Protein kinase C α and ε phosphorylation of troponin and myosin binding protein C reduce Ca2+ sensitivity in human myocardium

Previous studies indicated that the increase in protein kinase C (PKC)-mediated myofilament protein phosphorylation observed in failing myocardium might be detrimental for contractile function. This study was designed to reveal and compare the effects of PKCα- and PKCε-mediated phosphorylation on myofilament function in human myocardium. Isometric force was measured at different [Ca²⁺] in single permeabilized cardiomyocytes from failing human left ventricular tissue. Activated PKCα and PKCε equally reduced Ca²⁺ sensitivity in failing cardiomyocytes (ΔpCa₅₀ = 0.08 ± 0.01). Both PKC isoforms increased phosphorylation of troponin I- (cTnI) and myosin binding protein C (cMyBP-C) in failing cardiomyocytes. Subsequent incubation of failing cardiomyocytes with the catalytic subunit of protein kinase A (PKA) resulted in a further reduction in Ca²⁺ sensitivity, indicating that the effects of both PKC isoforms were not caused by cross-phosphorylation of PKA sites. Both isozymes showed no effects on maximal force and only PKCα resulted in a modest significant reduction in passive force. Effects of PKCα were only minor in donor cardiomyocytes, presumably because of already saturated cTnI and cMyBP-C phosphorylation levels. Donor tissue could therefore be used as a tool to reveal the functional effects of troponin T (cTnT) phosphorylation by PKCα. Massive dephosphorylation of cTnT with alkaline phosphatase increased Ca²⁺ sensitivity. Subsequently, PKCα treatment of donor cardiomyocytes reduced Ca²⁺ sensitivity (ΔpCa₅₀ = 0.08 ± 0.02) and solely increased phosphorylation of cTnT, but did not affect maximal and passive force. PKCα- and PKCε-mediated phosphorylation of cMyBP-C and cTnI as well as cTnT decrease myofilament Ca²⁺ sensitivity and may thereby reduce contractility and enhance relaxation of human myocardium.     

Enhanced myofilament responsiveness upon β-adrenergic stimulation in post-infarct remodeled myocardium

Previously we showed that left ventricular (LV) responsiveness to exercise-induced increases in noradrenaline was blunted in pigs with a recent myocardial infarction (MI) [van der Velden et al. Circ Res. 2004], consistent with perturbed β-adrenergic receptor (β-AR) signaling. Here we tested the hypothesis that abnormalities at the myofilament level underlie impaired LV responsiveness to catecholamines in MI. Myofilament function and protein composition were studied in remote LV biopsies taken at baseline and during dobutamine stimulation 3 weeks after MI or sham. Single permeabilized cardiomyocytes demonstrated reduced maximal force (F_max) and higher Ca²⁺-sensitivity in MI compared to sham. F_max did not change during dobutamine infusion in sham, but markedly increased in MI. Moreover, the dobutamine-induced decrease in Ca²⁺-sensitivity was significantly larger in MI than sham. Baseline phosphorylation assessed by phosphostaining of β-AR target proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI) in MI and sham was the same. However, the dobutamine-induced increase in overall cTnI phosphorylation and cTnI phosphorylation at protein kinase A (PKA)-sites (Ser23/24) was less in MI compared to sham. In contrast, the dobutamine-induced phosphorylation of cMyBP-C at Ser282 was preserved in MI, and coincided with increased autophosphorylation (at Thr282) of the cytosolic Ca²⁺-dependent calmodulin kinase II (CaMKII-δC). In conclusion, in post-infarct remodeled myocardium myofilament responsiveness to dobutamine is significantly enhanced despite the lower increase in PKA-mediated phosphorylation of cTnI. The increased myofilament responsiveness in MI may depend on the preserved cMyBP-C phosphorylation possibly resulting from increased CaMKII-δC activity and may help to maintain proper diastolic performance during exercise.     

Slow skeletal troponin I gene transfer, expression, and myofilament incorporation enhances adult cardiac myocyte contractile function

The functional significance of the developmental transition from slow skeletal troponin I (ssTnI) to cardiac TnI (cTnI) isoform expression in cardiac myocytes remains unclear. We show here the effects of adenovirus-mediated ssTnI gene transfer on myofilament structure and function in adult cardiac myocytes in primary culture. Gene transfer resulted in the rapid, uniform, and nearly complete replacement of endogenous cTnI with the ssTnI isoform with no detected changes in sarcomeric ultrastructure, or in the isoforms and stoichiometry of other myofilament proteins compared with control myocytes over 7 days in primary culture. In functional studies on permeabilized single cardiac myocytes, the threshold for Ca²⁺-activated contraction was significantly lowered in adult cardiac myocytes expressing ssTnI relative to control values. The tension–Ca²⁺ relationship was unchanged from controls in primary cultures of cardiac myocytes treated with adenovirus containing the adult cardiac troponin T (TnT) or cTnI cDNAs. These results indicate that changes in Ca²⁺ activation of tension in ssTnI-expressing cardiac myocytes were isoform-specific, and not due to nonspecific functional changes resulting from overexpression of a myofilament protein. Further, Ca²⁺-activated tension development was enhanced in cardiac myocytes expressing ssTnI compared with control values under conditions mimicking the acidosis found during myocardial ischemia. These results show that ssTnI enhances contractile sensitivity to Ca²⁺ activation under physiological and acidic pH conditions in adult rat cardiac myocytes, and demonstrate the utility of adenovirus vectors for rapid and efficient genetic modification of the cardiac myofilament for structure/function studies in cardiac myocytes.     

PKC-betaII sensitizes cardiac myofilaments to Ca2+ by phosphorylating troponin I on threonine-144

Ventricular myocytes express Galphaq-coupled receptors that can mediate enhanced contractility by increasing the sensitivity of the contractile apparatus to Ca(2+). The precise mechanisms underlying this change have been difficult to define, in part because myofilament regulatory proteins contain multiple phosphorylation sites for protein kinase C (PKC), protein kinase A (PKA) and myosin light chain kinase (MLCK), with potentially opposing effects. MLCK increases whereas PKC and PKA have a strong tendency to decrease myofilament Ca(2+) sensitivity in myocardium. Here we show in mouse cardiac myocytes that PKC-betaII can increase Ca(2+) sensitivity of tension by a similar magnitude to MLCK but via a distinct mechanism. For PKC-betaII (32)P-incorporation occurred primarily into cardiac troponin I (cTnI) and functional effects were highly dependent upon mutations in phosphorylation sites of cTnI. Replacement of serines-23/24 (PKA sites) with alanine prevented cross-phosphorylation of these sites, reduced (32)P-incorporation into cTnI by half and resulted in myofilament Ca(2+) sensitization rather than desensitization in response to PKC-betaII. Replacement of three additional sites on cTnI, serines-43/45 and threonine-144, eliminated PKC-betaII-mediated Ca(2+) sensitization and the remaining (32)P-incorporation into cTnI. A preference for PKC-betaII phosphorylation of threonine-144 in the intact filament lattice was revealed by differential stable isotope labeling and supported by an analysis of peptide phosphorylation. The results suggest that threonine-144 within the critical inhibitory domain of cTnI represents a novel site of regulation of myofilament Ca(2+) sensitivity by PKC-betaII, with possible implications for chronically stressed or diseased hearts.     

Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation

The heart is remarkably adaptable in its ability to vary its function to meet the changing demands of the circulatory system. During times of physiological stress, cardiac output increases in response to increased sympathetic activity, which results in protein kinase A (PKA)-mediated phosphorylations of the myofilament proteins cardiac troponin (cTn)I and cardiac myosin-binding protein (cMyBP)-C. Despite the importance of this mechanism, little is known about the relative contributions of cTnI and cMyBP-C phosphorylation to increased cardiac contractility. Using engineered mouse lines either lacking cMyBP-C (cMyBP-C(-/-)) or expressing a non-PKA phosphorylatable cTnI (cTnI(ala2)), or both (cMyBP-C(-/-)/cTnI(ala2)), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnI(ala2) skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium; however, PKA had no effect on stretch activation in cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnI(ala2) myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca(2+) sensitivity of force, whereas phosphorylation of cMyBP-C accelerates the kinetics of force development. These results predict that PKA phosphorylation of myofibrillar proteins in living myocardium contributes to accelerated relaxation in diastole and increased rates of force development in systole.     

Effects of contractile protein phosphorylation on force development in permeabilized rat cardiac myocytes

The phosphorylation status of myofibrillar proteins influences the Ca²⁺ responsiveness of the myofilaments,but the contribution of and the interaction between the individual components is poorly characterized. Therefore, in Langendorff perfused rat hearts (n=30), the phosphorylation levels of cardiac myosin binding protein-C (cMyBP-C), troponin I and T (cTnI, cTnT) and myosin light chain 1 and 2 (MLC-1, MLC-2) were determined by 1- and 2-dimensional gel electrophoresis. Isometric force development, its Ca²⁺-sensitivity, the rate of tension redevelopment (k_tr) and passive force (F_pas) were studied at optimal sarcomere length (2.2 μm) in mechanically isolated,permeabilized cardiomyocytes at 15 °C. Protein phosphorylation was varied by: 1) blocking spontaneous cardiac activity by lidocaine (0.35 mM; Quiescence); 2) electrical stimulation of the hearts at 5 Hz (Contraction) and 3. treatment of contracting hearts with Isoprenaline (1 μM). MLC-2 phosphorylation was increased in the Contraction group almost 2-fold, relative to the Quiescence group, whereas cMyBP-C and cTnI phosphorylation remained the same. Isoprenaline resulted in 3.7-fold increases in both cMyBP-C and cTnI phosphorylation, but did not result in a further increase in MLC-2 phosphorylation.No significant differences were found in maximum force and k_tr between groups, both before and after protein kinase A (PKA) treatment. Ca²⁺-sensitivity in the Contraction and Isoprenaline groups was significantly reduced in comparison to the Quiescence group. These differences were largely abolished by PKA and F_pas was reduced. These results highlight the impact of PKA-dependent phosphorylation on Ca²⁺-sensitivity and provide evidence for an interaction between the effects of TnI and MLC-2 phosphorylation.     

Nitroxyl (HNO) for Treatment of Acute Heart Failure

The loss of contractile function is a hallmark of heart failure. Although increasing intracellular Ca²⁺ is a possible strategy for improving contraction, current inotropic agents that achieve this by raising intracellular cAMP levels, such as β-agonists and phosphodiesterase inhibitors, are generally deleterious when administered as long-term therapy due to arrhythmia and myocardial damage. Nitroxyl donors have been shown to improve cardiac function in normal and failing dogs, and in isolated cardiomyocytes they increase fractional shortening and Ca²⁺ transients, independently from cAMP/PKA or cGMP/PKG signaling. Instead, nitroxyl targets cysteines in the EC-coupling machinery and myofilament proteins, reversibly modifying them to enhance Ca²⁺ handling and myofilament Ca²⁺ sensitivity. Phase I–IIa trials with CXL-1020, a novel pure HNO donor, reported declines in left and right heart filling pressures and systemic vascular resistance, and increased cardiac output and stroke volume index. These findings support the concept of nitroxyl donors as attractive agents for the treatment of acute decompensated heart failure.     

Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.     

Binding of myosin binding protein-C to myosin subfragment S2 affects contractility independent of a tether mechanism

Mutations in the cardiac myosin binding protein-C gene (cMyBP-C) are among the most prevalent causes of inherited hypertrophic cardiomyopathy. Although most cMyBP-C mutations cause reading frameshifts that are predicted to encode truncated peptides, it is not known if or how expression of these peptides causes disease. One possibility is that because the N-terminus contains a unique binding site for the S2 subfragment of myosin, shortened cMyBP-C peptides could directly affect myosin contraction by binding to S2. To test this hypothesis, we compared the effects of a C1C2 protein containing the myosin S2 binding site on contractile properties in permeabilized myocytes from wild-type and cMyBP-C knockout mice. In wild-type myocytes, the C1C2 protein reversibly increased myofilament Ca2+ sensitivity of tension, but had no effect on resting tension. Identical results were observed in cMyBP-C knockout myocytes where C1C2 increased Ca2+ sensitivity of tension with the half-maximal response elicited at approximately 5 micromol/L C1C2. Maximum force was not affected by C1C2. However, phosphorylation of C1C2 by cAMP-dependent protein kinase reduced its ability to increase Ca2+ sensitivity. These results demonstrate that binding of the C1C2 peptide to S2 alone is sufficient to affect myosin contractile function and suggest that regulated binding of cMyBP-C to myosin S2 by phosphorylation directly influences myofilament Ca2+ sensitivity.     

Effect of troponin I Ser23/24 phosphorylation on Ca-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca²⁺-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca²⁺-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66 ± 1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca²⁺-sensitivity (pCa₅₀) to 5.50 ± 0.02 relative to the donor control value (pCa₅₀ = 5.43 ± 0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca²⁺-sensitivity relative to the failing control (pCa₅₀ = 5.60 ± 0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca²⁺-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca²⁺-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca²⁺-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca²⁺-sensitivity.     

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes

β₁-Adrenergic receptors (β₁ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. β₁ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, β₁AR activation enhanced the L-type Ca²⁺ current, intracellular Ca²⁺ transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-β-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by β₁ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. β₁AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected β₁AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between β₁AR and EPR responses. They also suggest that β₁AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.     

Allele and species dependent contractile defects by restrictive and hypertrophic cardiomyopathy-linked troponin I mutants

Restrictive cardiomyopathy (RCM) is a debilitating disease characterized by impaired ventricular filling, reduced ventricular volumes, and severe diastolic dysfunction. Hypertrophic cardiomyopathy (HCM) is characterized by ventricular hypertrophy and heightened risk of premature sudden cardiac death. These cardiomyopathies can result from mutations in the same gene that encodes for cardiac troponin I (cTnI). Acute genetic engineering of adult rat cardiac myocytes was used to ascertain whether primary physiologic outcomes could distinguish between RCM and HCM alleles at the cellular level. Co-transduction of cardiac myocytes with wild-type (WT) cTnI and RCM/HCM linked mutants in cTnI's inhibitory region (IR) demonstrated that WT cTnI preferentially incorporated into the sarcomere over IR mutants. The cTnI IR mutants exhibited minor effects in single myocyte Ca²⁺-activated tension assays yet prolonged relaxation and Ca²⁺ decay. In comparison RCM cTnI mutants in the helix-4/C-terminal region demonstrated a) hyper-sensitivity to Ca²⁺ under loaded conditions, b) slowed myocyte mechanical relaxation and Ca²⁺ transient decay, c) frequency-dependent Ca²⁺-independent diastolic tone, d) heightened myofilament incorporation and e) irreversible cellular contractile defects with acute diltiazem administration. For species comparison, a subset of cTnI mutants were tested in isolated adult rabbit cardiac myocytes. Here, RCM and HCM mutant cTnIs exerted similar effects of slowed myocyte relaxation and Ca²⁺ transient decay but did not show variable phenotypes by cTnI region. This study highlights cellular contractile defects by cardiomyopathy mutant cTnIs that are allele and species dependent. The species dependent results in particular raise important issues toward elucidating a unifying mechanistic pathway underlying the inherited cardiomyopathies.     

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin–myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca²⁺. We investigated whether ADP-stimulated force development (without Ca²⁺) can be used to reveal changes in actin–myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7_mut, MYBPC3_mut) and thin-filament (TNNT2_mut, TNNI3_mut) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCM_smn, and MYH7_mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2_mut sample corrected the abnormal actin–myosin blockade. In MYBPC3_trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C’s role in actin–myosin regulation. In the presence of Ca²⁺, ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca²⁺, pathologic [ADP] and low PKA-phosphorylation, high actin–myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.Heart failure (HF) is a syndrome clinically defined as the inability of the heart to sufficiently supply blood to organs and tissues (1). Systolic dysfunction is present in approximately one-half of the HF population, whereas diastolic dysfunction is a common feature in almost all HF patients (2). Moreover, in hypertrophic cardiomyopathy (HCM), which is caused by mutations in genes encoding thin- and thick-filament proteins, impaired diastolic function is frequently observed (3). Impaired relaxation of the heart may be caused by high myofilament Ca²⁺ sensitivity. This increased sensitivity for Ca²⁺ would result in residual myofilament activation at diastolic [Ca²⁺], which may delay the onset of ventricular relaxation and limit proper filling of the heart. High myofilament Ca²⁺ sensitivity has been observed in both acquired and genetic forms of cardiomyopathy (3, 4). In human idiopathic dilated cardiomyopathy (IDCM), high myofilament Ca²⁺ sensitivity has been associated with reduced β-adrenergic receptor-mediated phosphorylation by protein kinase A (PKA) (4). Reduced PKA phosphorylation of cardiac troponin I (cTnI) and cardiac myosin-binding protein C (cMyBP-C) increases myofilament Ca²⁺ sensitivity (5–8). Likewise, high myofilament Ca²⁺ sensitivity is a common characteristic of HCM and may be caused by the mutant protein or by reduced PKA-mediated protein phosphorylation secondary to HCM disease progression (3, 9).Contractile performance of the heart muscle may thus be perturbed by mutation-induced and phosphorylation-mediated protein changes that affect thin-filament transitions. Ca²⁺-induced cardiac muscle contraction is tightly modulated by the troponin–tropomyosin complex that regulates the interactions between the actin thin filament and myosin thick filament (i.e., cross-bridge formation). Accordingly, the myofilaments oscillate between three transitions termed the blocked (B-state), closed (C-state), and open (M-state) states of thin-filament regulation that represent the distinct position of tropomyosin on actin (10–12) (Fig. 1). In the absence of Ca²⁺ (B state), tropomyosin sterically blocks the myosin-binding sites on actin (Fig. 1A). Upon electrical activation of cardiomyocytes, the rise of cytosolic [Ca²⁺] alters the conformation of the troponin–tropomyosin complex, which moves tropomyosin on actin and exposes myosin-binding sites (C state). Weakly bound cross-bridges (myosin-ADP-Pi) populate the C state (10, 12) (Fig. 1B). Transition to the M state involves release of inorganic phosphate (Pi) from the cross-bridge and strong-binding cross-bridge formation (myosin-ADP) that induces additional movement of tropomyosin, resulting in myofilament contraction and sliding (Fig. 1C).Open in a separate windowFig. 1.Three-state model of thin-filament activation. Seven actin monomers (circles), spanned by one tropomyosin dimer (red strand), together with the troponin complex (not depicted) comprise one functional unit (A₇TmTn). Two functional units are depicted, and individual myosins are shown as triangles (weak, weak-binding cross-bridges; strong, strong-binding cross-bridges). (A) B state (blocked); when ATP is present and cytoplasmic [Ca²⁺] is low and is not bound to cardiac troponin C (cTnC), tropomyosin is sterically blocking the myosin-binding sites on actin. (B) C state (Ca²⁺-induced); upon rise in cytoplasmic [Ca²⁺], Ca²⁺ binds to cTnC, inducing conformational changes of the troponin complex, resulting in a ∼25° movement of tropomyosin on the thin filament, thereby exposing myosin-binding sites on actin. In the C state, the myofilament is not yet activated as non–tension-generating cross-bridges bind weakly to actin. (C) M state (myosin induced); the strong binding of tension-generating cross-bridges induces a ∼10° movement of tropomyosin on actin, resulting in myofilament activation and contraction.The three-state model of cross-bridge interaction implies that the main task of Ca²⁺ is to uncover myosin-binding sites on actin and that formation of myosin-ADP represents the main regulator of force development and contraction. Notably, solution (10) and cryo-electron microscopy (13) studies have shown that in the absence of Ca²⁺ the myofilaments are not entirely blocked, as ∼5% of the thin filaments have tropomyosin localized in the C-state position. This observation suggests that conditions that promote myosin-ADP formation can trigger myofilament contraction in Ca²⁺-free conditions and thereby impair relaxation. Indeed, in membrane-permeabilized rabbit skeletal muscle fibers (14), bovine myocardium (15, 16) and human cardiac muscle (17) millimolar levels of ADP stimulate force development in the absence of Ca²⁺.Because ADP-stimulated contraction is due to myosin-ADP binding to the nonblocked sites of the thin filament in the absence of Ca²⁺, it provides an experimental tool to assess changes in tropomyosin’s position in acquired and genetic cardiomyopathies in which altered protein phosphorylation and mutant proteins may alter myofilament activation. In addition, it could represent a pathomechanism underlying the diastolic dysfunction seen in both disease states. Solution studies with mutant troponin proteins, which are known to cause HCM, showed a reduction in the B state at low-Ca²⁺ conditions compared with wild-type troponin proteins (18, 19). Mutation-induced irregularities in troponin–tropomyosin interactions disrupt the B state and shift the thin filament to the C state, increasing the available myosin-binding sites on actin.In addition to Ca²⁺-induced changes of the thin filament, tropomyosin location may also be altered by the thick-filament protein cMyBP-C. Recent evidence supports that the N-terminal extension of cMyBP-C binds the low-Ca²⁺–state (B-state) position of tropomyosin on actin and interferes with tropomyosin–actin interactions, dislocating tropomyosin into the C-state position (i.e., the presence of cMyBP-C sensitizes the thin filament to Ca²⁺) (20, 21). Because it was previously shown that in Ca²⁺-free conditions (B state) ∼5% of the thin filaments (lacking cMyBP-C) have tropomyosin localized in the C-state position (10), more myofilaments may be in the C state in the presence of cMyBP-C. We (22) and others (23) have shown that cMyBP-C mutations, which are a major cause of HCM, have a reduced level of healthy cMyBP-C protein compared with nonfailing hearts (i.e., haploinsufficiency), which may alter tropomyosin position on the thin filament.To verify whether ADP-stimulated contraction provides an experimental tool to assess mutation-induced and phosphorylation-mediated changes in thin-filament transitions, which precede Ca²⁺ activation of myofilaments, we tested the following hypotheses: (i) that IDCM and HCM samples with thin-filament mutations are more sensitive to ADP, as a result of a higher accessibility of myosin-binding sites on actin, whereas (ii) cMyBP-C haploinsufficient HCM myocardium has a reduced ADP sensitivity (i.e., less cMyBP-C causes reduced displacement of tropomyosin from the B state) compared with cells from nonfailing hearts. To answer our hypotheses, we activated membrane-permeabilized human cardiomyocytes in ADP containing Ca²⁺-free solutions. Cells were isolated from HCM patients with mutations in genes encoding thick-filament (MYH7, MYBPC3) and thin-filament (TNNT2, TNNI3) proteins and patients with IDCM and compared with cells from sarcomere mutation-negative HCM (HCM_smn) and nonfailing donors. Finally, we investigated whether the ADP level as observed in diseased hearts, in the presence of Ca²⁺, increases myofilament force development in cardiomyocytes from human cardiomyopathy hearts.We conclude that, in HCM with thin-filament mutations, tropomyosin’s ability to block myosin-binding sites on actin is reduced. This effect is exacerbated in HCM samples by the low PKA phosphorylation of myofilament proteins, which is also observed in human IDCM. In contrast, cMyBP-C HCM-causing mutations reduce accessibility of myosin for actin. The findings in this study provide evidence that ADP-mediated activation can be used as an experimental tool to reveal mutation- and phosphorylation-mediated changes in tropomyosin location on the thin filament.     

Independent modulation of contractile performance by cardiac troponin I Ser43 and Ser45 in the dynamic sarcomere

Protein kinase C (PKC) targets cardiac troponin I (cTnI) S43/45 for phosphorylation in addition to other residues. During heart failure, cTnI S43/45 phosphorylation is elevated, and yet there is ongoing debate about its functional role due, in part, to the emergence of complex phenotypes in animal models. The individual functional influences of phosphorylated S43 and S45 also are not yet known. The present study utilizes viral gene transfer of cTnI with phosphomimetic S43D and/or S45D substitutions to evaluate their individual and combined influences on function in intact adult cardiac myocytes. Partial replacement (≤ 40%) with either cTnIS43D or cTnIS45D reduced the amplitude of contraction, and cTnIS45D slowed contraction and relaxation rates, while there were no significant changes in function with cTnIS43/45D. More extensive replacement (≥ 70%) with cTnIS43D, cTnIS45D, and cTnIS43/45D each reduced the amplitude of contraction. Additional experiments also showed cTnIS45D reduced myofilament Ca^2 + sensitivity of tension. At the same time, shortening rates returned toward control values with cTnIS45D and the later stages of relaxation also became accelerated in myocytes expressing cTnIS43D and/or S45D. Further studies demonstrated this behavior coincided with adaptive changes in myofilament protein phosphorylation. Taken together, the results observed in myocytes expressing cTnIS43D and/or S45D suggest these 2 residues reduce function via independent mechanism(s). The changes in function associated with the onset of adaptive myofilament signaling suggest the sarcomere is capable of fine tuning PKC-mediated cTnIS43/45 phosphorylation and contractile performance. This modulatory behavior also provides insight into divergent phenotypes reported in animal models with cTnI S43/45 phosphomimetic substitutions.     

Protein kinase A-mediated acceleration of the stretch activation response in murine skinned myocardium is eliminated by ablation of cMyBP-C

Beta-adrenergic agonists induce protein kinase A (PKA) phosphorylation of the cardiac myofilament proteins myosin binding protein C (cMyBP-C) and troponin I (cTnI), resulting in enhanced systolic function, but the relative contributions of cMyBP-C and cTnI to augmented contractility are not known. To investigate possible roles of cMyBP-C in this response, we examined the effects of PKA treatment on the rate of force redevelopment and the stretch activation response in skinned ventricular myocardium from both wild-type (WT) and cMyBP-C null (cMyBP-C(-/-)) myocardium. In WT myocardium, PKA treatment accelerated the rate of force redevelopment and the stretch activation response, resulting in a shorter time to the peak of delayed force development when the muscle was stretched to a new isometric length. Ablation of cMyBP-C accelerated the rate of force redevelopment and stretch activation response to a degree similar to that observed in PKA treatment of WT myocardium; however, PKA treatment had no effect on the rate of force development and the stretch activation response in null myocardium. These results indicate that ablation of cMyBP-C and PKA treatment of WT myocardium have similar effects on cross-bridge cycling kinetics and suggest that PKA phosphorylation of cMyBP-C accelerates the rate of force generation and thereby contributes to the accelerated twitch kinetics observed in living myocardium during beta-adrenergic stimulation.     

Calcium sensitivity, force frequency relationship and cardiac troponin I: Critical role of PKA and PKC phosphorylation sites

Transgenic models with pseudo phosphorylation mutants of troponin I, PKA sites at Ser 22 and 23 (cTnIDD_22,23 mice) or PKC sites at Ser 42 and 44 (cTnIAD_22,23DD_42,44) displayed differential force-frequency relationships and afterload relaxation delay in vivo. We hypothesized that cTnI PKA and PKC phosphomimics impact cardiac muscle rate-related developed twitch force and relaxation kinetics in opposite directions. cTnIDD_22,23 transgenic mice produce a force frequency relationship (FFR) equivalent to control NTG albeit at lower peak [Ca²⁺]_i, while cTnIAD_22,23DD_42,44 TG mice had a flat FFR with normal peak systolic [Ca²⁺]_i, thus suggestive of diminished responsiveness to [Ca²⁺]_i at higher frequencies. Force-[Ca²⁺]_i hysteresis analysis revealed that cTnIDD_22,23 mice have a combined enhanced myofilament calcium peak response with an enhanced slope of force development and decline per unit of [Ca²⁺]_i, whereas cTnIAD_22,23DD_42,44 transgenic mice showed the opposite. The computational ECME model predicts that the TG lines may be distinct from each other due to different rate constants for association/dissociation of Ca²⁺ at the regulatory site of cTnC. Our data indicate that cTnI phosphorylation at PKA sites plays a critical role in the FFR by increasing relative myofilament responsiveness, and results in a distinctive transition between activation and relaxation, as displayed by force-[Ca²⁺]_i hysteresis loops. These findings may have important implications for understanding the specific contribution of cTnI to β-adrenergic inotropy and lusitropy and to adverse contractile effects of PKC activation, which is relevant during heart failure development.     

Melatonin triggers PKA activation in the rodent malaria parasite Plasmodium chabaudi

Abstract: Calcium (Ca²⁺) is a critical regulator of many aspects of the Plasmodium reproductive cycle. In particular, intra‐erythrocyte Plasmodium parasites respond to circulating levels of the melatonin in a process mediated partly by intracellular Ca²⁺. Melatonin promotes the development and synchronicity of parasites, thereby enhancing their spread and worsening the clinical implications. The signalling mechanisms underlying the effects of melatonin are not fully established, although both Ca²⁺ and cyclic AMP (cAMP) have been implicated. Furthermore, it is not clear whether different strains of Plasmodium use the same, or divergent, signals to control their development. The aim of this study was to explore the signalling mechanisms engaged by melatonin in P. chabaudi, a virulent rodent parasite. Using parasites at the throphozoite stage acutely isolated from mice erythrocytes, we demonstrate that melatonin triggers cAMP production and protein kinase A (PKA) activation. Interestingly, the stimulation of cAMP/PKA signalling by melatonin was dependent on elevation of Ca²⁺ within the parasite, because buffering Ca²⁺ changes using the chelator BAPTA prevented cAMP production in response to melatonin. Incubation with melatonin evoked robust Ca²⁺ signals within the parasite, as did the application of a membrane‐permeant analogue of cAMP. Our data suggest that P. chabaudi engages both Ca²⁺ and cAMP signalling systems when stimulated by melatonin. Furthermore, there is positive feedback between these messengers, because Ca²⁺ evokes cAMP elevation and vice versa. Melatonin more than doubled the observed extent of parasitemia, and the increase in cAMP concentration and PKA activation was essential for this effect. These data support the possibility to use melatonin antagonists or derivates in therapeutic approach.     

A CaMKII/PDE4D negative feedback regulates cAMP signaling

cAMP production and protein kinase A (PKA) are the most widely studied steps in β-adrenergic receptor (βAR) signaling in the heart; however, the multifunctional Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is also activated in response to βAR stimulation and is involved in the regulation of cardiac excitation-contraction coupling. Its activity and expression are increased during cardiac hypertrophy, in heart failure, and under conditions that promote arrhythmias both in animal models and in the human heart, underscoring the clinical relevance of CaMKII in cardiac pathophysiology. Both CaMKII and PKA phosphorylate a number of protein targets critical for Ca²⁺ handling and contraction with similar, but not always identical, functional consequences. How these two pathways communicate with each other remains incompletely understood, however. To maintain homeostasis, cyclic nucleotide levels are regulated by phosphodiesterases (PDEs), with PDE4s predominantly responsible for cAMP degradation in the rodent heart. Here we have reassessed the interaction between cAMP/PKA and Ca²⁺/CaMKII signaling. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP levels. Moreover, we show that these effects are mediated, at least in part, by CaMKII regulation of PDE4D. This regulation establishes a negative feedback loop necessary to maintain cAMP/CaMKII homeostasis, revealing a previously unidentified function for PDE4D as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.During cardiac excitation-contraction coupling (ECC), Ca²⁺ elevation throughout the cell promotes myofilament sliding, which generates contractile force. This process is highly regulated by positive and negative regulatory circuits, the most critical being the sympathetic nervous system that acts via activation of the β-adrenergic (βAR)/cAMP/PKA signaling pathway. During βAR stimulation, PKA phosphorylates and activates key proteins involved in ECC and Ca²⁺ handling. These proteins include L-type Ca²⁺ channels and ryanodine receptors (RyR), leading to enhanced Ca²⁺ influx and consequent sarcoplasmic reticulum (SR) Ca²⁺ release; phospholamban (PLB), increasing SR Ca²⁺ uptake by the Ca²⁺ ATPase (SERCA), thereby accelerating cardiac relaxation; and contractile proteins, increasing cell contraction. Collectively, these events produce the typical inotropic and lusitropic effects of βAR stimulation (1).This βAR/cAMP/PKA pathway is only one of the components involved in regulating cardiac function, however. Data accumulated in the last decade have revealed that Ca²⁺/calmodulin-dependent kinase II (CaMKII) is equally important to the regulation of cardiac function under physiological and pathological conditions (2–7). Most of the functions distal to cAMP and PKA are regulated by CaMKII as well (8). Proteins critical for ECC are substrates for both PKA and CaMKII, and phosphorylation at different sites often produces similar changes in protein function. Whereas CaMKII is directly regulated by Ca²⁺, its activity is indirectly regulated by cAMP (9). In response to βAR stimulation, Epac (Exchange Protein directly Activated by cAMP) activates CaMKII (10–14). This regulation is critical in pathophysiological conditions in which CaMKII expression and activation may be elevated, such as hypertrophy, heart failure, and arrhythmias (2, 3). Despite the wealth of data available, the exact mechanisms integrating CaMKII activity with cAMP/PKA signaling remain unclear.An established concept in cell signaling is that freely diffusible cAMP is not distributed uniformly throughout the cell, but is compartmentalized to generate specificity and to allow PKA regulation in distinct subdomains (15). Phosphodiesterases (PDEs), the enzymes that degrade cAMP, have emerged as ubiquitous and important modulators of cAMP/PKA signaling in specific cellular compartments, including cardiac myocytes (16, 17). They are part of macromolecular complexes that include PKAs and A-kinase anchoring proteins (AKAPs). In contrast to the large body of data linking PDEs to PKA regulation, whether PDEs regulate CaMKII is unclear. We and others have shown that genetic ablation of PDE4s, the isoform responsible for cAMP degradation in the heart, disrupts ECC via PKA-mediated alteration in Ca²⁺ handling (18–20); however, the possibility that some of the effects are mediated by CaMKII has not been investigated.For proper homeostasis, cAMP signals are constrained by feedback mechanisms required to tightly regulate cyclic nucleotide levels under basal or stimulated conditions, with the feedback regulation of PDE4s a preeminent example. PDE4s are activated by PKA phosphorylation, providing a negative feedback mechanism by which cAMP regulates its own level (21, 22). Given the cAMP-dependent, Epac-mediated CaMKII activation, we surmise that feedback mechanisms linking cAMP and CaMKII must be operating in cardiac myocytes as well. In the present study, we reassessed the interaction between cAMP signaling and CaMKII. We demonstrate that CaMKII activity constrains basal and βAR-activated cAMP signaling. These effects are mediated, at least in part, by CaMKII regulation of PDE4D, revealing an additional function of this enzyme as a critical integrator of cAMP/PKA and Ca²⁺/CaMKII signaling.     

Single amino acid substitutions define isoform-specific effects of troponin I on myofilament Ca2+ and pH sensitivity

Troponin I isoforms play a key role in determining myofilament Ca2+ sensitivity in cardiac muscle. The goal here was to identify domain clusters and residues that confer troponin I isoform-specific myofilament Ca2+ and pH sensitivities of contraction. Key domains/residues that contribute to troponin I isoform-specific Ca2+ and pH sensitivity were studied using gene transfer of a slow skeletal troponin I (ssTnI) template, with targeted cardiac troponin I (cTnI) residue substitutions. Substitutions in ssTnI with cognate cTnI residues R125Q, H132A, and V134E, studied both independently and together (ssTnIQAE), resulted in efficient stoichiometric replacement of endogenous myofilament cTnI in adult cardiac myocytes. In permeabilized myocytes, the pCa50 of tension ([Ca2+] required for half maximal force), and the acidosis-induced rightward shift of pCa50 were converted to the cTnI phenotype in myocytes expressing ssTnIQAE or ssTnIH132A, and there was no functionally additive effect of ssTnIQAE versus ssTnIH132A. Interestingly, only the acidosis-induced shift in Ca2+ sensitivity was comparable to cTnI in myocytes expressing ssTnIV134E, while ssTnIR125Q fully retained the ssTnI phenotype. An additional ssTnIN141H substitution, which lies within the same structural region of TnI as V134, produced a shift in myofilament Ca2+ sensitivity comparable to cTnI at physiological pH, while the acidic pH response was similar to the effect of wild-type ssTnI. Analysis of sarcomere shortening in intact adult cardiac myocytes was consistent with the force measurements. Targeted substitutions in the carboxyl portion of TnI produced residue-specific influences on myofilament Ca2+ and pH sensitivity of force and give new molecular insights into the TnI isoform dependence of myofilament function.