Peter VanBuren

Cardiac V1 And V3 Myosins Differ in Their Hydrolytic and Mechanical Activities In Vitro

The two mammalian cardiac myosin heavy chain isoforms, alpha and beta, have 93% amino acid homology, but hearts expressing these myosins exhibit marked differences in their mechanical activities. To further understand the function of these cardiac myosins as molecular motors, we compared the ability of these myosins to hydrolyze ATP and to both translocate actin filaments and generate force in an in vitro motility assay. V1 myosin has twice the actin-activated ATPase activity and three times the actin filament sliding velocity when compared with V3 myosin. In contrast, the force-generating ability of these myosins is quite different when the total force produced by a small population of myosin molecules (> 50) is examined. V1 myosin produces only one half the average cross-bridge force of V3 myosin. With discrete areas of primary structural heterogeneity known to exist between alpha and beta heavy chains, the differences we report in the hydrolytic and mechanical activities of the motors are explored in the context of potential structural and kinetic differences between the V1 and V3 myosins.

C-Terminal Truncation of Cardiac Troponin I Causes Divergent Effects on ATPase and Force Implications for the Pathophysiology of Myocardial Stunning

Abstract— Myocardial stunning is a form of reversible myocardial ischemia/reperfusion injury associated with systolic and diastolic contractile dysfunction. In the isolated rat heart model, myocardial stunning is characterized by specific C-terminal proteolysis of the myofilament protein, troponin I (cTnI) that yields cTnI1-193. To determine the effect of this particular C-terminal truncation of cTnI, without the confounding factor of other stunning-induced protein modifications, a series of solution biochemical assays has been undertaken using the human homologue of mouse/rat cTnI1-193, cTnI1-192. Affinity chromatography and actin sedimentation experiments detected little, or no, difference between the binding of cTnI (cTnI1-209) and cTnI1-192 to actin-tropomyosin, troponin T, or troponin C. Both cTnI and cTnI1-192 inhibit the actin-tropomyosin–activated ATPase activity of myosin subfragment 1 (S1), and this inhibition is released by troponin C in the presence of Ca2+. However, cTnI1-192, when reconstituted as part of the troponin complex (cTn1-192), caused a 54±11% increase in the maximum Ca2+-activated actin-tropomyosin-S1 ATPase activity, compared with troponin reconstituted with cTnI (cTn). Furthermore, cTn1-192 increased Ca2+ sensitivity of both the actin-tropomyosin-activated S1 ATPase activity and the Ca2+-dependent sliding velocity of reconstituted thin filaments, in an in vitro motility assay, compared with cTn. In an in vitro force assay, the actin-tropomyosin filaments bearing cTn1-192 developed only 76±4% (P <0.001) of the force obtained with filaments composed of reconstituted cTn. We suggest that cTnI proteolysis may contribute to the pathophysiology of myocardial stunning by altering the Ca2+-sensing and chemomechanical properties of the myofilaments.

Thin Filament-Based Modulation of Contractile Performance in Human Heart Failure

Background—The contribution of the sarcomere’s thin filament to the contractile dysfunction of human cardiomyopathy is not well understood. Methods and Results—We have developed techniques to isolate and functionally characterize intact (native) thin filaments obtained from failing and nonfailing human ventricular tissue. By use of in vitro motility and force assays, native thin filaments from failing ventricular tissue exhibited a 19% increase in maximal velocity but a 27% decrease in maximal contractile force compared with nonfailing myocardium. Native thin filaments isolated from human myocardium after left ventricular assist device support demonstrated a 37% increase in contractile force. Dephosphorylation of failing native thin filaments resulted in a near-normalization of thin-filament function, implying a phosphorylation-mediated mechanism. Tissue expression of the protein kinase C isoforms &agr;, &bgr;1, and &bgr;2 was increased in failing human myocardium and reduced after left ventricular assist device support. Conclusions—These novel findings demonstrate that (1) the thin filament is a key modulator of contractile performance in the failing human heart, (2) thin-filament function is restored to near normal levels after LVAD support, and (3) the alteration of thin-filament function in failing human myocardium is mediated through phosphorylation, most likely through activation of protein kinase C.

Myosin from failing and non-failing human ventricles exhibit similar contractile properties.

In non-failing human myocardium, V1 myosin comprises a small amount (<10%) of the total myosin content, whereas end-stage failing hearts contain nearly 100% V3 myosin. It has been suggested that this shift in V1 myosin isoform content may contribute to the contractile deficit in human myocardial failure. To test this hypothesis, myosin was isolated from human failing and non-failing ventricles, and non-failing atria. Performance was assessed in in vitro motility and isometric force assays. Consistent with prior reports, a small amount of V1 myosin was present in both non-failing (6.2 +/- 1.0%) and failing (3.5 +/- 1.4%) ventricular tissues. No difference in isometric force or unloaded shortening velocity was observed for failing and non-failing ventricular myosin irrespective of myosin isoform content. Atrial tissue expressing predominantly V1 myosin (66.7 +/- 4.1%) generated half the force but greater velocity compared with ventricular tissue, expressing predominantly V3 myosin. In additional experiments, rabbit cardiac myosin was used in a calcium regulated assay system to determine if V1 and V3 isoforms differentially affect thin filament activation. Half-maximal calcium activation was similar for the two cardiac isoforms. A 1:9 mixture of V1/V3 myosin, simulating isoform composition in non-failing human myocardium, was indistinguishable from 100% V3 myosin (simulating the failing state) with regard to velocity of shortening and average force. These data suggest that the myosin isoform shift reported in human myocardial failure does not significantly contribute to the contractile deficit of this disease.

Effect of Resistance Training on Physical Disability in Chronic Heart Failure

PURPOSE Patients with chronic heart failure (CHF) report difficulty performing activities of daily living. To our knowledge, however, no study has directly measured performance in activities of daily living in these patients to systematically assess their level of physical disability. Moreover, the contribution of skeletal muscle weakness to physical disability in CHF remains unclear. Thus, we measured performance in activities of daily living in CHF patients and controls, its relationship to aerobic capacity and muscle strength, and the effect of resistance exercise training to improve muscle strength and physical disability. METHODS Patients and controls were assessed for performance in activities of daily living, self-reported physical function, peak aerobic capacity, body composition, and muscle strength before and after an 18-wk resistance training program. To remove the confounding effects of several disease-related factors (muscle disuse, hospitalization, acute illness), we recruited controls with similar activity levels as CHF patients and tested patients >6 months after any disease exacerbation/hospitalization. RESULTS Performance in activities of daily living was 30% lower (P < 0.05) in CHF patients versus controls and was related to both reduced aerobic capacity (P < 0.001) and muscle strength (P < 0.01). Moreover, resistance training improved (P < 0.05 to P < 0.001) physical function and muscle strength in patients and controls similarly, without altering aerobic capacity. CONCLUSIONS CHF patients are characterized by marked physical disability compared with age- and physical activity-matched controls, which is related to reduced aerobic capacity and muscle strength. CHF patients respond to resistance training with normal strength/functional adaptations. Our results support muscle weakness as a determinant of physical disability in CHF and show that interventions that increase muscle strength (resistance training) reduce physical disability.

Chronic heart failure decreases cross-bridge kinetics in single skeletal muscle fibres from humans

Skeletal muscle function is impaired in heart failure patients due, in part, to loss of myofibrillar protein content, in particular myosin. In the present study, we utilized small‐amplitude sinusoidal analysis for the first time in single human skeletal muscle fibres to measure muscle mechanics, including cross‐bridge kinetics, to determine if heart failure further impairs contractile performance by altering myofibrillar protein function. Patients with chronic heart failure (n= 9) and controls (n= 6) were recruited of similar age and physical activity to diminish the potentially confounding effects of ageing and muscle disuse. Patients showed decreased cross‐bridge kinetics in myosin heavy chain (MHC) I and IIA fibres, partially due to increased myosin attachment time (ton). The increased ton compensated for myosin protein loss previously found in heart failure patients by increasing the fraction of the total cycle time myosin is bound to actin, resulting in a similar number of strongly bound cross‐bridges in patients and controls. Accordingly, isometric tension did not differ between patients and controls in MHC I or IIA fibres. Patients also had decreased calcium sensitivity in MHC IIA fibres and alterations in the viscoelastic properties of the lattice structure of MHC I and IIA fibres. Collectively, these results show that heart failure alters skeletal muscle contraction at the level of the myosin–actin cross‐bridge, leading to changes in muscle mechanics which could contribute to impaired muscle function. Additionally, we uncovered a unique kinetic property of MHC I fibres, a potential indication of two distinct populations of cross‐bridges, which may have important physiological consequences.

Mechanisms Underlying Skeletal Muscle Weakness in Human Heart Failure: alterations in Single Fiber Myosin Protein Content and Function

Background— Patients with chronic heart failure (HF) frequently experience skeletal muscle weakness that limits physical function. The mechanisms underlying muscle weakness, however, have not been clearly defined. Methods and Results— This study examined the hypothesis that HF promotes a loss of myosin protein from single skeletal muscle fibers, which in turn reduces contractile performance. Ten patients with chronic HF and 10 controls were studied. Muscle atrophy was not evident in patients, and groups displayed similar physical activity levels, suggesting that observed differences reflect the effects of HF and not muscle atrophy or disuse. In single muscle fibers, patients with HF showed reduced myosin heavy chain protein content ( P <0.05) that manifested as a reduction in functional myosin-actin cross-bridges ( P <0.05). No evidence was found for a generalized loss of myofilament protein, suggesting a selective loss of myosin. Accordingly, single muscle fiber maximal Ca2+-activated tension was reduced in myosin heavy chain I fibers in patients ( P <0.05). However, tension was maintained in myosin heavy chain IIA fibers in patients because a greater proportion of available myosin heads were bound to actin during Ca2+ activation ( P <0.01). Conclusions— Collectively, our results show that HF alters the quantity and functionality of the myosin molecule in skeletal muscle, leading to reduced tension in myosin heavy chain I fibers. Loss of single fiber myosin protein content represents a potential molecular mechanism underlying muscle weakness and exercise limitation in patients with HF. Received April 29, 2009; accepted August 7, 2009.Background—Patients with chronic heart failure (HF) frequently experience skeletal muscle weakness that limits physical function. The mechanisms underlying muscle weakness, however, have not been clearly defined. Methods and Results—This study examined the hypothesis that HF promotes a loss of myosin protein from single skeletal muscle fibers, which in turn reduces contractile performance. Ten patients with chronic HF and 10 controls were studied. Muscle atrophy was not evident in patients, and groups displayed similar physical activity levels, suggesting that observed differences reflect the effects of HF and not muscle atrophy or disuse. In single muscle fibers, patients with HF showed reduced myosin heavy chain protein content (P<0.05) that manifested as a reduction in functional myosin-actin cross-bridges (P<0.05). No evidence was found for a generalized loss of myofilament protein, suggesting a selective loss of myosin. Accordingly, single muscle fiber maximal Ca2+-activated tension was reduced in myosin heavy chain I fibers in patients (P<0.05). However, tension was maintained in myosin heavy chain IIA fibers in patients because a greater proportion of available myosin heads were bound to actin during Ca2+ activation (P<0.01). Conclusions—Collectively, our results show that HF alters the quantity and functionality of the myosin molecule in skeletal muscle, leading to reduced tension in myosin heavy chain I fibers. Loss of single fiber myosin protein content represents a potential molecular mechanism underlying muscle weakness and exercise limitation in patients with HF.

Human actin mutations associated with hypertrophic and dilated cardiomyopathies demonstrate distinct thin filament regulatory properties in vitro

Two cardiomyopathic mutations were expressed in human cardiac actin, using a Baculovirus/insect cell system; E99K is associated with hypertrophic cardiomyopathy whereas R312H is associated with dilated cardiomyopathy. The hypothesis that the divergent phenotypes of these two cardiomyopathies are associated with fundamental differences in the molecular mechanics and thin filament regulation of the underlying actin mutation was tested using the in vitro motility and laser trap assays. In the presence of troponin (Tn) and tropomyosin (Tm), beta-cardiac myosin moved both E99K and R312H thin filaments at significantly (p<0.05) slower velocities than wild type (WT) at maximal Ca(++). At submaximal Ca(++), R312H thin filaments demonstrated significantly increased Ca(++) sensitivity (pCa(50)) when compared to WT. Velocity as a function of ATP concentration revealed similar ATP binding rates but slowed ADP release rates for the two actin mutants compared to WT. Single molecule laser trap experiments performed using both unregulated (i.e. actin) and regulated thin filaments in the absence of Ca(++) revealed that neither actin mutation significantly affected the myosins unitary step size (d) or duration of strong actin binding (t(on)) at 20 microM ATP. However, the frequency of individual strong-binding events in the presence of Tn and Tm, was significantly lower for E99K than WT at comparable myosin surface concentrations. The cooperativity of a second myosin head binding to the thin filament was also impaired by E99K. In conclusion, E99K inhibits the activation of the thin filament by myosin strong-binding whereas R312H demonstrates enhanced calcium activation.

Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay.

The modulatory role of whole cardiac myosin binding protein-C (cMyBP-C) on myosin force and motion generation was assessed in an in vitro motility assay. The presence of cMyBP-C at an approximate molar ratio of cMyBP-C to whole myosin of 1:2, resulted in a 25% reduction in thin filament velocity (P<0.002) with no effect on relative isometric force under maximally activated conditions (pCa 5). Cardiac MyBP-C was capable of inhibiting actin filament velocity in a concentration-dependent manner using either whole myosin, HMM or S1, indicating that the cMyBP-C does not have to bind to myosin LMM or S2 subdomains to exert its effect. The reduction in velocity by cMyBP-C was independent of changes in ionic strength or excess inorganic phosphate. Co-sedimentation experiments demonstrated S1 binding to actin is reduced as a function of cMyBP-C concentration in the presence of ATP. In contrast, S1 avidly bound to actin in the absence of ATP and limited cMyBP-C binding, indicating that cMyBP-C and S1 compete for actin binding in an ATP-dependent fashion. However, based on the relationship between thin filament velocity and filament length, the cMyBP-C induced reduction in velocity was independent of the number of cross-bridges interacting with the thin filament. In conclusion, the effects of cMyBP-C on velocity and force at both maximal and submaximal activation demonstrate that cMyBP-C does not solely act as a tether between the myosin S2 and LMM subdomains but likely affects both the kinetics and recruitment of myosin cross-bridges through its direct interaction with actin and/or myosin head.

Resistance training alters skeletal muscle structure and function in human heart failure: effects at the tissue, cellular and molecular levels

•  Individuals suffering from chronic heart failure are less able to perform everyday tasks. •  This physical disability is explained, in part, by muscle weakness secondary to alterations in the proteins in muscles that are necessary for muscle contraction (myofilament proteins). •  Weight training exercise increases muscle strength and physical function in heart failure patients, but the mechanisms of these improvements is uncertain. •  We show that resistance training improves muscle strength through increased function of myofilament proteins. •  These studies are important because they identify the molecular and cellular mechanisms whereby this type of training may promote beneficial changes in physical function in elderly individuals with heart failure.