Cardiac Muscle Relaxation Research Articles

Novel heterozygous mutation of CACNA2D1 gene in a Chinese family with arrhythmia

BackgroundPrimary electrical disorders (PEDs) are a group of cardiac rhythm abnormalities that occur in the absence of detectable structural heart disease and are a significant cause of sudden cardiac death (SCD). The initiation of cardiac muscle contraction and relaxation is orchestrated by the action potential (AP), generated through ionic changes across the membrane. Mutations in the AP-related gene CACNA2D1 have been identified as a causative factor for PED.MethodsWe recruited a Chinese family with a history of arrhythmia. The proband has experienced palpitations and chest tightness for over 40 years, with symptoms worsening over the past year. Whole exome sequencing (WES) was used to determine the genetic etiologies in this family.ResultsA novel heterozygous missense mutation (NM_000722.3: c.1685G > C;p.G562A) of CACNA2D1 gene was detected. Genotyping of the proband’s parents indicated that the arrhythmia phenotype in the proband was caused by a de novo mutation.ConclusionsWES was utilized to explore the genetic etiology in a family with arrhythmia, leading to the identification of a novel mutation in the CACNA2D1 gene. This study not only expands the mutation spectrum of the CACNA2D1 gene but also contributes to genetic counseling and clinical diagnosis for this family.

Kinetics and mapping of Ca-driven calmodulin conformations on skeletal and cardiac muscle ryanodine receptors

Calmodulin transduces [Ca2+] information regulating the rhythmic Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during contraction and relaxation in cardiac and skeletal muscle. However, the structural dynamics by which calmodulin modulates the sarcoplasmic reticulum Ca2+ release channel, the ryanodine receptor, at physiologically relevant [Ca2+] is unknown. Using fluorescence lifetime FRET, we resolve different structural states of calmodulin and Ca2+-driven shifts in the conformation of calmodulin bound to ryanodine receptor. Skeletal and cardiac ryanodine receptor isoforms show different calmodulin-ryanodine receptor conformations, as well as binding and structural kinetics with 0.2-ms resolution, which reflect different functional roles of calmodulin. These FRET methods provide insight into the physiological calmodulin-ryanodine receptor structural states, revealing additional distinct structural states that complement cryo-EM models that are based on less physiological conditions. This technology will drive future studies on pathological calmodulin-ryanodine receptor interactions and dynamics with other important ryanodine receptor bound modulators.

Glutamate 139 of tropomyosin is critical for cardiac thin filament blocked-state stabilization

The cardiac thin filament proteins troponin and tropomyosin control actomyosin formation and thus cardiac contractility. Calcium binding to troponin changes tropomyosin position along the thin filament, allowing myosin head binding to actin required for heart muscle contraction. The thin filament regulatory proteins are hot spots for genetic mutations causing heart muscle dysfunction. While much of the thin filament structure has been characterized, critical regions of troponin and tropomyosin involved in triggering conformational changes remain unresolved. A poorly resolved region, helix-4 (H4) of troponin I, is thought to stabilize tropomyosin in a position on actin that blocks actomyosin interactions at low calcium concentrations during muscle relaxation. We have proposed that contact between glutamate 139 on tropomyosin and positively charged residues on H4 leads to blocking-state stabilization. In this study, we attempted to disrupt these interactions by replacing E139 with lysine (E139K) to define the importance of this residue in thin filament regulation. Comparison of mutant and wild-type tropomyosin was carried out using in-vitro motility assays, actin co-sedimentation, and molecular dynamics simulations to determine perturbations in troponin-tropomyosin function caused by the tropomyosin mutation. Motility assays revealed that mutant thin filaments moved at higher velocity at low calcium with increased calcium sensitivity demonstrating that tropomyosin residue 139 is vital for proper tropomyosin-mediated inhibition during relaxation. Similarly, molecular dynamic simulations revealed a mutation-induced decrease in interaction energy between tropomyosin-E139K and troponin I (R170 and K174). These results suggest that salt-bridge stabilization of tropomyosin position by troponin IH4 is essential to prevent actomyosin interactions during cardiac muscle relaxation.

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).

Cardiac Myosin Inhibitors: Expanding the Horizon for Hypertrophic Cardiomyopathy Management.

To review the current literature on the efficacy and safety of cardiac myosin inhibitors (CMIs) for the treatment of hypertrophic cardiomyopathy (HCM). A literature search was conducted on PubMed from origin to April 2023, using the search terms "MYK-461," "mavacamten," "CK-3773274," and "aficamten." Studies were limited to English-based literature, human subjects, and clinical trials resulting in the inclusion of 13 articles. ClinicalTrials.gov was also used with the same search terms for ongoing and completed trials. Only phase II and III studies were included in this review except for pharmacokinetic studies that were used to describe drug properties. CMIs enable cardiac muscle relaxation by decreasing the number of myosin heads that can bind to actin and form cross-bridges. Mavacamten, the first Food and Drug Administration (FDA)-approved drug in this class, has been shown to improve hemodynamic, functional, and quality of life measures in HCM with obstruction. In addition, aficamten is likely to become the next FDA-approved CMI with promising phase II data and an ongoing phase III trial expected to release results in the next year. CMIs provide a novel option for obstructive hypertrophic cardiomyopathy, particularly in those not suitable for septal reduction therapy. Utilization of these agents requires knowledge of drug interactions, dose titration schemes, and monitoring parameters for safety and efficacy. CMIs represent a new class of disease-specific drugs for treatment of HCM. Cost-effectiveness studies are needed to delineate the role of these agents in patient therapy.

Phosphate toxicity and SERCA2a dysfunction in sudden cardiac arrest.

Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one-third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion-channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end-product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end-product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low-phosphate dietary interventions to reduce the risk of sudden cardiac arrest.

Down syndrome patients with normal hearts: are they really normal?

Even though congenital heart disease is a common finding in down syndrome (DS) patients, some of them have anatomically normal hearts. However, the term "normal" might not be suitable, as these patients usually suffer from functional cardiac dysfunction. Several research highlighted that despite the absence of anatomical heart defects, subtle cardiac function derangements are present in DS patients. We aim to assess cardiac functions by Two-dimensional echocardiography and tissue Doppler imaging (TDI) in pediatric DS patients who have anatomically normal hearts. One hundred seventy-two patients with karyotyping confirmed DS with anatomically normal hearts and 165 healthy normal control children were enrolled in the current study. Their cardiac functions were assessed using both 2-dimensional echocardiography and TDI. Both patients and controls had structurally and anatomically normal hearts. In DS patients, the right side of the heart showed a significant reduction in both systolic and diastolic functions. Systolic dysfunction was evident by significantly decreased levels of Tricuspid annular plane systolic excursion and systolic wave by TDI. Diastolic dysfunction of the right ventricle was evident by prolonged deceleration time by conventional echocardiography and a significant decrease in annular tissue doppler velocity during early diastole/late diastole ratio by TDI. The E/De ratio was significantly increased. Even with anatomically normal hearts, DS patients should undergo cardiac function assessment by echocardiography & TDI. TDI is superior to conventional echocardiography in detecting subtle cardiac dysfunction especially left ventricular diastolic dysfunction in DS patients. TDI showed a significant decrease in the early/atrial ratio of mitral valve annulus and prolongation of left ventricle isometric relaxation time in DS children. Also, the left ventricle E/De ratio was prolonged denoting elevated filling pressures and diastolic dysfunction. This indicates that the TDI has higher sensitivity to detect diastolic dysfunction than conventional Echocardiography. Biventricular TDI-derived myocardial performance index was found to be significantly increased in DS children.

Kinetics and mapping of calcium-driven calmodulin conformations on ryanodine receptors.

Calmodulin (CaM) is an important Ca2+-sensor for Ca2+ cycling between the sarcoplasmic reticulum and cytoplasm during relaxation and contraction in cardiac and skeletal muscle. Although several high-resolution cryo-EM structures have been published recently, the physiologically relevant mechanism by which CaM modulates the SR Ca2+ release channel (ryanodine receptor, RyR) is still not completely understood. Using distances calculated from fluorescence lifetime (FLT)-detected FRET between RyR-bound FKBP to CaM, we have trilaterated the structural states of CaM and mapped Ca2+-driven shifts in the conformation of CaM bound to RyR. As the locations of both the N- and C-lobes can be determined, we found that CaM becomes more compact in contracting high-Ca2+ relative to the relaxing low-Ca2+ and that skeletal and cardiac RyR isoforms show different CaM-RyR conformations and binding. These structural observations correlate with the experimentally determined modulatory action of CaM. By integrating our FLT-FRET system in a stopped-flow experiment, we resolved CaM binding kinetics and millisecond Ca2+-driven structural transitions of CaM-bound to RyR. Distinct binding kinetics of CaM to RyR1 and RyR2 were observed, suggesting different CaM-RyR interactions. Specifically, CaM association to RyR2 is three-fold faster vs. RyR1 at 30 nM and 30 μM. Upon rapid Ca2+ transition from μM to nM, the structural transition is four-fold slower for CaM bound to RyR2 relative to RyR1. The results provide mechanistic insight into CaM's action to transduce Ca2+ signals for RyR channel function during muscle contraction, and furthermore the technology developed holds promise for determining structure-function relationships of other RyR modulators. This work was supported by NIH grants R01HL92097 (RLC, DMB) and R37AG26160 (DDT), and by American Heart Association Fellowship 16POST31010019 (RTR).

Suppression of lusitropy as a disease mechanism in cardiomyopathies.

In cardiac muscle the action of adrenaline on β1 receptors of heart muscle cells is essential to adjust cardiac output to the body's needs. Adrenergic activation leads to enhanced contractility (inotropy), faster heart rate (chronotropy) and faster relaxation (lusitropy), mainly through activation of protein kinase A (PKA). Efficient enhancement of heart output under stress requires all of these responses to work together. Lusitropy is essential for shortening the heartbeat when heart rate increases. It therefore follows that, if the lusitropic response is not present, heart function under stress will be compromised. Current literature suggests that lusitropy is primarily achieved due to PKA phosphorylation of troponin I (TnI) and phospholamban (PLB). It has been well documented that PKA-induced phosphorylation of TnI releases Ca2+ from troponin C faster and increases the rate of cardiac muscle relaxation, while phosphorylation of PLB increases SERCA activity, speeding up Ca2+ removal from the cytoplasm. In this review we consider the current scientific evidences for the connection between suppression of lusitropy and cardiac dysfunction in the context of mutations in phospholamban and thin filament proteins that are associated with cardiomyopathies. We will discuss what advances have been made into understanding the physiological mechanism of lusitropy due to TnI and PLB phosphorylation and its suppression by mutations and we will evaluate the evidence whether lack of lusitropy is sufficient to cause cardiomyopathy, and under what circumstances, and consider the range of pathologies associated with loss of lusitropy. Finally, we will discuss whether suppressed lusitropy due to mutations in thin filament proteins can be therapeutically restored.

Characterizing the Effects of Voluntary Wheel Running on Cardiac SERCA Function in Ovariectomized Mice

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) actively transports Ca2+ into the sarcoplasmic reticulum to facilitate cardiac muscle relaxation. Phospholamban (PLN) allosterically inhibits SERCA, and an imbalance of SERCA2a, dominant cardiac isoform, and PLN content disrupts Ca2+ homeostasis and cardiac contractility. A previous study has shown that ovariectomized (OVX) rats have reduced SERCA activity due to lowered SERCA2a and increased PLN content. Furthermore, it was found that forced treadmill running in OVX rats restored SERCA activity and protein content levels. Here, we investigated whether voluntary wheel running (VWR) would produce similar effects on cardiac SERCA function in OVX mice. Female mice were divided into the following groups for 8 weeks: SHAM; OVX; SHAM + VWR; and OVX + VWR (n = 10/group). SERCA activity and Ca2+ uptake assays were performed in cardiac muscle homogenates. Protein levels of SERCA2, PLN, and pPLN were determined via Western blot analysis. We found statistical interactions for Ca2+ uptake, maximal SERCA activity, and SERCA2a content where VWR increased these parameters in SHAM mice but not in OVX mice. We detected a main effect of OVX on PLN content, and main effects of OVX and VWR on pPLN content. The OVX mice ran significantly less than the SHAM mice, suggesting that estrogen deprivation and lack of regular exercise may blunt the effects of voluntary aerobic exercise on cardiac SERCA function.

Potential Value of Circular RNA circTBC1D4 in Gastrointestinal Stromal Tumors.

Aims To explore the expression of circular RNA (circRNA) in gastrointestinal stromal tumors. Background Gastrointestinal stromal tumors (GIST) are mainly distributed in the stomach and small intestine. Recently, it has been verified that circular RNA (circRNA) has an important function in the regulation of GIST. Nevertheless, detailed investigations of circRNA-miRNA-mRNA regulatory networks in GIST are lacking. Objective To analyze the gastrointestinal stromal tumor circRNA-miRNA-mRNA network, assessing the effect of circle RNA in gastrointestinal stromal tumors. Method All the differential circRNAs and mRNAs were obtained from Gene Expression Omnibus (GEO) microarray data (GSE131481 and GSE147303, GSE131481, and GSE13861). Furthermore, a circRNA-miRNA-mRNA network was established. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to reveal the correlation between the functions of signaling pathways and target genes. The hub genes of protein-protein interaction (PPI) network and cytoHubba were also defined. Quantitative real-time PCR (qRT-PCR) was used to measure the expression levels of hsa-circ-0002917 (circTBC1D4), hsa-miR-590-5p (miR-590-5p), and PLN. Results PPI network and Cytoscape showed that ATP1A2, PLN, KCNMA1, and SCNN1B were four central DEGs. GO analysis results revealed that DEGs were involved in negative management of myocardial contraction, regulation of myocardial cell contraction, ethanol oxidation, cellular potassium ion homeostasis, and relaxation of cardiac muscle, and KEGG analysis showed that major DEGs were with cGMP-PKG signaling pathway. Moreover, we obtained two pairs of axes, namely, hsa-circ-0039216/hsa-miR-338-3p/ATP1A2 and hsa-circ-0002917/hsa-miR-590-5p/PLN. The target of TBC1D4 is miR-590-5p, and miR-590-5p increased after knocking down TBC1D4. Moreover, PLN was the target of miR-590-5p, and miR-590-5p exerts antitumor effects by reducing PLN. Conclusions In this study, we constructed a circRNA-miRNA-mRNA management network interrelated with GIST and researched the potential roles of circRNA. Moreover, we discovered a new molecular landmarker for the prediction, diagnosis, and therapy of patients.

Remote Hind‐Limb Ischemia Mechanism of Preserved Ejection Fraction During Heart Failure

During acute heart failure (HF), remote ischemic conditioning (RIC) has proven to be beneficial; however, it is currently unclear whether it also extends benefits from chronic congestive, cardiopulmonary heart failure (CHF). Previous studies from our laboratory have shown three phases describing CHF viz. 1) HF with preserved ejection fraction (HFpEF), 2) HF with reduced EF (HFrEF), and 3) HF with reversed EF. Although reciprocal organ interaction, ablation of sympathetic, and calcium signaling genes are associated with HFpEF to HFrEF, the mechanism is unclear. The HFrEF ensues, in part, due to reduced angiogenesis, coronary reserve, and leakage of endocardial endothelial (EE) and finally breakdown of the blood‐heart barrier (BHB). In fact, our hypothesis states that a change in phenotype from compensatory HFpEF to decompensatory HFrEF is determined by a potential decrease in regenerative capacity, proangiogenic factors along with a concomitant increase in epigenetic memory, inflammation that combinedly cause oxidative, and proteolytic stress response. To test this hypothesis, we created CHF by aorta‐vena‐cava (AV) fistula in a group of mice that were subsequently treated with that of hind‐limb RIC. HFpEF vs. HFrEF transition was determined by serial or longitudinal echo measurements. Results revealed an increase in skeletal muscle musclin contents, bone‐marrow (CD71), and sympathetic activation (β2‐AR) by RIC. We also observed a decrease in vascular density, and attenuation of EE‐BHB function due to a corresponding increase in the activity of MMP‐2, VEGF, caspase, and calpain. This decrease was successfully mitigated by RIC‐released skeletal muscle derived exosomes that contain musclin; the myokine along with bone marrow, and sympathetic activation. In short, based on proteome (omics) analysis, ∼20 proteins that appear to be involved in the signaling pathway responsible for the synthesis, contraction, and relaxation of cardiac muscle were found to be the dominant features. Thus, our results support that the CHF phenotype causes dysfunction of cardiac metabolism affecting its contraction, and relaxation. Interestingly, RIC was able to mitigate many of the deleterious changes, as revealed by our multi‐omics findings.

Evolution of the N-Terminal Regulation of Cardiac Troponin I for Heart Function of Tetrapods: Lungfish Presents an Example of the Emergence of Novel Submolecular Structure to Lead the Capacity of Adaptation.

Troponin-based Ca2+ regulation of striated muscle contraction emerged approximately 700 million years ago with largely conserved functions during evolution. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI is specifically expressed in the adult heart and has a unique N-terminal extension implicating a specific value during natural selection. The N-terminal extension of cardiac TnI in higher vertebrates contains β-adrenergic-regulated protein kinase A (PKA) phosphorylation sites as a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Phylogenic studies showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. This apparently rapid evolution of β-adrenergic regulation of cardiac function suggests a high selection value for the heart of vertebrate animals on land to work under higher metabolic demands. Sequencing and PKA phosphorylation data showed that lungfish cardiac TnI has evolved with an amphibian-like N-terminal extension with prototype PKA phosphorylation sites while its overall structure remained fish like. The data demonstrate that the submolecular structure of TnI may evolve ahead of the whole protein for cardiac muscle contractility to adapt to new environmental conditions. Understanding the evolution of the β-adrenergic regulation of TnI and cardiac adaptation to the increased energetic demands of life on land adds knowledge for the treatment of human heart diseases and failure.

Remote Hind-Limb Ischemia Mechanism of Preserved Ejection Fraction During Heart Failure

During acute heart failure (HF), remote ischemic conditioning (RIC) has proven to be beneficial; however, it is currently unclear whether it also extends benefits from chronic congestive, cardiopulmonary heart failure (CHF). Previous studies from our laboratory have shown three phases describing CHF viz. (1) HF with preserved ejection fraction (HFpEF), (2) HF with reduced EF (HFrEF), and (3) HF with reversed EF. Although reciprocal organ interaction, ablation of sympathetic, and calcium signaling genes are associated with HFpEF to HFrEF, the mechanism is unclear. The HFrEF ensues, in part, due to reduced angiogenesis, coronary reserve, and leakage of endocardial endothelial (EE) and finally breakdown of the blood-heart barrier (BHB) integrity. In fact, our hypothesis states that a change in phenotype from compensatory HFpEF to decompensatory HFrEF is determined by a potential decrease in regenerative, proangiogenic factors along with a concomitant increase in epigenetic memory, inflammation that combinedly causes oxidative, and proteolytic stress response. To test this hypothesis, we created CHF by aorta-vena-cava (AV) fistula in a group of mice that were subsequently treated with that of hind-limb RIC. HFpEF vs. HFrEF transition was determined by serial/longitudinal echo measurements. Results revealed an increase in skeletal muscle musclin contents, bone-marrow (CD71), and sympathetic activation (β2-AR) by RIC. We also observed a decrease in vascular density and attenuation of EE-BHB function due to a corresponding increase in the activity of MMP-2, vascular endothelial growth factor (VEGF), caspase, and calpain. This decrease was successfully mitigated by RIC-released skeletal muscle exosomes that contain musclin, the myokine along with bone marrow, and sympathetic activation. In short, based on proteome (omics) analysis, ∼20 proteins that appear to be involved in signaling pathways responsible for the synthesis, contraction, and relaxation of cardiac muscle were found to be the dominant features. Thus, our results support that the CHF phenotype causes dysfunction of cardiac metabolism, its contraction, and relaxation. Interestingly, RIC was able to mitigate many of the deleterious changes, as revealed by our multi-omics findings.

Emergence of the N-Terminal Regulatory Domain of Cardiac Troponin I for Living on Land and the Function of Adult Heart of Higher Vertebrates

The troponin complex plays a central role in regulating the contraction and relaxation of striated muscles. Troponin-based thin filament regulation of muscle contraction emerged approximately 700 million years ago with largely conserved functions. Troponin I (TnI) is the inhibitory subunit of troponin and has evolved into three muscle type-specific isoforms in vertebrates. Cardiac TnI has an N-terminal extension that is specifically expressed in the adult heart, implicating a unique selection value. Cardiac TnI of higher vertebrate species contains beta-adrenergic regulated PKA phosphorylation sites in the N-terminal extension, which is a mechanism to enhance cardiac muscle relaxation and facilitate ventricular filling. Our data mining showed that the N-terminal extension of cardiac TnI first emerged in the genomes of early tetrapods as well as primordial lobe-finned fishes such as the coelacanth whereas it is absent in ray-finned fish. An intriguing finding is the lack of a transitional species that has an N-terminal extension in cardiac TnI without the PKA phosphorylation sites. This apparently rapid evolution of the beta-adrenergic regulation of cardiac function suggests a high selection value in the cardiac function of vertebrate animals on land. To understand the evolution and original function of the N-terminal extension in modulating cardiac muscle contractility, our study comparing the cardiac muscle of primitive lobe-finned fish with that of mammals and ray-finned fish investigates its role in cardiac adaptation to the increased energetic demands of life on land and significance in human health and the treatment of heart failure.

The C-Terminal End Peptide of Troponin I as a Myofilament Ca2+Desensitizer

The C-terminal end segment of troponin I (TnI) is a highly conserved structure. Mutation in this segment impairs cardiac muscle relaxation. This segment is a Ca2+-regulated allosteric structure in troponin complex and binds tropomyosin in low Ca2+state, indicating a role in the inhibitory activity of TnI during muscle relaxation. The C-terminal end peptide forms a conserved epitope recognized by a monoclonal antibody. We characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its effect on modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 free peptide in physiological solution retains the native epitope recognized by the anti-TnI C-terminus monoclonal antibody. HcTnI-C27 peptide also retains the binding affinity for tropomyosin as that residing in intact TnI. A restrictive cardiomyopathy mutation inHcTnI-C27 peptide (R192H) abolishes its bindings to the anti-TnI C-terminus monoclonal antibody and tropomyosin, demonstrating a pathogenic destruction with loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of free HcTnI-C27 peptide reduces Ca2+-sensitivity of myofibrils at high Ca2+state without decreasing the maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer a physiological effect on myofilament Ca2+-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation in the activated state as a potential treatment for diastolic dysfunction and heart failure. This notion is supported by previous findings that a conformational modulation of the T subunit of troponin (TnT) to add a TnI C-terminus-like element in the contractile machinery of transgenic mouse cardiac muscle selectively prolongs ventricular end systolic ejection time to increase stroke volume without increase in pressure development.

The relation between sarcomere energetics and the rate of isometric tension relaxation in healthy and diseased cardiac muscle

Full muscle relaxation happens when [Ca2+] falls below the threshold for force activation. Several experimental models, from whole muscle organs and intact muscle down to skinned fibers, have been used to explore the cascade of kinetic events leading to mechanical relaxation. The use of single myofibrils together with fast solution switching techniques, has provided new information about the role of cross-bridge (CB) dissociation in the time course of isometric force decay. Myofibril’s relaxation is biphasic starting with a slow seemingly linear phase, with a rate constant, slow kREL, followed by a fast mono-exponential phase. Sarcomeres remain isometric during the slow force decay that reflects CB detachment under isometric conditions while the final fast relaxation phase begins with a sudden give of few sarcomeres and is then dominated by intersarcomere dynamics. Based on a simple two-state model of the CB cycle, myofibril slow kREL represents the apparent forward rate with which CBs leave force generating states (gapp) under isometric conditions and correlates with the energy cost of tension generation (ATPase/tension ratio); in short slow kREL ~ gapp ~ tension cost. The validation of this relationship is obtained by simultaneously measuring maximal isometric force and ATP consumption in skinned myocardial strips that provide an unambiguous determination of the relation between contractile and energetic properties of the sarcomere. Thus, combining kinetic experiments in isolated myofibrils and mechanical and energetic measurements in multicellular cardiac strips, we are able to provide direct evidence for a positive linear correlation between myofibril isometric relaxation kinetics (slow kREL) and the energy cost of force production both measured in preparations from the same cardiac sample. This correlation remains true among different types of muscles with different ATPase activities and also when CB kinetics are altered by cardiomyopathy-related mutations. Sarcomeric mutations associated to hypertrophic cardiomyopathy (HCM), a primary cardiac disorder caused by mutations in genes encoding sarcomeric proteins, have been often found to accelerate CB turnover rate and increase the energy cost of myocardial contraction. Here we review data showing that faster CB detachment results in a proportional increase in the energetic cost of tension generation in heart samples from both HCM patients and mouse models of the disease.

The evolutionarily conserved C-terminal peptide of troponin I is an independently configured regulatory structure to function as a myofilament Ca2+-desensitizer

The C-terminal end segment of troponin subunit I (TnI) is a structure highly conserved among the three muscle type-specific isoforms and across vertebrate species. Partial deletion or point mutation in this segment impairs cardiac muscle relaxation. In the present study, we characterized the C-terminal 27 amino acid peptide of human cardiac TnI (HcTnI-C27) for its role in modulating muscle contractility. Biologically or chemically synthesized HcTnI-C27 peptide retains an epitope structure in physiological solutions similarly to that in intact TnI as recognized by an anti-TnI C-terminus monoclonal antibody (mAb TnI-1). Protein binding studies found that HcTnI-C27 retains the binding affinity for tropomyosin as previously shown with intact cardiac TnI. A restrictive cardiomyopathy mutation R192H in this segment abolishes the bindings to mAb TnI-1 and tropomyosin, demonstrating a pathogenic loss of function. Contractility studies using skinned muscle preparations demonstrated that addition of HcTnI-C27 peptide reduces the Ca2+-sensitivity of myofibrils without decreasing maximum force production. The results indicate that the C-terminal end segment of TnI is a regulatory element of troponin, which retains the native configuration in the form of free peptide to confer an effect on myofilament Ca2+-desensitization. Without negative inotropic impact, this short peptide may be developed into a novel reagent to selectively facilitate cardiac muscle relaxation at the activated state as a potential treatment for heart failure.

Analysis of cardiac troponin proteoforms by top-down mass spectrometry.

The cardiac troponin complex, composed of three regulatory proteins (cTnI, cTnT, TnC), functions as the critical regulator of cardiac muscle contraction and relaxation. Myofilament protein-protein interactions are regulated by post-translational modifications (PTMs) to the protein constituents of this complex. Dysregulation of troponin PTMs, particularly phosphorylation, results in altered cardiac contractility. Altered PTMs and isoforms have been increasingly recognized as the molecular mechanisms underlying heart diseases. Therefore, it is essential to comprehensively analyze cardiac troponin proteoforms that arise from PTMs, alternative splicing, and sequence variations. In this chapter, we described two detailed protocols for the enrichment and purification of endogenous cardiac troponin proteoforms from cardiac tissue. Subsequently, mass spectrometry (MS)-based top-down proteomics utilizing online liquid chromatography (LC)/quadrupole time-of-flight (Q-TOF) MS for separation, profiling, and quantification of the troponins was demonstrated. Characterization of troponin amino acid sequence and the localization of PTMs were shown using Fourier-transform ion cyclotron resonance (FT-ICR) MS with electron capture dissociation (ECD) and collisionally activated dissociation (CAD). Furthermore, we described the use of MASH software, a comprehensive and free software package developed in our lab, for top-down proteomics data analysis. The methods we described can be applied for the analysis of troponin proteoforms in cardiac tissues, from animal models to human clinical samples, for heart disease.

Opioidergic conditioning of the human heart muscle in nitric oxide-dependent mechanism.

Opioidergic conditioning is well documented to trigger cardioprotection against ischemia/ reperfusion (I/R) injury. Previous studies on animal models have suggested that nitric oxide (NO) mediates the beneficial effect of opioids, but the role of NO in humans seems to be controversial. The aim of the study was to assess the influence of NO modulators on opioid-induced cardioprotection in the human myocardium. Trabeculae of the human right atria were electrically driven in an organ bath and subjected to simulated I/R injury. The non-selective inhibitor of nitric oxide synthase (NOS) - N-methyl-l-arginine (LNMMA), the donor of NO - S-Nitroso-N-acetylpenicillamine (SNAP) or morphine (in the amount of 10-4 M) were used at the time of re-oxygenation. The additional trabecula was subjected to the hypoxia protocol only (control). The contractility of the myocardium was assessed as the maximal force of a contraction (Amax), the rate of rise of the force of a contraction (Slope L) and the cardiac muscle relaxation - as the rate of decay of the force of a contraction (Slope T). The application of 100 μM LNMMA resulted in the decrease of Amax, Slope L and Slope T during the re-oxygenation period as compared to control. The application of 10-4 M morphine and/or 100 μM SNAP resulted in a partial reversal of the detrimental influence of LNMMA. At the re-oxygenation period, the blockade of NO synthesis has a deleterious effect on the systolic and diastolic function of the human myocardium as well as attenuates the beneficial effect of morphine conditioning.