Posttranslational Modifications Of Sarcomeric Proteins Research Articles

Cardiac myosin regulatory light chain kinase modulates cardiac contractility by phosphorylating both myosin regulatory light chain and troponin I

Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been studied extensively in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In this study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically monophosphorylates Ser23 of human cardiac troponin I (cTnI) in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent in vitro and in situ, suggesting species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart.

Top-down Mass Spectrometry of Sarcomeric Protein Post-translational Modifications from Non-human Primate Skeletal Muscle.

Sarcomeric proteins, including myofilament and Z-disk proteins, play critical roles in regulating muscle contractile properties. A variety of isoforms and post-translational modifications (PTMs) of sarcomeric proteins have been shown to be associated with modulation of muscle functions and the occurrence of muscle diseases. Non-human primates (NHPs) are excellent research models for sarcopenia, a disease associated with alterations in sarcomeric proteins, due to their marked similarities to humans. However, the sarcomeric proteins in NHP skeletal muscle have not been well characterized. To gain a deeper understanding of sarcomeric proteins in NHP skeletal muscle, we employed top-down mass spectrometry (MS) to conduct a comprehensive analysis on isoforms and PTMs of sarcomeric proteins in rhesus macaque skeletal muscle. We identified 23 protein isoforms with 46 proteoforms of sarcomeric proteins, including 6 isoforms with 18 proteoforms from fast skeletal troponin T. Particularly, for the first time, a novel PDZ/LIM domain protein isoform, PDLIM7, was characterized with a newly identified protein sequence. Moreover, we also identified multiple PTMs on these proteins, including deamidation, methylation, acetylation, tri-methylation, phosphorylation, and S-glutathionylation. Most PTM sites were localized, including Asn13 deamidation on MLC-2S; His73 methylation on αactin; N-terminal acetylation on most identified proteins; N-terminal tri-methylation on MLC-1S, MLC-1F, MLC-2S, and MLC-2F; Ser14 phosphorylation on MLC-2S; and Ser15 and Ser16 phosphorylation on MLC-2F. In summary, a comprehensive characterization of sarcomeric proteins including multiple isoforms and PTMs in NHP skeletal muscle was achieved by analyzing intact proteins in the top-down MS approach.

Cardiac Disorders and Pathophysiology of Sarcomeric Proteins.

The sarcomeric proteins represent the structural building blocks of heart muscle, which are essential for contraction and relaxation. During recent years, it has become evident that posttranslational modifications of sarcomeric proteins, in particular phosphorylation, tune cardiac pump function at rest and during exercise. This delicate, orchestrated interaction is also influenced by mutations, predominantly in sarcomeric proteins, which cause hypertrophic or dilated cardiomyopathy. In this review, we follow a bottom-up approach starting from a description of the basic components of cardiac muscle at the molecular level up to the various forms of cardiac disorders at the organ level. An overview is given of sarcomere changes in acquired and inherited forms of cardiac disease and the underlying disease mechanisms with particular reference to human tissue. A distinction will be made between the primary defect and maladaptive/adaptive secondary changes. Techniques used to unravel functional consequences of disease-induced protein changes are described, and an overview of current and future treatments targeted at sarcomeric proteins is given. The current evidence presented suggests that sarcomeres not only form the basis of cardiac muscle function but also represent a therapeutic target to combat cardiac disease.

Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics

Despite advancements in symptom management for heart failure (HF), this devastating clinical syndrome remains the leading cause of death in the developed world. Studies using animal models have greatly advanced our understanding of the molecular mechanisms underlying HF; however, differences in cardiac physiology and the manifestation of HF between animals, particularly rodents, and humans necessitates the direct interrogation of human heart tissue samples. Nevertheless, an ever-present concern when examining human heart tissue samples is the potential for artefactual changes related to temperature changes during tissue shipment or sample processing. Herein, we examined the effects of temperature on the post-translational modifications (PTMs) of sarcomeric proteins, the proteins responsible for muscle contraction, under conditions mimicking those that might occur during tissue shipment or sample processing. Using a powerful top-down proteomics method, we found that sarcomeric protein PTMs were differentially affected by temperature. Specifically, cardiac troponin I and enigma homolog isoform 2 showed robust increases in phosphorylation when tissue was incubated at either 4 °C or 22 °C. The observed increase is likely due to increased cyclic AMP levels and activation of protein kinase A in the tissue. On the contrary, cardiac troponin T and myosin regulatory light chain phosphorylation decreased when tissue was incubated at 4 °C or 22 °C. Furthermore, significant protein degradation was also observed after incubation at 4 °C or 22 °C. Overall, these results indicate that temperature exerts various effects on sarcomeric protein PTMs and careful tissue handling is critical for studies involving human heart samples. Moreover, these findings highlight the power of top-down proteomics for examining the integrity of cardiac tissue samples.

Novel Sarcopenia-related Alterations in Sarcomeric Protein Post-translational Modifications (PTMs) in Skeletal Muscles Identified by Top-down Proteomics

Sarcopenia, the age-related loss of skeletal muscle mass and strength, is a significant cause of morbidity in the elderly and is a major burden on health care systems. Unfortunately, the underlying molecular mechanisms in sarcopenia remain poorly understood. Herein, we utilized top-down proteomics to elucidate sarcopenia-related changes in the fast- and slow-twitch skeletal muscles of aging rats with a focus on the sarcomeric proteome, which includes both myofilament and Z-disc proteins-the proteins that constitute the contractile apparatuses. Top-down quantitative proteomics identified significant changes in the post-translational modifications (PTMs) of critical myofilament proteins in the fast-twitch skeletal muscles of aging rats, in accordance with the vulnerability of fast-twitch muscles to sarcopenia. Surprisingly, age-related alterations in the phosphorylation of Cypher isoforms, proteins that localize to the Z-discs in striated muscles, were also noted in the fast-twitch skeletal muscle of aging rats. This represents the first report of changes in the phosphorylation of Z-disc proteins in skeletal muscle during aging. In addition, increased glutathionylation of slow skeletal troponin I, a novel modification that may help protect against oxidative damage, was observed in slow-twitch skeletal muscles. Furthermore, we have identified and characterized novel muscle type-specific proteoforms of myofilament proteins and Z-disc proteins, including a novel isoform of the Z-disc protein Enigma. The finding that the phosphorylation of Z-disc proteins is altered in response to aging in the fast-twitch skeletal muscles of aging rats opens new avenues for the investigation of the role of Z-discs in age-related muscle dysfunction.

Sarcomeric protein modification during adrenergic stress enhances cross-bridge kinetics and cardiac output.

Molecular adaptations to chronic neurohormonal stress, including sarcomeric protein cleavage and phosphorylation, provide a mechanism to increase ventricular contractility and enhance cardiac output, yet the link between sarcomeric protein modifications and changes in myocardial function remains unclear. To examine the effects of neurohormonal stress on posttranslational modifications of sarcomeric proteins, mice were administered combined α- and β-adrenergic receptor agonists (isoproterenol and phenylephrine, IPE) for 14 days using implantable osmotic pumps. In addition to significant cardiac hypertrophy and increased maximal ventricular pressure, IPE treatment accelerated pressure development and relaxation (74% increase in dP/dtmax and 14% decrease in τ), resulting in a 52% increase in cardiac output compared with saline (SAL)-treated mice. Accelerated pressure development was maintained when accounting for changes in heart rate and preload, suggesting that myocardial adaptations contribute to enhanced ventricular contractility. Ventricular myocardium isolated from IPE-treated mice displayed a significant reduction in troponin I (TnI) and myosin-binding protein C (MyBP-C) expression and a concomitant increase in the phosphorylation levels of the remaining TnI and MyBP-C protein compared with myocardium isolated from saline-treated control mice. Skinned myocardium isolated from IPE-treated mice displayed a significant acceleration in the rate of cross-bridge (XB) detachment (46% increase) and an enhanced magnitude of XB recruitment (43% increase) at submaximal Ca2+ activation compared with SAL-treated mice but unaltered myofilament Ca2+ sensitivity of force generation. These findings demonstrate that sarcomeric protein modifications during neurohormonal stress are molecular adaptations that enhance in vivo ventricular contractility through accelerated XB kinetics to increase cardiac output.NEW & NOTEWORTHY Posttranslational modifications to sarcomeric regulatory proteins provide a mechanism to modulate cardiac function in response to stress. In this study, we demonstrate that neurohormonal stress produces modifications to myosin-binding protein C and troponin I, including a reduction in protein expression within the sarcomere and increased phosphorylation of the remaining protein, which serve to enhance cross-bridge kinetics and increase cardiac output. These findings highlight the importance of sarcomeric regulatory protein modifications in modulating ventricular function during cardiac stress.

Contractile protein phosphorylation predicts human heart disease phenotypes

Human heart failure has been associated with a low level of thin-filament protein phosphorylation and an increase in calcium sensitivity of contraction relative to both "control" human heart tissue and tissue from small animal models. However, diverse strategies of human tissue procurement and the reliance on tissue obtained from subjects with end-stage heart failure suggest this may be an incomplete characterization. Therefore, we evaluated cardiac left ventricular (LV) biopsy samples from patients with aortic stenosis undergoing valve replacement who presented either with LV hypertrophy and preserved systolic function (Hyp) or with LV dilation and reduced ejection fraction (Dil). In Hyp, total troponin I (TnI) phosphorylation was markedly increased and myosin light chain 2 (MLC2) phosphorylation was unchanged relative to a control group of patients with normal LV function. Conversely, in Dil, total TnI phosphorylation was significantly reduced compared with control subjects and MLC2 phosphorylation was increased. Site-specific analysis of TnI phosphorylation revealed phenotype-specific differences such that Hyp samples demonstrated significant increases in phosphorylation at serine 22/23 and Dil samples had significant decreases at serine 43. The ratio of phosphorylation at the two sites was biased toward serine 22/23 in Hyp and toward serine 43/45 in Dil. Western blot analysis showed that protein phosphatase-1 was reduced in Hyp and protein phosphatase-2 was reduced in Dil. These data suggest that posttranslational modifications of sarcomeric proteins, both singly and in combination, are stage specific. Defining these changes in progressive heart disease may provide important diagnostic and treatment information.

Myocardial infarction in mice alters sarcomeric function via post-translational protein modification

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species. We surgically induced MI in 6-7-month-old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3-4 days after MI or sham (SH) surgery. MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent-extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca(2+) sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of Ca-ATP in myofibrillar preparations, which demonstrated a decline in Ca-ATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal non-reducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca(2+) sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process.

Reverse Remodeling With Left Ventricular Assist Devices

Over the last 2 decades, numerous advancements in medical therapies have improved patient outcomes in heart failure (HF). However, a significant number of patients still progress to end-stage HF, in which treatment options are largely limited to cardiac transplantation. As patient demands for transplant continue to exceed the supply of available organs, mechanical assist devices—specifically, the left ventricular assist device (LVAD)—were initially introduced as a bridge to cardiac transplantation. LVADs have 2 important beneficial effects. First, LVADs are placed in parallel to the native left ventricle (LV), causing pressure and volume unloading of the LV. Second, LVADs restore cardiac output and subsequent perfusion to the organs. As a result of these 2 effects, it became evident that some patients had actual improvement in LV function after LVAD placement. The term reverse remodeling was used to describe the improvement in myocardial function that was observed in patients with a seemingly end-stage disease. With reverse remodeling, a new hope for the treatment of HF was born—using LVADs as a bridge to recovery; however, to date, this promise has largely been unrealized. This probably is reflective of the fact that the sequela of mechanical ventricular unloading are quite complex and appear to involve the engagement of competing biological pathways including regression of cardiomyocyte hypertrophy as well as progressive cell atrophy. Although the promise of ventricular recovery still persists, its actualization will await a more comprehensive dissection of these competing biological processes. This review will discuss the beneficial clinical effects of LVAD support as well as review what is known about the cellular and molecular response to mechanical unloading and mechanisms of reverse remodeling. Key research findings have been summarized in the Table. View this table: Table. Summary of Research of LVAD Support on Clinical Effects and the Cellular and Molecular Changes That May Contribute to Reverse …

H2O2 alters rat cardiac sarcomere function and protein phosphorylation through redox signaling

ROS, such as H(2)O(2), are a component of pathological conditions in many organ systems and have been reported to be elevated in cardiac pathophysiology. The experiments presented here test the hypothesis that H(2)O(2) induces alterations in cardiac myofilament function by the posttranslational modification of sarcomeric proteins indirectly through PKC signaling. In vitro assessment of actomyosin Mg(2+)-ATPase activity of myofibrillar fractions showed blunted relative ATP consumption in the relaxed state (pCa 8.0) in response to treatment with 0.5 mM H(2)O(2) before myofilament isolation. The effect was attributable to downstream "redox signaling," inasmuch as the direct application of H(2)O(2) to isolated myofibrils did not alter Mg(2+)-ATPase activity. Ca(2+)-ATPase activity, which was used as a measure of myofibrillar myosin function, was unaffected by H(2)O(2). Functional experiments using rat cardiac trabeculae treated with 0.5 or 5 mM H(2)O(2) followed by detergent extraction of membranes demonstrated increased Ca(2+) sensitivity of force production, a faster rate of force redevelopment, and (for 5 mM) decreased maximum tension. Biochemical analysis of myocardial samples treated with 0.5 mM H(2)O(2) demonstrated increased phosphorylation of two sarcomeric proteins: cardiac troponin I and myosin-binding protein-C. These changes were eliminated by a general PKC inhibitor. However, H(2)O(2) and the general PKC activator PMA induced different phosphorylation patterns in cardiomyocytes in which PKC-delta was elevated by viral infection. These data provide evidence that PKC-dependent redox signaling affects the function of cardiac myofilaments and indicate modification of specific proteins through this signaling mechanism.

Functional Analysis of a Unique Troponin C Mutation, GLY159ASP, that Causes Familial Dilated Cardiomyopathy, Studied in Explanted Heart Muscle

Familial dilated cardiomyopathy can be caused by mutations in the proteins of the muscle thin filament. In vitro, these mutations decrease Ca(2+) sensitivity and cross-bridge turnover rate, but the mutations have not been investigated in human tissue. We studied the Ca(2+)-regulatory properties of myocytes and troponin extracted from the explanted heart of a patient with inherited dilated cardiomyopathy due to the cTnC G159D mutation. Mass spectroscopy showed that the mutant cTnC was expressed approximately equimolar with wild-type cTnC. Contraction was compared in skinned ventricular myocytes from the cTnC G159D patient and nonfailing donor heart. Maximal Ca(2+)-activated force was similar in cTnC G159D and donor myocytes, but the Ca(2+) sensitivity of cTnC G159D myocytes was higher (EC(50) G159D/donor=0.60). Thin filaments reconstituted with skeletal muscle actin and human cardiac tropomyosin and troponin were studied by in vitro motility assay. Thin filaments containing the mutation had a higher Ca(2+) sensitivity (EC(50) G159D/donor=0.55 + or - 0.13), whereas the maximally activated sliding speed was unaltered. In addition, the cTnC G159D mutation blunted the change in Ca(2+) sensitivity when TnI was dephosphorylated. With wild-type troponin, Ca(2+) sensitivity was increased (EC(50) P/unP=4.7 + or - 1.9) but not with cTnC G159D troponin (EC(50) P/unP=1.2 + or - 0.1). We propose that uncoupling of the relationship between phosphorylation and Ca(2+) sensitivity could be the cause of the dilated cardiomyopathy phenotype. The differences between these data and previous in vitro results show that native phosphorylation of troponin I and troponin T and other posttranslational modifications of sarcomeric proteins strongly influence the functional effects of a mutation.

Correlations between alterations in length-dependent Ca2+ activation of cardiac myofilaments and the end-systolic pressure–volume relation

We have tested the hypothesis that alterations in length dependent activation (LDA) of cardiac myofilaments represent an important regulatory mechanism affecting the Frank-Starling mechanism as determined by the slope (E(es)) of the relation between left ventricular (LV) volume and end-systolic pressure. We employed a transgenic (TG) mouse model in which the cardiac isoform of TnI (cTnI) has been completely replaced with slow skeletal TnI (ssTnI), the embryonic/neonatal isoform in the heart. Compared to non-transgenic (NTG) controls, myofilaments from TG-ssTnI hearts demonstrate an increase in Ca(2+) sensitivity and a substantially blunted LDA that is unaffected by PKA-dependent phosphorylation. We measured in situ LV pressure and volume relations during basal conditions and isoproterenol (ISO) stimulation. In the basal state in TG-ssTnI hearts there was significant increase in end-systolic pressure and slight decrease in heart rate. ISO stimulation resulted in a significant increase in heart rate, ejection fraction, maximum dP/dt, preload-recruitable stroke work, maximum dP/dt versus end diastolic volume and cardiac output in both groups. During basal conditions there was no difference in the E(es) relation between NTG and TG-ssTnI groups. However, during ISO stimulation the E(es) relation was significantly different between NTG and TG-ssTnI groups. Our study provides the first direct evidence that enhancement in differences in LDA between cardiac myofilaments from NTG and TG-ssTnI hearts induced by post-translational modifications of sarcomeric proteins are reflected in the in situ beating heart by a different change in E(es). Thus, changes in LDA should be considered in interpreting results from in situ experiments on inotropic effects associated with physiological and patho-physiological states of the heart.