Sensitivity Of Myofilaments Research Articles

Maturation attenuates the effects of cGMP on contraction, [Ca 2+] i and Ca 2+ sensitivity in ovine basilar arteries

The present study explores the hypothesis that age-related variations in cerebrovascular responses to vasodilators reflect corresponding age-dependent differences in the mechanisms coupling changes in cytosolic cGMP to vasorelaxation. The experiments focused on cGMP's ability to decrease either [Ca 2+] i or myofilament Ca 2+ sensitivity, because both effects can contribute to cGMP-induced vasodilation. Use of the cGMP analog 8-pCPT-cGMP minimized problems associated with limited cell permeation or cGMP hydrolysis. In fetal basilars contracted with 10 μM serotonin, the EC 30 for 8-pCPT-cGMP-induced relaxation was 6 μM. In fura-2 loaded fetal basilars, pretreatment with 6 μM 8-pCPT-cGMP significantly depressed the sensitivity of [Ca 2+] i to 5HT, and also myofilament sensitivity to calcium, but only in fetal arteries. In fetal basilar arteries contracted with 120 mM potassium, the EC 30 for 8-pCPT-cGMP-induced relaxation was 25 μM. In fura-2 loaded ovine arteries, pretreatment with 25 μM 8-pCPT-cGMP had no effect on the ability of graded concentrations of potassium to elevate [Ca 2+] i but reduced potassium's ability to induce contraction and attenuated myofilament calcium sensitivity; these latter effects were significant only in fetal arteries. In α-toxin permeabilized preparations, 25 μM 8-pCPT-cGMP significantly depressed both basal- and agonist-stimulated myofilament calcium sensitivity, only in fetal but not in adult basilars. Together, these results demonstrate that: (1) sensitivity to cGMP is greater in fetal than adult sheep arteries independent of method of contraction; (2) cGMP can reduce [Ca 2+] i but only in agonist-contracted and not in potassium-contracted arteries; (3) and cGMP attenuates myofilament calcium sensitivity regardless of method of contraction. Overall, the data demonstrate that variations in the ability of cGMP to produce vasodilatation reflect age-, artery-, and agonist-dependent differences in the combination of mechanisms mediating responses to cGMP.

Transgenic overexpression of constitutively active protein kinase C epsilon causes concentric cardiac hypertrophy.

To test the hypothesis that activation of the protein kinase C (PKC) epsilon isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression of a constitutively active mutant of the PKCepsilon isoform driven by an alpha-myosin heavy chain promoter. In transgenic mice, the protein level of PKCepsilon in heart tissue was increased 9-fold. There was a 6-fold increase of the membrane/cytosol ratio, and PKC activity in the membrane fraction was 4.2-fold compared with wild-type mice. The heart weight was increased by 28%, and upregulation of the mRNA for beta-myosin heavy chain and alpha-skeletal actin was observed in transgenic mouse hearts. Echocardiography demonstrated increased anterior and posterior wall thickness with normal left ventricular function and dimensions, indicating concentric cardiac hypertrophy. Isolated cardiomyocyte mechanical function was slightly decreased, and Ca(2+) signals were markedly depressed in transgenic mice, suggesting that myofilament sensitivity to Ca(2+) was increased. No differences were observed in either the levels of cardiac Ca(2+)-handling proteins or the degree of cardiac regulatory protein phosphorylation between wild-type and transgenic mice. Unlike mice with PKCbeta(2) overexpression, transgenic mice with cardiac-specific overexpression of the active PKCepsilon mutant demonstrated concentric hypertrophy with normal in vivo cardiac function. Thus, PKC isoforms may play differential functional roles in cardiac hypertrophy and failure.

The involvement of intracellular Ca(2+) in 5-HT(1B/1D) receptor-mediated contraction of the rabbit isolated renal artery.

5-Hydroxytryptamine(1B/1D) (5-HT(1B/1D)) receptor coupling to contraction was investigated in endothelium-denuded rabbit isolated renal arteries, by simultaneously measuring tension and intracellular [Ca(2+)], and tension in permeabilized smooth muscle cells. In intact arterial segments, 1 nM - 10 microM 5-HT failed to induce contraction or increase the fura-2 fluorescence ratio (in the presence of 1 microM ketanserin and prazosin to block 5-HT(2) and alpha(1)-adrenergic receptors, respectively). However, in vessels pre-exposed to either 20 mM K(+) or 30 nM U46619, 5-HT stimulated concentration-dependent increases in both tension and intracellular [Ca(2+)]. 1 nM - 10 microM U46619 induced concentration-dependent contractions. In the presence of nifedipine (0.3 and 1 microM) the maximal contraction to U46619 (10 microM) was reduced by around 70%. The residual contraction was abolished by the putative receptor operated channel inhibitor, SKF 96365 (2 microM). With 0.3 microM nifedipine present, 100 nM U46619 evoked similar contraction to 30 nM U46619 in the absence of nifedipine, but contraction to 5-HT (1 nM - 10 microM) was abolished. In permeabilized arterial segments, 10 mM caffeine, 1 microM IP(3) or 100 microM phenylephrine, each evoked transient contractions by releasing Ca(2+) from intracellular stores, whereas 5-HT had no effect. In intact arterial segments pre-stimulated with 20 mM K(+), 5-HT-evoked contractions were unaffected by 1 microM thapsigargin, which inhibits sarco- and endoplasmic reticulum calcium-ATPases. In vessels permeabilized with alpha-toxin and then pre-contracted with Ca(2+) and GTP, 5-HT evoked further contraction, reflecting increased myofilament Ca(2+)-sensitivity. Contraction linked to 5-HT(1B/1D) receptor stimulation in the rabbit renal artery can be explained by an influx of external Ca(2+) through voltage-dependent Ca(2+) channels and sensitization of the contractile myofilaments to existing levels of Ca(2+), with no release of Ca(2+) from intracellular stores.

PII: S0003-4975(00)01446-6

PII: S0003-4975(00)01446-6

Further Evidence for the Cardiac Troponin C Mediated Calcium Sensitization by Levosimendan: Structure-response and Binding Analysis with Analogs of Levosimendan

Levosimendan, an inodilatory drug discovered using troponin C as a target protein, has a cardiac effect deriving from the calcium sensitization of contractile proteins. The aim of this study was to give further evidence that levosimendan binds to cardiac troponin C and that the binding involves amino acid residues on helixϵ of the N-terminal domain of this calcium-binding protein. Nine organic molecules, obtained by chemical modification of levosimendan, were tested both for their calcium-dependent binding to troponin C and troponin complex affinity HPLC columns, and for their ability to increase the calcium sensitivity of myofilaments in cardiac skinned fibers. A good correlation between the calcium sensitization and the calcium-dependent binding to troponin complex (r=0.90) and to cardiac troponin C (r=0.91) for the analogs of levosimendan was shown. In addition, the effect of levosimendan on the calcium-induced conformational changes in native and point-mutated cTnC was studied. Cys84→Ser, Asp87→Lys and Asp88→Ala point-mutated cTnC were shown to maintain a high affinity to calcium, but their Ca2+titration curves were not influenced by levosimendan as for the native protein. Finally, it was demonstrated that the NMR chemical shifts of the terminal methyl groups of Met47, Met81, and Met85 on calcium-saturated cTnC were changed after addition of levosimendan in water solution at pH 7.4. This effect was not seen when adding an analog of levosimendan, which did not bind to the troponin C affinity HPLC column and did not increase the calcium-induced tension in cardiac skinned fibers.

Mechanisms Associated with the Negative Inotropic Effect of Deuterium Oxide in Single Rat Ventricular Myocytes

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

Mechanisms associated with the negative inotropic effect of deuterium oxide in single rat ventricular myocytes

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.

Functional consequences of the deletion mutation deltaGlu160 in human cardiac troponin T.

To explore the functional consequences of a deletion mutation of troponin T (DeltaGlu160) found in familial hypertrophic cardiomyopathy, the mutant human cardiac troponin T, and wild-type troponins T, I, and C were expressed in Escherichia coli and directly incorporated into isolated porcine cardiac myofibrils using our previously reported troponin exchange technique. The mutant troponin T showed a slightly reduced potency in replacing the endogenous troponin complex in myofibrils and did not affect the inhibitory action of troponin I but potentiated the neutralizing action of troponin C, suggesting that the deletion of a single amino acid, Glu-160, in the strong tropomyosin-binding region affects the tropomyosin binding affinity of the entire troponin T molecule and alters the interaction between troponin I and troponin C within ternary troponin complex in the thin filament. This mutation also increased the Ca(2+) sensitivity of the myofibrillar ATPase activity, as in the case of other mutations in troponin T with clinical phenotypes of poor prognosis similar to that of Glu160. These results provide strong evidence that the increased Ca(2+) sensitivity of cardiac myofilament is a typical functional consequence of the troponin T mutation associated with a malignant form of hypertrophic cardiomyopathy.

Urodilatin limits acute reperfusion injury in the isolated rat heart.

Hypercontracture is an important mechanism of myocyte death during reperfusion. cGMP modulates the sensitivity of contractile myofilaments to Ca2+, and increasing cGMP concentration during the last minutes of anoxia prevents reoxygenation-induced hypercontracture in isolated cardiomyocytes. The purpose of this study was to determine whether stimulation of particulate guanylyl cyclase with the natriuretic peptide urodilatin, given at the time of reperfusion, reduces myocardial necrosis in the rat heart submitted to transient ischemia. Isolated rat hearts (n = 38) were submitted to either 40 or 60 min of no-flow ischemia and 2 h of reperfusion, and were allocated to receive or not receive 0.05 microM urodilatin during the first 15 min of reperfusion or non-reperfusion treatment. A marked reduction in myocardial cGMP concentration was observed in control hearts during reperfusion after 40 or 60 min of ischemia. Urodilatin significantly attenuated cGMP depletion during initial reperfusion, markedly improved contractile recovery after 40 min of ischemia (P < 0.0309), and reduced reperfusion-induced increase in left ventricular end-diastolic pressure (P = 0.0139), LDH release (P = 0.0263), and contraction band necrosis (P = 0.0179) after 60 min of ischemia. The beneficial effect of urodilatin was reproduced by the membrane permeable cGMP analog 8-Bromo-cGMP. These results indicate that reduced cGMP concentration may impair myocyte survival during reperfusion. Stimulation of particulate guanylyl cyclase may appear as a new strategy to prevent immediate lethal reperfusion injury.

Estrogen supplement prevents the calcium hypersensitivity of cardiac myofilaments in ovariectomized rats

Our previous biochemical and mechanical studies have demonstrated an increase in Ca2+ sensitivity of cardiac myofilaments in ovariectomized rats. To test whether the body weight gain associated with ovariectomy contributed some effects to the changes in myofibrillar functions, the relations of pCa (-log Ca2+ molar concentration) to actomyosin adenosine triphosphatase (ATPase) activity of isolated myofibrillar preparations from 10-week pair-fed ovariectomized rats were compared with those from sham-operated controls. Despite similar body weights, the maximum myofibrillar ATPase activity was significantly lower in pair-fed ovariectomized rats as compared to that of sham-operated controls. In addition, the pCa-actomyosin ATPase relationship of pair-fed ovariectomized hearts still demonstrated a significant leftward shift in pCa50 (-log half-maximally Ca2+ activation) from that of sham-operated controls. To find out which hormone was responsible for the observed increase in myofibrillar Ca2+ sensitivity, different sex hormone supplemental regimens were administered to ovariectomized rats. Subcutaneous injection of estrogen (5 microg/rat) or estrogen plus progesterone (1 mg/rat) three times a week could effectively prevent the changes in body weight, heart weight, and uterine weight of the ovariectomized animals. Moreover, supplements of either estrogen or progesterone could prevent a decrease in maximum ATPase activity. In contrast, only the estrogen replacement could abolish the Ca2+ hypersensitivity of the myofilaments in these ovariectomized rats. These results suggest differential cardio-regulatory effects of ovarian sex hormones on the Ca2+ activation of the myofilaments.

Effects of genistein and daidzein on enhanced vascular contractile reactivity and Ca2+ sensitivity in cardiomyopathic hamsters.

We investigated the contribution of tyrosine kinase activity to the enhanced vasoreactivity in cardiomyopathic (CM) hamsters. The contractile response of the aorta to phenylephrine was greatly enhanced in CM in comparison to control hamsters. Genistein, a tyrosine kinase inhibitor, did not affect the maximum contractile response to phenylephrine in control aortas, although the sensitivity to phenylephrine was significantly diminished. In contrast, genistein markedly inhibited both the sensitivity and the maximum contractile response to phenylephrine in CM aortas. Daidzein, an inactive form of genistein, also inhibited the sensitivity to phenylephrine in both strains although the maximum contractile response was not altered even in the CM aorta. The Ca2+ sensitivity of tension was significantly augmented in alpha toxin-permeabilized smooth muscle from the mesenteric artery of CM hamsters. Furthermore, phenylephrine enhanced myofilament sensitivity to Ca2+ more in CM than control hamsters. The enhancement of myofilament Ca2+ sensitivity by phenylephrine was markedly inhibited by genistein only in CM hamsters. The inhibition by daidzein of the phenylephrine effect on the Ca2+ sensitivity in CM hamsters was less pronounced. These results suggest that an increase in tyrosine kinase activity may lead to enhanced vascular reactivity in CM hamsters possibly due to an increased Ca2+ sensitivity of the contractile apparatus.

Intracellular effects of nitric oxide on force production and Ca2+ sensitivity of cardiac myofilaments.

The gaseous free radical nitric oxide (NO*) has been implicated in a wide range of physiological functions and also has a role in the pathogenesis of cellular injury. It has been suggested that NO* and its congeners may exert effects on actin-myosin crossbridge cycling by modulating critical thiols on the myosin head. To understand the mode and site of actin of NO* in myofibrils, the effects of the NO* donor 3-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl-1-propanamine (NOC-7) have been studied in Triton X-100-treated rabbit cardiac fibers, in which isometric force was measured at controlled degrees of activation. Experiments were undertaken after previous exposure of the preparations to NOC-7 (for 30 min). We found that NO* induced several alterations of myofibrillar function, i.e., decrease in Ca2+ sensitivity and Hill coefficient and potentiation of rigor contracture. We attributed the effect on rigor contracture to strong inhibition of myofibrillar creatine kinase (CK) activity, because it could be prevented by exogeneously added CK; such CK inactivation afforded by NO* may result in the myofibrillar ATP-to-ADP ratio. In further experiments, concentration of NO* released from NOC-7 was determined by the electron spin resonance spin-trapping technique; N-(dithiocarboxy)sarcosine-Fe2+ complex was used as the spin-trap. NO* at cumulative concentration of 0.69 microM was effective in producing both enhancement of rigor contracture and decrease of myofibrillar-bound CK activity; however, Ca2+-sensitivity (pCa50) was significantly decreased at >5.6 microM of NO*, suggesting a result from different mechanisms. Thus, the observed decrease in Ca2+ sensitivity seems to be associated with direct modification of the regulatory proteins by a relatively higher concentration of NO*, and possibly not via inhibition of myofibrillar CK activity. The data reported here indicate that CK may be a pathophysiologically main target for increased NO* formation at low molecular range in the disease state in cardiac muscle.

Role of Ca2+ availability to myofilaments and their sensitivity to Ca2+ in myocyte contractile dysfunction in heart failure.

Contractile function is depressed at the isolated myocyte level in heart failure (HF), which could result from the decreased availability of intracellular calcium ([Ca2+]i) to the myofibrils and/or the depressed sensitivity of myofilaments to [Ca2+]i. However, the cellular basis of contractile dysfunction remains unestablished. We isolated left ventricular myocytes from dogs with rapid pacing-induced HF. Cell shortening and [Ca2+]i transients were measured by indo-1 fluorescence and the myofilament Ca2+ sensitivity was analyzed by the shortening-[Ca2+]i relation in intact myocytes as well as by the pCa tension relation in skinned cells. Peak cell shortening magnitude was depressed in HF, associated with a parallel decrease of [Ca2+]i transient amplitude. There was a significant positive correlation between these two variables (r = 0.71, P < 0.01). In contrast, myofibrillar sensitivity to Ca2+, determined by both intact and skinned myocytes, was comparable between control and HF. Further, there was no significant difference in Ca2+ sensitivity between control and HF even at shorter (1.8 microns) or longer (2.2 microns) sarcomere length. Using both intact and skinned cellular preparations, a potential defect in myocyte contractile function in HF was a reduction in Ca2+ availability to the myofilaments, rather than the inherent defects in myofilament sensitivity to Ca2+.

Relationship between pHi and ca 2+ sensitivity of myofilaments in rat ventricular myocytes

Relationship between pHi and ca 2+ sensitivity of myofilaments in rat ventricular myocytes

Positive lusitropic action of MCC-135, a new Ca 2+-handling modulator, is associated with modified Ca 2+-uptake by sarcoplasmic reticulum and Ca 2+ sensitivity of cardiac myofilaments

Positive lusitropic action of MCC-135, a new Ca 2+-handling modulator, is associated with modified Ca 2+-uptake by sarcoplasmic reticulum and Ca 2+ sensitivity of cardiac myofilaments

Dexamethasone inhibits endotoxin-induced changes in calcium and contractility in rat isolated papillary muscle

This study investigates whether endotoxin-induced contractile dysfunction is associated with a defect in the modulation of calcium homeostasis and the potential mechanisms involved. Treatment of rats in vivo with endotoxin significantly decreased the magnitude of contractile transients in electrically stimulated left ventricular papillary muscle isolated after an equilibration period of 6 hours. Although no significant difference was found in the peak intracellular calcium concentration ([Ca2+]i) between the endotoxin-treated and control groups, resting [Ca2+]iwas significantly elevated in the endotoxin-treated group, producing a smaller Ca2+transient (basal-peak difference) in this group. Pretreatment of rats with dexamethasone prevented the endotoxin-induced decrease in peak tension and inhibited the elevation in resting [Ca2+]i, with a resultant maintenance of Ca2+transient magnitude. Similar observations were made during stimulation of the muscles by the β-adrenoceptor agonist, isoprenaline. These results show that endotoxin-induced reduction of cardiac contractile performance is mediated, at least in part, by elevating resting [Ca2+]i, and a glucocorticoid protected from these negative effects. While endotoxin reduces the magnitude of the Ca2+transient it does not alter peak [Ca2+]iavailability. Further investigation is required to determine whether endotoxin decreases contractile performance by reducing the sensitivity of cardiac myofilaments to calcium.

Augmented acetylcholine‐induced, Rho‐mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen‐induced airway hyperresponsive rats

Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM. The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats. The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins. Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats. Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.

Pharmacomechanical coupling in rat vas deferens: Effects of agents that modulate intracellular release of calcium and protein kinase C activation

The effects of agents that modulate intracellular release of calcium and protein kinase C (PKC) activation on noradrenaline (NA)-induced contractions of epididymal vas deferens in calcium-free/EGTA (1 mM) medium were investigated. NA (100 μM) or methoxamine (100 μM) evoked repeatable contractions. Clonidine (100–300 μM) was ineffective. The contractions to NA were reduced by procaine (1–10 mM) but not by thapsigargin (0.1–30 μM), ryanodine (1–30 μM) or TMB-8 (1–30 μM). Contractions to cumulative additions of NA (1–100 μM) were enhanced in the presence of cyclopiazonic acid (10 & 30 μM) but not ryanodine (10 & 30 μM). Sequential contractions to NA were not blocked by PKC inhibitors, calphostin C (1 μM) or Ro 31-8220 (1–30 μM) but were reduced by H-7 (1–30 μM), a broad spectrum protein kinase inhibitor. Although RT-PCR experiments detected mRNA for some Ca 2+-dependent/DAG-activated and Ca 2+-independent/DAG-activated PKC isoforms in epididymal vas deferens, the PKC activators, phorbol 12, 13-dibutyrate (100 μM) or phorbol 12-myristate 13-acetate (100 μM) failed to activate the tissues in calcium-free medium but enhanced subsequent contractions to NA. These results indicate a limited role for intracellular calcium stores and phorbol ester/DAG-sensitive PKC isoforms in NA-induced contraction of epididymal rat vas deferens in calcium-free medium. The results suggest that pharmacomechanical coupling triggered by NA may involve the sensitization of contractile myofilaments to Ca 2+ or a Ca 2+-independent mechanism. The possible involvement of Ca 2+-independent/DAG-insensitive PKC isoforms and agonist-dependent but PKC-independent sensitization pathway is discussed.

Effects of levosimendan on cardiac arrhythmia: electrophysiologic and ambulatory electrocardiographic findings in phase II and phase III clinical studies in cardiac failure

Levosimendan is a novel inotropic and vasodilating agent that enhances the calcium sensitivity of myofilaments by binding to troponin C. Unlike other calcium sensitizers, it is highly dependent on the intracellular concentration of calcium. As a result of this unique property, levosimendan might be expected to increase cardiac contractile force without significantly impairing ventricular relaxation. The electrophysiologic effects of intravenously infused levosimendan were examined in healthy volunteers, and its effects on the surface electrocardiogram and on arrhythmia were recorded on 24-hour Holter recorders in patients with heart failure. Levosimendan had no significant effects on heart rate when data were pooled from the 24-hour electrocardiograms of patients receiving various dose levels, although increases were noted at high doses. The uncorrected QT interval remained unchanged, but the rate-corrected QT interval (QT c) was modestly prolonged at doses several-fold higher than that required for therapeutic effect. Atrial and ventricular effective refractory periods in patients with normal heart function were slightly shortened, although the average effect on the ventricles was only 2–5 msec at different pacing rates. No increase in the frequency of nonsustained ventricular tachycardia (VT) was found from the analysis of ambulatory electrocardiograph data from a total of 792 1-day recordings pooled from 10 studies, which included data from 386 heart failure patients. There was no evidence of any increase in the development of new supraventricular or ventricular tachyarrhythmias, including torsade de pointes, in patients who did not exhibit these abnormalities at baseline. The electrophysiologic actions of levosimendan in healthy volunteers and the effects of the drug on the QT interval of the electrocardiogram and on arrhythmia recorded during its intravenous use in patients with severe heart failure demonstrated little potential for the drug to provoke life-threatening proarrhythmic reactions.

Activation of Nitric Oxide Synthase (NOS3) by Mechanical Activity Alters Contractile Activity in a Ca2+-Independent Manner in Cardiac Myocytes: Role of Troponin I Phosphorylation

Cardiac myocytes express the calcium-responsive nitric oxide synthase (eNOS or NOS3). Activation of NOS3 by increased intracellular Ca2+concentration, [Ca2+]i, has been demonstrated to decrease myocyte contractile responsiveness, although this appears to occur in a Ca2+-independent manner. Therefore, the aim of this study was to examine the possibility that contractile activity could be modulated by an NO-mediated alteration in the phosphorylation status of troponin I, which is known to alter myofilament sensitivity to Ca2+. During pacing at 3 Hz,32P-labeled myocytes exhibited a 59 ± 9% increase in TnI phosphorylation compared to quiescent cells (p< 0.05), an effect that was significantly attenuated by either methylene blue orl-nitroarginine (l-NA). While exposure to methylene blue significantly increased the contractile amplitude of paced myocytes, this was not accompanied by an alteration in intracellular Ca2+. These data indicate that the NO-mediated effects on myocyte contraction may be elicited through an alteration in myofilament Ca2+sensitivity that results from an alteration in the phosphorylation status of troponin I.