Sensitivity Of Force Development Research Articles

Phosphorylation of the regulatory light chains of myosin affects Ca2+ sensitivity of skeletal muscle contraction.

The role of phosphorylation of the myosin regulatory light chains (RLC) is well established in smooth muscle contraction, but in striated (skeletal and cardiac) muscle its role is still controversial. We have studied the effects of RLC phosphorylation in reconstituted myosin and in skinned skeletal muscle fibers where Ca2+ sensitivity and the kinetics of steady-state force development were measured. Skeletal muscle myosin reconstituted with phosphorylated RLC produced a much higher Ca2+ sensitivity of thin filament-regulated ATPase activity than nonphosphorylated RLC (change in -log of the Ca2+ concentration producing half-maximal activation = approximately 0.25). The same was true for the Ca2+ sensitivity of force in skinned skeletal muscle fibers, which increased on reconstitution of the fibers with the phosphorylated RLC. In addition, we have shown that the level of endogenous RLC phosphorylation is a crucial determinant of the Ca2+ sensitivity of force development. Studies of the effects of RLC phosphorylation on the kinetics of force activation with the caged Ca2+, DM-nitrophen, showed a slight increase in the rates of force development with low statistical significance. However, an increase from 69 to 84% of the initial steady-state force was observed when nonphosphorylated RLC-reconstituted fibers were subsequently phosphorylated with exogenous myosin light chain kinase. In conclusion, our results suggest that, although Ca2+ binding to the troponin-tropomyosin complex is the primary regulator of skeletal muscle contraction, RLC play an important modulatory role in this process.

Invited Review: pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation.

Cardiac muscle contraction depends on the tightly regulated interactions of thin and thick filament proteins of the contractile apparatus. Mutations of thin filament proteins (actin, tropomyosin, and troponin), causing familial hypertrophic cardiomyopathy (FHC), occur predominantly in evolutionarily conserved regions and induce various functional defects that impair the normal contractile mechanism. Dysfunctional properties observed with the FHC mutants include altered Ca(2+) sensitivity, changes in ATPase activity, changes in the force and velocity of contraction, and destabilization of the contractile complex. One apparent tendency observed in these thin filament mutations is an increase in the Ca(2+) sensitivity of force development. This trend in Ca(2+) sensitivity is probably induced by altering the cross-bridge kinetics and the Ca(2+) affinity of troponin C. These in vitro defects lead to a wide variety of in vivo cardiac abnormalities and phenotypes, some more severe than others and some resulting in sudden cardiac death.

Hypercontractile Properties of Cardiac Muscle Fibers in a Knock-in Mouse Model of Cardiac Myosin-binding Protein-C

Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.

Altered Regulation of Cardiac Muscle Contraction by Troponin T Mutations That Cause Familial Hypertrophic Cardiomyopathy

To study the effect of troponin (Tn) T mutations that cause familial hypertrophic cardiomyopathy (FHC) on cardiac muscle contraction, wild-type, and the following recombinant human cardiac TnT mutants were cloned and expressed: I79N, R92Q, F110I, E163K, R278C, and intron 16(G(1) --> A) (In16). These TnT FHC mutants were reconstituted into skinned cardiac muscle preparations and characterized for their effect on maximal steady state force activation, inhibition, and the Ca(2+) sensitivity of force development. Troponin complexes containing these mutants were tested for their ability to regulate actin-tropomyosin(Tm)-activated myosin-ATPase activity. TnT(R278C) and TnT(F110I) reconstituted preparations demonstrated dramatically increased Ca(2+) sensitivity of force development, while those with TnT(R92Q) and TnT(I79N) showed a moderate increase. The deletion mutant, TnT(In16), significantly decreased both the activation and the inhibition of force, and substantially decreased the activation and the inhibition of actin-Tm-activated myosin-ATPase activity. ATPase activation was also impaired by TnT(F110I), while its inhibition was reduced by TnT(R278C). The TnT(E163K) mutation had the smallest effect on the Ca(2+) sensitivity of force; however, it produced an elevated activation of the ATPase activity in reconstituted thin filaments. These observed changes in the Ca(2+) regulation of force development caused by these mutations would likely cause altered contractility and contribute to the development of FHC.

Abnormal Cardiac Structure and Function in Mice Expressing Nonphosphorylatable Cardiac Regulatory Myosin Light Chain 2

A role for myosin phosphorylation in modulating normal cardiac function has long been suspected, and we hypothesized that changing the phosphorylation status of a cardiac myosin light chain might alter cardiac function in the whole animal. To test this directly, transgenic mice were created in which three potentially phosphorylatable serines in the ventricular isoform of the regulatory myosin light chain were mutated to alanines. Lines were obtained in which replacement of the endogenous species in the ventricle with the nonphosphorylatable, transgenically encoded protein was essentially complete. The mice show a spectrum of cardiovascular changes. As previously observed in skeletal muscle, Ca(2+) sensitivity of force development was dependent upon the phosphorylation status of the regulatory light chain. Structural abnormalities were detected by both gross histology and transmission electron microscopic analyses. Mature animals showed both atrial hypertrophy and dilatation. Echocardiographic analysis revealed that as a result of chamber enlargement, severe tricuspid valve insufficiency resulted in a detectable regurgitation jet. We conclude that regulated phosphorylation of the regulatory myosin light chains appears to play an important role in maintaining normal cardiac function over the lifetime of the animal.

The Role of the Four Ca2+ Binding Sites of Troponin C in the Regulation of Skeletal Muscle Contraction

In order to study the role of the Ca2+-specific sites (I and II) and the high affinity Ca2+-Mg2+ sites (III and IV) of TnC in the regulation of muscle contraction, we have constructed four mutants and the wild type (WTnC) of chicken skeletal TnC, with inactivated Ca2+ binding sites I and II (TnC1,2-), site III (TnC3-), site IV (TnC4-), and sites III and IV (TnC3,4C-). All Ca2+ binding site mutations were generated by replacing the Asp at the X-coordinating position of the Ca2+ binding loop with Ala. The binding of these mutated proteins to TnC-depleted skinned skeletal muscle fibers was investigated as well as the rate of their dissociation from these fibers. The proteins were also tested for their ability to restore steady state force to TnC-depleted fibers. We found that although the NH2-terminal mutant of TnC (TnC1,2-) bound to the TnC-depleted fibers (with a lower affinity than wild type TnC (WTnC)), it was unable to reactivate Ca2+-dependent force. This supports earlier findings that the low affinity Ca2+ binding sites (I and II) in TnC are responsible for the Ca2+-dependent activation of skeletal muscle contraction. All three COOH-terminal mutants of TnC bound to the TnC-depleted fibers, had different rates of dissociation, and could restore steady state force to the level of unextracted fibers. Although both high affinity Ca2+ binding sites (III and IV) are important for binding to the fibers, site III appears to be the primary determinant for maintaining the structural stability of TnC in the thin filament. Moreover, our results suggest an interaction between the NH2- and COOH-terminal domains of TnC, since alteration of sites I and II lowers the binding affinity of TnC to the fibers, and mutations in sites III and IV affect the Ca2+ sensitivity of force development.

Phosphorylation of Both Serine Residues in Cardiac Troponin I Is Required to Decrease the Ca2+ Affinity of Cardiac Troponin C

The phosphorylation of cardiac muscle troponin I (CTnI) at two adjacent N-terminal serine residues by cAMP-dependent protein kinase (PKA) has been implicated in the inotropic response of the heart to beta-agonists. Phosphorylation of these residues has been shown to reduce the Ca2+ affinity of the single Ca(2+)-specific regulatory site of cardiac troponin C (CTnC) and to increase the rate of Ca2+ dissociation from this site (Robertson, S. P., Johnson, J. D., Holroyde, M. J., Kranias, E. G., Potter, J. D., and Solaro, R. J. (1982) J. Biol. Chem. 257, 260-263). Recent studies (Zhang, R., Zhao, J., and Potter, J. D. (1995) Circ. Res. 76, 1028-1035) have correlated this increase in Ca2+ dissociation with a reduced Ca2+ sensitivity of force development and a faster rate of cardiac muscle relaxation in a PKA phosphorylated skinned cardiac muscle preparation. To further determine the role of the two PKA phosphorylation sites in mouse CTnI (serine 22 and 23), serine 22 or 23, or both were mutated to alanine. The wild type and the mutated CTnIs were expressed in Escherichia coli and purified. Using these mutants, it was found that serine 23 was phosphorylated more rapidly than serine 22 and that both serines are required to be phosphorylated in order to observe the characteristic reduction in the Ca2+ sensitivity of force development seen in a skinned cardiac muscle preparation. The latter result confirms that PKA phosphorylation of CTnI, and not other proteins, is responsible for this change in Ca2+ sensitivity. The results also suggest that one of the serines (23) may be constitutively phosphorylated and that serine 22 may be functionally more important.

G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation

The Ca2+ sensitivities of tonic (pulmonary and femoral artery) and phasic (portal vein and ileum) smooth muscles and the effects of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) and norepinephrine on Ca2+ sensitivity of force development and myosin light chain (MLC20) phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenous calmodulin. The Ca2+ sensitivity of force was higher (approximately 3-fold) in the tonic than in the phasic smooth muscles. The nucleotide specificity of Ca2+ sensitization was: GTP gamma S much greater than GTP greater than ITP much greater than CTP = UTP. Baseline phosphorylation (7% at pCa greater than 8) and maximal phosphorylation (58% at pCa 5.0) were both lower in portal vein than in femoral artery (20 and 97%). Norepinephrine and GTP gamma S increased phosphorylation at constant [Ca2+] (pCa 7.0-6.5). MLC20 phosphorylation induced by norepinephrine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S). In portal vein at pCa 5, GTP gamma S increased phosphorylation from 58%, the maximal Ca2(+)-activated value, to 75%, and at pCa greater than 8, from 7 to 13%. In femoral artery at pCa 5, neither phosphorylation (97%) nor force was affected by GTP gamma S, while at pCa greater than 8, GTP gamma S caused an increase in force (16% of maximum) with a borderline increase in MLC20 phosphorylation (from 20 to 27%). MLC20 phosphorylation (up to 100%) was positively correlated with force. The major results support the hypothesis that the G-protein coupled Ca2(+)-sensitizing effect of agonists on force development is secondary to increased MLC20 phosphorylation.