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The physiological acclimatisation and adaptation processes in skeletal muscle at high altitude are of high medical and social relevance not only to understand limitations in physical performance at high altitude but also to understand the consequences of hypoxemia and tissue hypoxia in critically ill patients. Of particular importance in these processes are the alterations in content and function of mitochondria and myoglobin. The majority of studies on oxygen delivery to the tissues and utilisation by the cellular metabolism at high altitude were performed after prolonged stay at high altitude and in altitude-adapted highlanders. However, these studies do not provide insight in the sequence of events during the physiological acclimatisation and adaptation processes. Therefore, it is important to identify the early alterations in structure and function of the major determinants of the oxygen transport via myoglobin and oxygen utilisation by the mitochondria in skeletal muscle at high altitude. To achieve this goal, it is of interest to collect, analyse and compare quadriceps muscle biopsies and venous blood samples of climbers, guides and porters before and after climbing Mount Kilimanjaro and in participants of the Kilimanjaro Marathon before and after the run. The samples will be carefully documented and stored in the Kilimanjaro Biobank and will be made available to other research groups.

The sarcomeric proteins represent the structural building blocks of heart muscle, which are essential for contraction and relaxation. During recent years, it has become evident that posttranslational modifications of sarcomeric proteins, in particular phosphorylation, tune cardiac pump function at rest and during exercise. This delicate, orchestrated interaction is also influenced by mutations, predominantly in sarcomeric proteins, which cause hypertrophic or dilated cardiomyopathy. In this review, we follow a bottom-up approach starting from a description of the basic components of cardiac muscle at the molecular level up to the various forms of cardiac disorders at the organ level. An overview is given of sarcomere changes in acquired and inherited forms of cardiac disease and the underlying disease mechanisms with particular reference to human tissue. A distinction will be made between the primary defect and maladaptive/adaptive secondary changes. Techniques used to unravel functional consequences of disease-induced protein changes are described, and an overview of current and future treatments targeted at sarcomeric proteins is given. The current evidence presented suggests that sarcomeres not only form the basis of cardiac muscle function but also represent a therapeutic target to combat cardiac disease.

AIM: Calcium ions play a pivotal role in matching energy supply and demand in cardiac muscle. Mitochondrial calcium concentration is lower in animal models of heart failure with reduced ejection fraction (HFrEF), but limited information is available about mitochondrial calcium handling in heart failure with preserved ejection fraction (HFpEF).METHODS: We assessed mitochondrial Ca2+ handling in intact cardiomyocytes from Zucker/fatty Spontaneously hypertensive F1 hybrid (ZSF1)-lean (control) and ZSF1-obese rats, a metabolic risk-related model of HFpEF. A mitochondrially targeted Ca2+ indicator (MitoCam) was expressed in cultured adult rat cardiomyocytes. Cytosolic and mitochondrial Ca2+ transients were measured at different stimulation frequencies. Mitochondrial respiration and swelling, and expression of key proteins were determined ex vivo.RESULTS: At rest, mitochondrial Ca2+ concentration in ZSF1-obese was larger than in ZSF1-lean. The diastolic and systolic mitochondrial Ca2+ concentrations increased with stimulation frequency, but the steady-state levels were larger in ZSF1-obese. The half-widths of the contractile responses, the resting cytosolic Ca2+ concentration and the decay half-times of the cytosolic Ca2+ transients were higher in ZSF1-obese, likely because of a lower SERCA2a/phospholamban ratio. Mitochondrial respiration was lower, particularly with nicotinamide adenine dinucleotide (NADH) (complex I) substrates, and mitochondrial swelling was larger in ZSF1-obese.CONCLUSION: The free mitochondrial calcium concentration is higher in HFpEF owing to alterations in mitochondrial and cytosolic Ca2+ handling. This coupling between cytosolic and mitochondrial Ca2+ levels may compensate for myocardial ATP supply in vivo under conditions of mild mitochondrial dysfunction. However, if mitochondrial Ca2+ concentration is sustainedly increased, it might trigger mitochondrial permeability transition pore opening.

Key points Fast sarcomere-level mechanics in intact trabeculae, which allows the definition of the mechano-kinetic properties of cardiac myosin in situ, is a fundamental tool not only for understanding the molecular mechanisms of heart performance and regulation, but also for investigating the mechanisms of the cardiomyopathy-causing mutations in the myosin and testing small molecules for therapeutic interventions. The approach has been applied to measure the stiffness and force of the myosin motor and the fraction of motors attached during isometric twitches of electrically paced trabeculae under different extracellular Ca2+ concentrations. Although the average force of the cardiac myosin motor (∼6 pN) is similar to that of the fast myosin isoform of skeletal muscle, the stiffness (1.07 pN nm-1 ) is 2- to 3-fold smaller. The increase in the twitch force developed in the presence of larger extracellular Ca2+ concentrations is fully accounted for by a proportional increase in the number of attached motors. Abstract The mechano-kinetic properties of the cardiac myosin were studied in situ, in trabeculae dissected from the right ventricle of the rat heart, by measuring the stiffness of the half-sarcomere both at the twitch force peak (Tp ) of an electrically paced intact trabecula at different extracellular Ca2+ concentrations ([Ca2+ ]o ), and in the same trabecula after skinning and induction of rigor. Taking into account the contribution of filament compliance to half-sarcomere compliance and the lattice geometry, we found that the stiffness of the cardiac myosin motor is 1.07 ± 0.09 pN nm-1 , which is slightly larger than that of the slow myosin isoform of skeletal muscle (0.6-0.8 pN nm-1 ) and 2- to 3-fold smaller than that of the fast skeletal muscle isoform. The increase in Tp from 61 ± 4 kPa to 93 ± 9 kPa, induced by raising [Ca2+ ]o from 1 to 2.5 mm at sarcomere length ∼2.2 μm, is accompanied by an increase of the half-sarcomere stiffness that is explained by an increase of the fraction of actin-attached motors from 0.08 ± 0.01 to 0.12 ± 0.02, proportional to Tp . Consequently, each myosin motor bears an average force of 6.14 ± 0.52 pN independently of Tp and [Ca2+ ]o . The application of fast sarcomere-level mechanics to intact trabeculae to define the mechano-kinetic properties of the cardiac myosin in situ represents a powerful tool for investigating cardiomyopathy-causing mutations in the myosin motor and testing specific therapeutic interventions.

The rate of oxidative phosphorylation depends on the contractile activity of the heart. Cardiac mitochondrial oxidative phosphorylation is determined by free ADP concentration, mitochondrial Ca2+accumulation, mitochondrial enzyme activities, and Krebs cycle intermediates. The purpose of the present study was to examine the factors that limit oxidative phosphorylation upon rapid changes in contractile activity in cardiac muscle. We tested the hypotheses that prior contractile performance enhances the changes in NAD(P)H and FAD concentration upon an increase in contractile activity and that this mitochondrial “priming” depends on pyruvate dehydrogenase activity. Intact rat cardiac trabeculae were electrically stimulated at 0.5 Hz for at least 30 min. Thereafter, two equal bouts at elevated stimulation frequency of 1, 2, or 3 Hz were applied for 3 min with 3 min of 0.5-Hz stimulation in between. No discernible time delay was observed in the changes in NAD(P)H and FAD fluorescence upon rapid changes in contractile activity. The amplitudes of the rapid changes in fluorescence upon an increase in stimulation frequency (the on-transients) were smaller than upon a decrease in stimulation frequency (the off-transients). A first bout in glucose-containing superfusion solution resulted, during the second bout, in an increase in the amplitudes of the on-transients, but the off-transients remained the same. No such priming effect was observed after addition of 10 mM pyruvate. These results indicate that mitochondrial priming can be observed in cardiac muscle in situ and that pyruvate dehydrogenase activity is critically involved in the mitochondrial adaptation to increases in contractile performance.NEW & NOTEWORTHY Mitochondrial respiration increases with increased cardiac contractile activity. Similar to mitochondrial “priming” in skeletal muscle, we hypothesized that cardiac mitochondrial activity is altered upon successive bouts of contractions and depends on pyruvate dehydrogenase activity. We found altered bioenergetics upon repeated contractile periods, indicative of mitochondrial priming in rat myocardium. No effect was seen when pyruvate was added to the perfusate. As such, pyruvate dehydrogenase activity is involved in the mitochondrial adaptation to increased contractile performance.

Objective Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. Methods To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Results Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Interpretation Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269-282.

The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5–10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent’s Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170–180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.

The mammalian heart pumps blood through the vessels, maintaining the dynamic equilibrium in a circulatory system driven by two pumps in series. This vital function is based on the fine-tuning of cardiac performance by the Frank-Starling mechanism that relates the pressure exerted by the contracting ventricle (end systolic pressure) to its volume (end systolic volume). At the level of the sarcomere, the structural unit of the cardiac myocytes, the Frank-Starling mechanism consists of the increase in active force with the increase of sarcomere length (length-dependent activation). We combine sarcomere mechanics and micrometer-nanometer-scale X-ray diffraction from synchrotron light in intact ventricular trabeculae from the rat to measure the axial movement of the myosin motors during the diastole-systole cycle under sarcomere length control. We find that the number of myosin motors leaving the off, ATP hydrolysis-unavailable state characteristic of the diastole is adjusted to the sarcomere length-dependent systolic force. This mechanosensing-based regulation of the thick filament makes the energetic cost of the systole rapidly tuned to the mechanical task, revealing a prime aspect of the Frank-Starling mechanism. The regulation is putatively impaired by cardiomyopathy-causing mutations that affect the intramolecular and intermolecular interactions controlling the off state of the motors.

Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. Little is known about the magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiomyocytes. Using adenoviral infection, a ratiometric mitochondrially-targeted FRET-based calcium indicator (4 mtD3cpv, MitoCam) was expressed in cultured adult rat cardiomyocytes and the free mitochondrial calcium concentration ([Ca2+]m) was measured at different stimulation frequencies (0.1-4 Hz) and external calcium concentrations (1.8-3.6 mM) at 37°C. Cytosolic calcium concentrations were assessed under the same experimental conditions in separate experiments using Fura-4AM. The increases in [Ca2+]m during electrical stimulation at 0.1 Hz were rapid (rise time = 49 ± 2 ms), while the decreases in [Ca2+]m occurred more slowly (decay half time = 1.17 ± 0.07 s). Model calculations confirmed that this asymmetry caused the rise in [Ca2+]m during diastole observed at elevated stimulation frequencies. Inhibition of the mitochondrial sodium-calcium exchanger (mNCE) resulted in a rise in [Ca2+]m at baseline and -paradoxically- in an acceleration of Ca2+-release. In conclusion: rapid increases in [Ca2+]m allow for fast adjustment of mitochondrial ATP production to increases in myocardial demand on a beat-to-beat basis and mitochondrial calcium release depends on mNCE activity and mitochondrial calcium buffering. This article is protected by copyright. All rights reserved

RATIONALE: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity, duration of hospital stay and health care costs. The mechanisms underlying diaphragm weakness are unknown, but might include mitochondrial dysfunction and oxidative stress.OBJECTIVES: We hypothesized that weakness of diaphragm muscle fibers in critically ill patients is accompanied by impaired mitochondrial function, structure, and increased markers of oxidative stress.METHODS: To test these hypotheses, we studied contractile force, mitochondrial function, and mitochondrial structure in diaphragm muscle fibers. Fibers were isolated from diaphragm biopsies of thirty-six mechanically ventilated critically ill patients and compared to those isolated from biopsies of twenty-seven patients with suspected early-stage lung malignancy (controls).MEASUREMENTS AND MAIN RESULTS: Diaphragm muscle fibers from critically ill patients displayed significant atrophy and contractile weakness, but lacked impaired mitochondrial respiration and increased levels of oxidative stress markers. Mitochondrial energy status and morphology were not altered, despite a lower content of fusion proteins.CONCLUSIONS: Critically ill patients have manifest diaphragm muscle fiber atrophy and weakness, in the absence of mitochondrial dysfunction and oxidative stress. Thus, mitochondrial dysfunction and oxidative stress do not play a causative role in the development of atrophy and contractile weakness of the diaphragm in critically ill patients.

Item does not contain fulltext AIMS: In cardiac hypertrophy (CH) and heart failure (HF), alterations occur in mitochondrial enzyme content and activities but the origin and implications of these changes for mitochondrial function need to be resolved. METHODS AND RESULTS: Right ventricular CH or HF was induced by monocrotaline injection, which causes pulmonary artery hypertension, in rats. Results were compared with saline injection (CON). NAD(P)H and FAD autofluorescence were recorded in thin intact cardiac trabeculae during transitions in stimulation frequency, to assess mitochondrial complex I and complex II function, respectively. Oxygen consumption, mitochondrial morphology, protein content, and enzymatic activity were assessed. NAD(P)H autofluorescence upon an increase in stimulation frequency showed a rapid decline followed by a slow recovery. FAD autofluorescence followed a similar time course, but in opposite direction. The amplitude of the early rapid change in NAD(P)H autofluorescence was severely depressed in CH and HF compared with CON. The rapid changes in FAD autofluorescence in CH and HF were reduced to a lesser extent. Complex I-coupled respiration showed an approximately 3.5-fold reduction in CH and HF; complex II-coupled respiration was depressed two-fold in HF. Western blot analyses revealed modest reductions in complex I protein content in CH and HF and in complex I activity in supercomplexes in HF. Mitochondrial volume density was similar, but mitochondrial remodelling was evident from changes in ultrastructure and fusion/fission indices in CH and HF. CONCLUSION: These results suggest that the alterations in mitochondrial function observed in right ventricular CH and HF can be mainly attributed to complex I dysfunction.

Nemaline myopathy and congenital fiber type disproportion are among the most common nondystrophic congenital muscular disorders.1 Genes that are implicated in these myopathies encode proteins that are either components of the skeletal muscle thin filament, including nebulin (NEB), skeletal muscle alpha-actin1 (ACTA1), beta-tropomyosin 2 (TPM2), alpha-tropomyosin 3 (TPM3), troponin T type 1 (TNNT1), cofilin-2 (CFL2), and leiomodin-3 (LMOD3), or are thought to contribute to stability or turnover of thin filament proteins, such as kelch repeat and BTB (POZ) domain containing 13 (KBTBD13), and kelchlike family members 40 (KLHL40) and 41 (KLHL41).2–6 Hence, muscle diseases caused by mutations in these genes are here referred to as thin filament myopathies. For a schematic of the thin filament and its associated proteins, see Figure 1. Patients with thin filament myopathy suffer from muscle weakness, but the underlying mechanisms are poorly understood. FIGURE 1 Schematic of the skeletal muscle thin filament. The thin filament is an essential structure in the sarcomere, the smallest contractile unit in skeletal muscle. The actin-based backbone of the thin filament is decorated with proteins that are involved ... The thin filament is a major constituent of the sarcomere, the smallest contractile unit in muscle, and is essential for force generation; its length determines the overlap between the thin and thick filament, and thereby the number of force-generating interactions that can be formed between actin and myosin. In healthy human muscle, the length of the thick filament is 1.6μm and that of the thin filament is regulated at 1.1 to 1.3μm.7 Accordingly, force depends on sarcomere length, with increasing force as the overlap between thick and thin filaments increases (up to a sarcomere length of ~2.6μm; ie, the ascending limb of the force–sarcomere length relation), and decreasing force at longer sarcomere lengths as the overlap between thick and thin filaments decreases (ie, the descending limb; Fig 2A8,9). Hence, appropriate length of the thin filament is important for muscle fiber strength; a shorter length causes lower force generation by shifting the descending limb of the force–sarcomere length relation downward. FIGURE 2 The force–sarcomere length relation in muscle fibers from thin filament myopathy patients and control (CTRL) subjects. (A) The shape of the force–sarcomere length relation is determined by the amount of overlap between the thick and thin ... Whether mutations in genes implicated in thin filament myopathy contribute to force loss by affecting thin filament length is unclear. Mouse models with mutations in Neb exhibit a shorter thin filament length associated with lower force generation that becomes more prominent as sarcomere length increases.10–12 Preliminary studies on a small number of human biopsies were largely in agreement with this observation.13,14 Whether these findings translate to a large group of patients, and whether shorter thin filament length is a general mechanism underlying force loss in thin filament myopathies with a variety of different gene defects, is unknown. Therefore, we studied muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. In these fibers, we determined the sarcomere length-dependence of force, a functional assay that provides insight into the contractile strength of muscle fibers as well as the length of the thin filaments. In a novel, conditional Neb knockout mouse model that recapitulates thin filament myopathy—including shorter thin filament length—we studied whether muscle possesses mechanisms that compensate for shorter thin filament length, and we specifically focused on the addition of sarcomeres in series.

The power in the myocardium sarcomere is generated by two bipolar arrays of the motor protein cardiac myosin II extending from the thick filament and pulling the thin, actin-containing filaments from the opposite sides of the sarcomere. Despite the interest in the definition of myosin-based cardiomyopathies, no study has yet been able to determine the mechanokinetic properties of this motor protein in situ. Sarcomere-level mechanics recorded by a striation follower is used in electrically stimulated intact ventricular trabeculae from the rat heart to determine the isotonic velocity transient following a stepwise reduction in force from the isometric peak force TP to a value T(0.8-0.2 TP). The size and the speed of the early rapid shortening (the isotonic working stroke) increase by reducing T from ∼3 nm per half-sarcomere (hs) and 1,000 s(-1) at high load to ∼8 nm⋅hs(-1) and 6,000 s(-1) at low load. Increases in sarcomere length (1.9-2.2 μm) and external [Ca(2+)]o (1-2.5 mM), which produce an increase of TP, do not affect the dependence on T, normalized for TP, of the size and speed of the working stroke. Thus, length- and Ca(2+)-dependent increase of TP and power in the heart can solely be explained by modulation of the number of myosin motors, an emergent property of their array arrangement. The motor working stroke is similar to that of skeletal muscle myosin, whereas its speed is about three times slower. A new powerful tool for investigations and therapies of myosin-based cardiomyopathies is now within our reach.

The functional properties of cardiac mitochondria in intact preparations have been mainly studied by measurements of nicotinamide adenine dinucleotide (NADH) autofluorescence, which reflects mitochondrial complex I function. To assess complex II function, we extended this method by measuring flavin adenine dinucleotide (FAD)-related autofluorescence in electrically stimulated cardiac trabeculae isolated from the right ventricle from the rat at 27°C. NADH and FAD autofluorescence and tension responses were measured when stimulation frequency was increased from 0.5 Hz to 1, 2 or 3 Hz for 3 min, and thereafter decreased to 0.5 Hz. Maximal complex I and complex II activity in vitro were determined in saponin-permeabilized right ventricular tissue by respirometry. NADH responses upon an increase in stimulation frequency showed a rapid decline, followed by a slow recovery towards the initial level. FAD responses followed a similar time course, but in the opposite direction. The amplitudes of early rapid changes in the NADH and FAD concentration correlated well with the change in tension time integral per second (R2 = 0.833 and 0.660 for NADH and FAD, respectively), but with different slopes for the up and down transient. Maximal velocity of the increase in FAD concentration (16 ± 4 μm s−1), measured upon an increase in stimulation frequency from 0.5 to 3 Hz was considerably smaller than that of the decrease in NADH (78 ± 13 μm s−1). The respiration measurements indicated that the maximal velocity of NADH utilization (143 ± 14 μm s−1) was 2 times smaller than that of FADH2 (291 ± 19 μm s−1). This indicates that in cardiac mitochondria considerable complex II activity reserve is present.

Variations of free calcium concentration ([Ca2+]) are powerful intracellular signals, controlling contraction as well as metabolism in muscle cells. To fully understand the role of calcium redistribution upon excitation and contraction in skeletal muscle cells, the local [Ca2+] in different compartments needs to be taken into consideration. Fluorescent probes allow the determination of [Ca2+] in the cytosol where myofibrils are embedded, the lumen of the sarcoplasmic reticulum (SR) and the mitochondrial matrix. Previously, models have been developed describing intracellular calcium handling in skeletal and cardiac muscle cells. However, a comprehensive model describing the kinetics of the changes in free calcium concentration in these three compartments is lacking. We designed a new 3D compartmental model of the half sarcomere with radial symmetry, which accounts for diffusion of Ca2+ into the three compartments and simulates its dynamics at rest and at various rates of stimulation in mice skeletal muscle fibers. This model satisfactorily reproduces both the amplitude and time course of the variations of [Ca2+] in the three compartments in mouse fast fibers. As an illustration of the applicability of the model, we investigated the effects of Calsequestrin (CSQ) ablation. CSQ is the main Ca2+ buffer in the SR, localized in close proximity of its calcium release sites and near to the mitochondria. CSQ knock-out mice muscles still preserve a near-normal contractile behavior, but it is unclear whether this is caused by additional SR calcium buffering or a significant contribution of calcium entry from extracellular space, via stored-operated calcium entry (SOCE). The model enabled quantitative assessment of these two scenarios by comparison to measurements of local calcium in the cytosol, the SR and the mitochondria. In conclusion, the model represents a useful tool to investigate the impact of protein ablation and of pharmacological interventions on intracellular calcium dynamics in mice skeletal muscle.

Aims This study aimed to elucidate the molecular background of increased Ca2+ sensitivity of force production in cardiomyocytes of end-stage human heart failure. Methods and results Ca2+-activated isometric force and the cross-bridge specific rate of force redevelopment (ktr) were determined in Triton-skinned myocytes from end-stage failing and non-failing donor hearts. Measurements (control: pH 7.2, 0 mM inorganic phosphate (Pi)) were performed under test conditions that probed either the Ca2+-regulatory function of the thin filaments (pH 6.5), the kinetics of the actin-myosin cross-bridge cycle (10 mM Pi), or both (pH 6.5, 10 mM Pi). The control maximal Ca2+-activated force (Fo) and ktrmax did not differ between failing and non-failing myocytes. At submaximal [Ca2+], however, both force and ktr were higher in failing than in donor myocytes. The difference in the Ca2+ sensitivities of force production was preserved when the thin filament regulatory function was perturbed by acidosis (pH 6.5) but was abolished by cross-bridge modulation (i.e. by Pi) both at pH 7.2 and at pH 6.5. Pi induced a larger reduction in force but a smaller increase in ktr in the failing myocytes than in the non-failing myocytes at submaximal [Ca2+]. Conclusion The enhanced Pi sensitivity of the actin-myosin interaction suggests that the Pi release step of the actin-myosin cross-bridge cycle is modified during end-stage human heart failure. This might be of functional importance when Pi accumulates (e.g. during cardiac ischaemia). Moreover, this alteration can influence cardiac energetics and the clinical efficacy of sarcomere targeted agents in human heart failure.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding β-myosin heavy chain (β-MyHC). R403Q locates in the globular head of myosin (S1), responsible for interaction with actin, and thus motor function of myosin. Increased cross-bridge relaxation kinetics caused by the R403Q mutation might underlie increased energetic cost of tension generation; however, direct evidence is absent. Here we studied to what extent cross-bridge kinetics and energetics are related in single cardiac myofibrils and multicellular cardiac muscle strips of three HCM patients with the R403Q mutation and nine sarcomere mutation-negative HCM patients (HCMsmn). Expression of R403Q was on average 41 ± 4% of total MYH7 mRNA. Cross-bridge slow relaxation kinetics in single R403Q myofibrils was significantly higher (P < 0.0001) than in HCMsmn myofibrils (0.47 ± 0.02 and 0.30 ± 0.02 s-1, respectively). Moreover, compared to HCMsmn, tension cost was significantly higher in the muscle strips of the three R403Q patients (2.93 ± 0.25 and 1.78 ± 0.10 μmol l-1 s-1 kN-1 m-2, respectively) which showed a positive linear correlation with relaxation kinetics in the corresponding myofibril preparations. This correlation suggests that faster cross-bridge relaxation kinetics results in an increase in energetic cost of tension generation in human HCM with the R403Q mutation compared to HCMsmn. Therefore, increased tension cost might contribute to HCM disease in patients carrying the R403Q mutation.

Frank-Starling's law reflects the ability of the heart to adjust the force of its contraction to changes in ventricular filling, a property based on length-dependent myofilament activation (LDA). The threonine at amino acid 143 of cardiac troponin I (cTnI) is prerequisite for the length-dependent increase in Ca2+sensitivity. Thr143 is a known target of protein kinase C (PKC) whose activity is increased in cardiac disease. Thr143 phosphorylation may modulate length-dependent myofilament activation in failing hearts. Therefore, we investigated if pseudo-phosphorylation at Thr143 modulates length dependence of force using troponin exchange experiments in human cardiomyocytes. In addition, we studied effects of protein kinase A (PKA)-mediated cTnI phosphorylation at Ser23/24, which has been reported to modulate LDA. Isometric force was measured at various Ca2+concentrations in membrane-permeabilized cardiomyocytes exchanged with recombinant wild-type (WT) troponin or troponin mutated at the PKC site Thr143 or Ser23/24 into aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. In troponin-exchanged donor cardiomyocytes experiments were repeated after incubation with exogenous PKA. Pseudo-phosphorylation of Thr143 increased myofilament Ca2+sensitivity compared with WT without affecting LDA in failing and donor cardiomyocytes. Subsequent PKA treatment enhanced the length-dependent shift in Ca2+sensitivity after WT and 143D exchange. Exchange with Ser23/24 variants demonstrated that pseudo-phosphorylation of both Ser23 and Ser24 is needed to enhance the length-dependent increase in Ca2+sensitivity. cTnI pseudo-phosphorylation did not alter length-dependent changes in maximal force. Thus phosphorylation at Thr143 enhances myofilament Ca2+sensitivity without affecting LDA, while Ser23/24 bisphosphorylation is needed to enhance the length-dependent increase in myofilament Ca2+sensitivity.

Mutations in the MYBPC3 gene, encoding cardiac myosin binding protein C (cMyBP-C) are frequent causes of hypertrophic cardiomyopathy (HCM). Previously, we have presented evidence for reduced cMyBP-C expression (haploinsufficiency), in patients with a truncation mutation in MYBPC3. In mice, lacking cMyBP-C cross-bridge kinetics was accelerated. In this study, we investigated whether cross-bridge kinetics was altered in myectomy samples from HCM patients harboring heterozygous MYBPC3 mutations (MYBPC3mut). Isometric force and the rate of force redevelopment (k tr) at different activating Ca2+ concentrations were measured in mechanically isolated Triton-permeabilized cardiomyocytes from MYBPC3mut (n = 18) and donor (n = 7) tissue. Furthermore, the stretch activation response of cardiomyocytes was measured in tissue from eight MYBPC3mut patients and five donors to assess the rate of initial force relaxation (k 1) and the rate and magnitude of the transient increase in force (k 2 and P 3, respectively) after a rapid stretch. Maximal force development of the cardiomyocytes was reduced in MYBPC3mut (24.5 ± 2.3 kN/m2) compared to donor (34.9 ± 1.6 kN/m2). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca2+ concentrations were similar (k tr at maximal activation: 0.63 ± 0.03 and 0.75 ± 0.09 s−1, respectively). Moreover, the stretch activation parameters did not differ significantly between MYBPC3mut and donor (k 1: 8.5±0.5 and 8.8 ± 0.4 s−1; k 2: 0.77 ± 0.06 and 0.74 ± 0.09 s−1; P 3: 0.08 ± 0.01 and 0.09 ± 0.01, respectively). Incubation with protein kinase A accelerated k 1 in MYBPC3mut and donor to a similar extent. Our experiments indicate that, at the cMyBP-C expression levels in this patient group (63 ± 6 % relative to donors), cross-bridge kinetics are preserved and that the depressed maximal force development is not explained by perturbation of cross-bridge kinetics.

Cardiac troponin I (cTnI) is well known as a biomarker for the diagnosis of myocardial damage. However, because of its central role in the regulation of contraction and relaxation in heart muscle, cTnI may also be a potential target for the treatment of heart failure. Studies in rodent models of cardiac disease and human heart samples showed altered phosphorylation at various sites on cTnI (i.e. site-specific phosphorylation). This is caused by altered expression and/or activity of kinases and phosphatases during heart failure development. It is not known whether these (transient) alterations in cTnI phosphorylation are beneficial or detrimental. Knowledge of the effects of site-specific cTnI phosphorylation on cardiomyocyte contractility is therefore of utmost importance for the development of new therapeutic strategies in patients with heart failure. In this review we focus on the role of cTnI phosphorylation in the healthy heart upon activation of the beta-adrenergic receptor pathway (as occurs during increased stress and exercise) and as a modulator of the Frank-Starling mechanism. Moreover, we provide an overview of recent studies which aimed to reveal the functional consequences of changes in cTnI phosphorylation in cardiac disease.

BACKGROUND: After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function. OBJECTIVES: The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients. METHODS: LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated. RESULTS: Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (∼30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (∼25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ∼30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation. CONCLUSIONS: The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients. © 2014 by the American College of Cardiology Foundation.

Aims Familial hypertrophic cardiomyopathy (HCM), frequently caused by sarcomeric gene mutations, is characterized by cellular dysfunction and asymmetric left-ventricular (LV) hypertrophy. We studied whether cellular dysfunction is due to an intrinsic sarcomere defect or cardiomyocyte remodelling. Methods and results Cardiac samples from 43 sarcomere mutation-positive patients (HCMmut: mutations in thick ( MYBPC3 , MYH7 ) and thin ( TPM1 , TNNI3 , TNNT2 ) myofilament genes) were compared with 14 sarcomere mutation-negative patients (HCMsmn), eight patients with secondary LV hypertrophy due to aortic stenosis (LVHao) and 13 donors. Force measurements in single membrane-permeabilized cardiomyocytes revealed significantly lower maximal force generating capacity (Fmax) in HCMmut (21 ± 1 kN/m2) and HCMsmn (26 ± 3 kN/m2) compared with donor (36 ± 2 kN/m2). Cardiomyocyte remodelling was more severe in HCMmut compared with HCMsmn based on significantly lower myofibril density (49 ± 2 vs. 63 ± 5%) and significantly higher cardiomyocyte area (915 ± 15 vs. 612 ± 11 μm2). Low Fmax in MYBPC3 mut, TNNI3 mut, HCMsmn, and LVHao was normalized to donor values after correction for myofibril density. However, Fmax was significantly lower in MYH7 mut, TPM1 mut, and TNNT2 mut even after correction for myofibril density. In accordance, measurements in single myofibrils showed very low Fmax in MYH7 mut, TPM1 mut, and TNNT2 mut compared with donor (respectively, 73 ± 3, 70 ± 7, 83 ± 6, and 113 ± 5 kN/m2). In addition, force was lower in MYH7 mut cardiomyocytes compared with MYBPC3 mut, HCMsmn, and donor at submaximal [Ca2+]. Conclusion Low cardiomyocyte Fmax in HCM patients is largely explained by hypertrophy and reduced myofibril density. MYH7 mutations reduce force generating capacity of sarcomeres at maximal and submaximal [Ca2+]. These hypocontractile sarcomeres may represent the primary abnormality in patients with MYH7 mutations.

Objective: To investigate whether sarcomeric dysfunction contributes to muscle weakness in facioscapulohumeral muscular dystrophy (FSHD). Methods: Sarcomeric function was evaluated by contractile studies on demembranated single muscle fibers obtained from quadriceps muscle biopsies of 4 patients with FSHD and 4 healthy controls. The sarcomere length dependency of force was determined together with measurements of thin filament length using immunofluorescence confocal scanning laser microscopy. X-ray diffraction techniques were used to study myofilament lattice spacing. Results: FSHD muscle fibers produced only 70% of active force compared to healthy controls, a reduction which was exclusive to type II muscle fibers. Changes in force were not due to changes in thin filament length or sarcomere length. Passive force was increased 5- to 12-fold in both fiber types, with increased calcium sensitivity of force generation and decreased myofilament lattice spacing, indicating compensation by the sarcomeric protein titin. Conclusions: We have demonstrated a reduction in sarcomeric force in type II FSHD muscle fibers, and suggest compensatory mechanisms through titin stiffening. Based on these findings, we propose that sarcomeric dysfunction plays a critical role in the development of muscle weakness in FSHD. © 2013 American Academy of Neurology.

Introduction Hypertrophic cardiomyopathy (HCM) is an autosomal dominant heart disease mostly due to mutations in genes encoding sarcomeric proteins. HCM is characterised by asymmetric hypertrophy of the left ventricle (LV) in the absence of another cardiac or systemic disease. At present it lacks specific treatment to prevent or reverse cardiac dysfunction and hypertrophy in mutation carriers and HCM patients. Previous studies have indicated that sarcomere mutations increase energetic costs of cardiac contraction and cause myocardial dysfunction and hypertrophy. By using a translational approach, we aim to determine to what extent disturbances of myocardial energy metabolism underlie disease progression in HCM. Methods Hypertrophic obstructive cardiomyopathy (HOCM) patients and aortic valve stenosis (AVS) patients will undergo a positron emission tomography (PET) with acetate and cardiovascular magnetic resonance imaging (CMR) with tissue tagging before and 4 months after myectomy surgery or aortic valve replacement + septal biopsy. Myectomy tissue or septal biopsy will be used to determine efficiency of sarcomere contraction in-vitro, and results will be compared with in-vivo cardiac performance. Healthy subjects and non-hypertrophic HCM mutation carriers will serve as a control group. Endpoints Our study will reveal whether perturbations in cardiac energetics deteriorate during disease progression in HCM and whether these changes are attributed to cardiac remodelling or the presence of a sarcomere mutation per se. In-vitro studies in hypertrophied cardiac muscle from HOCM and AVS patients will establish whether sarcomere mutations increase ATP consumption of sarcomeres in human myocardium. Our follow-up imaging study in HOCM and AVS patients will reveal whether impaired cardiac energetics are restored by cardiac surgery.

Background— The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. Methods and Results— Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean±SEM. RV diastolic stiffness (β) was significantly increased in PAH patients (PAH, 0.050±0.005 versus control, 0.029±0.003; P 2 versus control, 218±21 μm 2 ; P P 200%; P interaction P Conclusions— RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Background: Nemaline myopathy-the most common non-dystrophic congenital myopathy-is caused by mutations in thin filament genes, of which the nebulin gene is the most frequently affected one. The nebulin gene codes for the giant sarcomeric protein nebuli which plays a crucial role in skeletal muscle contractile performance. Muscle weakness is a hallmark feature of nemaline myopathy patients with nebulin mutations, and is caused by changes in contractile protein function, including a lower calcium-sensitivity of force generation. To date no therapy exists to treat muscle weakness in nemaline myopathy. Here, we studied the ability of the novel fast skeletal muscle troponin activator, CK- 2066260, to augment force generation at submaximal calcium levels in muscle cells from nemaline myopathy patients with nebulin mutations. Methods: Contractile protein function was determined in permeabilised muscle cells isolated from frozen patient biopsies. The effect of 5 μM CK-2066260 on force production was assessed. Results: Nebulin protein concentrations were severely reduced in muscle cells from these patients compared to controls, while myofibrillar ultrastructure was largely preserved. Both maximal active tension and the calciumsensitivity of force generation were lower in patients compared to controls. Importantly, CK-2066260 greatly increased the calcium-sensitivity of force generation- without affecting the cooperativity of activation-in patients to levels that exceed those observed in untreated control muscle. Conclusions: Fast skeletal troponin activation is a therapeutic mechanism to augment contractile protein function in nemaline myopathy patients with nebulin mutations and with other neuromuscular diseases.

PKA-mediated phosphorylation of contractile proteins upon β-adrenergic stimulation plays an important role in the regulation of cardiac performance. Phosphorylation of the PKA sites (Ser23/Ser24) of cardiac troponin (cTn)I results in a decrease in myofilament Ca2+sensitivity and an increase in the rate of relaxation. However, the relation between the level of phosphorylation of the sites and the functional effects in the human myocardium is unknown. Therefore, site-directed mutagenesis was used to study the effects of phosphorylation at Ser23and Ser24of cTnI on myofilament function in human cardiac tissue. Serines were replaced by aspartic acid (D) or alanine (A) to mimic phosphorylation and dephosphorylation, respectively. cTnI-DD mimics both sites phosphorylated, cTnI-AD mimics Ser23unphosphorylated and Ser24phosphorylated, cTnI-DA mimics Ser23phosphorylated and Ser24unphosphorylated, and cTnI-AA mimics both sites unphosphorylated. Force development was measured at various Ca2+concentrations in permeabilized cardiomyocytes in which the endogenous troponin complex was exchanged with these recombinant human troponin complexes. In donor cardiomyocytes, myofilament Ca2+sensitivity (pCa50) was significantly lower in cTnI-DD (pCa50: 5.39 ± 0.01) compared with cTnI-AA (pCa50: 5.50 ± 0.01), cTnI-AD (pCa50: 5.48 ± 0.01), and cTnI-DA (pCa50: 5.51 ± 0.01) at ∼70% cTn exchange. No effects were observed on the rate of tension redevelopment. In cardiomyocytes from idiopathic dilated cardiomyopathic tissue, a linear decline in pCa50with cTnI-DD content was observed, saturating at ∼55% bisphosphorylation. Our data suggest that in the human myocardium, phosphorylation of both PKA sites on cTnI is required to reduce myofilament Ca2+sensitivity, which is maximal at ∼55% bisphosphorylated cTnI. The implications for in vivo cardiac function in health and disease are detailed in the discussion in this article.

We previously demonstrated that diaphragm muscle weakness is present in experimental pulmonary arterial hypertension (PH). However, the nature of this diaphragm weakness is still unknown. Therefore, the aim of this study was to investigate whether changes at the sarcomeric level contribute to diaphragm weakness in PH. For this purpose, in control rats and rats with monocrotaline-induced PH, contractile performance and myosin heavy chain content of demembranated single diaphragm fibers were determined. We observed a reduced maximal tension of 20% ( P < 0.05), whereas tension cost was preserved in type 2X and 2B diaphragm fibers in PH compared with control. The reduced maximal tension was associated with a reduction of force generated per half-sarcomeric myosin heavy chain content. Additionally, reduced Ca2+ sensitivity of force generation was found in type 2X fibers compared with control, which could exacerbate diaphragm muscle weakness at submaximal activation. No changes in maximal tension and Ca2+ sensitivity of force generation were observed in fibers from the nonrespiratory extensor digitorum longus muscle. Together, these findings indicate that diaphragm weakness in PH is at least partly caused by sarcomeric dysfunction, which appears to be specific for the diaphragm.

Background— Hypertrophic cardiomyopathy (HCM), typically characterized by asymmetrical left ventricular hypertrophy, frequently is caused by mutations in sarcomeric proteins. We studied if changes in sarcomeric properties in HCM depend on the underlying protein mutation. Methods and Results— Comparisons were made between cardiac samples from patients carrying a MYBPC3 mutation (MYBPC3 mut ; n=17), mutation negative HCM patients without an identified sarcomere mutation (HCM mn ; n=11), and nonfailing donors (n=12). All patients had normal systolic function, but impaired diastolic function. Protein expression of myosin binding protein C (cMyBP-C) was significantly lower in MYBPC3 mut by 33±5%, and similar in HCM mn compared with donor. cMyBP-C phosphorylation in MYBPC3 mut was similar to donor, whereas it was significantly lower in HCM mn . Troponin I phosphorylation was lower in both patient groups compared with donor. Force measurements in single permeabilized cardiomyocytes demonstrated comparable sarcomeric dysfunction in both patient groups characterized by lower maximal force generating capacity in MYBPC3 mut and HCM mn, compared with donor (26.4±2.9, 28.0±3.7, and 37.2±2.3 kN/m 2 , respectively), and higher myofilament Ca 2+ -sensitivity (EC 50 =2.5±0.2, 2.4±0.2, and 3.0±0.2 μmol/L, respectively). The sarcomere length-dependent increase in Ca 2+ -sensitivity was significantly smaller in both patient groups compared with donor (ΔEC 50 : 0.46±0.04, 0.37±0.05, and 0.75±0.07 μmol/L, respectively). Protein kinase A treatment restored myofilament Ca 2+ -sensitivity and length-dependent activation in both patient groups to donor values. Conclusions— Changes in sarcomere function reflect the clinical HCM phenotype rather than the specific MYBPC3 mutation. Hypocontractile sarcomeres are a common deficit in human HCM with normal systolic left ventricular function and may contribute to HCM disease progression.

Background— Aortic stenosis (AS) and diabetes mellitus (DM) are frequent comorbidities in aging populations. In heart failure, DM worsens diastolic left ventricular (LV) dysfunction, thereby adversely affecting symptoms and prognosis. Effects of DM on diastolic LV function were therefore assessed in aortic stenosis, and underlying myocardial mechanisms were identified. Methods and Results— Patients referred for aortic valve replacement were subdivided into patients with AS and no DM (AS; n=46) and patients with AS and DM (AS-DM; n=16). Preoperative Doppler echocardiography and hemodynamics were implemented with perioperative LV biopsies. Histomorphometry and immunohistochemistry quantified myocardial collagen volume fraction and myocardial advanced glycation end product deposition. Isolated cardiomyocytes were stretched to 2.2-μm sarcomere length to measure resting tension (F passive ). Expression and phosphorylation of titin isoforms were analyzed with gel electrophoresis with ProQ Diamond and SYPRO Ruby stains. Reduced LV end-diastolic distensibility in AS-DM was evident from higher LV end-diastolic pressure (21±1 mm Hg for AS versus 28±4 mm Hg for AS-DM; P =0.04) at comparable LV end-diastolic volume index and attributed to higher myocardial collagen volume fraction (AS, 12.9±1.1% versus AS-DM, 18.2±2.6%; P 2 versus AS-DM, 31.4±6.1 score per 1 mm 2 ; P =0.03), and higher F passive (AS, 3.5±1.7 kN/m 2 versus AS-DM, 5.1±0.7 kN/m 2 ; P =0.04). Significant hypophosphorylation of the stiff N2B titin isoform in AS-DM explained the higher F passive and normalization of F passive after in vitro treatment with protein kinase A. Conclusions— Worse diastolic LV dysfunction in AS-DM predisposes to heart failure and results from more myocardial fibrosis, more intramyocardial vascular advanced glycation end product deposition, and higher cardiomyocyte F passive , which was related to hypophosphorylation of the N2B titin isoform.

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed beta-adrenergic receptor (beta-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F(max)) and higher Ca(2+)-sensitivity in MI compared to sham. F(max), did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca(2+)-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of beta-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca(2+)-dependent calmodulin kinase II (CaMKII-delta C). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-delta C activity and may help to maintain proper diastolic performance during exercise. (C) 2010 Elsevier Ltd. All rights reserved.

Hypertrophic cardiomyopathy (HCM) is a familial disorder characterized by left ventricular hypertrophy in the absence of other cardiac or systemic disease likely to cause this hypertrophy. HCM is considered a disease of the sarcomere as most causal mutations are identified in genes encoding sarcomeric proteins, although several other disorders have also been linked to the HCM phenotype. The clinical course of HCM is characterized by a large inter- and intrafamilial variability, ranging from severe symptomatic HCM to asymptomatic individuals. The general picture emerges that the underlying pathophysiology of HCM is complex and still scarcely clarified. Recent findings indicated that both functional and morphological (macroscopic and microscopic) changes of the HCM muscle are present before the occurrence of HCM phenotype. This review aims to provide an overview of the myocardial alterations that occur during the gradual process of wall thickening in HCM on a myofilament level, as well as the structural and functional abnormalities that can be observed in genetically affected individuals prior to the development of HCM with state of the art imaging techniques, such as tissue Doppler echocardiography and cardiovascular magnetic resonance imaging. Additionally, present and future therapeutic options will be briefly discussed.

Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is clinically characterized by muscle weakness. However, the mechanisms underlying this weakness are poorly understood. Here, we studied the contractile phenotype of skeletal muscle from NM patients with nebulin mutations (NEM2). SDS-PAGE and Western blotting studies revealed markedly reduced nebulin protein levels in muscle from NM patients, whereas levels of other thin filament-based proteins were not significantly altered. Muscle mechanics studies indicated significantly reduced calcium sensitivity of force generation in NM muscle fibers compared to control fibers. In addition, we found slower rate constant of force redevelopment, as well as increased tension cost, in NM compared to control fibers, indicating that in NM muscle the rate of cross-bridge attachment is reduced, whereas the rate of cross-bridge detachment is increased. The resulting reduced fraction of force generating cross-bridges is expected to greatly impair the force generating capacity of muscle from NM patients. Thus, the present study provides important novel insights into the pathogenesis of muscle weakness in nebulin-based NM.

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca(2+)-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca(2+)-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66+/-1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca(2+)-sensitivity (pCa(50)) to 5.50+/-0.02 relative to the donor control value (pCa(50)=5.43+/-0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca(2+)-sensitivity relative to the failing control (pCa(50)=5.60+/-0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca(2+)-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca(2+)-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca(2+)-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca(2+)-sensitivity.

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCalpha- and PKCepsilon-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca2+] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCalpha and PKCepsilon equally reduced Ca2+ sensitivity in failing cardiomyocytes (DeltapCa50 = 0.08 +/- 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca2+ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCalpha resulted in a modest significant reduction in passive force. Effects of PKCalpha were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCalpha. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca2+ sensitivity. Subsequently, PKCalpha treatment of donor cardiomyocytes reduced Ca2+ sensitivity (DeltapCa50 = 0.08 +/- 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCalpha- and PKCepsilon-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca2+ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.

Background— Mutations in the MYBPC3 gene, encoding cardiac myosin-binding protein C (cMyBP-C), are a frequent cause of familial hypertrophic cardiomyopathy. In the present study, we investigated whether protein composition and function of the sarcomere are altered in a homogeneous familial hypertrophic cardiomyopathy patient group with frameshift mutations in MYBPC3 (MYBPC3 mut ). Methods and Results— Comparisons were made between cardiac samples from MYBPC3 mutant carriers (c.2373dupG, n=7; c.2864_2865delCT, n=4) and nonfailing donors (n=13). Western blots with the use of antibodies directed against cMyBP-C did not reveal truncated cMyBP-C in MYBPC3 mut . Protein expression of cMyBP-C was significantly reduced in MYBPC3 mut by 33±5%. Cardiac MyBP-C phosphorylation in MYBPC3 mut samples was similar to the values in donor samples, whereas the phosphorylation status of cardiac troponin I was reduced by 84±5%, indicating divergent phosphorylation of the 2 main contractile target proteins of the β-adrenergic pathway. Force measurements in mechanically isolated Triton-permeabilized cardiomyocytes demonstrated a decrease in maximal force per cross-sectional area of the myocytes in MYBPC3 mut (20.2±2.7 kN/m 2 ) compared with donor (34.5±1.1 kN/m 2 ). Moreover, Ca 2+ sensitivity was higher in MYBPC3 mut (pCa 50 =5.62±0.04) than in donor (pCa 50 =5.54±0.02), consistent with reduced cardiac troponin I phosphorylation. Treatment with exogenous protein kinase A, to mimic β-adrenergic stimulation, did not correct reduced maximal force but abolished the initial difference in Ca 2+ sensitivity between MYBPC3 mut (pCa 50 =5.46±0.03) and donor (pCa 50 =5.48±0.02). Conclusions— Frameshift MYBPC3 mutations cause haploinsufficiency, deranged phosphorylation of contractile proteins, and reduced maximal force-generating capacity of cardiomyocytes. The enhanced Ca 2+ sensitivity in MYBPC3 mut is due to hypophosphorylation of troponin I secondary to mutation-induced dysfunction.

Background— Myofilament contractility of individual cardiomyocytes is depressed in remote noninfarcted myocardium and contributes to global left ventricular pump dysfunction after myocardial infarction (MI). Here, we investigated whether β-blocker therapy could restore myofilament contractility. Methods and Results— In pigs with a MI induced by ligation of the left circumflex coronary artery, β-blocker therapy (bisoprolol, MI+β) was initiated on the first day after MI. Remote left ventricular subendocardial biopsies were taken 3 weeks after sham or MI surgery. Isometric force was measured in single permeabilized cardiomyocytes. Maximal force (F max ) was lower, whereas Ca 2+ sensitivity was higher in untreated MI compared with sham (both P 2+ sensitivity was abolished by treatment of cells with the β-adrenergic kinase, protein kinase A. β-blocker therapy partially reversed F max and Ca 2+ sensitivity to sham values and significantly reduced passive force. Despite the lower myofilament Ca 2+ sensitivity in MI+β compared with untreated myocardium, the protein kinase A induced reduction in Ca 2+ sensitivity was largest in cardiomyocytes from myocardium treated with β-blockers. Phosphorylation of β-adrenergic target proteins (myosin binding protein C and troponin I) did not differ among groups, whereas myosin light chain 2 phosphorylation was reduced in MI, which coincided with increased expression of protein phosphatase 1. β-blockade fully restored the latter alterations and significantly reduced expression of protein phosphatase 2a. Conclusions— β-blockade reversed myofilament dysfunction and enhanced myofilament responsiveness to protein kinase A in remote myocardium after MI. These effects likely contribute to the beneficial effects of β-blockade on global left ventricular function after MI.

Background— Excessive diastolic left ventricular stiffness is an important contributor to heart failure in patients with diabetes mellitus. Diabetes is presumed to increase stiffness through myocardial deposition of collagen and advanced glycation end products (AGEs). Cardiomyocyte resting tension also elevates stiffness, especially in heart failure with normal left ventricular ejection fraction (LVEF). The contribution to diastolic stiffness of fibrosis, AGEs, and cardiomyocyte resting tension was assessed in diabetic heart failure patients with normal or reduced LVEF. Methods and Results— Left ventricular endomyocardial biopsy samples were procured in 28 patients with normal LVEF and 36 patients with reduced LVEF, all without coronary artery disease. Sixteen patients with normal LVEF and 10 with reduced LVEF had diabetes mellitus. Biopsy samples were used for quantification of collagen and AGEs and for isolation of cardiomyocytes to measure resting tension. Diabetic heart failure patients had higher diastolic left ventricular stiffness irrespective of LVEF. Diabetes mellitus increased the myocardial collagen volume fraction only in patients with reduced LVEF (from 14.6±1.0% to 22.4±2.2%, P 2 , P =0.006). Diabetes increased myocardial AGE deposition in patients with reduced LVEF (from 8.8±2.5 to 24.1±3.8 score/mm 2 ; P =0.005) and less so in patients with normal LVEF (from 8.2±2.5 to 15.7±2.7 score/mm 2 , P =NS). Conclusions— Mechanisms responsible for the increased diastolic stiffness of the diabetic heart differ in heart failure with reduced and normal LVEF: Fibrosis and AGEs are more important when LVEF is reduced, whereas cardiomyocyte resting tension is more important when LVEF is normal.

Many changes in morphology, biochemical properties and myocyte function occur during development to heart failure. Most changes may be compensatory, yet unable to prevent cardiac dysfunction in the long run. This illustrates that it is important to carefully dissect the disease causing modifications from cardiac adaptation, in order to obtain a better understanding of the pathophysiological processes leading to heart failure.

Cardiac performance depends on power developed by the myocardium and mutations of cardiac myosin, which affect power output, have been proposed to be responsible for various forms of cardiomyopathy. Precise measurements of force and filament sliding generated by cardiac myosin are lacking. Here sarcomere-level mechanics are applied for the first time to intact trabeculae from the right ventricle of the rat to determine the size and the speed of the working stroke of cardiac myosin in situ and their dependency on the load. The isotonic shortening transients elicited by stepwise drops in force to 0.2-0.8 of peak force (Tp) are recorded at sarcomere length of 1.9 and 2.2 μm and external Ca2+-concentrations ([Ca2+]o) 1 and 2.5 mM (temperature 27 oC). The size and the speed of the early rapid phase of shortening (the mechanical manifestation of the myosin working stroke) increase with the reduction of the load from approximately 3 nm per half-sarcomere and 1000 s−1 at high load to 6 nm per half-sarcomere and 7000 s−1 at low load, but are not affected by the increase of sarcomere length or [Ca2+]o. Thus the two-fold increase of force and power in the range of sarcomere length and [Ca2+]o explored are solely due to the increase in number of myosin motors. These results demonstrate the possibility for in situ studies of the molecular basis of cardiomyopathies. Supported by MIUR-PRIN and Telethon (Italy).

The dependence of contractile properties on intracellular calcium in cardiac tissue is a highly cooperative process. Here, the temperature and calcium dependence of contractile and energetical properties in permeabilized cardiac trabeculae from rat were studied to provide novel insights into the underlying kinetic processes. Myofilament Ca2+ sensitivity significantly increased with temperature between 15 and 25°C, whereas its steepness was independent of temperature. A direct proportionality between active tension and Ca2+-activated rate of ATP hydrolysis was observed; the slope of this relationship (tension cost) was highly temperature dependent. The rate of tension redevelopment following a quick release–restretch manoeuvre (ktr) depended in a complex manner on the level of contractile activation and on temperature. At saturating calcium levels, the temperature dependence (Q10) of ktr and Ca2+-activated ATP hydrolysis rate were similar (Q10∼3.5), and significantly higher than the Q10 for maximum tension (Tmax; Q10∼1.3) or tension cost (Q10∼2.5). In contrast, at a low level of contractile activation (∼5% of Tmax), the Q10 of ktr was similar to that of tension cost, and significantly lower than the Q10 of Ca2+-activated ATP hydrolysis at that level of contractile activation. Our results are consistent with the hypothesis that at high levels of contractile activation, the rates of tension redevelopment and Ca2+-activated ATP hydrolysis are determined by both apparent cross-bridge attachment and detachment rates, while at low levels, ktr is limited by cross-bridge detachment rate. Tension cost, on the other hand, is determined solely by cross-bridge detachment kinetics at all temperatures and levels of contractile activation.

Phosphorylation of cardiac myofilament proteins represents one of the main post-translational mechanisms that regulate cardiac pump function. Human studies are often limited by the amount of available tissue as biopsies taken during cardiac catheterization weigh only 1 mg (dry weight). Similarly, investigation of time- (or dose-) dependent changes in protein phosphorylation in animal studies is often hampered by tissue availability. The present study describes quantitative analysis of phosphorylation status of multiple myofilament proteins by 2-DE and Pro-Q® Diamond stained gradient gels using minor amounts (-0.5 mg dry weight) of human and pig cardiac tissue.

The positive force-frequency relation, one of the key factors modulating performance of healthy myocardium, has been attributed to an increased Ca(2+) influx per unit of time. In failing hearts, a blunted, flat or negative force-frequency relation has been found. In healthy and failing hearts frequency-dependent alterations in Ca(2+) sensitivity of the myofilaments, related to different phosphorylation levels of contractile proteins, could contribute to this process. Therefore, the frequency dependency of force, intracellular free Ca(2+) ([Ca(2+)](i)), Ca(2+) sensitivity and contractile protein phosphorylation were determined in control and monocrotaline-treated, failing rat hearts. An increase in frequency from 0.5 to 6 Hz resulted in an increase in force in control (14.3 +/- 3.0 mN mm(-2)) and a decrease in force in failing trabeculae (9.4 +/- 3.2 mN mm(-2)), whereas in both groups the amplitude of [Ca(2+)](i) transient increased. In permeabilized cardiomyocytes, isolated from control hearts paced at 0 and 9 Hz, Ca(2+) sensitivity remained constant with frequency (pCa(50): 5.55 +/- 0.02 and 5.58 +/- 0.01, respectively, P>0.05), whereas in cardiomyocytes from failing hearts Ca(2+) sensitivity decreased with frequency (pCa(50): 5.62 +/- 0.01 and 5.57 +/- 0.01, respectively, P

The increase in Ca2+ sensitivity of isometric force development along with sarcomere length (SL) is considered as the basis of the Frank-Starling law of the heart, possibly involving the regulation of cross-bridge turnover kinetics. Therefore, the Ca2+ dependencies of isometric force production and of the cross-bridge-sensitive rate constant of force redevelopment ( ktr) were determined at different SLs (1.9 and 2.3 μm) in isolated human, murine, and porcine permeabilized cardiomyocytes. ktr was also determined in the presence of 10 mM inorganic phosphate (Pi), which interfered with the force-generating cross-bridge transitions. The increases in Ca2+ sensitivities of force with SL were very similar in human, murine, and porcine cardiomyocytes (ΔpCa50: ∼0.11). ktr was higher ( P < 0.05) in mice than in humans or pigs at all Ca2+ concentrations ([Ca2+]) [maximum ktr ( ktr,max) at a SL of 1.9 μm and pCa 4.75: 1.33 ± 0.11, 7.44 ± 0.15, and 1.02 ± 0.05 s−1, in humans, mice, and pigs, respectively] but ktr did not depend on SL in any species. Moreover, when the ktr values for each species were expressed relative to their respective maxima, similar Ca2+ dependencies were obtained. Ten millimolar Pi decreased force to ∼60–65% and left ΔpCa50 unaltered in all three species. Pi increased ktr,max by a factor of ∼1.6 in humans and pigs and by a factor of ∼3 in mice, independent of SL. In conclusion, species differences exert a major influence on ktr, but SL does not appear to modulate the cross-bridge turnover rates in human, murine, and porcine hearts.

The contractile dysfunction of the hibernating myocardium in situ results from local environmental factors, but also from intrinsic cellular remodelling that may determine reversibility. Previous studies have suggested defects in myofilament Ca 2+ responsiveness. We prepared single myocytes from control (CTRL, n pigs =7) and from hibernating myocardium (HIB, n pigs =8), removed the membranes and measured isometric force development during direct activation of the myofilaments. One- and 2-dimensional polyacrylamide gel electrophoresis and specific phosphoprotein immunoblotting were performed on tissue homogenates from matched samples. Cellular ultrastructure was evaluated using electron microscopy. Normalized for cross-sectional area, passive force was not different but maximal isometric force was significantly reduced in myocytes from HIB (11.6±1.5 kN/m 2 versus 18.7±1.6 kN/m 2 in CTRL, P 2+ sensitivity and steepness of the normalized force-pCa relationship were not different, and neither was the rate of force redevelopment (K tr ). No alterations were observed in isoform expression, phosphorylation or degradation of specific myofibrillar proteins. However, in HIB samples the total protein volume density was decreased by 23% ( P P

The extent and mechanism of the cardiac benefit of early exercise training following myocardial infarction (MI) is incompletely understood, but may involve blunting of abnormalities in Ca 2+ -handling and myofilament function. Consequently, we investigated the effects of 8-weeks of voluntary exercise, started early after a large MI, on left ventricular (LV) remodeling and dysfunction in the mouse. Exercise had no effect on survival, MI size or LV dimensions, but improved LV fractional shortening from 8±1 to 12±1%, and LVdP/dt P30 from 5295±207 to 5794±207 mm Hg/s (both P 2+ -sensitivity (ΔpCa 50 =0.037). This effect of exercise was PKA-mediated and likely because of improved β 1 -adrenergic signaling, as suggested by the increased β 1 -adrenoceptor protein (48%) and cAMP levels (36%; all P max increased from 14.3±0.7 to 18.3±0.8 kN/m 2 P P 2+ -concentrations (by ∼30%, P 2+ -transient amplitude, indicating that the improved LV and cardiomyocyte shortening were principally because of improved myofilament function. In conclusion, early exercise in mice after a large MI has no effect on LV remodeling, but attenuates global LV dysfunction. The latter can be explained by the exercise-induced improvement of myofilament function.

Background: Mechanically ventilated Intensive Care (IC) patients develop diaphragm weakness, which may delay weaning and increases duration of hospitalization and morbidity. This weakness is caused by many factors. The activation mechanism of one of these factors, the ubiquitin-proteasome pathway, might involve mitochondrial changes. Aims: To investigate mitochondrial dynamics, morphology, and function in diaphragm fibers of IC-patients. Methods: Muscle fibers obtained by diaphragm biopsies of mechanically ventilated IC-patients (n=28) and control subjects (n=27) were analyzed by electron microscopy (EM), Western blotting, immunohistochemistry and respirometry. Results: Control and IC-patients were mechanically ventilated for 1.4±0.1 hours and 153±32.0 hours respectively, (p Conclusion: These data suggests altered mitochondrial dynamics and a disturbed network in IC-patients, which may contribute to diaphragm weakness and weaning failure.

Contains fulltext : 155008.pdf (Publisher’s version ) (Closed access) RATIONALE: The clinical significance of diaphragm weakness in critically ill patients is evident: it prolongs ventilator dependency, and increases morbidity and duration of hospital stay. To date, the nature of diaphragm weakness and its underlying pathophysiologic mechanisms are poorly understood. OBJECTIVES: We hypothesized that diaphragm muscle fibers of mechanically ventilated critically ill patients display atrophy and contractile weakness, and that the ubiquitin-proteasome pathway is activated in the diaphragm. METHODS: We obtained diaphragm muscle biopsies from 22 critically ill patients who received mechanical ventilation before surgery and compared these with biopsies obtained from patients during thoracic surgery for resection of a suspected early lung malignancy (control subjects). In a proof-of-concept study in a muscle-specific ring finger protein-1 (MuRF-1) knockout mouse model, we evaluated the role of the ubiquitin-proteasome pathway in the development of contractile weakness during mechanical ventilation. MEASUREMENTS AND MAIN RESULTS: Both slow- and fast-twitch diaphragm muscle fibers of critically ill patients had approximately 25% smaller cross-sectional area, and had contractile force reduced by half or more. Markers of the ubiquitin-proteasome pathway were significantly up-regulated in the diaphragm of critically ill patients. Finally, MuRF-1 knockout mice were protected against the development of diaphragm contractile weakness during mechanical ventilation. CONCLUSIONS: These findings show that diaphragm muscle fibers of critically ill patients display atrophy and severe contractile weakness, and in the diaphragm of critically ill patients the ubiquitin-proteasome pathway is activated. This study provides rationale for the development of treatment strategies that target the contractility of diaphragm fibers to facilitate weaning.

Background Nemaline myopathy (NM), the most common non-dystrophic congenital myopathy, is characterized by generalized skeletal muscle weakness, often from birth. To date, no therapy exists that enhances the contractile strength of muscles of NM patients. Mutations in NEB, encoding the giant protein nebulin, are the most common cause of NM. The pathophysiology of muscle weakness in NM patients with NEB mutations (NEB-NM) includes a lower calcium-sensitivity of force generation. We propose that the lower calcium-sensitivity of force generation in NEB-NM offers a therapeutic target. Levosimendan is a calcium sensitizer that is approved for use in humans and has been developed to target cardiac muscle fibers. It exerts its effect through binding to slow skeletal/cardiac troponin C. As slow skeletal/cardiac troponin C is also the dominant troponin C isoform in slow-twitch skeletal muscle fibers, we hypothesized that levosimendan improves slow-twitch muscle fiber strength at submaximal levels of activation in patients with NEB-NM. Methods To test whether levosimendan affects force production, permeabilized slow-twitch muscle fibers isolated from biopsies of NEB-NM patients and controls were exposed to levosimendan and the force response was measured. Results No effect of levosimendan on muscle fiber force in NEB-NM and control skeletal muscle fibers was found, both at a submaximal calcium level using incremental levosimendan concentrations, and at incremental calcium concentrations in the presence of levosimendan. In contrast, levosimendan did significantly increase the calcium-sensitivity of force in human single cardiomyocytes. Protein analysis confirmed that the slow skeletal/cardiac troponin C isoform was present in the skeletal muscle fibers tested. Conclusions These findings indicate that levosimendan does not improve the contractility in human skeletal muscle fibers, and do not provide rationale for using levosimendan as a therapeutic to restore muscle weakness in NEB-NM patients. We stress the importance of searching for compounds that improve the calcium-sensitivity of force generation of slow-twitch muscle fibers. Such compounds provide an appealing approach to restore muscle force in patients with NEB-NM, and also in patients with other neuromuscular disorders. Electronic supplementary material The online version of this article (doi:10.1186/s13395-015-0037-7) contains supplementary material, which is available to authorized users.