Excitation-contraction Coupling Research Articles

Loose-patch clamp analysis applied to voltage-gated ionic currents following pharmacological ryanodine receptor modulation in murine hippocampal cornu ammonis-1 pyramidal neurons.

The loose-patch clamp technique was first developed and used in native amphibian skeletal muscle (SkM), offering useful features complementing conventional sharp micro-electrode, gap, or conventional patch voltage clamping. It demonstrated the feedback effects of pharmacological modification of ryanodine receptor (RyR)-mediated Ca2+ release on the Na+ channel (Nav1.4) currents, initiating excitation-contraction coupling in native murine SkM. The effects of the further RyR and Ca2+-ATPase (SERCA) antagonists, dantrolene and cyclopiazonic acid (CPA), additionally implicated background tubular-sarcoplasmic Ca2+ domains in these actions. We extend the loose-patch clamp approach to ion current measurements in murine hippocampal brain slice cornu ammonis-1 (CA1) pyramidal neurons. We explored the effects on Na+ currents of pharmacologically manipulating RyR and SERCA-mediated intracellular store Ca2+ release and reuptake. We adopted protocols previously applied to native skeletal muscle. These demonstrated Ca2+-mediated feedback effects on the Na+ channel function. Experiments applying depolarizing 15ms duration loose-patch clamp steps to test voltages ranging from -40 to 120mV positive to the resting membrane potential demonstrated that 0.5mM caffeine decreased inward current amplitudes, agreeing with the previous SkM findings. It also decreased transient but not prolonged outward current amplitudes. However, 2mM caffeine affected neither inward nor transient outward but increased prolonged outward currents, in contrast to its increasing inward currents in SkM. Furthermore, similarly and in contrast to previous SkM findings, both dantrolene (10μM) and CPA (1μM) pre-administration left both inward and outward currents unchanged. Nevertheless, dantrolene pretreatment still abrogated the effects of subsequent 0.5- and 2-mM caffeine challenges on both inward and outward currents. Finally, CPA abrogated the effects of 0.5mM caffeine on both inward and outward currents, but with 2mM caffeine, inward and transient outward currents were unchanged, but sustained outward currents increased. We, thus, extend loose-patch clamping to establish pharmacological properties of murine CA1 pyramidal neurons and their similarities and contrasts with SkM. Here, evoked though not background Ca2+-store release influenced Nav and Kv excitation, consistent with smaller contributions of background store Ca2+ release to resting [Ca2+]. This potential non-canonical mechanism could modulate neuronal membrane excitability or cellular firing rates.

On the role of dysferlin in striated muscle: membrane repair, t-tubules and Ca2+ handling.

Dysferlin is a 237kDa membrane-associated protein characterised by multiple C2 domains with a diverse role in skeletal and cardiac muscle physiology. Mutations in DYSF are known to cause various types of human muscular dystrophies, known collectively as dysferlinopathies, with some patients developing cardiomyopathy. A myriad of in vitro membrane repair studies suggest that dysferlin plays an integral role in the membrane repair complex in skeletal muscle. In comparison, less is known about dysferlin in the heart, but mounting evidence suggests that dysferlin's role is similar in both muscle types. Recent findings have shown that dysferlin regulates Ca2+ handling in striated muscle via multiple mechanisms and that this becomes more important in conditions of stress. Maintenance of the transverse (t)-tubule network and the tight coordination of excitation-contraction coupling are essential for muscle contractility. Dysferlin regulates the maintenance and repair of t-tubules, and it is suspected that dysferlin regulates t-tubules and sarcolemmal repair through a similar mechanism. This review focuses on the emerging complexity of dysferlin's activity in striated muscle. Such insights will progress our understanding of the proteins and pathways that regulate basic heart and skeletal muscle function and help guide research into striated muscle pathology, especially that which arises due to dysferlin dysfunction.

Effects of electro-mechanical uncouplers, hormonal stimulation and pacing rate on the stability and function of cultured rabbit myocardial slices.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5Hz, 1Hz, and 2Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7d. Pacing rates above 1Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1d and 7d, while Ca2+ transient duration was prolonged after 7d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Mixture of Doxycycline, ML-7 and L-NAME Restores the Pro- and Antioxidant Balance during Myocardial Infarction-In Vivo Pig Model Study.

The restoration of blood flow to the ischemic myocardium inflicts ischemia/reperfusion (I/R) heart injury (IRI). The main contributors to IRI are increased oxidative stress and subsequent excessive production of ROS, increased expression of NOS and peroxinitate, activation of MMPs, and enhanced posttranslational modifications of contractile proteins, which make them more susceptible to proteolytic degradation. Since the pathophysiology of IRI is a complex issue, and thus, various therapeutic strategies are required to prevent or reduce IRI and microvascular dysfunction, in the current study we proposed an innovative multi-drug therapy using low concentrations of drugs applied intracoronary to reach microvessels in order to stabilize the pro- and antioxidant balance during a MI in an in vivo pig model. The ability of a mixture of doxycycline (1 μM), ML-7 (0.5 μM), and L-NAME (2 μM) to modulate the pro- and antioxidative balance was tested in the left ventricle tissue and blood samples. Data showed that infusion of a MIX reduced the total oxidative status (TOS), oxidative stress index (OSI), and malondialdehyde (MDA). It also increased the total antioxidant capacity, confirming its antioxidative properties. MIX administration also reduced the activity of MMP-2 and MMP-9, and then decreased the release of MLC1 and BNP-26 into plasma. This study demonstrated that intracoronary administration of low concentrations of doxycycline in combination with ML-7 and L-NAME is incredibly efficient in regulating pro- and antioxidant balance during MI.

Taurine activates the AKT-mTOR axis to restore muscle mass and contractile strength in human 3D in vitro models of steroid myopathy.

Steroid myopathy is a clinically challenging condition exacerbated by prolonged corticosteroid use or adrenal tumors. In this study, we engineered a functional three-dimensional (3D) in vitro skeletal muscle model to investigate steroid myopathy. By subjecting our bioengineered muscle tissues to dexamethasone treatment, we reproduced the molecular and functional aspects of this disease. Dexamethasone caused a substantial reduction in muscle force, myotube diameter and induced fatigue. We observed nuclear translocation of the glucocorticoid receptor (GCR) and activation of the ubiquitin-proteasome system within our model, suggesting their coordinated role in muscle atrophy. We then examined the therapeutic potential of taurine in our 3D model for steroid myopathy. Our findings revealed an upregulation of phosphorylated AKT by taurine, effectively countering the hyperactivation of the ubiquitin-proteasomal pathway. Importantly, we demonstrate that discontinuing corticosteroid treatment was insufficient to restore muscle mass and function. Taurine treatment, when administered concurrently with corticosteroids, notably enhanced contractile strength and protein turnover by upregulating the AKT-mTOR axis. Our model not only identifies a promising therapeutic target, but also suggests combinatorial treatment that may benefit individuals undergoing corticosteroid treatment or those diagnosed with adrenal tumors.

Benefits and challenges of reconstituting the actin cortex.

The cell's ability to change shape is a central feature in many cellular processes, including cytokinesis, motility, migration, and tissue formation. The cell constructs a network of contractile proteins underneath the cell membrane to form the cortex, and the reorganization of these components directly contributes to cellular shape changes. The desire to mimic these cell shape changes to aid in the creation of a synthetic cell has been increasing. Therefore, membrane-based reconstitution experiments have flourished, furthering our understanding of the minimal components the cell uses throughout these processes. Although biochemical approaches increased our understanding of actin, myosin II, and actin-associated proteins, using membrane-based reconstituted systems has further expanded our understanding of actin structures and functions because membrane-cortex interactions can be analyzed. In this review, we highlight the recent developments in membrane-based reconstitution techniques. We examine the current findings on the minimal components needed to recapitulate distinct actin structures and functions and how they relate to the cortex's impact on cellular mechanical properties. We also explore how co-processing of computational models with wet-lab experiments enhances our understanding of these properties. Finally, we emphasize the benefits and challenges inherent to membrane-based, reconstitution assays, ranging from the advantage of precise control over the system to the difficulty of integrating these findings into the complex cellular environment.

In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle.

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

Cardiotoxicity Assessment through a Polymer-Based Cantilever Platform: An Integrated Electro-Mechanical Screening Approach.

Preclinical drug screening for cardiac toxicity has traditionally relied on observing changes in cardiomyocytes' electrical activity, primarily through invasive patch clamp techniques or non-invasive microelectrode arrays (MEA). However, relying solely on field potential duration (FPD) measurements for electrophysiological assessment can miss the full spectrum of drug-induced toxicity, as different drugs affect cardiomyocytes through various mechanisms. A more comprehensive approach, combining field potential and contractility measurements, is essential for accurate toxicity profiling, particularly for drugs targeting contractile proteins without affecting electrophysiology. However, previously proposed platform has significant limitations in terms of simultaneous measurement. The novel platform addresses these issues, offering enhanced, non-invasive evaluation of drug-induced cardiotoxicity. It features eight cantilevers with patterned strain sensors and MEA, enabling real-time monitoring of both cardiomyocyte contraction force and field potential. This system can detect minimum cardiac contraction force of ≈2 µN and field potential signals with 50µm MEA diameter, using the same cardiomyocytes in measurements of two parameters. Testing with six drugs of varied mechanisms of action, the platform successfully identifies these mechanisms and accurately assesses toxicity profiles, including drugs not inhibiting potassium channels. This innovative approach presents a comprehensive, non-invasive method for cardiac function assessment, poised to revolutionize preclinical cardiotoxicity screening.

Evaluation of novel open-source software for cardiac optical mapping

KairoSight-3.0 is a recently released Python-based, open-source software for cardiac optical mapping analysis. Addressing challenges in high-resolution electrophysiological data analysis, KairoSight-3.0 facilitates comprehensive studies of cardiac conduction and excitation-contraction coupling. We compared its performance with ElectroMap, focusing on action potential duration and conduction velocity measurements in mouse heart models subjected to ischaemia and flecainide treatment. Our findings reveal that while both software are effective, inherent methodological differences impact measurement outcomes. KairoSight-3.0's robust analysis capabilities make it a valuable tool in cardiac research. Additionally, future directions for KairoSight-3.0 and other mapping analysis tools are explored. Statement of importanceOpen-source methods for analysis of cardiac optical mapping are vital tools in electrophysiological research. Our work directly evaluates the latest version of KarioSight, recently published in JMCC plus, with ElectroMap, an established and widely used tool. Our results show both software are effective in analysis of changes in both conduction and repolarisation. Considering the new features of KairoSight-3.0 and python implementation, our study importantly demonstrates the effectiveness of the software, highlights potential discrepancies between it and ElectroMap, and provides a perspective on future directions for KairoSight-3.0 and other software.

Rhizoctonia solani disease suppression: addition of keratin-rich soil amendment leads to functional shifts in soil microbial communities.

Promoting soil suppressiveness against soil borne pathogens could be a promising strategy to manage crop diseases. One way to increase the suppression potential in agricultural soils is via the addition of organic amendments. This microbe-mediated phenomenon, although not fully understood, prompted our study to explore the microbial taxa and functional properties associated with Rhizoctonia solani disease suppression in sugar beet seedlings after amending soil with a keratin-rich waste stream. Soil samples were analyzed using shotgun metagenomics sequencing. Results showed that both amended soils were enriched in bacterial families found in disease suppressive soils before, indicating that the amendment of keratin-rich material can support the transformation into a suppressive soil. On a functional level, genes encoding keratinolytic enzymes were found to be abundant in the keratin-amended samples. Proteins enriched in amended soils were those potentially involved in the production of secondary metabolites/antibiotics, motility, keratin-degradation, and contractile secretion system proteins. We hypothesize these taxa contribute to the amendment-induced suppression effect due to their genomic potential to produce antibiotics, secrete effectors via the contractile secretion system, and degrade oxalate-a potential virulence factor of R. solani-while simultaneously possessing the ability to metabolize keratin.

The role of protein kinase A signaling in cardiomyopathy

Cardiomyopathy, a diverse group of heart muscle diseases, poses a significant challenge to global cardiovascular health. However, the intricate molecular mechanisms underlying cardiomyopathy, especially the pivotal role of protein kinase A (PKA) signaling pathways in its pathogenesis, remains unclear. The review synthesizes current research findings and highlights the multifaceted interactions between PKA and key cellular components implicated in cardiomyopathy progression. Moreover, this paper unfolds with an exploration of the normal physiological functions of PKA in cardiac cells, emphasizing its role in regulating crucial processes such as excitation-contraction coupling and cellular energy homeostasis. The article navigates through the dysregulation of PKA signaling observed in various forms of cardiomyopathy, including hypertrophic, dilated, and restrictive subtypes. Insightful analyses into the impact of PKA-mediated phosphorylation events on cardiac contractility, ion channel function, and mitochondrial dynamics shed light on the intricate interplay between signaling cascades and disease manifestation. Future clinical trials targeting the PKA signaling pathway are required to seek more effective and precise therapeutic approaches of cardiomyopathy.

Phosphodiesterases 4B and 4D Differentially Regulate cAMP Signaling in Calcium Handling Microdomains of Mouse Hearts.

The ubiquitous second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates cardiac excitation-contraction coupling (ECC) by signaling in discrete subcellular microdomains. Phosphodiesterase subfamilies 4B and 4D are critically involved in the regulation of cAMP signaling in mammalian cardiomyocytes. Alterations of PDE4 activity in human hearts has been shown to result in arrhythmias and heart failure. Here, we sought to systematically investigate specific roles of PDE4B and PDE4D in the regulation of cAMP dynamics in three distinct subcellular microdomains, one of them located at the caveolin-rich plasma membrane which harbors the L-type calcium channels (LTCCs), as well as at two sarco/endoplasmic reticulum (SR) microdomains centered around SR Ca2+-ATPase (SERCA2a) and cardiac ryanodine receptor type 2 (RyR2). Transgenic mice expressing Förster Resonance Energy Transfer (FRET)-based cAMP-specific biosensors targeted to caveolin-rich plasma membrane, SERCA2a and RyR2 microdomains were crossed to PDE4B-KO and PDE4D-KO mice. Direct analysis of the specific effects of both PDE4 subfamilies on local cAMP dynamics was performed using FRET imaging. Our data demonstrate that all three microdomains are differentially regulated by these PDE4 subfamilies. Whereas both are involved in cAMP regulation at the caveolin-rich plasma membrane, there are clearly two distinct cAMP microdomains at the SR formed around RyR2 and SERCA2a, which are preferentially controlled by PDE4B and PDE4D, respectively. This correlates with local cAMP-dependent protein kinase (PKA) substrate phosphorylation and arrhythmia susceptibility. Immunoprecipitation assays confirmed that PDE4B is associated with RyR2 along with PDE4D. Stimulated Emission Depletion (STED) microscopy of immunostained cardiomyocytes suggested possible co-localization of PDE4B with both sarcolemmal and RyR2 microdomains. In conclusion, our functional approach could show that both PDE4B and PDE4D can differentially regulate cardiac cAMP microdomains associated with calcium homeostasis. PDE4B controls cAMP dynamics in both caveolin-rich plasma membrane and RyR2 vicinity. Interestingly, PDE4B is the major regulator of the RyR2 microdomain, as opposed to SERCA2a vicinity, which is predominantly under PDE4D control, suggesting a more complex regulatory pattern than previously thought, with multiple PDEs acting at the same location.

Cardiac arrhythmogenesis: roles of ion channels and their functional modification.

Cardiac arrhythmias cause significant morbidity and mortality and pose a major public health problem. They arise from disruptions in the normally orderly propagation of cardiac electrophysiological activation and recovery through successive cardiomyocytes in the heart. They reflect abnormalities in automaticity, initiation, conduction, or recovery in cardiomyocyte excitation. The latter properties are dependent on surface membrane electrophysiological mechanisms underlying the cardiac action potential. Their disruption results from spatial or temporal instabilities and heterogeneities in the generation and propagation of cellular excitation. These arise from abnormal function in their underlying surface membrane, ion channels, and transporters, as well as the interactions between them. The latter, in turn, form common regulatory targets for the hierarchical network of diverse signaling mechanisms reviewed here. In addition to direct molecular-level pharmacological or physiological actions on these surface membrane biomolecules, accessory, adhesion, signal transduction, and cytoskeletal anchoring proteins modify both their properties and localization. At the cellular level of excitation-contraction coupling processes, Ca2+ homeostatic and phosphorylation processes affect channel activity and membrane excitability directly or through intermediate signaling. Systems-level autonomic cellular signaling exerts both acute channel and longer-term actions on channel expression. Further upstream intermediaries from metabolic changes modulate the channels both themselves and through modifying Ca2+ homeostasis. Finally, longer-term organ-level inflammatory and structural changes, such as fibrotic and hypertrophic remodeling, similarly can influence all these physiological processes with potential pro-arrhythmic consequences. These normal physiological processes may target either individual or groups of ionic channel species and alter with particular pathological conditions. They are also potentially alterable by direct pharmacological action, or effects on longer-term targets modifying protein or cofactor structure, expression, or localization. Their participating specific biomolecules, often clarified in experimental genetically modified models, thus constitute potential therapeutic targets. The insights clarified by the physiological and pharmacological framework outlined here provide a basis for a recent modernized drug classification. Together, they offer a translational framework for current drug understanding. This would facilitate future mechanistically directed therapeutic advances, for which a number of examples are considered here. The latter are potentially useful for treating cardiac, in particular arrhythmic, disease.

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCMassay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Exploring contractile protein mechanisms and target medications for cardiomyopathic patients with diastolic dysfunction

AbstractGenetic defects have been increasingly found in cardiomyopathies, which are often present with mutations in cardiac contractile proteins. These congenital defects involve numerous intracellular pathways and share several critical clinical features, such as systolic or diastolic dysfunction fostering the various cardiomyopathic phenotypes. Hypertrophic cardiomyopathy and restrictive cardiomyopathy (RCM) share a common pathological feature, that is, diastolic dysfunction. Studies have shown that mutations of contractile proteins, especially myosin heavy chain and troponin, are tightly associated with diastolic dysfunction in patients with Cardiomyopathies (CMs), including pediatric patients with CM. Therapeutics, including green tea extract (epigallocatechin gallate) and mavacamten, interact directly with these contractile proteins and have shown promising results. This article will review recent and contemporary research on diastolic dysfunction in CMs, especially hypertrophic cardiomyopathy and RCM, which include their target proteins, mechanisms, clinical diagnosis, and potential therapies.

Reorganization of mitochondria-organelle interactions during postnatal development in skeletal muscle.

Skeletal muscle cellular development requires the integrated assembly of mitochondria and other organelles adjacent to the sarcomere in support of muscle contractile performance. However, it remains unclear how interactions among organelles and with the sarcomere relates to the development of muscle cell function. Here, we combine 3D volume electron microscopy, proteomic analyses, and live cell functional imaging to investigate the postnatal reorganization of mitochondria-organelle interactions in skeletal muscle. We show that while mitochondrial networks are disorganized and loosely associated with the contractile apparatus at birth, contact sites among mitochondria, lipid droplets and the sarcoplasmic reticulum are highly abundant in neonatal muscles. The maturation process is characterized by a transition to highly organized mitochondrial networks wrapped tightly around the muscle sarcomere but also to less frequent interactions with both lipid droplets and the sarcoplasmic reticulum. Concomitantly, expression of proteins involved in mitochondria-organelle membrane contact sites decreases during postnatal development in tandem with a decrease in abundance of proteins associated with sarcomere assembly despite an overall increase in contractile protein abundance. Functionally, parallel measures of mitochondrial membrane potential, NADH redox status, and NADH flux within intact cells revealed that mitochondria in adult skeletal muscle fibres maintain a more activated electron transport chain compared with neonatal muscle mitochondria. These data demonstrate a developmental redesign reflecting a shift from muscle cell assembly and frequent inter-organelle communication toward a muscle fibre with mitochondrial structure, interactions, composition and function specialized to support contractile function. KEY POINTS: Mitochondrial network organization is remodelled during skeletal muscle postnatal development. The mitochondrial outer membrane is in frequent contact with other organelles at birth and transitions to more close associations with the contractile apparatus in mature muscles. Mitochondrial energy metabolism becomes more activated during postnatal development. Understanding the developmental redesign process within skeletal muscle cells may help pinpoint specific areas of deficit in muscles with developmental disorders.

Abstract 1525 Structural and functional diversity of calcium-triggered contractile proteins in ciliates

Abstract 1525 Structural and functional diversity of calcium-triggered contractile proteins in ciliates

UNDERSTANDING MUSCLE CONTRACTION: MECHANISMS AND IMPLICATIONS

Muscle contraction is a fundamental physiological process critical for movement, stability, and various bodily functions. This research paper delves into the intricate mechanisms underlying muscle contraction, exploring the role of actin, myosin, calcium ions, and regulatory proteins in the sliding filament theory. Additionally, it discusses the physiological and biochemical aspects of muscle contraction, including the excitation-contraction coupling process and the energy requirements for muscle activity. Furthermore, the paper explores the implications of muscle contraction dysfunction in conditions such as muscular dystrophy and the potential therapeutic interventions. By comprehensively understanding muscle contraction, researchers and clinicians can advance treatments for muscle-related disorders and optimize performance in various fields.

Native Top-Down Mass Spectrometry for Characterizing Sarcomeric Proteins Directly from Cardiac Tissue Lysate.

Native top-down mass spectrometry (nTDMS) has emerged as a powerful structural biology tool that can localize post-translational modifications (PTMs), explore ligand-binding interactions, and elucidate the three-dimensional structure of proteins and protein complexes in the gas-phase. Fourier-transform ion cyclotron resonance (FTICR) MS offers distinct capabilities for nTDMS, owing to its ultrahigh resolving power, mass accuracy, and robust fragmentation techniques. Previous nTDMS studies using FTICR have mainly been applied to overexpressed recombinant proteins and protein complexes. Here, we report the first nTDMS study that directly analyzes human heart tissue lysate by direct infusion FTICR MS without prior chromatographic separation strategies. We have achieved comprehensive nTDMS characterization of cardiac contractile proteins that play critical roles in heart contraction and relaxation. Specifically, our results reveal structural insights into ventricular myosin light chain 2 (MLC-2v), ventricular myosin light chain 1 (MLC-1v), and alpha-tropomyosin (α-Tpm) in the sarcomere, the basic contractile unit of cardiac muscle. Furthermore, we verified the calcium (Ca2+) binding domain in MLC-2v. In summary, our nTDMS platform extends the application of FTICR MS to directly characterize the structure, PTMs, and metal-binding of endogenous proteins from heart tissue lysate without prior separation methods.

Synchronous force and Ca2+ measurements for repeated characterization of excitation-contraction coupling in human myocardium

Dysfunctional Ca2+ signaling affects the myocardial systole and diastole, may trigger arrhythmia and cause transcriptomic and proteomic modifications in heart failure. Thus, synchronous real-time measurement of Ca2+ and force is essential to investigate the relationship between contractility and Ca2+ signaling and the alteration of excitation-contraction coupling (ECC) in human failing myocardium. Here, we present a method for synchronized acquisition of intracellular Ca2+ and contraction force in long-term cultivated slices of human failing myocardium. Synchronous time series of contraction force and intracellular Ca2+ were used to calculate force-calcium loops and to analyze the dynamic alterations of ECC in response to various pacing frequencies, post-pause potentiation, high mechanical preload and pharmacological interventions in human failing myocardium. We provide an approach to simultaneously and repeatedly investigate alterations of contractility and Ca2+ signals in long-term cultured myocardium, which will allow detecting the effects of electrophysiological or pharmacological interventions on human myocardial ECC.