50 Years of the steric-blocking mechanism in vertebrate skeletal muscle: a retrospective.

Fifty years have now passed since Parry and Squire proposed a detailed structural model that explained how tropomyosin, mediated by troponin, played a steric-blocking role in the regulation of vertebrate skeletal muscle. In this Special Issue dedicated to the memory of John Squire it is an opportune time to look back on this research and to appreciate John's key contributions. A review is also presented of a selection of the developments and insights into muscle regulation that have occurred in the years since this proposal was formulated.

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.

Myofilaments: Movers and Rulers of the Sarcomere.

Striated cardiac and skeletal muscles play very different roles in the body, but they are similar at the molecular level. In particular, contraction, regardless of the type of muscle, is a precise and complex process involving the integral protein myofilaments and their associated regulatory components. The smallest functional unit of muscle contraction is the sarcomere. Within the sarcomere can be found a sophisticated ensemble of proteins associated with the thick filaments (myosin, myosin binding protein-C, titin, and obscurin) and thin myofilaments (actin, troponin, tropomyosin, nebulin, and nebulette). These parallel thick and thin filaments slide across one another, pulling the two ends of the sarcomere together to regulate contraction. More specifically, the regulation of both timing and force of contraction is accomplished through an intricate network of intra- and interfilament interactions belonging to each myofilament. This review introduces the sarcomere proteins involved in striated muscle contraction and places greater emphasis on the more recently identified and less well-characterized myofilaments: cardiac myosin binding protein-C, titin, nebulin, and obscurin. © 2017 American Physiological Society. Compr Physiol 7:675-692, 2017.

Cardiac troponin structure-function and the influence of hypertrophic cardiomyopathy associated mutations on modulation of contractility.

Cardiac troponin (cTn) acts as a pivotal regulator of muscle contraction and relaxation and is composed of three distinct subunits (cTnC: a highly conserved Ca(2+) binding subunit, cTnI: an actomyosin ATPase inhibitory subunit, and cTnT: a tropomyosin binding subunit). In this mini-review, we briefly summarize the structure-function relationship of cTn and its subunits, its modulation by PKA-mediated phosphorylation of cTnI, and what is known about how these properties are altered by hypertrophic cardiomyopathy (HCM) associated mutations of cTnI. This includes recent work using computational modeling approaches to understand the atomic-based structural level basis of disease-associated mutations. We propose a viewpoint that it is alteration of cTnC-cTnI interaction (rather than the Ca(2+) binding properties of cTn) per se that disrupt the ability of PKA-mediated phosphorylation at cTnI Ser-23/24 to alter contraction and relaxation in at least some HCM-associated mutations. The combination of state of the art biophysical approaches can provide new insight on the structure-function mechanisms of contractile dysfunction resulting cTnI mutations and exciting new avenues for the diagnosis, prevention, and even treatment of heart diseases.

[High-sensitivity troponin in patients with stable coronary artery disease]. / Hoogsensitief troponine bij stabiel coronairlijden.

With the introduction of more sensitive markers of myocardial necrosis, it was shown that the diagnosis of (acute) myocardial infarction could be improved. However, many patients without myocardial infarction have elevated troponin levels (low sensitivity), resulting in additional diagnostic testing and longer admission times to find the cause of the troponin rise. Elevated troponin levels in these patients were found to have limited diagnostic but strong prognostic value. This led to studies assessing the value of high-sensitivity troponin in patients with stable disease, e.g. coronary artery disease, or even in patients without apparent (clinical) disease. These studies consistently showed a strong association with long-term (cardiac) mortality. What is the value for the general physician in daily practice? Although troponin levels may help in finding (healthy?) patients at higher risk, it remains unclear whether interventions (medication, lifestyle) are more (cost-)effective in this high-risk subgroup. The role of troponin levels as a prognostic marker in stable patients therefore remains to be determined.

Roles of the troponin isoforms during indirect flight muscle development in Drosophila.

Troponin proteins in cooperative interaction with tropomyosin are responsible for controlling the contraction of the striated muscles in response to changes in the intracellular calcium concentration. Contractility of the muscle is determined by the constituent protein isoforms, and the isoforms can switch over from one form to another depending on physiological demands and pathological conditions. In Drosophila, amajority of themyofibrillar proteins in the indirect flight muscles (IFMs) undergo post-transcriptional and post-translational isoform changes during pupal to adult metamorphosis to meet the high energy and mechanical demands of flight. Using a newly generated Gal4 strain (UH3-Gal4) which is expressed exclusively in the IFMs, during later stages of development, we have looked at the developmental and functional importance of each of the troponin subunits (troponin-I, troponin-T and troponin-C) and their isoforms. We show that all the troponin subunits are required for normal myofibril assembly and flight, except for the troponin-C isoform 1 (TnC1). Moreover, rescue experiments conducted with troponin-I embryonic isoform in the IFMs, where flies were rendered flightless, show developmental and functional differences of TnI isoforms and importance of maintaining the right isoform.

Cardiac troponins: bench to bedside interpretation in cardiac disease.

Cardiac troponins are the preferred biomarkers for the determination of acute myocardial necrosis. The high sensitivity of the available assays has significantly increased the detection of microscopic amounts of myocardial damage. Although compelling evidence indicates that elevated cardiac troponins are markers of poor prognosis and increased mortality, irrespective of the clinical scenario, small elevations can be seen in protean conditions and may confound the diagnosis of acute coronary syndromes. Emerging evidence suggests multiple different cellular mechanisms leading to cardiac troponin release, which challenge long held paradigms such as equivalency between troponin release into the circulation and irreversible cell death. Hence, knowledge of the physiology and pathophysiology of these cardiac biomarkers is essential for their accurate interpretation and consequent correct clinical diagnosis. Herein, the current relevant information about cardiac troponins is discussed, with special emphasis on pathophysiology and clinical correlates.

Nuclear tropomyosin and troponin in striated muscle: new roles in a new locale?

Tropomyosin and troponin have well known Ca(2+)-regulatory functions in the striated muscle sarcomere. In this review, we summarize experimental evidence that tropomyosin and troponin are localized, with as yet unidentified functional roles, in the striated muscle cell nucleus. We also apply bioinformatics approaches that predict localization of some tropomyosin and troponin to the nucleus, and that SUMOylation could be a covalent modification that modulates their nuclear localization and function. Further, we provide examples of cardiomyopathy mutations that alter the predicted likelihood of nuclear localization and SUMOylation of tropomyosin. These observations suggest novel mechanisms by which cardiomyopathy mutations in tropomyosin and troponin might alter not only cardiac contractility but also nuclear function.

High sensitivity troponin in cardiovascular disease. Is there more than a marker of myocardial death?

Cardiovascular disease is the leading cause of death worldwide and coronary artery disease is its most prevalent manifestation, associated with high mortality and morbidity. In clinical practice cardiac troponins (cTn) are the cornerstone of the diagnosis, risk stratification and thus selection of the optimal treatment strategy in patients with acute coronary syndrome. According to the third update of the universal definition of myocardial infarction (MI) cTn is the preferred cardiac biomarker of myocardial necrosis in the setting of acute myocardial ischemia. Over the last years newer high sensitivity cardiac troponin (hs-cTn) assays have been developed that are more sensitive than conventional assays, have low limit of detection, low imprecision and low reference limits, but due to variability, the deployment of a standardization and harmonization method is required before their wide use in clinical practice. Recent studies have shown that their utilization seems to improve the diagnostic accuracy detecting MI in patients presenting with chest pain. However, the improved sensitivity comes along with a decreased specificity, though serial cTn measurements and the detection of early changes could improve the specificity and the overall diagnostic performance. Moreover, apart from their use in the diagnosis and risk stratification of MI and acute coronary syndromes, hs-cTn assays seem to have a key role in risk stratification and short and long-term prognosis in a variety of cardiovascular modalities such as stable coronary disease, heart failure and acute pulmonary embolism. In addition, studies have suggested that cTns may be used as a biomarker in the primary prevention of cardiovascular disease leading to the identification of high-risk populations or individuals with silent heart disease.

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.

Oxidative stress and cardiomyocyte necrosis with elevated serum troponins: pathophysiologic mechanisms.

The progressive nature of heart failure is linked to multiple factors, including an ongoing loss of cardiomyocytes and necrosis. Necrotic cardiomyocytes leave behind several footprints: the spillage of their contents leading to elevations in serum troponins; and morphologic evidence of tissue repair with scarring. The pathophysiologic origins of cardiomyocyte necrosis relates to neurohormonal activation, including the adrenergic nervous system. Catecholamine-initiated excessive intracellular Ca accumulation and mitochondria Ca overloading in particular initiate a mitochondriocentric signal-transducer-effector pathway to necrosis and which includes the induction of oxidative stress and opening of their inner membrane permeability transition pore. Hypokalemia, ionized hypocalcemia and hypomagnesemia, where consequent elevations in parathyroid hormone further account for excessive intracellular Ca accumulation, hypozincemia and hyposelenemia each compromise metalloenzyme-based antioxidant defenses. The necrotic loss of cardiomyocytes and adverse structural remodeling of myocardium is related to the central role played by a mitochondriocentric pathway initiated by neurohormonal activation.

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.

New sensitive cardiac troponin assays for the early diagnosis of myocardial infarction.

The objective of the current article is to review and evaluate the diagnostic and prognostic value of a new generation of sensitive assays for cardiac troponin I and troponin T. Cardiac-specific troponins I and T are the preferred diagnostic biomarker in patients presenting with suspected acute coronary syndromes. One important limitation of previous generation assays has been the relative insensitivity in detecting myocardial injury in patients with a short duration from symptom onset to presentation in the emergency room. Recently, sensitive assays for cardiac troponins I and T have been introduced as research tools and in clinical practice. Clinical trials evaluating these assays have demonstrated that sensitivity and overall diagnostic accuracy for acute myocardial infarction, defined by the 99th percentile cardiac troponin concentration in a healthy population, is enhanced, although at the cost of reduced specificity. Not surprisingly, the relative benefit compared to previous generation assays is greatest for those patients presenting early after symptom onset. A number of cardiac conditions other than acute coronary syndromes, as well as several noncardiac conditions, are associated with elevation of circulating cardiac troponins. Thus, with the use of more sensitive assays, clinical context and serial testing to document a rise and/or fall in concentrations will be increasingly important for correct interpretation of troponin results.

Extraction and replacement of the tropomyosin-troponin complex in isolated myofibrils.

Tropomyosin (Tm) is an essential component in the regulation of striated muscle contraction. Questions about Tm functional role have been difficult to study because sarcomere Tm content is not as easily manipulated as Troponin (Tn). Here we describe the method we recently developed to replace Tm-Tn of skeletal and cardiac myofibrils from animals and humans to generate an experimental model of homogeneous Tm composition and giving the possibility to measure a wide range of mechanical parameters of contraction (e.g. maximal force and kinetics of force generation). The success of the exchange was determined by SDS-PAGE and by mechanical measurements of calcium dependent force activation on the reconstituted myofibrils. In skeletal and cardiac myofibrils, the percentage of Tm replacement was higher than 90%. Maximal isometric tension was 30-35% lower in the reconstituted myofibrils than in control myofibrils but the rate of force activation (k(ACT)) and that of force redevelopment (k(TR)) were not significantly changed. Preliminary results show the effectiveness of Tm replacement in human cardiac myofibrils. This approach can be used to test the functional impact of Tm mutations responsible for human cardiomyopathies.

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.