Myofilament Proteins Research Articles

Thick filament activation is different in fast- and slow-twitch skeletal muscle.

The contractile properties of fast-twitch and slow-twitch skeletal muscles are primarily determined by the myosin isoform content and modulated by a variety of sarcomere proteins. X-ray diffraction studies of regulatory mechanisms in muscle contraction have focused predominately on fast- or mixed-fibre muscle with slow muscle being much less studied. Here, we used time-resolved X-ray diffraction to investigate the dynamic behaviour of the myofilament proteins in relatively pure slow-twitch-fibre rat soleus (SOL) and pure fast-twitch-fibre rat extensor digitorum longus (EDL) muscle during twitch and tetanic contractions at optimal length. During twitch contractions the diffraction signatures indicating a transition in the myosin heads from ordered OFF states, where heads are held close to the thick filament backbone, to disordered ON states, where heads are free to bind to thin filaments, were found in EDL and not in SOL muscle. During tetanic contraction, changes in the disposition of myosin heads as active tension develops is a quasi-stepwise process in EDL muscle whereas in SOL muscle this relationship appears to be linear. The observed reduced extensibility of the thick filaments in SOL muscle as compared to EDL muscles indicates a molecular basis for this behaviour. These data indicate that for the EDL, thick filament activation is a cooperative strain-induced mechano-sensing mechanism, whereas for the SOL, thick filament activation has a more graded response. These different approaches to thick filament regulation in fast- and slow-twitch muscles may be adaptations for short-duration, strong contractions versus sustained, finely controlled contractions, respectively. KEY POINTS: Fast-twitch muscle and slow-twitch muscle are optimized for strong, short-duration contractions and for tonic postural activity, respectively. Structural events (OFF to ON transitions) in the myosin-containing thick filaments in fast muscle help determine the timing and strength of contractions, but these have not been studied in slow-twitch muscle. The X-ray diffraction signatures of structural OFF to ON transitions are different in fast extensor digitorum longus (EDL) and slow soleus (SOL) muscle, being completely absent during twitches in soleus muscle and blunted during tetanic contractions SOL as compared to EDL Quasi-stepwise thick filament structural OFF to ON transitions in fast twitch muscle may be an adaptation for rapid, ballistic movements, whereas more graded OFF to ON structural transitions in slow-twitch muscle may be an adaptation for slower, finer motions.

Fast and slow skeletal myosin binding protein-C and aging

Aging is associated with skeletal muscle strength decline and cardiac diastolic dysfunction. The structural arrangements of the sarcomeric proteins, such as myosin binding protein-C (MyBP-C) are shown to be pivotal in the pathogenesis of diastolic dysfunction. Yet, the role of fast (fMyBP-C) and slow (sMyBP-C) skeletal muscle MyBP-C remains to be elucidated. Herein, we aimed to characterize MyBP-C and its paralogs in the fast tibialis anterior (TA) muscle from adult and old mice. Immunoreactivity preparations showed that the relative abundance of the fMyBP-C paralog was greater in the TA of both adult and old, but no differences were noted between groups. We further found that the expression level of cardiac myosin binding protein-C (cMyBP-C), an important modulator of cardiac output, was lowered by age. Standard SDS-PAGE along with Pro-Q Diamond phosphoprotein staining did not identify age-related changes in phosphorylated MyBP-C proteins from TA and cardiac muscles; however, it revealed that MyBP-C paralogs in fast skeletal and cardiac muscle were highly phosphorylated. Mass spectrometry further identified glycogen phosphorylase, desmin, actin, troponin T, and myosin regulatory light chain 2 as phosphorylated myofilament proteins in both ages. MyBP-C protein-bound carbonyls were determined using anti-DNP immunostaining and found the carbonyl level of fMyBP-C, sMyBP-C, and cMyBP-C to be similar between old and adult animals. In summary, our data showed some differences regarding the MyBP-C paralog expression and identified an age-related reduction of cMyBP-C expression. Future studies are needed to elucidate which are the age-driven post-translational modifications in the MyBP-C paralogs.

Alteration in tyrosine phosphorylation of cardiac proteome and EGFR pathway contribute to hypertrophic cardiomyopathy

Alterations of serine/threonine phosphorylation of the cardiac proteome are a hallmark of heart failure. However, the contribution of tyrosine phosphorylation (pTyr) to the pathogenesis of cardiac hypertrophy remains unclear. We use global mapping to discover and quantify site-specific pTyr in two cardiac hypertrophic mouse models, i.e., cardiac overexpression of ErbB2 (TgErbB2) and α myosin heavy chain R403Q (R403Q-αMyHC Tg), compared to control hearts. From this, there are significant phosphoproteomic alterations in TgErbB2 mice in right ventricular cardiomyopathy, hypertrophic cardiomyopathy (HCM), and dilated cardiomyopathy (DCM) pathways. On the other hand, R403Q-αMyHC Tg mice indicated that the EGFR1 pathway is central for cardiac hypertrophy, along with angiopoietin, ErbB, growth hormone, and chemokine signaling pathways activation. Surprisingly, most myofilament proteins have downregulation of pTyr rather than upregulation. Kinase-substrate enrichment analysis (KSEA) shows a marked downregulation of MAPK pathway activity downstream of k-Ras in TgErbB2 mice and activation of EGFR, focal adhesion, PDGFR, and actin cytoskeleton pathways. In vivo ErbB2 inhibition by AG-825 decreases cardiomyocyte disarray. Serine/threonine and tyrosine phosphoproteome confirm the above-described pathways and the effectiveness of AG-825 Treatment. Thus, altered pTyr may play a regulatory role in cardiac hypertrophic models.

Myofilaments promote wing expansion and maintain genitalia morphology in the American cockroach, Periplaneta americana.

Insects are the most widely distributed and successful animals on the planet. A large number of insects are capable of flight with functional wings. Wing expansion is an important process for insects to achieve functional wings after eclosion and healthy genital morphology is crucial for adult reproduction. Myofilaments are functional units that constitute sarcomeres and trigger muscle contraction. Here, we identified four myofilament proteins, including Myosin, Paramyosin, Tropomyosin and Troponin T, from the wing pads of nymphs in the American cockroach, Periplaneta americana. RNAi-mediated knockdown of Myosin, Paramyosin, Tropomyosin and Troponin T in the early stage of final instar nymphs caused a severely curly wing phenotype in the imaginal moult, especially in the Paramyosin and Troponin T knockdown groups, indicating that these myofilament proteins are involved in controlling wing expansion behaviours during the nymph-adult transition. In addition, the knockdown resulted in abnormal external genitalia, caused ovulation failure, and affected male accessory gland development. Interestingly, the expression of myofilament genes was induced by methoprene, a juvenile hormone (JH) analogue, and decreased by the depletion of the JH receptor gene Met. Altogether, we have determined that myofilament genes play an important role in promoting wing expansion and maintaining adult genitalia morphology, and their expression is induced by JH signalling. Our data reveal a novel mechanism by which wing expansion is regulated by myofilaments and the functions of myofilaments are involved in maintaining genitalia morphology.

Sarco/endoplasmic reticulum calcium ATPase activity is unchanged despite increased myofilament calcium sensitivity in Zucker type 2 diabetic fatty rat heart

Systolic and diastolic dysfunction in diabetes have frequently been associated with abnormal calcium (Ca2+) regulation. However, there is emerging evidence that Ca2+ mishandling alone is insufficient to fully explain diabetic heart dysfunction, with focus shifting to the properties of the myofilament proteins. Our aim was to examine the effects of diabetes on myofilament Ca2+ sensitivity and Ca2+ handling in left ventricular tissues isolated from the same type 2 diabetic rat hearts. We measured the force-pCa relationship in skinned left ventricular cardiomyocytes isolated from 20-week-old type 2 diabetic and non-diabetic rats. Myofilament Ca2+ sensitivity was greater in the diabetic relative to non-diabetic cardiomyocytes, and this corresponded with lower phosphorylation of cardiac troponin I (cTnI) at ser23/24 in the diabetic left ventricular tissues. Protein expression of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), phosphorylation of phospholamban (PLB) at Ser16, and SERCA/PLB ratio were lower in the diabetic left ventricular tissues. However, the maximum SERCA Ca2+ uptake rate was not different between the diabetic and non-diabetic myocardium. Our data suggest that impaired contractility in the diabetic heart is not caused by SERCA Ca2+ mishandling. This study highlights the important role of the cardiac myofilament and provides new insight on the pathophysiology of diabetic heart dysfunction.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by β-adrenergic stimulation

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B′-family in the heart. Its role in regulating the cardiac response to β-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B′ subunits (β and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show β-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to β-adrenergic stimulation in the myocardium.

Abstract P1104: Lower Troponin Expression In The Right Ventricle Of Rats Explains Interventricular Differences In E-c Coupling

Despite distinctive functional and anatomical differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, IonOptix system and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single cell contractility and cytosolic Ca 2+ ([Ca 2+ ] i ), respectively. Myofilament proteins were analyzed by immunoblotting. RVCMs showed significantly shorter action potential duration (APD) and higher density of transient outward K + current ( I to ). However, the triggered [Ca 2+ ] i change (Ca 2+ transient) was not different, while the decay rate of the Ca 2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was approximately 60% lower in RVCM, partly responsible for the slower decay of the Ca 2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca 2+ buffering capacity ( κ S ), which was proved by in situ analysis. The introduction of these new levels of cTn, I to and SERCA into a mathematical model of rat LVCM reproduced the similar Ca 2+ transient, slower Ca 2+ decay, shorter APD and smaller ΔSL of RVCM. Taken together, we show reduced expression of cTn proteins in the RVCMs, which provides an explanation for the inter-ventricular difference in the E-C coupling kinetics.

Abstract GS108: Troponin I Tyrosine-26 Phosphorylation Improves Relaxation And Is Beneficial During Pathological Diastolic Dysfunction

Diastolic dysfunction contributes to disease in both heart failure with preserved and reduced ejection fraction. There are currently no approved therapies to accelerate impaired relaxation for heart failure patients with diastolic dysfunction. A healthy heart can modify its ability to relax through post-translational modifications (e.g., phosphorylation) of myofilament proteins. The inhibitory subunit of the troponin complex, troponin I (TnI), is a key regulator of cardiac contraction and relaxation and is endogenously phosphorylated at Tyr-26. We previously demonstrated that this novel tyrosine phosphorylation decreases calcium sensitivity and accelerates calcium dissociation from troponin C. We therefore hypothesize that increasing TnI Tyr-26 phosphorylation will accelerate relaxation and be beneficial during pathological diastolic dysfunction. To determine the effects of TnI Tyr-26 phosphorylation in vivo , we generated a phospho-mimetic mouse with TnI Tyr-26 mutated to Glu. Structural and functional echocardiography and hemodynamics measurements demonstrate that TnI Tyr-26 phosphorylation accelerates relaxation and increases diastolic function in vivo . To determine the effect of this TnI Tyr-26 phosphorylation mediated acceleration of diastolic function during disease, we performed unilateral nephrectomy with DOCA-salt (neph/DOCA) to induce diastolic dysfunction. Wild-type mice subjected to neph/DOCA develop left ventricular hypertrophy, enlarged left atria, elevated diastolic pressure, and slowed relaxation, indicative of diastolic dysfunction. In contrast with wild-type, TnI Tyr-26 phosphorylation mice subjected to neph/DOCA display normal diastolic function without any cardiac remodeling. Overall, we demonstrate that TnI Tyr-26 phosphorylation inhibits the development of diastolic dysfunction and is therefore a novel mechanism to accelerate myocardial relaxation in vivo.

Abstract P1061: Crosstalk Between Acetylation And Methylglyoxal Modifications On Myofilament Proteins Affects Sarcomere Function

Diabetes doubles the risk of developing heart failure, independent of coronary artery disease and hypertension. The mechanisms connecting these two diseases are poorly understood, although we have previously shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis byproduct that can irreversibly modify lysine (K) and arginine (R) residues in a process called glycation. We found MGO glycation of the myofilament decreased calcium sensitivity and maximum calcium activated force in diabetes. Here we hypothesized MGO may further impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to impact cardiac function. To assess this hypothesis, non-failing human left ventricular (LV) myocardial tissue was exposed to MGO (100 μM) or vehicle and then the treated with acetic anhydride in acetonitrile (300 μM, Ac 2 O-ACN) overnight. Ac 2 O-ACN increased myofilament protein acetylation in both groups, but this effect was greatly reduced in samples pre-treated with MGO, suggesting these two PTMs do compete for a subset of lysine residues. To discover the specific sites of this acetylation-glycation crosstalk, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes, which revealed sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Importantly, these glycation-acetylation crosstalk sites were found in human samples and the data suggest the balance of these may change in disease states. To explore the functional consequence(s) of this crosstalk, we incubated human LV isolated skinned myocytes with Ac 2 O-ACN (300 μM, overnight), which enhanced function by significantly increasing calcium sensitivity. However, pre-treatment with MGO (100 μM) completely blocked this functional effect of acetylation. Together, these data demonstrate that glycation and acetylation compete for certain myofilament lysine residues, and that glycation can block the beneficial functional effects of acetylation. These results provide insight into the molecular mechanisms behind diabetic cardiomyopathy and the utility of targeting acetylation clinically.

SUMOylation does not affect cardiac troponin I stability but alters indirectly the development of force in response to Ca2.

Post-translational modification of the myofilament protein troponin I by phosphorylation is known to trigger functional changes that support enhanced contraction and relaxation of the heart. We report for the first time that human troponin I can also be modified by SUMOylation at lysine 177. Functionally, TnI SUMOylation is not a factor in the development of passive and maximal force generation in response to calcium, however this modification seems to act indirectly by preventing SUMOylation of other myofilament proteins to alter calcium sensitivity and cooperativity of myofilaments. Utilising a novel, custom SUMO site-specific antibody that recognises only the SUMOylated form of troponin I, we verify that this modification occurs in human heart and that it is upregulated during disease.

Ca2+/calmodulin‐dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model

AimsVolume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression.Methods and resultsTherefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO‐induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age‐matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4‐ and 8‐month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated.ResultsThe model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4‐ and 8‐month, which indicates VO‐induced hypertrophy. In addition, 8‐months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity‐time index compared with their age‐matched controls. These changes were accompanied by increased Ca2+ sensitivity and titin‐based cardiomyocyte stiffness in 8‐month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8‐month VO rats compared with age‐matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca2+ sensitivity and titin‐based cardiomyocyte stiffness and perhaps the development of hypertrophy.ConclusionsOur findings showed VO‐induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Age‐ and sex‐specific differences in activation of the cardiac adrenergic cascade

Heart failure (HF) is a costly and devastating public health problem, which differentially impacts male and female patients of all ages across the life‐course. The adrenergic system is dysregulated with HF and is the target of front‐line therapeutics. However, the majority of the work aimed at elucidating the adrenergic signaling response has been conducted in adult males. Consequently, mechanisms of adrenergic signaling that differ by sex and age remain unclear. Here, we set out to identify mechanistic differences in the adrenergic pathway in response to isoproterenol (ISO) stimulation in juvenile (4 weeks), adult (4‐6 months) and aged (18 months) C57Bl6 mice of both sexes. ISO was delivered by mini‐osmotic pump (30 mg/kg/day) for one week. We assessed changes in β‐adrenergic receptor (β‐AR) expression, protein kinase activity, and myofilament protein phosphorylation and function. β1‐AR and β2‐AR protein expression were significantly lower in response to ISO in juvenile males with no difference in expression in juvenile females. In contrast, ISO stimulation in aged males significantly upregulated β1‐AR and β2‐AR protein expression without changes in expression in aged females. Analysis of sarcomeric proteins indicated that phosphorylation of troponin varied in an age‐ and sex‐dependent manner. Phosphorylation of the inhibitory subunit of troponin (TnI) at Ser23/24 was upregulated in response to ISO in females of all ages with no differences in males. These changes in phosphorylation decreased myofilament Ca2+ sensitivity in female hearts in response to ISO. Together, this work suggests that male hearts responded robustly to ISO stimulation as evident by activation throughout the adrenergic cascade while female hearts demonstrated activation of downstream mechanisms at the level of the sarcomere. Ongoing experiments will characterize calcium handling and myofilament mechanics to mechanistically link upstream signals to downstream changes in contractility. These data demonstrate that age and sex are significant biological factors which contribute to adrenergic‐mediated changes in cardiac performance. Understanding these differences is critical for successful therapeutic intervention within the adrenergic signaling cascade for male and female patients of all ages.

Treadmill running increases the calcium sensitivity of myofilaments in diabetic rats.

The cardiovascular benefits of regular exercise are unequivocal, yet patients with type 2 diabetes respond poorly to exercise due to a reduced cardiac reserve. The contractile response of diabetic cardiomyocytes to β-adrenergic stimulation is attenuated, which may result in altered myofilament calcium sensitivity and posttranslational modifications of cardiac troponin I (cTnI). Treadmill running increases myofilament calcium sensitivity in nondiabetic rats, and thus we hypothesized that endurance training would increase calcium sensitivity of diabetic cardiomyocytes and alter site-specific phosphorylation of cTnI. Calcium sensitivity, or pCa50, was measured in Zucker diabetic fatty (ZDF), nondiabetic (nDM), and diabetic (DM) rat hearts after 8 wk of either a sedentary (SED) or progressive treadmill running (TR) intervention. Skinned cardiomyocytes were connected to a capacitance-gauge transducer and a torque motor to measure force as a function of pCa (-log[Ca2+]). Specific phospho-sites on cTnI and O-GlcNAcylation were quantified by immunoblot and total protein phosphorylation by fluorescent gel staining (ProQ Diamond). The novel finding in this study was that training increased pCa50 in both DM and nDM cardiomyocytes (P = 0.009). Phosphorylation of cTnI amino acid residues Ser23/24, a crucial protein kinase A site, and Threonine (Thr)144 was lower in DM hearts, but there was no effect of training on site-specific phosphorylation. In addition, total phosphorylation and O-GlcNAcylation levels were not different between SED and TR groups. These findings suggest that regular exercise may benefit the diabetic heart by specifically targeting myofilament contractile function.NEW & NOTEWORTHY We examined the effects of training on the myofilament calcium in diabetic rat hearts. After 8 wk of treadmill running, both nondiabetic and diabetic cardiomyocytes had increased myofilament calcium sensitivity compared with their sedentary counterparts, but there was no effect of training on the phosphorylation or O-GlcNAcylation status of myofilament proteins measured in this study. These data highlight one potential mechanism capable of reversing, in part, reduced cardiac reserve in the diabetic heart.

Lower troponin expression in the right ventricle of rats explains interventricular differences in E-C coupling.

Despite distinctive functional and anatomic differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, the IonOptix system, and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single-cell contractility, and cytosolic Ca2+ ([Ca2+]i), respectively. Myofilament proteins were analyzed by immunoblotting. RVCM showed significantly shorter action potential duration (APD) and higher density of transient outward K+ current (Ito). However, the triggered [Ca2+]i change (Ca2+ transient) was not different, while the decay rate of the Ca2+ transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was ∼60% lower in RVCM, which is partly responsible for the slower decay of the Ca2+ transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca2+ buffering capacity (κS), which was proved by in situ analysis. The introduction of these new levels of cTn, Ito, and SERCA into a mathematical model of rat LVCM reproduced the similar Ca2+ transient, slower Ca2+ decay, shorter APD, and smaller ΔSL of RVCM. Taken together, these data show reduced expression of cTn proteins in the RVCM, which provides an explanation for the interventricular difference in the E-C coupling kinetics.

Abstract 11592: Glycogen Synthase Kinase 3β Localizes to the Z-Disc to Maintain Length Dependent Activation

Glycogen Synthase Kinase 3β (GSK-3β) can modulate myofilament function in vitro . However, it’s in vivo role, mechanism, and translational relevance are unknown, which we investigated here using inducible cardiomyocyte specific GSK-3β KO mice. Compared to tamoxifen-treated GSK-3β fl/fl Cre- mice (Con), skinned myocytes from KO mice had reduced calcium sensitivity at long sarcomere lengths (SL = 2.2 μm), but there were no differences at short SL (~1.9 μm). Thus, myocytes from KO mice did not sensitize to calcium with stretch, a mechanism called length dependent activation (LDA) that underlies the organ-level Frank-Starling law. LDA has been attributed to (1) phosphorylation of myofilament proteins, (2) altered lattice-spacing, and (3) changes to titin’s elastic properties. Using mass spectrometry and small energy x-ray diffraction we ruled out the first two mechanisms, however we did find that GSK-3β KO myocytes had decreased passive tension – indicating the loss of LDA was due to loss of titin as a length sensor. Interestingly, immunofluorescence showed that GSK-3β localized to the z-disc when phosphorylated at Y216 and, via mass spectrometry, phosphorylated primarily z-disc proteins including several sites on the structural protein Ablim-1, which we showed also localizes to the z-disc. These data suggested that GSK-3β’s effect on titin is likely through altering its ability to anchor to the z-disc through targeting of these z-disc proteins. To provide further evidence that GSK-3β is specifically altering LDA, we genetically removed a downstream effecter of LDA, cardiac myosin binding protein-C (cMyBP-C). In vitro treatment with exogenous GSK-3β was able to increase calcium sensitivity at long SLs in both KO and WT mice but had no effect on cMyBPC KO mice or mice lacking the c-terminal domains of cMyBPC that are known to be important for LDA. Lastly, we found that human heart failure patients had less myofilament GSK-3β compared to non-failing patients, and that these same samples had a depressed LDA. This work has identified a novel mechanism by which GSK-3β localizes to the myofilament to modulate LDA and indicates that z-disc localized GSK-3β may be a possible therapeutic target to restore the Frank-Starling mechanism in heart failure patients.

Abstract 11855: Disruption of β-arrestin-2 Signaling Resulted in Impaired Systolic and Diastolic Function During Aging

Introduction: Angiotensin II (ang II) signaling at ang II receptor type 1 (AT1R), which is a G-protein coupled receptor (GPCR), promotes both G-protein signaling as well as β-arrestin-2 signaling. β-arrestin-2 participates in GPCR desensitization, internalization, but also acts a scaffold for adaptive signal transduction that may occur independently or in parallel to G-protein signaling. Hypothesis: We tested the hypothesis that β-arrestin-2 is an important element in the preservation of cardiac function during aging. Methods: We tested this hypothesis by aging β-arrestin-2 knock-out (KO) mice, and wild-type equivalent (WT) to 12-16 months. We performed echocardiography studies, and Western blot analysis. Results: By echocardiography we found that compared to controls aged KO mice exhibited enlarged left atria (2.2±0.1 vs 2.5±0.1 mm) and left ventricular diameters (4.2±0.2 vs 4.5 ±0.1mm) as well as depressed contractility parameters such as s’ (23.2±1.4 vs 21.4±0.8 mm/s) and decreased circumferential fiber shortening velocity (5.5±0.4 vs 4.5±0.2) with preserved ejection fraction (EF>50%). Aged KO also exhibited depressed relaxation parameters such as e’ (29.2±1.4 vs 21.4±0,6 mm/s) and E/e’ (27.4±1.6 vs 35.6±2.0) when compared to WT controls at the same age. Cardiac dysfunction in aged KO mice was correlated with decreased phosphorylation of RLC and increased phosphorylation of cMyBP-C at Ser302. Among other kinases, cMyBP-C phosphorylation at Ser302 is a PKCε target which activation is negatively regulated by β-arrestin-2 signaling. To determine the direct effects of PKCε over-activation on cMyBP-C phosphorylation, we used mice that over-expresses constitutively active PKC ε in the heart. We found that similarly to our aged KO mice, PKCε overexpression model exhibited significantly higher levels of cMyBP-C phosphorylation at Ser302, when aged to 12 - 15 months. Conclusions: Deletion of β-arrestin-2 signaling in KO mice affected aged mice, resulting in changes in the phosphorylation of myofilament proteins consisting of decreased phosphorylation of RLC and increased phosphorylation of cMyBP-C at Ser302 through the unrestricted activity of PKCε. These changes negatively affected systolic and diastolic function in aged KO mice.

Long-Term PDE-5A Inhibition Improves Myofilament Function in Left and Right Ventricular Cardiomyocytes through Partially Different Mechanisms in Diabetic Rat Hearts.

Heart failure with preserved ejection fraction (HFpEF) and right ventricular (RV) dysfunction are frequent complications of diabetic cardiomyopathy. Here we aimed to characterize RV and left ventricular (LV) remodeling and its prevention by vardenafil (a long-acting phosphodiesterase-5A (PDE-5A) inhibitor) administration in a diabetic HFpEF model. Zucker Diabetic Fatty (ZDF) and control, ZDF Lean (Lean) male rats received 10 mg/kg vardenafil (ZDF + Vard; Lean + Vard) per os, on a daily basis for a period of 25 weeks. In vitro force measurements, biochemical and histochemical assays were employed to assess cardiomyocyte function and signaling. Vardenafil treatment increased cyclic guanosine monophosphate (cGMP) levels and decreased 3-nitrotyrosine (3-NT) levels in the left and right ventricles of ZDF animals, but not in Lean animals. Cardiomyocyte passive tension (Fpassive) was higher in LV and RV cardiomyocytes of ZDF rats than in those receiving preventive vardenafil treatment. Levels of overall titin phosphorylation did not differ in the four experimental groups. Maximal Ca2+-activated force (Fmax) of LV and RV cardiomyocytes were preserved in ZDF animals. Ca2+-sensitivity of isometric force production (pCa50) was significantly higher in LV (but not in RV) cardiomyocytes of ZDF rats than in their counterparts in the Lean or Lean + Vard groups. In accordance, the phosphorylation levels of cardiac troponin I (cTnI) and myosin binding protein-C (cMyBP-C) were lower in LV (but not in RV) cardiomyocytes of ZDF animals than in their counterparts of the Lean or Lean + Vard groups. Vardenafil treatment normalized pCa50 values in LV cardiomyocytes, and it decreased pCa50 below control levels in RV cardiomyocytes in the ZDF + Vard group. Our data illustrate partially overlapping myofilament protein alterations for LV and RV cardiomyocytes in diabetic rat hearts upon long-term PDE-5A inhibition. While uniform patterns in cGMP, 3-NT and Fpassive levels predict identical effects of vardenafil therapy for the diastolic function in both ventricles, the uneven cTnI, cMyBP-C phosphorylation levels and pCa50 values implicate different responses for the systolic function.

Alterations in ACE and ACE2 Activities and Cardiomyocyte Signaling Underlie Improved Myocardial Function in a Rat Model of Repeated Remote Ischemic Conditioning.

Post-ischemic left ventricular (LV) remodeling and its hypothetical prevention by repeated remote ischemic conditioning (rRIC) in male Sprague–Dawley rats were studied. Myocardial infarction (MI) was evoked by permanent ligation of the left anterior descending coronary artery (LAD), and myocardial characteristics were tested in the infarcted anterior and non-infarcted inferior LV regions four and/or six weeks later. rRIC was induced by three cycles of five-minute-long unilateral hind limb ischemia and five minutes of reperfusion on a daily basis for a period of two weeks starting four weeks after LAD occlusion. Sham operated animals served as controls. Echocardiographic examinations and invasive hemodynamic measurements revealed distinct changes in LV systolic function between four and six weeks after MI induction in the absence of rRIC (i.e., LV ejection fraction (LVEF) decreased from 52.8 ± 2.1% to 50 ± 1.6%, mean ± SEM, p < 0.05) and in the presence of rRIC (i.e., LVEF increased from 48.2 ± 4.8% to 55.2 ± 4.1%, p < 0.05). Angiotensin-converting enzyme (ACE) activity was about five times higher in the anterior LV wall at six weeks than that in sham animals. Angiotensin-converting enzyme 2 (ACE2) activity roughly doubled in post-ischemic LVs. These increases in ACE and ACE2 activities were effectively mitigated by rRIC. Ca2+-sensitivities of force production (pCa50) of LV permeabilized cardiomyocytes were increased at six weeks after MI induction together with hypophosphorylation of 1) cardiac troponin I (cTnI) in both LV regions, and 2) cardiac myosin-binding protein C (cMyBP-C) in the anterior wall. rRIC normalized pCa50, cTnI and cMyBP-C phosphorylations. Taken together, post-ischemic LV remodeling involves region-specific alterations in ACE and ACE2 activities together with changes in cardiomyocyte myofilament protein phosphorylation and function. rRIC has the potential to prevent these alterations and to improve LV performance following MI.

Protein Crotonylation Expert Review: A New Lens to Take Post-Translational Modifications and Cell Biology to New Heights.

Genome regulation, temporal and spatial variations in cell function, continues to puzzle and interest life scientists who aim to unravel the molecular basis of human health and disease, not to mention plant biology and ecosystem diversity. Despite important advances in epigenomics and protein post-translational modifications over the past decade, there is a need for new conceptual lenses to understand biological mechanisms that can help unravel the fundamental regulatory questions in genomes and the cell. To these ends, lys crotonylation (Kcr) is a reversible protein modification catalyzed by protein crotonyl transferases and decrotonylases. First identified on histones, Kcr regulates cellular processes at the chromatin level. Research thus far has revealed that Kcr marks promoter sites of active genes and potential enhancers. Eventually, Kcr on a number of nonhistone proteins was reported. The abundance of Kcr on ribosomal and myofilament proteins indicates its functional roles in protein synthesis and muscle contraction. Kcr has also been associated with pluripotency, spermiogenesis, and DNA repair. In plants, large-scale mass spectrometry-based experiments validated the roles of Kcr in photosynthesis. In this expert review, we present the latest thinking and findings on lys crotonylation with an eye to regulation of cell biology. We discuss the enrichment techniques, putative biological functions, and challenges associated with studying this protein modification with vast biological implications. Finally, we reflect on the future outlook about the broader relevance of Kcr in animals, microbes, and plant species.

Troponin Variants as Markers of Skeletal Muscle Health and Diseases.

Ca2+-regulated contractility is a key determinant of the quality of muscles. The sarcomeric myofilament proteins are essential players in the contraction of striated muscles. The troponin complex in the actin thin filaments plays a central role in the Ca2+-regulation of muscle contraction and relaxation. Among the three subunits of troponin, the Ca2+-binding subunit troponin C (TnC) is a member of the calmodulin super family whereas troponin I (TnI, the inhibitory subunit) and troponin T (TnT, the tropomyosin-binding and thin filament anchoring subunit) are striated muscle-specific regulatory proteins. Muscle type-specific isoforms of troponin subunits are expressed in fast and slow twitch fibers and are regulated during development and aging, and in adaptation to exercise or disuse. TnT also evolved with various alternative splice forms as an added capacity of muscle functional diversity. Mutations of troponin subunits cause myopathies. Owing to their physiological and pathological importance, troponin variants can be used as specific markers to define muscle quality. In this focused review, we will explore the use of troponin variants as markers for the fiber contents, developmental and differentiation states, contractile functions, and physiological or pathophysiological adaptations of skeletal muscle. As protein structure defines function, profile of troponin variants illustrates how changes at the myofilament level confer functional qualities at the fiber level. Moreover, understanding of the role of troponin modifications and mutants in determining muscle contractility in age-related decline of muscle function and in myopathies informs an approach to improve human health.