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Biomedical subjects

Richard L Moss

Publications and source records attributed to Richard L Moss.

At least 19 recordsLinked to original sources
PubMed2006

Role of myosin heavy chain composition in the stretch activation response of rat myocardium.

The speed and force of myocardial contraction during systolic ejection is largely dependent on the intrinsic contractile properties of cardiac myocytes. As the myosin heavy chain (MHC) isoform of cardiac muscle is an important determinant of the contractile properties of individual myocytes, we studied the effects of altered MHC isoform expression in rat myocardium on the mechanical properties of skinned ventricular preparations. Skinned myocardium from thyroidectomized rats expressing only the beta MHC isoform displayed rates of force redevelopment that were about 2.5-fold slower than in myocardium from hyperthyroid rats expressing only the alpha MHC isoform, but the amount of force generated at a given level of Ca2+ activation was not different. Because recent studies suggest that the stretch activation response in myocardium has an important role in systolic function, we also examined the effect of MHC isoform expression on the stretch activation response by applying a rapid stretch (1% of muscle length) to an otherwise isometrically contracting muscle fibre. Sudden stretch of myocardium resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (i.e. stretch activation) to levels greater than prestretch force. beta MHC expression dramatically slowed the overall rate of the stretch activation response compared to expression of alpha MHC isoform; specifically, the rate of force decay was approximately 2-fold slower and the rate of delayed force development was approximately 2.5-fold slower. In contrast, MHC isoform had no effect on the amplitude of the stretch activation response. Collectively, these data show that expression of beta MHC in myocardium dramatically slows rates of cross-bridge recruitment and detachment which would be expected to decrease power output and contribute to depressed systolic function in end-stage heart failure.

Animals↗
PubMed2006

Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C.

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.

Animals↗
PubMed2006

Acceleration of stretch activation in murine myocardium due to phosphorylation of myosin regulatory light chain.

The regulatory light chains (RLCs) of vertebrate muscle myosins bind to the neck region of the heavy chain domain and are thought to play important structural roles in force transmission between the cross-bridge head and thick filament backbone. In vertebrate striated muscles, the RLCs are reversibly phosphorylated by a specific myosin light chain kinase (MLCK), and while phosphorylation has been shown to accelerate the kinetics of force development in skeletal muscle, the effects of RLC phosphorylation in cardiac muscle are not well understood. Here, we assessed the effects of RLC phosphorylation on force, and the kinetics of force development in myocardium was isolated in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC, subsequently skinned, and then treated with MLCK to phosphorylate RLC. Since RLC phosphorylation may be an important determinant of stretch activation in myocardium, we recorded the force responses of skinned myocardium to sudden stretches of 1% of muscle length both before and after treatment with MLCK. MLCK increased RLC phosphorylation, increased the Ca(2+) sensitivity of isometric force, reduced the steepness of the force-pCa relationship, and increased both Ca(2+)-activated and Ca(2+)-independent force. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force, i.e., stretch activation, to levels greater than pre-stretch force. MLCK had profound effects on the stretch activation responses during maximal and submaximal activations: the amplitude and rate of force decay after stretch were significantly reduced, and the rate of delayed force recovery was accelerated and its amplitude reduced. These data show that RLC phosphorylation increases force and the rate of cross-bridge recruitment in murine myocardium, which would increase power generation in vivo and thereby enhance systolic function.

Animals↗
PubMed2006

Ablation of cardiac myosin-binding protein-C accelerates stretch activation in murine skinned myocardium.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament accessory protein that binds tightly to myosin, but despite evidence that mutations in the cMyBP-C gene comprise a frequent cause of hypertrophic cardiomyopathy, relatively little is known about the role(s) of cMyBP-C in myocardium. Based on earlier studies demonstrating the potential importance of stretch activation in cardiac contraction, we examined the effects of cMyBP-C on the stretch activation responses of skinned ventricular preparations from wild-type (WT) and homozygous cMyBP-C knockout mice (cMyBP-C(-/-)) previously developed in our laboratory. Sudden stretch of skinned myocardium during maximal or submaximal Ca2+ activations resulted in an instantaneous increase in force that quickly decayed to a minimum and was followed by a delayed redevelopment of force (ie, stretch activation) to levels greater than prestretch force. Ablation of cMyBP-C dramatically altered the stretch activation response, ie, the rates of force decay and delayed force transient were accelerated compared with WT myocardium. These results suggest that cMyBP-C normally constrains the spatial position of myosin cross-bridges, which, in turn, limits both the rate and extent of interaction of cross-bridges with actin. We propose that ablation of cMyBP-C removes this constraint, increases the likelihood of cross-bridge binding to actin, and speeds the rate of delayed force development following stretch. Regardless of the specific mechanism, acceleration of cross-bridge cycling in cMyBP-C(-/-) myocardium could account for the abbreviation of systolic ejection in this mouse as a direct consequence of premature stretch activation of ventricular myocardium.

Animals↗
PubMed2006

Ablation of myosin-binding protein-C accelerates force development in mouse myocardium.

Myosin-binding protein-C (MyBP-C) is a thick filament-associated protein that binds tightly to myosin. Given that cMyBP-C may act to modulate cooperative activation of the thin filament by constraining the availability of myosin cross-bridges for binding to actin, we investigated the role of MyBP-C in the regulation of cardiac muscle contraction. We assessed the Ca(2+) sensitivity of force (pCa(50)) and the activation dependence of the rate of force redevelopment (k(tr)) in skinned myocardium isolated from wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) mice. Mechanical measurements were performed at 22 degrees C in the absence and presence of a strong-binding, nonforce-generating analog of myosin subfragment-1 (NEM-S1). In the absence of NEM-S1, maximal force and k(tr) and the pCa(50) of isometric force did not differ between WT and cMyBP-C(-/-) myocardium; however, ablation of cMyBP-C-accelerated k(tr) at each submaximal force. Treatment of WT and cMyBP-C(-/-) myocardium with 3 muM NEM-S1 elicited similar increases in pCa(50,) but the effects of NEM-S1 to increase k(tr) at submaximal forces and thereby markedly reduce the activation dependence of k(tr) occurred to a greater degree in cMyBP-C(-/-) myocardium. Together, these results support the idea that cMyBP-C normally acts to constrain the interaction between myosin and actin, which in turn limits steady-state force development and the kinetics of cross-bridge interaction.

Actins↗
PubMed2006

Cytoplasmic gamma-actin is not required for skeletal muscle development but its absence leads to a progressive myopathy.

Nonmuscle gamma(cyto)-actin is expressed at very low levels in skeletal muscle but uniquely localizes to costameres, the cytoskeletal networks that couple peripheral myofibrils to the sarcolemma. We generated and analyzed skeletal muscle-specific gamma(cyto)-actin knockout (Actg1-msKO) mice. Although muscle development proceeded normally, Actg1-msKO mice presented with overt muscle weakness accompanied by a progressive pattern of muscle fiber necrosis/regeneration. Functional deficits in whole-body tension and isometric twitch force were observed, consistent with defects in the connectivity between muscle fibers and/or myofibrils or at the myotendinous junctions. Surprisingly, gamma(cyto)-actin-deficient muscle did not demonstrate the fibrosis, inflammation, and membrane damage typical of several muscular dystrophies but rather presented with a novel progressive myopathy. Together, our data demonstrate an important role for minimally abundant but strategically localized gamma(cyto)-actin in adult skeletal muscle and describe a new mouse model to study the in vivo relevance of subcellular actin isoform sorting.

Actins↗
PubMed2006

Activation dependence of stretch activation in mouse skinned myocardium: implications for ventricular function.

Recent evidence suggests that ventricular ejection is partly powered by a delayed development of force, i.e., stretch activation, in regions of the ventricular wall due to stretch resulting from torsional twist of the ventricle around the apex-to-base axis. Given the potential importance of stretch activation in cardiac function, we characterized the stretch activation response and its Ca2+ dependence in murine skinned myocardium at 22 degrees C in solutions of varying Ca2+ concentrations. Stretch activation was induced by suddenly imposing a stretch of 0.5-2.5% of initial length to the isometrically contracting muscle and then holding the muscle at the new length. The force response to stretch was multiphasic: force initially increased in proportion to the amount of stretch, reached a peak, and then declined to a minimum before redeveloping to a new steady level. This last phase of the response is the delayed force characteristic of myocardial stretch activation and is presumably due to increased attachment of cross-bridges as a consequence of stretch. The amplitude and rate of stretch activation varied with Ca2+ concentration and more specifically with the level of isometric force prior to the stretch. Since myocardial force is regulated both by Ca2+ binding to troponin-C and cross-bridge binding to thin filaments, we explored the role of cross-bridge binding in the stretch activation response using NEM-S1, a strong-binding, non-force-generating derivative of myosin subfragment 1. NEM-S1 treatment at submaximal Ca2+-activated isometric forces significantly accelerated the rate of the stretch activation response and reduced its amplitude. These data show that the rate and amplitude of myocardial stretch activation vary with the level of activation and that stretch activation involves cooperative binding of cross-bridges to the thin filament. Such a mechanism would contribute to increased systolic ejection in response to increased delivery of activator Ca2+ during excitation-contraction coupling.

Actin Cytoskeleton↗
PubMed2006

Contributions of stretch activation to length-dependent contraction in murine myocardium.

The steep relationship between systolic force production and end diastolic volume (Frank-Starling relationship) in myocardium is a potentially important mechanism by which the work capacity of the heart varies on a beat-to-beat basis, but the molecular basis for the effects of myocardial fiber length on cardiac work are still not well understood. Recent studies have suggested that an intrinsic property of myocardium, stretch activation, contributes to force generation during systolic ejection in myocardium. To examine the role of stretch activation in length dependence of activation we recorded the force responses of murine skinned myocardium to sudden stretches of 1% of muscle length at both short (1.90 microm) and long (2.25 microm) sarcomere lengths (SL). Maximal Ca(2+)-activated force and Ca(2+) sensitivity of force were greater at longer SL, such that more force was produced at a given Ca(2+) concentration. Sudden stretch of myocardium during an otherwise isometric contraction resulted in a concomitant increase in force that quickly decayed to a minimum and was followed by a delayed development of force, i.e., stretch activation, to levels greater than prestretch force. At both maximal and submaximal activations, increased SL significantly reduced the initial rate of force decay following stretch; at submaximal activations (but not at maximal) the rate of delayed force development was accelerated. This combination of mechanical effects of increased SL would be expected to increase force generation during systolic ejection in vivo and prolong the period of ejection. These results suggest that sarcomere length dependence of stretch activation contributes to the steepness of the Frank-Starling relationship in living myocardium.

Animals↗
PubMed2005

Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition.

Rat cardiac titin undergoes developmental changes in isoform expression during the period from late embryonic through the first 20-25 days of life. At least five size classes of titin isoforms have been identified using SDS agarose gel electrophoresis. The longest normal isoform is expressed in the embryonic stages, and it is progressively replaced with increasingly smaller versions. The isoform switching is consistent with changes in resting tension from lower values in one-day neonates to higher levels in adult myocytes. Considerable micro-heterogeneity in alternative splicing patterns also was found, particularly in the N2BA PEVK region of human, rat, and dog ventricle. A rat mutation has been identified in which the embryonic-neonatal titin isoform transitions are markedly delayed. These mutant animals may prove useful for examining the role of titin in stretch-activated signal transduction and in the Frank-Starling relationship.

Alternative Splicing↗
PubMed2004

Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism.

Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeabilized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca2+ sensitivity of tension with the half-maximal response elicited at approximately 5 micromol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca2+ sensitivity.

Amino Acid Sequence↗
PubMed2004

Basal myosin light chain phosphorylation is a determinant of Ca2+ sensitivity of force and activation dependence of the kinetics of myocardial force development.

It is generally recognized that ventricular myosin regulatory light chains (RLC) are approximately 40% phosphorylated under basal conditions, and there is little change in RLC phosphorylation with agonist stimulation of myocardium or altered stimulation frequency. To establish the functional consequences of basal RLC phosphorylation in the heart, we measured mechanical properties of rat skinned trabeculae in which approximately 7% or approximately 58% of total RLC was phosphorylated. The protocol for achieving approximately 7% phosphorylation of RLC involved isolating trabeculae in the presence of 2,3-butanedione monoxime (BDM) to dephosphorylate RLC from its baseline level. Subsequent phosphorylation to approximately 58% of total was achieved by incubating BDM-treated trabeculae in solution containing smooth muscle myosin light chain kinase, calmodulin, and Ca2+ (i.e., MLCK treatment). After MLCK treatment, Ca2+ sensitivity of force increased by 0.06 pCa units and maximum force increased by 5%. The rate constant of force development (ktr) increased as a function of Ca2+ concentration in the range between pCa 5.8 and pCa 4.5. When expressed versus pCa, the activation dependence of ktr appeared to be unaffected by MLCK treatment; however, when activation was expressed in terms of isometric force-generating capability (as a fraction of maximum), MLCK treatment slowed ktr at submaximal activations. These results suggest that basal phosphorylation of RLC plays a role in setting the kinetics of force development and Ca2+ sensitivity of force in cardiac muscle. Our results also argue that changes in RLC phosphorylation in the range examined here influence actin-myosin interaction kinetics differently in heart muscle than was previously reported for skeletal muscle.

Animals↗
PubMed2004

Myosin crossbridge activation of cardiac thin filaments: implications for myocardial function in health and disease.

At the level of the myofibrillar proteins, activation of myocardial contraction is thought to involve switch-like regulation of crossbridge binding to the thin filaments. A central feature of this view of regulation is that Ca2+ binding to the low-affinity (approximately 3 micromol/L) site on troponin C alters the interactions of proteins in the thin filament regulatory strand, which leads to movement of tropomyosin from its blocking position on the thin filament and binding of crossbridges to actin. Although Ca2+ binding is a critical step in initiating contraction, this event alone does not account for the activation dependence of contractile properties of myocardium. Instead, activation is a highly cooperative process in which initial crossbridge binding to the thin filaments recruits additional crossbridge binding to actin as well as increased Ca2+ binding to troponin C. This review addresses possible roles of thin filament cooperativity in myocardium as a process that modulates the activation dependence of force and the rate of force development and also possible mechanisms by which cooperative signals are transmitted along the thick filament. Emerging evidence suggests that such mechanisms could contribute to the regulation of fundamental mechanical properties of myocardium and alterations in regulation that underlie contractile disorders in diseases such as cardiomyopathies.

Actin Cytoskeleton↗
PubMed2004

Expression of cardiac troponin T with COOH-terminal truncation accelerates cross-bridge interaction kinetics in mouse myocardium.

Transgenic mice expressing an allele of cardiac troponin T (cTnT) with a COOH-terminal truncation (cTnT(trunc)) exhibit severe diastolic and mild systolic dysfunction. We tested the hypothesis that contractile dysfunction in myocardium expressing low levels of cTnT(trunc) (i.e., <5%) is due to slowed cross-bridge kinetics and reduced thin filament activation as a consequence of reduced cross-bridge binding. We measured the Ca(2+) sensitivity of force development [pCa for half-maximal tension generation (pCa(50))] and the rate constant of force redevelopment (k(tr)) in cTnT(trunc) and wild-type (WT) skinned myocardium both in the absence and in the presence of a strong-binding, non-force-generating derivative of myosin subfragment-1 (NEM-S1). Compared with WT mice, cTnT(trunc) mice exhibited greater pCa(50), reduced steepness of the force-pCa relationship [Hill coefficient (n(H))], and faster k(tr) at submaximal Ca(2+) concentration ([Ca(2+)]), i.e., reduced activation dependence of k(tr). Treatment with NEM-S1 elicited similar increases in pCa(50) and similar reductions in n(H) in WT and cTnT(trunc) myocardium but elicited greater increases in k(tr) at submaximal activation in cTnT(trunc) myocardium. Contrary to our initial hypothesis, cTnT(trunc) appears to enhance thin filament activation in myocardium, which is manifested as significant increases in Ca(2+)-activated force and the rate of cross-bridge attachment at submaximal [Ca(2+)]. Although these mechanisms would not be expected to depress systolic function per se in cTnT(trunc) hearts, they would account for slowed rates of myocardial relaxation during early diastole.

Actin Cytoskeleton↗
PubMed2004

Role of cardiac myosin binding protein C in sustaining left ventricular systolic stiffening.

Despite advances in the molecular biology of cardiac myosin binding protein-C (cMyBP-C), little is understood about its precise role in muscle contraction, particularly in the intact heart. We tested the hypothesis that cMyBP-C is central to the time course and magnitude of left ventricular systolic elastance (chamber stiffening), and assessed mechanisms for this influence in intact hearts, trabeculae, and skinned fibers from wild-type (+/+) and homozygous truncated cMyBP-C (t/t) male mice. cMyBP-C protein was not detected by gel electrophoresis or Western blot in t/t myocardium. cMyBP-C t/t ventricles displayed reduced peak elastance, but more strikingly a marked abbreviation of the systolic elastance time course, which peaked earlier (27.6+/-2.1 ms) than in +/+ controls (47.8+/-1.6 ms). Control hearts reached only 42+/-4% of maximum elastance at the onset of ejection, with substantial further stiffening during ejection. This contrasted to t/t mutants, which reached 77+/-3% of peak elastance before ejection of peak. These unusual findings were not observed in alternative models involving severe cardiomyopathy, but were recapitulated in a cMyBP-C null mouse. The abbreviated elastance time course and lower peak were consistent with earlier time-to-peak trabecular tension, increased unloaded shortening velocity in t/t skinned muscle strips, and dramatically reduced myofilament stiffness at diastolic calcium concentrations. These results provide novel insights into the role of cMyBP-C in myocardial systolic mechanics. Abnormal sarcomere shortening velocity and abbreviated muscle stiffening may underlie development of cardiac dysfunction associated with deficient incorporation of cMyBP-C.

Animals↗
PubMed2004

Titin isoform changes in rat myocardium during development.

Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710 kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990 kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1 microm, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.

Alternative Splicing↗
PubMed2003

Loaded shortening, power output, and rate of force redevelopment are increased with knockout of cardiac myosin binding protein-C.

Myosin binding protein-C (MyBP-C) is localized to the thick filaments of striated muscle where it appears to have both structural and regulatory functions. Importantly, mutations in the cardiac MyBP-C gene are associated with familial hypertrophic cardiomyopathy. The purpose of this study was to examine the role that MyBP-C plays in regulating force, power output, and force development rates in cardiac myocytes. Skinned cardiac myocytes from wild-type (WT) and MyBP-C knockout (MyBP-C-/-) mice were attached between a force transducer and position motor. Force, loaded shortening velocities, and rates of force redevelopment were measured during both maximal and half-maximal Ca2+ activations. Isometric force was not different between the two groups with force being 17.0+/-7.2 and 20.5+/-3.1 kN/m2 in wild-type and MyBP-C-/- myocytes, respectively. Peak normalized power output was significantly increased by 26% in MyBP-C-/- myocytes (0.15+/-0.01 versus 0.19+/-0.03 P/Po x ML/sec) during maximal Ca2+ activations. Interestingly, peak power output in MyBP-C-/- myocytes was increased to an even greater extent (46%, 0.09+/-0.03 versus 0.14+/-0.02 P/Po x ML/sec) during half-maximal Ca2+ activations. There was also an effect on the rate constant of force redevelopment (ktr) during half-maximal Ca2+ activations, with ktr being significantly greater in MyBP-C-/- myocytes (WT=5.8+/-0.9 s(-1) versus MyBP-C-/-=7.7+/-1.7 s(-1)). These results suggest that cMyBP-C is an important regulator of myocardial work capacity whereby MyBP-C acts to limit power output.

Animals↗