Myofilament Length-dependent Activation Research Articles

Mechanistic study of mechanical stretch induced increase in Ca2+ sensitivity of troponin C.

The Frank-Starling Law of the heart is a direct consequence of myofilament length dependent activation (LDA) whereby increased sarcomere length (SL) leads to both increased myocardial Ca2+ sensitivity and maximum force. The molecular mechanisms underlying LDA remain elusive. Since troponin complex binds Ca2+ and regulates myosin-actin interaction during muscle contraction, it has been considered the key element involved LDA. This study aims to explore the molecular mechanism underlying sarcomere LDA and to investigate the functional role of each troponin protein in the LDA regulation. A mutant troponin-I, in which the SP-region was replaced with a G-linker (cTnI-ΔSP), was constructed to study the molecular interaction between troponin-I, troponin-C, and myosin during LDA regulation. The cTnI-ΔSP was used to prevent the functional effect from crossbridge (XB) on the SL-induced Ca2+ sensitivity changes of troponin complex. The SL-induced Ca2+ sensitivity change was monitored by in situ FRET which represents the Ca2+-induced cTnC structural change. Our results show that there are no significant changes of the SL-induced pCa50 shift of troponin in the absence of strong XB, suggesting that besides the XB feedback, the LDA of thin filament may have an alternative pathway involving troponin itself. To further understand the mechanism underlying LDA, recombinant cTnT and cTnI mutants carrying an enzyme cutting site at one end of the IT arm region, respectively, were used in our study. By selectively breaking T1 and T2 of cTnT or separating the C-domain of cTnI from the N-domain, we were able to examine the mechanistic pathway of SL-stretch on the Ca2+-induced structural change of cTnC. Our results provide novel information on the molecular mechanism of how troponin-Ca2+ activation senses myocardial sarcomere stretches to promote myosin-actin interactions at the diastolic level of Ca2+.

The Super-Relaxed State and Length Dependent Activation in Porcine Myocardium.

[Figure: see text].

Dilated cardiomyopathy mutation (R174W) in troponin T attenuates the length-mediated increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Alterations in length-dependent activation (LDA) may constitute a mechanism by which cardiomyopathy mutations lead to deleterious phenotypes and compromised heart function, because LDA underlies the molecular basis by which the heart tunes myocardial force production on a beat-to-beat basis (Frank-Starling mechanism). In this study, we investigated the effect of DCM-linked mutation (R173W) in human cardiac troponin T (TnT) on myofilament LDA. R173W mutation is associated with left ventricular dilatation and systolic dysfunction and is found in multiple families. R173W mutation is in the central region (residues 80-180) of TnT, which is known to be important for myofilament cooperativity and cross-bridge (XB) recruitment. Steady-state and dynamic contractile parameters were measured in detergent-skinned guinea pig left ventricular muscle fibers reconstituted with recombinant guinea pig wild-type TnT (TnTWT) or mutant TnT (TnTR174W; guinea pig analog of human R173W mutation) at two different sarcomere lengths (SL): short (1.9 µm) and long (2.3 µm). TnTR174W decreased pCa50 (-log [Ca2+]free required for half-maximal activation) to a greater extent at long than at short SL; for example, pCa50 decreased by 0.12 pCa units at long SL and by 0.06 pCa units at short SL. Differential changes in pCa50 at short and long SL attenuated the SL-dependent increase in myofilament Ca2+ sensitivity (ΔpCa50) in TnTR174W fibers; ΔpCa50 was 0.10 units in TnTWT fibers but only 0.04 units in TnTR174W fibers. Furthermore, TnTR174W blunted the SL-dependent increase in the magnitude of XB recruitment. Our observations suggest that the R173W mutation in human cardiac TnT may impair Frank-Starling mechanism.NEW & NOTEWORTHY This work characterizes the effect of dilated cardiomyopathy mutation in cardiac troponin T (TnTR174W) on myofilament length-dependent activation. TnTR174W attenuates the length-dependent increase in cross-bridge recruitment and myofilament Ca2+ sensitivity.

Cardiac-specific knockout of Lmod2 results in a severe reduction in myofilament force production and rapid cardiac failure

Leiomodin-2 (Lmod2) is a striated muscle-specific actin binding protein that is implicated in assembly of thin filaments. The necessity of Lmod2 in the adult mouse and role it plays in the mechanics of contraction are unknown. To answer these questions, we generated cardiac-specific conditional Lmod2 knockout mice (cKO). These mice die within a week of induction of the knockout with severe left ventricular systolic dysfunction and little change in cardiac morphology. Cardiac trabeculae isolated from cKO mice have a significant decrease in maximum force production and a blunting of myofilament length-dependent activation. Thin filaments are non-uniform and substantially reduced in length in cKO hearts, affecting the functional overlap of the thick and thin filaments. Remarkably, we also found that Lmod2 levels are directly linked to thin filament length and cardiac function in vivo, with a low amount (<20%) of Lmod2 necessary to maintain cardiac function. Thus, Lmod2 plays an essential role in maintaining proper cardiac thin filament length in adult mice, which in turn is necessary for proper generation of contractile force. Dysregulation of thin filament length in the absence of Lmod2 contributes to heart failure.

Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres

The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation.

Titin strain contributes to the Frank–Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins

The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

β-Adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both cardiac myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length-dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA- or DDD-permeabilized myocytes (n = 12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca(2+)] relationships were determined at two sarcomere lengths (SL = 1.9 μm and SL = 2.3 μm). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca(2+)] required to achieve 50% force activation at the two SLs. We found that PKA-mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67 and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA-mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL while accelerating cross-bridge detachment and relaxation at short SLs.

Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte

The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

Impact of Troponin-I Phosphorylation on Human Cardiac Myofilament Function

A long-term goal of work in our laboratories is to understand the structural basis of myofilament function, i.e. cross-bridge dynamics (CD) and myofilament length dependent activation (LDA) in striated muscle in heath and disease. Moreover, in cardiac muscle LDA underlies the Frank Starling Law of the Heart. Troponin phosphorylation, and in particular troponin-I (cTnI), has been suggested to be a pivotal modulator of myofilament function. Here we examined the impact of phosphorylation of distinct cTnI domains on CD and LDA in isolated human myocardium. Site specific phosphorylation was accomplished by charge mutation of hcTnI phosphomimics on the PKA (S23/24D), PKC (S42/44E; T143E), AMPK (S150D), and novel (S5/6D) sites, followed by recombinant protein exchange into skinned non-failing human LV cardiac muscle strips (∼2mm long, and ∼150um diameter. Force and ATPase activity was measured as function of [Ca2+] at short and long sarcomere length (SL=2.0&2.3um). We found, LDA: hcTnI-S150D attenuated, hcTnI-S42/43E increase, and no effect for the other sites. CD as indexed by tension cost was: decreased for cTnI-S42/44E and hcTnI-S150D, and no effect for the other sites. We conclude that cTnI phosphorylation at distinct sites differentially affect cross-bridge cycling and length dependent activation in human myocardium. Structural analysis employing x-ray diffraction is underway to determine the structural basis for these phenomena. Supported by NIH HL075494, HL62426, GM103622.

Titin-Based Modulation of Sarcomere Structure as Revealed by Equatorial X-Ray Diffraction

Myofilament Length Dependent Activation (LDA) is the cellular mechanism underlying the Frank-Starling Law of the Heart. LDA is defined as an increase in force for a given amount of calcium when the muscle is stretched to a longer sarcomere length. Despite its physiological importance, the sensor that underlies LDA is not known. To address this issue we use X-ray diffraction of intact twitching rat papillary muscle where muscle initially at maximum sarcomere length (Lmax) is quickly released to slack length (Slack). X-ray patterns are taken is a 10 ms window just prior to force generation.Previous analyses of the data did not support the hypothesis that there was a radially outward movement of myosin heads at Lmax requiring some other explanation. The equatorial portion of our X-ray patterns show 5 pairs of well-resolved diffraction spots from the 1,0 to the 3, 0 equatorial reflection. This allowed calculation of two dimensional electron density maps (ED maps) comparing Lmax and Slack from both wild type rats and mutant rats with exceptionally long titin (J Mol Cell Cardiol. 44:983-91 2008) where the passive tension generated at Lmax, as well as LDA, is much reduced. Difference ED maps (Lmax-Slack) from WT rats indicate a better localization of both thick and thin filaments at Lmax plus the existence of bridging density joining the thick and thin filaments at Lmax and not at slack. In difference ED maps from the titin mutant rats, the bridging structures are absent and only the thick filaments appeared better localized. These results support a model where a small number of crossbridges transmit titin-based strain from the thick filament to the thin filament as part of the mechanism of LDA. Supported by NIH R01HL075494 and 9 P41 GM103622.

Cardiac Thin Filament Structural Modulation by Sarcomere Length

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. LDA has been studied intensively and appears to be modulated through various mechanisms, such as contractile proteins composition and their phosphorylation status. However, the cellular molecular mechanisms that underlie this phenomenon are still not well characterized.The aim of our study is to determine whether LDA is regulated through cTnC structural changes upon stretch. Accordingly, we used a single attached skinned cardiac myocyte in combination with confocal fluorescent measurement. Using this technique, we found that stretch of a relaxed cell, in the absence of Ca2+, resulted in marked alterations of cTnC structure as reported by cTnC-T53C-IAF confocal fluorescence. Moreover, titin mutant cells show a drastic alteration of both passive tension and myofilament sensitivity to calcium (pCa50) upon stretch. Consistent with this finding, by employing time-resolved x-ray diffraction of intact, electrically stimulated rat myocardium, we found marked changes in troponin and myosin structure upon stretch in the diastolic phase (i.e. when cross-bridges are not active). Moreover, we repeated these x-ray experiments using rat myocardium that expresses an unusually long titin molecule and found that, when compared to WT, diastolic stretch in these muscles did not induce the structural changes in troponin and myosin observed in WT. These results strongly suggest a direct effect of sarcomere length on thin and thick filament and implicate titin to be the molecule underling the length signal transduction for LDA.

Cardiac Myosin Binding Protein-C Phosphorylation and Sarcomere Function

Rationale: Cardiac myosin binding protein-C (cMyBP-C) has three phosphorylatable serines at N-terminus (Ser-273, Ser-282, and Ser-302) and phosphorylation states of these serines may alter the thick filament structure and function. Phosphorylation of these serines in cMyBP-C may also alter sarcomere length-dependent modulation of contractile force development (LDA), which would be expected to modulate the Frank-Starling Law of the Heart.Objective: To analyze length dependent activation in cMyBP-C for nonphosphorylated and phosphomimetic states of serines 273, 282, 302 and to study the contribution of cMyBP-C in regulating cardiac contraction at different sarcomere lengths.Methods & Results: We studied the force-Ca2+ relationship in skinned myocytes isolated from (a) nonphosphorylated alanine transgenic mice (cMyBP-CAAA), (b) phosphomimetic aspartic acid transgenic mice (cMyBP-CDDD), and normal cMyBP-CWT mice at 2.0 and 2.3 μm sarcomere length (SL).At short SL, Ca2+ sensitivity was similar in all groups. However, Ca2+ sensitivity was increased in the cMyBP-CAAA group compared to either the WT or the cMyBP-CDDD group. Similar results were obtained for maximum force development. As a consequence, LDA was blunted (∼40%) in cMyBP-CAAA myocardium. There were no differences in the level of cooperativety as indexed by the Hill coefficient in any group.Conclusion: Phosphorylated cMyBP-C has been shown to contribute to regulation of cardiac sarcomere function via modulation of the cMyBP-C-actin interaction as well as the disposition of the cross-bridges in relation to the thin filament. Moreover removal of cMyBP-C results in blunted LDA, and a cardiac dysfunction that can be prevented by cMyBP-CDDD but not cMyBP-CAAA. Our data showed that lack of cMyBP-C phosphorylation results in blunted LDA, similar to that found previously in the absence cMyBP-C. We conclude that cMyBP-C phosphorylation modulates myofilament length dependent activation, possibly via modulation of the cMyBP-C interaction with actin.

Zebrafish Heart as a Model System to Study Structure-Function Relationships

The zebrafish (Danio rerio) has been intensively used in cardiovascular biology mainly for study of heart development. This is a promising cost-effective model system to study structure-function due to the ease of genetic manipulations. However, the basic contractile physiology of zebrafish heart is still incompletely understood. Moreover, the applicability of the fish heart as a model system for study mammalian heart contractility needs to be established.The aim of our work was to establish the zebrafish as a model system to approach structure-function cardiac myocyte biology. Accordingly, we performed experiments with a focus on the Frank-Starling mechanisms at two levels: cellular (intact cells) and myofilament level (permeabilized cells). In single cell experiments we were able to attach a single enzymatically isolated zebrafish ventricular myocytes to myotak coated carbon probes and measure force, intracellular calcium, cell length, and sarcomere length in electrically paced cells over a range (20%) of cell lengths. In skinned cell experiments, we used a single myofibril technique to measure activation/relaxation kinetics and force-Ca2+ relations at short and long sarcomere lengths (2.2 and 2.4 um). Zebrafish intact myocytes displayed robust length induced increase in twitch force in the absence of calcium transient alteration. In skinned zebrafish muscle we found robust myofilament length dependent activation as well as characteristic activation/relaxation dynamics. We conclude that the zebrafish heart is an appropriate model to study cardiac structure-function relationships.

Cardiac Thin Filament Activation Modulation by Stretch

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. LDA has been studied intensively and appears to be modulated through various mechanisms, such as the composition of the contractile proteins and their phosphorylation status. However, the cellular molecular mechanisms that underlie this phenomenon are still not well characterized.The aim of our study is to determine whether LDA is regulated through cTnC structural changes upon stretch. Accordingly, we used a single attached skinned cardiac myocyte in combination with confocal fluorescent measurement. using this technique, we found that stretch of a relaxed cell, in the absence of Ca2+, resulted in marked alterations of cTnC structure as reported by cTnC-T53C-IAF confocal fluorescence. Moreover, titin mutant cells show a drastic alteration of both passive tension and myofilament sensitivity to calcium (pCa50) upon stretch. Consistent with this finding, by employing time-resolved x-ray diffraction of intact, electrically stimulated rat myocardium, we found marked changes in troponin and myosin structure upon stretch in the diastolic phase (i.e. when cross-bridges are not active). Moreover, we repeated these x-ray experiments using rat myocardium that expresses an unusually long titin molecule and found that, when compared to WT, diastolic stretch in these muscles did not cause structural changes in troponin and myosin structures. These results strongly implicate titin to be the molecule that transmits the length signal for LDA.

Blunted Length-Dependent Activation Caused by the Homozygous TNNT2 Mutation K280N

Background Recently, we observed reduced myofilament length-dependent activation in cardiomyocytes from a patient harboring a homozygous mutation (K280N) in the TNNT2 gene encoding cardiac troponin T (cTnT) compared to non-failing donors. Protein kinase A (PKA)-mediated phosphorylation of cardiac troponin I (cTnI) did not rescue impaired length-dependence. Moreover, cTnI phosphorylation did not differ between TNNT2mut and donor myocardium. In this study we present direct evidence that the K280N mutation in TNNT2 blunts length-dependent activation.Methods Force measurements were performed in single permeabilized cardiomyocytes isolated from the TNNT2mut heart at various [Ca2+] and sarcomere lengths of 1.8 and 2.2 μm, with and without PKA-pretreatment. To investigate if mutant cTnT underlies impaired length-dependent activation, the endogenous mutant troponin complex was partially exchanged with recombinant whole human wild-type troponin (Tnwt) complex. TNNT2mut sample was exchanged with 0.25, 0.5 and 1 mg/mL Tnwt complex, which in accordance with previous studies from our group is predicted to yield ∼40%, ∼50% and ∼70% of troponin exchange, respectively.Results The length-dependent increase in myofilament Ca2+-sensitivity (ΔEC50) was not significantly different between TNNT2mut, TNNT2∼40%, TNNT2∼50%, TNNT2∼70%, (ΔEC50=0.35±0.16, 0.23±0.06, −0.02 ±0.02 and 0.22±0.06 μmol/L, respectively), but was significantly reduced compared to donor (ΔEC50=0.77±0.06 μmol/L). PKA-pretreatment did not restore the reduced length-dependent activation of TNNT2mut and TNNT2∼40% (ΔEC50=0.26±0.19 and 0.32±0.15), but did recover the blunted length-dependent activation of TNNT2∼50% and TNNT2∼70% (ΔEC50=0.71±0.04 and 0.79±0.14) to control values (ΔEC50=0.82±0.09).Conclusions Length-dependent activation of myofilaments is corrected to donor values only when ∼50% of mutant cTnT is replaced by wild-type troponin and subsequent phosphorylation with PKA. Our data show that the TNNT2 mutation K280N underlies impaired length-dependent activation in a dose-dependent manner.

Impact of titin isoform on length dependent activation and cross-bridge cycling kinetics in rat skeletal muscle

The magnitude of length dependent activation in striated muscle has been shown to vary with titin isoform. Recently, a rat that harbors a homozygous autosomal mutation (HM) causing preferential expression of a longer, giant titin isoform was discovered (Greaser et al. 2005). Here, we investigated the impact of titin isoform on myofilament force development and cross-bridge cycling kinetics as function of sarcomere length (SL) in tibialis anterior skeletal muscle isolated from wild type (WT) and HM. Skeletal muscle bundles from HM rats exhibited reductions in passive tension, maximal force development, myofilament calcium sensitivity, maximal ATP consumption, and tension cost at both short and long sarcomere length (SL=2.8μm and SL=3.2μm, respectively). Moreover, the SL-dependent changes in these parameters were attenuated in HM muscles. Additionally, myofilament Ca2+ activation–relaxation properties were assessed in single isolated myofibrils. Both the rate of tension generation upon Ca2+ activation (kACT) as well as the rate of tension redevelopment following a length perturbation (kTR) were reduced in HM myofibrils compared to WT, while relaxation kinetics were not affected. We conclude that presence of a long isoform of titin in the striated muscle sarcomere is associated with reduced myofilament force development and cross-bridge cycling kinetics, and a blunting of myofilament length dependent activation. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Myofilament Length-Dependent Activation Develops within 5 ms in Guinea-Pig Myocardium

Myofilament length-dependent activation is a universal property of striated muscle, yet the molecular mechanisms that underlie this phenomenon are incompletely understood. Additionally, the rate by which sarcomere length (SL) is sensed and then transduced to form length-dependent activation is unknown. Here, using isolated guinea-pig myocardium, we employed a rapid solution-switch single myofibril technique that allows for the study of contractile action/relaxation dynamics in the virtual absence of diffusion delays. We compared contraction kinetics obtained at submaximal activation at steady-state SL with contractions observed after rapid SL ramps to that same SL just before activation. Neither the activation and relaxation kinetics nor the final submaximal force development differed significantly between the two contraction modes for SL ramps as fast as 5 ms. We conclude that the transduction of the length signal by the cardiac sarcomere to modulate thin filament activation levels occurs virtually instantaneously, possibly resulting from structural rearrangements of the contractile proteins.

Bradykinin restores left ventricular function, sarcomeric protein phosphorylation, and e/nNOS levels in dogs with Duchenne muscular dystrophy cardiomyopathy

Cardiomyopathy is a lethal result of Duchenne muscular dystrophy (DMD), but its characteristics remain elusive. The golden retriever muscular dystrophy (GRMD) dogs produce DMD pathology and mirror DMD patient's symptoms, including cardiomyopathy. We previously showed that bradykinin slows the development of pacing-induced heart failure. Therefore, the goals of this research were to characterize dystrophin-deficiency cardiomyopathy and to examine cardiac effects of bradykinin in GRMD dogs. At baseline, adult GRMD dogs had reduced fractional shortening (28 ± 2 vs. 38 ± 2% in control dogs, P < 0.001) and left ventricular (LV) subendocardial dysfunction leading to impaired endo-epicardial gradient of radial systolic velocity (1.3 ± 0.1 vs. 3.8 ± 0.2 cm/s in control dogs, P < 0.001) measured by echocardiography. These changes were normalized by bradykinin infusion (1 µg/min, 4 weeks). In isolated permeabilized LV subendocardial cells of GRMD dogs, tension-calcium relationships were shifted downward and force-generating capacity and transmural gradient of myofilament length-dependent activation were impaired compared with control dogs. Concomitantly, phosphorylation of sarcomeric regulatory proteins and levels of endothelial and neuronal nitric oxide synthase (e/nNOS) in LV myocardium were significantly altered in GRMD dogs. All these abnormalities were normalized in bradykinin-treated GRMD dogs. Cardiomyopathy in GRMD dogs is characterized by profound LV subendocardial dysfunction, abnormal sarcomeric protein phosphorylation, and impaired e/nNOS, which can be normalized by bradykinin treatment. These data provide new insights into the pathophysiological mechanisms accounting for DMD cardiomyopathy and open new therapeutic perspectives.

Cardiac Thin Filament Activation Modulation by Sarcomere Length

Myofilament Length Dependent Activation (LDA) forms the cellular basis of the Frank-Starling law observed on the heart. It has been previously shown in isolated myocardium that the responsiveness of cardiac myofilaments to activating calcium ions is increased with stretch. However, the molecular mechanism underlying LDA is still unknown. The aim of our study was to investigate whether LDA is modulated by increased calcium binding to the thin filaments, and whether or not thick filaments modulate this mechanism. Accordingly, experiments were performed under strict sarcomere length control on skinned guinea pig multicellular preparations isolated from the left ventricle. Myofilament calcium sensitivity was measured at both short (1.9 μm) and long (2.3 μm) sarcomere length. Simultaneously, calcium binding to cTnC was monitored using fluorescently labeled cTnC (cTnCT53C∗) with an environmentally sensitive probe (2-(4′-iodoadcetamidoanilo)-napthalene-6-sulfonic acid (IAANS)). The involvement of thick filaments in the regulation of calcium binding to cTnC was evaluated either in the presence of Blebbistatin to inhibit cross bridges formation or in rigor solution to inhibit cross-bridges detachment. Calcium dependent modulation of IAANS fluorescence showed calcium dependent decrease in IAANS fluorescence, indicating that the label was correctly incorporated into a functional TnC. The labeled TnC was then incorporated into recombinant Tn complex (Tn∗) and endogenous Tn was exchanged for Tn∗. Preliminary data show that Tn exchange did not alter the myofilament properties as demonstrated by preserved Tension-pCa curves at both short and long SL. Our data also show alteration of IAANS emitted fluorescence upon calcium activation in exchanged skinned muscles. Moreover, fluorescence was also altered by stretch in relaxed muscles. These data suggest that LDA may be due to altered thin filament activation in response to stretch secondary to a direct influence of sarcomere length on troponin structure.

Structural Changes in Both the Troponin Complex and the Thick Filament May Underlie Myofilament Length Dependent Activation

The main cellular mechanism that underlies the so-called “Frank-Starling Law of the Heart” is an increase in the responsiveness of cardiac myofilaments to activating Ca2+ at longer sarcomere lengths (SL). The structural basis of this “Length Dependent Activation” (LDA) is not known. 2D X-ray diffraction patterns were obtained using the BioCAT beamline 18ID at the Advanced Photon Source from electrically stimulated (0.2 Hz) intact, twitching papillary muscle isolated from rat hearts during a 10 ms time window in diastole just prior to electrical stimulation. Diffraction patterns were compared from muscles that were stretched to Lmax (SL= ∼2.3 μm) to those taken following a quick release to slack length (SL=∼1.9 μm). We previously reported that myosin heads moved radially inwards at longer SL suggesting that an increased radial extent of crossbridges at longer length cannot explain increased calcium sensitivity so other explanations must be sought. It is known that changes in isoform composition of the troponin complex can markedly affect calcium sensitivity but the role of troponin in the length sensing mechanism underlying LDA is not clear. Here we analyzed the meridional patterns which showed that the 3rd order troponin repeat distance, the 3rd - order troponin reflection intensity and the 2nd order myosin (“forbidden”) meridional reflection all increased significantly (P < 0.01) at Lmax as compared to slack length. Thus, stretching intact heart muscle in diastole induces changes in the structure of both the thick filaments and the thin filaments. It appears, then, that the length sensing mechanism underlying LDA must involve connections of some kind that transmit strain between the thick and thin filament that alter the structure of the troponin complex and, presumably, myofilament contractile properties. Supported by NIH HL075494 and RR08630.