. Papillary muscles were exposed to 8 µM lead acetate and evaluated 60 min later. Isometric contractions increased (control: 0.546 ± 0.07 vs lead: 0.608 ± 0.06 g/mg) and time to peak tension decreased (control: 268 ± 13 vs lead: 227 ± 5.58 ms), but relaxation time was unchanged. Post-pause potentiation was similar between groups (n = 6 per group), suggesting no change in sarcoplasmic reticulum activity, evaluated indirectly by this protocol. After 1-h exposure to lead acetate, the papillary muscles became hyperactive in response to a β-adrenergic agonist (10 µM isoproterenol). In addition, post-rest contractions decreased, suggesting a reduction in sarcolemmal calcium influx. The heart samples treated with 8 µM lead acetate presented increased Na+,K+-ATPase (approximately 140%, P < 0.05 for control vs lead) and myosin ATPase (approximately 30%, P < 0.05 for control vs lead) activity. Our results indicated that acute exposure to low lead concentrations produces direct positive inotropic and lusitropic effects on myocardial contractility and increases the right and left ventricular systolic pressure, thus potentially contributing to the early development of hypertension.

Sarcoplasmic Reticulum Calcium Leak in Normal and Dystrophic Skeletal Muscle

Under resting conditions, external calcium is known to enter skeletal muscle cells while calcium stored in the sarcoplasmic reticulum (SR) leaks into the cytosol. The nature of the pathways involved in the resting sarcolemmal calcium entry and in the SR calcium leak are still debated but several lines of evidence suggest that an up-regulation of these calcium fluxes occurs in Duchenne Muscular Dystrophy (DMD). We investigated here SR calcium permeation at resting potential and in response to depolarization in voltage-controlled skeletal muscle fibers from control and mdx mice, the murine model of DMD. Using the cytosolic calcium dye Fura2, we first demonstrated that the rate of calcium increase induced by CPA (cyclopiazonic acid) inhibition of SR Ca2+-ATPases at resting potential is significantly higher in mdx fibers suggesting an elevated SR passive calcium leak. However, in these experiments, sarcolemmal calcium influx may contribute to the CPA-induced calcium increase and another series of experiments indicated that CPA-induced SR calcium leak was deeply modified in the absence of external calcium. Fibers were then loaded with the low affinity calcium dye Fluo5N-AM and dialyzed with 50 mM EGTA to measure intraluminal SR calcium changes. Depolarization pulses evoked voltage-dependent Fluo5N fluorescence decreases followed by a recovery phase which was inhibited by CPA, demonstrating that Fluo5N actually reports intraluminal SR calcium changes. Voltage-dependence and magnitude of depolarization-induced SR calcium depletion were found to be unchanged in mdx fibers but the rate of the recovery phase that followed depletion was found to be faster, suggesting a higher SR calcium reuptake capacity in mdx fibers. Finally, CPA-induced SR calcium leak at −80 mV was found to be significantly higher in mdx fibers. The elevated SR passive calcium leak may participate to the muscle degenerative process in mdx muscle.

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

Lead (Pb2+) poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g) before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 microM) to the bath. Changes in rate of stimulation (0.1-1.5 Hz), relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM), and the effect of isoproterenol (20 ng/mL) were determined before and after the addition of 100 microM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz) and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 microM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 microM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 microM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 microM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Effects of temperature and calcium availability on ventricular myocardium from rainbow trout.

We studied the mechanical and electrophysiological properties of ventricular myocardium from rainbow trout (Oncorhynchus mykiss) in vitro at 4, 10, and 18 degrees C from fish acclimated at 10 degrees C. Temperature alone did not significantly alter the contractile force of the myocardium, but the time to peak tension and time to 80% relaxation were prolonged at 4 degrees C and shortened at 18 degrees C. The duration of the action potential was also prolonged at 4 degrees C and progressively shortened at higher temperatures. An alteration of the stimulation frequency did not affect contraction amplitude at any temperature. Calcium influx via L-type calcium channels was increased by raising extracellular calcium concentration (¿Ca(2+)(o)) or including Bay K 8644 (Bay K) and isoproterenol in the bathing medium. These treatments significantly enhanced the contractile force at all temperatures. Calcium channel blockers had a reverse-negative inotropic effect. Unexpectedly, the duration of the action potential at 10 degrees C was shortened as ¿Ca(2+)(o) increased. However, Bay K prolonged the plateau phase at 4 degrees C. Caffeine, which promotes the release of sarcoplasmic reticulum (SR) calcium, increased contractile force eightfold at all three temperatures, but the SR blocker ryanodine was only inhibitory at 4 degrees C. Our results suggest that contractile force in ventricular myocardium from Oncorhynchus mykiss is primarily regulated by sarcolemmal calcium influx and that ventricular contractility is maintained during exposure to a wide range of temperatures.

Numerical simulation of local calcium movements during L-type calcium channel gating in the cardiac diad

Computer simulation was used to investigate the calcium levels after sarcolemmal calcium influx through L-type calcium channels (DHPRs) into the narrow diadic space of cardiac muscle. The effect of various cytosolic and membranebound buffers, diad geometry, DHPR properties (open time and current), and surface charge were examined. The simulations showed that phospholipid binding sites on the sarcolemmal membrane are the major buffer affecting free calcium ([Ca2+]) levels in the diad. The inclusion of surface charge effects calculated from Gouy-Chapman theory resulted in a marked decrease in [Ca2+] levels at all times and a faster decay of [Ca2+] after termination of DHPR influx. For a DHPR current of 200 fA, [Ca2+] at the center of the diad reached peak levels of approximately 73 microM. In larger diads (> or = 400 nm diameter), [Ca2+] decayed more slowly than in smaller diads (100-200 nm diameter), although peak [Ca2+] levels reached during typical DHPR open times were similar. For a wide range of DHPR single-channel current magnitudes (Ica = 25-200 fA), [Ca2+] levels in the diad were approximately proportional to ICa. The decrease in calculated [Ca2+] levels due to the effects of surface charge can be interpreted as resulting from an effective "volume expansion" of the diad space. Furthermore, the layer of increased [Ca2+] close to the sarcolemmal membrane can act as a fast buffer.

Numerical analysis of ryanodine receptor activation by L-type channel activity in the cardiac muscle diad

Computer simulations were used to examine the response of ryanodine receptors (RyRs) to the sarcolemmal calcium influx via L-type calcium channels (DHPRs). The effects of ryanodine receptor organization, diad geometry, DHPR single-channel current, and DHPR gating were examined. In agreement with experimental findings, the simulations showed that RyRs can respond rapidly (approximately 0.4 ms) to calcium influx via DHPRs. The responsiveness of the RyR depends on the geometrical arrangement between the RyRs and the DHPR in the diad, with wider diads being generally less responsive. When the DHPR single-channel current is small (approximately 25 fA), the organization of RyRs into small clusters results in an improved responsiveness. With experimentally observed DHPR mean open and closed times (0.17 ms and 4 ms, respectively) it is the first opening of the DHPR that is most likely to activate the RyR. A measure of the efficiency (Q) by which DHPR gating evokes sarcoplasmic reticulum release is defined. Q is at maximum for tau approximately 0.3 ms, and we interpret this finding in terms of the "tuning" of DHPR gating to RyR response. If certain cardiac myopathies are associated with a mismatch in the "tuning," then modification of DHPR gating with drugs to "retune" calcium-induced calcium release should be possible.

Role of intracellular calcium handling in force-interval relationships of human ventricular myocardium.

Experiments were performed in human working myocardium to investigate the relationship of intracellular calcium handling and availability to alterations in the strength of contraction produced by changes in stimulation rate and pattern. Both control and myopathic muscles exhibited potentiation of peak isometric force during the postextrasystolic contraction which was associated with an increase in the peak intracellular calcium transient. Frequency-related force potentiation was attenuated in myopathic muscles compared to controls. This occurred despite an increase in resting intracellular calcium and in the peak amplitude of the calcium transient as detected with aequorin. Therefore, abnormalities in contractile function of myopathic muscles during frequency-related force potentiation are not due to decreased availability of intracellular calcium, but more likely reflect differences in myofibrillar calcium responsiveness. Sarcolemmal calcium influx may also contribute to frequency-related changes in contractile force in myopathic muscles as suggested by a decrease in action potential duration with increasing stimulation frequency which is associated with fluctuations in peak calcium transient amplitude.

Effects of divalent cation ionophore A23187 on cardiac contractile parameters.

The divalent cation ionophore A23187, when added to guinea pig papillary muscle, produced contractile effects that were similar to those produced by isoproterenol or histamine, but the ionophore's effects did not appear to result from the release of endogenous transmitters or prostaglandin production. Optimally effective concentrations of A23187 (6 microM) and isoproterenol (1 microM) more than doubled the peak contractile force and the rates of force development and relaxation and caused a 25% decrease in the duration of the contraction. Both A23187 and isoproterenol decreased the resting tension by approximately 0.15 g and significantly diminished the magnitude of a potassium-induced contracture. The positive inotropic effect of A23187 was prevented by incubating the tissue in calcium-depleted medium and antagonized by D 600, a blocker of sarcolemmal calcium influx, and acetylcholine. The contractile effects of A23187 appear to be related, in part, to its ability to increase the movement of calcium across the sarcolemma electroneutrally, since no change of the action potential occurred. In addition, possible intracellular actions of this ionophore may produce contractile effects that resemble those produced by isoproterenol and that reflect an increased sequestration of calcium within the myocardial cell.