Heart failure (HF) is a leading cause of morbidity and mortality worldwide and is most often precipitated by myocardial infarction. However, the molecular changes driving cardiac dysfunction immediately after myocardial infarction remain poorly understood. Myofilament proteins, responsible for cardiac contraction and relaxation, play critical roles in signal reception and transduction in HF. Post-translational modifications of myofilament proteins afford a mechanism for the beat-to-beat regulation of cardiac function. Thus it is of paramount importance to gain a comprehensive understanding of post-translational modifications of myofilament proteins involved in regulating early molecular events in the post-infarcted myocardium. We have developed a novel liquid chromatography–mass spectrometry-based top-down proteomics strategy to comprehensively assess the modifications of key cardiac proteins in the myofilament subproteome extracted from a minimal amount of myocardial tissue with high reproducibility and throughput. The entire procedure, including tissue homogenization, myofilament extraction, and on-line LC/MS, takes less than three hours. Notably, enabled by this novel top-down proteomics technology, we discovered a concerted significant reduction in the phosphorylation of three crucial cardiac proteins in acutely infarcted swine myocardium: cardiac troponin I and myosin regulatory light chain of the myofilaments and, unexpectedly, enigma homolog isoform 2 (ENH2) of the Z-disc. Furthermore, top-down MS allowed us to comprehensively sequence these proteins and pinpoint their phosphorylation sites. For the first time, we have characterized the sequence of ENH2 and identified it as a phosphoprotein. ENH2 is localized at the Z-disc, which has been increasingly recognized for its role as a nodal point in cardiac signaling. Thus our proteomics discovery opens up new avenues for the investigation of concerted signaling between myofilament and Z-disc in the early molecular events that contribute to cardiac dysfunction and progression to HF.
Cardiac resynchronization therapy (CRT), the application of biventricular stimulation to correct discoordinate contraction, is the only heart failure treatment that enhances acute and chronic systolic function, increases cardiac work, and reduces mortality. Resting myocyte function also increases after CRT despite only modest improvement in calcium transients, suggesting that CRT may enhance myofilament calcium responsiveness. To test this hypothesis, we examined adult dogs subjected to tachypacing-induced heart failure for 6 weeks, concurrent with ventricular dyssynchrony (HFdys) or CRT. Myofilament force-calcium relationships were measured in skinned trabeculae and/or myocytes. Compared with control, maximal calcium-activated force and calcium sensitivity declined globally in HFdys; however, CRT restored both. Phosphatase PP1 induced calcium desensitization in control and CRT-treated cells, while HFdys cells were unaffected, implying that CRT enhances myofilament phosphorylation. Proteomics revealed phosphorylation sites on Z-disk and M-band proteins, which were predicted to be targets of glycogen synthase kinase-3β (GSK-3β). We found that GSK-3β was deactivated in HFdys and reactivated by CRT. Mass spectrometry of myofilament proteins from HFdys animals incubated with GSK-3β confirmed GSK-3β–dependent phosphorylation at many of the same sites observed with CRT. GSK-3β restored calcium sensitivity in HFdys, but did not affect control or CRT cells. These data indicate that CRT improves calcium responsiveness of myofilaments following HFdys through GSK-3β reactivation, identifying a therapeutic approach to enhancing contractile function.
Protein phosphorylation is reversibly regulated by the interplay between kinases and phosphatases. Recent developments within the field of proteomics have revealed the extent of this modification in nature. To date there is still a lack of information about phosphatase specificity for different proteomes and their conditions to achieve maximum enzyme activity. This information is important per se, and in addition often requested in functional and biochemical in vitro studies, where a dephosphorylated sample is needed as a negative control to define baseline conditions. In this study, we have addressed the effectiveness of two phosphatases endogenously present in the heart (protein phosphatases 1 and 2A) and two generic phosphatases (alkaline phosphatase and lambda protein phosphatase) on three cardiac subproteomes known to be regulated by phosphorylation. We optimized the dephoshorylating conditions on a cardiac tissue fraction comprising cytosolic and myofilament proteins using 2-DE and MS. The two most efficient conditions were further investigated on a mitochondrial-enriched fraction. Dephosphorylation of specific proteins depends on the phosphatase, its concentration, as well as sample preparation including buffer composition. Finally, we analyzed the efficiency of alkaline phosphatase, the phosphatase with the broadest substrate specificity, using TiO2 peptide enrichment and 2DLC-MS/MS. Under these conditions, 95% of the detected cardiac cytoplasmic-enriched phospho-proteome was dephosphorylated. In summary, targeting dephosphorylation of the cardiac muscle subproteome or a specific protein will drive the selection of the specific phosphatase, and each requires different conditions for optimal performance.
dephosphorylation; phosphatase; phosphorylation; cardiac muscle; 2-DE DIGE
This study was conducted to identify molecular mechanisms which explain interventricular differences in myofilament function in experimental congestive heart failure (CHF). CHF was induced in rats by chronic aortic banding or myocardial infarction for 32–36 weeks. Right and left ventricular (RV, LV) myocytes were mechanically isolated, triton-skinned, and attached to a force transducer and motor arm. Myofilament force–[Ca2+] relations assessed maximal Ca2+-saturated force (Fmax) and the [Ca2+] at 50% of Fmax (EC50). Myofilament protein phosphorylation was determined via ProQ diamond phospho-staining. Protein kinase C (PKC)-α expression/activation and site-specific phosphorylation of cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were measured via immunoblotting. Relative to controls, failing RV myocytes displayed a ~45% decrease in Fmax with no change in EC50, whereas failing LV myocytes displayed a ~45% decrease in Fmax and ~50% increase in EC50. Failing LV myofilaments were less Ca2+-sensitive (37% increase in EC50) than failing RV myofilaments. Expression and activation of PKC-α was increased twofold in failing RV myocardium and relative to the RV, PKC-α was twofold higher in the failing LV, while PKC-β expression was unchanged by CHF. PKC-α-dependent phosphorylation and PP1-mediated dephosphorylation of failing RV myofilaments increased EC50 and increased Fmax, respectively. Phosphorylation of cTnI and cTnT was greater in failing LV myofilaments than in failing RV myofilaments. RV myofilament function is depressed in experimental CHF in association with increased PKC-α signaling and myofilament protein phosphorylation. Furthermore, myofilament dysfunction is greater in the LV compared to the RV due in part to increased PKC-α activation and phosphorylation of cTnI and cTnT.
Heart; Muscle mechanics; Force–pCa relation; Myofilaments; Protein kinase C; Hypertrophy
Endothelial-dependent regulation of vascular tone occurs in part via protein kinase G1α-mediated changes in smooth muscle myofilament sensitivity to Ca2+. Tissue-specific differences in PKG-dependent relaxation have been attributed to altered expression of myofilament-associated proteins that are substrates for PKG binding. These include the alternative splicing of the myosin targeting subunit (MYPT1) of myosin light chain phosphatase to yield leucine zipper positive (LZ+) and negative (LZ−) isovariants, with the former being required for PKG-mediated relaxation, and/or altered expressions of telokin, vasodilator-stimulated phosphoprotein (VASP) or heat shock protein Hsp20. During human pregnancy the uterine and placental circulations remain distinct entities and, as such, their mechanisms of vascular tone regulation may differ. Indeed, the sensitivity of myometrial arteries to endothelial-dependent agonists has been suggested to be greater than that of placental arteries. We tested the hypothesis that this was related to tissue-specific changes in PKG-mediated myofilament Ca2+-desensitization and/or the expressions of PKG-interacting myofilament-associated proteins. Permeabilized human placental and myometrial arteries were constricted with maximal activating Ca2+ (pCa 4.5), or sub-maximal Ca2+ (pCa 6.7) and the thrombane mimetic U46619, and exposed to 8-Br-cGMP. In each case, relaxation was significantly greater in myometrial arteries (e.g. relaxation in pCa 4.5 to 8-Br-cGMP was 49 ± 9.7%, n = 7) than placental arteries (relaxation of 23 ± 6.6%, n = 6, P < 0.05). MYPT1 protein levels, or MYPT1 LZ+/LZ− mRNA ratios, were similar for both artery types. Of other proteins examined, only Hsp20 expression was significantly elevated in myometrial arteries than placental arteries. These results demonstrate that the reduced human placental artery relaxation to PKG stimulation lies partly at the level of myofilament (de)activation and may be related to a lower expression of Hsp20 than in myometrial arteries.
human placental arteries; human myometrial arteries; PKG; Hsp20
Sepsis-associated cardiac dysfunction represents an intrinsic impairment of cardiomyocyte function due in part to a decrease in myofilament Ca2+ sensitivity associated with a sustained increase in cardiac troponin I (cTnI) phosphorylation at Ser23/24. Dephosphorylation of cTnI is under regulatory control. Thus, muscarinic and adenosine A1-receptor agonists antagonize β-adrenergic stimulation via activation of protein phosphatase 2A (PP2A). The aim of this study was to determine whether modulation of PP2A and thus cTnI phosphorylation could improve sepsis-induced contractile dysfunction.
Methods and results
Cardiomyocytes were isolated from control or septic mice 16–18 h after an injection of vehicle or lipopolysaccharide (LPS; 9 mg/kg ip) respectively. Protein expression and phosphatase activity were determined in homogenates of control and septic hearts. Our data showed that LPS significantly increased cTnI phosphorylation at Ser23/24 in cardiomyocytes and reduced contraction amplitude without affecting Ca2+-transients. Treatment of cardiomyocytes with the A1 agonist cyclopentyladenosine (CPA) or the protein kinase A inhibitor H89 significantly attenuated the LPS-induced contractile dysfunction without effect on Ca2+-transients. Co-treatment with CPA and H89 completely reversed the contractile dysfunction. Increased cTnI phosphorylation in septic hearts was associated with a significant reduction in the protein expression of both the catalytic and regulatory subunits (B56α) of PP2A and a decrease in PP2A activity. CPA treatment of septic hearts increased PP2A activity. An increase in the protein expression of demethylated PP2A and a decrease in the PP2A-methyltransferase (PPMT; the methyltransferase that catalyses this reaction) were also observed.
These data support the hypothesis that sustained cTnI phosphorylation underlies the contractile dysfunction seen in sepsis.
Troponin I; Cardiomyocytes; Myofilaments; Phosphorylation; Protein phosphatase 2A
SR33805, a potent Ca2+ channel blocker, increases cardiac myofilament Ca2+ sensitivity in healthy rat cardiomyocytes. Therefore, the aim of the present study was to evaluate the effects of SR33805 on contractile properties in ischaemic failing hearts after myocardial infarction (MI) in vivo and in vitro at the cellular level.
Methods and results
The effect of SR33805 (10 µM) was tested on the excitation–contraction coupling of cardiomyocytes isolated from rat with end-stage heart failure. Cell shortening and Ca2+ transients were measured in intact cardiomyocytes, while contractile properties were determined in Triton X-100 permeabilized myocytes. Acute treatment with SR33805 restored the MI-altered cell shortening without affecting the Ca2+ transient amplitude, suggesting an increase of myofilament Ca2+ sensitivity in MI myocytes. Indeed, a SR33805-induced sensitization of myofilament activation was found to be associated with a slight increase in myosin light chain-2 phosphorylation and a more significant decrease on troponin I (TnI) phosphorylation. Decreased TnI phosphorylation was related to inhibition of protein kinase A activity by SR33805. Finally, administration of a single intra-peritoneal bolus of SR33805 (20 mg/kg) improved end-systolic strain and fractional shortening of MI hearts.
The present study indicates that treatment with SR33805 improved contractility of ischaemic failing hearts after MI in the rat by selectively modulating the phosphorylation status of sarcomeric regulatory proteins, which then sensitized the myofilaments to Ca2+. Our results gave a proof of concept that manipulation of the Ca2+ sensitivity of sarcomeric regulatory proteins can be used to improve contractility of a failing heart.
Myocytes; Heart failure; Contractile function; Sarcomere; Ventricular function
Increased vascular smooth muscle contractility has an important role in the development of cerebral vasospasm after subarachnoid hemorrhage (SAH). Myofilament Ca2+ sensitivity is a major determinant of smooth muscle contractility. We investigated changes in the Ca2+-sensitizing effect of endothelin-1 (ET-1) and the mechanisms underlying ET-1-induced Ca2+ sensitization after SAH using a rabbit SAH model. After SAH, the contractile response to ET-1 was enhanced, and the ETA receptor expression was upregulated in the basilar artery. In α-toxin-permeabilized preparations, ET-1 induced enhanced and prolonged contraction after SAH, suggesting that ET-1-induced Ca2+ sensitization is potentiated after SAH. Endothelin-1-induced Ca2+ sensitization became less sensitive to inhibitors of Rho-associated coiled-coil protein kinase (ROCK) and protein kinase C (PKC) after SAH. The expression of PKCα, ROCK2, PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17) and myosin phosphatase target subunit 1 (MYPT1) was upregulated, and the level of phosphorylation of CPI-17 and MYPT1 was elevated after SAH. This study demonstrated for the first time that the Ca2+-sensitizing effect of ET-1 on myofilaments is potentiated after SAH. The increased expression and activity of PKCα, ROCK2, CPI-17, and MYPT1, as well as the upregulation of ETA receptor expression are suggested to underlie the enhanced and prolonged Ca2+ sensitization induced by ET-1.
calcium; pharmacology; physiology; smooth muscle; subarachnoid hemorrhage; vasospasm
Estrogens have well-recognized and complex cardiovascular effects, including altering myocardial contractility through changes in myofilament function. The presence of multiple estrogen receptor (ER) isoforms in the heart may explain some discrepant findings about the cardiac effects of estrogens. Most studies examining the impact of estrogens on the heart have focused on chronic changes in estrogen levels, and have not investigated rapid, non-genomic pathways. The first objective of this study was to determine how acute activation of ERα impacts cardiac myofilaments. Nongenomic myocardial estrogen signaling is associated with the activation of a variety of signaling pathways. p38 MAPK has been implicated in acute ER signaling in the heart, and is known to affect myofilament function. Thus, the second objective of this study was to determine if acute ERα activation mediates its myofilament effects through p38 MAPK recruitment. Hearts from female C57Bl/6 mice were perfused with the ERα agonist PPT and myofilaments isolated. Activation of ERα depressed actomyosin MgATPase activity and decreased myofilament calcium sensitivity. Inhibition of p38 MAPK attenuated the myofilament effects of ERα activation. ERα stimulation did not affect global myofilament protein phosphorylation, but troponin I phosphorylation at the putative PKA phosphorylation sites was decreased. Changes in myofilament activation did not translate into alterations in whole heart function. The present study provides evidence supporting rapid, non-genomic changes in cardiac myofilament function following acute ERα stimulation mediated by the p38 MAPK pathway.
Our previous studies demonstrated a relation between glutathionylation of cardiac myosin binding protein C (cMyBP-C) and diastolic dysfunction in a hypertensive mouse model stressed by treatment with salt, deoxycorticosterone acetate, and unilateral nephrectomy. Although these results strongly indicated an important role for S-glutathionylation of myosin binding protein C as a modifier of myofilament function, indirect effects of other post-translational modifications may have occurred. Moreover, we did not determine the sites of thiol modification by glutathionylation. To address these issues, we developed an in vitro method to mimic the in situ S-glutathionylation of myofilament proteins and determined direct functional effects and sites of oxidative modification employing Western blotting and mass spectrometry. We induced glutathionylation in vitro by treatment of isolated myofibrils and detergent extracted fiber bundles (skinned fibers) with oxidized glutathione (GSSG). Immuno-blotting results revealed increased glutathionylation with GSSG treatment of a protein band around 140 kDa. Using tandem mass spectrometry, we identified the 140 kDa band as cMyBP-C and determined the sites of glutathionylation to be at cysteines 655, 479, and 627. Determination of the relation between Ca2+-activation of myofibrillar acto-myosin ATPase rate demonstrated an increased Ca2+-sensitivity induced by the S-glutathionylation. Force generating skinned fiber bundles also showed an increase in Ca-sensitivity when treated with oxidized glutathione, which was reversed with the reducing agent, dithiothreitol (DTT). Our data demonstrate that a specific and direct effect of S-glutathionylation of myosin binding protein C is a significant increase in myofilament Ca2+-sensitivity. Our data also provide new insights into the functional significance of oxidative modification of myosin binding protein C and the potential role of domains not previously considered to be functionally significant as controllers of myofilament Ca2+-responsiveness and dynamics.
oxidative stress; sarcomeres; C-protein; cardiac relaxation
There is over-whelming evidence that protein phosphorylations regulate cardiac function and remodeling. A wide variety of protein kinases, e.g., phosphoinositide 3-kinase (PI3K), Akt, GSK-3, TGFβ, and PKA, MAPKs, PKC, Erks, and Jaks, as well as phosphatases, e.g., phosphatase I (PP1) and calcineurin, control cardiomyocyte growth and contractility. In the present work, we used global phosphoprotein profiling to identify phosphorylated proteins associated with pressure overload (PO) cardiac hypertrophy and heart failure. Phosphoproteins from hypertrophic and systolic failing hearts from male hypertensive Dahl salt-sensitive rats, trans-aortic banded (TAC), and spontaneously hypertensive heart failure (SHHF) rats were analyzed. Profiling was performed by 2-dimensional difference in gel electrophoresis (2D-DIGE) on phospho-enriched proteins. A total of 25 common phosphoproteins with differences in abundance in (1) the 3 hypertrophic and/or (2) the 2 systolic failure heart models were identified (CI>99%) by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) and Mascot analysis. Among these were (1) myofilament proteins, including alpha-tropomyosin and myosin regulatory light chain 2, cap Z interacting protein (cap ZIP), and tubulin β5; (2) mitochondrial proteins, including pyruvate dehydrogenase α, branch chain ketoacid dehydrogenase E1, and mitochondrial creatine kinase; (3) phosphatases, including protein phosphatase 2A and protein phosphatase 1 regulatory subunit; and (4) other proteins including proteosome subunits α type 3 and β type 7, and eukaryotic translation initiation factor 1A (eIF1A). The results include previously described and novel phosphoproteins in cardiac hypertrophy and systolic failure.
Hypertrophy; Heart failure; Pressure overload; Phospho-profiling; 2D-DIGE; Dahl rats
Protein phosphatase (PP) type 2A is a multifunctional serine/threonine phosphatase that is involved in cardiac excitation–contraction coupling. The PP2A core enzyme is a dimer, consisting of a catalytic C and a scaffolding A subunit, which is targeted to several cardiac proteins by a regulatory B subunit. At present, it is controversial whether PP2A and its subunits play a critical role in end-stage human heart failure. Here we report that the application of purified PP2AC significantly increased the Ca2+-sensitivity (ΔpCa50 = 0.05 ± 0.01) of the contractile apparatus in isolated skinned myocytes of non-failing (NF) hearts. A higher phosphorylation of troponin I (cTnI) was found at protein kinase A sites (Ser23/24) in NF compared to failing myocardium. The basal Ca2+-responsiveness of myofilaments was enhanced in myocytes of ischemic (ICM, ΔpCa50 = 0.10 ± 0.03) and dilated (DCM, ΔpCa50 = 0.06 ± 0.04) cardiomyopathy compared to NF. However, in contrast to NF myocytes the treatment with PP2AC did not shift force-pCa relationships in failing myocytes. The higher basal Ca2+-sensitivity in failing myocytes coincided with a reduced protein expression of PP2AC in left ventricular tissue from patients suffering from ICM and DCM (by 50 and 56% compared to NF, respectively). However, PP2A activity was unchanged in failing hearts despite an increase of both total PP and PP1 activity. The expression of PP2AB56α was also decreased by 51 and 62% in ICM and DCM compared to NF, respectively. The phosphorylation of cTnI at Ser23/24 was reduced by 66 and 49% in ICM and DCM compared to NF hearts, respectively. Our results demonstrate that PP2A increases myofilament Ca2+-sensitivity in NF human hearts, most likely via cTnI dephosphorylation. This effect is not present in failing hearts, probably due to the lower baseline cTnI phosphorylation in failing compared to non-failing hearts.
Protein phosphatase 2A; Myofilament function; Protein phosphorylation; Cardiomyocyte; Troponin I
The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure–function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.
hypertrophy; protein kinase A (PKA); A kinase anchoring protein (AKAP); contractility
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.
Protein kinase C; Cardiac; Heart failure; Myofilament function; Contractile proteins; Phosphorylation
In the heart, lysine acetylation has been implicated in processes ranging from transcriptional control of pathological remodeling, to cardioprotection arising from caloric restriction. Given the emerging importance of this post-translational modification, we used a proteomic approach to investigate the broader role of lysine acetylation in the heart using a guinea pig model. Briefly, hearts were fractionated into myofilament-, mitochondrial- and cytosol-enriched fractions prior to proteolysis and affinity-enrichment of acetylated peptides. LC-MS/MS analysis identified 1075 acetylated peptides, harboring 994 acetylation sites that map to 240 proteins with a global protein false discovery rate <0.8%. Mitochondrial targets account for 59% of identified proteins and 64% of sites. The majority of the acetyl-proteins are enzymes involved in fatty acid metabolism, oxidative phosphorylation or the TCA cycle. Within the cytosolic fraction, the enzymes of glycolysis, fatty acid synthesis and lipid binding are prominent. Nuclear targets included histones and the transcriptional regulators E1A(p300) and CREB binding protein. Comparison of our dataset with three previous global acetylomic studies uniquely revealed 53 lysine-acetylated proteins. Specifically, newly-identified acetyl-proteins include Ca2+-handling proteins, RyR2 and SERCA2, and the myofilament proteins, myosin heavy chain, myosin light chains and subunits of the Troponin complex, among others. These observations were confirmed by anti-acetyl-lysine immunoblotting. In summary, cardiac lysine acetylation may play a role in cardiac substrate selection, bioenergetic performance, and maintenance of redox balance. New sites suggest a host of potential mechanisms by which excitation-contraction coupling may also be modulated.
Aim: Transmural differences in sarcomeric protein composition and function across the left ventricular (LV) wall have been reported. We studied in pigs sarcomeric function and protein phosphorylation in subepicardial (EPI) and subendocardial (ENDO) layers of remote LV myocardium after myocardial infarction (MI), induced by left circumflex coronary artery ligation. Methods: EPI and ENDO samples were taken 3 weeks after sham surgery (n = 12) or induction of MI (n = 12) at baseline (BL) and during β-adrenergic receptor (βAR) stimulation with dobutamine. Isometric force was measured in single cardiomyocytes at various [Ca2+] and 2.2 μm sarcomere length. Results: In sham hearts, no significant transmural differences were observed in myofilament function or protein phosphorylation. Myofilament Ca2+-sensitivity was significantly higher in both EPI and ENDO of MI compared to sham hearts. Maximal force was significantly reduced in MI compared to sham, but solely in ENDO cells. A higher passive force was observed in MI hearts, but only in EPI cells. The proportion of stiff N2B isoform was higher in EPI than in ENDO in both sham and MI hearts, and a trend toward increased N2B-proportion appeared in MI EPI, but not MI Endo. Analysis of myofilament protein phosphorylation did not reveal significant transmural differences in phosphorylation of myosin binding protein C, desmin, troponin T, troponin I (cTnI), and myosin light chain 2 (MLC-2) both at BL and during βAR stimulation with dobutamine infusion. A significant increase in MLC-2 phosphorylation was observed during dobutamine only in sham. In addition, the increase in cTnI phosphorylation upon dobutamine was twofold lower in MI than in sham. Conclusion: Myofilament dysfunction is present in both EPI and ENDO in post-MI remodeled myocardium, but shows a high degree of qualitative heterogeneity across the LV wall. These heterogeneous transmural changes in sarcomeric properties likely contribute differently to systolic vs. diastolic global LV dysfunction after MI.
sarcomere function; myocardial infarction; heart; subendocardium; subepicardium; protein phosphorylation
The highly organized contractile machinery in skeletal and cardiac muscles requires an assembly of myofilament proteins with stringent stoichiometry. To understand the maintenance of myofilament protein stoichiometry under dynamic protein synthesis and catabolism in muscle cells, we investigated the equilibrium of troponin I (TnI) in mouse cardiac muscle during developmental isoform switching and in under- and over-expression models. Compared with the course of developmental TnI isoform switching in normal hearts, the postnatal presence of slow skeletal muscle TnI lasted significantly longer in the hearts of cardiac TnI (cTnI) knockout (cTnI-KO) mice, in which the diminished synthesis was compensated by prolonging the life of myofilamental TnI. Transgenic postnatal expression of an N-terminal truncated cTnI (cTnI-ND) using α-myosin heavy chain promoter effectively rescued the lethality of cTnI-KO mice and shortened the postnatal presence of slow TnI in cardiac muscle. cTnI-KO mice rescued with different levels of cTnI-ND over-expression exhibited similar levels of myocardial TnI comparable to that in wild type hearts, demonstrating that excessive synthesis would not increase TnI stoichiometry in the myofilaments. Consistently, haploid under-expression of cTnI in heterozygote cTnI-KO mice was sufficient to sustain the normal level of myocardial cTnI, indicating that cTnI is synthesized in excess in wild type cardiomyocytes. Altogether, these observations suggest that under wide ranges of protein synthesis and turnover, myofilament incorporation determines the stoichiometry of troponin subunits in muscle cells.
myofilament; troponin; stoichiometry; cardiac troponin I knockout mice; cardiac muscle
High-myofilament Ca2+-sensitivity has been proposed as trigger of disease pathogenesis in familial hypertrophic cardiomyopathy (HCM) based on in vitro and transgenic mice studies. However, myofilament Ca2+-sensitivity depends on protein phosphorylation and muscle length, and at present, data in human are scarce.
To investigate whether high-myofilament Ca2+-sensitivity and perturbed length-dependent activation are characteristics for human HCM with mutations in thick- and thin-filament proteins.
Methods and Results
Cardiac samples from patients with HCM harboring mutations in genes encoding thick (MYH7, MYBPC3) and thin (TNNT2, TNNI3, TPM1) filament proteins were compared with sarcomere mutation-negative HCM and nonfailing donors. Cardiomyocyte force measurements showed higher myofilament Ca2+-sensitivity in all HCM samples and low phosphorylation of protein kinase A (PKA)-targets compared with donors. After exogenous PKA treatment, myofilament Ca2+-sensitivity was either similar (MYBPC3mut, TPM1mut, sarcomere mutation-negative HCM), higher (MYH7mut, TNNT2mut), or even significantly lower (TNNI3mut) compared with donors. Length-dependent activation was significantly smaller in all HCM than in donor samples. PKA treatment increased phosphorylation of PKA-targets in HCM myocardium and normalized length-dependent activation to donor values in sarcomere mutation-negative HCM and HCM with truncating MYBPC3 mutations, but not in HCM with missense mutations. Replacement of mutant by wild-type troponin in TNNT2mut and TNNI3mut corrected length-dependent activation to donor values.
High-myofilament Ca2+-sensitivity is a common characteristic of human HCM and partly reflects hypophosphorylation of PKA-targets compared with donors. Length-dependent sarcomere activation is perturbed by missense mutations, possibly via post-translational modifications other than PKA-hypophosphorylation or altered protein–protein interactions, and represents a common pathomechanism in HCM.
calcium; cardiomyopathy; contractility; hypertrophy; myocardium
The phosphorylation state of several cardiac myofilament proteins changes with the level of stretch in intact, twitch-contracting cardiac muscles. It remains unclear which kinases are involved in the length-dependent phosphorylation of these proteins. We set out to investigate which kinases are involved after a step-wise change in cardiac muscle length. We hypothesize that myofilament protein phosphorylation by PKCβII and PKA alters contractile kinetics during length-dependent activation. Right ventricular intact trabeculae were isolated from New Zealand White rabbit hearts and stimulated to contract at 1 Hz. Twitch force recordings where taken at taut and optimal muscle lengths before and after administration of kinase inhibitors at 37 °C. PKCβII inhibition significantly decreased time from stimulation to peak force (TTP), time from peak force to 50% relaxation (RT50), and 90% relaxation (RT90) at optimal muscle length. This led to a loss in the length-dependent increase of RT50 and RT90 in the presence of the PKCβII inhibitor, whereas the length-dependent increase in RT50 and RT90 was seen in the controls. PKA inhibition using H-89 significantly decreased TTP at both taut and optimal muscle lengths. Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicates a role for PKCβII in the phosphorylation of tropomyosin and myosin light chain-2 (MLC2) and PKA for tropomyosin, troponin-I, MLC2, myosin binding protein-C, troponin-T (TnT) 3 and TnT4. Our data provide evidence for two signaling kinases acting upon myofilament proteins during length-dependent activation, and provide further insight for length-dependent myofilament function.
Frank-Starling mechanism; Contraction; Kinetics; Protein kinase C; Protein kinase A; Phosphorylation; Rabbit
In the myocardium, redox/cysteine modification of proteins regulating Ca2+ cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one electron reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery.
To determine the effects of HNO modification in cardiac myofilament proteins.
Methods and Results
The HNO-donor, 1-nitrosocyclohexyl acetate (NCA), was found to act directly on the myofilament proteins increasing maximum force (Fmax) and reducing the concentration of Ca2+ for 50% activation (Ca50) in intact and skinned cardiac muscles. The effects of NCA are reversible by reducing agents and distinct from those of another HNO-donor Angeli’s salt (AS), which was previously reported to increase Fmax without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide linked actin-tropomyosin and myosin heavy chain (MHC)-myosin light chain 1 (MLC1). Comparison of the NCA and AS effects with the modifications induced by each donor indicated the actin-tropomyosin and MHC-MLC1 interactions independently correlated with increased Ca2+ sensitivity and force generation, respectively.
HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca2+ responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.
contractility; nitroxyl; redox-switch; oxidation; calcium; oxidant signalling; redox
Troponin C (TnC) is implicated in the initiation of myocyte contraction via binding of cytosolic and subsequent recognition of the Troponin I switch peptide. Mutations of the cardiac TnC N-terminal regulatory domain have been shown to alter both calcium binding and myofilament force generation. We have performed molecular dynamics simulations of engineered TnC variants that increase or decrease sensitivity, in order to understand the structural basis of their impact on TnC function. We will use the distinction for mutants that are associated with increased affinity and for those mutants with reduced affinity. Our studies demonstrate that for GOF mutants V44Q and L48Q, the structure of the physiologically-active site II binding site in the -free (apo) state closely resembled the -bound (holo) state. In contrast, site II is very labile for LOF mutants E40A and V79Q in the apo form and bears little resemblance with the holo conformation. We hypothesize that these phenomena contribute to the increased association rate, , for the GOF mutants relative to LOF. Furthermore, we observe significant positive and negative positional correlations between helices in the GOF holo mutants that are not found in the LOF mutants. We anticipate these correlations may contribute either directly to affinity or indirectly through TnI association. Our observations based on the structure and dynamics of mutant TnC provide rationale for binding trends observed in GOF and LOF mutants and will guide the development of inotropic drugs that target TnC.
Muscle cells contract using a network of thread-like protein assemblies called myofilaments. Contraction is preceded by a signal that causes calcium to rush into the cell cytosol, where it can freely diffuse to and bind the myofilament proteins. Troponin C, a calcium sensor located on the thin filament, initiates and regulates the cascade of changes resulting in the generation of force by the thin and thick filaments comprising the myofilament lattice. In heart tissue, pathological conditions known as dilated and hypertrophic cardiomyopathies (DCM and HCM, respectively) are in part associated with abnormalities in the ability of the myofilaments to generate force at normal calcium concentrations. Manipulation of Troponin C calcium-binding through protein engineering and pharmaceutical intervention has thus attracted considerable attention as a therapeutic strategy for ameliorating these cardiac defects. In this study, we uncover a molecular basis of altered calcium handling for several engineered Troponin C variants, which provides further insight into tuning its control of myofilament contraction.
Protein kinase Cα (PKCα) is one of the predominant PKC isoforms that phosphorylate cardiac troponin. PKCα is implicated in heart failure and serves as a potential therapeutic target, however, the exact consequences for contractile function in human myocardium are unclear. This study aimed to investigate the effects of PKCα phosphorylation of cardiac troponin (cTn) on myofilament function in human failing cardiomyocytes and to resolve the potential targets involved.
Methods and Results
Endogenous cTn from permeabilized cardiomyocytes from patients with end-stage idiopathic dilated cardiomyopathy was exchanged (∼69%) with PKCα-treated recombinant human cTn (cTn (DD+PKCα)). This complex has Ser23/24 on cTnI mutated into aspartic acids (D) to rule out in vitro cross-phosphorylation of the PKA sites by PKCα. Isometric force was measured at various [Ca2+] after exchange. The maximal force (Fmax) in the cTn (DD+PKCα) group (17.1±1.9 kN/m2) was significantly reduced compared to the cTn (DD) group (26.1±1.9 kN/m2). Exchange of endogenous cTn with cTn (DD+PKCα) increased Ca2+-sensitivity of force (pCa50 = 5.59±0.02) compared to cTn (DD) (pCa50 = 5.51±0.02). In contrast, subsequent PKCα treatment of the cells exchanged with cTn (DD+PKCα) reduced pCa50 to 5.45±0.02. Two PKCα-phosphorylated residues were identified with mass spectrometry: Ser198 on cTnI and Ser179 on cTnT, although phosphorylation of Ser198 is very low. Using mass spectrometry based-multiple reaction monitoring, the extent of phosphorylation of the cTnI sites was quantified before and after treatment with PKCα and showed the highest phosphorylation increase on Thr143.
PKCα-mediated phosphorylation of the cTn complex decreases Fmax and increases myofilament Ca2+-sensitivity, while subsequent treatment with PKCα in situ decreased myofilament Ca2+-sensitivity. The known PKC sites as well as two sites which have not been previously linked to PKCα are phosphorylated in human cTn complex treated with PKCα with a high degree of specificity for Thr143.
The extracellular matrix (ECM) protein-integrin-cytoskeleton axis plays a central role as a mechanotransducing protein assemblage in many cell types. However, how the process of mechanotransduction and the mechanical generated signals arising from this axis affect myofilament function in cardiac muscle are not completely understood. We hypothesize that ECM proteins can regulate cardiac function through integrin binding, and thereby alter the intracellular calcium concentration ([Ca2+]i) and/or modulate myofilament activation processes. Force measurements made in mouse papillary muscle demonstrated that in the presence of the soluble form of the ECM protein, fibronectin (FN), active force was increased significantly by 40% at 1 Hz, 54% at 2 Hz, 35% at 5 Hz and 16% at 9 Hz stimulation frequencies. Furthermore, increased active force in the presence of FN was associated with 12–33% increase in [Ca2+]i and 20–50% increase in active force per unit Ca2+. A function blocking antibody for α5 integrin prevented the effects of the FN on the changes in force and [Ca2+]i, whereas a function blocking α3 integrin antibody did not reverse the effects of FN. The effects of FN were reversed by an L-type Ca2+ channel blocker, verapamil or PKA inhibitor. Freshly isolated cardiomyocytes exhibited a 39% increase in contraction force and a 36% increase in L-type Ca2+ current in the presence of FN. Fibers treated with FN showed a significant increase in the phosphorylation of phospholamban; however, the phosphorylation of troponin I was unchanged. These results demonstrate that FN acts via α5β1 integrin to increase force production in myocardium and that this effect is partly mediated by increases in [Ca2+]i and Ca2+ sensitivity, PKA activation and phosphorylation of phospholamban.
Fibronectin; Integrin; Extracellular matrix proteins; Calcium; Force–[Ca2+]i relationship; Phospholamban; Cardiac muscle
Activation of the β-adrenergic receptor (βAR) pathway is the main mechanism of the heart to increase cardiac output via protein kinase A (PKA)-mediated phosphorylation of cellular target proteins, and perturbations therein may contribute to cardiac dysfunction in heart failure. In the present study a comprehensive analysis was made of mediators of the βAR pathway, myofilament properties and cardiac structure in patients with idiopathic (IDCM; n = 13) and ischemic (ISHD; n = 10) cardiomyopathy in comparison to non-failing hearts (donor; n = 10) for the following parameters: βAR density, G-coupled receptor kinases 2 and 5, stimulatory and inhibitory G-proteins, phosphorylation of myofilament targets of PKA, protein phosphatase 1, phospholamban, SERCA2a and single myocyte contractility. All parameters exhibited the expected alterations of heart failure, but for most of them the extent of alteration was greater in IDCM than in ISHD. Histological analysis also revealed higher collagen in IDCM compared to ISHD. Alterations in the βAR pathway are more pronounced in IDCM than in ISHD and may reflect sequential changes in cellular protein composition and function. Our data indicate that cellular dysfunction is more severe in IDCM than in ISHD.
β-Adrenergic receptor; Protein phosphorylation; Myofilament function; Cardiomyocyte; Collagen
Following activation by G-protein-coupled receptor agonists, protein kinase C (PKC) modulates cardiac myocyte function by phosphorylation of intracellular targets including myofilament proteins cardiac troponin I (cTnI) and cardiac myosin binding protein C (cMyBP-C). Since PKC phosphorylation has been shown to decrease myofibril ATPase activity, we hypothesized that PKC phosphorylation of cTnI and cMyBP-C will lower myocyte power output and, in addition, attenuate the elevation in power in response to protein kinase A (PKA)-mediated phosphorylation. We compared isometric force and power generating capacity of rat skinned cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of cMyBP-C and cTnI and decreased both maximal Ca2+ activated force and Ca2+ sensitivity of force. Moreover, during submaximal Ca2+ activations PKC decreased power output by 62 %, which arose from both the fall in force and slower loaded shortening velocities since depressed power persisted even when force levels were matched before and after PKC. In addition, PKC blunted the phosphorylation of cTnI by PKA, reduced PKA-induced spontaneous oscillatory contractions, and diminished PKA-mediated elevations in myocyte power. To test whether altered thin filament function plays an essential role in these contractile changes we investigated the effects of chronic cTnI pseudo-phosphorylation on myofilament function using myocyte preparations from transgenic animals in which either only PKA phosphorylation sites (Ser-23/Ser-24) (PP) or both PKA and PKC phosphorylation sites (Ser-23/Ser-24/Ser-43/Ser-45/T-144) (All-P) were replaced with aspartic acid. Cardiac myocytes from All-P transgenic mice exhibited reductions in maximal force, Ca2+ sensitivity of force, and power. Similarly diminished power generating capacity was observed in hearts from All-P mice as determined by in situ pressure–volume measurements. These results imply that PKC-mediated phosphorylation of cTnI plays a dominant role in depressing contractility, and, thus, increased PKC isozyme activity may contribute to maladaptive behavior exhibited during the progression to heart failure.
PKC; PKA; Cardiac myocyte; Cardiac troponin I; Power output