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.     

Characterization of the IgG-Fc receptor on human platelets

To determine quantitatively the number and avidity of receptors for the Fc portion of IgG on human platelets, we have measured the binding to platelets of human monomeric monoclonal IgG, and of small covalently crosslinked polymers of IgG1 labeled with 125I. The binding of labeled IgG1 monomers to platelets is too weak to permit quantitation. The binding of dimers or larger polymers of IgG1 is much more avid (greater at 4 degrees C than 37 degrees C), is readily reversible, and is saturable. The number of receptor sites ranges from 400 to 2000 per platelet and the mean equilibrium association constant (Ka) for the binding of dimers at 4 degrees C is 2.2 x 10(7) M-1 +/- 0.9 x 10(7) M- 1. The binding is specific for the Fc portion of IgG, and IgG1 and IgG3 bind to the receptors much more avidly than IgG2 or IgG4. Unlabeled IgG1 dimers are about 7--8-fold more potent in inhibiting binding than are IgG1 monomers, and larger polymers are even more potent than dimers. Thus, the Fc receptors on platelets bind human IgG1 with the same specificity and similar avidity as Fc receptors on polymorphonuclear leukocytes (PMNs), but PMNs have about 300-fold more receptors per unit of surface area than platelets.     

The response of red cell hexose monophosphate shunt after sulfhydryl inhibition

In this investigation, we studied the importance of cellular glutathione (GSH) in the hexose monophosphate shunt (HMPS) activity of unstimulated human erythrocytes and the mechanism by which pyruvate stimulates the HMPS. The rate of HMPS activity was measured by the production of radioactive CO2 from 14C-1-glucose or 14C-1-ribose using a vibrating reed electrometer and ionization chamber. HMPS activity was not significantly impaired by N-ethylmaleimide (NEM) in concentrations which bound all red cell GSH. Red cells incubated under carbon monoxide (CO), an experimental condition which eliminates peroxide production, still had HMPS activity which was 44% of the value under air. Pyruvate stimulation of the HMPS was unaffected by doses of NEM which bound all cellular GSH or by incubation under CO. These data indicated that pyruvate stimulation of the HMPS occurs by pathways which do not involve peroxide formation, GSH, or oxygen. This study indicates that sulfhydryl blockade of GSH does not necessarily inhibit HMPS activity and that HMPS activity in red cells may respond to reactions not linked directly to glutathione reduction.     

Changes in protein turnover, IGF-I and IGF binding proteins in children with cancer

Changes in insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding proteins (IGFBPs) were correlated with protein synthesis and breakdown using [1- ¹³C]leucine before chemotherapy and during subsequent febrile neutropenia (FN) in eight children with cancer, aged 6.3–17.5 y. IGF-I levels were similar to age-matched controls before chemotherapy (mean ±SEM: 250 ±28 and 228 ±22 μg l^-1, respectively). During FN, IGF-I fell to 156 ±22 /ng l ^-1(p= 0:02), and rose to 276 ±27 μ g l ^-1 with recovery at 6 months (p = 0:004). Similarly, IGFBP-3 decreased from 4.0 ±0.2mgl^-1 before chemotherapy to 3.0 ±0.3 mgl^-1 during FN (p= 0:01), and returned to 4.1 ±0.2mgl ^-1 at 6 months (p= 0:01). IGF-I correlated with IGFBP-3 (r=+0:7, p <0:001). Scanning densitometry showed a decrease in IGFBP-3 from 94 to 54% during FN, when the presence of IGFBP-3 protease activity was observed. Compared with normal human serum, IGFBP-2 was elevated throughout the study. IGFBP-1 increased from 14.6 ±3.5 to 30.6 ±2.8/ngl^-1 (p = 0:004), whereas serum insulin decreased from 26.5 ±6.8 to 7.8 ±0.8 mUl^-1 (p= 0:03) before and during FN, respectively. Whilst IGF-I and IGFBP-3 fell, daytime growth hormone increased from 3.3 ±0.6 to 6.7±0.8mUl ^-1 (p= 0:01), and cortisol from 197 ±48 to 594±98nmoll ^-1 (p = 0:005). Albumin decreased from 47 ±2 to 38 ±2gl^-1 (p= 0:004) and improved to 47 ±2gl^-1 with recovery (p= 0:003). Protein synthesis increased from 4.5 ±0.4 to 5.0 ±0.6gkg^-1 d^-1 before chemotherapy and during FN, while protein breakdown rose from 5.4 ±0.4 to 6.3 ±0.4kg^-1d^-1. Increasing protein breakdown was related to falling IGF-I and IGFBP-3 levels. Modification of IGFBP-3 by circulating proteolytic activity may alter IGF bioavailability, allowing protein synthesis to increase during periods of severe catabolic stress.