Motivated by the need for an analytical tool that can be used routinely to analyze data collected from isolated, detergent-skinned cardiac muscle fibers, we developed a mathematical model for representing the force response to step changes in muscle length (i.e., quick stretch and release). Our proposed model is reasonably simple, consisting of only five parameters representing: (1) the rate constant by which length change–induced distortion of elastic elements is dissipated; (2) the stiffness of the muscle fiber; (3) the amplitude of length-mediated recruitment of stiffness elements; (4) the rate constant by which this length-mediated recruitment takes place; and (5) the magnitude of the nonlinear interaction term by which distortion of elastic elements affects the number of recruited stiffness elements. Fitting this model to a family of force recordings representing responses to eight amplitudes of step length change (±2.0% baseline muscle length in 0.5% increments) enabled four things: (1) reproduction of all the identifiable features seen in a family of force responses to both positive and negative length changes; (2) close fitting of all records from the whole family of these responses with very little residual error; (3) estimation of all five model parameters with a great degree of certainty; and (4) importantly, ready discrimination between cardiac muscle fibers with different contractile regulatory proteins but showing only subtly different contractile function. We recommend this mathematical model as an analytic tool for routine use in studies of cardiac muscle fiber contractile function. Such model-based analysis gives novel insight to the contractile behavior of cardiac muscle fibers, and it is useful for characterizing the mechanistic effects that alterations of cardiac contractile proteins have on cardiac contractile function.

Nebulin is a giant filamentous F-actin-binding protein (ϳ800 kDa) that binds along the thin filament of the skeletal muscle sarcomere. Nebulin is one of the least well understood major muscle proteins. Although nebulin is usually viewed as a structural protein, here we investigated whether nebulin plays a role in muscle contraction by using skinned muscle fiber bundles from a nebulin knock-out (NEB KO) mouse model. We measured force-pCa (؊log[Ca 2؉ ]) and force-ATPase relations, as well as the rate of tension re-development (k tr ) in tibialis cranialis muscle fibers. To rule out any alterations in troponin (Tn) isoform expression and/or status of Tn phosphorylation, we studied fiber bundles that had been reconstituted with bacterially expressed fast skeletal muscle recombinant Tn. We also performed a detailed analysis of myosin heavy chain, myosin light chain, and myosin light chain 2 phosphorylation, which showed no significant differences between wild type and NEB KO. Our mechanical studies revealed that NEB KO fibers had increased tension cost (5.9 versus 4.4 pmol millinewtons ؊1 mm ؊1 s ؊1 ) and reductions in k tr (4.7 versus 7.3 s ؊1 ), calcium sensitivity (pCa 50 5.74 versus 5.90), and cooperativity of activation (n H 3.64 versus 4.38). Our findings indicate the following: 1) in skeletal muscle nebulin increases thin filament activation, and 2) through altering cross-bridge cycling kinetics, nebulin increases force and efficiency of contraction. These novel properties of nebulin add a new level of understanding of skeletal muscle function and provide a mechanism for the severe muscle weakness in patients with nebulin-based nemaline myopathy.Muscle contraction is based on cyclic interactions between the myosin cross-bridges that are part of the thick filaments and actin, the major protein of the thin filament (1). The thin filament contains troponin C (TnC), 2 troponin I (TnI), and troponin T (TnT), which together make up the troponin (Tn) complex. The Tn complex and tropomyosin (Tm) together participate in the calcium-dependent regulation of thin filament activation (2). In addition to Tn and Tm, thin filaments of vertebrate skeletal muscle also contain nebulin (3). Nebulin is a giant filamentous protein that spans the entire thin filament, with its C terminus anchored in the Z-disk and its N-terminal region located near the thin filament pointed end (4, 5). The majority of the nebulin sequence is composed of ϳ35-amino acid modules, with the central module 9 (M9) and module 162 (M162) modules arranged into seven-module super-repeats (6 -8). This arrangement enables a single nebulin module to interact with a single actin monomer, and each nebulin super-repeat to associate with a single Tm⅐Tn complex (8 -10). Previous work has shown that nebulin plays structural roles (11,12), and this work is focused on the role of nebulin in regulating muscle contraction.Evidence for the role of nebulin in regulating thin filament activation has been obtained in the following: 1) in in vitro studies that showed that nebul...

Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in vitro motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured ktr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (krel) and XB recruitment (kdf) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow β-myosin heavy chain (β-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca2+] activations, suggesting that OM cooperatively recruits additional XB’s into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by ktr were not observed, and there was a significant OM-induced decrease in krel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in krel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the kdf was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB’s at low Ca2+-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.

Key pointsr Phosphorylation of cardiac myosin binding protein-C Ser282 has been proposed to modulate the phosphorylation of Ser273 and Ser302, and thereby the contractile response to increased β-adrenergic stimulation, yet the precise functional role of Ser282 is unknown.r Protein kinase A phosphorylation of Ser273 and Ser302 was unaffected by Ser282 phospho-ablation, suggesting that Ser282 phosphorylation is not required for full phosphorylation of neighbouring residues.r Mice with Ser282 phospho-ablation (TG S282A ) displayed normal basal in vivo cardiac function but impaired rates of pressure development in response to β-adrenergic stimulation.r Basal rates of cross-bridge kinetics were unaffected by Ser282 phospho-ablation; however, the protein kinase A-mediated acceleration of cross-bridge recruitment was blunted in TG S282A myocardium.r Collectively, our data suggests that Ser282 phosphorylation is critical to achieve complete acceleration of cardiac contractile function in response to increased β-adrenergic stimulation, but also implicates Ser273 and Ser302 phosphorylation as important modulators of the cardiac myosin binding protein-C-mediated contractile response.Abstract Cardiac myosin binding protein-C phosphorylation plays an important role in modulating cardiac muscle function and accelerating contraction. It has been proposed that Ser282 phosphorylation may serve as a critical molecular switch that regulates the phosphorylation of neighbouring Ser273 and Ser302 residues, and thereby govern myofilament contractile acceleration in response to protein kinase A (PKA). Therefore, to determine the regulatory roles of Ser282 we generated a transgenic (TG) mouse model expressing cardiac myosin binding protein-C with a non-phosphorylatable Ser282 (i.e. serine to alanine substitution, TG S282A ). Myofibrils isolated from TG S282A hearts displayed robust PKA-mediated phosphorylation of Ser273 and Ser302, and the increase in phosphorylation was identical to TG wild-type (TG WT ) controls. No signs of pathological cardiac hypertrophy were detected in TG S282A hearts by either histological examination of cardiac sections or echocardiography. Baseline fractional shortening, ejection fraction, isovolumic relaxation time, rate of pressure development and rate of relaxation (τ) were unaltered in TG S282A mice. However, the increase in cardiac contractility as well as the acceleration of pressure development observed in response to β-adrenergic stimulation was attenuated in TG S282A mice. In agreement with our in vivo data, in vitro force measurements revealed that PKA-mediated acceleration of cross-bridge kinetics in TG S282A myocardium was significantly attenuated compared to TG WT myocardium. Taken together, our data suggest that Abbreviations cMyBP-C, cardiac myosin binding protein-C; dP/dt, rate of LV pressure development; dP/dt max , peak rate of left ventricular pressure development; EDP, end-diastolic pressure; F max , maximum Ca 2+ activated force; F min , Ca 2+ independent force; H&E, haematoxylin and eosin...

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is an important regulator of contractile function, however, its contributions to length-dependent changes in cross-bridge (XB) kinetics is unknown. Therefore, we performed mechanical experiments to quantify contractile function in detergent-skinned ventricular preparations isolated from wild-type (WT) hearts, and hearts expressing non-phosphorylatable cMyBP-C [Ser to Ala substitutions at residues Ser273, Ser282, and Ser302 (i.e., 3SA)], at sarcomere length (SL) 1.9 μm or 2.1μm, prior and following protein kinase A (PKA) treatment. Steady-state force generation measurements revealed a blunting in the length-dependent increase in myofilament Ca2+-sensitivity of force generation (pCa50) following an increase in SL in 3SA skinned myocardium compared to WT skinned myocardium. Dynamic XB behavior was assessed at submaximal Ca2+-activations by imposing an acute rapid stretch of 2% of initial muscle length, and measuring both the magnitudes and rates of resultant phases of force decay due to strain-induced XB detachment and delayed force rise due to recruitment of additional XBs with increased SL (i.e., stretch activation). The magnitude (P2) and rate of XB detachment (krel) following stretch was significantly reduced in 3SA skinned myocardium compared to WT skinned myocardium at short and long SL, and prior to and following PKA treatment. Furthermore, the length-dependent acceleration of krel due to decreased SL that was observed in WT skinned myocardium was abolished in 3SA skinned myocardium. PKA treatment accelerated the rate of XB recruitment (kdf) following stretch at both SL's in WT but not in 3SA skinned myocardium. The amplitude of the enhancement in force generation above initial pre-stretch steady-state levels (P3) was not different between WT and 3SA skinned myocardium at any condition measured. However, the magnitude of the entire delayed force phase which can dip below initial pre-stretch steady-state levels (Pdf) was significantly lower in 3SA skinned myocardium under all conditions, in part due to a reduced magnitude of XB detachment (P2) in 3SA skinned myocardium compared to WT skinned myocardium. These findings demonstrate that cMyBP-C phospho-ablation regulates SL- and PKA-mediated effects on XB kinetics in the myocardium, which would be expected to contribute to the regulation of the Frank-Starling mechanism.