Cardiac Myofibrils Research Articles

Preventive impact of probiotic supplements on heart injury and inflammatory indices in a rat model of myocardial infarction: histopathological and gene expression evaluation.

Although there is a bulk of evidence on the favorable effect of probiotics on the cardiac system, their role in the management of myocardial infarction is not clear. Three viable probiotic bacterial strains, namely Lactobacillus reuteri, Bifidobacterium longum, and Bifidobacterium lactis, were gavaged to the rats daily for 28 days prior to the induction of myocardial injury. Myocardial injury was induced by the use of isoproterenol (ISO) in the probiotics, control and sham groups. The heart tissues were catheterized to evaluate the histopathological parameters and measure the expression of genes related to inflammation. Treatment with ISO caused subendocardial necrosis and rupture of cardiac myofibrils. Pretreatment with probiotics reduced the size of myocardial infarction caused by ISO. Also, in the probiotic group, a relative decrease in the amount of tissue fibrosis and rupture of cardiomyocytes fibers was seen. Pretreatment with probiotics partially ameliorated myocardial necrosis, edema and leukocyte infiltration. Also, a remarkable decrease was detected in the expression of tissue proinflammatory genes in the pretreated group with probiotics. Thus, viable probiotic supplementation may ameliorate or prevent cardiac injury. Additional preclinical and clinical studies are required to clarify the impact of probiotics in the prevention and management of cardiovascular disease.

Genetic excision of the regulatory cardiac troponin I extension in high-heart rate mammal clades.

Mammalian cardiac troponin I (cTnI) contains a highly conserved amino-terminal extension harboring protein kinase A targets [serine-23 and -24 (Ser23/24)] that are phosphorylated during β-adrenergic stimulation to defend diastolic filling by means of an increased cardiomyocyte relaxation rate. In this work, we show that the Ser23/24-encoding exon 3 of TNNI3 was pseudoexonized multiple times in shrews and moles to mimic Ser23/24 phosphorylation without adrenergic stimulation, facilitating the evolution of exceptionally high resting heart rates (~1000 beats per minute). We further reveal alternative exon 3 splicing in distantly related bat families and confirm that both cTnI splice variants are incorporated into cardiac myofibrils. Because exon 3 of human TNNI3 exhibits a relatively low splice strength score, our findings offer an evolutionarily informed strategy to excise this exon to improve diastolic function during heart failure.

Mechanisms of a novel regulatory light chain-dependent cardiac myosin inhibitor.

Hypertrophic cardiomyopathy (HCM) is a genetic disease of the heart characterized by thickening of the left ventricle (LV), hypercontractility, and impaired relaxation. HCM is caused primarily by heritable mutations in sarcomeric proteins, such as β myosin heavy chain. Until recently, medications in clinical use for HCM did not directly target the underlying contractile changes in the sarcomere. Here, we investigate a novel small molecule, RLC-1, identified in a bovine cardiac myofibril high-throughput screen. RLC-1 is highly dependent on the presence of a regulatory light chain to bind to cardiac myosin and modulate its ATPase activity. In demembranated rat LV trabeculae, RLC-1 decreased maximal Ca2+-activated force and Ca2+ sensitivity of force, while it increased the submaximal rate constant for tension redevelopment. In myofibrils isolated from rat LV, both maximal and submaximal Ca2+-activated force are reduced by nearly 50%. Additionally, the fast and slow phases of relaxation were approximately twice as fast as DMSO controls, and the duration of the slow phase was shorter. Structurally, x-ray diffraction studies showed that RLC-1 moved myosin heads away from the thick filament backbone and decreased the order of myosin heads, which is different from other myosin inhibitors. In intact trabeculae and isolated cardiomyocytes, RLC-1 treatment resulted in decreased peak twitch magnitude and faster activation and relaxation kinetics. In conclusion, RLC-1 accelerated kinetics and decreased force production in the demembranated tissue, intact tissue, and intact whole cells, resulting in a smaller cardiac twitch, which could improve the underlying contractile changes associated with HCM.

Mechanisms of vibration-induced structural myocardial remodeling

The review analyzes literature data on structural changes in the heart of patients with vibration disease, as detected by echocardiographic methods. Particularly, it highlights concentric remodeling of the left ventricle chambers and disturbances in diastolic function. The review also discusses a 1.2-fold decrease in heart structure intensity compared to healthy individuals (p 0.05). Furthermore, it examines changes in morphometric and bioenergetic parameters of cardiomyocytes under different experimental vibration modes (7 and 56 sessions at a frequency of 8 Hz), confirming the disruptions in the relationship between the spatial configuration of the heart cavities, contractile ability, and energy supply potential. Loss of cardiac myofibrils represents the transition from myocardial hypertrophy to decompensation, accompanied by an increase in degenerative (dystrophic) signs such as the loss of sarcomeres in cardiomyocytes. Understanding these pathological (morphological) processes requires consideration of various mediators that regulate cell metabolism, proliferation, growth, and survival, including stromal interaction molecule, calcium ATPase of the endo(sarco)plasmic reticulum, inositol-1,4,5-triphosphate receptor, protein that forms CRAC channels, and transient receptor potential canonical. The degradation system of the extracellular matrix, including matrix metalloproteinases and tissue inhibitors, plays a crucial role in structural cardiac remodeling. This system regulates the rate of mRNA synthesis on the DNA matrix by binding to specific DNA regions that control cardiac nutrition and plasticity. The review suggests that these findings can help explain some patterns of cardiac remodeling development in patients with vibration disease and determine the direction of pathogenetically based therapy. This therapy should consider not only the vibration-protective effect of drugs but also their ability to inhibit and regress myocardial remodeling.

Fennel seeds extract prevents fructose-induced cardiac dysfunction in a rat model of metabolic syndrome via targeting abdominal obesity, hyperuricemia and NF-κβ inflammatory pathway

BackgroundMetabolic syndrome (MetS) is commonly associated with increased risk of cardiac disease that affects a large number of world populations. ObjectiveThis research attempted to investigate the efficacy of fennel seeds extract (FSE) in preventing development of cardiac dysfunction in rats on fructose enriched diet for 3 months, as a model of MetS. Materials & methodsThirty adult Wistar male rats (160–170 g) were assigned into 5 groups including control, vehicle, FSE (200 mg/kg BW) and fructose (60%) fed rats with and without FSE. Following the last treatment, blood pressure, ECG and heart rate were measured. Next, blood and cardiac tissues were taken for biochemical and histological investigations. ResultsFeeding fructose exhibited characteristic features of MetS involving, hypertension, abnormal ECG, elevated heart rate, serum glucose, insulin, lipids and insulin resistance, accompanied by abdominal obesity, cardiac hypertrophy and hyperuricemia. Fructose fed rats also showed significant reduction in cardiac antioxidants (GSH, SOD, CAT) with elevation in oxidative stress indices (NADPH oxidase, O2.-, H2O2, MDA, PCO), NF-κβ, pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), adhesion molecules (ICAM-1, VCAM-1) and serum cardiac biomarkers (AST, LDH, CK-MB, cTn-I). Histopathological changes evidenced by destruction of cardiac myofibrils, cytoplasmic vacuolization, and aggregation of inflammatory cells were also detected. Consumption of FSE showed high ability to alleviate fructose-induced hypertension, ECG abnormalities, cardiac hypertrophy, metabolic alterations, oxidative stress, inflammation and histological injury. ConclusionFindings could suggest FSE as a complementary supplement for preventing MetS and associated cardiac outcomes. However, well controlled clinical studies are still needed.

Distinguish response of low-dose radiation with different dose-rate on gene expression of human coronary artery endothelial cells: a bioinformatic study based on transcriptomic sequencing

Purpose People are exposed to low-dose radiation in medical diagnosis, occupational, or life circumstances, but the effect of low-dose radiation on human health is still controversial. The biological effects of radiation below 100 mGy are still unproven. In this study, we observed the effects of low-dose radiation (100 mGy) on gene expression in human coronary artery endothelial cells (HCAECs) and its effect on molecular signaling. Materials and methods HCAECs were exposed to 100 mGy ionizing radiation at 6 mGy/h (low-dose-rate) or 288 mGy/h (high-dose-rate). After 72 h, total RNA was extracted from sham or irradiated cells for Quant-Seq 3'mRNA-Seq, and bioinformatic analyses were performed using Metascape. Gene profiling was validated using qPCR. Results Compared to the non-irradiated control group, 100 mGy of ionizing radiation at 6 mGy/h altered the expression of 194 genes involved in signaling pathways related to heart contraction, blood circulation, and cardiac myofibril assembly differentially. However, 100 mGy at 288 mGy/h altered expression of 450 genes involved in cell cycle-related signaling pathways, including cell division, nuclear division, and mitosis differentially. Additionally, gene signatures responding to low-dose radiation, including radiation dose-specific gene profiles (HIST1H2AI, RAVER1, and POTEI) and dose-rate-specific gene profiles (MYL2 for the low-dose-rate and DHRS9 and CA14 for the high-dose-rate) were also identified. Conclusions We demonstrated that 100 mGy low-dose radiation could alter gene expression and molecular signaling pathways at the low-dose-rate and the high-dose-rate differently. Our findings provide evidence for further research on the potential impact of low-dose radiation on cardiovascular function.

Cardiac Troponin Activator CK-963 Increases Cardiac Contractility in Rats.

Novel cardiac troponin activators were identified using a high throughput cardiac myofibril ATPase assay and confirmed using a series of biochemical and biophysical assays. HTS hit 2 increased rat cardiomyocyte fractional shortening without increasing intracellular calcium concentrations, and the biological target of 1 and 2 was determined to be the cardiac thin filament. Subsequent optimization to increase solubility and remove PDE-3 inhibition led to the discovery of CK-963 and enabled pharmacological evaluation of cardiac troponin activation without the competing effects of PDE-3 inhibition. Rat echocardiography studies using CK-963 demonstrated concentration-dependent increases in cardiac fractional shortening up to 95%. Isothermal calorimetry studies confirmed a direct interaction between CK-963 and a cardiac troponin chimera with a dissociation constant of 11.5 ± 3.2 μM. These results provide evidence that direct activation of cardiac troponin without the confounding effects of PDE-3 inhibition may provide benefit for patients with cardiovascular conditions where contractility is reduced.

Lung-to-Heart Nano-in-Micro Peptide Promotes Cardiac Recovery in a Pig Model of Chronic Heart Failure

BackgroundThe lack of disease-modifying drugs is one of the major unmet needs in patients with heart failure (HF). Peptides are highly selective molecules with the potential to act directly on cardiomyocytes. However, a strategy for effective delivery of therapeutics to the heart is lacking. ObjectivesIn this study, the authors sought to assess tolerability and efficacy of an inhalable lung-to-heart nano-in-micro technology (LungToHeartNIM) for cardiac-specific targeting of a mimetic peptide (MP), a first-in-class for modulating impaired L-type calcium channel (LTCC) trafficking, in a clinically relevant porcine model of HF. MethodsHeart failure with reduced ejection fraction (HFrEF) was induced in Göttingen minipigs by means of tachypacing over 6 weeks. In a setting of overt HFrEF (left ventricular ejection fraction [LVEF] 30% ± 8%), animals were randomized and treatment was started after 4 weeks of tachypacing. HFrEF animals inhaled either a dry powder composed of mannitol-based microparticles embedding biocompatible MP-loaded calcium phosphate nanoparticles (dpCaP-MP) or the LungToHeartNIM only (dpCaP without MP). Efficacy was evaluated with the use of echocardiography, invasive hemodynamics, and biomarker assessment. ResultsDpCaP-MP inhalation restored systolic function, as shown by an absolute LVEF increase over the treatment period of 17% ± 6%, while reversing cardiac remodeling and reducing pulmonary congestion. The effect was recapitulated ex vivo in cardiac myofibrils from treated HF animals. The treatment was well tolerated, and no adverse events occurred. ConclusionsThe overall tolerability of LungToHeartNIM along with the beneficial effects of the LTCC modulator point toward a game-changing treatment for HFrEF patients, also demonstrating the effective delivery of a therapeutic peptide to the diseased heart.

Investigating Viscoelastic Properties of Myofibrils Isolated From hiPSC-CMs Using Atomic Force Microscopy and Quasi-Linear Viscoelastic Model

Abstract Knowing the mechanical properties of cardiac myofibrils isolated from human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can provide valuable insight into the structure and function of the heart muscle. Previous studies focused mostly on studying myofibrillar stiffness using simplified elastic models. In this study, the mechanical properties of myofibrils isolated from hiPSC-CMs were measured using atomic force microscopy (AFM). The quasi-linear viscoelastic (QLV) model was used to interpret the elastic and viscous properties of myofibrils. Since there have been no previous studies on the viscoelastic properties of myofibrils extracted from hiPSC-CMs, myofibrils extracted from porcine left-ventricular (LV) tissue were used to compare and verify experimental processes and QLV model parameters. The elastic modulus of myofibrils extracted from porcine LV tissue was determined to be 8.82 ± 6.09 kPa which is consistent with previous studies which reported that porcine LV tissue is less stiff on average than mouse and rat cardiac myofibrils. The elastic modulus of myofibrils extracted from hiPSC-CMs was found to be 9.78 ± 5.80 kPa, which is consistent with the range of 5–20 kPa reported for myofibrils extracted from the adult human heart. We found that myofibrils isolated from hiPSC-CMs relax slower than myofibrils extracted from porcine LV tissue, particularly in the first 0.25 s after the peak stress in the stress relaxation test. These findings provide important insights into the mechanical behavior of hiPSC-CMs and have implications for the development of treatments for heart diseases.

Galangin Attenuates Myocardial Ischemic Reperfusion-Induced Ferroptosis by Targeting Nrf2/Gpx4 Signaling Pathway.

Myocardial ischemic reperfusion injury (MIRI) is a crucial clinical problem globally. The molecular mechanisms of MIRI need to be fully explored to develop new therapeutic methods. Galangin (Gal), which is a natural flavonoid extracted from Alpinia Officinarum Hance and Propolis, possesses a wide range of pharmacological activities, but its effects on MIRI remain unclear. This study aimed to determine the pharmacological effects of Gal on MIRI. C57BL/6 mice underwent reperfusion for 3 h after 45 min of ischemia, and neonatal rat cardiomyocytes (NRCs) subjected to hypoxia and reoxygenation (HR) were cultured as in vivo and in vitro models. Echocardiography and TTC-Evans Blue staining were performed to evaluate the myocardial injury. Transmission electron microscope and JC-1 staining were used to validate the mitochondrial function. Additionally, Western blot detected ferroptosis markers, including Gpx4, FTH, and xCT. Gal treatment alleviated cardiac myofibril damage, reduced infarction size, improved cardiac function, and prevented mitochondrial injury in mice with MIRI. Gal significantly alleviated HR-induced cell death and mitigated mitochondrial membrane potential reduction in NRCs. Furthermore, Gal significantly inhibited ferroptosis by preventing iron overload and lipid peroxidation, as well as regulating Gpx4, FTH, and xCT expression levels. Moreover, Gal up-regulated nuclear transcriptive factor Nrf2 in HR-treated NRCs. Nrf2 inhibition by Brusatol abolished the protective effect of Gal against ferroptosis. This study revealed that Gal alleviates myocardial ischemic reperfusion-induced ferroptosis by targeting Nrf2/Gpx4 signaling pathway.

Characterizing residual and passive force enhancements in cardiac myofibrils

Residual force enhancement (RFE), an increase in isometric force after active stretching of a muscle compared with the purely isometric force at the corresponding length, has been consistently observed throughout the structural hierarchy of skeletal muscle. Similar to RFE, passive force enhancement (PFE) is also observable in skeletal muscle and is defined as an increase in passive force when a muscle is deactivated after it has been actively stretched compared with the passive force following deactivation of a purely isometric contraction. These history-dependent properties have been investigated abundantly in skeletal muscle, but their presence in cardiac muscle remains unresolved and controversial. The purpose of this study was to investigate whether RFE and PFE exist in cardiac myofibrils and whether the magnitudes of RFE and PFE increase with increasing stretch magnitudes. Cardiac myofibrils were prepared from the left ventricles of New Zealand White rabbits, and the history-dependent properties were tested at three different final average sarcomere lengths (n = 8 for each), 1.8, 2, and 2.2 μm, while the stretch magnitude was kept at 0.2 μm/sarcomere. The same experiment was repeated with a final average sarcomere length of 2.2 μm and a stretching magnitude of 0.4 μm/sarcomere (n = 8). All 32 cardiac myofibrils exhibited increased forces after active stretching compared with the corresponding purely isometric reference conditions (p < 0.05). Furthermore, the magnitude of RFE was greater when myofibrils were stretched by 0.4 compared with 0.2 μm/sarcomere (p < 0.05). We conclude that, like in skeletal muscle, RFE and PFE are properties of cardiac myofibrils and are dependent on stretch magnitude.

HFpEF animal models display differences in myofibril mechanics.

Heart Failure with preserved Ejection Fraction (HFpEF) is a complex, multi-organ disease historically defined by cardiac aberrations including diastolic dysfunction with normal resting systolic function and depressed systolic reserve, hypertrophy, and impaired relaxation. Though diastolic dysfunction is a hallmark of HFpEF, it is unknown whether organ-level deficiencies in relaxation persist down to the myofibrils, the contractile organelles within cells. Here, we performed mechanics analysis on cardiac myofibrils from three common HFpEF animal models to assess potential differences in myofibrillar force and relaxation kinetics, independent of higher-order deficits associated with the disease. The Göttingen minipig (GM) HFpEF model recapitulates the spectrum of human HFpEF multiorgan pathophysiology, including severe diastolic dysfunction, myocardial hypertrophy, fibrosis, and pulmonary and systemic hypertension. We found that GM-HFpEF myofibrils produced significantly less maximal force versus controls, with no change in either the slow or fast relaxation phases. Therefore, impaired myofibril relaxation likely does not contribute to the diastolic dysfunction in this model. Another model assessed was the Zucker spontaneously hypertensive heart failure F1 (ZSF1) rat, which exhibits elevated E/e′, insulin resistance, elevated end-diastolic pressure, and preserved ejection fraction. Our preliminary data indicate that ZSF1 myofibrils produce normal maximal force, but have a slower initial rate of relaxation compared to controls. These data suggest that impaired myofibril relaxation may contribute to diastolic dysfunction in the ZSF1 model. Moreover, the distinct responses of myofibrils from these models appear to recapitulate cell-level behavior in human cardiomyocytes isolated from patients with different HFpEF subphenotypes. Therefore, certain animal models may mimic unique HFpEF characteristics at the cellular and/or myofibril levels and be better-suited for investigating specific aspects of this extremely complex syndrome, and aid in the design of effective pharmacological or device-based treatments.

Elucidating the mechanism of Danicamtiv on force, kinetics, and myosin structure and function.

Myosin modulators are a novel class of small molecules that alter cardiac contractility. Omecamtiv mecarbil, the first identified myosin activator, showed only modest clinical benefits in systolic heart failure patients. Thus, there is an urgency to develop alternative myosin activators. Danicamtiv (Dani) has emerged as a potential candidate; however, a detailed mechanism is not known. Here, we aim to elucidate the mechanism of Dani on contractile function in pig cardiac muscle. Demembranated ventricular tissues show a significant 0.1 pCa unit increase in calcium sensitivity and 10% increase in maximal force after incubation in 1 µM Dani. The most potent effects occur in submaximal calcium concentrations, leading to a flattening of the force-calcium relationship, suggesting decreased cooperativity. Maximal rates of tension redevelopment are decreased by approximately 60% with Dani. Isolated cardiac myofibrils provide details about contractile kinetics. Experiments with 1 µM Dani show a 49% decrease in fast-phase relaxation kinetics. Slow-phase isometric relaxation exhibits 47% slower crossbridge detachment rate and 34% longer thin filament deactivation. Next, we assess ATP utilization in the crossbridge cycle. Filament sliding velocity slows 55% on addition of 0.5 µM Dani, similar to the effect of ADP on velocity. The effects of Dani and ADP are not additive suggesting a similar mode of action. ATP binding is unaltered up to 10 µM Dani using stopped flow spectroscopy. Results of X-ray diffraction studies of porcine myocardium at rest show an increase in equatorial intensity ratio (I1,1/I1,0) in response to 50 µM Dani, reflecting an increased proximity of myosin heads to the thin filament. In conclusion, we hypothesize that Dani primes the thick filament for activation and alters relaxation through inhibited ATP hydrolysis product release. Future studies will test these hypotheses.

Truncated titin is integrated into the human dilated cardiomyopathic sarcomere.

Heterozygous truncating mutations in the TTN gene (TTNtv) encoding the giant protein titin are the most common genetic cause of dilated cardiomyopathy (DCM). However, the molecular mechanisms by which TTNtv mutations induce DCM are controversial. Here we investigated 118 clinically identified DCM human cardiac samples with next-generation sequencing, high-resolution gel electrophoresis, Western blot analysis and super-resolution microscopy in order to dissect the structural and functional consequences of TTNtv mutations. The occurrence of TTNtv was found to be 15% in the DCM cohort. Truncated titin proteins matching, by molecular weight, the gene-sequence predictions were detected in the majority of the TTNtv samples. The total amount of expressed titin, which includes the truncated fragments, was comparable in the TTNtv+ and TTNtv- samples, indicating that titin haploinsufficiency is not the leading cause of the molecular pathogenesis. Proteomic analysis of washed cardiac myofibrils and STED super-resolution microscopy of myocardial sarcomeres labeled with sequence-specific anti-titin antibodies revealed that truncated titin is structurally integrated in the sarcomere. Sarcomere length-dependent anti-titin epitope position, shape and intensity analysis pointed at structural disarrangements in the I/A junction and the M-band of TTNtv+ sarcomeres, which may play a role, via faulty mechanosensor function, in the development of manifest DCM.

Myosin-binding protein C stabilizes, but is not the sole determinant of SRX myosin in cardiac muscle.

The myosin super-relaxed (SRX) state is central to striated muscle metabolic and functional regulation. In skeletal muscle, SRX myosin are predominantly colocalized with myosin-binding protein C (MyBP-C) in the sarcomere C-zone. To define how cardiac MyBP-C (cMyBP-C) and its specific domains contribute to stabilizing the SRX state in cardiac muscle, we took advantage of transgenic cMyBP-C null mice and those expressing cMyBP-C with a 271-residue N-terminal truncation. Utilizing super-resolution microscopy, we determined the lifetime and subsarcomeric location of individual fluorescent-ATP turnover events within isolated cardiac myofibrils. The proportion of SRX myosin demonstrated a gradient along the half-thick filament, highest in the P- and C-zones (72 ± 9% and 71 ± 6%, respectively) and lower in the D-zone (45 ± 10%), which lies farther from the sarcomere center and lacks cMyBP-C, suggesting a possible role for cMyBP-C in stabilizing the SRX. However, myofibrils from cMyBP-C null mice demonstrated an ∼40% SRX reduction, not only within the now cMyBP-C-free C-zone (49 ± 9% SRX), but also within the D-zone (22 ± 5% SRX). These data suggest that the influence of cMyBP-C on the SRX state is not limited to the C-zone but extends along the thick filament. Interestingly, myofibrils with N-terminal truncated cMyBP-C had an SRX content and spatial gradient similar to the cMyBP-C null, indicating that the N terminus of cMyBP-C is necessary for cMyBP-C's role in enhancing the SRX gradient along the entire thick filament. Given that SRX myosin exist as a gradient along the thick filament that is highest in the C-zone, even in the absence of cMyBP-C or its N-terminus, an inherent bias must exist in the structure of the thick filament to stabilize the SRX state.

Fluorescence lifetime-based assay reports structural changes in cardiac muscle mediated by effectors of contractile regulation.

Cardiac muscle contraction is regulated by Ca2+-induced structural changes of the thin filaments to permit myosin cross-bridge cycling driven by ATP hydrolysis in the sarcomere. In congestive heart failure, contraction is weakened, and thus targeting the contractile proteins of the sarcomere is a promising approach to therapy. However, development of novel therapeutic interventions has been challenging due to a lack of precise discovery tools. We have developed a fluorescence lifetime-based assay using an existing site-directed probe, N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenediamine (IANBD) attached to human cardiac troponin C (cTnC) mutant cTnCT53C, exchanged into porcine cardiac myofibrils. We hypothesized that IANBD-cTnCT53C fluorescence lifetime measurements provide insight into the activation state of the thin filament. The sensitivity and precision of detecting structural changes in cTnC due to physiological and therapeutic modulators of thick and thin filament functions were determined. The effects of Ca2+ binding to cTnC and myosin binding to the thin filament were readily detected by this assay in mock high-throughput screen tests using a fluorescence lifetime plate reader. We then evaluated known effectors of altered cTnC-Ca2+ binding, W7 and pimobendan, and myosin-binding drugs, mavacamten and omecamtiv mecarbil, used to treat cardiac diseases. Screening assays were determined to be of high quality as indicated by the Z' factor. We conclude that cTnC lifetime-based probes allow for precise evaluation of the thin filament activation in functioning myofibrils that can be used in future high-throughput screens of small-molecule modulators of function of the thin and thick filaments.

Epicardial adipose tissue thickness is related to early subclinical myocardial dysfunction, particularly in patients with type 2 diabetes mellitus: a case control study

BackgroundCardiac myofibrillary dysfunction, which can be measure by echocardiographical strain value, represents an early subclinical manifestation of heart failure. Epicardial Adipose tissue (EAT) is related to low degree inflammation and oxidative damage in the adjacent tissue.AimTo explore whether EAT affects early myocardial dysfunction, as assessed strain values.MethodsCase–Control design. Patients lacking clinical significant heart failure, thyroid or renal disease or malignant abnormalities were included. Clinical-demographic and biochemical data were collected. EAT and myofibril deformation were measured by echocardiography.ResultsA total of 71 patients were analyzed, and further subdivided according to type 2 Diabetes Mellitus (t2DM). Higher strain value (higher than -22.4%cut-off value) was associated with male sex and higher anthropometric and metabolic risk measures; particularly those with t2DM. Higher EAT was also associated higher strain value (AUC = 0.92 ± 0.06, p = 0.004), and further correlation was evidenced (rho = 0.488, p < 0.001), with significant influence of t2DM.ConclusionEAT was related to strain value, suggesting the influence of cardiac adipose tissue on the deformability of cardiac myofibril, with a more significant effect in the population with t2DM.

Truncated titin in the cardiac myofibril in dilated cardiomyopathy

Truncated titin in the cardiac myofibril in dilated cardiomyopathy

Abstract 14927: Real-Time Super-Resolution Analysis of Sarcomere Motion Reveals Context-Dependent Stochastic Heterogeneity in Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Studies of sarcomere dynamics in cardiac myofibrils are key to understand how myosin-actin contractility translates to whole heart contractions. Such analyses require high spatial (nm) and temporal (ms) resolution of multiple sarcomeres in parallel. Here, we present a new imaging and computational method to analyze sarcomere structure and function in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes. For this we (1) engineered a fluorescent z-band tag (ACTN2-Citrine) by CRISPR-editing, (2) developed micropatterned soft (7:1, 10 kPa) substrate cultures to mimic in vivo conditions, (3) made use of high-speed microscopy (66 fps), and (4) designed an automated AI-based sarcomere detection and analysis tool (SarcAsM - Sarcomere Analysis Multitool). Cardiomyocytes were differentiated from the ACTN2-Citrine iPSC-line and purified using a staged growth factor and metabolic selection protocol (&gt;95% purity with a ventricular myocyte phenotype). Our analysis revealed highly heterogeneous single sarcomere motion, i.e., shortening and lengthening patterns during the same time of the contraction cycle. Notably, the observed rich dynamics on single sarcomere-level are not apparent from whole cell analyses. Interestingly, culture on stiffer substrates (&gt;20 kPa; mimicking cardiac fibrosis) resulted in reduced cell-level contraction, but constant sarcomere level contractility with however enhanced heterogeneity. Analysis of a large data set (&gt;1,200 cells) in conjunction with a biophysical model showed that heterogeneous sarcomere motion is not caused by static non-uniformity (e.g., strong/weak sarcomeres), but can be primarily attributed to the stochastic and non-linear nature of sarcomere dynamics and thus occurs intrinsically during cardiomyocyte beating. Our findings show that that sarcomere heterogeneity is an important feature of cardiomyocyte contractility. The introduced tools for unbiased high-throughput analysis of sarcomere-level dynamics can be exploited to gain a better understanding on how sarcomere level contractility translates to whole cardiomyocyte contractions under simulated physiological and pathological conditions.

An increase in force after stretch of diaphragm fibers and myofibrils is accompanied by an increase in sarcomere length nonuniformities and Ca2+ sensitivity.

When muscle fibers from limb muscles are stretched while activated, the force increases to a steady-state level that is higher than that produced during isometric contractions at a corresponding sarcomere length, a phenomenon known as residual force enhancement (RFE). The mechanisms responsible for the RFE are an increased stiffness of titin molecules that may lead to an increased Ca2+ sensitivity of the contractile apparatus, and the development of sarcomere length nonuniformities. RFE is not observed in cardiac myofibrils, which makes this phenomenon specific to certain preparations. The aim of this study was to investigate whether the RFE is present in the diaphragm, and its potential association with an increased Ca2+ sensitivity and the development of sarcomere length nonuniformities. We used two preparations: single intact fibers and myofibrils isolated from the diaphragm of mice. We investigated RFE in a variety of lengths across the force-length relationship. RFE was observed in both preparations at all lengths investigated and was larger with increasing magnitudes of stretch. RFE was accompanied by an increased Ca2+ sensitivity as shown by a change in the force-pCa2+ curve, and increased sarcomere length nonuniformities. Therefore, RFE is a phenomenon commonly observed in skeletal muscles, with mechanisms that are similar across preparations.