OXIDATIVE STRESS AND SARCOMERIC PROTEINS

Abstract

Oxidative stress accompanies a wide spectrum of clinically important cardiac disorders, including ischemia/reperfusion, diabetes, and hypertensive heart disease. While reactive oxygen species (ROS) can activate signaling pathways that contribute to ischemic preconditioning and cardioprotection, high levels of ROS induce structural modifications of the sarcomere that impact on pump function and the pathogenesis of heart failure. However, the precise nature of the redox-dependent change in contractility is determined by the source/identity of the oxidant species, the level of oxidative stress, and the chemistry/position of oxidant-induced post-translational modifications on individual proteins within the sarcomere. This review focuses on various ROS-induced post-translational modifications of myofilament proteins (including direct oxidative modifications of myofilament proteins, myofilament protein phosphorylation by ROS-activated signaling enzymes, and myofilament protein cleavage by ROS-activated proteases) that have been implicated in the control of cardiac contractility.

INTRODUCTION

Reactive oxygen species (ROS) production increases in the context of many cardiac disorders. ROS-activated mechanisms that contribute to ischemic preconditioning are cardioprotective. However, high levels of ROS production that overwhelm cellular antioxidant defense systems generally produce deleterious changes in contractile performance and lead to adverse cardiac remodeling. Some cardiodepressive actions of ROS have been attributed to the activation of signaling pathways that influence the expression, phosphorylation, or function of calcium regulatory proteins (such as sarcoplasmic reticular Ca-ATPase 2a and ryanodine receptor 2), leading to changes in the magnitude or timing of the calcium transient and an inadequate calcium-induced contractile response ^1–3. However, oxidative stress-induced modifications of contractile proteins - that are not associated with changes in intracellular calcium homeostasis - also may contribute to contractile dysfunction and the evolution of heart failure. This review focuses on ROS-induced structural modifications of the sarcomere – due to direct oxidative modifications of myofilament proteins, myofilament protein phosphorylation by ROS-activated kinases, or myofilament protein cleavage by ROS-activated proteases – that interfere with the transduction of calcium-dependent contractile responses. Modifications that are not the obvious or direct target of a ROS-regulated processes are beyond the scope of this review and are not covered.

Oxidative modifications of sarcomeric proteins

The contractile apparatus consists of a parallel array of interdigitating thick and thin filaments that form the molecular motor that powers cardiac contraction (Fig 1). The thin filament backbone is made up of two helical strands of actin monomers, the elongated tropomyosin molecule that associates end-to-end to form a continuous strand along the actin filament, and troponin complexes (consisting of the calcium-binding cTnC subunit, the inhibitory cTnI subunit, and the tropomyosin-binding cTnT subunit) positioned every 7^th actin monomer along the thin filament. The thick filament is formed by two myosin heavy chain (MHC) molecules complexed with two molecules of myosin light chain 1 (MLC-1 or essential light chain) and two molecules of myosin light chain 2 (MLC-2 or regulatory light chain). The smaller light chain proteins are positioned at the myosin lever arm, between the rod portion of the molecule that forms the thick filament backbone and the head region that contains the actin- and nucleotide-binding sites. Cardiac contraction is powered by cyclical interactions between the myosin motor and actin-containing thin filaments – with additional regulation provided by cardiac myosin binding protein-C (cMyBP-C), a large multidomain thick filament protein located in the C-zone of the sarcomere. Titin - the third giant filament protein - runs from the Z disc to the M-band at the center of the sarcomere. Titin plays a role in the structural organization and assembly of myofibrillar proteins and it functions as a molecular scaffold to recruit signaling molecules that influence mechanotransduction. Titin also contains three serially-linked spring-like segments in an elastic I-band (the immunoglobulin-like domains, a Proline, Glutamate, Valine, and Lysine rich PEVK element, and an N2B element) that control the passive tension of the heart. The extensible elements in titin’s I-band region are targets for molecular events (isoform splicing and post-translational modifications) that ‘fine-tune’ titin’s elasticity.

Fig 1.

Schematic showing the arrangement of the major contractile and regulatory proteins in the sarcomere.

Oxidative stress and increased formation of ROS (or reactive nitrogen species) can result in direct chemical oxidation (or nitrosylation) of many contractile proteins, leading to changes in their structural conformation and/or functional activity. Protein oxidation or nitrosylation generally occurs at reactive thiol moieties in cysteine (or to a lesser extent methionine) residues. The reactivity of any particular cysteine residue is determined by the pKa of its thiol moiety; cysteine residues adjacent to basic amino acids such as Arg or Lys, aromatic amino acids, or metal centers have a relatively low pK_a (<6.5), are prone to deprotonate, and tend to be more susceptible to oxidation (Fig 2). The reaction between the cysteine thiolate anion and H₂O₂ results in the formation of sulfenic acid (R-SOH), a relatively unstable structure that typically reacts with other thiol groups to form intra- or intermolecular disulfide bonds. A reaction between the cysteine thiolate anion and NO (S-nitrosylation) or glutathione (S-glutathionylation) leads to the formation of mixed disulfides. The formation of mixed disulfides prevents further more irreversible peroxidation of sulfenic acid to more highly oxidized sulfinic (R-SO₂H) or sulfonic (R-SO₃H) acid species - that typically are more disruptive to protein structure and function. Other residues also can be targets for oxidative modification. When oxidative stress and superoxide formation is associated with increased formation of NO (for example during early post-ischemic reperfusion or in the context of inflammation, where proinflammatory cytokines increase the expression of inducible nitric oxide synthase ^{4, 5}), a near diffusion-limited reaction results in the formation of peroxynitrite (ONOO^-); peroxynitrite is a highly reactive compound that promotes protein tyrosine nitration, the addition of a nitro group (NO₂) to the 3-position of the tyrosine phenolic ring. Peroxynitrite also can oxidize Cys residues and promote protein carbonylation, the addition of a carbonyl group to susceptible Lys, Arg, or Pro residues.

Fig 2.

Major redox modifications of cysteine and tyrosine side chains.

Cardiac contraction typically is reduced following treatment with oxidizing agents such as superoxide anion or H₂O₂ (the more stable reactive species formed endogenously through spontaneous or SOD-catalyzed dismutation of superoxide). Early studies in chemically “skinned” rat cardiac muscle fibers showed that superoxide anion depresses maximal calcium-activated force without changing calcium-sensitivity or influencing rigor contracture in ATP-free solutions ⁶. These results were interpreted as evidence that superoxide anion acts in a very specific manner to alter some aspect of cross-bridge cycling (rather than some more nonspecific mechanism, for example a proteolytic event that disrupts the structural integrity of the sarcomere). Initial attempts to expose mechanism showed that H₂O₂ treatment of isolated rat heart leads to the oxidation of thin filament proteins - both cysteinyl oxidation of tropomyosin and cysteinyl oxidation/carbonylation of actin ^{7, 8}. Oxidative modifications of tropomyosin also have been detected - in association with the development of contractile dysfunction - in ischemic microembolized pig hearts and in the early post-myocardial infarction period in mouse hearts ^{9, 10}. Oxidative modifications of actin (and protein kinase C-α) are detected during reperfusion of ischemic rat hearts ¹¹. There is evidence that oxidative modifications of cardiac tropomyosin (at its single cysteine residue at position 190) leads to the formation of dimers that alter tropomyosin’s flexibility and interfere with tropomyosin’s interactions with other thin filament proteins. While some investigators have argued that these structural events contribute to oxidative stress- and heart-failure-dependent changes in contractility ¹², this formulation ignores the many other ROS-dependent modifications of sarcomeric proteins that are detected in end stage human heart failure, that correlate with contractile dysfunction, and also may be contributory ⁸.

Peroxynitrite also decreases maximal force development of the intact heart and it decreases contractility in isolated human ventricular myocytes. Some of the cardiodepressant actions of peroxynitrite have been attributed to an increase in cGMP and the activation of a protein kinase G- (PKG-) dependent pathway that decreases myofilament responsiveness to calcium ^{13, 14}. However, peroxynitrite decreases force generation in isolated rat cardiac trabeculae in association with the nitration of MHC (and the myofibrillar isoform of creatine kinase, an additional target for a post-translational modification that impairs contractility by disrupting myofibrillar energetic mechanisms ^{15, 16}). In vitro studies performed on purified MHC show that peroxynitrite promotes myosin nitration, cysteine oxidation and/or carbonylation at several highly-reactive solvent-exposed sites in the catalytic subfragment-1 (S1) globular head region ¹⁷. Functional studies suggest that these redox-induced post-translational modifications (in particular, myosin oxidation at Cys⁷⁰⁷/Cys⁶⁹⁷ and myosin carbonylation at Lys⁸⁴_, which sits at a domain interface in close proximity to the reactive Cys⁷⁰⁷/Cys⁶⁹⁷ residues) lead to a partial unfolding of the myosin subfragment-1, enhanced susceptibility to proteolytic cleavage by trypsin, and changes in Mg-ATPase activity (both increased intrinsic Mg²⁺-ATPase activity and decreased actin-stimulated Mg²⁺-ATPase activity ^{15, 17–19}). However, there is reason to interpret the results of studies performed on purified myosin preparations in solution with caution, since some oxidative modifications of myosin (for example Cys⁷⁰⁷ oxidation) are not detected in more physiologically relevant preparations (i.e., in isolated cardiac myofibrils), where incorporation of myosin into the myofilament lattice leads to decreased cysteine reactivity ^{20, 21}. In this regard, studies in an aging rat heart model identify myosin nitration at Tyr¹¹⁴, Tyr¹¹⁶, Tyr¹³⁴, and Tyr¹⁴² and pharmacologic studies suggest that peroxynitrite decreases force generation by increasing myosin carbonylation; these studies conclude that nitration is not contributory and cysteine oxidation may actually be protective, since cysteine residues might act as peroxynitrite scavengers and prevent the peroxynitrite-induced modifications elsewhere in the protein that disrupt functional activity ^{17, 22}). The singular focus on myosin as the primary target of oxidative modifications also may be misguided, since ischemia/reperfusion injury leads to a decrease in maximum force per cross-sectional area and a decrease in rate of tension redevelopment in association with S-glutathionylation of actin in rat heart ²³; pro-oxidants such as glutathione+H₂O₂ or glutathione+diamide induce a high level of α-actin (not myosin) S-glutathionylation in isolated human cardiac myofibrils ²⁰. Actin S-glutathionylation at Cys³⁷⁴ (a site at the physiologically labile C-terminus) slows the kinetics of α-actin polymerization in vitro, destabilizes actin filaments in vivo, influences actin’s role as a myosin binding partner in the sarcomere, and decreases contractility; substitution of a glutathionylated form of actin for unmodified actin decreases maximal actomyosin-S1 ATPase activity ²⁴.

Oxidative modifications of other sarcomeric proteins also have been identified. Peroxynitrite treatment or aging has been linked to increased nitration of tyrosine residues in a range of sarcomeric proteins, including cardiac troponin I (cTnI), cardiac troponin T (cTnT), MHC, myosin light chain, tropomyosin, cMyBP-C, actin desmin, and α-actinin ^{22, 25,26}. Studies in human cardiomyocytes link α-actinin nitration to changes in cellular ultrastructure (disruption of the myofibrillar cross-striation pattern) and a defect in contractile function (reduced isometric force generation) ¹³. The less compliant titin N2B isoform also has been characterized as a redox-sensor. Titin is co-expressed in the heart as N2BA and N2B isoforms that arise through alternative splicing of the transcript of a single gene. The principal difference between titin N2BA and N2B isoforms is in the length of their elastic I-band spring segment; N2B has a relatively short I-band segment and is very stiff, whereas N2BA has a longer I-band region and is more compliant. The shorter titin N2B isoform contains six cysteine residues that form one or more disulfide bonds under oxidizing conditions; disulfide bonding decreases the extensibility of N2B and leads to an increase cardiac stiffness ²⁷.

While oxidizing agents such as H₂O₂, superoxide, or peroxynitrite typically reduce force generation in skinned muscle preparations, nitroxyl (HNO, an electron reduction product of NO that displays very distinct chemistry and reactivity) acts in an antithetical fashion to increase force generation by increasing myofilament calcium sensitivity ²⁸. HNO reacts chiefly with cysteine thiols, forming either a N-hydroxlsulfenamide (RSNHOH) or (if there is a second cysteine in close proximity) inter- or intramolecular disulfide bonds (Fig 2). A recent study mapped HNO-dependent redox modifications in the sarcomere to strategically located cysteine thiols in actin, tropomyosin, MHC, and MLC1. The HNO-dependent formation of actin-tropomyosin dimers (due to disulfide bridging between Cys²⁵⁷ in actin and Cys¹⁹⁰ in tropomyosin) is predicted to tether tropomyosin to a position that is more permissive for Ca²⁺-induced myofilament activation, thereby increasing contractility. The HNO-dependent formation of dimers between MHC and Cys⁸¹ in MLC1 is predicted to enhance myofilament calcium sensitivity and would also improve cardiac contractility ²⁹. These recently identified redox-dependent modifications of myofilament proteins that enhance force generation represent promising targets for novel classes of inotropic agents that could be developed for the therapy of heart failure.

REDOX-REGULATION OF MYOFILAMENT PROTEIN PHOSPHORYLATION

Cardiac contraction must be dynamically regulated on a beat-to-beat basis to accommodate to changes in hemodynamic load and to respond to neurohumeral stresses. Much of this control is accomplished by signal-regulated protein kinases (or phosphatases) that regulate the phosphorylation state of strategically located Ser or Thr residues in various myofilament proteins (i.e., myofibrillar protein phosphorylation is almost exclusively on Ser/Thr - and not Tyr - residues). Of note, many protein kinases that contribute to mechanical or neurohumoral control of cardiac contraction also are regulated by oxidative stress. This section focuses on phosphorylation events on the thin filament proteins cTnI and cTnT, the thick filament accessory protein cMyBP-C, and titin that are targets for redox-regulated enzymes.

Redox-regulation of thin filament protein phosphorylation

cTnI is the inhibitory component of the troponin complex that functions to ‘fine-tune’ myofilament function to hemodynamic load; cTnI contains three well described phosphorylation clusters at Ser²³/Ser²⁴, Ser⁴³/Ser⁴⁵, and Thr¹⁴⁴. cTnI phosphorylation at Ser²³/Ser²⁴ (in the N-terminal region unique to cardiac TnI) is generally attributed to the beta-adrenergic receptor pathway involving protein kinase A (PKA) ³⁰. cTnI-Ser²³/Ser²⁴ phosphorylation accelerates the off-rate for calcium binding to cTnC, leading to a faster rate of cardiac relaxation (which is crucial to accommodate the beta-adrenergic receptor dependent positive chronotropic response). PKA is a heterotetramer enzyme consisting of two catalytic (C) subunits that are maintained in an inactive conformation by two cAMP-binding regulatory (R) subunits. cAMP activates PKA by binding to the R subunits; this interaction leads to the dissociation of the enzyme complex and frees the C subunit to phosphorylate target substrates. PKA holoenzymes are classified as type I or II based upon the identity of the R subunit (RI or RII) in the enzyme complex. Cardiomyocytes co-express both PKAI and PKAII enzymes that display distinct biochemical properties and subcellular localization patterns; PKAII is primarily recovered in the particulate cell fraction (in association with membrane scaffolding proteins, or A-kinase Anchoring Proteins [AKAPs]) whereas the type I PKA holoenzyme is recovered primarily as a cytosolic enzyme. While RI and RII subunits share similar domain organization, there is genetic and biochemical evidence that RI and RII are not functionally non-redundant. In particular, PKAI functions as a redox-activated enzyme (Table 2). RI subunits contain a pair of redox-sensitive cysteine thiols within the N-terminal AKAP binding region of the protein; these redox-sensitive cysteine thiols are not present in RII. The redox-sensitive cysteine thiols in RI form interprotein disulfide dimers that stabilize a conformation that binds AKAP proteins with higher affinity ³¹. In cardiomyocytes, this is detected as a redox-dependent increase in PKAI binding to α-MHC, which has been characterized as a putative AKAP in the myofilament fraction ³². In theory, RI dimerization might also control binding to cTnT, another myofilament protein recently identified as a sarcomeric AKAP ³³, but this has not been considered. Since the PKAI holoenzyme is activated by substrate-induced sensitization to cAMP (i.e. it displays activity at low cAMP concentrations that do not support activation of type II PKA), the redox-dependent redistribution of PKAI to the sarcomere could allow for the phosphorylation of cTnI (and other sarcomeric substrates such as cMyBP-C, see below) and an increased cardiac contractility under conditions that are not associated with a beta-adrenergic receptor-dependent increase in cAMP ³².

TABLE 2.

OXIDATIVE MODIFICATIONS OF PROTEIN KINASES

Kinase	Post-Translational Modification	Functional effects	Ref
PKA	RI subunit oxidation	↑ RI binding to AKAPs (α-MHC) ↑ PKAI kinase activity	32
	Catalytic domain Cys199 -S-glutathionylation	↓kinase activity	34–36
PKG1α	Oxidation of Cys42 in the homodimerization domain	↑ affinity for substrates ↑ cGMP-independent catalytic activity	38
PKC	Oxidation of C1 domain Cys residues	↓ autoinhibition, ↑ kinase activity	59
	Calpain-dependent cleavage – release of a constitutively active catalytic domain fragment	↑ PKCα catalytic activity, ↑ PKCδ-dependent phosphorylation of 14-3-3	68, 85–87
	Oxidation of a conserved activation loop Cys	↓ kinase activity	36, 59
	Src-dependent phosphorylation of PKCδ at Y311	Altered substrate specificity-acquisition of cTnI-T144 kinase activity	40
PKD	c-Abl- and Src-dependent phosphorylations of PKD at Tyr463 and Tyr95 that relieve autoinhibition, promote PKCδ-dependent PKD phosphorylation at Ser744/Ser748.	↑ kinase activity	50, 51
CaMKII	Met281/Met282 oxidation	↑ Ca2+-independent catalytic activity	76
ASK-1	Mechanisms that disrupt a C-terminal interaction with 14-3-3: Dephosphorylation of Ser967 at the ASK-1 C-terminus or phosphorylation of 14-3-3 by ROS-regulated kinases (PKD, MST-1, catalytic fragment of PKCδ). Mechanisms that disrupt an N-terminal interaction with Trx-1 (Trx-1 oxidation)	↑ kinase activity	66–68
MST-1	Caspase-dependent cleavage of an autoinhibitory domain	↑ kinase activity	60

The presence of distinct PKAI and PKAII activation mechanisms at the sarcomere allows for dynamic and nuanced control of myofilament function in response to various physiologic and pathologic stimuli. However, a redox-dependent mechanism that activates PKAI (via the RI subunit) may be counterbalanced by oxidative modifications involving a strategic located cysteine residue in the PKA catalytic subunit (at position 199 in the activation loop); S-glutathionylation at Cys¹⁹⁹ (or the formation of internal disulfide between Cys¹⁹⁹ and Cys³⁴³) leads to a decrease in kinase activity ^{34, 35}. Structural models suggest that the redox-dependent decrease in catalytic activity is due to a steric effect and reduced affinity for substrate ³⁴, but there is biochemical evidence that the cysteine thiol modification also decreases catalytic activity indirectly by facilitating the dephosphorylation of an adjacent threonine residue in the activation loop (a post-translational modification that is required for kinase activity ³⁶).

Most studies have focused on cTnI-Ser²³/Ser²⁴ phosphorylation as a post-translational modification regulated by PKA, but this site also is a target for phosphorylation by other ROS-regulated Ser/Thr kinases. For example, autocrine/paracrine stimuli that activate the NO/cGMP pathway can promote cTnI-Ser²³/Ser²⁴ phosphorylation by protein kinase G (PKG) ³⁷. PKG1α (a major PKG isoform in cardiomyocytes) contains a reactive cysteine at position 42 in the N-terminal homodimerization domain that abuts in the enzyme homodimer; oxidative stress leads to the formation of interprotein disulfide bonds that increase the enzyme’s affinity for substrate and leads to a high level of cGMP-independent PKG1α catalytic activity. The N-terminus of PKG1β (the other major PKG splice variant in cardiomyocytes) does not contain a reactive cysteine at this position and is not activated by oxidative stress ³⁸. The redox-dependent mechanism for PKG1α activation appears to be important in the vasculature, where it provides for stimulus-specific mechanisms to control vasodilatation in response to NO and oxidative stress ³⁹; the functional consequences of a redox-dependent PKG1α activation mechanism in cardiomyocytes warrant further study.

Other redox-regulated signaling enzymes that can function as cTnI-Ser²³/Ser²⁴ kinases include protein kinase D (PKD), p90 ribosomal S6 kinase (RSK), and certain isoforms of protein kinase C (PKC) ^40–44 (Table 2). PKD is a signal-regulated Ser/Thr kinase that phosphorylates sarcomeric proteins (cTnI, cMyBP-C) and regulates cardiac contractility; PKD also activates signal transduction pathways that regulate gene expression and contribute to cardiac hypertrophy ⁴⁵. The canonical pathway for PKD activation involves the growth factor receptor-dependent hydrolysis of membrane phosphoinositides leading to the formation of diacylglycerol and the co-localization of PKD with allosterically-activated PKC isoforms at diacyglycerol-enriched membranes; this facilitates PKC-dependent trans-phosphorylation of PKD at Ser⁷⁴⁴/Ser⁷⁴⁸ (two highly conserved serine residues in the activation loop that regulate catalytic activity). The activated PKD enzyme then phosphorylates target substrates - typically at LxRxxpS consensus phosphorylation motifs ⁴⁶. In this regard, it is interesting to note that PKD displays a high level of in vitro cTnI-Ser²³/Ser²⁴ catalytic activity, although rodent (PVRRRs²³s²⁴) and human (PIRRRS²³S²⁴) cTnI sequences diverge somewhat from an optimal PKD consensus phosphorylation motif. However, there is ample evidence that PKD can phosphorylate substrates (such as c-Jun, β-catenin, and type IIα PI4P kinase) that do not conform to a LxRxxpS/T motif and there are hints in the literature that the flexibility of target substrate recognition may be enhanced during oxidative stress (due to the somewhat different ROS-dependent mechanism for PKD activation ^47–49). PKD is activated during oxidative stress via a mechanism involving c-Abl, which phosphorylates PKD at Tyr⁴⁶³ in its autoinhibitory pleckstrin homology domain ⁵⁰. This induces a conformational change that relieves autoinhibition and permits Src-dependent PKD phosphorylation at Tyr^{95 51}. Since the phospho-tyrosine at position 95 is a consensus-binding motif for the C2 domain of PKCδ, this leads to a docking interaction between PKD and PKCδ and PKCδ-dependent PKD phosphorylation at Ser⁷⁴⁴/Ser⁷⁴⁸. Stimulus-specific differences in PKD activation mechanisms (in response to growth factor receptors and during oxidative stress) have been linked to distinct functional responses in the vasculature; the prediction that the activation mode might also dictate the in vivo actions of PKD in cardiomyocytes has not been considered. Rather, studies to date show that endothelin-1 receptors recruit a PKD-dependent mechanism that promotes cTnI-Ser²³/Ser²⁴ phosphorylation, decreases myofilament Ca²⁺ sensitivity, and enhances contraction in adult cardiomyocytes ^42–44, but the endothelin-1 receptor-dependent increase in PKD1 activity does not couple to changes in cTnI-Ser²³/Ser²⁴ phosphorylation in cultured neonatal rat cardiomyocytes ⁵². These divergent results suggest that stimulus-, age-, or disease-dependent differences in the cellular signaling machinery might influence PKD’s signaling repertoire (and PKD-dependent control of contraction) in cardiomyocytes.

cTnI contains additional phosphorylation sites at Ser⁴³/Ser⁴⁵ and Thr¹⁴⁴ that are traditionally viewed as a target for PKC ^53–56. While the functional importance of Ser⁴³/Ser⁴⁵ phosphorylation remains uncertain, Thr¹⁴⁴ is strategically positioned in the inhibitory region of cTnI where it can regulate calcium sensitivity and cross-bridge cycling rates. Thr¹⁴⁴ phosphorylation has been attributed to PKCβ or the Tyr³¹¹-phosphorylated form of PKCδ (a form of PKCδ that accumulates during oxidative stress ^{57, 58}). Of note, cTnI is phosphorylated by PKCδ in a stimulus-specific manner ⁴⁰ PKCδ phosphorylates cTnI exclusively at Ser²³/Ser²⁴ when it is allosterically activated by lipid cofactors. However, oxidative stress activates Src which phosphorylates PKCδ at Tyr³¹¹; the Tyr³¹¹-phosphorylated form of PKCδ displays a high level of Thr¹⁴⁴ kinase activity – it executes coordinate cTnI phosphorylations at Ser²³/Ser²⁴ and Thr^{144 40}. This distinct cTnI phosphorylation pattern (i.e., involving a dual phosphorylation at Ser²³/Ser²⁴ and Thr¹⁴⁴) is functionally important, since cTnI-Thr¹⁴⁴ phosphorylation alone has little effect on force generation or calcium sensitivity; Thr¹⁴⁴ phosphorylation becomes functionally important in a Ser²³/Ser²⁴-phosphorylated background, where it prevents calcium desensitization due to cTnI-Ser²³/Ser²⁴ phosphorylation ⁵⁷. While these studies focus on a very specific ROS-dependent mechanism that ‘fine tunes’ the enzymology of PKCδ other redox modifications play a more general role to regulate PKC activity. For example, the lipid-binding C1 domain (that is conserved module in the regulatory domain of all phorbol ester-sensitive PKCs) contains redox-sensitive cysteine residues; oxidative modifications at these sites lead to conformational changes that relieve autoinhibition and induce a high level of cofactor-independent catalytic activity. Redox-modifications of the highly conserved cysteine residues in the catalytic domain activation loop have an opposite effect and disrupt PKC catalytic activity⁵⁹.

Mst1 (mammalian sterile 20-like kinase 1) is a pro-apoptotic kinase that is activated via autophosphorylation and caspase-dependent cleavage of its autoinhibitory domain. Mst1 is activated in the context of ischemia/reperfusion injury and it contributes to adverse cardiac remodeling ⁶⁰. Recent studies indicate that Mst1 interacts with and phosphorylates cTnI; phosphorylation has been mapped primarily to Thr³² (with some additional phosphorylation at Thr⁵², and Thr¹³⁰ at Thr¹⁴⁴). Mst1 also phosphorylates cTnT, but only when it is incorporated into the troponin complex; Mst1 does not phosphorylate free cTnT. There is evidence that the Mst1-dependent phosphorylation induces a conformational change in cTnI that alters its binding affinity for cTnT and cTnC ⁶¹.

cTnT is another thin filament proteins that contains phosphorylation clusters at Thr¹⁹⁷/Ser²⁰¹/Thr²⁰⁶ and Ser²⁷⁸/Thr²⁸⁷ - although studies to date suggest that T²⁰⁶ is the only phosphorylation site that directly influences contractility. cTnT-Thr²⁰⁶ phosphorylation has been attributed to PKC or Raf-1 (but not PKA or PKG) and is implicated as a mechanism that desensitized the myofilament response to calcium, decreases actomyosin Mg-ATPase, and depressed cardiac contractility ^{62, 63}. cTnT also is phosphorylated by apoptosis signal-regulated kinase-1 (ASK-1), a ROS-regulated stress activated MAPK kinase kinase that is abundant in cardiomyocytes ^{64, 65}. ASK-1 contains a central kinase domain flanked by regulatory domains that engage in intermolecular interactions that limit ASK-1 catalytic activity. The interaction between the Ser⁹⁶⁷-phosphorylated C-terminal regulatory domain of ASK-1 and 14-3-3 proteins – and the interaction between the ASK-1 N-terminal regulatory domain and reduced thioredoxin-1 – clamps ASK-1 in a configuration that maintains low basal catalytic activity. ASK-1 is activated during oxidative stress as a result of molecular events that disrupt these intermolecular interactions, including the ROS-dependent increase in the activity of cellular phosphatases that dephosphorylate the ASK-1 C-terminal Ser⁹⁶⁷ residue, 14-3-3 protein phosphorylation by ROS-regulated kinases (such as PKD, Mst family kinases, or the catalytic fragment of PKCδ ^66–68), or Trx-1 oxidation. ASK-1 activation has been linked to the activation of JNK or NF-κB pathways that influence apoptotic/necrotic cell death and adverse cardiac remodeling ^{64, 65}. However, the activated form of ASK-1 also is detected in the sarcomere, where it phosphorylates cTnT ⁶⁹. ASK-1 activation leads to decreased cardiomyocyte contractility, but the link between cTnT phosphorylation and the cardiodepressant actions of ASK-1 remain uncertain, both because ASK-1 phosphorylates cTnT at Thr¹⁹⁷ and Ser²⁰¹ –not Thr²⁰⁶ (the site that has been implicated in the control of thin filament function ⁶³) and the activated form of ASK-1 also decreases the amplitude of Ca²⁺ transient (providing an alternate mechanism to explain the decrease in cardiomyocyte contractility ⁶⁹).

Redox-regulation of Thick filament protein phosphorylation

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that is required for sarcomeric integrity, the regulation of cardiac contraction, and cardioprotection. cMyBP-C contains multiple phosphorylation sites in a linker region located between the Ig-like C1 and C2 domains in the N-terminal myosin-binding region of the protein (a region unique to the cardiac isoform of MyBP-C). An interaction between this region of cMyBP-C and the myosin subfragment 2 (S2) domain (a region close to the lever arm) influences thick filament packing and the kinetics of cross-bridge cycling; phosphorylation disrupts this interaction and accelerates cross-bridge kinetics. The three best-characterized cMyBP-C phosphorylation sites in this region are at RRTSer²⁷³, RR(I/T)Ser²⁸², and LKKRDSer³⁰²; these three serine residues are flanked by sequences that support phosphorylation by basophilic kinases (and Ser³⁰² resides in an optimal phosphorylation motif for PKD ⁷⁰). Early studies established that all three sites are phosphorylated by PKA and that PKA also targets an additional in vitro phosphorylation site at Ser^{307 71,72}. However, current literature suggests that phosphorylation is regulated in a hierarchical manner and that individual sites on cMyBP-C are differentially phosphorylated by PKC, PKD, RSK, and Ca²⁺- and calmodulin-dependent protein kinase II (CaMKII). In particular, Ser²⁸² is a phosphoacceptor site for PKA, CaMKII, or RSK; phosphorylation at this site primes cMyBP-C for subsequent PKA-, PKC-, CaMKII- or PKD-dependent phosphorylation at Ser³⁰² (and PKA- or PKC-dependent phosphorylation at Ser^{273 71, 73}). Mutagenesis studies suggest cMyBP-C phosphorylation at Ser²⁸² is sufficient to accelerate cross-bridge cycle kinetics (i.e., that under certain circumstances this post-translational modification can result in functional changes even without an increase in cMyBP-C phosphorylation at Ser³⁰² or cTnI phosphorylation at Ser²²/Ser²³). However, there also is evidence that the physiologic control of cardiac contractile function requires reversible phosphorylations at all three sites ^{73, 74} and that electrical field stimulation leads to an increase in Ca²⁺-activated contractility at least in part through a mechanism involving PKD-dependent cMyBP-C phosphorylation at Ser^{302 75}.

The ROS-dependent mechanisms that activate PKA, PKC, and PKD - that might underlie a redox-dependent increase in cMyBP-C phosphorylation - were considered in previous sections. CaMKII also has been characterized as a ROS-activated protein kinase (Table 2). CaMKII functions as dodecameric enzyme that is comprised of individual monomers containing three key structural elements: an association domain that controls assembly of the holoenzyme, a kinase domain that phosphorylates target substrates, and a regulatory domain containing an autoinhibitory motif that regulates catalytic activity. Stimuli that increase intracellular calcium and promote Ca²⁺/CaM binding to CaMKII induce a conformational change that relieves autoinhibition. With prolonged increases in intracellular calcium, CaMKII executes an intersubunit phosphorylation at Thr²⁸⁷ in the autoinhibitory domain that prevents re-association of the regulatory and catalytic domains and confers Ca²⁺-independent catalytic activity. Recent studies indicate that the methionine residues at positions 281/282 in CaMKII’s autoinhibitory domain (adjacent to the Thr²⁸⁷ phosphorylation site) are targets for oxidative modifications ⁷⁶. Oxidation at these sites leads to a high level of Ca²⁺/CaM-independent CaMKII activity. Since oxidized and autophosphorylated forms of CaMKII share many cellular actions, a role for the redox-activated form of CaMKII as a cMyBP-C-Ser²⁸² kinase is plausible and warrants future consideration.

Redox-regulation of Titin phosphorylation

Changes in titin isoform expression during development and in disease provide a mechanism to regulate cardiac stiffness on a relatively long time scale. The relatively high elastic recoil of the perinatal heart is attributable to a low N2BA/N2B ratio, whereas an increase in the N2BA/N2B ratio in chronic heart failure leads to a decrease in passive tension. The spring-like segments in titin’s elastic I-band also are targets for phosphorylation events that lead to more dynamic changes in cardiac elasticity. The serially-linked spring-like segments in titin’s I band are differentially phosphorylated by PKA/PKG and PKC. PKA and PKG both phosphorylate a single serine residue at position 469 in the N2B segment, leading to a decrease in passive tension ^{77, 78}. Since this residue is conserved in human cardiac N2BA and N2B isoforms, this post-translational modification constitutes a general mechanism to regulate cardiac stiffness. PKCα phosphorylates cardiac and skeletal muscle titin isoforms primarily at different serine residues (Ser¹¹⁸⁷⁸ and Ser¹²⁰²²) in the PEVK domain; phosphorylation in the PEVK domain has an antithetical effect to increase passive tension ⁷⁹. Phosphorylation sites in other regions of the titin protein that do not regulate mechanical function also have been identified; some have speculated that these post-translational modifications may regulate docking interactions and influence titin’s role as a molecular scaffold.

MYOFILAMENT PROTEIN CLEAVAGE

Cardiac injury and oxidative stress also can lead to the degradation of sarcomeric proteins. Early studies showed that cTnI degradation is a prominent feature of ischemic damage, that degraded forms of cTnI remain associated with the myofilament lattice, and that cTnI cleavage may contribute to ischemia-induced changes in force generation and myofibrillar calcium sensitivity ^{80, 81}. Some studies attribute myofilament protein degradation to μ-calpain, a calcium-dependent myofibril-associated protease that is activated in ischemic cardiomyocytes ⁸². There is evidence that cTnI is degraded to progressively smaller cleavage products with increasingly severe or prolonged intervals of ischemia/reperfusion injury. A brief/mild episode of ischemia/reperfusion injury leads to the conversion of cTnI (a 210 amino acid protein) to a smaller degradation product (residues 1-193) that forms covalent complexes with cleaved forms of cTnT and cTnC ⁸³. More severe ischemia/reperfusion injury leads to further degradation of cTnI and the accumulation of shorter catalytic fragments (consisting of residues 63 193 and 73 193) that lack the N-terminal PKA phosphorylation sites and do not form these covalent complexes. Some studies suggest that cTnI may be protected from this form of proteolytic degradation by PKA-dependent phosphorylation of cTnI at Ser²³/Ser^{24 82, 83}. cTnT also appears to be vulnerable to calpain-dependent proteolytic cleavage with even very brief episodes of ischemia/reperfusion injury. Calpain cleaves cTnT at a site that removes the NH₂-terminal modulatory domain, leaving a conformationally altered cTnT core structure (residues 72-291) that displays altered binding to cTnI, cTnC and Tm ⁸⁴. Finally, MLC1 also is degraded during prolonged/severe episodes of ischemia/reperfusion injury; this also contributes to a decrease in force generation and calcium sensitivity ⁸¹.

While most studies have focused on calpain-mediated proteolytic events that are localized to the sarcomere, calpain could in theory influence contractile function by proteolytically activating protein kinases that phosphorylate myofibrillar proteins ⁸⁵. For example, calpain cleaves PKCα at the V3 hinge region, freeing the C-terminal catalytic domain from the autoinhibitory constraints imposed by the N-terminal regulatory domain. There is recent evidence that the PKCα catalytic domain fragment displays a high level of constitutive activity; it acts as a ‘rogue’ kinase to phosphorylate cellular substrates – including those that are not (or are only weakly) phosphorylated by full-length PKCα. Receptor-independent proteolytic activation mechanisms are not specific for PKCα – since calpain cleaves other PKC isoforms ^{86, 87} and other Ser/Thr kinases such as PKD ⁸⁸. A role for unregulated/mislocalized catalytic domain fragments generated during oxidative stress, as mediators of pathological cardiac remodeling and changes in contractile performance, has not been considered.

Calpain may not be the only (or even the primary) mediator of sarcomeric protein breakdown in the ischemic heart, since pro-apoptotic stimuli and oxidative stress also increase the activity of other proteolytic enzymes. For example, caspase-3 is activated by pro-apoptotic stimuli and it cleaves actin, α-actinin, and cTnT. Caspase-3 cleaves cTnT at a consensus site at DFD↓D⁹⁷, but only when the protein is incorporated into the myofilament lattice; caspase-3 does not cleave free cTnT ⁸⁹. Functional studies link caspase-3 treatment of skinned fiber bundles to defects in force/Ca²⁺ relationships and myofibrillar ATPase activity. These results suggest that caspase-induced myofilament protein breakdown may contribute to mechanical dysfunction and the evolution of heart failure ⁸⁹. However, the importance of caspase-3 as a general mediator of myofibrillar protein breakdown in the setting oxidative stress remains uncertain, since caspase-3 contains a redox-sensitive catalytic cysteine (Cys¹⁶³); oxidative modifications (S-glutathionylation or S-nitrosylation) at this site have been linked to a decrease in caspase-3 activity ^90–92.

Matrix metalloproteinase-2 (MMP-2, an abundant MMP in cardiomyocytes and many other cell types) also has recently emerged as a functionally important redox-activated endopeptidase that cleaves sarcomeric proteins. In fact, some have argued that at least some proteolytic events previously attributed to calpain may actually be mediated by MMP2, since [a] calpain and MMP-2 cleave many common substrates, [b] many inhibitors (MDL-28170, ALLN, ALLM, and PD-150606) that have been used to define the cardiac actions of calpain are also effective inhibitors of MMP-2 ⁹³, and [c] degraded forms of sarcomeric proteins such as cTnI are not detected in transgenic mice with cardiac-specific calpain overexpression ⁹⁴. MMPs are zinc-dependent endoproteinases that are synthesized as latent, inactive zymogens that are maintained in an inactive state by an interaction between a cysteine thiol in the propeptide domain and the Zn²⁺-containing catalytic domain. MMP-2 is activated in the pericellular or extracellular compartment by upstream proteases (such as MMP-14) that cleave the inhibitory pro-peptide domain and expose the active site. This leads to the degradation of extracellular matrix and underlies MMP-2 widely recognized roles in tissue remodeling (including embryogenesis, angiogenesis, myocardial infarction, and various forms of wound healing). However, there is recent evidence that the highly conserved Cys in the propeptide domain is a target for oxidative modifications (specifically, peroxynitrite-dependent S-glutathiolation); an oxidative modification at this site disrupts the intramolecular autoinhibitory interaction and provides a non-proteolytic mechanism to activate MMP-2 ⁹⁵. The redox-activated form of MMP-2 is recovered in the sarcomere, where it anchors to proteolytic targets such as cTnI and MLC-1. MMP-2 cleaves cTnI, MLC-1, and MLC-2 during ischemia/reperfusion injury; some studies suggest that these sarcomeric protein cleavage events contribute to oxidative stress-dependent defects in cardiac contractility ^96–98. Moreover, there is increasing evidence that the controls of redox-induced events in the sarcomere can be rather elaborate and multi-factorial, since MLC-1 and MLC-2 are primed for MMP-2-dependent degradation by redox-induced post-translational modifications. For example, MMP-2-dependent cleavage of MLC-1 (at Y¹⁸⁹↓E¹⁹⁰ in its accessible C-terminus) is enhanced by a peroxynitrite-induced increase in MLC-1-Tyr⁷⁸/Tyr¹⁹⁰ nitration and Cys⁸¹ nitrosylation ^97,99. Similarly, MMP-2-dependent cleavage of MLC-2 is facilitated by nitration at Tyr^{150 98}. Finally, there is evidence that MMP-2 localizes to the Z-disk where it might play a role in Z-disk assembly and the maintenance of sarcomeric integrity by binding and cleaving α-actinin and titin ^{100, 101}.

CONCLUSIONS, CAVEATS, AND FUTURE DIRECTIONS

This review summarizes recent advances in our knowledge of ROS-regulated post-translational modifications in sarcomeric proteins. The lengthy list and spectrum of the redox-regulated events summarized in Table 1 is a testament to recent advances in methodologies for proteomic profiling and the growing recognition that redox biology plays a fundamentally important role in the control of cardiac contraction. While there is considerable evidence that many protein redox modifications lead to functionally important changes in sarcomeric protein structure, stability, interactivity, and/or activity, our current understanding of the redox-dependent mechanisms that control contractility in vivo in the intact heart remains rather rudimentary in large part because biochemical studies have focused primarily on redox-dependent modifications on single purified contractile proteins or preparations that contain selected components of the contractile apparatus; the large size and limited solubility of many myofibrillar proteins makes some types of biochemical analysis quite challenging. Extrapolations from these more reductionist systems to the in vivo context may be misleading for several reasons. First, the conformation and/or exposed surfaces of a contractile protein may be altered by interactions with binding partners in the myofilament lattice in a manner that influences the accessibility of post-translational modification sites and either facilitates or prevents reactivity. Second, ROS-dependent modifications of sarcomeric proteins seldom occur in isolation – and structural modifications of one protein can have far-reaching effects on molecular interactions between sarcomeric proteins elsewhere in the complex. Hence, the ensemble effects of all post-translational modifications in the sarcomere determine the nature of the ROS-induced change in cardiac contractility in vivo in the intact heart. Finally, generalizations regarding ROS-dependent changes in cardiac contraction ignore the fact that oxidative stress represents a spectrum of responses that depend on the precise chemical nature of the oxidant species and level/severity of oxidative stress; this review provides numerous examples of oxidant species and ROS-activated enzymes that trigger different (in some cases diametrically opposite) effects on cardiac contractility. The complexities inherent in these redox-regulated mechanisms that control pump function present both challenges and opportunities for the development of more specific therapeutic strategies for heart disease.

TABLE 1.

ROS-INDUCED MODIFICATIONS OF CARDIAC SARCOMERIC PROTEINS

Protein	Phosphorylation	Ref	Functional Effect	Oxidation	Functional effects	Ref
cTnI	Ser23/Ser24 PKA, PKC, PKG, PKD, p90RSK	30, 37 40–44	↑ Ca2+ dissociation from TnC ↓ myofilament Ca2+ sensitivity ↑ rate of relaxation	Tyr nitration		25, 26
	Ser43/Ser45
	Thr144 PKCβ MST-1 Y311-phosphorylated PKCδ	57, 58 61 40	↑ myofilament Ca2+ sensitivity Altered cTnI conformation and cTnI binding to cTnT/cTnC
	Thr32/Thr52/Thr130 MST-1	61
cTnT	Ser206 PKC Raf-1	63 62	↓ maximal force ↓ myofilament Ca2+ sensitivity	Tyr nitration		25, 26
	Thr197/Ser201 PKC ASK-1	63 69
	Ser274/Thr287 PKC	63
Tm				Cys190 Oxidation	↓ contractile function Formation of Tm dimers ↓ binding to actin ↓ formation of actin-TM complexes	7–10, 12
				HNO-dependent Cys190 Oxidation	Formation of dimers Cys257 in actin Tethers Tm to a position permissive for Ca2+- induced myofilament activation	29
				Cys190 Carbonylation		8
				Tyr nitration	↓ contractile function	25, 26
MHC				Cys697/Cys707 Oxidation	↓ Maximal force	17–19
				HNO-dependent Cys oxidation	Dimerization with Cys81 in MLC1 ↑myofilament Ca2+ sensitivity ↑Contractility	29
				Lys84 carbonylation		17
				Nitration Tyr114, Tyr116, Tyr134, Tyr142	↓ contractile function	15, 17, 22
MLC1				Tyr78/Tyr190 nitration Cys81 nitrosylation	↑ degradation by MMP-2 ↓ contractility	25, 99
MLC2				Tyr152 nitration	↑ degradation by MMP-2 ↓ contractility	98
cMyBP-C	Ser282 PKA, PKC, CaMK, PKD Ser302 PKA, PKC, PKD Ser273 PKA,PKC	30,31 75	Primes MyBP-C for subsequent phosphorylation at Ser302 and Ser273 Accelerates cross-bridge cycle kinetics	Tyr nitration		25, 26
Actin				Cys374 oxidation	↓Tm-actin binding ↓ maximal force ↓ contractile function ↑ F-actin depolymerization ↓ Myosin ATPase activity ↓Actin filament sliding velocity	7, 8, 11
				Cys374 glutathionylation	↓ α-actin polymerization kinetics Destabilizes actin filaments Decreases contractility	20, 23, 24
				Cys257 oxidation by HNO	Formation of dimers Cys190 in Tm Tethers Tm to a position that is permissive for Ca2+-induced myofilament activation ↑ contractility	29
				Carbonylation	↓ contractile function	8
				Tyr nitration	↓ contractile function	25
α-Actinin				Tyr nitration	Deterioration of cross-striated pattern ↓ longitudinal force transmission	13, 25, 26
Titin	Ser469 PKA PKG	77, 78	↓ passive tension	Cys oxidation	S-S bond formation that decreases the extensibility of titin N2B ↑ cardiac stiffness	27
	Ser11878/Ser12022 PKCα	79	↑ passive tension

Non-standard Abbreviations

AKAP

A-kinase anchoring proteins

ASK-1

apoptosis signal-regulated kinase-1

CaMKII

Ca²⁺- and calmodulin-dependent protein kinase II

cMyBP

cardiac myosin binding protein

cTnC

cardiac troponin C

cTnI

cardiac troponin I

cTnT

cardiac troponin T

HNO

nitroxyl

MHC

myosin heavy chain

MLC-1

myosin light chain 1 (or essential light chain)

MLC-2

myosin light chain 2 (or regulatory light chain)

MMP

matrix metalloproteinase

Mst1

mammalian sterile 20-like kinase 1

NO

nitric oxide

ONOO^-

peroxynitrite

PKA

protein kinase A

PKC

protein kinase C

PKD

protein kinase D

PKG

Protein kinase G

PTM

Post-translational modification

RNS

reactive nitrogen species

ROS

reactive oxygen species

Tm

Tropomyosin

Trx-1

thioredoxin-1