Daniel P. Fitzsimons

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 (≈3 μmol/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.

Differential roles of regulatory light chain and myosin binding protein-C phosphorylations in the modulation of cardiac force development

Phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) and myosin binding protein‐C (cMyBP‐C) by protein kinase A (PKA) independently accelerate the kinetics of force development in ventricular myocardium. However, while MLCK treatment has been shown to increase the Ca2+ sensitivity of force (pCa50), PKA treatment has been shown to decrease pCa50, presumably due to cardiac troponin I phosphorylation. Further, MLCK treatment increases Ca2+‐independent force and maximum Ca2+‐activated force, whereas PKA treatment has no effect on either force. To investigate the structural basis underlying the kinase‐specific differential effects on steady‐state force, we used synchrotron low‐angle X‐ray diffraction to compare equatorial intensity ratios (I1,1/I1,0) to assess the proximity of myosin cross‐bridge mass relative to actin and to compare lattice spacings (d1,0) to assess the inter‐thick filament spacing in skinned myocardium following treatment with either MLCK or PKA. As we showed previously, PKA phosphorylation of cMyBP‐C increases I1,1/I1,0 and, as hypothesized, treatment with MLCK also increased I1,1/I1,0, which can explain the accelerated rates of force development during activation. Importantly, interfilament spacing was reduced by ∼2 nm (Δ 3.5%) with MLCK treatment, but did not change with PKA treatment. Thus, RLC or cMyBP‐C phosphorylation increases the proximity of cross‐bridges to actin, but only RLC phosphorylation affects lattice spacing, which suggests that RLC and cMyBP‐C modulate the kinetics of force development by similar structural mechanisms; however, the effect of RLC phosphorylation to increase the Ca2+ sensitivity of force is mediated by a distinct mechanism, most probably involving changes in interfilament spacing.

Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium

1 The effects of ventricular myosin heavy chain (MHC) composition on the kinetics of activation and relaxation were examined in both chemically skinned and intact myocardial preparations from adult rats. Thyroid deficiency was induced to alter ventricular MHC isoform expression from ∼80 %α‐MHC/20 %β‐MHC in euthyroid rats to 100 %β‐MHC, without altering the expression of thin‐filament‐associated regulatory proteins. 2 In single skinned myocytes, increased expression of β‐MHC did not significantly affect either maximal Ca2+‐activated tension (P0) or the Ca2+ sensitivity of tension (pCa50). However, unloaded shortening velocity (V0) decreased by 80 % due to increased β‐MHC expression. 3 The kinetics of activation and relaxation were examined in skinned multicellular preparations using the caged Ca2+ compound DM‐nitrophen and caged Ca2+ chelator diazo‐2, respectively. Myocardium expressing 100 %β‐MHC exhibited apparent rates of submaximal and maximal tension development (kCa) that were 60 % lower than in control myocardium, and a 2‐fold increase in the half‐time for relaxation from steady‐state submaximal force. 4 The time courses of cell shortening and intracellular Ca2+ transients were assessed in living, electrically paced myocytes, both with and without β‐adrenergic stimulation (70 nm isoproterenol (isoprenaline)). Thyroid deficiency had no affect on either the extent of myocyte shortening or the resting or peak fura‐2 fluorescence ratios. However, induction of β‐MHC expression by thyroid deficiency was associated with increased half‐times for myocyte shortening and relengthening and increased half‐time for the decay of the fura‐2 fluorescence ratio. Qualitatively similar results were obtained in both the absence and the presence of β‐adrenergic stimulation although the β‐agonist accelerated the kinetics of the twitch and the Ca2+ transient. 5 Collectively, these data provide evidence that increased β‐MHC expression contributes significantly to the observed depression of contractile function in thyroid deficient myocardium by slowing the rates of both force development and force relaxation.

Strong Binding of Myosin Modulates Length-Dependent Ca2+ Activation of Rat Ventricular Myocytes

Reductions in sarcomere length (SL) and concomitant increases in interfilament lattice spacing have been shown to decrease the Ca2+ sensitivity of tension in myocardium. We tested the idea that increased lattice spacing influences the SL dependence of isometric tension by reducing the probability of strong interactions of myosin crossbridges with actin, thereby decreasing cooperative activation of the thin filament. Single ventricular myocytes were isolated by enzymatic digestion of rat hearts and were subsequently rapidly skinned. Maximal tension and Ca2+ sensitivity of tension (ie, pCa50) were measured in the absence and presence of N-ethylmaleimide-modified myosin subfragment 1 (NEM-S1) at both short and long SLs. NEM-S1, a strong-binding non-tension-generating derivative of the myosin head, was applied to single skinned myocytes to cooperatively promote strong binding of endogenous myosin crossbridges. Compared with control myocytes at SL of approximately 1.90 microm, application of NEM-S1 markedly increased submaximal Ca2+-activated tensions and thereby increased Ca2+ sensitivity; ie, pCa50 increased from 5.40+/-0.02 to 5.52+/-0.02 pCa units in the presence of NEM-S1. Furthermore, NEM-S1 treatment reversibly eliminated the SL dependence of the Ca2+ sensitivity of tension, in that the DeltapCa50 between short and long lengths was 0. 02+/-0.01 pCa units in the presence of NEM-S1 compared with a DeltapCa50 of 0.10+/-0.01 pCa units in control myocytes. From these results we conclude that the decrease in the Ca2+ sensitivity of tension at short SL results predominantly from decreased cooperative activation of the thin filament due to reductions in the number of strong-binding crossbridges.

Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament

1 To determine the ability of strong‐binding myosin cross‐bridges to activate the myocardial thin filament, we examined the Ca2+ dependence of force and cross‐bridge interaction kinetics at 15°C in the absence and presence of a strong‐binding, non‐force‐generating derivative of myosin subfragment‐1 (NEM‐S1) in chemically skinned myocardium from adult rats. 2 Relative to control conditions, application of 6 μM NEM‐S1 significantly increased Ca2+‐independent tension, measured at pCa 9.0, from 0.8 ± 0.3 to 3.7 ± 0.8 mN mm−2. Furthermore, NEM‐S1 potentiated submaximal Ca2+‐activated forces and thereby increased the Ca2+ sensitivity of force, i.e. the [Ca2+] required for half‐maximal activation (pCa50) increased from pCa 5.85 ± 0.05 to 5.95 ± 0.04 (change in pCa50 (ΔpCa50) = 0.11 ± 0.02). The augmentation of submaximal force by NEM‐S1 was accompanied by a marked reduction in the steepness of the force‐pCa relationship for forces less than 0.50 Po (maximum Ca2+‐activated force), i.e. the Hill coefficient (n2) decreased from 4.72 ± 0.38 to 1.54 ± 0.07. 3 In the absence of NEM‐S1, the rate of force redevelopment (ktr) was found to increase from 1.11 ± 0.21 s−1 at submaximal [Ca2+] (pCa 6.0) to 9.28 ± 0.41 s−1 during maximal Ca2+ activation (pCa 4.5). Addition of NEM‐S1 reduced the Ca2+ dependence of ktr by eliciting maximal values at low levels of Ca2+, i.e. ktr was 9.38 ± 0.30 s−1 at pCa 6.6 compared to 9.23 ± 0.27 s−1 at pCa 4.5. At intermediate levels of Ca2+, ktr was less than maximal but was still greater than values obtained at the same pCa in the absence of NEM‐S1. 4 NEM‐S1 dramatically reduced both the extent and rate of relaxation from steady‐state submaximal force following flash photolysis of the caged Ca2+ chelator diazo‐2. 5 These data demonstrate that strongly bound myosin cross‐bridges increase the level of thin filament activation in myocardium, which is manifested by an increase in the rate of cross‐bridge attachment, potentiation of force at low levels of free Ca2+, and slowed rates of relaxation.

Protein Kinase A–Mediated Phosphorylation of cMyBP-C Increases Proximity of Myosin Heads to Actin in Resting Myocardium

Protein kinase A-mediated (PKA) phosphorylation of cardiac myosin binding protein C (cMyBP-C) accelerates the kinetics of cross-bridge cycling and may relieve the tether-like constraint of myosin heads imposed by cMyBP-C. We favor a mechanism in which cMyBP-C modulates cross-bridge cycling kinetics by regulating the proximity and interaction of myosin and actin. To test this idea, we used synchrotron low-angle x-ray diffraction to measure interthick filament lattice spacing and the equatorial intensity ratio, I11/I10, in skinned trabeculae isolated from wild-type and cMyBP-C null (cMyBP-C−/−) mice. In wild-type myocardium, PKA treatment appeared to result in radial or azimuthal displacement of cross-bridges away from the thick filaments as indicated by an increase (approximately 50%) in I11/I10 (0.22±0.03 versus 0.33±0.03). Conversely, PKA treatment did not affect cross-bridge disposition in mice lacking cMyBP-C, because there was no difference in I11/I10 between untreated and PKA-treated cMyBP-C−/− myocardium (0.40±0.06 versus 0.42±0.05). Although lattice spacing did not change after treatment in wild-type (45.68±0.84 nm versus 45.64±0.64 nm), treatment of cMyBP-C−/− myocardium increased lattice spacing (46.80±0.92 nm versus 49.61±0.59 nm). This result is consistent with the idea that the myofilament lattice expands after PKA phosphorylation of cardiac troponin I, and when present, cMyBP-C, may stabilize the lattice. These data support our hypothesis that tethering of cross-bridges by cMyBP-C is relieved by phosphorylation of PKA sites in cMyBP-C, thereby increasing the proximity of cross-bridges to actin and increasing the probability of interaction with actin on contraction.

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 mum without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.

Cardiac MyBP-C Regulates the Rate and Force of Contraction in Mammalian Myocardium

Cardiac myosin-binding protein-C (cMyBP-C) is a thick filament-associated protein that seems to contribute to the regulation of cardiac contraction through interactions with either myosin or actin or both. Several studies over the past several years have suggested that the interactions of cardiac myosin-binding protein-C with its binding partners vary with its phosphorylation state, binding predominantly to myosin when dephosphorylated and to actin when it is phosphorylated by protein kinase A or other kinases. Here, we summarize evidence suggesting that phosphorylation of cardiac myosin binding protein-C is a key regulator of the kinetics and amplitude of cardiac contraction during β-adrenergic stimulation and increased stimulus frequency. We propose a model for these effects via a phosphorylation-dependent regulation of the kinetics and extent of cooperative recruitment of cross bridges to the thin filament: phosphorylation of cardiac myosin binding protein-C accelerates cross bridge binding to actin, thereby accelerating recruitment and increasing the amplitude of the cardiac twitch. In contrast, enhanced lusitropy as a result of phosphorylation seems to be caused by a direct effect of phosphorylation to accelerate cross-bridge detachment rate. Depression or elimination of one or both of these processes in a disease, such as end-stage heart failure, seems to contribute to the systolic and diastolic dysfunction that characterizes the disease.

Aging-dependent depression in the kinetics of force development in rat skinned myocardium

Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of beta-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 x Brown Norway F1 hybrid rats, we examined both the rate of tension development (kCa) and unloaded shortening velocity in chemically skinned myocardium. Although kCa in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal kCa were significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in beta-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity.Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of β-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 × Brown Norway F1hybrid rats, we examined both the rate of tension development ( k Ca) and unloaded shortening velocity in chemically skinned myocardium. Although k Ca in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal k Cawere significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in β-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity.

Myosin binding protein-C phosphorylation is the principal mediator of protein kinase A effects on thick filament structure in myocardium

Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) is a regulator of pump function in healthy hearts. However, the mechanisms of regulation by cAMP-dependent protein kinase (PKA)-mediated cMyBP-C phosphorylation have not been completely dissociated from other myofilament substrates for PKA, especially cardiac troponin I (cTnI). We have used synchrotron X-ray diffraction in skinned trabeculae to elucidate the roles of cMyBP-C and cTnI phosphorylation in myocardial inotropy and lusitropy. Myocardium in this study was isolated from four transgenic mouse lines in which the phosphorylation state of either cMyBP-C or cTnI was constitutively altered by site-specific mutagenesis. Analysis of peak intensities in X-ray diffraction patterns from trabeculae showed that cross-bridges are displaced similarly from the thick filament and toward actin (1) when both cMyBP-C and cTnI are phosphorylated, (2) when only cMyBP-C is phosphorylated, and (3) when cMyBP-C phosphorylation is mimicked by replacement with negative charge in its PKA sites. These findings suggest that phosphorylation of cMyBP-C relieves a constraint on cross-bridges, thereby increasing the proximity of myosin to binding sites on actin. Measurements of Ca(2+)-activated force in myocardium defined distinct molecular effects due to phosphorylation of cMyBP-C or co-phosphorylation with cTnI. Echocardiography revealed that mimicking the charge of cMyBP-C phosphorylation protects hearts from hypertrophy and systolic dysfunction that develops with constitutive dephosphorylation or genetic ablation, underscoring the importance of cMyBP-C phosphorylation for proper pump function.