Remote ischemic preconditioning-induced late cardioprotection: possible role of melatonin-mitoKATP-H2S signaling pathway

Purpose: Remote ischemic preconditioning (RIPC) confers cardioprotection against ischemia reperfusion (IR) injury. However, the precise mechanisms involved in RIPC-induced cardioprotection are not fully explored. The present study was aimed to identify the role of melatonin in RIPC-induced late cardioprotective effects in rats and to explore the role of H2 S, TNF-α and mitoKATP in melatoninmediated effects in RIPC. Methods: Wistar rats were subjected to RIPC in which hind limb was subjected to four alternate cycles of ischemia and reperfusion of 5 min duration by using a neonatal blood pressure cuff. After 24 h of RIPC or ramelteon-induced pharmacological preconditioning, hearts were isolated and subjected to IR injury on the Langendorff apparatus. Results: RIPC and ramelteon preconditioning protected the hearts from IR injury and it was assessed by a decrease in LDH-1, cTnT and increase in left ventricular developed pressure (LVDP). RIPC increased the melatonin levels (in plasma), H2 S (in heart) and decreased TNF-α levels. The effects of RIPC were abolished in the presence of melatonin receptor blocker (luzindole), ganglionic blocker (hexamethonium) and mitochondrial KATP blocker (5-hydroxydecanoic acid). Conclusion: RIPC produce delayed cardioprotection against IR injury through the activation of neuronal pathway, which may increase the plasma melatonin levels to activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels. Ramelteon-induced pharmacological preconditioning may also activate the cardioprotective signaling pathway involving the opening of mitochondrial KATP channels, decrease in TNF-α production and increase in H2 S levels.

Contribuições e limitações dos biomarcadores no diagnóstico e prognóstico da insuficiência cardíaca com fração de ejeção preservada / Contributions and limitations of biomarkers in the diagnosis of heart failure with preserved ejection fraction

A insuficiência cardíaca é uma síndrome clínica de prevalência crescente. Aproximadamente 30-50% dos pacientes com insuficiência cardíaca congestiva têm disfunção diastólica como causa de seus sintomas. No entanto, o diagnóstico de insuficiência cardíaca de fração de ejeção preservada não é tão simples. Neste contexto, cresce o interesse em encontrarmos biomarcadores que possam auxiliar no diagnóstico e na avaliação prognóstica da insuficiência cardíaca de fração de ejeção preservada. As evidências atuais são provenientes de estudos que avaliaram pacientes com disfunção sistólica e diastólica. No entanto, alguns biomarcadores parecem promissores na avaliação de pacientes com disfunção diastólica. Os peptídeos natriuréticos são os biomarcadores mais amplamente estudados. Níveis de peptídeo natriurético do tipo B e da fração N-terminal do peptídeo natriurético do tipo B são mais elevados entre pacientes com disfunção diastólica quando comparados a indivíduos saudáveis e parecem estar relacionados a maior mortalidade intra-hospitalar e maior risco de evento combinado de morte e re-hospitalização. Assim como entre pacientes com disfunção sistólica, pacientes com insuficiência cardíaca de fração de ejeção preservada com troponina elevada também parecem apresentar maior risco de morte e re-hospitalização. Mais recentemente, tem sido descritos biomarcadores de fibrose como galectina-3 e ST2 solúvel e dados da literatura sugerem que quando elevados podem refletir pior prognóstico. De forma geral, biomarcadores que identificam injúria miocárdica; alterações no turnover celular e marcadores de fibrose parecem promissores por refletirem mecanismos fisiopatológicos da insuficiência cardíaca de fração de ejeção preservada. Assim, estudos prospectivos envolvendo especificamente esta população ainda são necessários para que o real papel destes biomarcadores seja estabelecido.

Heart Failure is a disease whose prevalence has been increasing in the last years. About 30-50% of patients with congestive heart failure have heart failure with preserved ejection fraction (HF-pEF) as the cause of their symptoms. However, the diagnosis of HF-pEF is not so easy to be established. In this context, multiple biomarkers of heart failure have emerged recently and have been used to help in the diagnosis and prognosis of HF-pEF. Current evidence about biomarkers in heart failure comes from trials that involved patients with systolic and diastolic dysfunction. There are few studies involving exclusively patients with HF-pEF. Nevertheless, some biomarkers seem to be promising in patients with HF-pEF. Natriuretic peptides are the most common biomarkers of heart failure, B-type natriuretic peptide and N-terminal B-type natriuretic peptide are higher in patient with diastolic dysfunction when compared to healthy people and seem to be related with higher in-hospital mortality and higher risk of death and re-hospitalization. Patients with HF-pEF, who have high levels of troponin, have also higher risk of death and re-hospitalization. Recently, new biomarkers of fibrosis as 3-galectin and soluble ST2 have been described and data suggest that high levels of these biomarkers should reflect worse prognosis. In summary, biomarkers of myocardial injury; cellular turnover changes and fibrosis seem to be promising biomarkers of the heart failure with preserved ejection fraction since they could reflect their physiopathological mechanisms. So, we believe that prospective thats involving patients with heart failure with preserved ejection fraction are still necessary to establish the importance of these biomarkers.

The effects of phosphate and acidosis on regulated thin-filament velocity in an in vitro motility assay.

Muscle fatigue from intense contractile activity is thought to result, in large part, from the accumulation of inorganic phosphate (P(i)) and hydrogen ions (H(+)) acting to directly inhibit the function of the contractile proteins; however, the molecular basis of this process remain unclear. We used an in vitro motility assay and determined the effects of elevated H(+) and P(i) on the ability of myosin to bind to and translocate regulated actin filaments (RTF) to gain novel insights into the molecular basis of fatigue. At saturating Ca(++), acidosis depressed regulated filament velocity (V(RTF)) by ≈ 90% (6.2 ± 0.3 vs. 0.5 ± 0.2 µm/s at pH 7.4 and 6.5, respectively). However, the addition of 30 mM P(i) caused V(RTF) to increase fivefold, from 0.5 ± 0.2 to 2.6 ± 0.3 µm/s at pH 6.5. Similarly, at all subsaturating Ca(++) levels, acidosis slowed V(RTF), but the addition of P(i) significantly attenuated this effect. We also manipulated the [ADP] in addition to the [P(i)] to probe which specific step(s) of cross-bridge cycle of myosin is affected by elevated H(+). The findings are consistent with acidosis slowing the isomerization step between two actomyosin ADP-bound states. Because the state before this isomerization is most vulnerable to P(i) rebinding, and the associated detachment from actin, this finding may also explain the P(i)-induced enhancement of V(RTF) at low pH. These results therefore may provide a molecular basis for a significant portion of the loss of shortening velocity and possibly muscular power during fatigue.

Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases.

Limited neural input results in muscle weakness in neuromuscular disease because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission. We developed a small-molecule fast-skeletal-troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neural input is otherwise diminished secondary to neuromuscular disease. Binding selectively to the fast-skeletal-troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards, as does the force-frequency relationship of a nerve-muscle pair, so that CK-2017357 increases the production of muscle force in situ at sub-maximal nerve stimulation rates. Notably, we show that sensitization of the fast-skeletal-troponin complex to calcium improves muscle force and grip strength immediately after administration of single doses of CK-2017357 in a model of the neuromuscular disease myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.

Acrylodan-labeled smooth muscle tropomyosin reports differences in the effects of troponin and caldesmon in the transition from the active state to the inactive state.

Changes in the orientation of tropomyosin on actin are important for the regulation of striated muscle contraction and could also be important for smooth muscle regulation. We showed earlier that acrylodan-labeled skeletal muscle tropomyosin reports the kinetics of the reversible transitions among the active, intermediate, and inactive states when S1 is rapidly detached from actin-tropomyosin. We now show that acrylodan-labeled smooth muscle tropomyosin reports similar transitions among states of actin-tropomyosin. When S1 was rapidly detached from actin-smooth muscle tropomyosin, there was a rapid decrease in acrylodan-tropomyosin fluorescence as the intermediate state became populated. The rate constant for this process was >600 s(-1) at temperatures near 5 °C. In the presence of skeletal troponin and EGTA, the decrease in fluorescence was followed by the redevelopment of fluorescence as the inactive state became populated. The apparent rate constant for the fluorescence increase was 14 s(-1) at 5 °C. Substituting smooth muscle caldesmon for skeletal muscle troponin produced a similar decrease and re-increase in fluorescence, but the apparent rate constant for the increase was >10 times that observed with troponin. Furthermore, the fluorescence increase was correlated with an increase in the extent of caldesmon attachment as S1-ATP dissociated. Although the measured rate constant appeared to reflect the rate-limiting transition for inactivation, it is unclear if the fluorescence change resulted from caldesmon binding, the movement of tropomyosin over actin, or both.

Troponin in newborns and pediatric patients.

Cardiac troponin represents a sensitive and specific marker of ischemic myocardial damage in adult and neonatal populations. Cardiac function in neonates could be influenced by the severity of respiratory distress and its ventilatory management. This short review summarizes the experimental and clinical evidence regarding the role of cardiac troponin in assessment of cardiac function, in following findings: neonatal intensive care, respiratory distress syndrome, asphyxia, congenital heart disease and post cardiac surgery.

Insights into the kinetics of Ca2+-regulated contraction and relaxation from myofibril studies.

Muscle contraction results from force-generating interactions between myosin cross-bridges on the thick filament and actin on the thin filament. The force-generating interactions are regulated by Ca(2+) via specialised proteins of the thin filament. It is controversial how the contractile and regulatory systems dynamically interact to determine the time course of muscle contraction and relaxation. Whereas kinetics of Ca(2+)-induced thin-filament regulation is often investigated with isolated proteins, force kinetics is usually studied in muscle fibres. The gap between studies on isolated proteins and structured fibres is now bridged by recent techniques that analyse the chemical and mechanical kinetics of small components of a muscle fibre, subcellular myofibrils isolated from skeletal and cardiac muscle. Formed of serially arranged repeating units called sarcomeres, myofibrils have a complete fully structured ensemble of contractile and Ca(2+) regulatory proteins. The small diameter of myofibrils (few micrometres) facilitates analysis of the kinetics of sarcomere contraction and relaxation induced by rapid changes of [ATP] or [Ca(2+)]. Among the processes studied on myofibrils are: (1) the Ca(2+)-regulated switch on/off of the troponin complex, (2) the chemical steps in the cross-bridge adenosine triphosphatase cycle, (3) the mechanics of force generation and (4) the length dynamics of individual sarcomeres. These studies give new insights into the kinetics of thin-filament regulation and of cross-bridge turnover, how cross-bridges transform chemical energy into mechanical work, and suggest that the cross-bridge ensembles of each half-sarcomere cooperate with each other across the half-sarcomere borders. Additionally, we now have a better understanding of muscle relaxation and its impairment in certain muscle diseases.

Caldesmon inhibits both force development and transition of actin monomers from "OFF" to "ON" conformational state by changing its position in thin filaments.

We have investigated the effect of caldesmon on the actin conformational state and its position at force generation in glycerinated fibers upon transformation from relaxation to rigor. F-actin and caldesmon were labeled with TRITC-phalloidin or acrylodan, respectively, and the orientation and mobility of the probes were calculated. Transition from relaxation to rigor was accompanied by force development and by the changes in orientation and mobility of TRITC-phalloidin that were typical for actin monomer transformation from the "OFF" to the "ON" conformational state. In the presence of caldesmon, both the force developed by the fibers and the changes in the orientation and mobility of TRITC-phalloidin were markedly decreased. In contrast, the orientation and mobility of acrylodan change essentially showed the displacement of the caldesmon molecules and the changes in its mobility. The results are evidence that structure and/or mode of the attachment of caldesmon to actin modulates both the force production and transition of actin monomers from "OFF" to "ON" conformations in the ATPase cycle.

Use of thin filament reconstituted muscle fibres to probe the mechanism of force generation.

The technique of selective removal of the thin filament by gelsolin in bovine cardiac muscle fibres, and reconstitution of the thin filament from isolated proteins is reviewed, and papers that used reconstituted preparations are discussed. By comparing the results obtained in the absence/presence of regulatory proteins tropomyosin (Tm) and troponin (Tn), it is concluded that the role of Tm and Tn in force generation is not only to expose the binding site of actin to myosin, but also to modify actin for better stereospecific and hydrophobic interaction with myosin. This conclusion is further supported by experiments that used a truncated Tm mutant and the temperature study of reconstituted fibres. The conclusion is consistent with the hypothesis that there are three states in the thin filament: blocked state, closed state, and open state. Tm is the major player to produce these effects, with Tn playing the role of Ca2+ sensing and signal transmission mechanism. Experiments that changed the number of negative charges at the N-terminal finger of actin demonstrates that this part of actin is essential to promote the strong interaction between actin and myosin molecules, in addition to the well-known weak interaction that positions the myosin head at the active site of actin prior to force generation.

Single-myosin crossbridge interactions with actin filaments regulated by troponin-tropomyosin.

Striated muscle contraction is governed by the thin filament regulatory proteins troponin and tropomyosin. Here, we investigate the molecular mechanisms by which troponin-tropomyosin inhibits myosin's interactions with the thin filament in the absence of calcium by using a laser trap. The displacement events for a single-myosin molecule interacting with a reconstituted thin filament were shorter (step size = 5 nm) and prolonged (69 ms) compared with actin alone (11 nm and 26 ms, respectively). However, these changes alone do not account for the degree of inhibition of thin filament movement observed in an ensemble assay. Our investigations of single- and multiple-myosin molecules with regulated thin filaments suggest the primary basis for this inhibition derives from an approximately 100-fold decrease in the probability of myosin attaching to actin. At higher myosin concentrations, short bursts of motility are observed in a laser trap consistent with the strong binding of a single-myosin crossbridge, resulting in cooperative binding of other cycling crossbridges. We confirmed this cooperativity in the in vitro motility assay by observing thin filament translocation in the absence of calcium but at low [ATP], consistent with rigor activation. We have developed a simple mechanistic model that reproduces and provides insight into both the observed single-myosin molecule and ensemble data in the absence of Ca(2+). These data support the hypothesis that thin filament inhibition in the absence of Ca(2+) is largely achieved by modulating the rate of attachment and/or transition from the weakly to strongly bound state.

Mini-thin filaments regulated by troponin-tropomyosin.

Striated muscle thin filaments contain hundreds of actin monomers and scores of troponins and tropomyosins. To study the cooperative mechanism of thin filaments, "mini-thin filaments" were generated by isolating particles nearly matching the minimal structural repeat of thin filaments: a double helix of actin subunits with each strand approximately seven actins long and spanned by a troponin-tropomyosin complex. One end of the particles was capped by a gelsolin (segment 1-3)-TnT fusion protein (substituting for normal TnT), and the other end was capped by tropomodulin. EM showed that the particles were 46 +/- 9 nm long, with a knob-like mass attributable to gelsolin at one end. Average actin, tropomyosin, and gelsolin-troponin composition indicated one troponin-tropomyosin attached to each strand of the two-stranded actin filament. The minifilaments thus nearly represent single regulatory units of thin filaments. The myosin S1 MgATPase rate stimulated by the minifilaments was Ca2+-sensitive, indicating that single regulatory length particles are sufficient for regulation. Ca2+ bound cooperatively to cardiac TnC in conventional thin filaments but noncooperatively to cardiac TnC in minifilaments in the absence of myosin. This suggests that thin filament Ca2+-binding cooperativity reflects indirect troponin-troponin interactions along the long axis of conventional filaments, which do not occur in minifilaments. Despite noncooperative Ca2+ binding to minifilaments in the absence of myosin, Ca2+ cooperatively activated the myosin S1-particle ATPase rate. Two-stranded single regulatory units therefore may be sufficient for myosin-mediated Ca2+-binding cooperativity. Functional mini-thin filaments are well suited for biochemical and structural analysis of thin-filament regulation.

Hypergravity from conception to adult stage: effects on contractile properties and skeletal muscle phenotype.

This study examined the effects of an elevation of the gravity factor (hypergravity--2 g) on the molecular and functional characteristics of rat soleus and plantaris muscles. Long Evans rats were conceived, born and reared (CBR) continuously in hypergravity conditions until the age of 100 days. Whole muscle morphological parameters, Ca2+ activation characteristics from single skinned fibers, troponin (Tn) subunit and myosin heavy (MHC) and light (MLC) chains isoform compositions were examined in CBR and control muscles from age-paired terrestrial rats. Decreases in body and muscle mass in soleus and plantaris muscles were observed and associated, in the soleus, with a decrease in fiber diameter. The specific force of CBR soleus fibers was increased, and correlated with the elevation of Ca2+ affinity. This was accompanied by slow-to-slower TnC and TnI isoform transitions and a rearrangement in TnT fast isoform content. The MHC transformations of the soleus after hypergravity were associated with the up (down)-regulation of the MHCI (MHCIIa) mRNA isoforms. The MLC2 phosphorylation state remained unchanged in the soleus muscle. The results suggested that the gravity factor could interact with rat muscle development and that hypergravity experiments could provide good tools for the study of myofibrillar protein plasticity and their associated pathways of regulation.

Conformational switching in muscle.

Muscles can be studied as complex systems of many interacting proteins and investigated at many different levels of organization. This talk will describe how we modeled the mechanism of Ca activation, the structure of the muscle proteins, and protein complexes (including actin monomers, tropomyosin and troponin complexes, and myosin) to examine two different scientific problems: the mechano-chemical energy transduction mechanism, and the control system of that mechanism. The methods we used--saturation transfer electron paramagnetic resonance, phosphorescence anisotropy, and fluorescence resonance energy transfer--reveal two specific structures: a hinge between the motor and regulatory domains, and a stiff regulatory domain. This indicates that the structure of the myosin head is capable of generating translating conformational changes within the motor domain to the swing of the regulatory domain, and that the regulatory domain is rigid enough to act as a lever arm.

Markers to define ischemia: are they ready for prime time use in patients with acute coronary syndromes?

Optimal treatment of patients who present with chest pain is predicated on accurate identification of those patients with a cardiac etiology of their discomfort. Serial troponins and electrocardiograms are very sensitive for the detection of myocardial infarction but they are insensitive for the detection of ischemia. There are many analytes that are being actively evaluated for routine use to facilitate the identification of patients with myocardial ischemia. At present, only one assay is US Food and Drug Administration-approved for the exclusion of ischemia; many other analytes are under clinical evaluation and are briefly reviewed. At present, none of these analytes are yet appropriate for routine clinical use.

Cardiomiopatia hipertrófica / Hypertrophic cardiomyopathy

A cardiomiopatia hipertrófica é doença genética autossômica dominante em mais da metade dos casos. Foram descritas, até o momento, mais de 270 mutações em dez genes que codificam proteínas do sarcômero cardíaco. Para cada gene existem diversas mutações, cada qual com particularidades quanto a hipertrofia miocárdica, penetrância e prognóstico, principalmente em relação a morte súbita. Há evidência de que outro fatores genéticos têm papel na hipertrofia da cardiomiopatia hpertrófica, como o polimorfismo no gene da enzima conversora da angiotensina e em outros genes modificadores, ambos podendo influenciar o grau de hipertrofia e, eventualmente, a ocorrência de morte súbita. Neste artigo são descritas as mutações relatadas na literatura, assim como nossa experiência no Instituto do Coração, que é a primeira no Brasil nessa moléstia

Covalent and noncovalent modification of thin filament action: the essential role of troponin in cardiac muscle regulation.

Troponin is essential for the regulation of cardiac contraction. Troponin is a sarcomeric molecular switch, directly regulating the contractile event in concert with intracellular calcium signals. Troponin isoform switching, missense mutations, proteolytic cleavage, and posttranslational modifications are known to directly affect sarcomeric regulation. This review focuses on physiologically relevant covalent and noncovalent modifications in troponin as part of a thematic series on cardiac thin filament function in health and disease.

The effect of tropomyosin on force and elementary steps of the cross-bridge cycle in reconstituted bovine myocardium.

The role of tropomyosin (Tm) in the elementary steps of the cross-bridge cycle in bovine myocardium was investigated. The thin filament was selectively removed using gelsolin (thin filament severing protein), and the actin filament was reconstituted from G-actin. Tm was further reconstituted without troponin (Tn), and the kinetic constants of the elementary steps of the cross-bridge cycle were deduced using sinusoidal analysis at pCa

Skeletal regulatory proteins enhance thin filament sliding speed and force by skeletal HMM.

At saturating calcium and nucleotide concentrations, troponin (Tn) and tropomyosin (Tm) enhance the in vitro motility speed of individual actin filaments, suggesting the roles of these thin filament proteins in regulating contraction may include a modulation of crossbridge kinetics. Using a homogeneous complement of fast rabbit skeletal proteins, we examined if Tn and Tm modify specific transitions in the crossbridge cycle by varying skeletal muscle crossbridge kinetics and measuring actin filament sliding speed and steady-state force using the in vitro motility and microneedle assays, respectively. Skeletal regulatory proteins increased the force and sliding speed of actin filaments sliding on skeletal HMM. Faster crossbridge cycling with increased temperature or with substitution of dATP as the contractile substrate resulted in both increased sliding speed and force of unregulated filaments, while the addition of regulatory proteins diminished or eliminated this increase. In contrast, regulatory proteins did not influence filament mechanics when crossbridge cycling was slowed with lowered ATP concentration. The results are most simply explained if addition of the Tn and Tm complex to actin enhances both the transition rate of the force-generating actomyosin isomerization (or the preceding transition) and the apparent crossbridge detachment rate, but that the relative influence of Tn and Tm is dependent on the external load.

Cooperative regulation of myosin-actin interactions by a continuous flexible chain II: actin-tropomyosin-troponin and regulation by calcium.

The model of myosin regulation by a continuous tropomyosin chain is generalized to a chain of tropomyosin-troponin units. Myosin binding to regulated actin is cooperative and initially inhibited by the chain as before. In the absence of calcium, myosin is further inhibited by the binding of troponin-I to actin, which through the whole of troponin pins the tropomyosin chain in a blocking position; myosin and TnI compete for actin and induce oppositely-directed chain kinks. The model predicts equilibrium binding curves for myosin-S1 and TnI as a function of their first-order affinities K(S1) and L(TI). Myosin is detached by the actin binding of TnI, but TnI is more efficiently detached by myosin when the kink size (typically nine to ten actin sites) spans the seven-site spacing between adjacent TnI molecules. An allosteric mechanism is used for coupling the detachment of TnI to calcium binding by TnC. With thermally activated TnI kinks (kink energy B approximately k(B)T), TnI also binds cooperatively to actin, producing cooperative detachment of myosin and biphasic myosin-calcium Hill plots, with Hill coefficients of 2 at high calcium and 4-6 at low calcium as observed in striated muscle. The theory also predicts the cooperative effects observed in the calcium loading of TnC.