ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin–myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca²⁺. We investigated whether ADP-stimulated force development (without Ca²⁺) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7_mut, MYBPC3_mut) and thin-filament (TNNT2_mut, TNNI3_mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCM_smn, and MYH7_mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2_mut sample corrected the abnormal actin–myosin blockade. In MYBPC3_trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C’s role in actin–myosin regulation. In the presence of Ca²⁺, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca²⁺, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.Heart failure (HF) is a syndrome clinically defined as the inability of the heart to sufficiently supply blood to organs and tissues (1). Systolic dysfunction is present in approximately one-half of the HF population, whereas diastolic dysfunction is a common feature in almost all HF patients (2). Moreover, in hypertrophic cardiomyopathy (HCM), which is caused by mutations in genes encoding thin- and thick-filament proteins, impaired diastolic function is frequently observed (3). Impaired relaxation of the heart may be caused by high myofilament Ca²⁺ sensitivity. This increased sensitivity for Ca²⁺ would result in residual myofilament activation at diastolic [Ca²⁺], which may delay the onset of ventricular relaxation and limit proper filling of the heart. High myofilament Ca²⁺ sensitivity has been observed in both acquired and genetic forms of cardiomyopathy (3, 4). In human idiopathic dilated cardiomyopathy (IDCM), high myofilament Ca²⁺ sensitivity has been associated with reduced β-adrenergic receptor-mediated phosphorylation by protein kinase A (PKA) (4). Reduced PKA phosphorylation of cardiac troponin I (cTnI) and cardiac myosin-binding protein C (cMyBP-C) increases myofilament Ca²⁺ sensitivity (5–8). Likewise, high myofilament Ca²⁺ sensitivity is a common characteristic of HCM and may be caused by the mutant protein or by reduced PKA-mediated protein phosphorylation secondary to HCM disease progression (3, 9).Contractile performance of the heart muscle may thus be perturbed by mutation-induced and phosphorylation-mediated protein changes that affect thin-filament transitions. Ca²⁺-induced cardiac muscle contraction is tightly modulated by the troponin–tropomyosin complex that regulates the interactions between the actin thin filament and myosin thick filament (i.e., cross-bridge formation). Accordingly, the myofilaments oscillate between three transitions termed the blocked (B-state), closed (C-state), and open (M-state) states of thin-filament regulation that represent the distinct position of tropomyosin on actin (10–12) (Fig. 1). In the absence of Ca²⁺ (B state), tropomyosin sterically blocks the myosin-binding sites on actin (Fig. 1A). Upon electrical activation of cardiomyocytes, the rise of cytosolic [Ca²⁺] alters the conformation of the troponin–tropomyosin complex, which moves tropomyosin on actin and exposes myosin-binding sites (C state). Weakly bound cross-bridges (myosin-ADP-Pi) populate the C state (10, 12) (Fig. 1B). Transition to the M state involves release of inorganic phosphate (Pi) from the cross-bridge and strong-binding cross-bridge formation (myosin-ADP) that induces additional movement of tropomyosin, resulting in myofilament contraction and sliding (Fig. 1C).Open in a separate windowFig. 1.Three-state model of thin-filament activation. Seven actin monomers (circles), spanned by one tropomyosin dimer (red strand), together with the troponin complex (not depicted) comprise one functional unit (A₇TmTn). Two functional units are depicted, and individual myosins are shown as triangles (weak, weak-binding cross-bridges; strong, strong-binding cross-bridges). (A) B state (blocked); when ATP is present and cytoplasmic [Ca²⁺] is low and is not bound to cardiac troponin C (cTnC), tropomyosin is sterically blocking the myosin-binding sites on actin. (B) C state (Ca²⁺-induced); upon rise in cytoplasmic [Ca²⁺], Ca²⁺ binds to cTnC, inducing conformational changes of the troponin complex, resulting in a ∼25° movement of tropomyosin on the thin filament, thereby exposing myosin-binding sites on actin. In the C state, the myofilament is not yet activated as non–tension-generating cross-bridges bind weakly to actin. (C) M state (myosin induced); the strong binding of tension-generating cross-bridges induces a ∼10° movement of tropomyosin on actin, resulting in myofilament activation and contraction.The three-state model of cross-bridge interaction implies that the main task of Ca²⁺ is to uncover myosin-binding sites on actin and that formation of myosin-ADP represents the main regulator of force development and contraction. Notably, solution (10) and cryo-electron microscopy (13) studies have shown that in the absence of Ca²⁺ the myofilaments are not entirely blocked, as ∼5% of the thin filaments have tropomyosin localized in the C-state position. This observation suggests that conditions that promote myosin-ADP formation can trigger myofilament contraction in Ca²⁺-free conditions and thereby impair relaxation. Indeed, in membrane-permeabilized rabbit skeletal muscle fibers (14), bovine myocardium (15, 16) and human cardiac muscle (17) millimolar levels of ADP stimulate force development in the absence of Ca²⁺.Because ADP-stimulated contraction is due to myosin-ADP binding to the nonblocked sites of the thin filament in the absence of Ca²⁺, it provides an experimental tool to assess changes in tropomyosin’s position in acquired and genetic cardiomyopathies in which altered protein phosphorylation and mutant proteins may alter myofilament activation. In addition, it could represent a pathomechanism underlying the diastolic dysfunction seen in both disease states. Solution studies with mutant troponin proteins, which are known to cause HCM, showed a reduction in the B state at low-Ca²⁺ conditions compared with wild-type troponin proteins (18, 19). Mutation-induced irregularities in troponin–tropomyosin interactions disrupt the B state and shift the thin filament to the C state, increasing the available myosin-binding sites on actin.In addition to Ca²⁺-induced changes of the thin filament, tropomyosin location may also be altered by the thick-filament protein cMyBP-C. Recent evidence supports that the N-terminal extension of cMyBP-C binds the low-Ca²⁺–state (B-state) position of tropomyosin on actin and interferes with tropomyosin–actin interactions, dislocating tropomyosin into the C-state position (i.e., the presence of cMyBP-C sensitizes the thin filament to Ca²⁺) (20, 21). Because it was previously shown that in Ca²⁺-free conditions (B state) ∼5% of the thin filaments (lacking cMyBP-C) have tropomyosin localized in the C-state position (10), more myofilaments may be in the C state in the presence of cMyBP-C. We (22) and others (23) have shown that cMyBP-C mutations, which are a major cause of HCM, have a reduced level of healthy cMyBP-C protein compared with nonfailing hearts (i.e., haploinsufficiency), which may alter tropomyosin position on the thin filament.To verify whether ADP-stimulated contraction provides an experimental tool to assess mutation-induced and phosphorylation-mediated changes in thin-filament transitions, which precede Ca²⁺ activation of myofilaments, we tested the following hypotheses: (i) that IDCM and HCM samples with thin-filament mutations are more sensitive to ADP, as a result of a higher accessibility of myosin-binding sites on actin, whereas (ii) cMyBP-C haploinsufficient HCM myocardium has a reduced ADP sensitivity (i.e., less cMyBP-C causes reduced displacement of tropomyosin from the B state) compared with cells from nonfailing hearts. To answer our hypotheses, we activated membrane-permeabilized human cardiomyocytes in ADP containing Ca²⁺-free solutions. Cells were isolated from HCM patients with mutations in genes encoding thick-filament (MYH7, MYBPC3) and thin-filament (TNNT2, TNNI3) proteins and patients with IDCM and compared with cells from sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Finally, we investigated whether the ADP level as observed in diseased hearts, in the presence of Ca²⁺, increases myofilament force development in cardiomyocytes from human cardiomyopathy hearts.We conclude that, in HCM with thin-filament mutations, tropomyosin’s ability to block myosin-binding sites on actin is reduced. This effect is exacerbated in HCM samples by the low PKA phosphorylation of myofilament proteins, which is also observed in human IDCM. In contrast, cMyBP-C HCM-causing mutations reduce accessibility of myosin for actin. The findings in this study provide evidence that ADP-mediated activation can be used as an experimental tool to reveal mutation- and phosphorylation-mediated changes in tropomyosin location on the thin filament.     

Weight information labels on media models reduce body dissatisfaction in adolescent girls

PurposeTo examine how weight information labels on variously sized media models affect (pre)adolescent girls' body perceptions and how they compare themselves with media models.MethodsWe used a three (body shape: extremely thin vs. thin vs. normal weight) × three (information label: 6-kg underweight vs. 3-kg underweight vs. normal weight) experimental design in three age-groups (9–10 years, 12–13 years, and 15–16 years; n = 184). The girls completed questionnaires after exposure to media models.ResultsWeight information labels affected girls' body dissatisfaction, social comparison with media figures, and objectified body consciousness. Respondents exposed to an extremely thin body shape labeled to be of “normal weight” were most dissatisfied with their own bodies and showed highest levels of objectified body consciousness and comparison with media figures. An extremely thin body shape combined with a corresponding label (i.e., 6-kg underweight), however, induced less body dissatisfaction and less comparison with the media model. Age differences were also found to affect body perceptions: adolescent girls showed more negative body perceptions than preadolescents.ConclusionsWeight information labels may counteract the generally media-induced thin-body ideal. That is, when the weight labels appropriately informed the respondents about the actual thinness of the media model's body shape, girls were less affected. Weight information labels also instigated a normalization effect when a “normal-weight” label was attached to underweight-sized media models. Presenting underweight as a normal body shape, clearly increased body dissatisfaction in girls. Results also suggest age between preadolescence and adolescence as a critical criterion in responding to media models' body shape.     

Dietary non-tocopherol antioxidants present in extra virgin olive oil increase the resistance of low density lipoproteins to oxidation in rabbits

Consumption of a range of dietary antioxidants may be beneficial in protecting low density lipoprotein (LDL) against oxidative modification, as studies have demonstrated that antioxidants other than vitamin E may also function against oxidation of LDL in vitro. In the present study, the effect of polyphenol antioxidants on the susceptibility of LDL to copper-mediated oxidation was investigated after feeding semi-purified diets to 3 groups of New Zealand white (NZW) rabbits. All diets comprised 40% energy as fat with 17% energy as oleic acid. Dietary fatty acid compositions were identical. Oils with different polyphenol contents were used to provide the dietary source of oleic acid — refined olive oil, extra virgin olive oil and Trisun high oleic sunflower seed oil. Polyphenolic compounds (hydroxytyrosol and p-tyrosol) could only be detected in the extra virgin olive oil. Vitamin E was equalised in all diets. LDL oxidizability in vitro was determined by continuously monitoring the copper-induced formation of conjugated dienes after 6 weeks of experimental diet feeding. The lag phase before demonstrable oxidation occurred was significantly increased in the high polyphenol, extra virgin olive oil group (P < 0.05) when compared with combined results from the low polyphenol group (refined olive oil and Trisun), even though the LDL vitamin E concentration in the high polyphenol group was significantly lower. The rate of conjugated diene formation was not influenced by the presence of dietary polyphenols. Results demonstrate that antioxidants, possibly phenolic compounds which are present only in extra virgin olive oil, may contribute to the endogenous antioxidant capacity of LDL, resulting in an increased resistance to oxidation as determined in vitro.     

Exercise Capacity, Muscle Strength, and Fatigue in Sarcoidosis: A Follow-Up Study

Purpose

The purpose of this study was to examine changes in the prevalence of exercise intolerance, reduced muscle strength, and fatigue and the changes in these parameters in individual patients during a 2-year follow-up study.

Methods

Ninety sarcoidosis patients (62 males and 28 females; mean age: 46.0 ± 10.2 years) participated in a 2-year follow-up study. At the baseline and follow-up measurements, patients performed a 6-min walk test and elbow flexor muscle strength, quadriceps peak torque, and hamstrings peak torque tests. Maximal inspiratory pressure was recorded. All patients completed the Fatigue Assessment Scale.

Results

Both at baseline and follow-up, a substantial proportion of the patients showed a reduced 6-minute walk test (41.6 and 34.8 %, respectively), elbow flexor muscle strength (6.7 and 14.6 %), quadriceps peak torque (21.3 and 18 %), hamstrings peak torque (13.5 and 12.4 %), and maximal inspiratory pressure (45.9 and 48.6 %). The majority of the patients reported fatigue (86 and 77 %). These physical impairments remained stable during the follow-up period. The prevalence of these physical impairments in patients diagnosed with sarcoidosis <2 years before inclusion in this study was similar to that in patients with a longer history of the disease.

Conclusions

Exercise intolerance, muscle weakness, and fatigue are frequent problems in symptomatic sarcoidosis patients with a stable and persistent character. This study highlights that beyond medical treatment a rehabilitation program should be considered as adjunct therapy in the multidisciplinary management of sarcoidosis patients even though the achieved benefit needs future studies.