S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding.

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF)-the most common sustained cardiac arrhythmia-increases thromboembolic stroke risk 5-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C (protein phosphatase 1 regulatory subunit 12C)-the PP1 (protein phosphatase 1) regulatory subunit targeting MLC2a (atrial myosin light chain 2)-causes hypophosphorylation of MLC2a and results in atrial hypocontractility. METHODS: Right atrial appendage tissues were isolated from human patients with AF versus sinus rhythm controls. Western blots, coimmunoprecipitation, and phosphorylation studies were performed to examine how the PP1c (PP1 catalytic subunit)-PPP1R12C interaction causes MLC2a dephosphorylation. In vitro studies of pharmacological MRCK (myotonic dystrophy kinase-related Cdc42-binding kinase) inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with electrophysiology studies. RESULTS: In human patients with AF, PPP1R12C expression was increased 2-fold versus sinus rhythm controls (P=2.0×10-2; n=12 and 12 in each group) with >40% reduction in MLC2a phosphorylation (P=1.4×10-6; n=12 and 12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=2.9×10-2 and 6.7×10-3, respectively; n=8 and 8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a and dephosphorylation of MLC2a. Mice treated with lentiviral PPP1R12C vector demonstrated a 150% increase in left atrial size versus controls (P=5.0×10-6; n=12, 8, and 12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in mice treated with lentiviral PPP1R12C vector was significantly higher than in controls (P=1.8×10-2 and 4.1×10-2, respectively; n=6, 6, and 5). CONCLUSIONS: Patients with AF exhibit increased levels of PPP1R12C protein compared with controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments.

We tested the hypothesis that isoform shifts in sarcomeres of the immature heart modify the effect of cardiac myosin-directed sarcomere inhibitors and activators. Omecamtiv mecarbil (OM) activates tension and is in clinical trials for the treatment of adult acute and chronic heart failure. Mavacamten (Mava) inhibits tension and is in clinical trials to relieve hypercontractility and outflow obstruction in advanced genetic hypertrophic cardiomyopathy (HCM), which is often linked to mutations in sarcomeric proteins. To address the effect of these agents in developing sarcomeres, we isolated heart fiber bundles, extracted membranes with Triton X-100, and measured tension developed over a range of Ca2+ concentrations with and without OM or Mava treatment. We made measurements in fiber bundles from hearts of adult nontransgenic (NTG) controls expressing cardiac troponin I (cTnI), and from hearts of transgenic (TG-ssTnI) mice expressing the fetal/neonatal form, slow skeletal troponin I (ssTnI). We also compared fibers from 7- and 14-day-old NTG mice expressing ssTnI and cTnI. These studies were repeated with 7- and 14-day-old transgenic mice (TG-cTnT-R92Q) expressing a mutant form of cardiac troponin T (cTnT) linked to HCM. OM increased Ca2+-sensitivity and decreased cooperative activation in both ssTnI- and cTnI-regulated myofilaments with a similar effect: reducing submaximal tension in immature and mature myofilaments. Although Mava decreased tension similarly in cTnI- and ssTnI-regulated myofilaments controlled either by cTnT or cTnT-R92Q, its effect involved a depressed Ca2+-sensitivity in the mature cTnT-R92 myofilaments. Our data demonstrate an influence of myosin and thin-filament associated proteins on the actions of myosin-directed agents such as OM and Mava. SIGNIFICANCE STATEMENT: The effects of myosin-targeted activators and inhibitors on Ca2+-activated tension in developing cardiac sarcomeres presented here provide novel, ex vivo evidence as to their actions in early-stage cardiac disorders. These studies advance understanding of the molecular mechanisms of these agents, which are important in preclinical studies employing sarcomere Ca2+-response as a screening approach. The data also inform the use of commonly immature cardiac myocytes generated from human-inducible pluripotent stem cells in screening for sarcomere activators and inhibitors.

Truncation of the N-terminus of cardiac troponin I initiates adaptive remodeling of the myocardial proteosome via phosphorylation of mechano-sensitive signaling pathways.

The cardiac isoform of troponin I has a unique N-terminal extension (~ 1-30 amino acids), which contributes to the modulation of cardiac contraction and relaxation. Hearts of various species including humans produce a truncated variant of cardiac troponin I (cTnI-ND) deleting the first ~ 30 amino acids as an adaption in pathophysiological conditions. In this study, we investigated the impact of cTnI-ND chronic expression in transgenic mouse hearts compared to wildtype (WT) controls (biological n = 8 in each group). We aimed to determine the global phosphorylation effects of cTnI-ND on the cardiac proteome, thereby determining the signaling pathways that have an impact on cardiac function. The samples were digested and isobarically labeled and equally mixed for relative quantification via nanoLC-MS/MS. The peptides were then enriched for phospho-peptides and bioinformatic analysis was done with Ingenuity Pathway Analysis (IPA). We found approximately 77% replacement of the endogenous intact cTnI with cTnI-ND in the transgenic mouse hearts with 1674 phospho-proteins and 2971 non-modified proteins. There were 73 significantly altered phospho-proteins; bioinformatic analysis identified the top canonical pathways as associated with integrin, protein kinase A, RhoA, and actin cytoskeleton signaling. Among the 73 phospho-proteins compared to controls cTnI-ND hearts demonstrated a significant decrease in paxillin and YAP1, which are known to play a role in cell mechano-sensing pathways. Our data indicate that cTnI-ND modifications in the sarcomere are sufficient to initiate changes in the phospho-signaling profile that may underly the chronic-adaptive response associated with cTnI cleavage in response to stressors by modifying mechano-sensitive signaling pathways.

Deletion of P21-activated kinase-1 induces age-dependent increased visceral adiposity and cardiac dysfunction in female mice.

It is known that there is an age-related progression in diastolic dysfunction, especially prevalent in postmenopausal women, who develop heart failure with preserved ejection fraction (HFpEF, EF > 50%). Mechanisms and therapies are poorly understood, but there are strong correlations between obesity and HFpEF. We have tested the hypothesis that P21-activated kinase-1 (PAK1) preserves cardiac function and adipose tissue homeostasis during aging in female mice. Previous demonstrations in male mice by our lab that PAK1 activity confers cardio-protection against different stresses formed the rationale for this hypothesis. Our studies compared young (3-6 months) and middle-aged (12-15 months) female and male PAK1 knock-out mice (PAK1-/-) and wild-type (WT) equivalent. Female WT mice exhibited increased cardiac PAK1 abundance during aging. By echocardiography, compared to young WT female mice, middle-aged WT female mice showed enlargement of the left atrium as well as thickening of posterior wall and increased left ventricular mass; however, all contraction and relaxation parameters were preserved during aging. Compared to WT controls, middle-aged PAK1-/- female mice demonstrated worsening of cardiac function involving a greater enlargement of the left atrium, ventricular hypertrophy, and diastolic dysfunction. Moreover, with aging PAK1-/- female mice, unlike male PAK1-/- mice, exhibited increased adiposity with increased accumulation of visceral adipose tissue. Our data provide evidence for the significance of PAK1 signaling as an element in the preservation of cardiac function and adipose tissue homeostasis in females during aging.

A Perspective on Personalized Therapies in Hypertrophic Cardiomyopathy.

ABSTRACT: A dominant mechanism of sudden cardiac death in the young is the progression of maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy. Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. We discuss approaches for personalized treatments that are indicated by recent advanced understanding of the progression.

Tropomyosin pseudo-phosphorylation results in dilated cardiomyopathy.

Phosphorylation of cardiac sarcomeric proteins plays a major role in the regulation of the physiological performance of the heart. Phosphorylation of thin filament proteins, such as troponin I and T, dramatically affects calcium sensitivity of the myofiber and systolic and diastolic functions. Phosphorylation of the regulatory protein tropomyosin (Tpm) results in altered biochemical properties of contraction; however, little is known about the physiological effect of Tpm phosphorylation on cardiac function. To address the in vivo significance of Tpm phosphorylation, here we generated transgenic mouse lines having a phosphomimetic substitution in the phosphorylation site of α-Tpm (S283D). High expression of Tpm S283D variant in one transgenic mouse line resulted in an increased heart:body weight ratio, coupled with a severe dilated cardiomyopathic phenotype resulting in death within 1 month of birth. Moderate Tpm S283D mice expression in other lines caused mild myocyte hypertrophy and fibrosis, did not affect lifespan, and was coupled with decreased expression of extracellular signal-regulated kinase 1/2 kinase signaling. Physiological analysis revealed that the transgenic mice exhibit impaired diastolic function, without changes in systolic performance. Surprisingly, we observed no alterations in calcium sensitivity of the myofibers, cooperativity, or calcium-ATPase activity in the myofibers. Our experiments also disclosed that casein kinase 2 plays an integral role in Tpm phosphorylation. In summary, increased expression of pseudo-phosphorylated Tpm impairs diastolic function in the intact heart, without altering calcium sensitivity or cooperativity of myofibers. Our findings provide the first extensive in vivo assessment of Tpm phosphorylation in the heart and its functional role in cardiac performance.

Interplay of actin, ADP and Mg2+ interactions with striated muscle myosin: Implications of their roles in ATPase.

The effects of Mg2+ on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg2+ inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg2+ were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg2+ bound to a fluorescent ADP analog about five-times tighter than without Mg2+. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg2+. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (Kd) without Mg2+ was more than five-fold smaller than with Mg2+. Further, we examined effects of F-actin, ADP and Mg2+ binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg2+-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg2+ affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.

ß-Arrestin mediates the Frank-Starling mechanism of cardiac contractility.

The Frank-Starling law of the heart is a physiological phenomenon that describes an intrinsic property of heart muscle in which increased cardiac filling leads to enhanced cardiac contractility. Identified more than a century ago, the Frank-Starling relationship is currently known to involve length-dependent enhancement of cardiac myofilament Ca2+ sensitivity. However, the upstream molecular events that link cellular stretch to the length-dependent myofilament Ca2+ sensitivity are poorly understood. Because the angiotensin II type 1 receptor (AT1R) and the multifunctional transducer protein ß-arrestin have been shown to mediate mechanosensitive cellular signaling, we tested the hypothesis that these two proteins are involved in the Frank-Starling mechanism of the heart. Using invasive hemodynamics, we found that mice lacking ß-arrestin 1, ß-arrestin 2, or AT1R were unable to generate a Frank-Starling force in response to changes in cardiac volume. Although wild-type mice pretreated with the conventional AT1R blocker losartan were unable to enhance cardiac contractility with volume loading, treatment with a ß-arrestin-biased AT1R ligand to selectively activate ß-arrestin signaling preserved the Frank-Starling relationship. Importantly, in skinned muscle fiber preparations, we found markedly impaired length-dependent myofilament Ca2+ sensitivity in ß-arrestin 1, ß-arrestin 2, and AT1R knockout mice. Our data reveal ß-arrestin 1, ß-arrestin 2, and AT1R as key regulatory molecules in the Frank-Starling mechanism, which potentially can be targeted therapeutically with ß-arrestin-biased AT1R ligands.

Peroxisome proliferator-activated receptor-α expression induces alterations in cardiac myofilaments in a pressure-overload model of hypertrophy.

Although alterations in fatty acid (FA) metabolism have been shown to have a negative impact on contractility of the hypertrophied heart, the targets of action remain elusive. In this study we compared the function of skinned fiber bundles from transgenic (Tg) mice that overexpress a relatively low level of the peroxisome proliferator-activated receptor α (PPARα), and nontransgenic (NTg) littermates. The mice (NTg-T and Tg-T) were stressed by transverse aortic constriction (TAC) and compared with shams (NTg-S and Tg-S). There was an approximate 4-fold increase in PPARα expression in Tg-S compared with NTg-S, but Tg-T hearts showed the same PPARα expression as NTg-T. Expression of PPARα did not alter the hypertrophic response to TAC but did reduce ejection fraction (EF) in Tg-T hearts compared with other groups. The rate of actomyosin ATP hydrolysis was significantly higher in Tg-S skinned fiber bundles compared with all other groups. Tg-T hearts showed an increase in phosphorylation of specific sites on cardiac myosin binding protein-C (cMyBP-C) and ß-myosin heavy chain isoform. These results advance our understanding of potential signaling to the myofilaments induced by altered FA metabolism under normal and pathological states. We demonstrate that chronic and transient PPARα activation during pathological stress alters myofilament response to Ca2+ through a mechanism that is possibly mediated by MyBP-C phosphorylation and myosin heavy chain isoforms.NEW & NOTEWORTHY Data presented here demonstrate novel signaling to sarcomeric proteins by chronic alterations in fatty acid metabolism induced by PPARα. The mechanism involves modifications of key myofilament regulatory proteins modifying cross-bridge dynamics with differential effects in controls and hearts stressed by pressure overload.

Ceramide-mediated depression in cardiomyocyte contractility through PKC activation and modulation of myofilament protein phosphorylation.

Although ceramide accumulation in the heart is considered a major factor in promoting apoptosis and cardiac disorders, including heart failure, lipotoxicity and ischemia-reperfusion injury, little is known about ceramide's role in mediating changes in contractility. In the present study, we measured the functional consequences of acute exposure of isolated field-stimulated adult rat cardiomyocytes to C6-ceramide. Exogenous ceramide treatment depressed the peak amplitude and the maximal velocity of shortening without altering intracellular calcium levels or kinetics. The inactive ceramide analog C6-dihydroceramide had no effect on myocyte shortening or [Ca(2+)]i transients. Experiments testing a potential role for C6-ceramide-mediated effects on activation of protein kinase C (PKC) demonstrated evidence for signaling through the calcium-independent isoform, PKCε. We employed 2-dimensional electrophoresis and anti-phospho-peptide antibodies to test whether treatment of the cardiomyocytes with C6-ceramide altered myocyte shortening via PKC-dependent phosphorylation of myofilament proteins. Compared to controls, myocytes treated with ceramide exhibited increased phosphorylation of myosin binding protein-C (cMyBP-C), specifically at Ser273 and Ser302, and troponin I (cTnI) at sites apart from Ser23/24, which could be attenuated with PKC inhibition. We conclude that the altered myofilament response to calcium resulting from multiple sites of PKC-dependent phosphorylation contributes to contractile dysfunction that is associated with cardiac diseases in which elevations in ceramides are present.

The ß-arrestin-biased ligand TRV120023 inhibits angiotensin II-induced cardiac hypertrophy while preserving enhanced myofilament response to calcium.

In the present study, we compared the cardioprotective effects of TRV120023, a novel angiotensin II (ANG II) type 1 receptor (AT1R) ligand, which blocks G protein coupling but stimulates ß-arrestin signaling, against treatment with losartan, a conventional AT1R blocker in the treatment of cardiac hypertrophy and regulation of myofilament activity and phosphorylation. Rats were subjected to 3 wk of treatment with saline, ANG II, ANG II + losartan, ANG II + TRV120023, or TRV120023 alone. ANG II induced increased left ventricular mass compared with rats that received ANG II + losartan or ANG II + TRV120023. Compared with saline controls, ANG II induced a significant increase in pCa50 and maximum Ca(2+)-activated myofilament tension but reduced the Hill coefficient (nH). TRV120023 increased maximum tension and pCa50, although to lesser extent than ANG II. In contrast to ANG II, TRV120023 increased nH. Losartan blocked the effects of ANG II on pCa50 and nH and reduced maximum tension below that of saline controls. ANG II + TRV120023 showed responses similar to those of TRV120023 alone; compared with ANG II + losartan, ANG II + TRV120023 preserved maximum tension and increased both pCa50 and cooperativity. Tropomyosin phosphorylation was lower in myofilaments from saline-treated hearts compared with the other groups. Phosphorylation of cardiac troponin I was significantly reduced in ANG II + TRV120023 and TRV120023 groups versus saline controls, and myosin-binding protein C phosphorylation at Ser(282) was unaffected by ANG II or losartan but significantly reduced with TRV120023 treatment compared with all other groups. Our data indicate that TRV120023-related promotion of ß-arrestin signaling and enhanced contractility involves a mechanism promoting the myofilament response to Ca(2+) via altered protein phosphorylation. Selective activation of ß-arrestin-dependent pathways may provide advantages over conventional AT1R blockers.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

Altered coronary artery function, arteriogenesis and endothelial YAP signaling in postnatal hypertrophic cardiomyopathy.

Introduction: Hypertrophic cardiomyopathy (HCM) is a cardiovascular genetic disease caused largely by sarcomere protein mutations. Gaps in our understanding exist as to how maladaptive sarcomeric biophysical signals are transduced to intra- and extracellular compartments leading to HCM progression. To investigate early HCM progression, we focused on the onset of myofilament dysfunction during neonatal development and examined cardiac dynamics, coronary vascular structure and function, and mechano-transduction signaling in mice harboring a thin-filament HCM mutation. Methods: We studied postnatal days 7-28 (P7-P28) in transgenic (TG) TG-cTnT-R92Q and non-transgenic (NTG) mice using skinned fiber mechanics, echocardiography, biochemistry, histology, and immunohistochemistry. Results: At P7, skinned myofiber bundles exhibited an increased Ca2+-sensitivity (pCa50 TG: 5.97 ± 0.04, NTG: 5.84 ± 0.01) resulting from cTnT-R92Q expression on a background of slow skeletal (fetal) troponin I and α/ß myosin heavy chain isoform expression. Despite the transition to adult isoform expressions between P7-P14, the increased Ca2+- sensitivity persisted through P28 with no apparent differences in gross morphology among TG and NTG hearts. At P7 significant diastolic dysfunction was accompanied by coronary flow perturbation (mean diastolic velocity, TG: 222.5 ± 18.81 mm/s, NTG: 338.7 ± 28.07 mm/s) along with localized fibrosis (TG: 4.36% ± 0.44%, NTG: 2.53% ± 0.47%). Increased phosphorylation of phospholamban (PLN) was also evident indicating abnormalities in Ca2+ homeostasis. By P14 there was a decline in arteriolar cross-sectional area along with an expansion of fibrosis (TG: 9.72% ± 0.73%, NTG: 2.72% ± 0.2%). In comparing mechano-transduction signaling in the coronary arteries, we uncovered an increase in endothelial YAP expression with a decrease in its nuclear to cytosolic ratio at P14 in TG hearts, which was reversed by P28. Conclusion: We conclude that those early mechanisms that presage hypertrophic remodeling in HCM include defective biophysical signals within the sarcomere that drive diastolic dysfunction, impacting coronary flow dynamics, defective arteriogenesis and fibrosis. Changes in mechano-transduction signaling between the different cellular compartments contribute to the pathogenesis of HCM.

Myosin Light Chain Dephosphorylation by PPP1R12C Promotes Atrial Hypocontractility in Atrial Fibrillation.

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls ( P =2.0×10 -2 , n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation ( P =1.4×10 -6 , n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF ( P =2.9×10 -2 and 6.7×10 -3 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls ( P =5.0×10 -6 , n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls ( P =1.8×10 -2 and 4.1×10 -2 respectively, n= 6,6,5). Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key determinant of atrial contractility in AF.

Maintenance of adult cardiac function requires the chromatin factor Asxl2.

During development and differentiation, cell type-specific chromatin configurations are set up to facilitate cell type-specific gene expression. Defects in the establishment or the maintenance of the correct chromatin configuration have been associated with diseases ranging from leukemia to muscular dystrophy. The heart expresses many chromatin factors, and we are only beginning to understand their roles in heart development and function. We have previously shown that the chromatin regulator Asxl2 is highly expressed in the murine heart both during development and adulthood. In the absence of Asxl2, there is a significant reduction in trimethylation of histone H3 lysine 27 (H3K27), a histone mark associated with lineage-specific silencing of developmental genes. Here we present evidence that Asxl2 is required for the long-term maintenance of ventricular function and for the maintenance of normal cardiac gene expression. Asxl2(-/-) hearts displayed progressive deterioration of ventricular function. By 10 months of age, there was ~37% reduction in fractional shortening in Asxl2(-/-) hearts compared to wild-type. Analysis of the expression of myofibril proteins suggests that Asxl2 is required for the repression of ß-MHC. Asxl2(-/-) hearts did not exhibit hypertrophy, suggesting that the de-repression of ß-MHC was not the result of hypertrophic response. Instead, Asxl2 and the histone methyltansferase Ezh2 co-localize to ß-MHC promoter, suggesting that Asxl2 directly represses ß-MHC. Interrogation of the CardioGenomics database revealed that ASXL2 is down-regulated in the hearts of patients with ischemic or idiopathic dilated cardiomyopathy. We propose that chromatin factors like Asxl2 function in the adult heart to regulate cell type- and stage-specific patterns of gene expression, and the disruption of such regulation may be involved in the etiology and/or development of certain forms of human heart disease.

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.

Mechano-growth factor E-domain modulates cardiac contractile function through 14-3-3 protein interactomes.

In the heart, alternative splicing of the igf-I gene produces two isoforms: IGF-IEa and IGF-IEc, (Mechano-growth factor, MGF). The sequence divergence between their E-domain regions suggests differential isoform function. To define the biological actions of MGF's E-domain, we performed in silico analysis of the unique C-terminal sequence and identified a phosphorylation consensus site residing within a putative 14-3-3 binding motif. To test the functional significance of Ser 18 phosphorylation, phospho-mimetic (S/E18) and phospho-null (S/A18) peptides were delivered to mice at different doses for 2 weeks. Cardiovascular function was measured using echocardiography and a pressure-volume catheter. At the lowest (2.25 mg/kg/day) and highest (9 mg/kg/day) doses, the peptides produced a depression in systolic and diastolic parameters. However, at 4.5 mg/kg/day the peptides produced opposing effects on cardiac function. Fractional shortening analysis also showed a similar trend, but with no significant change in cardiac geometry. Microarray analysis discovered 21 genes (FDR p < 0.01), that were expressed accordant with the opposing effects on contractile function at 4.5 mg/kg/day, with the nuclear receptor subfamily 4 group A member 2 (Nr4a2) identified as a potential target of peptide regulation. Testing the regulation of the Nr4a family, showed the E-domain peptides modulate Nr4a gene expression following membrane depolarization with KCl in vitro. To determine the potential role of 14-3-3 proteins, we examined 14-3-3 isoform expression and distribution. 14-3-3γ localized to the myofilaments in neonatal cardiac myocytes, the cardiac myocytes and myofilament extracts from the adult heart. Thermal shift analysis of recombinant 14-3-3γ protein showed the S/A18 peptide destabilized 14-3-3γ folding. Also, the S/A18 peptide significantly inhibited 14-3-3γ's ability to interact with myosin binding protein C (MYPC3) and phospholamban (PLN) in heart lysates from dobutamine injected mice. Conversely, the S/E18 peptide showed no effect on 14-3-3γ stability, did not inhibit 14-3-3γ's interaction with PLN but did inhibit the interaction with MYPC3. Replacing the glutamic acid with a phosphate group on Ser 18 (pSer18), significantly increased 14-3-3γ protein stability. We conclude that the state of Ser 18 phosphorylation within the 14-3-3 binding motif of MGF's E-domain, modulates protein-protein interactions within the 14-3-3γ interactome, which includes proteins involved in the regulation of contractile function.

Expression of slow skeletal TnI in adult mouse hearts confers metabolic protection to ischemia.

Changes in metabolic and myofilament phenotypes coincide in developing hearts. Posttranslational modification of sarcomere proteins influences contractility, affecting the energetic cost of contraction. However, metabolic adaptations to sarcomeric phenotypes are not well understood, particularly during pathophysiological stress. This study explored metabolic adaptations to expression of the fetal, slow skeletal muscle troponin I (ssTnI). Hearts expressing ssTnI exhibited no significant ATP loss during 5 min of global ischemia, while non-transgenic littermates (NTG) showed continual ATP loss. At 7 min ischemia TG-ssTnI hearts retained 80±12% of ATP versus 49±6% in NTG (P<0.05). Hearts expressing ssTnI also had increased AMPK phosphorylation. The mechanism of ATP preservation was augmented glycolysis. Glycolytic end products (lactate and alanine) were 38% higher in TG-ssTnI than NTG at 2 min and 27% higher at 5 min. This additional glycolysis was supported exclusively by exogenous glucose, and not glycogen. Thus, expression of a fetal myofilament protein in adult mouse hearts induced elevated anaerobic ATP production during ischemia via metabolic adaptations consistent with the resistance to hypoxia of fetal hearts. The general findings hold important relevance to both our current understanding of the association between metabolic and contractile phenotypes and the potential for invoking cardioprotective mechanisms against ischemic stress. This article is part of a Special Issue entitled "Possible Editorial".

Expression of tropomyosin-κ induces dilated cardiomyopathy and depresses cardiac myofilament tension by mechanisms involving cross-bridge dependent activation and altered tropomyosin phosphorylation.

Tropomyosin-kappa (TPM1-κ) is a newly discovered tropomyosin (TM) isoform that is exclusively expressed in the human heart and generated by an alternative splicing of the α-TM gene. We reported that TPM1-κ expression was increased in the hearts of patients with chronic dilated cardiomyopathy (DCM). To increase our understanding of the significance of this shift in isoform population, we generated transgenic (TG) mice expressing TPM1-κ in the cardiac compartment where TPM1-κ replaces 90% of the native TM. We previously showed that there was a significant inhibition of the ability of strongly bound cross-bridges to induce activation of TG myofilaments (Rajan et al., Circulation 121:410-418, 2010). Here, we compared the force-Ca(2+) relations in detergent extracted (skinned) fiber bundles isolated from non-transgenic (NTG) and TG-TPM1-κ hearts at two sarcomere lengths (SLs). Our data demonstrated a significant decrease in the Ca(2+) sensitivity of the myofilaments from TG-TPM1-κ hearts with no change in the maximum developed tension, length-dependent activation, and the ratio of ATPase rate to tension. There was also no difference in the affinity and cooperativity of Ca(2+)-binding to troponin in thin filaments reconstituted with either TPM1-κ or α-TM. We also compared protein phosphorylation in NTG and TG-TPM1-κ myofilaments. There was a decrease in the total phosphorylation of TPM1-κ compared to α-TM, but no significant change in other major sarcomeric proteins. Our results identify a novel mode of myofilament desensitization to Ca(2+) associated with a DCM linked switch in TM isoform population.