Regulation Of Cardiac Contraction Research Articles

MicroRNA-1 Deficiency Is a Primary Etiological Factor Disrupting Cardiac Contractility and Electrophysiological Homeostasis.

MicroRNA-1 (miR1), encoded by the genes miR1-1 and miR1-2, is the most abundant microRNA in the heart and plays a critical role in heart development and physiology. Dysregulation of miR1 has been associated with various heart diseases, where a significant reduction (>75%) in miR1 expression has been observed in patient hearts with atrial fibrillation or acute myocardial infarction. However, it remains uncertain whether miR1-deficiency acts as a primary etiological factor of cardiac remodeling. miR1-1 or miR1-2 knockout mice were crossbred to produce 75%-miR1-knockdown (75%KD; miR1-1+/-:miR1-2-/- or miR1-1-/-:miR1-2+/-) mice. Cardiac pathology of 75%KD cardiomyocytes/hearts was investigated by ECG, patch clamping, optical mapping, transcriptomic, and proteomic assays. In adult 75%KD hearts, the overall miR1 expression was reduced to ≈25% of the normal wild-type level. These adult 75%KD hearts displayed decreased ejection fraction and fractional shortening, prolonged QRS and QT intervals, and high susceptibility to arrhythmias. Adult 75%KD cardiomyocytes exhibited prolonged action potentials with impaired repolarization and excitation-contraction coupling. Comparatively, 75%KD cardiomyocytes showcased reduced Na+ current and transient outward potassium current, coupled with elevated L-type Ca2+ current, as opposed to wild-type cells. RNA sequencing and proteomics assays indicated negative regulation of cardiac muscle contraction and ion channel activities, along with a positive enrichment of smooth muscle contraction genes in 75%KD cardiomyocytes/hearts. miR1 deficiency led to dysregulation of a wide gene network, with miR1's RNA interference-direct targets influencing many indirectly regulated genes. Furthermore, after 6 weeks of bi-weekly intravenous tail-vein injection of miR1 mimics, the ejection fraction and fractional shortening of 75%KD hearts showed significant improvement but remained susceptible to arrhythmias. miR1 deficiency acts as a primary etiological factor in inducing cardiac remodeling via disrupting heart regulatory homeostasis. Achieving stable and appropriate microRNA expression levels in the heart is critical for effective microRNA-based therapy in cardiovascular diseases.

T2D triggers distinct cardiac transcriptomic signatures in patients with severe aortic stenosis

Abstract Type 2 diabetes (T2D) is often associated to left ventricular (LV) pressure overload, as in aortic stenosis (AS). We reported, on DIAPASON cohort, that T2D increases adverse cardiac remodelling and dysfunction in patients with severe AS referred for aortic valve replacement. We aimed to study the underlying molecular mechanisms by a transcriptomic approach. We realised a transcriptomic analysis of LV cardiac biopsies obtained during aortic valve surgery from 17 T2D and 30 non-diabetic patients. Principal component analysis (PCA), on the 1029 significantly differentially expressed genes, shows a general segregation between T2D and non-diabetic patients. Functional enrichment maps reveal that 4 major biological processes are upregulated in T2D patients: regulation of inflammation, adaptation to cardiac hypertrophy, regulation of endothelial function/angiogenesis, and organisation of extracellular matrix. Decreased gene expression by T2D gathers 4 major biological processes: regulation of cardiac muscle contraction, mitochondrial organization, organization of mitochondrial respiratory chain and amino acid metabolism. After adjustment with other T2D comorbidities (age, sex, hypertension, BMI or the 4 combined), T2D effect remains mainly significant on the upregulated biological processes whereas the downregulation of amino acid metabolism, regulation of cardiac muscle contraction and mitochondrial transport processes seems to depend on hypertension and age. As an overlap was still present between T2D and non-diabetics transcriptomes in the PCA, we decided to use Factor Analysis of Mixed Data to identify potential subgroups of patients among the whole cohort. 84 patients have been clustered into 3 groups. Cluster 1 is associated with a patient profile without T2D and displaying a mild cardiac dysfunction, contrary to cluster 3 which includes mainly male T2D patients with a pronounced structural and functional cardiac alteration. Cluster 2 is only composed of female patients with higher BMI and leptin serum level, diastolic alteration and a moderately increased hypertrophy. Biological processes were differently affected between cluster 1 and cluster 2 or 3. Cluster 2 presents a decrease in gene expression involved in histone and protein demethylation. As for cluster 3, the expression of genes involved in immune response processes is increased while the one for the regulation of cardiac muscle organisation is decreased. To conclude, T2D is specifically associated with a reduction of gene expression involved in mitochondrial function, and an upregulation of angiogenesis, inflammation, and organisation of extracellular matrix. The unsupervised analysis revealed different clusters with specific gene expression and more specifically an association of female sex and metabolic syndrome with demethylation processes, and an association of T2D in men with inflammation and dysregulation of cardiac muscle contraction processes.Biological process in T2D patients

Mouse Models of Cardiomyopathies Caused by Mutations in Troponin C.

Cardiac muscle contraction is regulated via Ca2+ exchange with the hetero-trimeric troponin complex located on the thin filament. Binding of Ca2+ to cardiac troponin C, a Ca2+ sensing subunit within the troponin complex, results in a series of conformational re-arrangements among the thin filament components, leading to an increase in the formation of actomyosin cross-bridges and muscle contraction. Ultimately, a decline in intracellular Ca2+ leads to the dissociation of Ca2+ from troponin C, inhibiting cross-bridge cycling and initiating muscle relaxation. Therefore, troponin C plays a crucial role in the regulation of cardiac muscle contraction and relaxation. Naturally occurring and engineered mutations in troponin C can lead to altered interactions among components of the thin filament and to aberrant Ca2+ binding and exchange with the thin filament. Mutations in troponin C have been associated with various forms of cardiac disease, including hypertrophic, restrictive, dilated, and left ventricular noncompaction cardiomyopathies. Despite progress made to date, more information from human studies, biophysical characterizations, and animal models is required for a clearer understanding of disease drivers that lead to cardiomyopathies. The unique use of engineered cardiac troponin C with the L48Q mutation that had been thoroughly characterized and genetically introduced into mouse myocardium clearly demonstrates that Ca2+ sensitization in and of itself should not necessarily be considered a disease driver. This opens the door for small molecule and protein engineering strategies to help boost impaired systolic function. On the other hand, the engineered troponin C mutants (I61Q and D73N), genetically introduced into mouse myocardium, demonstrate that Ca2+ desensitization under basal conditions may be a driving factor for dilated cardiomyopathy. In addition to enhancing our knowledge of molecular mechanisms that trigger hypertrophy, dilation, morbidity, and mortality, these cardiomyopathy mouse models could be used to test novel treatment strategies for cardiovascular diseases. In this review, we will discuss (1) the various ways mutations in cardiac troponin C might lead to disease; (2) relevant data on mutations in cardiac troponin C linked to human disease, and (3) all currently existing mouse models containing cardiac troponin C mutations (disease-associated and engineered).

Phospholamban pentamerization increases sensitivity and dynamic range of cardiac relaxation.

A key event in the regulation of cardiac contraction and relaxation is the phosphorylation of phospholamban (PLN) that relieves the inhibition of the sarco/endoplasmic Ca2+-ATPase SERCA2a. PLN exists in an equilibrium between monomers and pentamers. While only monomers can inhibit SERCA2a by direct interaction, the functional role of pentamers is still unclear. This study investigates the functional consequences of PLN pentamerization. We generated transgenic mouse models expressing either a PLN mutant that cannot form pentamers (TgAFA-PLN) or wildtype PLN (TgPLN) in a PLN-deficient background. TgAFA-PLN hearts demonstrated 3-fold stronger phosphorylation of monomeric PLN, accelerated Ca2+ cycling of cardiomyocytes and enhanced contraction and relaxation of sarcomeres and whole hearts in vivo. All of these effects were observed under baseline conditions and abrogated upon inhibition of protein kinase A (PKA). Mechanistically, far western kinase assays revealed that PLN pentamers are phosphorylated by PKA directly and independent of any subunit exchange for free monomers. In vitro-phosphorylation of synthetic PLN demonstrated that pentamers even provide a preferred PKA substrate and compete with monomers for the kinase, thereby reducing monomer phosphorylation and maximizing SERCA2a inhibition. However, β-adrenergic stimulation induced strong PLN monomer phosphorylation in TgPLN hearts and sharp acceleration of cardiomyocyte Ca2+ cycling and hemodynamic values that now were indistinguishable from TgAFA-PLN and PLN-KO hearts. The pathophysiological relevance of PLN pentamerization was evaluated using transverse aortic constriction (TAC) to induce left ventricular pressure overload. Compared to TgPLN, TgAFA-PLN mice demonstrated reduced survival after TAC, impaired cardiac hemodynamics, failure to respond to adrenergic stimulation, higher heart weight, and increased myocardial fibrosis. The findings show that PLN pentamerization greatly impacts on SERCA2a activity as it mediates the full range of PLN effects from maximum inhibition to full release of SERCA2a. function. This regulation is important for myocardial adaptation to sustained pressure overload. Pentamerization of PLN adds to the regulation of cardiac contractile function and facilitates myocardial transition to an energy saving mode during resting phases. Thus, PLN pentamers would protect cardiomyocytes from energetic deficits, and they improve stress adaptation of the heart as shown for sustained pressure overload in this study. Strategies that target PLN pentamerization promise therapeutic potential in the treatment of myocardial maladaptation to stress as well as cardiac pathologies associated with altered monomer-to-pentamer ratios, e.g., cardiomyopathies due to PLN mutations, certain types of heart failure, and aged hearts.

Network pharmacology and LC-MS approachs to explore the active compounds and mechanisms of Yuanjiang decoction for treating bradyarrhythmia

BackgroundYuanjiang decoction (YJD), a traditional Chinese medicinal prescription, has been found to have a significant heart rate-increasing effect and is effective in the treatment of symptomatic bradyarrhythmia in previous studies. However, its specific components and potential mechanisms remain unclear. MethodsIn this study, we detected and identified the main compounds of YJD using liquid chromatography-mass spectrometry (LC-MS). Through the approach of network pharmacology, we predicted the core targets of the active components, bradyarrhythmia targets, and obtained potential anti-bradyarrhythmia targets of YJD. We further performed protein to protein interaction (PPI), gene ontology (GO) enrichment analyses and kyoto encyclopedia of genes and genomes (KEGG) signaling pathway analyses for core targets, and constructed network of key active ingredients-core targets of YJD. Finally, molecular docking and molecular dynamics simulation were performed for key active ingredients and core targets. ResultsThe YJD contains a total of 35 main chemical components. The key active ingredients-core targets network contains 36 nodes and 90 edges, including 20 key active ingredients and 16 core targets. The core targets in the PPI network were TP53, TNF, HRAS, PPARG, IL1B, KCNH2, SCN5A, IDH1, LMNA, ACHE, F2, DRD2, CALM1, KCNQ1, TNNI3, IDH2 and TNNT2. KEGG pathway analysis showed that YJD treatment of bradyarrhythmia mainly involves neuroactive ligand-receptor interaction, adrenergic signaling in cardiomyocytes, cAMP signaling pathway, calcium signaling pathway, cholinergic synaptic and serotonergic synapse signaling pathway. The biological processes mainly include regulation of hormone levels, regulation of cardiac contraction, chemical synaptic transmission, circadian rhythm, positive regulation of heart rate, smooth muscle contraction, response to metal ion, oxidation-reduction process, neurotransmitter transport and import across plasma membrane. Molecular docking and molecular dynamics simulation results showed that hesperidin and tetrahydropalmatine had higher affinity with DRD2 and KCNQ1, respectively. ConclusionThis study reveals the pharmacodynamic material basis of YJD and its potential multicomponent–multitarget–multipathway pharmacological effects, predicted its potential anti-bradyarrhythmia mechanism may be related to the regulation of myocardial autonomic nervous function and related ion channels. Our work demonstrates that YJD has great potential for treating bradyarrhythmias as a complementary medicine, and the results can provide a theoretical basis for the development and clinical application of YJD.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

G Protein-Coupled Receptors-Receptors With New Tricks Up Their Sleeves.

The Screening and Identification of Key Biomarkers in Adrenocortical Carcinoma: Evidence from a Bioinformatics Analysis

<sec> <title>Objective:</title> This study aimed to identify the potential key genes associated with the progression and prognosis of adrenocortical carcinoma (ACC). </sec> <sec> <title>Methods:</title> Differentially expressed genes (DEGs) in ACC cells and normal adrenocortical cells were assessed by microarray from the Gene Expression Omnibus database. The biological functions of the classified DEGs were examined by Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) enrichment analyses and a protein–protein interaction (PPI) network was mapped using Cytoscape software. MCODE software was also used for the module analysis and then 4 algorithms of cytohubba software were used to screen hub genes. The overall survival (OS) examination of the hub genes was then performed by the ualcan online tool. </sec> <sec> <title>Results:</title> Two GSEs (GSE12368, GSE33371) were downloaded from GEO including 18 and 43 cases, respectively. One hundred and sixty-nine DEGs were identified, including 57 upregulated genes and 112 downregulated genes. The Gene Ontology (GO) analyses showed that the upregulated genes were significantly enriched in the mitotic cytokines is, nucleus and ATP binding, while the downregulated genes were involved in the positive regulation of cardiac muscle contraction, extracellular space, and heparin-binding (P < 0.05). The Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) pathway examination showed significant pathways including the cell cycle and the complement and coagulation cascades. The protein– protein interaction (PPI) network consisted of 162 nodes and 847 edges, including mitotic nuclear division, cytoplasmic, protein kinase binding, and cell cycle. All 4 identified hub genes (FOXM1, UBE2C, KIF11, and NDC80) were associated with the prognosis of adrenocortical carcinoma (ACC) by survival analysis. </sec> <sec> <title>Conclusions:</title> The present study offered insights into the molecular mechanism of adrenocortical carcinoma (ACC) that may be beneficial in further analyses. </sec>

Abstract 14011: Myomegalin, a 250 Kilodalton Phosphodiesterase Interacting Protein (PDE4DIP), Shows a Novel Protein Interaction With Cardiac Myosin Binding Protein C

Cardiac myosin binding protein C (cMyBP-C) is a sarcomeric protein important in the regulation of cardiac contraction through its phosphorylation-dependent interactions with myosin heavy chain and actin. MYBPC3, the gene encoding cMyBP-C, contains the largest number of hypertrophic cardiomyopathy (HCM)-causing mutations. Interestingly, the region of MYBPC3 containing the greatest number of HCM-causing missense mutations, the central domains, have an unknown physiological relevance to the overall function of the protein. Intriguingly, central domain C3 contains a positively charged, putative surface binding interface for an as-yet unidentified ligand. Two of the most common HCM-causing mutations, R495Q and R502W, located in C3, appear to alter this charged interface. We have previously shown that these two mutations lead to altered contractile kinetics and hypophosphorylation of cMyBP-C. We hypothesize that this region of C3 contains an important binding region that is disrupted by both the R495Q and R502W mutations. To reveal candidate binding proteins, we performed a pull-down assay using mouse cardiac homogenate and a GST-tagged C2-C4 fragment of cMyBP-C, with or without the R495Q mutation. Mass spectrometry revealed that myomegalin, a 250 kDa phosphodiesterase-4D-interacting protein (PDE4DIP), bound the WT, but not the R495Q C2-C4 fragment. To confirm the interaction between cMyBP-C and myomegalin in situ , we performed Duolink proximity ligase assay (PLA) in cultured neonatal mouse cardiomyocytes which revealed a positive interaction in cMyBP-C +/+ but not in cMyBP-C -/- cells (n>20) (Figure). Our data strongly suggest that a large isoform of myomegalin, separately known to form a protein complex with PKA/AKAP, forms a novel interaction with cMyBP-C. We hypothesize that perturbations in this interaction lead to altered PKA localization and the observed changes in R495Q and R502W cMyBP-C phosphorylation, and may contribute to the HCM phenotype.

Molecular dynamics provides new insights into the mechanism of calcium signal transduction and interdomain interactions in cardiac troponin.

Understanding the regulation of cardiac muscle contraction at a molecular level is crucial for the development of therapeutics for heart conditions. Despite the availability of atomic structures of the protein components of cardiac muscle thin filaments, detailed insights into their dynamics and response to calcium are yet to be fully depicted. In this study, we used molecular dynamics simulations of the core domains of the cardiac muscle protein troponin to characterize the equilibrium dynamics of its calcium-bound and calcium-free forms, with a focus on elements of cardiac muscle contraction activation and deactivation, that is, calcium binding to the cardiac troponin Ca2+ -binding subunit (TnC) and the release of the switch region of the troponin inhibitory subunit (TnI) from TnC. The process of calcium binding to the TnC binding site is described as a three-step process commencing with calcium capture by the binding site residues, followed by cooperative residue interplay bringing the calcium ion to the binding site, and finally, calcium-water exchange. Furthermore, we uncovered a set of TnC-TnI interdomain interactions that are critical for TnC N-lobe hydrophobic pocket dynamics. Absence of these interactions allows the closure of the TnC N-lobe hydrophobic pocket while the TnI switch region remains expelled, whereas if the interactions are maintained, the hydrophobic pocket remains open. Modification of these interactions may fine-tune the ability of the TnC N-lobe hydrophobic pocket to close or remain open, modulate cardiac contractility and present potential therapy-relevant targets.

Phosphorylation of Troponin I finely controls the positioning of Troponin for the optimal regulation of cardiac muscle contraction

Troponin is the Ca2+ molecular switch that regulates striated muscle contraction. In the heart, troponin Ca2+ sensitivity is also modulated by the PKA-dependent phosphorylation of a unique 31-residue N-terminal extension region of the Troponin I subunit (NH2-TnI). However, the detailed mechanism for the propagation of the phosphorylation signal through Tn, which results in the enhancement of the myocardial relaxation rate, is difficult to examine within whole Tn. Several models exist for how phosphorylation modulates the troponin response in cardiac cells but these are mostly built from peptide-NMR studies and molecular dynamics simulations. Here we used a paramagnetic spin labeling approach to position and track the movement of the NH2-TnI region within whole Tn. Through paramagnetic relaxation enhancement (PRE)-NMR experiments, we show that the NH2-TnI region interacts with a broad surface area on the N-domain of the Troponin C subunit. This region includes the Ca2+ regulatory Site II and the TnI switch-binding site. Phosphorylation of the NH2-TnI both weakens and shifts this region to an adjacent site on TnC. Interspin EPR distances between NH2-TnI and TnC further reveal a phosphorylation induced re-orientation of the TnC N-domain under saturating Ca2+ conditions. We propose an allosteric model where phosphorylation triggered cooperative changes in both the interaction of the NH2-TnI region with TnC, and the re-orientation of the TnC interdomain orientation, together promote the release of the TnI switch-peptide. Enhancement of the myocardial relaxation rate then occurs. Knowledge of this unique role of phosphorylation in whole Tn is important for understanding pathological processes affecting the heart.

Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+ Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+-dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At pCa 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at pCa 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steady-state isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.

C0 Ig-Domain of Cardiac Myosin Binding Protein-C Interacts with the Regulatory Light Chain of Myosin-S1 Bound to the Native Cardiac Thin Filament

Muscle contraction, relies on sliding of myosin-based thick filaments and thin filaments comprised of actin, tropomyosin and troponin. Cardiac myosin binding protein-C (cMyBP-C), an intrinsic protein of the thick filament, is a key regulator of actomyosin interactions, essential for both normal cardiac contraction and for increased cardiac contractility in response to inotropic stimuli. Mutations in MYBPC3, the gene encoding cMyBP-C, are the single most common genetic cause of hypertrophic cardiomyopathy (HCM). It has been proposed that the interaction of the N-terminal domains (NTDs) of cMyBP-C (e.g. C0, C1, M and C2) with myosin heads may stabilize the super-relaxed state (SRX-state) of the thick filament, while NMR experiments directly confirmed the binding of the C0 Ig-domain of cMyBP-C to the isolated regulatory light chain (RLC) of myosin. We recently demonstrated that C1 can activate the thin filament to the same extent as rigor myosin-S1, while C0 significantly enhances the activating effect of C1. Our data also show that C0 competes with myosin-S1 for binding to the thin filament. In order to understand how C0 interacts with the thick and thin filaments upon active cross-bridge formation we used cryo electron microscopy and image analysis of native cardiac thin filaments decorated with rigor myosin-S1 and the C0-domain of cMyBP-C. We show that C0 readily binds to the RLC of myosin-S1 bound to the thin filament in rigor state. Taking into account that the C0-domain is specific to the cardiac muscle and is absent in the skeletal MyBP-C we suggest a model of how the C0-domain of cMyBP-C participates in the regulation of cardiac contraction.

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament

Muscle contraction relies on interaction between myosin-based thick filaments and actin-based thin filaments. Myosin binding protein C (MyBP-C) is a key regulator of actomyosin interactions. Recent studies established that the N'-terminal domains (NTDs) of MyBP-C can either activate or inhibit thin filaments, but the mechanism of their collective actionis poorly understood. Cardiac MyBP-C (cMyBP-C) harbors an extra NTD, which is absent in skeletal isoforms of MyBP-C, and its role in regulation of cardiac contraction is unknown. Here we show that the first two domains of human cMyPB-C (i.e., C0 and C1) cooperate to activate the thin filament. We demonstrate that C1 interacts with tropomyosin via a positively charged loop and that this interaction, stabilized by the C0 domain, is required for thin filament activation by cMyBP-C. Our data reveal a mechanism by which cMyBP-C can modulate cardiac contraction and demonstrate a function of the C0 domain.

HCM and DCM cardiomyopathy-linked α-tropomyosin mutations influence off-state stability and crossbridge interaction on thin filaments

Calcium regulation of cardiac muscle contraction is controlled by the thin-filament proteins troponin and tropomyosin bound to actin. In the absence of calcium, troponin-tropomyosin inhibits myosin-interactions on actin and induces muscle relaxation, whereas the addition of calcium relieves the inhibitory constraint to initiate contraction. Many mutations in thin filament proteins linked to cardiomyopathy appear to disrupt this regulatory switching. Here, we tested perturbations caused by mutant tropomyosins (E40K, DCM; and E62Q, HCM) on intra-filament interactions affecting acto-myosin interactions including those induced further by myosin association. Comparison of wild-type and mutant human α-tropomyosin (Tpm1.1) behavior was carried out using in vitro motility assays and molecular dynamics simulations. Our results show that E62Q tropomyosin destabilizes thin filament off-state function by increasing calcium-sensitivity, but without apparent affect on global tropomyosin structure by modifying coiled-coil rigidity. In contrast, the E40K mutant tropomyosin appears to stabilize the off-state, demonstrates increased tropomyosin flexibility, while also decreasing calcium-sensitivity. In addition, the E40K mutation reduces thin filament velocity at low myosin concentration while the E62Q mutant tropomyosin increases velocity. Corresponding molecular dynamics simulations indicate specific residue interactions that are likely to redefine underlying molecular regulatory mechanisms, which we propose explain the altered contractility evoked by the disease-causing mutations.

Using Statistical Mechanics to Understand Calcium Sparks

The crucial importance of local Ca signaling for regulation of cardiac muscle contraction was theoretically predicted in 1992 as part of local control theory and validated by the experimental discovery of Ca sparks. Sparks are generated by release channel clusters in the form of localized Ca releases from junctional sarcoplasmic reticulum (SR). As release channels interact, the spark is an emergent behavior of the collective, rather than a property of an individual channel. This nanoscale system of interacting molecules which generates elementary biological signals is particularly interesting because this is where physics meets biology. We have constructed an exact mapping of the release channel cluster to a famous model of an interacting particle system in statistical physics, namely the Ising model. We demonstrated that the channel synchronization that corresponds to termination of signals is described by the same equation as the phase transition associated to the change of magnetic field in an Ising ferromagnet. Our Ising-based theory predicts the critical level of SR [Ca] that is required for spark termination. If the SR depletion does not reach this level the spark does not terminate and becomes metastable. The prediction was confirmed by numerical simulations using 2013 Stern et al. spark model. In an ongoing work, we explore the fine structure of calcium sparks using Peierl's contour analysis to investigate the famous Onsager's phase transition to describe the transition from ordered to disordered sparks as a function of β (effective inverse temperature). The disordered sparks were manifested in numerical simulations by the presence of Ca embers, i.e. small clusters of opened channels. In summary, we describe deterministically the behavior of a system on a coarser scale (release unit) that is random on a finer scale (release channels), bridging the gap between scales.

Research Progress of Cardiac Myosin Binding Protein C in Dilated Cardiomyopathy and Other Cardiac Conditions

Since its discovery, myosin-binding protein C (cMyBP-C) has become a protein of interest clinically. With emergence of new methodologies and technologies, the structure and functions of cMyBP-C from different aspects can be studied, enabling us to better understand its involvement in certain cardiac conditions. Studying its kinetics of release and clearance from the circulation and by comparing to other conventional biomarkers, it has been reported that cMyBP-C is eligible to be a novel biomarker for several cardiac conditions. Moreover, studying the genetics and their involvement in pathogenic mechanisms has opened the ideas for potential therapeutic strategies. More and more researches are constantly being done to better understand the role of cMyBP-C in dilated cardiomyopathy (DCM). The importance of cMyBP-C to the heart is still actively being investigated. Its presence is however crucial for sarcomere organization and proper regulation of cardiac contraction during systole and complete relaxation during diastole. Genetic mutation in cMyBP-C has been linked to cardiac conditions including hypertrophic and dilated cardiomyopathies. Around 350 types of mutations have already been documented leading to various cardiac conditions and abnormalities. Analyzing human heart samples has enabled us to better understand the importance of cMyBP-C and how its mutations lead to inherited cardiomyopathies. It is therefore necessary to have an update about the research progress of cMyBP-C in relation to DCM and other cardiac conditions.

Identification of molecular characteristics induced by radiotherapy in rectal cancer based on microarray data.

The present study aimed to reveal the molecular characteristics induced by radiotherapy in rectal cancer at the transcriptome level. Microarray data (ID, GSE26027) downloaded from the Gene Expression Omnibus database were re-analyzed to identify differentially expressed genes (DEGs) between rectal cancer tissues during and prior to radiotherapy. The DEGs were then inputted into the database for annotation, visualization and integrated discovery, an online tool to perform enrichment analyses, and into the search tool for the retrieval of interacting genes/proteins database to identify protein-protein interactions (PPIs). Subsequently, a PPI network was constructed, which was screened for densely connected modules. Furthermore, protein domain enrichment analysis was performed. In total, 690 DEGs, including 179 upregulated and 511 downregulated DEGs, were found in rectal cancer tissues during and prior to radiotherapy. The upregulated DEGs were significantly enriched in 'positive regulation of transport' and 'regulation of cardiac muscle contraction', while the downregulated DEGs were most markedly enriched in 'cell migration', 'cell-cell signaling', 'extracellular matrix organization' and 'blood vessel development', including prostaglandin-endoperoxide synthase 2, transforming growth factor β-induced, 68 kDa endothelin receptor type A, brain-derived neurotrophic factor, TIMP metallopeptidase inhibitor 1, and serpin family E member 1, which were the top 6 hub nodes in the PPI network. Furthermore, 2 protein domains were significantly enriched by PPI modules, including: The collagen triple helix repeat (CTHR) family members collagen type (COL) 5A2, COL9A3, COL6A3, COL21A1, COL5A3, COL11A1, COL7A1 and CTHR-containing-1; and the olfactory receptor family (OR) members OR7E24, OR7A17, OR6A2, OR1F1, OR10H3 and OR7A10. A total of 7 upregulated DEGs were characterized as tumor suppressor genes, and 8 downregulated DEGs were characterized as oncogenes. The biological processes or protein domains enriched by upregulated or downregulated DEGs may improve the understanding of molecular characteristics in response to radiotherapy.

Abstract 85: Interactions Between Cardiac Myosin Binding Protein C and Actin Contribute to the Regulation of Cardiac Contraction

Cardiac myosin binding protein C (cMyBP-C) is a regulatory muscle protein that is essential for proper cardiac contraction, and mutations in cMyBP-C are commonly associated with hypertrophic cardiomyopathy. cMyBP-C not only interacts with myosin, but with actin as well. In vitro studies have demonstrated that multiple functions of cMyBP-C could readily be explained by an interaction between cMyBP-C and actin, but the in vivo significance of cMyBP-C binding to either myosin or actin is not well understood. Here we created transgenic mice with a single point mutation (L348P) in a key binding domain of cMyBP-C that enhances the binding affinity of cMyBP-C for actin in vitro (Bezold et al , JBC 2013) to gain insights into the relevance of cMyBP-C binding to actin in working hearts. Echocardiograms from 3 month old male L348P-Tg mice (N=23) and non-transgenic (nTg, N=17) controls were used to assess systolic and diastolic function. Results showed significantly prolonged isovolumetric relaxation time (L348P-Tg: 18.5±0.6 vs nTg: 10.2±0.3 ms) and slower movement of the mitral valve annulus (E’: -17.2±1.5 vs -32.2±1.6 and A’: -9.1±1.7 vs -17.6±1.0 mm/s, p<0.05), accompanied by slower inflow of blood into the left ventricle (reduced E and A, prolonged mitral valve deceleration time). Pressure-volume measurements showed significantly reduced rates of pressure decay in L348P-Tg mice (Tau Glantz: 39.1±2.6 vs 12.7±0.9 ms) and an increased end-diastolic pressure volume relationship (0.13±0.02 vs 0.07±0.01). We challenged mice with acute beta-adrenergic stimulation (isoprenaline injection) to determine whether the L348P mutation affected contractile reserve. Isoprenaline had little effect on diastolic parameters, but revealed systolic dysfunction in L348P-Tg mice as evident from a blunted increase in contraction (e.g. fractional shortening after isoprenaline: 38.5±1.3 vs 44.2±1.7%). Taken together our results show for the first time that interactions between cMyBP-C and actin are relevant for functioning of the whole heart. Increasing the cMyBP-C-actin interaction by the L348P mutation caused increased stiffness of the left ventricle, slowed relaxation and diastolic dysfunction. Results also suggested that the L348P mutation reduces contractile reserve.

Impact of disease-causing mutations on inter-domain interactions in cMyBP-C: a steered molecular dynamics study

The molecular interactions of the sarcomeric proteins are essential in the regulation of various cardiac functions. Mutations in the gene MYBPC3 coding for cardiac myosin-binding protein-C (cMyBP-C), a multi-domain protein, are the most common cause of hypertrophic cardiomyopathy (HCM). The N-terminal complex, C1-motif-C2 is a central region in cMyBP-C for the regulation of cardiac muscle contraction. However, the mechanism of binding/unbinding of this complex during health and disease is unknown. Here, we study possible mechanisms of unbinding using steered molecular dynamics simulations for the complex in the wild type, in single mutations (E258K in C1, E441K in C2), as well as in a double mutation (E258K in C1 + E441K in C2), which are associated with severe HCM. The observed molecular events and the calculation of force utilized for the unbinding suggest the following: (i) double mutation can encourage the formation of rigid complex that required large amount of force and long-time to unbind, (ii) C1 appears to start to unbind ahead of C2 regardless of the mutation, and (iii) unbinding of C2 requires larger amount of force than C1. This molecular insight suggests that key HCM-causing mutations might significantly modify the native affinity required for the assembly of the domains in cMyBP-C, which is essential for normal cardiac function.

Functional Divergence in Teleost Cardiac Troponin Paralogs Guides Variation in the Interaction of TnI Switch Region with TnC.

Gene duplication results in extra copies of genes that must coevolve with their interacting partners in multimeric protein complexes. The cardiac troponin (Tn) complex, containing TnC, TnI, and TnT, forms a distinct functional unit critical for the regulation of cardiac muscle contraction. In teleost fish, the function of the Tn complex is modified by the consequences of differential expression of paralogs in response to environmental thermal challenges. In this article, we focus on the interaction between TnI and TnC, coded for by genes that have independent evolutionary origins, but the co-operation of their protein products has necessitated coevolution. In this study, we characterize functional divergence of TnC and TnI paralogs, specifically the interrelated roles of regulatory subfunctionalization and structural subfunctionalization. We determined that differential paralog transcript expression in response to temperature acclimation results in three combinations of TnC and TnI in the zebrafish heart: TnC1a/TnI1.1, TnC1b/TnI1.1, and TnC1a/TnI1.5. Phylogenetic analysis of these highly conserved proteins identified functionally divergent residues in TnI and TnC. The structural and functional effect of these Tn combinations was modeled with molecular dynamics simulation to link divergent sites to changes in interaction strength. Functional divergence in TnI and TnC were not limited to the residues involved with TnC/TnI switch interaction, which emphasizes the complex nature of Tn function. Patterns in domain-specific divergent selection and interaction energies suggest that substitutions in the TnI switch region are crucial to modifying TnI/TnC function to maintain cardiac contraction with temperature changes. This integrative approach introduces Tn as a model of functional divergence that guides the coevolution of interacting proteins.