Titin-truncating mutations associated with dilated cardiomyopathy alter length-dependent activation and its modulation via phosphorylation.

AIMS: Dilated cardiomyopathy (DCM) is associated with mutations in many genes encoding sarcomere proteins. Truncating mutations in the titin gene TTN are the most frequent. Proteomic and functional characterizations are required to elucidate the origin of the disease and the pathogenic mechanisms of TTN-truncating variants. METHODS AND RESULTS: We isolated myofibrils from DCM hearts carrying truncating TTN mutations and measured the Ca2+ sensitivity of force and its length dependence. Simultaneous measurement of force and adenosine triphosphate (ATP) consumption in skinned cardiomyocytes was also performed. Phosphorylation levels of troponin I (TnI) and myosin binding protein-C (MyBP-C) were manipulated using protein kinase A and λ phosphatase. mRNA sequencing was employed to overview gene expression profiles. We found that Ca2+ sensitivity of myofibrils carrying TTN mutations was significantly higher than in myofibrils from donor hearts. The length dependence of the Ca2+ sensitivity was absent in DCM myofibrils with TTN-truncating variants. No significant difference was found in the expression level of TTN mRNA between the DCM and donor groups. TTN exon usage and splicing were also similar. However, we identified down-regulation of genes encoding Z-disk proteins, while the atrial-specific regulatory myosin light chain gene, MYL7, was up-regulated in DCM patients with TTN-truncating variants. CONCLUSION: Titin-truncating mutations lead to decreased length-dependent activation and increased elasticity of myofibrils. Phosphorylation levels of TnI and MyBP-C seen in the left ventricles are essential for the length-dependent changes in Ca2+ sensitivity in healthy donors, but they are reduced in DCM patients with TTN-truncating variants. A decrease in expression of Z-disk proteins may explain the observed decrease in myofibril passive stiffness and length-dependent activation.

Distinct hypertrophic cardiomyopathy genotypes result in convergent sarcomeric proteoform profiles revealed by top-down proteomics.

Hypertrophic cardiomyopathy (HCM) is the most common heritable heart disease. Although the genetic cause of HCM has been linked to mutations in genes encoding sarcomeric proteins, the ability to predict clinical outcomes based on specific mutations in HCM patients is limited. Moreover, how mutations in different sarcomeric proteins can result in highly similar clinical phenotypes remains unknown. Posttranslational modifications (PTMs) and alternative splicing regulate the function of sarcomeric proteins; hence, it is critical to study HCM at the level of proteoforms to gain insights into the mechanisms underlying HCM. Herein, we employed high-resolution mass spectrometry-based top-down proteomics to comprehensively characterize sarcomeric proteoforms in septal myectomy tissues from HCM patients exhibiting severe outflow track obstruction (n = 16) compared to nonfailing donor hearts (n = 16). We observed a complex landscape of sarcomeric proteoforms arising from combinatorial PTMs, alternative splicing, and genetic variation in HCM. A coordinated decrease of phosphorylation in important myofilament and Z-disk proteins with a linear correlation suggests PTM cross-talk in the sarcomere and dysregulation of protein kinase A pathways in HCM. Strikingly, we discovered that the sarcomeric proteoform alterations in the myocardium of HCM patients undergoing septal myectomy were remarkably consistent, regardless of the underlying HCM-causing mutations. This study suggests that the manifestation of severe HCM coalesces at the proteoform level despite distinct genotype, which underscores the importance of molecular characterization of HCM phenotype and presents an opportunity to identify broad-spectrum treatments to mitigate the most severe manifestations of this genetically heterogenous disease.

Author Correction: Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Cardiomyopathies and Related Changes in Contractility of Human Heart Muscle.

About half of hypertrophic and dilated cardiomyopathies cases have been recognized as genetic diseases with mutations in sarcomeric proteins. The sarcomeric proteins are involved in cardiomyocyte contractility and its regulation, and play a structural role. Mutations in non-sarcomeric proteins may induce changes in cell signaling pathways that modify contractile response of heart muscle. These facts strongly suggest that contractile dysfunction plays a central role in initiation and progression of cardiomyopathies. In fact, abnormalities in contractile mechanics of myofibrils have been discovered. However, it has not been revealed how these mutations increase risk for cardiomyopathy and cause the disease. Much research has been done and still much is being done to understand how the mechanism works. Here, we review the facts of cardiac myofilament contractility in patients with cardiomyopathy and heart failure.

Abnormal contractility in human heart myofibrils from patients with dilated cardiomyopathy due to mutations in TTN and contractile protein genes.

Dilated cardiomyopathy (DCM) is an important cause of heart failure. Single gene mutations in at least 50 genes have been proposed to account for 25-50% of DCM cases and up to 25% of inherited DCM has been attributed to truncating mutations in the sarcomeric structural protein titin (TTNtv). Whilst the primary molecular mechanism of some DCM-associated mutations in the contractile apparatus has been studied in vitro and in transgenic mice, the contractile defect in human heart muscle has not been studied. In this study we isolated cardiac myofibrils from 3 TTNtv mutants, and 3 with contractile protein mutations (TNNI3 K36Q, TNNC1 G159D and MYH7 E1426K) and measured their contractility and passive stiffness in comparison with donor heart muscle as a control. We found that the three contractile protein mutations but not the TTNtv mutations had faster relaxation kinetics. Passive stiffness was reduced about 38% in all the DCM mutant samples. However, there was no change in maximum force or the titin N2BA/N2B isoform ratio and there was no titin haploinsufficiency. The decrease in myofibril passive stiffness was a common feature in all hearts with DCM-associated mutations and may be causative of DCM.

Instrumentation to study myofibril mechanics from static to artificial simulations of cardiac cycle.

Many causes of heart muscle diseases and skeletal muscle diseases are inherited and caused by mutations in genes of sarcomere proteins which play either a structural or contractile role in the muscle cell. Tissue samples from human hearts with mutations can be obtained but often samples are only a few milligrams and it is necessary to freeze them for storage and transportation. Myofibrils are the fundamental contractile components of the muscle cell and retain all structural elements and contractile proteins performing in contractile event; moreover viable myofibrils can be obtained from frozen tissue.•We are describing a versatile technique for measuring the contractility and its Ca(2+) regulation in single myofibrils. The control of myofibril length, incubation medium and data acquisition is carried out using a digital acquisition board via computer software. Using computer control it is possible not only to measure contractile and mechanical parameters but also simulate complex protocols such as a cardiac cycle to vary length and medium independently.•This single myofibril force assay is well suited for physiological measurements. The system can be adapted to measure tension amplitude, rates of contraction and relaxation, Ca(2+) dependence of these parameters in dose-response measurements, length-dependent activation, stretch response, myofibril elasticity and response to simulated cardiac cycle length changes. Our approach provides an all-round quantitative way to measure myofibrils performance and to observe the effect of mutations or posttranslational modifications. The technique has been demonstrated by the study of contraction in heart with hypertrophic or dilated cardiomyopathy mutations in sarcomere proteins.

Uncoupling of myofilament Ca2+ sensitivity from troponin I phosphorylation by mutations can be reversed by epigallocatechin-3-gallate.

AIMS: Heart muscle contraction is regulated via the ß-adrenergic response that leads to phosphorylation of Troponin I (TnI) at Ser22/23, which changes the Ca(2+) sensitivity of the cardiac myofilament. Mutations in thin filament proteins that cause dilated cardiomyopathy (DCM) and some mutations that cause hypertrophic cardiomyopathy (HCM) abolish the relationship between TnI phosphorylation and Ca(2+) sensitivity (uncoupling). Small molecule Ca(2+) sensitizers and Ca(2+) desensitizers that act upon troponin alter the Ca(2+) sensitivity of the thin filament, but their relationship with TnI phosphorylation has never been studied before. METHODS AND RESULTS: Quantitative in vitro motility assay showed that 30 µM EMD57033 and 100 µM Bepridil increase Ca(2+) sensitivity of phosphorylated cardiac thin filaments by 3.1- and 2.8-fold, respectively. Additionally they uncoupled Ca(2+) sensitivity from TnI phosphorylation, mimicking the effect of HCM mutations. Epigallocatechin-3-gallate (EGCG) decreased Ca(2+) sensitivity of phosphorylated and unphosphorylated wild-type thin filaments equally (by 2.15 ± 0.45- and 2.80 ± 0.48-fold, respectively), retaining the coupling. Moreover, EGCG also reduced Ca(2+) sensitivity of phosphorylated but not unphosphorylated thin filaments containing DCM and HCM-causing mutations; thus, the dependence of Ca(2+) sensitivity upon TnI phosphorylation of uncoupled mutant thin filaments was restored in every case. In single mouse heart myofibrils, EGCG reduced Ca(2+) sensitivity of force and kACT and also preserved coupling. Myofibrils from the ACTC E361G (DCM) mouse were uncoupled; EGCG reduced Ca(2+) sensitivity more for phosphorylated than for unphosphorylated myofibrils, thus restoring coupling. CONCLUSION: We conclude that it is possible to both mimic and reverse the pathological defects in troponin caused by cardiomyopathy mutations pharmacologically. Re-coupling by EGCG may be of potential therapeutic significance for treating cardiomyopathies.

The dilated cardiomyopathy-causing mutation ACTC E361G in cardiac muscle myofibrils specifically abolishes modulation of Ca(2+) regulation by phosphorylation of troponin I.

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca(2+) sensitivity and increases the rate of Ca(2+) release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca(2+)-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca(2+) regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca(2+) sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca(2+) sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca(2+) sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca(2+) sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca(2+) sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.

Mechanical and energetic properties of papillary muscle from ACTC E99K transgenic mouse models of hypertrophic cardiomyopathy.

We compared the contractile performance of papillary muscle from a mouse model of hypertrophic cardiomyopathy [α-cardiac actin (ACTC) E99K mutation] with nontransgenic (non-TG) littermates. In isometric twitches, ACTC E99K papillary muscle produced three to four times greater force than non-TG muscle under the same conditions independent of stimulation frequency and temperature, whereas maximum isometric force in myofibrils from these muscles was not significantly different. ACTC E99K muscle relaxed slower than non-TG muscle in both papillary muscle (1.4×) and myofibrils (1.7×), whereas the rate of force development after stimulation was the same as non-TG muscle for both electrical stimulation in intact muscle and after a Ca²âº jump in myofibrils. The EC50 for Ca²âº activation of force in myofibrils was 0.39 ± 0.33 µmol/l in ACTC E99K myofibrils and 0.80 ± 0.11 µmol/l in non-TG myofibrils. There were no significant differences in the amplitude and time course of the Ca²âº transient in myocytes from ACTC E99K and non-TG mice. We conclude that hypercontractility is caused by higher myofibrillar Ca²âº sensitivity in ACTC E99K muscles. Measurement of the energy (work + heat) released in actively cycling heart muscle showed that for both genotypes, the amount of energy turnover increased with work done but with decreasing efficiency as energy turnover increased. Thus, ACTC E99K mouse heart muscle produced on average 3.3-fold more work than non-TG muscle, and the cost in terms of energy turnover was disproportionately higher than in non-TG muscles. Efficiency for ACTC E99K muscle was in the range of 11-16% and for non-TG muscle was 15-18%.

The in vitro motility assay parameters of actin filaments from Mytilus edulis exposed in vivo to copper ions.

One important aspect of oxidative stress is chemical modification of lipids, proteins and nucleic acids. Copper has been shown to be one of the agents causing oxidative stress. In muscles copper binds to Cys-374 of the actin monomer and catalyzes interchain S-S bond formation in F-actin. The aim of the present work was to study the functional consequences of actin modifications, induced by copper treatment of Mytilus edulis in vivo, on the in vitro motility parameters of isolated actin filaments from foot and adductor muscles. CuCl(2) treatment reduced the sliding velocity of actin filaments extracted from foot muscle by about 22% and increased their flexibility by 1.7 times, while had no effect on the motility and flexibility of adductor actin. Using immunoblotting techniques we found that copper ions induced carbonylation in foot but not in adductor actin. In samples of foot actin an increase in cross-linked oligomers and truncated monomers was detected. Carbonylated structures of actin and corresponding changes in its functional properties may be considered as biomarkers for environmental monitoring.

Diffusion dynamics of motor-driven transport: gradient production and self-organization of surfaces.

The interaction between cytoskeletal filaments (e.g., actin filaments) and molecular motors (e.g., myosin) is the basis for many aspects of cell motility and organization of the cell interior. In the in vitro motility assay (IVMA), cytoskeletal filaments are observed while being propelled by molecular motors adsorbed to artificial surfaces (e.g., in studies of motor function). Here we integrate ideas that cytoskeletal filaments may be used as nanoscale templates in nanopatterning with a novel approach for the production of surface gradients of biomolecules and nanoscale topographical features. The production of such gradients is challenging but of increasing interest (e.g., in cell biology). First, we show that myosin-induced actin filament sliding in the IVMA can be approximately described as persistent random motion with a diffusion coefficient (D) given by a relationship analogous to the Einstein equation (D = kT/gamma). In this relationship, the thermal energy (kT) and the drag coefficient (gamma) are substituted by a parameter related to the free-energy transduction by actomyosin and the actomyosin dissociation rate constant, respectively. We then demonstrate how the persistent random motion of actin filaments can be exploited in conceptually novel methods for the production of actin filament density gradients of predictable shapes. Because of regularly spaced binding sites (e.g., lysines and cysteines) the actin filaments act as suitable nanoscale scaffolds for other biomolecules (tested for fibronectin) or nanoparticles. This forms the basis for secondary chemical and topographical gradients with implications for cell biological studies and biosensing.

Bending flexibility of actin filaments during motor-induced sliding.

Muscle contraction and other forms of cell motility occur as a result of cyclic interactions between myosin molecules and actin filaments. Force generation is generally attributed to ATP-driven structural changes in myosin, whereas a passive role is ascribed to actin. However, some results challenge this view, predicting structural changes in actin during motor activity, e.g., when the actin filaments slide on a myosin-coated surface in vitro. Here, we analyzed statistical properties of the sliding filament paths, allowing us to detect changes of this type. It is interesting to note that evidence for substantial structural changes that led to increased bending flexibility of the filaments was found in phalloidin-stabilized, but not in phalloidin-free, actin filaments. The results are in accordance with the idea that a high-flexibility structural state of actin is a prerequisite for force production, but not the idea that a low-to-high flexibility transition of the actin filament should be an important component of the force-generating step per se. Finally, our data challenge the general view that phalloidin-stabilized filaments behave as native actin filaments in their interaction with myosin. This has important implications, since phalloidin stabilization is a routine procedure in most studies of actomyosin function.