Inhibition of Angiotensin II-Induced Cardiac Hypertrophy and Associated Ventricular Arrhythmias by a p21 Activated Kinase 1 Bioactive Peptide

Cardiac hypertrophy increases the risk of morbidity and mortality of cardiovascular disease and thus inhibiting such hypertrophy is beneficial. In the present study, we explored the effect of a bioactive peptide (PAP) on angiotensin II (Ang II)-induced hypertrophy and associated ventricular arrhythmias in in vitro and in vivo models. PAP enhances p21 activated kinase 1 (Pak1) activity by increasing the level of phosphorylated Pak1 in cultured neonatal rat ventricular myocytes (NRVMs). Such PAP-induced Pak1 activation is associated with a significant reduction of Ang II-induced hypertrophy in NRVMs and C57BL/6 mice, in vitro and in vivo, respectively. Furthermore, PAP antagonizes ventricular arrhythmias associated with Ang II-induced hypertrophy in mice. Its antiarrhythmic effect is likely to be involved in multiple mechanisms to affect both substrate and trigger of ventricular arrhythmogenesis. Thus our results suggest that Pak1 activation achieved by specific bioactive peptide represents a potential novel therapeutic strategy for cardiac hypertrophy and associated ventricular arrhythmias.     

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

We review development of evidence and current perceptions of the multiple and significant functions of cardiac troponin I in regulation and modulation of cardiac function. Our emphasis is on the unique structure function relations of the cardiac isoform of troponin I, especially regions containing sites of phosphorylation. The data indicate that modifications of specific regions cardiac troponin I by phosphorylations either promote or reduce cardiac contractility. Thus, a homeostatic balance in these phosphorylations is an important aspect of control of cardiac function. A new concept is the idea that the homeostatic mechanisms may involve modifications of intra-molecular interactions in cardiac troponin I.     

Tyrosine phosphorylation modifies protein kinase C delta-dependent phosphorylation of cardiac troponin I

Our study identifies tyrosine phosphorylation as a novel protein kinase Cdelta (PKCdelta) activation mechanism that modifies PKCdelta-dependent phosphorylation of cardiac troponin I (cTnI), a myofilament regulatory protein. PKCdelta phosphorylates cTnI at Ser23/Ser24 when activated by lipid cofactors; Src phosphorylates PKCdelta at Tyr311 and Tyr332 leading to enhanced PKCdelta autophosphorylation at Thr505 (its activation loop) and PKCdelta-dependent cTnI phosphorylation at both Ser23/Ser24 and Thr144. The Src-dependent acquisition of cTnI-Thr144 kinase activity is abrogated by Y311F or T505A substitutions. Treatment of detergent-extracted single cardiomyocytes with lipid-activated PKCdelta induces depressed tension at submaximum but not maximum [Ca2+] as expected for cTnI-Ser23/Ser24 phosphorylation. Treatment of myocytes with Src-activated PKCdelta leads to depressed maximum tension and cross-bridge kinetics, attributable to a dominant effect of cTnI-Thr144 phosphorylation. Our data implicate PKCdelta-Tyr311/Thr505 phosphorylation as dynamically regulated modifications that alter PKCdelta enzymology and allow for stimulus-specific control of cardiac mechanics during growth factor stimulation and oxidative stress.     

Regulation of cardiac excitation and contraction by p21 activated kinase-1

Cardiac excitation and contraction are regulated by a variety of signaling molecules. Central to the regulatory scheme are protein kinases and phosphatases that carry out reversible phosphorylation of different effectors. The process of β-adrenergic stimulation mediated by cAMP dependent protein kinase (PKA) forms a well-known pathway considered as the most significant control mechanism in excitation and contraction as well as many other regulatory mechanisms in cardiac function. However, although dephosphorylation pathways are critical to these regulatory processes, signaling to phosphatases is relatively poorly understood. Emerging evidence indicates that regulation of phosphatases, which dampen the effect of β-adrenergic stimulation, is also important. We review here functional studies of p21 activated kinase-1 (Pak1) and its potential role as an upstream signal for protein phosphatase PP2A in the heart. Pak1 is a serine/threonine protein kinase directly activated by the small GTPases Cdc42 and Rac1. Pak1 is highly expressed in different regions of the heart and modulates the activities of ion channels, sarcomeric proteins, and other phosphoproteins through up-regulation of PP2A activity. Coordination of Pak1 and PP2A activities is not only potentially involved in regulation of normal cardiac function, but is likely to be important in patho-physiological conditions.     

Calcium regulation of cardiac myofibrillar activation: Effects of MgATP

In 2 mM MgATP, 0.08 ionic strength and 1 mM free Mg⁺⁺ cardiac myofibrils bound 3.5 nmoles Ca/mg protein at maximal ATPase activation. Significant amounts of Ca were also bound to cardiac myosin with these same conditions. By subtraction of this myosin-bound Ca we obtained an estimate of 4 moles Ca bound per mole of myofibrillar troponin at maximal ATPase. We found, however, that Ca activation of myofibrillar ATPase could be estimated assuming that only two of troponin's Ca-binding sites are engaged in regulation of crossbridge activity. Increase in MgATP from 0.3 to 5.0 mM raised the free Ca, giving half-maximal isometric tension or ATPase. Although part of this shift is most probably due to changes in the number of rigor (nucleotidefree) actin-myosin linkages, the rightward shift of the free Ca⁺⁺-activation relation with increase in MgATP from 2 to 5 mM appears to be due to effects of active (nucleotide-containing) actin-myosin linkages.     

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity

When the (pH_i) surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca²⁺-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca²⁺-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca²⁺. This effect of acidic pH depends not only on a direct effect of protons on Ca²⁺-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using flourescent probes that report Ca²⁺-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca²⁺-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca²⁺-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe postioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca²⁺-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca²⁺-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic (pH_i) appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

A Novel,In-solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.Modulation of sarcomeric protein function ensures that the heart ejects blood against a systemic resistance to supply peripheral tissues with oxygen and nutrients and to remove carbon dioxide and wastes. Muscle contraction occurs by myosin motors of the thick filament reacting with actin and propelling thin filaments toward the center of the sarcomere. In cardiac muscle, contraction is activated by a release of sarcoplasmic reticular Ca²⁺ that binds to troponin C, which is positioned on the actin thin filament. It was long assumed that regulation of Ca²⁺ levels and cycling were the sole avenue by which contraction was modulated. It is now evident, however, that post-translational modifications (PTMs)¹ are important in regulating the function of ejecting ventricles as mechanisms downstream of Ca²⁺ fluxes at the level of the sarcomere appear to dominate ejection and sustain ventricular elastance during systole (1). Intra- and intermolecular interactions of sarcomeric thick and thin filament proteins are modifiable by PTMs, and it has been demonstrated that the intensity and dynamics of contraction and relaxation can be finely tuned via PTMs, particularly those of thin filament proteins (2, 3). Mechanisms by which PTMs tune molecular interactions of the thick filament are less well understood. Extrapolating from molecular mechanisms to the cardiac disorder requires an understanding of the endogenous charge state of proteins that can be governed by phosphorylation, acetylation, oxidation, and other post-translational modifications.The thick filament is composed mainly of myosin, a motor protein that interacts with neighboring proteins, including the essential (ELC) and regulatory light chains (RLC), and myosin-binding protein-C (MyBP-C) (Fig. 1). RLC binds the S1-S2 lever arm of the myosin motor and is optimally positioned at the fulcrum to modulate interactions between the globular myosin heavy chain (MHC) head, the coiled coil light meromyosin thick filament backbone, and the neighboring MyBP-C (Fig. 1). Cardiac RLC is phosphorylated in large part by myosin light chain kinase (4), which induces an increase in tension at submaximally activating levels of Ca²⁺ (5–7) in skinned cardiac fibers bathed in exogenous myosin light chain kinase. Furthermore, mice expressing a transgenic cardiac RLC with alanine residues in place of N-terminal serines have significantly impaired systolic kinetics in vivo (8). Separation of endogenous RLC by two-dimensional electrophoresis (2DE) clearly illustrates multiple phosphorylated spots of RLC (8). The most abundant phosphorylated spot of RLC was found to represent phosphorylation at Ser-15 (9). The second, more acidic phosphorylated spot is 3–5 times lower in abundance and has been difficult to capture using in-gel digestion and mass spectrometry likely due to protein loss during in-gel digestion combined with poor ionization potential of the phosphopeptide. A possible approach to overcome this limitation is the use of a gel-free (in-solution) technique for separating charged species of RLC that is designed to minimize loss and increase sample load.Open in a separate windowFig. 1.Schematic illustrating cardiac sarcomeric proteins. The myofibrillar lattice is composed mainly of actin thin filaments and myosin thick filaments, each bound to regulatory proteins. Activation of cardiac contraction during systole proceeds by calcium binding to troponin C (TnC), which induces conformational changes and altered interactions among troponin I (TnI), troponin T (TnT), and tropomyosin (Tm), resulting in the removal of steric inhibition over the myosin binding site on actin. An activated thin filament allows the binding of myosin heads, which then propel the actin filaments toward the center of the sarcomere. The thick filament is composed mainly of MHC, which binds two light chains, ELC and RLC, and associates with MyBP-C in the hinge and light meromyosin (LMM) regions.Biochemically, proteins comprising the thick filament present unique purification challenges in part due to their large size (MHC, 223 kDa; MyBP-C, 150 kDa), producing difficulties in the isolation of intact proteins (10–12). In contrast, non-covalently bound ELC and RLC (i.e. the light chains of myosin) are more amenable to separation using standard biochemical methods due in part to their moderate sizes (22.4 and 18.9 kDa, respectively); however, because purification methods for either MHC or ELC/RLC are not time- and cost-effective, novel strategies for preparing/enriching these proteins in a single step are warranted.When the research objective is to identify all proteins in a sample, a general, unbiased method is appropriate as two peptide ions with high quality, comprehensive MS/MS spectra are sufficient to reliably identify a protein. However, in targeted proteomics approaches where the objective is to distinguish functionally relevant charge variants (isoforms or post-translationally modified proteins) in a smaller subset of proteins, cleaving proteins into constituent peptides results in a loss of a significant amount of information and produces protein inference problems when assigning identities to modified versions of the same protein (13). A reliable and advantageous strategy to combat the issue of protein inference would be to discriminate charged variants at the intact protein level. Reverse-phase (RP) HPLC and OFFGEL electrophoresis (OGE) are two solution-based separation methods that exploit hydrophobicity and isoelectric point, respectively. Both techniques have been optimized for discriminatory separation at the peptide level (14, 15) but in the past have been underutilized in proteomics work flows for separation of intact proteins.We report here a rapid solution-based method for purifying endogenous sarcomeric proteins, allowing for the enrichment and identification of the low abundance phosphospecies of cardiac RLC. The approach uses a tandem OGE/HPLC work flow that discriminately separates RLC at the intact protein level in a quantifiable manner. The novelty and strengths of this method are that 1) milligram quantities of sarcomeric subproteome are focused with resolution qualitatively similar to that of 2DE, 2) quantifiable separation of post-translationally modified variants is achieved at the whole protein level, 3) high sequence coverage of the protein under study, RLC, is achieved due to the substantial enrichment of proteins, and 4) separation of high molecular weight cardiac thick filament proteins can be achieved while maintaining proteins in solution in their intact forms. This work flow was used to identify a novel doubly charged phosphospecies of mouse RLC phosphorylated at adjacent Ser-14 and Ser-15. Additionally, we successfully used our method for identifying and quantifying post-translational modifications of RLC occurring in human heart failure, thus demonstrating clinical utility for future studies.     

Increased Ca2+ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I

To understand the molecular mechanisms whereby cardiomyopathy-related cardiac troponin I (cTnI) mutations affect myofilament activity, we have investigated the Ca2+ binding properties of various assemblies of the regulatory components that contain one of the cardiomyopahty-related mutant cTnI. Acto-S1 ATPase activities in reconstituted systems were also determined. We investigated R145G and R145W mutations from the inhibitory region and D190H and R192H mutations from the second actin-tropomyosin-binding site. Each of the four mutations sensitized the acto-S1 ATPase to Ca2+. Whereas the mutations from the inhibitory region increased the basal level of ATPase activity, those from the second actin-tropomyosin-binding site did not. The effects on the Ca2+ binding properties of the troponin ternary complex and the troponin-tropomyosin complex with one of four mutations were either desensitization or no effect compared with those with wild-type cTnI. All of the mutations, however, affected the Ca2+ sensitivities of the reconstituted thin filaments in the same direction as the acto-S1 ATPase activity. Also the thin filaments with one of the mutant cTnIs bound Ca2+ with less cooperativity compared with those with wild-type cTnI. These data indicate that the mutations found in the inhibitory region and those from the second actin-tropomyosin site shift the equilibrium of the states of the thin filaments differently. Moreover, the increased Ca2+ bound to myofilaments containing the mutant cTnIs may be an important factor in triggered arrhythmias associated with the cardiomyopathy.     

Sub-proteomic fractionation,iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1 h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of < 5%. Compared to no fractionation prior to LC–MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four-fold. We found that approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.