Cardiac Disorders and Pathophysiology of Sarcomeric Proteins.

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.

Myosin filament activation in the heart is tuned to the mechanical task.

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.

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3).

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.

Size and speed of the working stroke of cardiac myosin in situ.

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 force and stiffness of myosin motors in the isometric twitch of a cardiac trabecula and the effect of the extracellular calcium concentration.

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.

Diaphragm Atrophy and Weakness in the Absence of Mitochondrial Dysfunction in the Critically Ill.

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 and structure, and by 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 36 mechanically ventilated critically ill patients and compared with those isolated from biopsies of 27 patients with suspected early-stage lung malignancy (control subjects). 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.

Empagliflozin decreases myocardial cytoplasmic Na+ through inhibition of the cardiac Na+/H+ exchanger in rats and rabbits.

AIMS/HYPOTHESIS: Empagliflozin (EMPA), an inhibitor of the renal sodium-glucose cotransporter (SGLT) 2, reduces the risk of cardiovascular death in patients with type 2 diabetes. The underlying mechanism of this effect is unknown. Elevated cardiac cytoplasmic Na+ ([Na+]c) and Ca2+ ([Ca2+]c) concentrations and decreased mitochondrial Ca2+ concentration ([Ca2+]m) are drivers of heart failure and cardiac death. We therefore hypothesised that EMPA would directly modify [Na+]c, [Ca2+]c and [Ca2+]m in cardiomyocytes. METHODS: [Na+]c, [Ca2+]c, [Ca 2+]m and Na+/H+ exchanger (NHE) activity were measured fluorometrically in isolated ventricular myocytes from rabbits and rats. RESULTS: An increase in extracellular glucose, from 5.5 mmol/l to 11 mmol/l, resulted in increased [Na+]c and [Ca2+]c levels. EMPA treatment directly inhibited NHE flux, caused a reduction in [Na+]c and [Ca2+]c and increased [Ca2+]m. After pretreatment with the NHE inhibitor, Cariporide, these effects of EMPA were strongly reduced. EMPA also affected [Na+]c and NHE flux in the absence of extracellular glucose. CONCLUSIONS/INTERPRETATION: The glucose lowering kidney-targeted agent, EMPA, demonstrates direct cardiac effects by lowering myocardial [Na+]c and [Ca2+]c and enhancing [Ca2+]m, through impairment of myocardial NHE flux, independent of SGLT2 activity.

Rapid frequency-dependent changes in free mitochondrial calcium concentration in rat cardiac myocytes.

KEY POINTS: Calcium ions regulate mitochondrial ATP production and contractile activity and thus play a pivotal role in matching energy supply and demand in cardiac muscle. The magnitude and kinetics of the changes in free mitochondrial calcium concentration in cardiac myocytes are largely unknown. Rapid stimulation frequency-dependent increases but relatively slow decreases in free mitochondrial calcium concentration were observed in rat cardiac myocytes. This asymmetry caused a rise in the mitochondrial calcium concentration with stimulation frequency. These results provide insight into the mechanisms of mitochondrial calcium uptake and release that are important in healthy and diseased myocardium. ABSTRACT: 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 Förster resonance energy transfer (FRET)-based calcium indicator (4mtD3cpv, 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.

Mutation-specific effects on thin filament length in thin filament myopathy.

OBJECTIVE: Thin filament myopathies are among the most common nondystrophic congenital muscular disorders, and are caused by mutations in genes encoding proteins that are associated with the skeletal muscle thin filament. Mechanisms underlying muscle weakness are poorly understood, but might involve the length of the thin filament, an important determinant of force generation. METHODS: We investigated the sarcomere length-dependence of force, a functional assay that provides insights into the contractile strength of muscle fibers as well as the length of the thin filaments, in muscle fibers from 51 patients with thin filament myopathy caused by mutations in NEB, ACTA1, TPM2, TPM3, TNNT1, KBTBD13, KLHL40, and KLHL41. RESULTS: Lower force generation was observed in muscle fibers from patients of all genotypes. In a subset of patients who harbor mutations in NEB and ACTA1, the lower force was associated with downward shifted force-sarcomere length relations, indicative of shorter thin filaments. Confocal microscopy confirmed shorter thin filaments in muscle fibers of these patients. A conditional Neb knockout mouse model, which recapitulates thin filament myopathy, revealed a compensatory mechanism; the lower force generation that was associated with shorter thin filaments was compensated for by increasing the number of sarcomeres in series. This allowed muscle fibers to operate at a shorter sarcomere length and maintain optimal thin-thick filament overlap. INTERPRETATION: These findings might provide a novel direction for the development of therapeutic strategies for thin filament myopathy patients with shortened thin filament lengths. Ann Neurol 2016;79:959-969.

Reduced force of diaphragm muscle fibers in patients with chronic thromboembolic pulmonary hypertension.

Patients with pulmonary hypertension (PH) suffer from inspiratory muscle weakness. However, the pathophysiology of inspiratory muscle dysfunction in PH is unknown. We hypothesized that weakness of the diaphragm, the main inspiratory muscle, is an important contributor to inspiratory muscle dysfunction in PH patients. Our objective was to combine ex vivo diaphragm muscle fiber contractility measurements with measures of in vivo inspiratory muscle function in chronic thromboembolic pulmonary hypertension (CTEPH) patients. To assess diaphragm muscle contractility, function was studied in vivo by maximum inspiratory pressure (MIP) and ex vivo in diaphragm biopsies of the same CTEPH patients (N = 13) obtained during pulmonary endarterectomy. Patients undergoing elective lung surgery served as controls (N = 15). Muscle fiber cross-sectional area (CSA) was determined in cryosections and contractility in permeabilized muscle fibers. Diaphragm muscle fiber CSA was not significantly different between control and CTEPH patients in both slow-twitch and fast-twitch fibers. Maximal force-generating capacity was significantly lower in slow-twitch muscle fibers of CTEPH patients, whereas no difference was observed in fast-twitch muscle fibers. The maximal force of diaphragm muscle fibers correlated significantly with MIP. The calcium sensitivity of force generation was significantly reduced in fast-twitch muscle fibers of CTEPH patients, resulting in a ∼40% reduction of submaximal force generation. The fast skeletal troponin activator CK-2066260 (5 µM) restored submaximal force generation to levels exceeding those observed in control subjects. In conclusion, diaphragm muscle fiber contractility is hampered in CTEPH patients and contributes to the reduced function of the inspiratory muscles in CTEPH patients.

Diaphragm muscle fiber weakness and ubiquitin-proteasome activation in critically ill patients.

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.

Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension.

Our objective was to investigate the role of creatine kinase in the contractile dysfunction of right ventricular failure caused by pulmonary artery hypertension. Pulmonary artery hypertension and right ventricular failure were induced in rats by monocrotaline and compared to saline-injected control animals. In vivo right ventricular diastolic pressure-volume relationships were measured in anesthetized animals; diastolic force-length relationships in single enzymatically dissociated myocytes and myocardial creatine kinase levels by Western blot. We observed diastolic dysfunction in right ventricular failure indicated by significantly steeper diastolic pressure-volume relationships in vivo and diastolic force-length relationships in single myocytes. There was a significant reduction in creatine kinase protein expression in failing right ventricle. Dysfunction also manifested as a shorter diastolic sarcomere length in failing myocytes. This was associated with a Ca(2+)-independent mechanism that was sensitive to cross-bridge cycling inhibition. In saponin-skinned failing myocytes, addition of exogenous creatine kinase significantly lengthened sarcomeres, while in intact healthy myocytes, inhibition of creatine kinase significantly shortened sarcomeres. Creatine kinase inhibition also changed the relatively flat contraction amplitude-stimulation frequency relationship of healthy myocytes into a steeply negative, failing phenotype. Decreased creatine kinase expression leads to diastolic dysfunction. We propose that this is via local reduction in ATP:ADP ratio and thus to Ca(2+)-independent force production and diastolic sarcomere shortening. Creatine kinase inhibition also mimics a definitive characteristic of heart failure, the inability to respond to increased demand. Novel therapies for pulmonary artery hypertension are needed. Our data suggest that cardiac energetics would be a potential ventricular therapeutic target.

A novel phosphorylation site, Serine 199, in the C-terminus of cardiac troponin I regulates calcium sensitivity and susceptibility to calpain-induced proteolysis.

Phosphorylation of cardiac troponin I (cTnI) by protein kinase C (PKC) is implicated in cardiac dysfunction. Recently, Serine 199 (Ser199) was identified as a target for PKC phosphorylation and increased Ser199 phosphorylation occurs in end-stage failing compared with non-failing human myocardium. The functional consequences of cTnI-Ser199 phosphorylation in the heart are unknown. Therefore, we investigated the impact of phosphorylation of cTnI-Ser199 on myofilament function in human cardiac tissue and the susceptibility of cTnI to proteolysis. cTnI-Ser199 was replaced by aspartic acid (199D) or alanine (199A) to mimic phosphorylation and dephosphorylation, respectively, with recombinant wild-type (Wt) cTn as a negative control. Force development was measured at various [Ca(2+)] and at sarcomere lengths of 1.8 and 2.2 µm in demembranated cardiomyocytes in which endogenous cTn complex was exchanged with the recombinant human cTn complexes. In idiopathic dilated cardiomyopathy samples, myofilament Ca(2+)-sensitivity (pCa50) at 2.2 µm was significantly higher in 199D (pCa50 = 5.79 ± 0.01) compared to 199A (pCa50 = 5.65 ± 0.01) and Wt (pCa50 = 5.66 ± 0.02) at ~63% cTn exchange. Myofilament Ca(2+)-sensitivity was significantly higher even with only 5.9 ± 2.5% 199D exchange compared to 199A, and saturated at 12.3 ± 2.6% 199D exchange. Ser199 pseudo-phosphorylation decreased cTnI binding to both actin and actin-tropomyosin. Moreover, altered susceptibility of cTnI to proteolysis by calpain I was found when Ser199 was pseudo-phosphorylated. Our data demonstrate that low levels of cTnI-Ser199 pseudo-phosphorylation (~6%) increase myofilament Ca(2+)-sensitivity in human cardiomyocytes, most likely by decreasing the binding affinity of cTnI for actin-tropomyosin. In addition, cTnI-Ser199 pseudo-phosphorylation or mutation regulates calpain I mediated proteolysis of cTnI.

Rapid changes in NADH and flavin autofluorescence in rat cardiac trabeculae reveal large mitochondrial complex II reserve capacity.

KEY POINTS: A photometry-based technique was developed to measure nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence and contractile properties simultaneously in intact rat trabeculae at a high time resolution. This provides insight into the function of mitochondrial complex I and II. Maximal complex I and complex II activities were determined in saponin-permeabilized right ventricular tissue by respirometry. In trabeculae, complex II function was considerably smaller than the maximal complex II activity, suggesting large complex II reserve capacity. Up-down asymmetry in NADH and FAD kinetics suggests a complex interaction between mitochondrial and contractile function. These data show that simultaneous measurement of contractile properties and NADH and FAD kinetics in cardiac trabeculae provides a mean to study the differences in complex I and II function in intact preparations in health and disease. ABSTRACT: 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 (R(2)  = 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.

Synergistic role of ADP and Ca(2+) in diastolic myocardial stiffness.

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin-related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca(2+) ] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction. Heart failure (HF) with diastolic dysfunction has been attributed to increased myocardial stiffness that limits proper filling of the ventricle. Altered cross-bridge interaction may significantly contribute to high diastolic stiffness, but this has not been shown thus far. Cross-bridge interactions are dependent on cytosolic [Ca(2+) ] and the regeneration of ATP from ADP. Depletion of myocardial energy reserve is a hallmark of HF leading to ADP accumulation and disturbed Ca(2+) handling. Here, we investigated if ADP elevation in concert with increased diastolic [Ca(2+) ] promotes diastolic cross-bridge formation and force generation and thereby increases diastolic stiffness. ADP dose-dependently increased force production in the absence of Ca(2+) in membrane-permeabilized cardiomyocytes from human hearts. Moreover, physiological levels of ADP increased actomyosin force generation in the presence of Ca(2+) both in human and rat membrane-permeabilized cardiomyocytes. Diastolic stress measured at physiological lattice spacing and 37°C in the presence of pathological levels of ADP and diastolic [Ca(2+) ] revealed a 76 ± 1% contribution of cross-bridge interaction to total diastolic stress in rat membrane-permeabilized cardiomyocytes. Inhibition of creatine kinase (CK), which increases cytosolic ADP, in enzyme-isolated intact rat cardiomyocytes impaired diastolic re-lengthening associated with diastolic Ca(2+) overload. In isolated Langendorff-perfused rat hearts, CK inhibition increased ventricular stiffness only in the presence of diastolic [Ca(2+) ]. We propose that elevations of intracellular ADP in specific types of cardiac disease, including those where myocardial energy reserve is limited, contribute to diastolic dysfunction by recruiting cross-bridges, even at low Ca(2+) , and thereby increase myocardial stiffness.

The striated muscles in pulmonary arterial hypertension: adaptations beyond the right ventricle.

Pulmonary arterial hypertension (PAH) is a fatal lung disease characterised by progressive remodelling of the small pulmonary vessels. The daily-life activities of patients with PAH are severely limited by exertional fatigue and dyspnoea. Typically, these symptoms have been explained by right heart failure. However, an increasing number of studies reveal that the impact of the PAH reaches further than the pulmonary circulation. Striated muscles other than the right ventricle are affected in PAH, such as the left ventricle, the diaphragm and peripheral skeletal muscles. Alterations in these striated muscles are associated with exercise intolerance and reduced quality of life. In this Back to Basics article on striated muscle function in PAH, we provide insight into the pathophysiological mechanisms causing muscle dysfunction in PAH and discuss potential new therapeutic strategies to restore muscle dysfunction.

Perturbed length-dependent activation in human hypertrophic cardiomyopathy with missense sarcomeric gene mutations.

RATIONALE: High-myofilament Ca(2+) sensitivity has been proposed as a trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) on the basis of in vitro and transgenic mice studies. However, myofilament Ca(2+) sensitivity depends on protein phosphorylation and muscle length, and at present, data in humans are scarce. OBJECTIVE: To investigate whether high myofilament Ca(2+) sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick and thin filament proteins. METHODS AND RESULTS: Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca(2+) sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA) targets compared with donors. After exogenous PKA treatment, myofilament Ca(2+) sensitivity was similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values. CONCLUSIONS: High-myofilament Ca(2+) sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via posttranslational modifications other than PKA hypophosphorylation or altered protein-protein interactions, and represents a common pathomechanism in HCM.

Faster cross-bridge detachment and increased tension cost in human hypertrophic cardiomyopathy with the R403Q MYH7 mutation.

The first mutation associated with hypertrophic cardiomyopathy (HCM) is the R403Q mutation in the gene encoding ß-myosin heavy chain (ß-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.

Preserved cross-bridge kinetics in human hypertrophic cardiomyopathy patients with MYBPC3 mutations.

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 Ca(2+) 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/m(2)) compared to donor (34.9 ± 1.6 kN/m(2)). The rates of force redevelopment in MYBPC3mut and donor over a range of Ca(2+) 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.

Unaffected contractility of diaphragm muscle fibers in humans on mechanical ventilation.

Several studies have indicated that diaphragm dysfunction develops in patients on mechanical ventilation (MV). Here, we tested the hypothesis that the contractility of sarcomeres, i.e., the smallest contractile unit in muscle, is affected in humans on MV. To this end, we compared diaphragm muscle fibers of nine brain-dead organ donors (cases) that had been on MV for 26 ± 5 h with diaphragm muscle fibers from nine patients (controls) undergoing surgery for lung cancer that had been on MV for less than 2 h. In each diaphragm specimen we determined 1) muscle fiber cross-sectional area in cryosections by immunohistochemical methods and 2) the contractile performance of permeabilized single muscle fibers by means of maximum specific force, kinetics of cross-bridge cycling by rate of tension redevelopment, myosin heavy chain content and concentration, and calcium sensitivity of force of slow-twitch and fast-twitch muscle fibers. In case subjects, we noted no statistically significant decrease in outcomes compared with controls in slow-twitch or fast-twitch muscle fibers. These observations indicate that 26 h of MV of humans is not invariably associated with changes in the contractile performance of sarcomeres in the diaphragm.