Effects of troponin I phosphorylation on conformational exchange in the regulatory domain of cardiac troponin C.

Conformational exchange has been demonstrated within the regulatory domain of calcium-saturated cardiac troponin C when bound to the NH2-terminal domain of cardiac troponin I-(1-80), and cardiac troponin I-(1-80)DD, having serine residues 23 and 24 mutated to aspartate to mimic the phosphorylated form of the protein. Binding of cardiac troponin I-(1-80) decreases conformational exchange for residues 29, 32, and 34. Comparison of average transverse cross correlation rates show that both the NH2- and COOH-terminal domains of cardiac troponin C tumble with similar correlation times when bound to cardiac troponin I-(1-80). In contrast, the NH2- and COOH-terminal domains in free cardiac troponin C and cardiac troponin C bound cardiac troponin I-(1-80)DD tumble independently. These results suggest that the nonphosphorylated cardiac specific NH2 terminus of cardiac troponin I interacts with the NH2-terminal domain of cardiac troponin C.     

The effects of modifiers on enzyme catalysis: A non-classical nearest neighbor approach

We present a nearest neighbor lattice model of the effects of modifiers on two-state enzyme catalysis of the reaction s ? p-We do not in general make the assumptions of the classical approach to cooperative catalysis that yield (1) adsorption isotherms of the same form as those for the corresponding equilibrium system and (2) a rate of the catalyzed reaction proportional to the number of occupied catalytic sites. Closed form results are obtained for two approximations, the Bragg-Williams and the quasi-chemical. The latter requires (l),but is exact for several simple cases, including the concerted model, under this condition. Under (1) it is found that an interaction between modifier and catalytic sites, whether attractive or repulsive, increases the magnitudes of the slopes of the adsorption isotherms but that interactions between identical sites (catalytic or modifier) increase these magnitudes if attractive and decrease them if repulsive. Thus, the former interaction allows for phase transitions if sufficiently attractive or repulsive, but the latter only if sufficiently attractive. Herein also lies the explanation for why the concerted model displays only “positive cooperativity”. It is further seen that it is not possible to classify a modifier as an activator or inhibitor of the catalyzed reaction solely on the basis of the sign of the interaction energy between catalytic and modifier sites. For agiven energy, the rate of the reaction may increase or decrease in response to the modifier, or it may respond biphasically. Similarly, the rate may respond biphasically to the activities of s or p, lead- ing to instabilities. Thus, possibilities of multiple nonequilibrium stationary states or spatio-temporal patterns are raised-     

Identification of a region of troponin I important in signaling cross-bridge-dependent activation of cardiac myofilaments

Force generating strong cross-bridges are required to fully activate cardiac thin filaments, but the molecular signaling mechanism remains unclear. Evidence demonstrating differential extents of cross-bridge-dependent activation of force, especially at acidic pH, in myofilaments in which slow skeletal troponin I (ssTnI) replaced cardiac TnI (cTnI) indicates the significance of a His in ssTnI that is an homologous Ala in cTnI. We compared cross-bridge-dependent activation in myofilaments regulated by cTnI, ssTnI, cTnI(A66H), or ssTnI(H34A). A drop from pH 7.0 to 6.5 induced enhanced cross-bridge-dependent activation in cTnI myofilaments, but depressed activation in cTnI(A66H) myofilaments. This same drop in pH depressed cross-bridge-dependent activation in both ssTnI myofilaments and ssTnI(H34A) myofilaments. Compared with controls, cTnI(A66H) myofilaments were desensitized to Ca(2+), whereas there was no difference in the Ca(2+)-force relationship between ssTnI and ssTnI(H34A) myofilaments. The mutations in cTnI and ssTnI did not affect Ca(2+) dissociation rates from cTnC at pH 7.0 or 6.5. However, at pH 6.5, cTnI(A66H) had lower affinity for cTnT than cTnI. We also probed cross-bridge-dependent activation in myofilaments regulated by cTnI(Q56A). Myofilaments containing cTnI(Q56A) demonstrated cross-bridge-dependent activation that was similar to controls containing cTnI at pH 7.0 and an enhanced cross-bridge-dependent activation at pH 6.5. We conclude that a localized N-terminal region of TnI comprised of amino acids 33-80, which interacts with C-terminal regions of cTnC and cTnT, is of particular significance in transducing signaling of thin filament activation by strong cross-bridges.     

In Situ Time-Resolved FRET Reveals Effects of Sarcomere Length on Cardiac Thin-Filament Activation

During cardiac thin-filament activation, the N-domain of cardiac troponin C (N-cTnC) binds to Ca²⁺ and interacts with the actomyosin inhibitory troponin I (cTnI). The interaction between N-cTnC and cTnI stabilizes the Ca²⁺-induced opening of N-cTnC and is presumed to also destabilize cTnI–actin interactions that work together with steric effects of tropomyosin to inhibit force generation. Recently, our in situ steady-state FRET measurements based on N-cTnC opening suggested that at long sarcomere length, strongly bound cross-bridges indirectly stabilize this Ca²⁺-sensitizing N-cTnC–cTnI interaction through structural effects on tropomyosin and cTnI. However, the method previously used was unable to determine whether N-cTnC opening depends on sarcomere length. In this study, we used time-resolved FRET to monitor the effects of cross-bridge state and sarcomere length on the Ca²⁺-dependent conformational behavior of N-cTnC in skinned cardiac muscle fibers. FRET donor (AEDANS) and acceptor (DDPM)-labeled double-cysteine mutant cTnC(T13C/N51C)_AEDANS-DDPM was incorporated into skinned muscle fibers to monitor N-cTnC opening. To study the structural effects of sarcomere length on N-cTnC, we monitored N-cTnC opening at relaxing and saturating levels of Ca²⁺ and 1.80 and 2.2-μm sarcomere length. Mg²⁺-ADP and orthovanadate were used to examine the structural effects of noncycling strong-binding and weak-binding cross-bridges, respectively. We found that the stabilizing effect of strongly bound cross-bridges on N-cTnC opening (which we interpret as transmitted through related changes in cTnI and tropomyosin) become diminished by decreases in sarcomere length. Additionally, orthovanadate blunted the effect of sarcomere length on N-cTnC conformational behavior such that weak-binding cross-bridges had no effect on N-cTnC opening at any tested [Ca²⁺] or sarcomere length. Based on our findings, we conclude that the observed sarcomere length-dependent positive feedback regulation is a key determinant in the length-dependent Ca²⁺ sensitivity of myofilament activation and consequently the mechanism underlying the Frank-Starling law of the heart.     

Removal of the Cardiac Troponin I N-terminal Extension Improves Cardiac Function in Aged Mice

The cardiac troponin I (cTnI) isoform contains a unique N-terminal extension that functions to modulate activation of cardiac myofilaments. During cardiac remodeling restricted proteolysis of cTnI removes this cardiac specific N-terminal modulatory extension to alter myofilament regulation. We have demonstrated expression of the N-terminal-deleted cTnI (cTnI-ND) in the heart decreased the development of the cardiomyopathy like phenotype in a β-adrenergic-deficient transgenic mouse model. To investigate the potential beneficial effects of cTnI-ND on the development of naturally occurring cardiac dysfunction, we measured the hemodynamic and biochemical effects of cTnI-ND transgenic expression in the aged heart. Echocardiographic measurements demonstrate cTnI-ND transgenic mice exhibit increased systolic and diastolic functions at 16 months of age compared with age-matched controls. This improvement likely results from decreased Ca²⁺ sensitivity and increased cross-bridge kinetics as observed in skinned papillary bundles from young transgenic mice prior to the effects of aging. Hearts of cTnI-ND transgenic mice further exhibited decreased β myosin heavy chain expression compared to age matched non-transgenic mice as well as altered cTnI phosphorylation. Finally, we demonstrated cTnI-ND expressed in the heart is not phosphorylated indicating the cTnI N-terminal is necessary for the higher level phosphorylation of cTnI. Taken together, our data suggest the regulated proteolysis of cTnI during cardiac stress to remove the unique cardiac N-terminal extension functions to improve cardiac contractility at the myofilament level and improve overall cardiac function.     

Sexual dimorphism in rat left atrial function and response to adrenergic stimulation

A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca²⁺ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca²⁺ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca²⁺ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca²⁺ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca²⁺-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.     

Identification of a functionally critical protein kinase C phosphorylation residue of cardiac troponin T

Cardiac Troponin T (cTnT) is one prominent substrate through which protein kinase C (PKC) exerts its effect on cardiomyocyte function. To determine the specific functional effects of the cTnT PKC-dependent phosphorylation sites (Thr197, Ser201, Thr206, and Thr287) we first mutated these residues to glutamate (E) or alanine (A). cTnT was selectively mutated to generate single, double, triple, and quadruple mutants. Bacterially expressed mutants were evaluated in detergent-treated mouse left ventricular papillary muscle fiber bundles where the endogenous troponin was replaced with a recombinant troponin complex containing either cTnT phosphorylated by PKC-alpha or a mutant cTnT. We simultaneously determined isometric tension development and actomyosin Mg-ATPase activity of the exchanged fiber bundles as a function of Ca2+ concentration. Our systematic analysis of the functional role of the multiple PKC phosphorylation sites on cTnT identified a localized region that controls maximum tension, ATPase activity, and Ca2+ sensitivity of the myofilaments. An important and novel finding of our study was that Thr206 is a functionally critical cTnT PKC phosphorylation residue. Its exclusive phosphorylation by PKC-alpha or replacement by Glu (mimicking phosphorylation) significantly decreased maximum tension, actomyosin Mg-ATPase activity, myofilament Ca2+ sensitivity, and cooperativity. On the other hand the charge modification of the other three residues together (T197/S201/T287-E) had no functional effect. Fibers bundles containing phosphorylated cTnT-wt (but not the T197/S201/T206/T287-E) exhibited a significant decrease of tension cost as compared with cTnT-wt.     

Phosphatidylinositol 4,5-bisphosphate and intracellular pH regulate the ROMK1 potassium channel via separate but interrelated mechanisms

ROMK channels are responsible for K(+) secretion in kidney. The activity of ROMK is regulated by intracellular pH (pH(i)) with acidification causing channel closure (effective pK(a) approximately 6.9). Recently, we and others reported that a direct interaction of the channels with phosphatidyl-4,5-bisphosphate (PIP(2)) is critical for opening of the inwardly rectifying K(+) channels. Here, we investigate the relationship between the mechanisms for regulation of ROMK by PIP(2) and by pH(i). We find that disruption of PIP(2)-ROMK1 interaction not only decreases single-channel open probability (P(o)) but gives rise to a ROMK1 subconductance state. This state has an increased sensitivity to intracellular protons (effective pK(a) shifted to pH approximately 7.8), such that the subconductance channels are relatively quiescent at physiological pH(i). Open probability for the subconductance channels can then be increased by intracellular alkalinization to supra-physiological pH. This increase in P(o) for the subconductance channels by alkalinization is not associated with an increase in PIP(2)-channel interaction. Thus, direct interaction with PIP(2) is critical for ROMK1 to open at full conductance. Disruption of this interaction increases pH(i) sensitivity for the channels via emergence of the subconductance state. The control of open probability of ROMK1 by pH(i) occurs via a mechanism distinct from the regulation by PIP(2).     

PKCepsilon increases phosphorylation of the cardiac myosin binding protein C at serine 302 both in vitro and in vivo

Cardiac myosin binding protein C (cMyBPC) phosphorylation is essential for normal cardiac function. Although PKC was reported to phosphorylate cMyBPC in vitro, the relevant PKC isoforms and functions of PKC-mediated cMyBPC phosphorylation are unknown. We recently reported that a transgenic mouse model with cardiac-specific overexpression of PKCepsilon (PKCepsilon TG) displayed enhanced sarcomeric protein phosphorylation and dilated cardiomyopathy. In the present study, we have investigated cMyBPC phosphorylation in PKCepsilon TG mice. Western blotting and two-dimensional gel electrophoresis demonstrated a significant increase in cMyBPC serine (Ser) phosphorylation in 12-month-old TG mice compared to wild type (WT). In vitro PKCepsilon treatment of myofibrils increased the level of cMyBPC Ser phosphorylation in WT mice to that in TG mice, whereas treatment of TG myofibrils with PKCepsilon showed only a minimal increase in cMyBPC Ser phosphorylation. Three peptide motifs of cMyBPC were identified as the potential PKCepsilon consensus sites including a 100% matched motif at Ser302 and two nearly matched motifs at Ser811 and Ser1203. We treated synthetic peptides corresponding to the sequences of these three motifs with PKCepsilon and determined phosphorylation by mass spectrometry and ELISA assay. PKCepsilon induced phosphorylation at the Ser302 site but not at the Ser811 or Ser1203 sites. A S302A point mutation in the Ser302 peptide abolished the PKCepsilon-dependent phosphorylation. Taken together, our data show that the Ser302 on mouse cMyBPC is a likely PKCepsilon phosphorylation site both in vivo and in vitro and may contribute to the dilated cardiomyopathy associated with increased PKCepsilon activity.