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.

Myopathy-inducing mutation H40Y in ACTA1 hampers actin filament structure and function.

In humans, more than 200 missense mutations have been identified in the ACTA1 gene. The exact molecular mechanisms by which, these particular mutations become toxic and lead to muscle weakness and myopathies remain obscure. To address this, here, we performed a molecular dynamics simulation, and we used a broad range of biophysical assays to determine how the lethal and myopathy-related H40Y amino acid substitution in actin affects the structure, stability, and function of this protein. Interestingly, our results showed that H40Y severely disrupts the DNase I-binding-loop structure and actin filaments. In addition, we observed that normal and mutant actin monomers are likely to form distinctive homopolymers, with mutant filaments being very stiff, and not supporting proper myosin binding. These phenomena underlie the toxicity of H40Y and may be considered as important triggering factors for the contractile dysfunction, muscle weakness and disease phenotype seen in patients.

Mutations in troponin T associated with Hypertrophic Cardiomyopathy increase Ca(2+)-sensitivity and suppress the modulation of Ca(2+)-sensitivity by troponin I phosphorylation.

We investigated the effect of 7 Hypertrophic Cardiomyopathy (HCM)-causing mutations in troponin T (TnT) on troponin function in thin filaments reconstituted with actin and human cardiac tropomyosin. We used the quantitative in vitro motility assay to study Ca(2+)-regulation of unloaded movement and its modulation by troponin I phosphorylation. Troponin from a patient with the K280N TnT mutation showed no difference in Ca(2+)-sensitivity when compared with donor heart troponin and the Ca(2+)-sensitivity was also independent of the troponin I phosphorylation level (uncoupled). The recombinant K280N TnT mutation increased Ca(2+)-sensitivity 1.7-fold and was also uncoupled. The R92Q TnT mutation in troponin from transgenic mouse increased Ca(2+)-sensitivity and was also completely uncoupled. Five TnT mutations (Δ14, Δ28 + 7, ΔE160, S179F and K273E) studied in recombinant troponin increased Ca(2+)-sensitivity and were all fully uncoupled. Thus, for HCM-causing mutations in TnT, Ca(2+)-sensitisation together with uncoupling in vitro is the usual response and both factors may contribute to the HCM phenotype. We also found that Epigallocatechin-3-gallate (EGCG) can restore coupling to all uncoupled HCM-causing TnT mutations. In fact the combination of Ca(2+)-desensitisation and re-coupling due to EGCG completely reverses both the abnormalities found in troponin with a TnT HCM mutation suggesting it may have therapeutic potential.

Troponin structure: its modulation by Ca(2+) and phosphorylation studied by molecular dynamics simulations.

The only available crystal structure of the human cardiac troponin molecule (cTn) in the Ca(2+) activated state does not include crucial segments, including the N-terminus of the cTn inhibitory subunit (cTnI). We have applied all-atom molecular dynamics (MD) simulations to study the structure and dynamics of cTn, both in the unphosphorylated and bis-phosphorylated states at Ser23/Ser24 of cTnI. We performed multiple microsecond MD simulations of wild type (WT) cTn (6, 5 µs) and bisphosphorylated (SP23/SP24) cTn (9 µs) on a 419 amino acid cTn model containing human sequence cTnC (1-161), cTnI (1-171) and cTnT (212-298), including residues not present in the crystal structure. We have compared our results to previous computational studies, and proven that longer simulations and a water box of at least 25 Å are needed to sample the interesting conformational shifts both in the native and bis-phosphorylated states. As a consequence of the introduction into the model of the C-terminus of cTnT that was missing in previous studies, cTnC-cTnI interactions that are responsible for the cTn dynamics are altered. We have also shown that phosphorylation does not increase cTn fluctuations, and its effects on the protein-protein interaction profiles cannot be assessed in a significant way. Finally, we propose that phosphorylation could provoke a loss of Ca(2+) by stabilizing out-of-coordination distances of the cTnC's EF hand II residues, and in particular Ser 69.

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.

Z-band alternatively spliced PDZ motif protein (ZASP) is the major O-linked ß-N-acetylglucosamine-substituted protein in human heart myofibrils.

We studied O-linked ß-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.

Tropomyosin isoform expression and phosphorylation in the human heart in health and disease.

We determined the isoforms of tropomyosin expressed and the level of tropomyosin phosphorylation in donor, end-stage failing and hypertrophic obstructive cardiomyopathy samples of human heart muscle. Western blots and isoform-specific antibodies showed that α-tropomyosin was the only significant isoform expressed and that tropomyosin was 25-30% phosphorylated at serine 283. Mass spectrometry confirmed directly that α-tropomyosin made up over 95% of tropomyosin but also indicated the presence of up to 4% κ-tropomyosin and much smaller amounts of ß-, γ- and smooth ß-tropomyosin and about 26% phosphorylation. Neither the isoform distribution nor the level of phosphorylation changed significantly in the pathological heart muscle samples.

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 ß-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 mol Pi/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.

Investigation of changes in skeletal muscle alpha-actin expression in normal and pathological human and mouse hearts.

We have developed a quantitative antibody-based assay to measure the content of skeletal muscle alpha-actin relative to cardiac alpha-actin. We found 21 +/- 2% skeletal muscle alpha-actin content in normal heart muscle of adult man and mouse. In end stage failing heart 53 +/- 5% of striated actin was skeletal muscle alpha-actin and in samples of inter-ventricular septum from patients with hypertrophic obstructive cardiomyopathy (HOCM) skeletal muscle alpha-actin was 72 +/- 2% of sarcomeric actin. Thin filaments containing actin isolated from normal and HOCM heart muscle were functionally indistinguishable when studied by quantitative in vitro motility assay. We also found elevated skeletal muscle alpha-actin (60 +/- 7%) in a mouse model of dilated cardiomyopathy.

The molecular phenotype of human cardiac myosin associated with hypertrophic obstructive cardiomyopathy.

AIM: The aim of the study was to compare the functional and structural properties of the motor protein, myosin, and isolated myocyte contractility in heart muscle excised from hypertrophic cardiomyopathy patients by surgical myectomy with explanted failing heart and non-failing donor heart muscle. METHODS: Myosin was isolated and studied using an in vitro motility assay. The distribution of myosin light chain-1 isoforms was measured by two-dimensional electrophoresis. Myosin light chain-2 phosphorylation was measured by sodium dodecyl sulphate-polyacrylamide gel electrophoresis using Pro-Q Diamond phosphoprotein stain. RESULTS: The fraction of actin filaments moving when powered by myectomy myosin was 21% less than with donor myosin (P = 0.006), whereas the sliding speed was not different (0.310 +/- 0.034 for myectomy myosin vs. 0.305 +/- 0.019 microm/s for donor myosin in six paired experiments). Failing heart myosin showed 18% reduced motility. One myectomy myosin sample produced a consistently higher sliding speed than donor heart myosin and was identified with a disease-causing heavy chain mutation (V606M). In myectomy myosin, the level of atrial light chain-1 relative to ventricular light chain-1 was 20 +/- 5% compared with 11 +/- 5% in donor heart myosin and the level of myosin light chain-2 phosphorylation was decreased by 30-45%. Isolated cardiomyocytes showed reduced contraction amplitude (1.61 +/- 0.25 vs. 3.58 +/- 0.40%) and reduced relaxation rates compared with donor myocytes (TT(50%) = 0.32 +/- 0.09 vs. 0.17 +/- 0.02 s). CONCLUSION: Contractility in myectomy samples resembles the hypocontractile phenotype found in end-stage failing heart muscle irrespective of the primary stimulus, and this phenotype is not a direct effect of the hypertrophy-inducing mutation. The presence of a myosin heavy chain mutation causing hypertrophic cardiomyopathy can be predicted from a simple functional assay.

Myosin binding protein C phosphorylation in normal, hypertrophic and failing human heart muscle.

Phosphorylation of myosin binding protein C (MyBP-C) was investigated in intraventricular septum samples taken from patients with hypertrophic cardiomyopathy undergoing surgical septal myectomy. These samples were compared with donor heart muscle, as a well-characterised control tissue, and with end-stage failing heart muscle. MyBP-C was partly purified from myofibrils using a modification of the phosphate-EDTA extraction of Hartzell and Glass. MyBP-C was separated by SDS-PAGE and stained for phosphoproteins using Pro-Q Diamond followed by total protein staining using Coomassie Blue. Relative phosphorylation level was determined from the ratio of Pro-Q Diamond to Coomassie Blue staining of MyBP-C bands as measured by densitometry. We compared 9 myectomy samples and 9 failing heart samples with 9 donor samples. MyBP-C phosphorylation in pathological muscle was lower than in donor (myectomy 40+/-2% of donor, P<0.0001; failing 45+/-3% of donor, P<0.0001). 6 myectomy samples were identified with MYBPC3 mutations, one with MYH7 mutation and two remained unknown, but there was no correlation between MYBPC3 mutation and MyBP-C phosphorylation level. In order to determine the number of phosphorylated sites in human cardiac MyBP-C samples, we phosphorylated the recombinant MyBP-C fragment, C0-C2 (1-453) with PKA using (gamma32)P-ATP up to 3.5 mol Pi/mol C0-C2. This measurement of phosphorylation was used to calibrate measurements of phosphorylation in SDS-PAGE using Pro-Q Diamond stain. The level of phosphorylation in donor heart MyBP-C was calculated to be 4.6+/-0.6 mol Pi/mol and 2.0+/-0.3 mol Pi/mol in myectomy samples. We conclude that MyBP-C is a highly phosphorylated protein in vivo and that diminished MyBP-C phosphorylation is a feature of both end-stage heart failure and hypertrophic cardiomyopathy.

Myofilament dysfunction in cardiac disease from mice to men.

In healthy human myocardium a tight balance exists between receptor-mediated kinases and phosphatases coordinating phosphorylation of regulatory proteins involved in cardiomyocyte contractility. During heart failure, when neurohumoral stimulation increases to compensate for reduced cardiac pump function, this balance is perturbed. The imbalance between kinases and phosphatases upon chronic neurohumoral stimulation is detrimental and initiates cardiac remodelling, and phosphorylation changes of regulatory proteins, which impair cardiomyocyte function. The main signalling pathway involved in enhanced cardiomyocyte contractility during increased cardiac load is the beta-adrenergic signalling route, which becomes desensitized upon chronic stimulation. At the myofilament level, activation of protein kinase A (PKA), the down-stream kinase of the beta-adrenergic receptors (beta-AR), phosphorylates troponin I, myosin binding protein C and titin, which all exert differential effects on myofilament function. As a consequence of beta-AR down-regulation and desensitization, phosphorylation of the PKA-target proteins within the cardiomyocyte may be decreased and alter myofilament function. Here we discuss involvement of altered PKA-mediated myofilament protein phosphorylation in different animal and human studies, and discuss the roles of troponin I, myosin binding protein C and titin in regulating myofilament dysfunction in cardiac disease. Data from the different animal and human studies emphasize the importance of careful biopsy procurement, and the need to investigate localization of kinases and phosphatases within the cardiomyocyte, in particular their co-localization with cardiac myofilaments upon receptor stimulation.

Molecular Defects in Cardiac Myofilament Ca2+-Regulation Due to Cardiomyopathy-Linked Mutations Can Be Reversed by Small Molecules Binding to Troponin.

The inherited cardiomyopathies, hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are relatively common, potentially life-threatening and currently untreatable. Mutations are often in the contractile proteins of cardiac muscle and cause abnormal Ca2+ regulation via troponin. HCM is usually linked to higher myofilament Ca2+-sensitivity whilst in both HCM and DCM mutant tissue there is often an uncoupling of the relationship between troponin I (TnI) phosphorylation by PKA and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline. The adrenergic response is blunted, and this may predispose the heart to failure under stress. At present there are no compounds or interventions that can prevent or treat sarcomere cardiomyopathies. There is a need for novel therapies that act at a more fundamental level to affect the disease process. We demonstrated that epigallocatechin-3 gallate (EGCG) was found to be capable of restoring the coupled relationship between Ca2+-sensitivity and TnI phosphorylation in mutant thin filaments to normal in vitro, independent of the mutation (15 mutations tested). We have labeled this property "re-coupling." The action of EGCG in vitro to reverse the abnormality caused by myopathic mutations would appear to be an ideal pharmaceutical profile for treatment of inherited HCM and DCM but EGCG is known to be promiscuous in vivo and is thus unsuitable as a therapeutic drug. We therefore investigated whether other structurally related compounds can re-couple myofilaments without these off-target effects. We used the quantitative in vitro motility assay to screen 40 compounds, related to C-terminal Hsp90 inhibitors, and found 23 that can re-couple mutant myofilaments. There is no correlation between re-couplers and Hsp90 inhibitors. The Ca2+-sensitivity shift due to TnI phosphorylation was restored to 2.2 ± 0.01-fold (n = 19) compared to 2.0 ± 0.24-fold (n = 7) in wild-type thin filaments. Many of these compounds were either pure re-couplers or pure desensitizers, indicating these properties are independent; moreover, re-coupling ability could be lost with small changes of compound structure, indicating the possibility of specificity. Small molecules that can re-couple may have therapeutic potential. HIGHLIGHTS - Inherited cardiomyopathies are common diseases that are currently untreatable at a fundamental level and therefore finding a small molecule treatment is highly desirable.- We have identified a molecular level dysfunction common to nearly all mutations: uncoupling of the relationship between troponin I phosphorylation and modulation of myofilament Ca2+-sensitivity, essential for normal responses to adrenaline.- We have identified a new class of drugs that are capable of both reducing Ca2+-sensitivity and/or recouping the relationship between troponin I phosphorylation and Ca2+-sensitivity.- The re-coupling phenomenon can be explained on the basis of a single mechanism that is testable.- Measurements with a wide range of small molecules of varying structures can indicate the critical molecular features required for recoupling and allows the prediction of other potential re-couplers.

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.

Investigations into the Sarcomeric Protein and Ca2+-Regulation Abnormalities Underlying Hypertrophic Cardiomyopathy in Cats (Felix catus).

Hypertrophic cardiomyopathy (HCM) is the most common single gene inherited cardiomyopathy. In cats (Felix catus) HCM is even more prevalent and affects 16% of the outbred population and up to 26% in pedigree breeds such as Maine Coon and Ragdoll. Homozygous MYBPC3 mutations have been identified in these breeds but the mutations in other cats are unknown. At the clinical and physiological level feline HCM is closely analogous to human HCM but little is known about the primary causative mechanism. Most identified HCM causing mutations are in the genes coding for proteins of the sarcomere. We therefore investigated contractile and regulatory proteins in left ventricular tissue from 25 cats, 18 diagnosed with HCM, including a Ragdoll cat with a homozygous MYBPC3 R820W, and 7 non-HCM cats in comparison with human HCM (from septal myectomy) and donor heart tissue. Myofibrillar protein expression was normal except that we observed 20-44% MyBP-C haploinsufficiency in 5 of the HCM cats. Troponin extracted from 8 HCM and 5 non-HCM cat hearts was incorporated into thin filaments and studied by in vitro motility assay. All HCM cat hearts had a higher (2.06 ± 0.13 fold) Ca2+-sensitivity than non-HCM cats and, in all the HCM cats, Ca2+-sensitivity was not modulated by troponin I phosphorylation. We were able to restore modulation of Ca2+-sensitivity by replacing troponin T with wild-type protein or by adding 100 µM Epigallocatechin 3-gallate (EGCG). These fundamental regulatory characteristics closely mimic those seen in human HCM indicating a common molecular mechanism that is independent of the causative mutation. Thus, the HCM cat is a potentially useful large animal model.

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.

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.

OBSCN Mutations Associated with Dilated Cardiomyopathy and Haploinsufficiency.

BACKGROUND: Studies 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. RESULTS: We 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.

Investigating the role of uncoupling of troponin I phosphorylation from changes in myofibrillar Ca(2+)-sensitivity in the pathogenesis of cardiomyopathy.

Contraction in the mammalian heart is controlled by the intracellular Ca(2+) concentration as it is in all striated muscle, but the heart has an additional signaling system that comes into play to increase heart rate and cardiac output during exercise or stress. ß-adrenergic stimulation of heart muscle cells leads to release of cyclic-AMP and the activation of protein kinase A which phosphorylates key proteins in the sarcolemma, sarcoplasmic reticulum and contractile apparatus. Troponin I (TnI) and Myosin Binding Protein C (MyBP-C) are the prime targets in the myofilaments. TnI phosphorylation lowers myofibrillar Ca(2+)-sensitivity and increases the speed of Ca(2+)-dissociation and relaxation (lusitropic effect). Recent studies have shown that this relationship between Ca(2+)-sensitivity and TnI phosphorylation may be unstable. In familial cardiomyopathies, both dilated and hypertrophic (DCM and HCM), a mutation in one of the proteins of the thin filament often results in the loss of the relationship (uncoupling) and blunting of the lusitropic response. For familial dilated cardiomyopathy in thin filament proteins it has been proposed that this uncoupling is causative of the phenotype. Uncoupling has also been found in human heart tissue from patients with hypertrophic obstructive cardiomyopathy as a secondary effect. Recently, it has been found that Ca(2+)-sensitizing drugs can promote uncoupling, whilst one Ca(2+)-desensitizing drug Epigallocatechin 3-Gallate (EGCG) can reverse uncoupling. We will discuss recent findings about the role of uncoupling in the development of cardiomyopathies and the molecular mechanism of the process.

Familial dilated cardiomyopathy mutations uncouple troponin I phosphorylation from changes in myofibrillar Ca²âº sensitivity.

AIMS: The pure form of familial dilated cardiomyopathy (DCM) is mainly caused by mutations in genes encoding sarcomeric proteins. Previous measurements using recombinant proteins suggested that DCM mutations in thin filament proteins decreased myofibrillar Ca(2+) sensitivity, but exceptions were reported. We re-investigated the molecular mechanism of familial DCM using native proteins. METHODS AND RESULTS: We used the quantitative in vitro motility assay and native troponin and tropomyosin to study DCM mutations in troponin I, troponin T, and α-tropomyosin. Four mutations reduced myofilament Ca(2+) sensitivity, but one mutation (TPM1 E54K) did not alter Ca(2+) sensitivity and another (TPM1 D230N) increased Ca(2+) sensitivity. In thin filaments from normal human and mouse heart, protein kinase A (PKA) phosphorylation of troponin I caused a two- to three-fold decrease in myofibrillar Ca(2+) sensitivity. However, Ca(2+) sensitivity did not change with the level of troponin I phosphorylation in any of the DCM-mutant containing thin filaments (E40K, E54K, and D230N in α-tropomyosin; R141W and ΔK210 in cardiac troponin T; K36Q in cardiac troponin I; G159D in cardiac troponin C, and E361G in cardiac α-actin). This 'uncoupling' was observed with native mutant protein from human and mouse heart and with recombinant mutant protein expressed in baculovirus/Sf9 systems. Uncoupling was independent of the fraction of mutated protein present above 0.55. CONCLUSION: We conclude that DCM-causing mutations in thin filament proteins abolish the relationship between myofilament Ca(2+) sensitivity and troponin I phosphorylation by PKA. We propose that this blunts the response to ß-adrenergic stimulation and could be the cause of DCM in the long term.