ABSTRACT
In this review, we present our current understanding of peripartum cardiomyopathy (PPCM) based on reports of the incidence, diagnosis and current treatment options. We summarise opinions on whether PPCM is triggered by vascular and/or hormonal causes and examine the influence of comorbidities such as preeclampsia. Two articles published in 2021 strongly support the hypothesis that PPCM may be a familial disease. Using large cohorts of PPCM patients, they summarised the available genomic DNA sequence data that are expressed in human cardiomyocytes. While PPCM is considered a disease predominately affecting the left ventricle, there are data to suggest that some cases also involve right ventricular failure. Finally, we conclude that there is sufficient evidence to warrant an RNAseq investigation and that this would be most informative if performed at the cardiomyocytes level rather than analysing genomic DNA from the peripheral circulation. Given the rarity of PPCM, the combined resources of international human heart tissue biobanks have assembled 30 ventricular tissue samples from PPCM patients, and we are actively seeking to enlarge this patient base by collaborating with human heart tissue banks and research laboratories who would like to join this endeavour.
ABSTRACT
AIMS: We investigated newly generated immortalized heterozygous and homozygous R349P desmin knock-in myoblasts in conjunction with the corresponding desminopathy mice as models for desminopathies to analyse major protein quality control processes in response to the presence of R349P mutant desmin. METHODS: We used hetero- and homozygous R349P desmin knock-in mice for analyses and for crossbreeding with p53 knock-out mice to generate immortalized R349P desmin knock-in skeletal muscle myoblasts and myotubes. Skeletal muscle sections and cultured muscle cells were investigated by indirect immunofluorescence microscopy, proteasomal activity measurements and immunoblotting addressing autophagy rate, chaperone-assisted selective autophagy and heat shock protein levels. Muscle sections were further analysed by transmission and immunogold electron microscopy. RESULTS: We demonstrate that mutant desmin (i) increases proteasomal activity, (ii) stimulates macroautophagy, (iii) dysregulates the chaperone assisted selective autophagy and (iv) elevates the protein levels of αB-crystallin and Hsp27. Both αB-crystallin and Hsp27 as well as Hsp90 displayed translocation patterns from Z-discs as well as Z-I junctions, respectively, to the level of sarcomeric I-bands in dominant and recessive desminopathies. CONCLUSIONS: Our findings demonstrate that the presence of R349P mutant desmin causes a general imbalance in skeletal muscle protein homeostasis via aberrant activity of all major protein quality control systems. The augmented activity of these systems and the subcellular shift of essential heat shock proteins may deleteriously contribute to the previously observed increased turnover of desmin itself and desmin-binding partners, which triggers progressive dysfunction of the extrasarcomeric cytoskeleton and the myofibrillar apparatus in the course of the development of desminopathies.
Subject(s)
Cardiomyopathies/genetics , Cardiomyopathies/physiopathology , Desmin/genetics , Muscle, Skeletal/physiopathology , Muscular Dystrophies/genetics , Muscular Dystrophies/physiopathology , Proteostasis/genetics , Animals , Autophagy/genetics , Disease Models, Animal , Mice , Muscle, Skeletal/metabolism , MutationABSTRACT
In the original version of this article, the name of one of the authors is not correct. The correct name should be W. A. Linke, which is shown correctly in the authorgroup section above.
ABSTRACT
The Sydney Heart Bank (SHB) is one of the largest human heart tissue banks in existence. Its mission is to provide high-quality human heart tissue for research into the molecular basis of human heart failure by working collaboratively with experts in this field. We argue that, by comparing tissues from failing human hearts with age-matched non-failing healthy donor hearts, the results will be more relevant than research using animal models, particularly if their physiology is very different from humans. Tissue from heart surgery must generally be used soon after collection or it significantly deteriorates. Freezing is an option but it raises concerns that freezing causes substantial damage at the cellular and molecular level. The SHB contains failing samples from heart transplant patients and others who provided informed consent for the use of their tissue for research. All samples are cryopreserved in liquid nitrogen within 40 min of their removal from the patient, and in less than 5-10 min in the case of coronary arteries and left ventricle samples. To date, the SHB has collected tissue from about 450 failing hearts (>15,000 samples) from patients with a wide range of etiologies as well as increasing numbers of cardiomyectomy samples from patients with hypertrophic cardiomyopathy. The Bank also has hearts from over 120 healthy organ donors whose hearts, for a variety of reasons (mainly tissue-type incompatibility with waiting heart transplant recipients), could not be used for transplantation. Donor hearts were collected by the St Vincent's Hospital Heart and Lung transplantation team from local hospitals or within a 4-h jet flight from Sydney. They were flushed with chilled cardioplegic solution and transported to Sydney where they were quickly cryopreserved in small samples. Failing and/or donor samples have been used by more than 60 research teams around the world, and have resulted in more than 100 research papers. The tissues most commonly requested are from donor left ventricles, but right ventricles, atria, interventricular system, and coronary arteries vessels have also been reported. All tissues are stored for long-term use in liquid N or vapor (170-180 °C), and are shipped under nitrogen vapor to avoid degradation of sensitive molecules such as RNAs and giant proteins. We present evidence that the availability of these human heart samples has contributed to a reduction in the use of animal models of human heart failure.
ABSTRACT
In the pathogenesis of dilated cardiomyopathy, cytoskeletal proteins play an important role. In this study, we analyzed titin expression in left ventricles of 19 control human donors and 9 severely diseased (nonischemic) dilated cardiomyopathy (DCM) transplant-patients, using gel-electrophoresis, immunoblotting, and quantitative RT-PCR. Both human-heart groups coexpressed smaller (approximately 3 MDa) N2B-isoform and longer (3.20 to 3.35 MDa) N2BA-isoforms, but the average N2BA:N2B-protein ratio was shifted from approximately 30:70 in controls to 42:58 in DCM hearts, due mainly to increased expression of N2BA-isoforms >3.30 MDa. Titin per unit tissue was decreased in some DCM hearts. The titin-binding protein obscurin also underwent isoform-shifting in DCM. Quantitative RT-PCR revealed a 47% reduction in total-titin mRNA levels in DCM compared with control hearts, but no differences in N2B, all-N2BA, and individual-N2BA transcripts. The reduction in total-titin transcripts followed from a decreased area occupied by myocytes and increased connective tissue in DCM hearts, as detected by histological analysis. Force measurements on isolated cardiomyofibrils showed that sarcomeric passive tension was reduced on average by 25% to 30% in DCM, a reduction readily predictable with a model of wormlike-chain titin elasticity. Passive-tension measurements on human-heart fiber bundles, before and after titin proteolysis, revealed a much-reduced relative contribution of titin to total passive stiffness in DCM. Results suggested that the titin-isoform shift in DCM depresses the proportion of titin-based stiffness by approximately 10%. We conclude that a lower-than-normal proportion of titin-based stiffness in end-stage failing hearts results partly from loss of titin and increased fibrosis, partly from titin-isoform shift. The titin-isoform shift may be beneficial for myocardial diastolic function, but could impair the contractile performance in systole.
Subject(s)
Cardiomyopathy, Dilated/pathology , Gene Expression Regulation/physiology , Muscle Proteins/physiology , Protein Kinases/physiology , Animals , Biomechanical Phenomena , Blotting, Western , Cardiomyopathy, Dilated/genetics , Cardiomyopathy, Dilated/metabolism , Connectin , Fibrosis , Guanine Nucleotide Exchange Factors/biosynthesis , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/physiology , Heart Failure/metabolism , Heart Failure/pathology , Heart Ventricles/chemistry , Heart Ventricles/pathology , Humans , Models, Biological , Molecular Weight , Muscle Proteins/biosynthesis , Muscle Proteins/chemistry , Muscle Proteins/genetics , Myocardium/pathology , Myofibrils/physiology , Pliability , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/physiology , Protein Kinases/biosynthesis , Protein Kinases/chemistry , Protein Kinases/genetics , Protein Serine-Threonine Kinases , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction , Rho Guanine Nucleotide Exchange Factors , Sus scrofaABSTRACT
Alcoholic myopathy is characterized by decreased protein synthesis and contents resulting in atrophy of muscle fibers. We investigated the effect of alcohol on the cytoskeletal muscle proteins, nebulin and titin. Because women are more susceptible than men to the toxic effects of alcohol, male and female rats were included. Four groups were investigated: alcoholic males, pair-fed males, alcoholic females, pair-fed females. Alcohol consumption per unit body weight was 12.9 g/kg.d, with no difference between males and females. After 10 wk, male and female rats fed alcohol had lower gastrocnemius and plantaris protein and RNA contents (P < 0.001), with no effect on soleus, indicating myopathy of type II fibers. The gastrocnemius was fractionated to measure myofibrillary protein contents. Low percentage SDS-gel electrophoresis was performed to determine myosin heavy chain (MHC), nebulin and titin contents. Alcohol reduced gastrocnemius myofibrillary protein and MHC contents, and the plantaris RNA/protein ratio (P < 0.01). The titin/MHC and nebulin/MHC ratios were unaffected, suggesting a concomitant reduction in titin and nebulin. The decreases in titin and nebulin contents may affect muscle function. An interaction between gender and alcohol was noted for the plantaris RNA/protein ratio (P < 0.025), suggesting a reduced capacity for muscle protein synthesis in females.
Subject(s)
Ethanol/adverse effects , Muscle Proteins/drug effects , Muscle, Skeletal/drug effects , Protein Kinases/drug effects , Animals , Connectin , Electrophoresis, Polyacrylamide Gel , Female , Male , Muscle, Skeletal/metabolism , Rats , Rats, Wistar , Sex FactorsABSTRACT
Passive stiffness was found to be increased in mouse soleus muscles lacking desmin. Because titin is considered to be the major source of muscle elasticity, the stiffening might be explainable by titin adaptation. To test this, passive mechanical properties of single skinned fibres of soleus muscles from desmin knockout and control mice were analysed by using various extension tests. Titin expression was studied by SDS-gel electrophoresis. Absence of desmin did not modify either electrophoretic mobility of the titin band (3700 kDa) or optical density-unit ratios between bands for titin and nebulin (congruent with 0.3) and bands for titin and myosin heavy chain (congruent with 0.08). Elastic properties of fibres were not altered in the absence of desmin since passive tensions were similar under quasi-static (56-66 kN m(-2)) and dynamic (100-118 kN m(-2)) conditions whatever the kind of fibre. Thus, titin is unlikely to be responsible for the large increase in passive stiffness observed in whole soleus muscles when desmin is lacking.
Subject(s)
Desmin/genetics , Muscle Contraction/physiology , Muscle Proteins/metabolism , Muscle, Skeletal/physiology , Protein Kinases/metabolism , Animals , Connectin , Elasticity , Electrophoresis, Polyacrylamide Gel , Mice , Mice, Knockout , Muscle Fibers, Fast-Twitch/chemistry , Muscle Fibers, Fast-Twitch/physiology , Muscle Fibers, Slow-Twitch/chemistry , Muscle Fibers, Slow-Twitch/physiology , Muscle Proteins/analysis , Muscle, Skeletal/cytology , Myosin Heavy Chains/analysis , Myosin Heavy Chains/metabolism , Protein Kinases/analysisABSTRACT
The giant muscle protein titin contains a unique sequence, the PEVK domain, the elastic properties of which contribute to the mechanical behavior of relaxed cardiomyocytes. Here, human N2-B-cardiac PEVK was expressed in Escherichia coli and tested-along with recombinant cardiac titin constructs containing immunoglobulin-like or fibronectin-like domains-for a possible interaction with actin filaments. In the actomyosin in vitro motility assay, only the PEVK construct inhibited actin filament sliding over myosin. The slowdown occurred in a concentration-dependent manner and was accompanied by an increase in the number of stationary actin filaments. High [Ca(2+)] reversed the PEVK effect. PEVK concentrations >/=10 microgram/mL caused actin bundling. Actin-PEVK association was found also in actin fluorescence binding assays without myosin at physiological ionic strength. In cosedimentation assays, PEVK-titin interacted weakly with actin at 0 degrees C, but more strongly at 30 degrees C, suggesting involvement of hydrophobic interactions. To probe the interaction in a more physiological environment, nonactivated cardiac myofibrils were stretched quickly, and force was measured during the subsequent hold period. The observed force decline could be fit with a three-order exponential-decay function, which revealed an initial rapid-decay component (time constant, 4 to 5 ms) making up 30% to 50% of the whole decay amplitude. The rapid, viscous decay component, but not the slower decay components, decreased greatly and immediately on actin extraction with Ca(2+)-independent gelsolin fragment, both at physiological sarcomere lengths and beyond actin-myosin overlap. Steady-state passive force dropped only after longer exposure to gelsolin. We conclude that interaction between PEVK-titin and actin occurs in the sarcomere and may cause viscous drag during diastolic stretch of cardiac myofibrils. The interaction could also oppose shortening during contraction.
Subject(s)
Actin Cytoskeleton/metabolism , Muscle Proteins/metabolism , Myocardium/metabolism , Myofibrils/metabolism , Protein Kinases/metabolism , Amino Acid Motifs/physiology , Animals , Binding, Competitive/physiology , Biological Assay , Chickens , Connectin , Humans , In Vitro Techniques , Macromolecular Substances , Muscle Proteins/genetics , Myocardial Contraction/physiology , Protein Binding/physiology , Protein Kinases/genetics , Protein Structure, Tertiary/physiology , Rabbits , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sarcomeres/physiology , Stress, Mechanical , Temperature , ViscosityABSTRACT
We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with alpha-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A-sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein-myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.
Subject(s)
Cell Nucleus/metabolism , Cytoplasm/metabolism , Microfilament Proteins/metabolism , Muscle Proteins/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Differentiation , Cell Line , Gene Expression , Green Fluorescent Proteins , Heat-Shock Response , Heating , Humans , Luminescent Proteins/genetics , Mice , Microfilament Proteins/classification , Microfilament Proteins/genetics , Microfilament Proteins/physiology , Molecular Sequence Data , Muscle Proteins/classification , Muscle Proteins/genetics , Muscle Proteins/physiology , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Muscle, Smooth/cytology , Muscle, Smooth/metabolism , Mutagenesis , Myocardium/metabolism , Nuclear Localization Signals/genetics , Nuclear Localization Signals/metabolism , Protein Transport , RNA, Messenger , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , Stress, Physiological , Thiazoles/pharmacology , ThiazolidinesABSTRACT
Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery. After extraction of the kettin-associated actin, the A-band edges were also stained. In contrast, the staining pattern of projectin, another IFM-I-band protein, was not altered by actin removal. Force measurements were performed on single IFM myofibrils to establish the passive length-tension relationship and record passive stiffness. Stiffness decreased within seconds during gelsolin incubation and to a similar degree upon kettin digestion with mu-calpain. Immunoblotting demonstrated the presence of kettin isoforms in normal Drosophila IFM myofibrils and in myofibrils from an actin-null mutant. Dotblot analysis revealed binding of COOH-terminal kettin domains to myosin. We conclude that kettin is attached not only to actin but also to the end of the thick filament. Kettin along with projectin may constitute the elastic filament system of insect IFM and determine the muscle's high stiffness necessary for stretch activation. Possibly, the two proteins modulate myofibrillar stiffness by expressing different size isoforms.
Subject(s)
Drosophila Proteins , Drosophila melanogaster/physiology , Insect Proteins/metabolism , Muscle Proteins/metabolism , Myofibrils/physiology , Sarcomeres/metabolism , Actins/metabolism , Animals , Biomechanical Phenomena , Calpain/pharmacology , Connectin , Flight, Animal , Gelsolin/pharmacology , Immunoblotting , Microscopy, Fluorescence , Protein Binding , Protein Isoforms , Sarcomeres/drug effects , Sarcomeres/ultrastructureABSTRACT
The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show that isolated psoas myofibrils respond to a stretch-hold protocol with a characteristic force decay that becomes more pronounced following stretch to above 2.6-microm sarcomere length. The force decay was readily reproducible by a Monte Carlo simulation taking into account both the kinetics of Ig-domain unfolding and the worm-like-chain model of entropic elasticity used to describe titin's elastic behavior. The modeling indicated that the force decay is explainable by the unfolding of only a very small number of Ig domains per titin molecule. The simulation also predicted that a unique sequence in titin, the PEVK domain, may undergo minor structural changes during sarcomere extension. Myofibrils subjected to 1-Hz cycles of stretch-release exhibited distinct hysteresis that persisted during repetitive measurements. Quick stretch-release protocols, in which variable pauses were introduced after the release, revealed a two-exponential time course of hysteresis recovery. The rate constants of recovery compared well with the refolding rates of Ig-like or fibronectin-like domains measured by single-protein mechanical analysis. These findings suggest that in the sarcomere, titin's Ig-domain regions may act as entropic springs capable of adjusting their contour length in response to a stretch.
Subject(s)
Muscle Proteins/chemistry , Muscle Proteins/physiology , Muscle, Skeletal/physiology , Myofibrils/physiology , Protein Kinases/chemistry , Protein Kinases/physiology , Amino Acid Sequence , Animals , Connectin , Elasticity , Kinetics , Models, Biological , Monte Carlo Method , Muscle Contraction/physiology , Myofibrils/ultrastructure , Protein Folding , Rats , Stress, Mechanical , Time Factors , ViscosityABSTRACT
Myosin-binding protein-C (MyBP-C) is a component of all striated-muscle sarcomeres, with a well established structural role and a possible function for force regulation. Multiple mutations within the gene for cardiac MyBP-C, one of three known isoforms, have been linked to familial hypertrophic cardiomyopathy. Here we generated a knock-in mouse model that carries N-terminal-shortened cardiac MyBP-C. The mutant protein was designed to have a similar size as the skeletal MyBP-C isoforms, whereas known myosin and titin binding sites as well as the phosphorylatable MyBP-C motif were not altered. We have shown that mutant cardiac MyBP-C is readily incorporated into the sarcomeres of both heterozygous and homozygous animals and can still be phosphorylated by cAMP-dependent protein kinase. Although histological characterization of wild-type and mutant hearts did not reveal obvious differences in phenotype, left ventricular fibers from homozygous mutant mice exhibited an increased Ca(2+) sensitivity of force development, particularly at lower Ca(2+) concentrations, whereas maximal active force levels remained unchanged. The results allow us to propose a model of how cMyBP-C may affect myosin-head mobility and to rationalize why N-terminal mutations of the protein in some cases of familial hypertrophic cardiomyopathy could lead to a hypercontractile state.
Subject(s)
Carrier Proteins/physiology , Gene Targeting , Heart/physiology , Myocardial Contraction , Animals , Calcium/metabolism , Carrier Proteins/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Heart/anatomy & histology , Mice , Models, Biological , Muscle Fibers, Skeletal/physiology , Myocardium/metabolism , Phosphorylation , RNA, Messenger/biosynthesis , Sequence DeletionABSTRACT
Skeletal-muscle titin contains in its I-band section two main elastic elements, stretches of Ig-like domains and the PEVK segment. Both elements contribute to the extensibility and passive force development of relaxed skeletal muscle fibers during stretch. To explore the nature of elasticity of the segments, their force-extension relation was determined with immunofluorescence and immunoelectron microscopy, combined with isolated myofibril mechanics. The results were then fitted with recent models of biopolymer elasticity. Whereas an entropic-spring mechanism may account for the elasticity of the Ig-domain segments, PEVK-titin elasticity appears to have both entropic and enthalpic origins. The modeling explains why the two elements extend sequentially upon stretch: elongation of the Ig-domain regions (with folded modules) is followed by unraveling of the PEVK domain. I-band titin in cardiac muscle is expressed in two main isoforms, N2-A and N2-B. The N2-A isoform is similar to that found in skeletal muscle, whereas the N2-B titin is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. By examining the extensibility of N2-B titin, it was found that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at low to modest stretch, and in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains.
Subject(s)
Muscle Proteins/chemistry , Muscle Proteins/physiology , Muscle, Skeletal/physiology , Myofibrils/physiology , Protein Kinases/chemistry , Protein Kinases/physiology , Sarcomeres/physiology , Animals , Connectin , Elasticity , Heart/physiology , Muscle, Skeletal/ultrastructure , Myocardium/ultrastructure , Protein Isoforms/chemistry , Protein Isoforms/physiology , ThermodynamicsABSTRACT
Titin, the giant protein of striated muscle, provides a continuous link between the Z-disk and the M-line of a sarcomere. The elastic I-band section of titin comprises two main structural elements, stretches of immunoglobulin-like domains and a unique sequence, the PEVK segment. Both elements contribute to the extensibility and passive force development of nonactivated muscle. Extensibility of the titin segments in skeletal muscle has been determined by immunofluorescence/immunoelectron microscopy of sarcomeres stained with sequence-assigned titin antibodies. The force developed upon stretch of titin has been measured on isolated molecules or recombinant titin fragments with the help of optical tweezers and the atomic force microscope. Force has also been measured in single isolated myofibrils. The force-extension relation of titin could be readily fitted with models of biopolymer elasticity. For physiologically relevant extensions, the elasticity of the titin segments was largely explainable by an entropic-spring mechanism. The modelling explains why during stretch of titin, the Ig-domain regions (with folded modules) extend before the PEVK domain. In cardiac muscle, I-band titin is expressed in different isoforms, termed N2-A and N2-B. The N2-A isoform resembles that of skeletal muscle, whereas N2-B titin is shorter and is distinguished by cardiac-specific Ig-motifs and nonmodular sequences within the central I-band section. Examination of N2-B titin extensibility revealed that this isoform extends by recruiting three distinct elastic elements: poly-Ig regions and the PEVK domain at lower stretch and, in addition, a unique 572-residue sequence insertion at higher physiological stretch. Extension of all three elements allows cardiac titin to stretch fully reversibly at physiological sarcomere lengths, without the need to unfold individual Ig domains. However, unfolding of a very small number of Ig domains remains a possibility.
Subject(s)
Muscle Proteins/physiology , Muscle, Skeletal/physiology , Protein Kinases/physiology , Animals , Connectin , Elasticity , Humans , Muscle Proteins/metabolism , Muscle, Skeletal/metabolism , Protein Kinases/metabolism , VertebratesABSTRACT
The study was aimed at determining both passive and Ca(2+)-activated forces of single skinned rat cardiac cells. Particular attention was paid to the descending limb of the active length-tension curve while the sarcomeric order of stretched cells was investigated before and during contraction. To analyse sarcomere length and sarcomere-length inhomogeneity, a fast Fourier transform (FFT) was employed. The fundamental frequency in the FFT spectrum is a measure of sarcomere length. The full-width-half-maximum of the first-order line is a measure of sarcomere-length inhomogeneity. In relaxing buffer, the sarcomere-length inhomogeneity of skinned cells increased linearly with mean sarcomere length. Upon Ca(2+)-dependent activation of skinned cells contracting isometrically, mean sarcomere length decreased slightly and inhomogeneity increased; both effects were greater at higher Ca(2+)concentrations. Maximum activation was reached at sarcomere lengths between 2.2 and 2.4 microm, whereas the descending limb of the active length-tension curve approached zero force already at approximately 2.8 microm. This steep force decline could not be explained by overly inhomogeneous sarcomere lengths in very long, contracting cells. Rather, the results of mechanical measurements on single cardiac myofibrils implied that high stretching is accompanied by irreversible structural alterations within cardiac sarcomeres, most likely thick-filament disarray and disruption of binding sites between myosin and titin due to changes in titin's tertiary structure. Loss of a regular thick-filament organization may then impair active force generation. We conclude that the descending limb of the cardiac length-tension curve is determined both by the degree of actin-myosin overlap and by the intrinsic properties of titin filaments.
Subject(s)
Myocardial Contraction , Sarcomeres/physiology , Stress, Mechanical , Actin Cytoskeleton/ultrastructure , Animals , Calcium/pharmacology , Calcium Signaling , Connectin , Muscle Proteins/chemistry , Myocardial Contraction/drug effects , Protein Kinases/chemistry , Protein Structure, Tertiary , Rats , Rats, Wistar , Sarcomeres/ultrastructureABSTRACT
In cardiac muscle, the giant protein titin exists in different length isoforms expressed in the molecule's I-band region. Both isoforms, termed N2-A and N2-B, comprise stretches of Ig-like modules separated by the PEVK domain. Central I-band titin also contains isoform-specific Ig-motifs and nonmodular sequences, notably a longer insertion in N2-B. We investigated the elastic behavior of the I-band isoforms by using single-myofibril mechanics, immunofluorescence microscopy, and immunoelectron microscopy of rabbit cardiac sarcomeres stained with sequence-assigned antibodies. Moreover, we overexpressed constructs from the N2-B region in chick cardiac cells to search for possible structural properties of this cardiac-specific segment. We found that cardiac titin contains three distinct elastic elements: poly-Ig regions, the PEVK domain, and the N2-B sequence insertion, which extends approximately 60 nm at high physiological stretch. Recruitment of all three elements allows cardiac titin to extend fully reversibly at physiological sarcomere lengths, without the need to unfold Ig domains. Overexpressing the entire N2-B region or its NH(2) terminus in cardiac myocytes greatly disrupted thin filament, but not thick filament structure. Our results strongly suggest that the NH(2)-terminal N2-B domains are necessary to stabilize thin filament integrity. N2-B-titin emerges as a unique region critical for both reversible extensibility and structural maintenance of cardiac myofibrils.
Subject(s)
Actin Cytoskeleton/metabolism , Muscle Proteins/metabolism , Myocardium/metabolism , Myofibrils/metabolism , Protein Kinases/metabolism , Actin Cytoskeleton/ultrastructure , Actins/metabolism , Animals , Antibodies/immunology , Cells, Cultured , Chickens , Connectin , Elasticity , Epitopes/immunology , Microscopy, Immunoelectron , Models, Biological , Molecular Motor Proteins/metabolism , Muscle Proteins/chemistry , Muscle Proteins/genetics , Myocardium/cytology , Myocardium/ultrastructure , Myofibrils/ultrastructure , Myosins/metabolism , Protein Folding , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Kinases/chemistry , Protein Kinases/genetics , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sarcomeres/metabolism , Sarcomeres/ultrastructure , TransfectionABSTRACT
The giant elastic protein titin is largely responsible for passive forces in cardiac myocytes. A number of different titin isoforms with distinctly different structural elements within their central I-band region are expressed in human myocardium. Their coexpression has so far prevented an understanding of the respective contributions of the isoforms to myocardial elasticity. Using isoform-specific antibodies, we find in the present study that rat myocardium expresses predominantly the small N2B titin isoform, which allows us to characterize the elastic behavior of this isoform. The extensibility and force response of N2B titin were studied by using immunoelectron microscopy and by measuring the passive force-sarcomere length (SL) relation of single rat cardiac myocytes under a variety of mechanical conditions. Experimental results were compared with the predictions of a mechanical model in which the elastic titin segment behaves as two wormlike chains, the tandem immunoglobulin (Ig) segments and the PEVK segment (rich in proline [P], glutamate [E], valine [V], and lysine [K] residues), connected in series. The overall contour length was predicted from the sequence of N2B cardiac titin. According to mechanical measurements, above approximately 2.2 microm SL titin's elastic segment extends beyond its predicted contour length. Immunoelectron microscopy indicates that a prominent source of this contour-length gain is the extension of the unique N2B sequence (located between proximal tandem Ig segment and PEVK), and that Ig domain unfolding is negligible. Thus, the elastic region of N2B cardiac titin consists of three mechanically distinct extensible segments connected in series: the tandem Ig segment, the PEVK segment, and the unique N2B sequence. Rate-dependent and repetitive stretch-release experiments indicate that both the contour-length gain and the recovery from it involve kinetic processes, probably unfolding and refolding within the N2B segment. As a result, the contour length of titin's extensible segment depends on the rate and magnitude of the preceding mechanical perturbations. The rate of recovery from the length gain is slow, ensuring that the adjusted length is maintained through consecutive cardiac cycles and that hysteresis is minimal. Thus, as a result of the extensible properties of the unique N2B sequence, the I-band region of the N2B cardiac titin isoform functions as a molecular spring that is adjustable.