Redox Signaling and Cardiac Sarcomeres*

Oxidative stress is common in many clinically important cardiac disorders, including ischemia/reperfusion, diabetes, and hypertensive heart disease. Oxidative stress leads to derangements in pump function due to changes in the expression or function of proteins that regulate intracellular Ca2+ homeostasis. There is growing evidence that the cardiodepressant actions of reactive oxygen species (ROS) also are attributable to ROS-dependent signaling events in the sarcomere. This minireview focuses on myofilament protein post-translational modifications induced by ROS or ROS-activated signaling enzymes that regulate cardiac contractility.

Oxidative stress is common in many clinically important cardiac disorders, including ischemia/reperfusion, diabetes, and hypertensive heart disease. Oxidative stress leads to derangements in pump function due to changes in the expression or function of proteins that regulate intracellular Ca 2؉ homeostasis. There is growing evidence that the cardiodepressant actions of reactive oxygen species (ROS) also are attributable to ROSdependent signaling events in the sarcomere. This minireview focuses on myofilament protein post-translational modifications induced by ROS or ROS-activated signaling enzymes that regulate cardiac contractility.
Cardiac myofilament activation is tightly regulated by protein-protein interactions that convert the chemical energy of ATP into the mechanical energy of muscle contraction. In the healthy heart, these protein-protein interactions are precisely tuned (largely through a series of orchestrated phosphorylations on myofilament proteins) to accommodate differences in hemodynamic load during rest and exercise. Disease-specific alterations in the post-translational modification of myofilament proteins lead to miscommunication between sarcomeric proteins and to contractile dysfunction.

Direct Reactive Oxygen Species-dependent Modifications of Sarcomeric Proteins
Many models of oxidative stress lead to heart failure syndromes that are not associated with changes in Ca 2ϩ homeostasis and are likely attributable to reactive oxygen species (ROS) 2 -dependent modifications of sarcomeric proteins (1)(2)(3)(4)(5)(6). ROS-dependent protein modifications typically map to reactive cysteines (cysteines flanked by basic or aromatic residues that form thiolate anions and are susceptible to redox modifications at physiological pH).
Studies in detergent-permeabilized cardiac fibers show that ROS reduce Ca 2ϩ -activated force, with no immediate effect on fibers in rigor (with inactive cross-bridges) (7), suggesting that ROS act on regions of the myofilaments exposed by Ca 2ϩ activation and inaccessible in attached cross-bridges (and that ROS do not induce nonspecific effects that disrupt the integrity of the sarcomeric lattice). Some studies identify the myosin heavy chain (MHC) as a redox sensor in the sarcomere because redox modifications at Cys 697 and Cys 707 decrease myosin ATPase activity and lead to myofilament dysfunction (6, 8 -10). Other redox modifications of proteins in the thin filament (actin and tropomyosin (Tm)) also lead to defects in actin-myosin crossbridge formation and thin filament activation by Ca 2ϩ (11). Cys 374 in actin (which indirectly interacts with myosin) may be particularly important because Cys 374 oxidation results in changes in maximum actomyosin ATPase activity and actin filament sliding velocity (12). Cys 374 also is the likely target of a glutathionylation reaction that decreases Tm-actin binding cooperativity and maximum developed force in permeabilized trabeculae (13). Redox modification of Tm (in this case, dimerization due Cys 190 oxidation) is detected in ischemic pig hearts and also may contribute to ROS-induced myofilament dysfunction because it alters Tm flexibility and Tm-thin filament interactions (14,15).
Titin (the giant sarcomeric protein that controls passive tension and functions as a molecular scaffold to recruit signaling proteins that regulate mechanotransduction) also is ROS-sensitive. Oxidative stress leads to the formation of one or more disulfide bonds involving the titin N2-B domain (which contains six Cys residues). This redox modification decreases the extensibility of titin and increases its passive tension (16,17). Desmin is the main intermediate filament protein that forms a network around sarcomeric Z-discs, links neighboring myofibrils, and connects myofilaments to other cellular structures (nuclei, cytoskeleton, and mitochondria) (11). Redox-modified (oxidized or nitrated) forms of desmin accumulate in insoluble aggregates that disrupt the sarcomeric lattice, have a toxic effect on the proteasome, and may contribute to contractile dysfunction (18,19).

Sarcomeric Protein Phosphorylation
Myofilament activation is modulated through sarcomeric protein phosphorylation. Because several sarcomeric proteins are phosphorylated by ROS-sensitive enzymes, stimuli that alter the intracellular redox state and shift the balance of cellular kinase versus phosphatase activity are predicted to alter cardiac contractility. Sarcomeric proteins cTnI, cTnT, cMyBP-C, and titin are phosphorylated by ROS-sensitive enzymes. cTnI contains functionally important phosphorylation clusters at Ser 23 /Ser 24 , Ser 43 /Ser 45 , and Thr 144 (as well as additional phosphorylation sites at Thr 32 , Thr 52 , Ser 76 /Ser 77 , Thr 130 , and Ser 150 that are less well characterized) (23)(24)(25). cTnI Ser 23 /Ser 24 phosphorylation is traditionally attributed to the ␤-adrenergic receptor (␤-AR)/cAMP pathway involving PKA. cTnI Ser 23 / Ser 24 phosphorylation reduces myofilament Ca 2ϩ sensitivity and is required for the ␤-AR-dependent lusitropic response. In some settings, cTnI Ser 23 /Ser 24 phosphorylation also is attributable to PKG, various PKC isoforms, or PKC-activated enzymes such as p90 RSK and PKD (26 -32). Ser 43 /Ser 45 and Thr 144 are traditionally viewed as sites for phosphorylation by PKC. Although recent studies show that Thr 144 is a good in vitro substrate for PKC␤ and Tyr-phosphorylated PKC␦ (27,28), PKCs with cTnI Ser 43 /Ser 45 kinase activity have not been identified. In fact, several laboratories have reported that Ser 43 / Ser 45 is a relatively poor in vitro substrate for many PKC isoforms. Nevertheless, WT cTnI replacement with a cTnI mutant harboring phosphomimetic substitutions at Ser 43 , Ser 45 , and Thr 144 leads to pronounced changes in contractile function in transgenic mice, suggesting that myofilaments may be very sensitive to PKC-dependent changes in cTnI phosphorylation (33).
cTnT phosphorylation at Thr 206 (by PKC or Raf-1 but not by PKA or PKG) (34, 35) results in decreased maximum force and myofilament Ca 2ϩ sensitivity. PKC and ASK-1 also phosphor-ylate cTnT at other sites (Table 1) (36,37). Because phosphorylation (or phosphomimetic substitutions) at sites other than Thr 206 does not lead to gross changes in mechanical function, some have speculated that other post-translational modifications (PTMs) on cTnT might regulate its scaffolding function (particularly because enzymes such as PKA and PKG phosphorylate cTnI only when anchored to cTnT) (38,80). cMyBP-C phosphorylation at Ser 273 , Ser 282 , and Ser 302 is generally attributed to PKA and viewed as a mechanism that decreases thick-thin filament interactions and increases force generation (39). There is recent evidence that Ser 302 (but not Ser 273 or Ser 282 ) also is phosphorylated by PKC⑀, PKC␦, and PKD and that Ser 302 phosphorylation alone may be sufficient to regulate contractile function (32,40).
The titin elastic region (consisting of serially linked immunoglobulin-like domains, the N2-B element, and a PEVK domain) is phosphorylated by PKA or PKG. PKA-or PKG-dependent phosphorylation of human titin at Ser 469 in the N2-B element decreases the passive tension of titin (41,42). PKG (but not PKA) also phosphorylates titin at other sites that do not influence its mechanical properties but could in theory control docking interactions on the titin scaffold (42). PKC␣ phosphorylates titin at two highly conserved sites in the PEVK region (Ser 11878 and Ser 12022 ), leading to an increase in the passive tension of titin (an effect opposite to the actions of PKA or PKG) (43). ROS-dependent Regulation of Sarcomeric Protein Phosphorylation-Oxidative stress typically increases protein phosphorylation by inhibiting protein phosphatases and stimulating protein kinases. The invariant Cys in the active sites of proteintyrosine phosphatases is highly susceptible to ROS-dependent inactivation (44). ROS-dependent inactivation of protein-tyrosine phosphatases is sufficient to increase protein Tyr phosphorylation. However, ROS-dependent increases in Src activity also are detected in cardiomyocytes and some other cell types (45)(46)(47). Changes in protein Tyr phosphorylation typically influence cell growth, survival, and differentiation rather than sarcomeric protein phosphorylation. However, ROS-dependent changes in protein Tyr phosphorylation indirectly influence sarcomeric protein phosphorylation by activating Ser/Thr kinases such as PKC and PKD (see below).
Oxidative stress also inactivates the Ser/Thr phosphatase calcineurin (or protein phosphatase 2B) (48). Redox regulation of calcineurin may impact transcriptional programs that regulate cardiac hypertrophy, but direct effects on the sarcomere are unlikely because myofilament protein phosphorylation is not disordered in transgenic mouse models of altered calcineurin activity (49). Rather, myofilament protein dephosphorylation is generally attributed to PP1 (protein phosphatase 1) or PP2A. Most studies have focused on PP2A, which co-immunoprecipitates with cTnT and cTnI, co-localizes to the Z-disc with ROSsensitive enzymes (PKC⑀, PKC, PAK-1, and p38 MAPK), and contributes to dynamic changes in cTnI and cMyBP-C phosphorylation (50 -53). The role of PP1-dependent sarcomeric protein dephosphorylation seems more tenuous because transgenic mouse models of altered PP1 activity display changes in phospholamban (but not cTnI) phosphorylation (54,55). In fact, the assumption that ROS inactivate PP1 and PP2A is not supported by in vitro biochemical studies or cell-based studies, which show that oxidative stress increases PP1 and/or PP2A activity (56 -59). This may be via an indirect mechanism because several ROS-activated kinases (PKC, PAK-1, and p38 MAPK) increase PP1 and/or PP2A activity (50,52,60). The functional consequences of ROS-dependent changes in PP1 or PP2A activity are difficult to predict because most studies have scrutinized kinase (and not phosphatase)-mediated mechanisms that regulate sarcomeric protein phosphorylation. This minireview focuses on the ROS-dependent mechanisms that regulate the various kinases that phosphorylate sarcomeric proteins.
ASK-1-ASK-1 (apoptosis signal-regulating kinase-1) is a ROS-regulated stress-activated MAPK kinase kinase that is abundant in cardiomyocytes and acts as a redox sensor to activate effector pathways that regulate apoptotic/necrotic cell death (61)(62)(63)(64)(65). ASK-1 contains a central kinase domain flanked by N-and C-terminal regulatory domains. In resting cells, ASK-1 activity is maintained at low basal levels as a result of inhibitory interactions between the Ser 967 -phosphorylated C-terminal regulatory domain and 14-3-3 proteins and between the N-terminal regulatory domain and reduced thioredoxin-1 (Trx1). Oxidation of Trx1 leads to the dissociation of ASK-1⅐Trx1 complexes. Oxidative stress also leads to the dissociation of the ASK-1⅐14-3-3 complex due to ASK-1 Ser 967 dephosphorylation (presumably due to the activation of a ROS-sensitive phosphatase) and/or 14-3-3 phosphorylation by a ROS-regulated kinase (PKD, Mst (mammalian sterile 20-like kinase) family kinases, or the catalytic fragment of PKC␦) (66 -68). Once released from these inhibitory constraints, ASK-1 is activated as a result of oligomerization and activation loop (Ser 845 ) autophosphorylation.
ASK-1 is activated by H 2 O 2 or agonists for G-protein-coupled receptors that increase ROS accumulation in vitro in cardiomyocyte cultures and by pressure overload or myocardial infarction in vivo in the intact heart (37,68,69). This increase in ASK-1 activity contributes to ventricular remodeling by activating pathways involving JNK or NF-B (69). Recent evidence indicates that ASK-1 co-localizes with sarcomeric structures, where it phosphorylates cTnT at Thr 197 and Ser 201 (37). Overexpression of the constitutively active ASK-1⌬N deletion mutant leads to increased cTnT phosphorylation and decreased fractional shortening in cardiomyocyte cultures (37). However, the role of cTnT phosphorylation in the cardiodepressant actions of ASK-1 remains uncertain because (a) cTnT phosphorylation at Thr 206 (not Thr 197 or Ser 201 ) has been implicated in the control of thin filament function, and (b) ASK-1⌬N overexpression also decreases the Ca 2ϩ transient amplitude (34,37).
Mst1-Mst1 is another ROS-activated Ser/Thr kinase that activates p38 MAPK/JNK and caspase-dependent mechanisms that amplify apoptosis (70,71). Mst1 also phosphorylates cTnI and cTnT; cTnT phosphorylation is detected only when Mst1 is anchored to cTnI. Mst1-dependent cTnI phosphorylation has been mapped to Thr 144 as well as novel sites (Thr 32 , Thr 52 , and Thr 130 ) that may influence the conformation of cTnI and its binding affinity for cTnT and cTnC (71).
PKA-PKA holoenzyme is a heterotetramer composed of two catalytic (C) subunits kept in an inactive conformation by two cAMP-binding regulatory (R) subunits. PKA activation is generally attributed to the ␤-AR/cAMP pathway; cAMP binding to the R subunit frees the C subunit to phosphorylate target substrates. However, ROS-dependent mechanisms that regulate PKA also influence myofilament protein phosphorylation.
PKA holoenzymes are classified as type I or II based upon the identity of their R subunit (RI or RII) that targets PKA to different subcellular compartments through interaction with PKAanchoring proteins. Both PKA RI and RII subunits are ubiquitously expressed in cardiac myocytes and were shown to interact with myofilaments (38). The presence of two distinct PKA isoforms anchored at the sarcomeres could impart a more dynamic modulation of myofilament function in response to varying cAMP levels, and the combined regulation could provide a more refined physiological response.
Oxidative modifications of the RI subunit (at a pair of reactive cysteines not found in the RII subunit) result in the formation of interprotein disulfide dimers that display increased affinity for ␣-MHC, translocate to the myofibrillar fraction, and phosphorylate cTnI and cMyBP-C (72). This ROS-dependent (cAMP-independent) mechanism involving type I PKA has been linked to an increase in cardiac contractility.
Oxidative modification of the C subunit at Cys 199 (one of two highly conserved Cys residues in the active sites of PKA and other Ser/Thr kinases such as PKC, PKG, and AKT) has the opposite effect to decrease PKA activity (73). Mutagenesis studies suggest that Cys 199 does not directly influence catalytic activity. Rather, the thiol modification at Cys 199 indirectly decreases catalytic activity by rendering the C subunit susceptible to phosphatase-mediated dephosphorylation at Thr 197 , a stable PTM at an adjacent site in the activation loop that is required for kinase activity (74). Cell-based studies suggest that similar thiol modifications may decrease activation loop phosphorylation and inactivate related kinases (such as PKC) (74).
PKG-PKG is activated by autocrine/paracrine stimuli that increase NO and cGMP. Mammalian PKGs are homodimers of identical subunits; each PKG monomer contains an N-terminal regulatory domain (consisting of an autoinhibitory pseudosubstrate sequence, tandem cGMP-binding cassettes, and a leucine zipper dimerization domain) and a C-terminal catalytic domain. An autoinhibitory interaction between the pseudosubstrate domain and the catalytic pocket maintains PKG in an inactive/resting state; cGMP binding induces a conformational change that relieves autoinhibition and permits activation.
Three molecular forms of mammalian PKG have been identified: PKGI␣ and PKGI␤ arise through alternative mRNA splicing and differ only at their extreme N-terminal ϳ100 amino acids (the dimerization domain), whereas PKGII is the product of a different gene locus. Because PKGI is the major isoform in cardiomyocytes, it is the focus of this discussion (75,76). The distinct N-terminal homodimerization domains of PKGI␣ and PKGI␤ underlie isoform-specific interactions with docking proteins and cell substrates. Elements within the dimerization domain influence the kinetics of cGMP binding and PKG activation; PKGI␣ and PKGI␤ have identical cGMPbinding cassettes, but PKGI␣ binds cGMP with 10-fold higher affinity than PKGI␤ (77,78). Native PKGI has traditionally been viewed as a constitutive dimer. However, a recent study challenged this assumption and showed that PKGI␣ dimerization is a ROS-regulated mechanism; this study concluded that the PKGI␣ dimers identified in previous studies are artifacts of oxidation during sample preparation (79). PKGI␣ dimerization results from disulfide bond formation between reactive Cys 42 residues that abut in the enzyme homodimer; PKGI␤ does not contain a reactive Cys at this position and is not ROS-sensitive. The PKGI␣ dimers that accumulate during oxidative stress display a high level of cGMP-independent catalytic activity (79). Moreover, whereas cGMP activates PKGI␣ by increasing its maximum velocity (V max and not the K m for substrate), ROSactivated (disulfide-linked) PKGI␣ dimers display a marked (Ͼ10-fold) increase in K m for substrate. Some have speculated that this ROS-induced increase in PKGI␣ affinity for substrate underlies the ROS-induced change in PKGI␣ subcellular compartmentation in smooth muscle cells; ROS-activated/disulfide-linked PKGI␣ translocates to membrane and myofilament fractions (which contain functionally important PKG substrates). Future studies that examine the subcellular com-partmentationandsubstratesofROS-activatedPKGI␣incardiomyocytes may be quite revealing, given evidence that a PKGI␣docking interaction (via its homodimerization domain) with cTnT is required for the rapid/efficient phosphorylation of cTnI (80). PKG-targeting mechanisms may be critical for substrate phosphorylation in cardiomyocytes, where PKG expres-sion is quite low (ϳ10-fold lower compared with PKA expression) (81), and the V max for cTnI phosphorylation by PKG is 12-fold lower than that for cTnI phosphorylation by PKA (82).
PKCs-PKCs are Ser/Thr kinases that are activated by growth factor-dependent pathways that mobilize Ca 2ϩ and promote diacylglycerol (DAG) accumulation. PKC isoforms contain a highly conserved C-terminal catalytic domain and are subdivided into three classes based on differences in their N-terminal regulatory domains. The regulatory domains of conventional or Ca 2ϩ -sensitive PKCs (␣, ␤I/␤II, and ␥) and novel PKCs (in cardiomyocytes, PKC␦ and PKC⑀) contain a C1 domain (consisting of tandem Cys-rich sequences) that binds lipid cofactors such as DAG and phorbol 12-myristate 13-acetate (PMA). Atypical PKCs ( and i/l) contain an abbreviated C1 domain (with only one Cys-rich motif) that binds phosphatidylinositol 1,4,5-trisphosphate or ceramide but not DAG or PMA. PKC activation is generally attributed to stimuli that promote DAG accumulation and anchor the enzyme in its active conformation to membranes. However, ROS-dependent mechanisms that activate PKCs by oxidizing C1 domain Cys residues (which disrupts autoinhibitory intramolecular constraints) also have been identified (83,84).
A ROS-dependent mechanism involving Tyr phosphorylation by Src specifically activates PKC␦ (and not other PKC isoforms). We recently demonstrated that Src phosphorylates PKC␦ at Tyr 311 in vitro and in H 2 O 2 -treated cardiomyocytes and that Tyr 311 -phosphorylated PKC␦ accumulates in the soluble fraction as a constitutively active lipid-independent enzyme; this form of the enzyme is poised to phosphorylate proteins in the sarcomere, not just on lipid membranes (85). We showed that 1) allosterically activated PKC␦ phosphorylates cTnI at Ser 23 /Ser 24 , 2) Tyr 311 -phosphorylated PKC␦ phosphorylates cTnI at Ser 23 /Ser 24 and Thr 144 , and 3) a PKC␦ mutant harboring a Y311F substitution selectively phosphorylates cTnI at Ser 23 /Ser 24 but not Thr 144 (28). Functional studies in detergent-skinned cardiomyocytes show that allosterically activated PKC␦ depresses tension at submaximum but not maximum Ca 2ϩ , as predicted for cTnI Ser 23 /Ser 24 phosphorylation. Src-phosphorylated PKC␦ (which phosphorylates cTnI at both Ser 23 /Ser 24 and Thr 144 ) depresses maximum tension and cross-bridge kinetics; under these conditions, the effect of cTnI Thr 144 phosphorylation predominates.
Stimulus-specific differences in PKC␦ phosphorylation of sarcomeric proteins have been identified in H 2 O 2 -and PMAtreated cardiomyocytes (86). Here, PMA and H 2 O 2 increase cTnI and cMyBP-C phosphorylation via a PKC-dependent mechanism, but only the H 2 O 2 -dependent increase in cTnI and cMyBP-C phosphorylation requires Src, presumably reflecting a role for Tyr-phosphorylated PKC␦. PMA and H 2 O 2 elicit distinct cTnI and cMyBP-C phosphorylation patterns in PKC␦-overexpressing cardiomyocytes, providing further evidence that stimulus-specific differences in the PTM of PKC␦ impact its enzymology and PKC␦-mediated sarcomeric protein phosphorylation. In a more general sense, these results caution against extrapolations regarding the cellular actions of ROS-activated enzymes based upon studies that examined myofilament protein regulation during signaling by G-protein-coupled receptors. Stimulus-specific differ-ences in the subcellular compartmentalization, binding partners, and enzymology of many signaling enzymes may impact sarcomeric protein phosphorylation.
PKD-PKD consists of a family of Ser/Thr kinases that exert important cardiac actions (87). PKDs contain an N-terminal regulatory C1 domain (which targets the enzyme to DAG-or phorbol ester-enriched membranes), an autoinhibitory pleckstrin homology domain, and a C-terminal kinase domain. Agonists that promote DAG accumulation activate PKD via a novel PKC-dependent pathway that leads to PKD phosphorylation at Ser 744 /Ser 748 in the activation loop. Ser 744 /Ser 748 -phosphorylated PKD then autophosphorylates at Ser 916 , and it displays a high level of activity toward heterologous substrates such as cTnI and cMyBP-C (30,31). PKD decreases myofilament Ca 2ϩ sensitivity by increasing cTnI Ser 23 /Ser 24 phosphorylation (similar to the actions of PKA). PKD also accelerates crossbridge cycle kinetics. This effect does not require cTnI Ser 23 / Ser 24 phosphorylation (because it is preserved in cardiomyocytes that express cTnI S23A/S24A in place of WT cTnI); it has been attributed to cMyBP-C phosphorylation at Ser 302 , the only site in cMyBP-C that is targeted by PKD (32).
PKD is activated during oxidative stress via a mechanism involving the ROS-activated form of PKC␦ and the tyrosine kinases Src and c-Abl; Src and c-Abl are not required for growth factor-dependent PKD activation (88,89). The current model holds that c-Abl phosphorylates PKD at Tyr 463 (in the pleckstrin homology domain), leading to a conformational change that permits Src-dependent PKD phosphorylation at Tyr 95 (90). This generates a consensus binding motif for the C2 domain of PKC␦, which activates PKD. ROS-activated PKD phosphorylates CREB (cAMP-responsive element-binding protein) in cardiomyocytes (91) and controls ASK-1, NF-B, and apoptotic cell death in other cell types (66,92). A potential role for PKD in redox regulation of sarcomeric protein phosphorylation has not been considered.

Conclusions
This minireview has summarized recent studies that identify ROS-induced PTMs of sarcomeric proteins that lead to contractile dysfunction (Fig. 1). Studies to date suggest that direct oxidative modifications of sarcomeric proteins lead to a decrease in force generation, whereas sarcomeric protein phosphorylation by ROS-activated enzymes decrease myofilament Ca 2ϩ sensitivity. However, these conclusions are based largely on experiments that rely on reductionist approaches to resolve the functional consequences of myofilament protein phosphorylation by a single ROS-activated enzyme or ROS-dependent PTMs of a single contractile protein. Extrapolations to the in vivo context must be made with caution for several reasons. . At high Ca 2ϩ , Ca 2ϩ binding to the regulatory site of cTnC induces a conformational change in cTnI causing disinhibition of actin. This conformational change is transmitted to cTnT, which moves Tm and exposes a weak myosin-binding site on actin. Myosin binding to actin enhances activation by pushing Tm farther away, leading to strong cross-bridge formation. This activation mechanism is regulated in a highly specific manner by phosphorylation reactions on individual sarcomeric proteins. Myofilament activation and contractile function also are altered during oxidative stress due to direct oxidative modifications (yellow stars) of specific sites on contractile proteins or ROS-induced changes in the activity of kinases or phosphatases that regulate sarcomeric protein phosphorylation (P). MLC, myosin light chain; mN, millinewtons.
1) An inherent assumption of studies in reductionist models is that kinases act in a stereotypical fashion to phosphorylate a fixed set of sarcomeric proteins (or consensus phosphorylation sites within a given sarcomeric protein). The stimulus-specific differences in cTnI phosphorylation by PKC␦ identified in our studies emphasize that standard in vitro approaches may not necessarily incorporate protocols that capture all regulatory phosphorylations that control in vivo enzyme activity (or substrate specificity). This approach also will not detect changes in phosphorylation due to ROS-dependent modifications of myofilament proteins that alter the accessibility of phosphorylation sites on individual substrate proteins.
2) ROS-dependent modifications of sarcomeric proteins do not occur in isolation. Rather, ROS-activated kinases typically phosphorylate multiple proteins in the sarcomere; many ROS-activated enzymes also sit upstream in signaling cascades that regulate effectors with Ser/Thr kinase activity. For example, PKC isoforms activate PKD, p90 RSK , and Raf-1; ROS-activated pathways involving PKD, PKC, or Mst phosphorylate 14-3-3 proteins, leading to decreased inhibitory interactions with ASK-1. Progress in understanding ROS-induced changes in contractile performance in vivo must consider the ensemble actions of multiple ROS-activated enzymes on multiple sarcomeric proteins.
3) Oxidative stress may induce a spectrum of responses that vary according to the identity of the free radical species, the location of the ROS signal, and/or the level of oxidative stress. This issue has been addressed directly in studies of redox regulation of PKA, where ROS shift the balance of cellular kinase versus phosphatase activity in a dose-dependent manner. Low levels of oxidant stress amplify PKA responses by inactivating phosphatases that counteract PKA-dependent phosphorylations; high levels of oxidative stress inactivate PKA (presumably due to direct Cys oxidation of the C subunit) and decrease substrate phosphorylation (93).
4) The importance of a particular ROS-dependent protein phosphorylation or oxidative modification may be context-dependent; it may be influenced by PTMs elsewhere in that particular protein or in other proteins in the sarcomere. For example, cTnI Thr 144 phosphorylation alone has little effect on force generation or Ca 2ϩ sensitivity, but Thr 144 phosphorylation prevents Ca 2ϩ desensitization due to cTnI Ser 23 /Ser 24 phosphorylation (i.e. Thr 144 phosphorylation becomes functionally important in a Ser 23 /Ser 24 -phosphorylated background) (94). Similarly, the functional consequences of PKC-dependent cTnI Ser 43 /Ser 45 /Thr 144 phosphorylation are amplified during acidosis (95). PTMs of sarcomeric proteins that are inert under normal physiological conditions and become functionally important only in a pathologic microenvironment may represent novel therapeutic targets.