Heart Myofibrils Research Articles

Titin truncating variant cardiomyopathy and related sarcomere insufficiency causes high energy demand resulting in mitochondrial dysfunction, autophagosome formation, and apoptosis

Abstract Background/Objectives Titin truncating variants (TTNtv) are the most prevalent genetic cause of dilated cardiomyopathy (DCM), resulting in upregulation of cardiac transcripts of oxidative phosphorylation (1,2). However, the underlying molecular mechanism(s) and cellular consequences of these findings remain unknown. Methods and results To gain insight into the metabolic changes and cellular consequences of a TTNtv, metabolic, mitochondrial, and survival pathways were studied in human TTNtv DCM hearts and isolated cardiomyocytes of TTNtv mice. TTNtv resulted in a significant increase of cardiac transcripts of glycolysis, citric acid cycle, mitochondrial fission, autophagy, and apoptosis when comparing RNAseq in 24 TTNtv and 27 mutation-negative DCM cardiac biopsies. Furthermore, a decrease in the area of myofibrils in human TTNtv hearts (TTNtv vs. mutation-negative DCM: 46%, and 62%, P=0.001), and an increase of mitochondrial (49% and 31%, P=0,001) and autophagosome areas (4% and 2%, P=0.002) was observed using transmission electron microscopy (TEM). Similar patterns of cardiomyocyte disorganization and stress could be seen in TTNtv hearts of mice even without a phenotype. Additionally, observed swollen mitochondria by TEM and decreased quantity of OXPHOS proteins by immunoblotting in murine TTNtv hearts indicate mitochondrial stress. Mitochondrial oxygen consumption at baseline and the maximum respiration in TTNtv cardiomyocytes of mice increased by a factor of 1.8 and 1.5 respectively (both P≤0.05), compared to WT. Furthermore, palmitate oxidation in TTNtv cardiomyocytes increased by 1.3 fold (P=0.005) compared to WT mice, suggestive of increased energy demand in TTNtv. Conclusion Myofibrillar insufficiency in human TTNtv DCM augments the cardiac oxygen and energy consumption, leading to pronounced morphological and functional mitochondrial decompensation. Swelling, damage and fission of mitochondria is further characterized by autophagosome formation and increased apoptosis pathways in TTNtv hearts. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Double-Dose consortium by Dutch Cardiovascular Alliance (DCVA)

Study of the influence of etoxidol on expression of follistatin-like protein-1 (FSTL-1) in myocardium after experimental infarction in rats

In heart attack, FSTL-1 is actively secreted by cardiomyocytes, accelerates growth of heart myofibrils and stimulates of vascular endothelial growth factor expression. The aim of this work was to investigate the effect of Etoxidol on synthesis of FSTL-1 in rats after myocardial infarction. The experiments were performed on Wistar rats weighing 250-350 g with simulated myocardial infarction or intact (group 5). Animals of control groups (groups 1, 2) were treated with saline for 7 and 14 days; Ethoxidol (24 mg/kg) was injected to animals of experimental groups (group 3, 4) (the daily dose was 6.36 mg/animal) for 6 or 14 days. The injection volume was 0.2 ml. At the beginning and at the end of the study plasma concentrations of FSTL-1 were determined by the ELISA method. Myocardial FSTL-1 gene expression was determined by real-time PCR. At the end of the experiments, the hearts were also used for histochemical analysis. To determine the size of the scar formed after the modeled heart attack, we used the classic Mallory staining method. The results show that the development of experimental acute myocardial infarction is accompanied by a significant increase in FSTL-1 expression in the heart, which was detected on the 7th day and stored increased by 14 days after a heart attack. After therapy with Ethoxidol, a tendency to a decrease in the expression of FSTL-1 by the 14th day was observed; it coincided with the dynamics of the plasma protein FSTL-1 level. It can be assumed that the downregulation trend in the FSTL-1 expression is associated with a more effective repair process after a heart attack, since FSTL-1 increases precisely in response to myocardial damage and decreases when the incentives for its expression from damaged heart tissue are reduced. Indirectly, this assumption is confirmed by the detected tendency to reduce the size of post-infarction fibrosis in the treatment with Ethoxidol. The results indicate the ability of Ethoxidol to influence FSTL-1 synthesis of in rats after myocardial infarction.

Beneficial Effect of Ethanol Extract from Black Rice on Endothelin-1 Expression and Histological Structure of Heart during Pre-eclampsia

In the case of pre-eclampsia, abnormality form also be found in the heart, which is later can be diagnosed as a particular symptom, hypertension. Endothelin-1, potential vasoconstrictor derived from heart, responsible to the mechanism of indirect impairment utero- spiral arteries into development of generalized endothelial dysfunction. This study was designed to identify the effect of ethanol extract from black rice in pre-eclamptic model rat, specifically related to its ability to maintain physiological activity of cardiovascular system. In this study, animal model was induced by multi-dose of tryponil sulphate 70mg/kgBW at 9th, 10th, 11th day of pregnancy, and the groups were devided into negative and positive control, treatment groups: P1, P2, P3 differed by ethanol extract dosages (12,5mg/kgBW, 25mg/kgBW, 50mg/kgBW respectively). Treatment started from day 12th to 18th of pregnancy, continued by statistical analysis to examined endothelin-1 immunohistochemistry expression using one way ANOVA and Tuckey-Test. This study indicated both the lowest expression of endothelin-1 and the normal structure of heart myofibrils, after histological examination, were identically displayed at P3 (50mg/kgBW ethanol extract) among groups. According to this study, we suggested that the ethanol extract of black rice possibly has certain compound either directly or indirectly induced cardiovascular changes during pre-eclamptic condition.

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.

Sept7b is required for the subcellular organization of cardiomyocytes and cardiac function in zebrafish.

Myofibrils made up of actin, myosin, and associated proteins generate the contractile force in muscle, and, consequently, mutations in these proteins may lead to heart failure. Septins are a conserved family of small GTPases that associate with actin filaments, microtubules, and cellular membranes. Despite the importance of septins in cytoskeleton organization, their role in cardiomyocyte organization and function is poorly characterized. Here, we show that septin 7 is expressed in both embryonic and adult zebrafish hearts and elucidate the physiological significance of sept7b, the zebrafish ortholog of human septin 7, in the heart in embryonic and larval zebrafish. Knockdown of sept7b reduced F-actin and α-cardiac actin expression in the heart and caused disorganization of actin filaments. Electron microscopy of sept7b-depleted larvae showed disorganization of heart myofibrils and partial detachment from Z-disks. Functional studies revealed that knockdown of sept7b leads to reduced ventricular dimensions, contractility, and cardiac output. Furthermore, we found that depletion of sept7b diminished the expression of retinaldehyde dehydrogenase 2, which catalyzes the synthesis of retinoic acid necessary for heart morphogenesis. We further observed that the sept7b and retinoic acid signaling pathways converge to regulate cardiac function. Together, these results specify an essential role for sept7b in the contractile function of the heart.NEW & NOTEWORTHY Knockdown of the zebrafish ortholog of human septin 7 (sept7b) destabilizes cardiac actin and reduces ventricular dimensions, contractility, and cardiac output in larval zebrafish, indicating that sept7b is essential for cardiac function. We further found that sept7b and retinoic acid signaling pathways converge to regulate cardiac function. These data prompt further studies defining the role of sept7b in cardiomyopathies.

OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency.

BackgroundStudies of the functional consequences of DCM-causing mutations have been limited to a few cases where patients with known mutations had heart transplants. To increase the number of potential tissue samples for direct investigation we performed whole exon sequencing of explanted heart muscle samples from 30 patients that had a diagnosis of familial dilated cardiomyopathy and screened for potentially disease-causing mutations in 58 HCM or DCM-related genes.ResultsWe identified 5 potentially disease-causing OBSCN mutations in 4 samples; one sample had two OBSCN mutations and one mutation was judged to be not disease-related. Also identified were 6 truncating mutations in TTN, 3 mutations in MYH7, 2 in DSP and one each in TNNC1, TNNI3, MYOM1, VCL, GLA, PLB, TCAP, PKP2 and LAMA4. The mean level of obscurin mRNA was significantly greater and more variable in healthy donor samples than the DCM samples but did not correlate with OBSCN mutations. A single obscurin protein band was observed in human heart myofibrils with apparent mass 960 ± 60 kDa. The three samples with OBSCN mutations had significantly lower levels of obscurin immunoreactive material than DCM samples without OBSCN mutations (45±7, 48±3, and 72±6% of control level).Obscurin levels in DCM controls, donor heart and myectomy samples were the same.Conclusions OBSCN mutations may result in the development of a DCM phenotype via haploinsufficiency. Mutations in the obscurin gene should be considered as a significant causal factor of DCM, alone or in concert with other mutations.

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

Heart muscle contraction is regulated via the β-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. 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. 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 Ca2+ Regulation by Phosphorylation of Troponin I

Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca2+ sensitivity and increases the rate of Ca2+ 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 Ca2+-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca2+ 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 Ca2+ 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 Ca2+ sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca2+ 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 Ca2+ 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 Ca2+ 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.

Top-Down Mass Spectrometry on Tissue Extracts and Biofluids with Isoelectric Focusing and Superficially Porous Silica Liquid Chromatography

Top-down mass spectrometry (MS) has emerged as a powerful complement to peptide-based proteomics. Despite advancements, the field has had limited application to clinical proteomics investigations due to the complexity and poor dynamic range of chromatography used to separate intact proteins from tissue and biofluids. To address these limitations, we developed a two-dimensional (2D) chromatography platform that includes isoelectric focusing (IEF) through immobilized pH gradient and superficially porous liquid chromatography (SPLC). Analysis of standard proteins demonstrates compatibility of IEF-SPLC processing and high resolving-power MS analysis with results showing ~7.0 femtomole detection limits and linear spectral response for proteins fractionated over ~4 log sample loads. For proteins from heart myofibrils and cerebrospinal fluid (CSF), compared to one-dimensional SPLC-MS, the 2D IEF-SPLC-MS platform resulted in a 5-6× increase in the number of unique monoisotopic masses observed <30 kDa and an ~4× improved mass range enabling the observation of proteins >200 kDa. In the heart myofibrils, common protein proteoforms observed were associated with phosphorylation of contractile proteins with results showing that quantitative evaluation of their PTM stoichiometry was possible despite differentially modified forms being fractionated into separate pI compartments. In CSF, diverse protein mutations and PTM classes were also observed, including differentially glycosylated protein forms separated to different pI. Results also demonstrate that by the generation of IEF-SPLC protein libraries by fraction collection, the platform enables prospective protein identification and proteoform analysis investigations by complementary top-down and bottom-up strategies. Overall, the 2D platform presented may provide the speed, dynamic range, and detection limits necessary for routine characterization of proteoform-based biomarkers from biofluids and tissues.

Z-band Alternatively Spliced PDZ Motif Protein (ZASP) Is the Major O-Linked β-N-Acetylglucosamine-substituted Protein in Human Heart Myofibrils

We studied O-linked β-N-acetylglucosamine (O-GlcNAc) modification of contractile proteins in human heart using SDS-PAGE and three detection methods: specific enzymatic conjugation of O-GlcNAc with UDP-N-azidoacetylgalactosamine (UDP-GalNAz) that is then linked to a tetramethylrhodamine fluorescent tag and CTD110.6 and RL2 monoclonal antibodies to O-GlcNAc. All three methods showed that O-GlcNAc modification was predominantly in a group of bands ~90 kDa that did not correspond to any of the major myofibrillar proteins. MALDI-MS/MS identified the 90-kDa band as the protein ZASP (Z-band alternatively spliced PDZ motif protein), a minor component of the Z-disc (about 1 per 400 α-actinin) important for myofibrillar development and mechanotransduction. This was confirmed by the co-localization of O-GlcNAc and ZASP in Western blotting and by immunofluorescence microscopy. O-GlcNAcylation of ZASP increased in diseased heart, being 49 ± 5% of all O-GlcNAc in donor, 68 ± 9% in end-stage failing heart, and 76 ± 6% in myectomy muscle samples (donor versus myectomy p < 0.05). ZASP is only 22% of all O-GlcNAcylated proteins in mouse heart myofibrils.

Cardiac remodeling and subcellular defects in heart failure due to myocardial infarction and aging

Although several risk factors including hypertension, cardiac hypertrophy, coronary artery disease, and diabetes are known to result in heart failure, elderly subjects are more susceptible to myocardial infarction and more likely to develop heart failure. This article is intended to discuss that cardiac dysfunction in hearts failing due to myocardial infarction and aging is associated with cardiac remodeling and defects in the subcellular organelles such as sarcolemma (SL), sarcoplasmic reticulum (SR), and myofibrils. Despite some differences in the pattern of heart failure due to myocardial infarction and aging with respect to their etiology and sequence of events, evidence has been presented to show that subcellular remodeling plays a critical role in the occurrence of intracellular Ca(2+)-overload and development of cardiac dysfunction in both types of failing heart. In particular, alterations in gene expression for SL and SR proteins induce Ca(2+)-handling abnormalities in cardiomyocytes, whereas those for myofibrillar proteins impair the interaction of Ca(2+) with myofibrils in hearts failing due to myocardial infarction and aging. In addition, different phosphorylation mechanisms, which regulate the activities of Ca(2+)-cycling proteins in SL and SR membranes as well as Ca(2+)-binding proteins in myofibrils, become defective in the failing heart. Accordingly, it is suggested that subcellular remodeling involving defects in Ca(2+)-handling and Ca(2+)-binding proteins as well as their regulatory mechanisms is intimately associated with cardiac remodeling and heart failure due to myocardial infarction and aging.

Analysis of cardiac myosin binding protein-C phosphorylation in human heart muscle

A unique feature of MyBP-C in cardiac muscle is that it has multiple phosphorylation sites. MyBP-C phosphorylation, predominantly by PKA, plays an essential role in modulating contractility as part of the cellular response to β-adrenergic stimulation. In vitro studies indicate MyBP-C can be phosphorylated at Serine 273, 282, 302 and 307 (mouse sequence) but little is known about the level of MyBP-C phosphorylation or the sites phosphorylated in heart muscle. Since current methodologies are limited in specificity and are not quantitative we have investigated the use of phosphate affinity SDS-PAGE together with a total anti MyBP-C antibody and a range of phosphorylation site-specific antibodies for the main sites (Ser-273, -282 and -302). With these newly developed methods we have been able to make a detailed quantitative analysis of MyBP-C phosphorylation in heart tissue in situ. We have found that MyBP-C is highly phosphorylated in non-failing human (donor) heart or mouse heart; tris and tetra-phosphorylated species predominate and less than 10% of MyBP-C is unphosphorylated (0, 9.3 ± 1%: 1P, 13.4 ± 2.7%: 2P, 10.5 ± 3.3%: 3P, 28.7 ± 3.7%: 4P, 36.4 ± 2.7%, n = 21). Total phosphorylation was 2.7 ± 0.07 molPi/mol MyBP-C. In contrast in failing heart and in myectomy samples from HCM patients the majority of MyBP-C was unphosphorylated. Total phosphorylation levels were 23% of normal in failing heart myofibrils (0, 60.1 ± 2.8%: 1P, 27.8 ± 2.8%: 2P, 4.8 ± 2.0%: 3P, 3.7 ± 1.2%: 4P, 2.8 ± 1.3%, n = 19) and 39% of normal in myectomy samples. The site-specific antibodies showed a distinctive distribution pattern of phosphorylation sites in the multiple phosphorylation level species. We found that phosphorylated Ser-273, Ser-282 and Ser-302 were all present in the 4P band of MyBP-C but none of them were significant in the 1P band, indicating that there must be at least one other site of MyBP-C phosphorylation in human heart. The pattern of phosphorylation at the three sites was not random, but indicated positive and negative interactions between the three sites. Phosphorylation at Ser-282 was not proportional to the number of sites available. The 2P band contained 302 but not 273; the 3P band contained 273 but not 302.

Molecular and Functional Characterization of a Novel Cardiac-Specific Human Tropomyosin Isoform

Tropomyosin (TM), an essential actin-binding protein, is central to the control of calcium-regulated striated muscle contraction. Although TPM1alpha (also called alpha-TM) is the predominant TM isoform in human hearts, the precise TM isoform composition remains unclear. In this study, we quantified for the first time the levels of striated muscle TM isoforms in human heart, including a novel isoform called TPM1kappa. By developing a TPM1kappa-specific antibody, we found that the TPM1kappa protein is expressed and incorporated into organized myofibrils in hearts and that its level is increased in human dilated cardiomyopathy and heart failure. To investigate the role of TPM1kappa in sarcomeric function, we generated transgenic mice overexpressing cardiac-specific TPM1kappa. Incorporation of increased levels of TPM1kappa protein in myofilaments leads to dilated cardiomyopathy. Physiological alterations include decreased fractional shortening, systolic and diastolic dysfunction, and decreased myofilament calcium sensitivity with no change in maximum developed tension. Additional biophysical studies demonstrate less structural stability and weaker actin-binding affinity of TPM1kappa compared with TPM1alpha. This functional analysis of TPM1kappa provides a possible mechanism for the consequences of the TM isoform switch observed in dilated cardiomyopathy and heart failure patients.

Quantitative Analysis of MyBP-C Phosphorylation in Human Heart using Phosphate Affinity SDS-Page

Phosphorylation sites in Cardiac MyBP-C have been predicted at Ser273,282 and 302 but studies in intact tissue have identified 5 phosphorylted sites and suggested up to 4.6molsPi/mol MyBP-C is present in human heart.We analysed MyBP-C phosphospecies in human heart myofibrils by phosphate affinity SDS-PAGE using a non-specific antibody raised against the MyBP-C peptide 2-14. We observed six bands corresponding to 0, 1P, 2P, 3P, 4P, 5P phospho-species. Control experiments with pure MyBP-C indicated that the antibody labelled all phosphospecies equally. The assigned phosphorylation levels were confirmed by staining western blots with PhosTools phosphoprotein stain. This separation permits direct quantitative determination of MyBP-C phosphospecies without need for calibration.In donor heart myofibrils the highly phosphorylated species predominated: 0, 7±3%: 1P, 1±1%: 2P, 23±7%: 3P, 41±2%: 4P, 20±8% (n=4) from which total phosphorylation of MyBP-C was calculated to be 3.4molsPi/mol. In failing heart unphosphorylated MyBP-C predominated (0, 48±4%: 1P, 4±4%: 2P, 27±5%: 1±1%: 3P, 17±4%: 4P, 4±2%, n=4) and calculated total phosphorylation was 1.5 molsPi/mol. Total phosphorylation in failing heart myofibrils was 44% of donor and in myectomy samples from HCM patients it was 29% of donor, compared with 45 and 40% respectively determined in previous assays.We conclude that MyBP-C is highly phosphorylated in vivo with significant phosphorylation of at least 5 sites and that phosphorylation is dynamic, being greatly reduced in pathological muscle. Initial tests using antibodies specific to Ser 273, 282 and 302 show distinct patterns on phosphate affinity SDS-PAGE indicating varying preferences for the highly phosphorylated species of MyBP-C in normal and pathological muscle.

Modulation of Titin-Based Stiffness by Disulfide Bonding in the Cardiac Titin N2-B Unique Sequence

The giant protein titin is responsible for the elasticity of nonactivated muscle sarcomeres. Titin-based passive stiffness in myocardium is modulated by titin-isoform switching and protein-kinase (PK)A- or PKG-dependent titin phosphorylation. Additional modulatory effects on titin stiffness may arise from disulfide bonding under oxidant stress, as many immunoglobulin-like (Ig-)domains in titin's spring region have a potential for S-S formation. Using single-molecule atomic force microscopy (AFM) force-extension measurements on recombinant Ig-domain polyprotein constructs, we show that titin Ig-modules contain no stabilizing disulfide bridge, contrary to previous belief. However, we demonstrate that the human N2-B-unique sequence (N2-Bus), a cardiac-specific, physiologically extensible titin segment comprising 572 amino-acid residues, contains up to three disulfide bridges under oxidizing conditions. AFM force spectroscopy on recombinant N2-Bus molecules demonstrated a much shorter contour length in the absence of a reducing agent than in its presence, consistent with intramolecular S-S bonding. In stretch experiments on isolated human heart myofibrils, the reducing agent thioredoxin lowered titin-based stiffness to a degree that could be explained (using entropic elasticity theory) by altered extensibility solely of the N2-Bus. We conclude that increased oxidant stress can elevate titin-based stiffness of cardiomyocytes, which may contribute to the global myocardial stiffening frequently seen in the aging or failing heart.

Knockdown of alpha myosin heavy chain disrupts the cytoskeleton and leads to multiple defects during chick cardiogenesis

Atrial septal defects are a common congenital heart defect in humans. Although mutations in different genes are now frequently being described, little is known about the processes and mechanisms behind the early stages of atrial septal development. By utilizing morpholino-induced knockdown in the chick we have analysed the role of alpha myosin heavy chain during early cardiogenesis in a temporal manner. Upon knockdown of alpha myosin heavy chain, three different phenotypes of the atrial septum were observed: (1) the atrial septum failed to initiate, (2) the septum was initiated but was growth restricted, or (3) incorrect specification occurred resulting in multiple septa forming. In addition, at a lower frequency, decreased alpha myosin heavy chain was found to give rise to an abnormally looped heart or an enlarged heart. Staining of the actin cytoskeleton indicated that many of the myofibrils in the knockdown hearts were not as mature as those observed in the controls, suggesting a mechanism for the defects seen. Therefore, these data suggest a role for alpha myosin heavy chain in modelling of the early heart and the range of defects to the atrial septum suggest roles in its initiation, specification and growth during development.

Arginyltransferase regulates alpha cardiac actin function, myofibril formation and contractility during heart development

Post-translational arginylation mediated by arginyltransferase (Ate1) is essential for cardiovascular development and angiogenesis in mammals and directly affects myocardium structure in the developing heart. We recently showed that arginylation exerts a number of intracellular effects by modifying proteins involved in the functioning of the actin cytoskeleton and in cell motility. Here, we investigated the role of arginylation in the development and function of cardiac myocytes and their actin-containing structures during embryogenesis. Biochemical and mass spectrometry analyses showed that alpha cardiac actin undergoes arginylation at four sites during development. Ultrastructural analysis of the myofibrils in wild-type and Ate1 knockout mouse hearts showed that the absence of arginylation results in defects in myofibril structure that delay their development and affect the continuity of myofibrils throughout the heart, predicting defects in cardiac contractility. Comparison of cardiac myocytes derived from wild-type and Ate1 knockout mouse embryos revealed that the absence of arginylation results in abnormal beating patterns. Our results demonstrate cell-autonomous cardiac myocyte defects in arginylation knockout mice that lead to severe congenital abnormalities similar to those observed in human disease, and outline a new function of arginylation in the regulation of the actin cytoskeleton in cardiac myocytes.

Induction of initial heart α‐actin, smooth muscle α‐actin, in chick pregastrula epiblast: The role of hypoblast and fibroblast growth factor‐8

During heart development at the gastrula stage, inhibition of bone morphogenetic protein (BMP) activity affects the heart specification but does not impair the expression of smooth muscle alpha-actin (SMA), which is first expressed in the heart mesoderm and recruited into initial heart myofibrils. Interaction of tissues between posterior epiblast and hypoblast at the early blastula stage is necessary to induce the expression of SMA, in which Nodal and Chordin are thought to be involved. Here we investigated the role of fibroblast growth factor-8 (FGF8) in the expression of SMA. In situ hybridization and reverse transcription-polymerase chain reaction showed that Fgf8b is expressed predominantly in the nascent hypoblast. Anti-FGF8b antibody inhibited the expression of SMA, cTNT, and Tbx5, which are BMP-independent heart mesoderm/early cardiomyocyte genes, but not Brachyury in cultured posterior blastoderm, and combined FGF8b and Nodal, but neither factor alone induced the expression of SMA in association with heart specific markers in cultured epiblast. Although FGF8b did not induce the upregulation of phospho-Smad2, anti-FGF8b properties suppressed phospho-Smad2 in cultured blastoderm. FGF8b was able to reverse the BMP-induced inhibition of cardiomyogenesis. The results suggest that FGF8b acts on the epiblast synergistically with Nodal at the pregastrula stage and may play a role in the expression of SMA during early cardiogenesis.

Measurement of troponin phosphatase activities in non-failing and failing human heart muscle

We have demonstrated that troponin isolated from failing heart confers a higher Ca 2+-sensitivity and slower crossbridge turnover rate on thin filaments compared with donor heart muscle. We have also shown that the difference is due to very low levels of protein kinase A (PKA) and protein kinase C (PKC) catalysed troponin I phosphorylation in failing heart. Previous investigations of phosphatases have used non-physiological substrates. We therefore investigated the troponin dephosphorylation activity in human heart muscle using pure human cardiac troponin as the substrate and whole tissue and myofibrillar extracts of human heart as the source of the phosphatase. Using the Pro-Q diamond phosphoprotein gel stain to detect phosphorylated proteins we found that the crude phosphatase specifically dephosphorylated troponin I and not troponin T. When measured with PKA-phosphorylated recombinant troponin I as the substrate, heart muscle myofibrils contained substantial phosphatase activity (up to 40,000 pmol/mg myofibril/min). Phosphatase activity was 94% inhibited by 2 nM okadaic acid, 30% inhibited by 0.2 μM inhibitor 2 and Ca2+-activated phosphatase activity was less than 5%. All this indicates that protein phosphatase 2a is the predominant phosphatase type present in human heart myofibrils. The specific activity of the troponin I phosphatase was increased by about 19.3±7.4% in failing heart muscle myofibrils compared with non-failing heart muscle myofibrils. Therefore, enhanced phosphatase activity may be responsible for the low levels of phosphorylation observed in failing heart muscle.

Rho kinase inhibitor Y27632 affects initial heart myofibrillogenesis in cultured chick blastoderm

During early vertebrate development, Rho-associated kinases (ROCKs) are involved in various developmental processes. Here, we investigated spatiotemporal expression patterns of ROCK1 protein and examined the role of ROCK during initial heart myofibrillogenesis in cultured chick blastoderm. Immunohistochemistry showed that ROCK1 protein was distributed in migrating mesendoderm cells, visceral mesoderm of the pericardial coelom (from which cardiomyocytes will later develop), and cardiomyocytes of the primitive heart tube. Pharmacological inhibition of ROCK by Y27632 did not alter the myocardial specification process in cultured posterior blastoderm. However, Y27632 disturbed the formation of striated heart myofibrils in cultured posterior blastoderm. Furthermore, Y27632 affected the formation of costamere, a vinculin/integrin-based rib-like cell adhesion site. In such cardiomyocytes, cell-cell adhesion was disrupted and N-cadherin was distributed in the perinuclear region. Pharmacological inactivation of myosin light chain kinase, a downstream of ROCK, by ML-9 perturbed the formation of striated myofibrils as well as costameres, but not cell-cell adhesion. These results suggest that ROCK plays a role in the formation of initial heart myofibrillogenesis by means of actin-myosin assembly, and focal adhesion/costamere and cell-cell adhesion.