pCa-tension Relationship Research Articles

P90RSK2, a new MLCK mediates contractility in myosin light chain kinase null smooth muscle.

Introduction: Phosphorylation of smooth muscle (SM) myosin regulatory light chain (RLC20) is a critical switch leading to SM contraction. The canonical view held that only the short isoform of myosin light chain kinase (MLCK1) catalyzed this reaction. It is now accepted that auxiliary kinases may contribute to vascular SM tone and contractility. We have previously reported that p90 ribosomal S6 kinase (RSK2) functions as such a kinase, in parallel with MLCK1, contributing ∼25% of the maximal myogenic force in resistance arteries. Thus, RSK2 may be instrumental in the regulation of basal vascular tone and blood pressure. Here, we take advantage of a MLCK1 null mouse (mylk1 -/-) to further test our hypothesis that RSK2 can function as an MLCK, playing a significant physiological role in SM contractility. Methods: Using fetal (E14.5-18.5) SM tissues, as embryos die at birth, we investigated the necessity of MLCK for contractility and fetal development and determined the ability of RSK2 kinase to compensate for the lack of MLCK and characterized its signaling pathway in SM. Results and Discussion: Agonists induced contraction and RLC20 phosphorylation in mylk1 -/- SM was attenuated by RSK2 inhibition. The pCa-tension relationships in permeabilized strips of bladder showed no difference in Ca2+ sensitivity in WT vs mylk1 -/- muscles, although the magnitude of force responses was considerably smaller in the absence of MLCK. The magnitude of contractile responses was similar upon addition of GTPγS to activate the RhoA/ROCK pathway or calyculinA to inhibit the myosin phosphatase. The Ca2+-dependent tyrosine kinase, Pyk2, contributed to RSK2-mediated contractility and RLC20 phosphorylation. Proximity-ligation and immunoprecipitation assays demonstrated an association of RSK2, PDK1 and ERK1/2 with MLCK and actin. RSK2, PDK1, ERK1/2 and MLCK formed a signaling complex on the actin filament, positioning them for interaction with adjacent myosin heads. The Ca2+-dependent component reflected the agonist mediated increases in Ca2+, which activated the Pyk2/PDK1/RSK2 signaling cascade. The Ca2+-independent component was through activation of Erk1/2/PDK1/RSK2 leading to direct phosphorylation of RLC20, to increase contraction. Overall, RSK2 signaling constitutes a new third signaling pathway, in addition to the established Ca2+/CaM/MLCK and RhoA/ROCK pathways to regulate SM contractility.

Myosin Rod Hypophosphorylation and CB Kinetics in Papillary Muscles from a TnC-A8V KI Mouse Model

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca2+-binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca2+-sensitivity and a decrease (0.8×) in cooperativity (nH) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP (K1) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate (k2) and its equilibrium constant (K2) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step (k4) decreased (0.5×), reversal step (k−4) increased (2×), and the phosphate-release step (1/K5) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the α-myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

Functional Significance of C-Terminal Mobile Domain of Cardiac Troponin I

C-terminal mobile domain of cTnI (cTnI-MD, residues 168-210) is a highly conserved region which plays important roles in regulating myofilament function. Due to the unresolved structure of the region, it is still unclear how cTnI-MD plays a role in modulating the sarcomere length (SL)-dependent and Ca2+-induced effects on cardiac contractility. In this study, the functional significance of the MD in contractile behavior of skinned rat papillary muscle fibers and SL dependent activation was studied by truncation of the entire MD (cTnI(1-167)) and the C-terminus of MD (cTnI(1-193)). We hypothesized that different truncations may have different impacts on SL-dependent Ca2+-regulation of tension development and cross-bridge cycling. To test this hypothesis, the tension-pCa relationships and myosin cross-bridge kinetics were measured as cTnI(1-167) and cTnI(1-193) were exchanged into skinned rat papillary muscle fibers at short (SL=1.9µm) and long SLs (SL=2.2µm). Our results indicate that the SL induced enhancement of Ca2+-sensitivity and cooperativity observed in wild-type fibers is preserved in cTnI(1-193) and is diminished in cTnI(1-167) fibers. cTnI(1-167) truncation resulted in enhanced maximum tension and cooperativity of Ca2+-activation compared to cTnI(WT) and cTnI(1-193) and enhanced Ca2+-sensitivity compared to cTnI(WT) at both short and long SLs. cTnI(1-193) truncation resulted in enhanced myofilament Ca2+-sensitivity at both SLs and a depressed maximum tension just at long SL. Both cTnI-MD truncations showed a faster cross-bridge attachment rate at both SLs. Compared to cTnI(WT), cTnI(1-167) showed a slower cross-bridge detachment rate while cTnI(1-193) showed a faster cross-bridge detachment rate at long SL. The results of our study indicate that the truncation of cTnI-MD at different regions causes destabilization of the closed-state position of tropomyosin suggested by the observed increase in the cross-bridge attachment rate to thin filament as well as Ca2+-sensitivity of tension development at both sarcomere lengths.

The effects of pH and Pi on tension and Ca2+ sensitivity of ventricular myofilaments from the anoxia-tolerant painted turtle

We aimed to determine how increases in intracellular H+ and inorganic phosphate (Pi) to levels observed during anoxic submergence affect contractility in ventricular muscle of the anoxia-tolerant Western painted turtle, Chrysemys picta bellii Skinned multicellular preparations were exposed to six treatments with physiologically relevant levels of pH (7.4, 7.0, 6.6) and Pi (3 and 8 mmol l-1). Each preparation was tested in a range of calcium concentrations (pCa 9.0-4.5) to determine the pCa-tension relationship for each treatment. Acidosis significantly decreased contractility by decreasing Ca2+ sensitivity (pCa50) and tension development (P<0.001). Increasing [Pi] also decreased contractility by decreasing tension development at every pH level (P<0.001) but, alone, did not affect Ca2+ sensitivity (P=0.689). Simultaneous increases in [H+] and [Pi] interacted to attenuate the decreased tension development and Ca2+ sensitivity (P<0.001), possibly reflecting a decreased sensitivity to Pi when it is present as the dihydrogen phosphate form, which increases as pH decreases. Compared with that of mammals, the ventricle of turtles exhibits higher Ca2+ sensitivity, which is consistent with previous studies of ectothermic vertebrates.

Increased Titin Compliance Reduced Length-Dependent Contraction and Slowed Cross-Bridge Kinetics in Skinned Myocardial Strips from Rbm (20ΔRRM) Mice.

Titin is a giant protein spanning from the Z-disk to the M-band of the cardiac sarcomere. In the I-band titin acts as a molecular spring, contributing to passive mechanical characteristics of the myocardium throughout a heartbeat. RNA Binding Motif Protein 20 (RBM20) is required for normal titin splicing, and its absence or altered function leads to greater expression of a very large, more compliant N2BA titin isoform in Rbm20 homozygous mice (Rbm20ΔRRM) compared to wild-type mice (WT) that almost exclusively express the stiffer N2B titin isoform. Prior studies using Rbm20ΔRRM animals have shown that increased titin compliance compromises muscle ultrastructure and attenuates the Frank-Starling relationship. Although previous computational simulations of muscle contraction suggested that increasing compliance of the sarcomere slows the rate of tension development and prolongs cross-bridge attachment, none of the reported effects of Rbm20ΔRRM on myocardial function have been attributed to changes in cross-bridge cycling kinetics. To test the relationship between increased sarcomere compliance and cross-bridge kinetics, we used stochastic length-perturbation analysis in Ca2+-activated, skinned papillary muscle strips from Rbm20ΔRRM and WT mice. We found increasing titin compliance depressed maximal tension, decreased Ca2+-sensitivity of the tension-pCa relationship, and slowed myosin detachment rate in myocardium from Rbm20ΔRRM vs. WT mice. As sarcomere length increased from 1.9 to 2.2 μm, length-dependent activation of contraction was eliminated in the Rbm20ΔRRM myocardium, even though myosin MgADP release rate decreased ~20% to prolong strong cross-bridge binding at longer sarcomere length. These data suggest that increasing N2BA expression may alter cardiac performance in a length-dependent manner, showing greater deficits in tension production and slower cross-bridge kinetics at longer sarcomere length. This study also supports the idea that passive mechanical characteristics of the myocardium influence ensemble cross-bridge behavior and maintenance of tension generation throughout the sarcomere.

Myosin MgADP release rate decreases at longer sarcomere length to prolong myosin attachment time in skinned rat myocardium.

Cardiac contractility increases as sarcomere length increases, suggesting that intrinsic molecular mechanisms underlie the Frank-Starling relationship to confer increased cardiac output with greater ventricular filling. The capacity of myosin to bind with actin and generate force in a muscle cell is Ca(2+) regulated by thin-filament proteins and spatially regulated by sarcomere length as thick-to-thin filament overlap varies. One mechanism underlying greater cardiac contractility as sarcomere length increases could involve longer myosin attachment time (ton) due to slowed myosin kinetics at longer sarcomere length. To test this idea, we used stochastic length-perturbation analysis in skinned rat papillary muscle strips to measure ton as [MgATP] varied (0.05-5 mM) at 1.9 and 2.2 μm sarcomere lengths. From this ton-MgATP relationship, we calculated cross-bridge MgADP release rate and MgATP binding rates. As MgATP increased, ton decreased for both sarcomere lengths, but ton was roughly 70% longer for 2.2 vs. 1.9 μm sarcomere length at maximally activated conditions. These ton differences were driven by a slower MgADP release rate at 2.2 μm sarcomere length (41 ± 3 vs. 74 ± 7 s(-1)), since MgATP binding rate was not different between the two sarcomere lengths. At submaximal activation levels near the pCa50 value of the tension-pCa relationship for each sarcomere length, length-dependent increases in ton were roughly 15% longer for 2.2 vs. 1.9 μm sarcomere length. These changes in cross-bridge kinetics could amplify cooperative cross-bridge contributions to force production and thin-filament activation at longer sarcomere length and suggest that length-dependent changes in myosin MgADP release rate may contribute to the Frank-Starling relationship in the heart.

Diversity and similarity of motor function and cross-bridge kinetics in papillary muscles of transgenic mice carrying myosin regulatory light chain mutations D166V and R58Q

Mechanical properties of skinned papillary muscle fibers from transgenic mice expressing familial hypertrophic cardiomyopathy associated mutations D166V and R58Q in myosin regulatory light chain were investigated. Elementary steps and the apparent rate constants of the cross-bridge cycle were characterized from the tension transients induced by sinusoidal length changes during maximal Ca2+ activation, together with ATP, ADP, and Pi studies. The tension–pCa relation was also tested in two sets of solutions with differing Pi and ionic strength. Our results showed that in both mutants the fast apparent rate constant 2πc and the rate constants of the cross-bridge detachment step (k2) were smaller than those of wild type (WT), demonstrating the slower cross-bridge kinetics. D166V showed significantly smaller ATP (K1) and ADP (K0) association constants than WT, displaying weaker ATP binding and easier ADP release, whereas those of R58Q were not significantly different from WT. In tension–pCa study, both D166V and R58Q mutations exhibited increased Ca2+ sensitivity and less cooperativity. We conclude that, while the two FHC mutations have similar clinical manifestations and prognosis, some of the mechanical parameters of cross-bridges (K0, K1) are differently modified, whereas some others (Ca2+-sensitivity, cooperativity, k2) are similarly modified by these two FHC associated mutations.

Notch Transcriptional Control of Vascular Smooth Muscle Regulatory Gene Expression and Function

Notch receptors and ligands mediate heterotypic cell signaling that is required for normal vascular development. Dysregulation of select Notch receptors in mouse vascular smooth muscle (VSM) and in genetic human syndromes causes functional impairment in some regional circulations, the mechanistic basis of which is undefined. In this study, we used a dominant-negative Mastermind-like (DNMAML1) to block signaling through all Notch receptors specifically in VSM to more broadly test a functional role for this pathway in vivo. Mutant DNMAML1-expressing mice exhibited blunted blood pressure responses to vasoconstrictors, and their aortic, femoral, and mesenteric arteries had reduced contractile responses to agonists and depolarization in vitro. The mutant arteries had significant and specific reduction in the expression and activity of myosin light chain kinase (MLCK), a primary regulator of VSM force production. Conversely, activated Notch signaling in VSM cells induced endogenous MLCK transcript levels. We identified MLCK as a direct target of activated Notch receptor as demonstrated by an evolutionarily conserved Notch-responsive element within the MLCK promoter that binds the Notch receptor complex and is required for transcriptional activity. We conclude that Notch signaling through the transcriptional control of key regulatory proteins is required for contractile responses of mature VSM. Genetic or pharmacological manipulation of Notch signaling is a potential strategy for modulating arterial function in human disease.

Fast Skeletal Muscle Troponin Activation Increases Force of Mouse Fast Skeletal Muscle and Ameliorates Weakness Due to Nebulin-Deficiency

The effect of the fast skeletal muscle troponin activator, CK-2066260, on calcium-induced force development was studied in skinned fast skeletal muscle fibers from wildtype (WT) and nebulin deficient (NEB KO) mice. Nebulin is a sarcomeric protein that when absent (NEB KO mouse) or present at low levels (nemaline myopathy (NM) patients with NEB mutations) causes muscle weakness. We studied the effect of fast skeletal troponin activation on WT muscle and tested whether it might be a therapeutic mechanism to increase muscle strength in nebulin deficient muscle. We measured tension–pCa relations with and without added CK-2066260. Maximal active tension in NEB KO tibialis cranialis fibers in the absence of CK-2066260 was ∼60% less than in WT fibers, consistent with earlier work. CK-2066260 shifted the tension-calcium relationship leftwards, with the largest relative increase (up to 8-fold) at low to intermediate calcium levels. This was a general effect that was present in both WT and NEB KO fiber bundles. At pCa levels above ∼6.0 (i.e., calcium concentrations <1 µM), CK-2066260 increased tension of NEB KO fibers to beyond that of WT fibers. Crossbridge cycling kinetics were studied by measuring ktr (rate constant of force redevelopment following a rapid shortening/restretch). CK-2066260 greatly increased ktr at submaximal activation levels in both WT and NEB KO fiber bundles. We also studied the sarcomere length (SL) dependence of the CK-2066260 effect (SL 2.1 µm and 2.6 µm) and found that in the NEB KO fibers, CK-2066260 had a larger effect on calcium sensitivity at the long SL. We conclude that fast skeletal muscle troponin activation increases force at submaximal activation in both wildtype and NEB KO fiber bundles and, importantly, that this troponin activation is a potential therapeutic mechanism for increasing force in NM and other skeletal muscle diseases with loss of muscle strength.

Pseudophosphorylation of Cardiac Troponin I Residues 23/24 Decreases Myofilament Ca2+ Sensitivity in Transgenic Mice Containing D230N Mutation in α-Tropomyosin

The role of sarcomeric mutations in pathological cardiac-remodeling is not well understood. Furthermore, the constant modulation of myofilament contractility by post-translational modifications (PTM) of regulatory proteins, to adapt to a frequently changing circulatory demand, adds to the complexity. Therefore, we investigated how such a PTM in cardiac troponin I (cTnI) interacted with D230N (a dilated cardiomyopathy-related α-tropomyosin mutation), to alter the contractile properties of the myofilament. We reconstituted the pseudophosphorylated form of cTnI (cTnID23/D24) into detergent-skinned papillary muscle fibers from non-transgenic (NTG) and transgenic D230N mice, to mimic PKA-mediated phosphorylation in response to β-adrenergic stimulation. We carried out mechanical and dynamic studies on cTnID23/D24-D230N and control fibers at sarcomere length 2.3 μm. The pCa-tension relationship revealed that cTnID23/D24 decreased the pCa50 (-log10 [Ca2+]free required for half maximal activation) from 5.61±0.02 to 5.34±0.01 in NTG fibers and from 5.49±0.02 to 5.22±0.01 in D230N fibers. Thus, cTnID23/D24 uniformly decreased pCa50 by 0.27 pCa units in both NTG and D230N fibers. Interestingly, D230N mutation also uniformly decreased pCa50 by 0.12 pCa units, regardless of the phosphorylation status of cTnI. This indicates that cTnID23/D24 and D230N act independently to exert an additive effect of decreasing myofilament Ca2+ sensitivity. Therefore, under sub-maximal concentrations of Ca2+, the amount of force produced would be greatly impaired, resulting in systolic dysfunction.

Calcium sensitivity and myofilament lattice structure in titin N2B KO mice

The cellular basis of the Frank–Starling “Law of the Heart” is the length-dependence of activation, but the mechanisms by which the sarcomere detects length changes and converts this information to altered calcium sensitivity has remained elusive. Here the effect of titin-based passive tension on the length-dependence of activation (LDA) was studied by measuring the tension–pCa relation in skinned mouse LV muscle at two sarcomere lengths (SLs). N2B KO myocardium, where the N2B spring element in titin is deleted and passive tension is elevated, was compared to WT myocardium. Myofilament lattice structure was studied with low-angle X-ray diffraction; the myofilament lattice spacing (d1,0) was measured as well as the ratio of the intensities of the 1,1 and 1,0 diffraction peaks (I1,1/I1,0) as an estimate of the degree of association of myosin heads with the thin filaments. Experiments were carried out in skinned muscle in which the lattice spacing was reduced with Dextran-T500. Experiments with and without lattice compression were also carried out following PKA phosphorylation of the skinned muscle. Under all conditions that were tested, LDA was significantly larger in N2B KO myocardium compared to WT myocardium, with the largest differences following PKA phosphorylation. A positive correlation between passive tension and LDA was found that persisted when the myofilament lattice was compressed with Dextran and that was enhanced following PKA phosphorylation. Low-angle X-ray diffraction revealed a shift in mass from thin filaments to thick filaments as sarcomere length was increased. Furthermore, a positive correlation was obtained between myofilament lattice spacing and passive tension and the change in I1,1/I1,0 and passive tension and these provide possible explanations for how titin-based passive tension might regulate calcium sensitivity.

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca(2+) dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca(2+) binding in the NH(2)-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin's detailed dynamics, troponin's regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca(2+) slowed HDX up to 2 orders of magnitude within the NH(2)-lobe and the NH(2)-lobe-associated TnI switch helix, implying that Ca(2+) greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca(2+) and actin. Ca(2+)-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca(2+) destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca(2+)-mediated regulation: the coiled-coil's NH(2)-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca(2+)-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca(2+), including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca(2+) release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin's dynamic features.

Myosin Cross-Bridge Dynamics in Patients With Hypertension and Concentric Left Ventricular Remodeling

Hypertension (HTN) causes concentric left ventricular remodeling, defined as an increased relative wall thickness or overt left ventricular hypertrophy, and associated diastolic dysfunction. HTN and concentric remodeling are also common precursors to heart failure with a preserved ejection fraction. It is not known whether the myofilament contributes to diastolic dysfunction in patients with concentric remodeling. Intraoperative myocardial biopsies were obtained in 15 male patients undergoing coronary bypass grafting, all with normal left ventricular ejection fraction and wall motion. Eight patients had a history of HTN and concentric remodeling. Seven without HTN or remodeling served as controls. Myocardial strips were dissected and demembranated with detergent. Isometric tension was measured and sinusoidal length perturbation analysis performed at sarcomere length 2.2 μm and pCa 8 to 4.5. Sinusoidal analysis provides estimates of cross-bridge dynamics, including rate constants of attachment and detachment and cross-bridge attachment time. The normalized isometric tension-pCa relation was similar in HTN and controls. However, cross-bridge attachment time was significantly prolonged at submaximal [Ca(2+)] (pCa ≥6.5) in HTN patients. Analysis of protein phosphorylation revealed ≈25% reduction in phosphorylation of troponin I in HTN patients (P<0.05). Compared with controls, patients with HTN and concentric remodeling display prolonged cross-bridge attachment time at submaximal [Ca(2+)] without a change in the tension-pCa relation. Prolonged cross-bridge attachment time implicates altered cross-bridge dynamics as a cause of slowed relaxation in these patients. This finding was associated with reduced phosphorylation of troponin I, suggesting decreased phosphorylation of protein kinase A/G sites as a mechanism.

Generation and Functional Characterization of Knock-in Mice Harboring the Cardiac Troponin I-R21C Mutation Associated with Hypertrophic Cardiomyopathy

The R21C substitution in cardiac troponin I (cTnI) is the only identified mutation within its unique N-terminal extension that is associated with hypertrophic cardiomyopathy (HCM) in man. Particularly, this mutation is located in the consensus sequence for β-adrenergic-activated protein kinase A (PKA)-mediated phosphorylation. The mechanisms by which this mutation leads to heart disease are still unclear. Therefore, we generated cTnI knock-in mouse models carrying an R21C mutation to evaluate the resultant functional consequences. Measuring the in vivo levels of incorporated mutant and WT cTnI, and their basal phosphorylation levels by top-down mass spectrometry demonstrated: 1) a dominant-negative effect such that, the R21C+/- hearts incorporated 24.9% of the mutant cTnI within the myofilament; and 2) the R21C mutation abolished the in vivo phosphorylation of Ser(23)/Ser(24) in the mutant cTnI. Adult heterozygous (R21C+/-) and homozygous (R21C+/+) mutant mice activated the fetal gene program and developed a remarkable degree of cardiac hypertrophy and fibrosis. Investigation of cardiac skinned fibers isolated from WT and heterozygous mice revealed that the WT cTnI was completely phosphorylated at Ser(23)/Ser(24) unless the mice were pre-treated with propranolol. After propranolol treatment (-PKA), the pCa-tension relationships of all three mice (i.e. WT, R21C+/-, and R21C+/+) were essentially the same. However, after treatment with propranolol and PKA, the R21C cTnI mutation reduced (R21C+/-) or abolished (R21C+/+) the well known decrease in the Ca(2+) sensitivity of tension that accompanies Ser(23)/Ser(24) cTnI phosphorylation. Altogether, the combined effects of the R21C mutation appear to contribute toward the development of HCM and suggest that another physiological role for the phosphorylation of Ser(23)/Ser(24) in cTnI is to prevent cardiac hypertrophy.

Cardiomyopathy-Related Mutations in Cardiac Troponin C, L29Q and G159D, Have Divergent Effects on Rat Cardiac Myofiber Contractile Dynamics.

Previous studies of cardiomyopathy-related mutations in cardiac troponin C (cTnC)—L29Q and G159D—have shown diverse findings. The link between such mutant effects and their divergent impact on cardiac phenotypes has remained elusive due to lack of studies on contractile dynamics. We hypothesized that a cTnC mutant-induced change in the thin filament will affect global myofilament mechanodynamics because of the interactions of thin filament kinetics with both Ca2+ binding and crossbridge (XB) cycling kinetics. We measured pCa-tension relationship and contractile dynamics in detergent-skinned rat cardiac papillary muscle fibers reconstituted with the recombinant wild-type rat cTnC (cTnCWT), cTnCL29Q, and cTnCG159D mutants. cTnCL29Q fibers demonstrated a significant decrease in Ca2+ sensitivity, but cTnCG159D fibers did not. Both mutants had no effect on Ca2+-activated maximal tension. The rate of XB recruitment dynamics increased in cTnCL29Q (26%) and cTnCG159D (25%) fibers. The rate of XB distortion dynamics increased in cTnCG159D fibers (15%). Thus, the cTnCL29Q mutant modulates the equilibrium between the non-cycling and cycling pool of XB by affecting the on/off kinetics of the regulatory units (Tropomyosin-Troponin); whereas, the cTnCG159D mutant increases XB cycling rate. Different effects on contractile dynamics may offer clue regarding how cTnCL29Q and cTnCG159D cause divergent effects on cardiac phenotypes.

Protein kinase A changes calcium sensitivity but not crossbridge kinetics in human cardiac myofibrils

We investigated the effect of PKA treatment (1 U/ml) on the mechanical properties of isolated human cardiac myofibrils. PKA treatment was associated with significant incorporation of radiolabeled phosphate into several sarcomeric proteins including troponin I and myosin binding protein C and was also associated with a right shift in the tension-pCa relation (ΔpCa(50) = 0.2 ± 0.1). PKA treatment also caused right shifts in the pCa dependence of the rate of tension development, tension redevelopment, and the linear and exponential phases of myofibril relaxation. However, there was no change in the same measures of crossbridge turnover when expressed as a function of tension. We conclude that the changes in crossbridge kinetics as a function of calcium concentration reflect a reduced tension due to a lower calcium sensitivity and that the relationship between crossbridge kinetics and tension was unchanged, indicating no direct effect of PKA treatment on crossbridge cycling.

Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function

The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.

Mechanical and contractile characteristics of rat thoracic duct and cervical lymphatics

Thoracic duct (TD) lymphatics exhibit an increase in tonic activity as transmural pressure increases; whereas cervical lymphatics show an increase in phasic contraction frequency. We hypothesize that mechanical and biochemical characteristics of these lymphatic myofilaments will be different. The length‐tension relationships were determined for cervical and TD lymphatics. Cervical and TD lymphatics exhibited a biphasic response to maximal activation with high K++PSS+Substance P. TD generated larger active peak and plateau tensions than did cervical lymphatics (in mN/mm; peak tension: 0.65±0.06 vs. 0.43±0.01 P&lt;0.03; plateau tension: 0.54±0.06 vs 0.25±0.04 p&lt;0.01). pCa‐tension relationships were also determined in a‐toxin permeabilized lymphatics. While maximal peak tension in a‐toxin permeabilized TD was greater than cervical lymphatics (0.76±0.08 vs. 0.50+0.07 p&lt;0.02), plateau tension was not significantly different (0.56±0.07 vs. 0.41±0.07 P&lt;0.15). The Ca2+sensitivity (pCa50) and cooperativity of TD and cervical lymphatics were not significantly different (5.93±0.02 vs 5.86±0.06 &amp; 1.73±0.11 vs. 1.49±0.09). Thus the differences in the mechanical characteristics between TD and cervical lymphatics are not attributable to variations in pCa50 and cooperativity; other Ca2+‐dependent or ‐independent mechanisms may play important roles in these lymphatics. Supported by NIH HL80526 &amp; LRF.

Determinants of Loaded Shortening in Cardiac Myocytes

Ventricular performance is dictated by stroke volume, which ultimately depends on the extent of myocyte shortening during loaded contractions. We propose that the extent of loaded shortening is determined by the balance between two processes: (i) Ca2+-cross-bridge-induced cooperative activation of the thin filament and (ii) shortening-induced cooperative deactivation of the thin filament. Accordingly, any modulator that augments contractility (i.e., stroke volume) should favor process (i) and diminish process (ii). Since β-adrenergic stimulation is known to increase contractility, we tested whether PKA (the myofibrillar ligand of β-adrenergic signaling) would increase cooperative activation and diminish shortening-induced deactivation in rat permeabilized cardiac myocytes during submaximal Ca2+ activations. Regarding cooperative activation, PKA increased the slope of tension-pCa relationships (nH = 3.85 ± 0.09 before versus nH = 5.03 ± 0.71 after PKA). PKA also slowed rates of force redevelopment, increased the transient force overshoot after a slack-restretch maneuver, and increased the rate and amplitude of spontaneous oscillatory contractions (SPOCs); all of which are consistent with greater cooperative activation of the thin filament. Regarding cooperative deactivation, PKA increased the curvature of myocyte length traces during lightly loaded shortening (kshortening = 6.41 ± 0.28 before versus kshortening = 9.45 ± 0.53 after PKA) and steepened sarcomere length-tension relationships; both of which implicate enhanced (rather than diminished) shortening-induced cooperative deactivation. Taken together, PKA-induced myofibrillar phosphorylation appears to augment both Ca2+-cross-bridge-induced cooperative activation of the thin filament and (ii) shortening-induced cooperative deactivation. Greater cooperative activation should lead to more cycling cross-bridges, which would speed loaded shortening against a given afterload. On the other hand, greater shortening-induced cooperative deactivation may be necessary to help accelerate relaxation and assist diastolic filling in the face of shorter systolic and diastolic times in the presence of higher heart rates induced by β-adrenergic stimulation.

Overexpression of Myosin Phosphatase Reduces Ca2+ Sensitivity of Contraction and Impairs Cardiac Function

Phosphorylation of the regulatory light chain of myosin (MLC) has roles in cardiac function. In vitro, myosin phosphatase target subunit 2 (MYPT2) is a strongly suspected regulatory subunit of cardiac myosin phosphatase (MP), but there is no in-vivo evidence regarding the functions of MYPT2 in the heart. Transgenic mice (Tg) overexpressing MYPT2 were generated using the alpha-MHC promoter. Tg hearts showed an increased expression of MYPT2 and concomitant increase of the endogenous catalytic subunit of type 1 phosphatase (PP1cdelta), resulting in an increase of the MP holoenzyme. The level of phosphorylation of ventricular MLC was reduced. The pCa-tension relationship, using beta-escin permeabilized fibers, revealed decreased Ca(2+) sensitization of contraction in the Tg heart. LV enlargement with associated impairment of function was observed in the Tg heart and ultrastructural examination showed cardiomyocyte degeneration. Overexpression of MYPT2 and the increase in PP1cdelta resulted in an increase of the MP holoenzyme and a decrease in the level of MLC phosphorylation. The latter induced Ca(2+) desensitization of contraction and decreased LV contractility, resulting in LV enlargement. Thus, MYPT2 is truly the regulatory subunit of cardiac MP in-vivo and plays a significant role in modulating cardiac function. (Circ J 2010; 74: 120 - 128).