Troponin I Phosphorylation Research Articles

When Is cAMP Not cAMP?

Many important cellular processes are controlled via stimulation (or inhibition) of signal transduction systems, among which heptahelical G protein–coupled receptors (GPCRs) figure prominently. A classical example in cardiac myocytes is the β-adrenergic receptor (β-AR) cascade (see Figure, panel A), which leads to positive inotropic and lusitropic effects.1 Occupation of the β-ARs by an agonist activates a GTP binding protein (Gs), such that the α subunit dissociates and activates adenylyl cyclase (AC), thereby producing cAMP. The increase in cAMP leads to the dissociation of the regulatory and catalytic subunits of protein kinase A (PKA). PKA can be tethered near its substrates by an A-kinase anchoring protein (AKAP). The PKA catalytic subunit phosphorylates several key myocyte proteins involved in excitation-contraction (E-C) coupling, including the L-type Ca2+ channel, phospholamban (PLB), ryanodine receptor (RyR), myosin binding protein C, and troponin I (TnI). These effects produce PKA-dependent increases in Ca2+ current ( I Ca), sarcoplasmic reticulum (SR) Ca2+ uptake and release, as well as a desensitization of the myofilaments to Ca2+. The net result is the characteristic positive inotropic and lusitropic effects of β-AR activation in cardiac myocytes. A, Local β-AR signaling cascade in cardiac myocytes. B, GLP-1 signaling cascade. In this pathway, cAMP may activate glycolysis but cannot activate I Ca, PLB, or TnI phosphorylation. Epi indicates epinephrine; PFK, phosphofructokinase; and ATPase, SR Ca2+-ATPase (see text for other abbreviations). The stimulatory effects of GPCR activation can be inhibited at several levels. The receptor can be desensitized by G protein receptor kinases (eg, β-ARK) and arrestins.2 The activation of AC by Gsα can be antagonized by an inhibitory G protein (Gi), which can be activated by muscarinic receptors (and may also be coactivated during β2-AR activation).3–5 The effects of …

Altered phosphorylation and calcium sensitivity of cardiac myofibrillar proteins during sepsis.

Altered phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins during different phases of sepsis were investigated. Sepsis was induced by cecal ligation and puncture (CLP). The results show that phosphorylation of troponin I (TnI) was increased by 268% during the early phase (9 h after CLP) but decreased by 46% during the late phase (18 h after CLP) of sepsis. Phosphorylation of C protein was increased by 76% during the early phase but decreased by 41% during the late phase of sepsis. Phosphorylation of myosin light chain-2 (MLC-2) remained unaltered during the early phase but was decreased by 38% during the late phase of sepsis. Phosphorylation of TnT was unaffected during the progression of sepsis. The increases in the phosphorylation of TnI and C protein during early sepsis were associated with the decrease in the Ca(2+) sensitivity of myofilaments and the increases in myocardial changes in tension development (+dP/dt(max)) and cAMP level. The decreases in the phosphorylation of TnI and C protein during late sepsis coincided with the declines in the activities of myofibrillar ATPase, Ca(2+) sensitivity of myofilaments, myocardial +/-dP/dt(max), and cAMP content. The increases and the decreases in the phosphorylation of TnI and C protein, +/-dP/dt(max), and the tissue cAMP level were sensitive to isoproterenol stimulation and propranolol inhibition. These findings suggest that alterations in the phosphorylation of myofibrillar proteins, such as TnI, C protein, and MLC-2, and changes in the activities and the Ca(2+) sensitivity of myofibrillar ATPase may contribute to the altered cardiac function during the progression of sepsis. Furthermore, the sepsis-induced alterations in the phosphorylation and Ca(2+) sensitivity of cardiac myofibrillar proteins were mediated via a beta-adrenergic receptor pathway.

Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle.

Phosphorylation of cardiac myofibrils by cAMP-dependent protein kinase (PKA) can increase the intrinsic rate of myofibrillar relaxation, which may contribute to the shortening of the cardiac twitch during beta-adrenoceptor stimulation. However, it is not known whether the acceleration of myofibrillar relaxation is due to phosphorylation of troponin I (TnI) or of myosin binding protein-C (MyBP-C). To distinguish between these possibilities, we used transgenic mice that overexpress the nonphosphorylatable, slow skeletal isoform of TnI in the myocardium and do not express the normal, phosphorylatable cardiac TNI: The intrinsic rate of relaxation of myofibrils from wild-type and transgenic mice was measured using flash photolysis of diazo-2 to rapidly decrease the [Ca(2+)] within skinned muscles from the mouse ventricles. Incubation with PKA nearly doubled the intrinsic rate of myofibrillar relaxation in muscles from wild-type mice (relaxation half-time fell from approximately 150 to approximately 90 ms at 22 degrees C) but had no effect on the relaxation rate of muscles from the transgenic mice. In parallel studies with intact muscles, we assessed crossbridge kinetics indirectly by determining f(min) (the frequency for minimum dynamic stiffness) during tetanic contractions. Stimulation of beta-adrenoceptors with isoproterenol increased f(min) from 1.9 to 3.1 Hz in muscles from wild-type mice but had no effect on f(min) in muscles from transgenic mice. We conclude that the acceleration of myofibrillar relaxation rate by PKA is due to phosphorylation of TnI, rather than MyBP-C, and that this may be due, at least in part, to faster crossbridge cycle kinetics.

Effect of Troponin I Phosphorylation by Protein Kinase A on Length-Dependence of Tension Activation in Skinned Cardiac Muscle Fibers

We examined the effect of troponin I (TnI) phosphorylation by cAMP-dependent protein kinase (PKA) on the length-dependent tension activation in skinned rat cardiac trabeculae. Increasing sarcomere length shifted the pCa (−log[Ca2+])–tension relation to the left. Treatment with PKA decreased the Ca2+ sensitivity of the myofilament and also decreased the length-dependent shift of the pCa–tension relation. Replacement of endogenous TnI with phosphorylated TnI directly demonstrated that TnI phosphorylation is responsible for the decreased length-dependence. When MgATP concentration was lowered in the absence of Ca2+, tension was elicited through rigorous cross-bridge-induced thin filament activation. Increasing sarcomere length shifted the pMgATP (−log[MgATP])–tension relation to the right, and either TnI phosphorylation or partial extraction of troponin C (TnC) abolished this length-dependent shift. We conclude that TnI phosphorylation by PKA attenuates the length-dependence of tension activation in cardiac muscle by decreasing the cross-bridge-dependent thin filament activation through a reduction of the interaction between TnI and TnC.

Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation.

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.

Altered phosphorylation of sarcoplasmic reticulum contributes to the diminished contractile response to isoproterenol in hypertrophied rat hearts.

We tested the hypothesis that changes in phosphorylation of the sarcoplasmic reticulum (SR) protein, phospholamban (PIB) and myofibrillar proteins troponin I (TnI) and C protein are responsible for the decreased relaxant response to isoproterenol in cardiac hypertrophy and failure induced by ascending aortic banding in rats. In isolated perfused heart preparations under maximal isoproterenol stimulation, the capacity for in vitro cAMP-dependent phosphorylation of PIB was significantly increased at the compensatory stage of hypertrophy (126-130%, P<0.001), but decreased with failure (70-76%, P<0.001). Phosphorylation of TnI also decreased in the failing hearts, however to a lesser extent (80-83%, P<0.05). No significant hypertrophy-related difference was evident in isoproterenol-induced phosphorylation of C protein. The relative tissue level of PIB was increased (150-168%, P<0.001) in hypertrophied and decreased (71-83.8%, P<0.05) in failing hearts compared with the respective age-matched sham-operated controls (100%). As a percentage above baseline, the maximal isoproterenol-induced increase in the EC50 of the SR Ca2+ pump in response to phosphorylation of PIB was 38.5+/-1.1% for sham-operated rats, and 26.0+/-3.8% and 15.4+/-4.2% for hypertrophied and failing hearts respectively. As a consequence, linear correlation was observed between the maximal increase in EC50 and the maximal rate of relaxation [(-dP/dt)/DevP] upon isoproterenol stimulation, declining with progressive hypertrophy to failure. These data suggest that hypertrophy-induced alterations in PIB phosphorylation and protein level contribute to the diminished relaxant response of the hypertrophied and failing heart to adrenergic agonists.

Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy.

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca(2+)-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca(2+) sensitivity on the Ca(2+)-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca(2+) sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.

CAPK-phosphorylation controls the interaction of the regulatory domain of cardiac myosin binding protein C with myosin-S2 in an on-off fashion

Myosin binding protein C is a protein of the myosin filaments of striated muscle which is expressed in isoforms specific for cardiac and skeletal muscle. The cardiac isoform is phosphorylated rapidly upon adrenergic stimulation of myocardium by cAMP-dependent protein kinase, and together with the phosphorylation of troponin-I and phospholamban contributes to the positive inotropy that results from adrenergic stimulation of the heart. Cardiac myosin binding protein C is phosphorylated by cAMP-dependent protein kinase on three sites in a myosin binding protein C specific N-terminal domain which binds to myosin-S2. This interaction with myosin close to the motor domain is likely to mediate the regulatory function of the protein. Cardiac myosin binding protein C is a common target gene of familial hypertrophic cardiomyopathy and most mutations encode N-terminal subfragments of myosin binding protein C. The understanding of the signalling interactions of the N-terminal region is therefore important for understanding the pathophysiology of myosin binding protein C associated cardiomyopathy. We demonstrate here by cosedimentation assays and isothermal titration calorimetry that the myosin-S2 binding properties of the myosin binding protein C motif are abolished by cAMP-dependent protein kinase-mediated tris-phosphorylation, decreasing the S2 affinity from a K d of ≈5 μM to undetectable levels. We show that the slow and fast skeletal muscle isoforms are no cAMP-dependent protein kinase substrates and that the S2 interaction of these myosin binding protein C isoforms is therefore constitutively on. The regulation of cardiac contractility by myosin binding protein C therefore appears to be a ‘brake-off’ mechanism that will free a specific subset of myosin heads from sterical constraints imposed by the binding to the myosin binding protein C motif.

Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy.

Most studies indicate that failing human hearts have greater baseline myofibrillar Ca2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca2+, thus altering the Ca2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism. Using in vitro back-phosphorylation with [gamma-32P]ATP, we demonstrated a significant (P<0.05) approximately 25% reduction in baseline PKA-dependent TnI phosphorylation in human hearts with dilated cardiomyopathy (DCM) compared with nonfailing (NF) human hearts. There was no significant difference in cAMP content or maximal PKA activity between DCM and NF hearts, but expression of the regulatory subunits of PKA-I (RI) and PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by approximately 40%, P<0.05; RII by approximately 30%, P<0.01). PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the beta-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca2+ sensitivity.

Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury.

Selective troponin I (TnI) modification has been demonstrated to be in part responsible for the contractile dysfunction observed with myocardial ischemia/reperfusion injury. We have isolated and characterized modified TnI products in isolated rat hearts after 0, 15, or 60 minutes of ischemia followed by 45 minutes of reperfusion using affinity chromatography with cardiac troponin C (TnC) and an anti-TnI antibody, immunological mapping, reversed-phase high-performance liquid chromatography, and mass spectrometry. Rat cardiac TnI becomes progressively degraded from 210 amino acid residues to residues 1-193, 63-193, and 73-193 with increased severity of injury. Degradation is accompanied by formation of covalent complexes between TnI 1-193 and, respectively, TnC residues 1-94 and troponin T (TnT) residues 191-298. The covalent complexes are likely a result of isopeptide bond formation between lysine 193 of TnI and glutamine 191 of TnT by the cross-linking enzyme transglutaminase. With severe ischemia, cellular necrosis results in specific release of TnI 1-193 into the reperfusion effluent and TnT degradation in the myocardium (25-, 27-, and 33-kDa products). Two-dimensional electrophoresis demonstrated that phosphorylation of TnI prevents ischemia-induced degradation. This study characterized the modified TnI products in isolated rat hearts reperfused after a brief or severe period of ischemia, revealing the progressive nature of TnI degradation, changes in phosphorylation, and covalent complexes with ischemia/reperfusion injury. Finally, we propose a model for ischemia/reperfusion injury in which the extent of proteolytic and transglutaminase activities ultimately determines whether apoptosis or necrosis is achieved.

Troponin I phosphorylation and myofilament calcium sensitivity during decompensated cardiac hypertrophy.

We have measured myocyte cell shortening, troponin-I (Tn-I) phosphorylation, Ca2+ dependence of actomyosin adenosinetriphosphatase (ATPase) activity, adenosine 3',5'-cyclic monophosphate (cAMP) levels, and myofibrillar isoform expression in the spontaneously hypertensive rat (SHR) during decompensated cardiac hypertrophy (76 wk old) and in age-matched Wistar-Kyoto rat (WKY) controls. The decreased inotropic response to beta-adrenergic stimulation previously observed in myocytes from 26-wk-old SHR was further reduced at 76 wk of age. In response to beta-adrenergic stimulation, Tn-I phosphorylation was greater in the 76-wk-old SHR than in the WKY, although cAMP-dependent protein kinase A (PKA)-dependent Tn-I phosphorylation in the SHR did not increase with progression from compensated (26 wk) to decompensated (76 wk) hypertrophy. We also observed a dissociation between the increased PKA-dependent Tn-I phosphorylation and decreased cAMP levels in the 76-wk-old SHR versus WKY during beta-adrenergic stimulation. Baseline Tn-I phosphorylation was significantly reduced in 76-wk-old SHR versus WKY and was associated with decreased basal cAMP levels and increased Ca2+ sensitivity of actomyosin ATPase activity. The change in myofilament Ca2+ sensitivity during beta-adrenergic stimulation in the 76-wk-old SHR (0.65 pCa units) was over twofold greater than in the 76-wk-old WKY (0.30 pCa units). We also determined whether embryonic troponin T isoforms were reexpressed in decompensated hypertrophy and observed significant reexpression of the embryonic cardiac troponin T isoforms in the 76-wk-old SHR. The significant decrease in Ca2+ sensitivity with beta-adrenergic stimulation in 76-wk-old SHR may contribute to the severely impaired inotropic response during decompensated hypertrophy in the SHR.

Experimental diabetes is associated with functional activation of protein kinase C epsilon and phosphorylation of troponin I in the heart, which are prevented by angiotensin II receptor blockade.

A cardiomyopathy that is characterized by an impairment in diastolic relaxation and a loss of calcium sensitivity of the isolated myofibril has been described in chronic diabetic animals and humans. To explore a possible role for protein kinase C (PKC)-mediated phosphorylation of myofibrillar proteins in this process, we characterized the subcellular distribution of the major PKC isoforms seen in the adult heart in cardiocytes isolated from diabetic rats and determined patterns of phosphorylation of the major regulatory proteins, including troponin I (TnI). Rats were made diabetic with a single injection of streptozotocin, and myocardiocytes were isolated and studied 3 to 4 weeks later. In nondiabetic animals, 76% of the PKC epsilon isoform was located in the cytosol and 24% was particulate, whereas in diabetic animals, 55% was cytosolic and 45% was particulate (P < .05). PKC delta, the other major PKC isoform seen in adult cardiocytes, did not show a change in subcellular localization. In parallel, TnI phosphorylation was increased 5-fold in cardiocytes isolated from the hearts of diabetic animals relative to control animals (P < .01). The change in PKC epsilon distribution and in TnI phosphorylation in diabetic animals was completely prevented by rendering the animals euglycemic with insulin or by concomitant treatment with a specific angiotensin II type-1 receptor (AT1) antagonist. Since PKC phosphorylation of TnI has been associated with a loss of calcium sensitivity of intact myofibrils, these data suggest that angiotensin II receptor-mediated activation of PKC may play a role in the contractile dysfunction seen in chronic diabetes.

Troponin I phosphorylation in spontaneously hypertensive rat heart: effect of beta-adrenergic stimulation.

We compared baseline and protein kinase A (PKA)-dependent troponin I (TnI) phosphorylation in 32Pi-labeled left ventricular myocytes from hearts of 26-wk spontaneously hypertensive rats (SHR) and Wistar-Kyoto controls (WKY). TnI phosphorylation was normalized to myosin light chain 2 phosphorylation, which was invariant. There was no difference in baseline TnI phosphorylation in SHR and WKY, but stimulation with isoproterenol, norepinephrine plus prazosin, forskolin, chloroadenosine 3',5'-cyclic monophosphate, or 3-isobutyl-1-methylxanthine caused a greater increase in TnI phosphorylation in the SHR than in the WKY. This was observed both in the presence and absence of the phosphatase inhibitor calyculin A; thus the differences in TnI phosphorylation between SHR and WKY are not due to decreased phosphatase activity in the SHR. After stimulation of the beta-adrenergic pathway, phospholamban phosphorylation was not different in SHR and WKY, indicating that the observed differences may be specific for PKA phosphorylation of TnI. The increased PKA-dependent TnI phosphorylation in the SHR resulted in decreased Ca2+ sensitivity of actomyosin adenosinetriphosphatase activity as compared with the WKY. We conclude that increased PKA-dependent TnI phosphorylation in the SHR may contribute to the impaired response to sympathetic stimulation.

Regulation of contractile proteins in diabetic heart.

Diabetes is one of the most prevalent chronic conditions that has a high association with death from cardiovascular disease(s). An impaired cardiac function independent of vascular disease suggests the existence of a primary myocardial defect in diabetes mellitus. We and others have documented that myocardial performance is impaired in the hearts of chronically diabetic rats and rabbits. Abnormalities in the contractile proteins and regulatory proteins could be responsible for the mechanical defects in streptozotocin (STZ)-diabetic hearts. The major focus of research on contractile proteins in the diabetic state has been on myosin ATPase and its isoenzymes. However, in the contractile protein system, this could be only one of the mechanisms that might be a controlling factor in myofilament contraction in diabetes. To define the role of cardiac contractile as well as regulatory proteins (troponin-tropomyosin) as a whole in the regulation of actomyosin system in diabetic cardiomyopathy, individual proteins of the cardiac system were reconstituted under controlled conditions. Enzymatic data confirmed a diminished calcium sensitivity in the regulation of the cardiac actomyosin system when regulatory protein(s) complex was recombined from diabetic hearts. This diminished calcium sensitivity along with shifts in cardiac myosin heavy chain (V1-->V3) could contribute to the impaired cardiac function in the hearts of chronic diabetic rats. It has also been reported that sarcomeric proteins such as myosin light chain-2 (MLC-2) and troponin I (TnI) could be involved in regulating muscle contraction and in calcium sensitivity. Since phosphorylation of cardiac TnI is associated with altered maximum enzymatic activity and calcium force relationship in isolated muscle preparations. TnI phosphorylation could contribute to depressed myocardial contractility in experimental diabetes. While we have yet to understand the exact function of each component in cardiac muscle and their behavior in concert where all of them act in tandem, we have focussed on the role of contractile proteins and their regulation in diabetes in this review. We have also included a brief discussions on other relevant intracellular components. In summary, there is substantial evidence to suggest that there are independent processes associated with diabetes which effect cardiac performance in experimental animals and in man. The focus of this review has been the explication of a biochemical defect which underlies cardiac contractile dysfunction in experimental models of diabetes.

Troponin I phosphorylation in heart homogenate from diabetic rat

Although cardiac myofibrillar ATPase activity has been shown to be depressed during the development of diabetic heart dysfunction, the mechanisms of this alteration are not fully understood. Since phosphorylation of troponin I (TnI) is known to decrease the myofibrillar ATPase activity, the present study was undertaken to examine the TnI phosphorylation capacity in the diabetic heart homogenate. For this purpose rats were made diabetic by injecting streptozotocin (65 mg/kg; i.v.) and the hearts were removed 8 wk later. Some 6 wk diabetic animals were injected with insulin (3 U/day) for 2 wk. TnI content in the heart homogenate was measured by immunoblot assay, the mRNA abundance for TnI gene was determined by Northern blot analysis and the in vitro phosphorylation level of TnI was estimated by the ratio of phosphorylated TnI and total (phosphorylated and unphosphorylated) TnI. No significant changes in TnI content and gene expression of TnI were observed in right and left ventricles from the diabetic rats. However, the phosphorylation of TnI was higher (∼ 40%) in the diabetic hearts; this change was reversible upon insulin treatment. These results regarding TnI phosphorylation measured under in vitro conditions suggest that increased phosphorylation of TnI may contribute toward the depression in cardiac myofibrillar ATPase activity in chronic diabetes.

Slowing of shortening velocity of rat cardiac myocytes by adenosine receptor stimulation regardless of beta-adrenergic stimulation.

1. Single ventricular myocytes were enzymatically isolated, incubated with the A1-purinergic and beta-adrenergic receptor-specific agonists N6-cyclopentyladenosine (CPA) and isoprenaline (Iso), and then rapidly skinned. Ca2+ sensitivity of isometric tension and unloaded shortening velocity (Vo) were measured, and protein kinase A (PKA)-specific phosphorylations of troponin I (TnI) and C-protein were assessed by back-phosphorylation of cell suspensions with [gamma-32P]-ATP. 2. Isoprenaline treatment decreased the Ca2+ sensitivity of isometric tension relative to propranolol-treated controls, as did simultaneous stimulation with Iso and CPA (Iso + CPA). CPA alone had no effect on Ca2+ sensitivity. Vo was greater in Iso-treated cells than in paired controls, while Vo was significantly less than control in both Iso + CPA-treated and CPA-treated cells. 3. Phosphorylation of TnI and C-protein was increased by Iso treatment and also when Iso and CPA were simultaneously applied. CPA alone caused a significant decrease in the phosphorylation state of these two proteins. 4. From these results we conclude that A1-purinergic receptor stimulation does not inhibit beta-adrenergic receptor-mediated phosphorylation of myofilament proteins, nor does it alter the Ca2+ sensitivity of isometric tension at the level of the myofilaments. However, A1-receptor stimulation does decrease Vo at the level of the myofilaments by a mechanism that is independent of beta-adrenergically mediated phosphorylation of TnI and C-protein.

Regulation of phospholamban and troponin-I phosphorylation in the intact rat cardiomyocytes by adrenergic and cholinergic stimuli: roles of cyclic nucleotides, calcium, protein kinases and phosphatases and depolarization.

Protein phosphorylation was investigated in [32P]-labeled cardiomyocytes isolated from adult rat heart ventricles. The beta-adrenergic stimulation (by isoproterenol, ISO) increased the phosphorylation of inhibitory subunit of troponin (TN-I), C-protein and phospholamban (PLN). Such stimulation was largely mediated by increased adenylyl cyclase (AC) activity, increased myoplasmic cyclic AMP and increased cyclic AMP dependent protein kinase (A-kinase)-catalyzed phosphorylation of these proteins in view of the following observations: (a) dibutyryl-and bromo-derivatives of cyclic AMP mimicked the stimulatory effect of ISO on protein phosphorylation while (b) Rp-cyclic AMP was found to attenuate ISO-dependent stimulation. Unexpectedly, 8-bromo cyclic GMP was found to markedly increase TN-I and PLN phosphorylation. Both beta 1- and beta 2-adrenoceptors were present and ISO binding to either receptor was found to stimulate myocyte AC. However, the stimulation of the beta 2-AR only marginally increased while the stimulation of beta 1-AR markedly increased PLN phosphorylation. Other stimuli that increase tissue cyclic AMP levels also increased PLN and TN-I phosphorylation and these included isobutylmethylxanthine (non-specific phosphodiesterase inhibitor), milrinone (inhibits cardiotonic inhibitable phosphodiesterase, sometimes called type III or IV) and forskolin (which directly stimulates adenylyl cyclase). Cholinergic agonists acting on cardiomyocyte M2-muscarinic receptors that are coupled to AC via pertussis toxin(PT)-sensitive G proteins inhibited AC and attenuated ISO-dependent increases in PLN and TN-I phosphorylation. The in vivo PT treatment, which ADP-ribosylated Gi-like protein(s) in the myocytes, markedly attenuated muscarinic inhibitory effect on PLN and TN-I phosphorylation on one hand and, increased the beta-adrenergic stimulation, on the other. Controlled exposure of isolated myocytes to N-ethyl maleimide, also led to the findings similar to those seen following the PT treatment. Exposure of myocytes to phorbol, 12-myristate, 13-acetate (PMA) increased the protein phosphorylation, augmenting the stimulation by ISO, and such augmentation was antagonized by propranolol suggesting modulation of the beta-adrenoceptor coupled AC pathway by PMA. Okadaic acid (OA) exposure of myocytes also increased protein phosphorylation with the results supporting the roles for type 1 and 2A protein phosphatases in the dephosphorylation of PLN and TN-I. Interestingly OA treatment attenuated the muscarinic inhibitory effect which was restored by subsequent brief exposure of myocytes to PMA. While the stimulation of alpha adrenoceptors exerted little effect on the phosphorylation of PLN and TN-I, inactivation of alpha adrenoceptors by chloroethylclonidine (CEC), augmented beta-adrenergically stimulated phosphorylation. KCl-dependent depolarization of myocytes was observed to potentiate ISO-dependent increase in phosphorylation (incubation period 15 sec to 1 min) as well as to accelerate the time-dependent decline in this phosphorylation seen upon longer incubation. Verapamil decreased ISO-stimulated protein phosphorylation in the depolarized myocytes. Depolarization was found to have little effect on the muscarinic inhibitory action on phosphorylation. Prior treatment of myocytes with PMA, was found to augment ISO-stimulated protein phosphorylation in the depolarized myocytes. Such augmented increases were completely blocked by propranolol. Forskolin also stimulated PLN and TN-I phosphorylation. Prior exposure of myocytes to forskolin followed by incubation in the depolarized and polarized media showed that PLN was dephosphorylated more rapidly in the depolarized myocytes. The results support the view that both cyclic AMP and calcium signals cooperatively increase the rates of phosphorylation of TN-I and PLN in the depolarized cardiomyocytes during beta-adrenergic stimulation. (ABSTRACT TRUNCATED)

The Effects of Phosphorylation of Cardiac Troponin-I on Its Interactions with Actin and Cardiac Troponin-C

It is known that the phosphorylation of two serine residues on the NH2-terminal extension specific to cardiac troponin-I (Tn-I) modulates the calcium-dependent activation of the myofilaments. The process by which this occurs remains an unsolved puzzle. We have applied a dissective approach to study the effect of this phosphorylation on the interactions between Tn-I and its partner proteins, actin and troponin-C (Tn-C). Using N-[14C]ethylmaleimide-labelled Tn-I in sedimentation assays with F-actin, we found that the dephosphorylated Tn-I binds to F-actin with pronounced positive cooperativity, both in the absence and the presence of tropomyosin. Phosphorylation of the protein slightly weakens the interaction in the absence of tropomyosin, but the cooperativity remained. In the presence of tropomyosin, phosphorylation of the Tn-I also appeared to slightly weaken the interaction as well but, more significantly, the cooperativity was eliminated. These data can only be explained simply by a cooperative interaction between the monomer units in the actin filament. The interactions between cardiac Tn-I and Tn-C were studied by labelling the Tn-C with the fluorescent probe dansyl aziridine. As expected, the binding of the dephosphorylated Tn-I to Tn-C was strengthened by over 20-fold upon the addition of calcium to the assay. Phosphorylation of the protein, however, had a dramatic effect on the interaction in that it appeared to desensitise the complex to the effect of calcium: the Ka values obtained for both interactions (+/- Ca2+) were almost identical. These results clearly indicate that the phosphorylation of Tn-I in the cardiac system has dramatic effects on the isolated inter-protein interactions. We also discuss the possible significance of such an effect on the interactions of the isolated proteins in their roles within the intact cardiac regulatory complex.

Protein Kinase C Phosphorylation of Cardiac Troponin I and Troponin T Inhibits Ca 2+-Stimulated MgATPase Activity in Reconstituted Actomyosin and Isolated Myofibrils, and Decreases Actin-Myosin Interactions

The inhibitory effects of the phosphorylation of bovine cardiac troponin I (TnI) and troponin T (TnT) by protein kinase C (PKC) on the activity of Ca 2+-stimulated MgATPase of reconstituted actomyosin complex, as a function of the concentration of myosin or myosin subfragment 1 (S-1), were investigated. Phosphorylation of TnI and/or TnT invariably decreased the Ca 2+-stimulated enzyme activity of reconstituted actomyosin or actomyosin S-1, regardless of the concentration of whole myosin or S-1. The inhibition due to phosphorylated TnI was partially overcome as the concentration of myosin or S-1 increased, suggesting simple competition of phosphorylated TnI with myosin or S-1 for actin binding sites. Inhibition due to phosphorylated TnT, however, remained constant at all concentrations of myosin or S-1, suggesting that phosphorylated TnT may inhibit full Ca 2+-activation of the thin filament. Both phosphorylated TnI and TnT inhibited the Ca 2+-stimulated binding of S-1 ADP to regulated actin, consistent with the notion that the effects of phosphorylation of TnI and TnT affected interactions of the thin filament with the thick filament. Effects of PKC phosphorylation of the contractile components in adult rat cardiac myofibrils were also investigated. PKC phosphorylation of TnI and TnT, as well as other proteins in the contractile complex, resulted in the inhibition of Ca 2+-stimulated MgATPase activity with little change in the Ca 2+-sensitivity. Thus, the negative inotropic effects attributable to activation of PKC by phorbol esters, as reported by others, could be explained in part through PKC mediated phosphorylation of components of the contractile apparatus.

Differential sensitivity to isoprenaline of troponin I and phospholamban phosphorylation in isolated rat hearts

Phosphorylation of phospholamban (PLB), a membrane-bound 15 kDa protein and troponin I (TNI) was studied in isolated perfused rat hearts by using the back-phosphorylation technique with [32P]ATP catalysed by an excess of exogenous catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase, followed by protein separation. This standardized method allows the quantitative detection of protein phosphorylation specifically stimulated by cAMP. In control hearts the extent of specific phosphorylation was equivalent to 3.3 nmol of PLB and 11.0 mumol of TNI per g of cardiac tissue. In hearts freeze-clamped 30 s after exposure to isoprenaline (10 pM-10 microM), there was a dose-dependent decrease in phosphate incorporation in vitro, indicating a phosphorylation of the respective proteins in vivo. A differential sensitivity of TNI and PLB phosphorylation towards the beta-adrenergic agonist and the subsequent increase in tissue cAMP was found, favouring TNI phosphorylation. K0.5 values for isoprenaline were 2.94 +/- 0.04 nM and 4.46 +/- 0.24 nM for PLB and the 15 kDa protein, but 0.13 +/- 0.01 nM for TNI phosphorylation in the intact tissue. At an isoprenaline-induced increase in cAMP less than 3 pmol/mg of protein there was no or only a small increase in PLB phosphorylation, whereas TNI phosphorylation was nearly maximal. By plotting phosphorylation data against changes in contractile parameters a strong correlation was obtained for TNI (r = 0.95), assuming a linear relationship. For PLB a complex relationship is likely to exist. Our data (i) indicate a functional compartmentalization of the cAMP signal cascade and (ii) confirm that phosphorylation of TNI rather than of PLB is related to changes in mechanical myocardial responses.