Cardiac Muscle Tissue Research Articles

Enhancing Regeneration and Functionality of Excitable Tissues via Integrating Bioelectronics and Bioengineered Constructs

Abstract The inherent complexities of excitable cardiac, nervous, and skeletal muscle tissues pose great challenges in constructing artificial counterparts with bioelectrical, structural, and mechanical properties that closely resemble their natural states. Recent advances have increasingly revealed the beneficial impact of bioelectrical microenvironments on cellular behaviors, functional tissue regeneration, and improved therapeutic outcomes for these excitable tissues. This review aims to unveil the mechanisms by which electrical microenvironments enhance the regeneration and functionality of excitable cells and tissues, considering both endogenous electrical cues from electroactive biomaterials and exogenous electrical stimuli from external electronic systems. We explore the synergistic effects of these electrical microenvironments, combined with structural and mechanical guidance, on the regeneration of excitable tissues using tissue engineering scaffolds. Additionally, the emergence of micro/nanoscale bioelectronics has significantly expanded the scope of this field, enabling intimate interactions between implantable bioelectronics and excitable tissues at the cellular, tissue, and organ levels. These interactions allow for precise data extraction and localized modulation of cell and tissue functionalities through intricately designed electronic components according to physiological needs. The integration of tissue engineering and bioelectronics holds the potential to achieve optimal outcomes, highlighting a growing trend in engineering living tissue construct-bioelectronic hybrids for restoring and monitoring damaged excitable tissues. Furthermore, we envision critical challenges in engineering the next generation of these hybrids, focusing on integrated fabrication strategies, the development of ionic conductive biomaterials, and their convergence with biosensors.

Mitochondrial Mutations in Cardiovascular Diseases: Preliminary Findings

Background/Objectives: Mitochondria are the main organelles for ATP synthesis able to produce energy for several different cellular activities. Cardiac cells require high amounts of energy and, thus, they contain a high number of mitochondria. Consequently, mitochondrial dysfunction in these cells is a crucial factor for the development of cardiovascular diseases. Mitochondria constitute central regulators of cellular metabolism and energy production, producing approximately 90% of the cells’ energy needs in the form of ATP via oxidative phosphorylation. The mitochondria have their own circular, double-stranded DNA encoding 37 genes. Any mitochondrial DNA sequence anomaly may result in defective oxidative phosphorylation and lead to cardiac dysfunction. Methods: In this study, we investigated the potential association between mitochondrial DNA mutation and cardiovascular disease. Cardiac tissue and serum samples were collected from seven patients undergoing coronary artery bypass grafting. Total DNA was extracted from cardiac muscle tissue specimens and serum and each sample was subjected to polymerase chain reaction (PCR) to amplify the NADH dehydrogenase 1 (ND1) gene, which is part of the mitochondrial complex I enzyme complex and was screened for mutations. Results: We identified one patient with a homoplasmic A to G substitution mutation in cardiac tissue DNA and two patients with heteroplasmic A3397G mutation in serum DNA. Specifically, amplicon sequence analysis revealed a homoplasmic A3397G substitution in the ND1 gene in a tissue sample of the patient with ID number 1 and a heteroplasmic mutation in A3397G in serum samples of patients with ID numbers 3 and 6, respectively. The A to G substitution changes the amino acid from methionine (ATA) to valine (GTA) at position 31 of the ND1 gene. Conclusions: The detection of this novel mutation in patients with coronary artery disease may contribute to our understanding of the association between mitochondrial dysfunction and the disease, implying that mitochondria may be key players in the pathogenesis of cardiovascular diseases.

Mechanical memory based biofabrication of hierarchical elastic cardiac tissue

Mimicking the multilayered, anisotropic, elastic structure of cardiac tissues for controlled guidiance of 3D cellular orientation is essential in designing bionic scaffolds for cardiac tissue biofabrication. Here, a hierarchically organized, anisotropic, wavy and conductive polycaprolactone/Au scaffold was created in a facile fashion based on mechanical memory during fabrication. The bionic 3D scaffold shows good biocompatibility, excellent biomimetic mechanical properties that guide myoblast alignment, support the hyperelastic behavior observed in native cardiac muscle tissue, and promote myotube maturation, which holds potential for cardiac muscle engineering and the establishment of anin vitroculture platform for drug screening.

Modeling the mechanism of Ca2+ release in skeletal muscle by DHPRs easing inhibition at RyR I1-sites.

Ca2+ release from the sarcoplasmic reticulum (SR) plays a central role in excitation-contraction coupling (ECC) in skeletal muscles. However, the mechanism by which activation of the voltage-sensors/dihydropyridine receptors (DHPRs) in the membrane of the transverse tubular system leads to activation of the Ca2+-release channels/ryanodine receptors (RyRs) in the SR is not fully understood. Recent observations showing that a very small Ca2+ leak through RyR1s in mammalian skeletal muscle can markedly raise the background [Ca2+] in the junctional space (JS) above the Ca2+ level in the bulk of the cytosol indicate that there is a diffusional barrier between the JS and the cytosol at large. Here, I use a mathematical model to explore the hypothesis that a sudden rise in Ca2+ leak through DHPR-coupled RyR1s, caused by reduced inhibition at the RyR1 Ca2+/Mg2+ inhibitory I1-sites when the associated DHPRs are activated, is sufficient to enable synchronized responses that trigger a regenerative rise of Ca2+ release that remains under voltage control. In this way, the characteristic response to Ca2+ of RyR channels is key not only for the Ca2+ release mechanism in cardiac muscle and other tissues, but also for the DHPR-dependent Ca2+ release in skeletal muscle.

Ginsenoside Rb1 reduces oxidative/carbonyl stress damage and dysfunction of RyR2 in the heart of streptozotocin-induced diabetic rats

BackgroundOxidative stress may contribute to cardiac ryanodine receptor (RyR2) dysfunction in diabetic cardiomyopathy. Ginsenoside Rb1 (Rb1) is a major pharmacologically active component of ginseng to treat cardiovascular diseases. Whether Rb1 treat diabetes injured heart remains unknown. This study was to investigate the effect of Rb1 on diabetes injured cardiac muscle tissue and to further investigate its possible molecular pharmacology mechanisms.MethodsMale Sprague-Dawley rats were injected streptozotocin solution for 2 weeks, followed 6 weeks Rb1 or insulin treatment. The activity of SOD, CAT, Gpx, and the levels of MDA was measured; histological and ultrastructure analyses, RyR2 activity and phosphorylated RyR2(Ser2808) protein expression analyses; and Tunel assay were performed.ResultsThere was decreased activity of SOD, CAT, Gpx and increased levels of MDA in the diabetic group from control. Rb1 treatment increased activity of SOD, CAT, Gpx and decreased the levels of MDA as compared with diabetic rats. Neutralizing the RyR2 activity significantly decreased in diabetes from control, and increased in Rb1 treatment group from diabetic group. The expression of phosphorylation of RyR2 Ser2808 was increased in diabetic rats from control, and were attenuated with insulin and Rb1 treatment. Diabetes increased the apoptosis rate, and Rb1 treatment decreased the apoptosis rate. Rb1 and insulin ameliorated myocardial injury in diabetic rats.ConclusionsThese data indicate that Rb1 could be useful for mitigating oxidative damage, reduced phosphorylation of RyR2 Ser2808 and decreased the apoptosis rate of cardiomyocytes in diabetic cardiomyopathy.

Мікрорівнева адаптація окремих органів і тканин стерляді (Acipenser ruthenus Linnaeus, 1758) в умовах теплового стресу

Purpose. To evaluate the variability of the histological parameters of individual organs and tissues of sterlet, to establish the level of effect of heat stress on the physiological status of fish and the degree of their adaptation. To determine the perspective of using histological monitoring data in fish farming practice. Methodology. The primary materials were obtained in aquarium conditions of the Aquatic Bioresources Laboratory of the Kherson State Agrarian and Economic University. The laboratory processing of histological samples was performed according to generally accepted methods and manuals. Histological studies were carried out using the author’s equipment and original methods, specially intended for the diagnostics of fish tissues. The obtained results were processed by methods of variational statistics using a package of appropriate MS Excel applications, with emphasis on the standard error. Findings. Microanatomical studies of sterlet showed that under conditions of heat stress, the most significant changes occur in adipose tissue, somatic and cardiac muscle tissue, thyroid gland and renal tissue, which can conditionally be considered as bioindicative zones. Under the potential influence of the thermal factor, the response of sterlet is expressed in a decrease in the lipid content in the accompanying muscles of adipose tissue. The effect is visualized in the fragmentation and subsequent shredding of lipid vacuoles, which leads to variability in adipocyte shape from stellate to spindle-shaped. The obtained results indicate a decrease in the excretion of thyroid hormones into the bloodstream by the thyroid gland and is expressed in a gradual increase in the diameter of the thyroid follicles (by 16.0 µm and 26.5 µm) and a corresponding decrease in the height of the follicular cells (by 1.1 µm and 2.0 µm), which is statistically significant. Intensive development of “additional” follicles inside existing ones indicates the prevention of thyroid intoxication of the body, which indicates the development of an adaptive reaction of the body. Comparative studies of the ventricular myocardium of fish allowed establishing certain morphological differences in the form of moderate interstitial edema of the tissue and intracellular edema of cardiomyocytes. In the final phase of the experiment, karyopyknosis was locally observed in the ventricular cardiomyocytes of 50% of stressed fish. Somatic muscle tissue is generally resistant to stress, but the rate of its development at elevated temperatures slows down. Characteristic signs of the histological picture of muscle stress are approximately the same ratio of stromal and parenchymal components, the central orientation of the nuclei within a single muscle fiber and insufficient filling of the fibers with the fibrillar component. Antidiuretic hormone is forcedly released in fish under conditions of heat stress, which leads to a decrease in the volume of the primary filtrate. Evidence is a narrow gap in the urinary space between the visceral and parietal layers of the renal corpuscle. Originality. New data are presented and existing data on the histological structure of individual organs and tissues of sterlet under stress loads are significantly expanded. For the first time, a detailed histological analysis of the structure of muscle, adipose tissue, as well as the kidney and thyroid gland of sterlet under heat stress conditions was carried out. The comparative analysis, current and updated information on compensatory changes in tissues demonstrate their adaptive potential, which was developed during the evolution of the species. Practical Value. The information obtained is of value in morphophysiological studies as well as in fish farming practice in connection with the modern problem of a decrease in the life status of fish under stress. The use of the obtained data allows a qualitative assessment of the physiological state and can be used in the process of developing corrective measures in order to consolidate hereditary resistance, which is ultimately aimed at preventing diseases of valuable aquaculture objects. Keywords: sterlet, heat stress, adaptation, muscle tissue, thyroid gland, myocardium, adipose tissue, renal tissue.

Long-range gene editing of LMNA

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Hartstichting (Dutch Heart Foundation) Background Mutations in the LMNA gene commonly affect the cardiac muscle tissue, which leads to the development of a severe dilated cardiomyopathy with life-threatening arrhythmias. The lack of response to conventional heart failure therapy in these patients indicates the urgent need for new therapies. Gene editing has the potential to become a curative treatment for these patients by correcting the genetic cause of the disease. Prime editing is a promising new gene editing technology, which uses a prime editing guide RNA (pegRNA) to target a specific genetic locus and creates edits using a reverse-transcriptase enzyme linked to a Cas9-nickase. Purpose Conventional prime-editing technology targets one mutation per pegRNA. However, with more than 600 disease-causing mutations described in LMNA, the feasibility of applying this technology to each mutation is limited. Therefore, we aim to target not a single, but a set of mutations with one single pegRNA. Methods The pegRNAs are optimised by selecting the optimal target regions, improving their stability and 3D structure. The in vitro editing efficiency is assessed using the fluoPEER reporter plasmid, transfected into HEK293T cells. Using flow cytometry, expression of mCherry fluorescent protein is detected following successful editing of the target mutation. Results Flow-cytometry analysis of treated cells revealed an editing efficiency of 12% with a long-range pegRNA targeting a full exon, compared to 15% with a conventional short pegRNA targeting the same mutation <10bp downstream of the Cas9 nick site. This editing efficiency using the same long-range pegRNA was retained at 9% when editing at a distant position >100bp downstream of the nick site. To increase the editing efficiency, we improved the stability of the pegRNAs by the addition of different 3’ structural motifs. This resulted in an increased editing efficiencies of 15% and 17% for nearby and distant mutations, respectively. Conclusion We show that it is possible to edit different mutations within a range of up to 120 bp with a single pegRNA. By optimisations of the pegRNA 3D structure and optimal selection of target locations we aim to further improve the editing efficiency of this system. This work could eventually provide a prime editing therapy able to cure disease in a subset of patients carrying different mutations in the same genetic region. For LMNA-associated diseases this would substantially reduce the number of pegRNAs that have to be developed to cover all mutations, greatly improving the feasibility of prime editing therapy.

#1228 MOTS-c improves CV and mortality risk prediction in chronic hemodialysis patients: a prospective, multicenter cohort study

Abstract Background and Aims Cardiovascular disease (CVD) is the leading cause of death in patients with end stage kidney disease (ESKD), especially in those undergoing chronic hemodialysis (HD), but correct risk prediction in this setting is often complicated. The mitochondrial open reading frame of 12S rRNA type-C (MOTS-s) is a peptide encoded by mitochondrial DNA, mainly expressed by cardiac and skeletal muscle tissues, which may prevent CVD thanks to its remarkable anti-oxidant capacities. From a clinical point of view, altered circulating MOTS-c levels predict worsen CV outcomes, particularly among diabetic individuals or post-AMI patients. Mitochondrial dysfunction is highly pervasive in HD patients. Starting from this background, we aimed at evaluating the clinical significance and predictive usefulness of measuring circulating MOTS-c in a multicenter cohort of chronic HD patients, beyond common cohort-related risk factors. Method We conducted a prospective, observational study on 94 chronic HD patients. The study endpoint was a composite of all-cause/CV mortality and non-fatal CV events. We identified the cohort-related predictors of the endpoint by logistic and Cox-regression analyses and built prognostic models based on such variables. Performance of such models was tested before and after the inclusion of MOTS-c. 31 healthy patients were employed as controls. MOTS-c was measured in serum using a commercially available ELISA kit. Results MOTS-c levels were more elevated in HD patients than in controls (36.7 [24.9-49.9] vs. 13.8 [10.4-28.7] ng/mL; p < 0.001; Fig. 1) and independently correlated with a history of ischemic heart disease (β = 0.213; p = 0.03), LVMi (β = 0.185; p = 0.05) and phosphate plasma concentration (β = −0.201; p = 0.05). Of note, MOTS-c levels were even more increased in the 53 individuals experiencing the endpoint during a median 26.5. months follow-up (p = 0.01; Fig. 2). MOTS-c predicted the endpoint at either multivariate logistic (OR 1.020; 95% CI 1.011-1.109; p = 0.03) or Cox-regression analyses (HR 1.004; 95% CI 1.000-1.025; p = 0.05) and the addition of this peptide to multivariate models including the other, cohort-related risk factors (age, LVMi, E/e’, diabetes, pulse pressure) remarkably improved the calibration, risk variability explanation (+3%), discrimination (ROC-AUC from 0.727 to 0.743; C-index from 0.658 to 0.700) and, particularly, the overall reclassification capacity (NRI 15.87%; p = 0.01). Conclusion Circulating MOTS-c balance is altered in chronic HD patients. In these individuals, MOTS-c may add significant information to improve CV and mortality risk prediction, on top of traditional and uremia-specific, cohort-related risk variables. Future investigations are needed to extend this preliminary observation in wider HD cohorts.

Molecular Pathways and Cellular Subsets Associated with Adverse Clinical Outcomes in Overlapping Immune-Related Myocarditis and Myositis.

Immune checkpoint therapies (ICT) can induce life-threatening immune-related adverse events, including myocarditis and myositis, which are rare but often concurrent. The molecular pathways and immune subsets underlying these toxicities remain poorly understood. To address this need, we performed single-cell RNA sequencing of heart and skeletal muscle biopsies obtained from living patients with cancers treated with ICTs and admitted to the hospital with myocarditis and/or myositis (overlapping myocarditis plus myositis, n = 10; myocarditis-only, n = 1) or ICT-exposed patients ruled out for toxicity utilized as controls (n = 9). All biopsies were obtained within 96 hours of clinical presentation. Analyses of 58,523 cells revealed CD8+ T cells with a cytotoxic phenotype expressing activation/exhaustion markers in both myocarditis and myositis. Furthermore, the analyses identified a population of myeloid cells expressing tissue-resident signatures and FcγRIIIa (CD16a), which is known to bind IgG and regulate complement activation. Immunohistochemistry of affected cardiac and skeletal muscle tissues revealed protein expression of pan-IgG and complement product C4d, which were associated with the presence of high-titer serum autoantibodies against muscle antigens in a subset of patients. We further identified a population of inflammatory IL1B+TNF+ myeloid cells specifically enriched in myocarditis and associated with greater toxicity severity and poorer clinical outcomes. These results provide insight into the myeloid subsets present in human immune-related myocarditis and myositis tissues and nominate new targets for investigation into rational treatments to overcome these high-mortality toxicities. See related Spotlight by Fankhauser et al., p. 954.

High-intensity exercise training using a rotarod instrument (RotaHIIT)significantly improves exercise capacity in mice.

Voluntary or forced exercise training in mice is used to assess functional capacity as well as potential disease-modifying effects of exercise over a range of cardiovascular disease phenotypes. Compared to voluntary wheel running, forced exercise training enables precise control of exercise workload and volume, and results in superior changes in cardiovascular performance. However, the use of a shock grid with treadmill-based training is associated with stress and risk of injury, and declining compliance with longer periods of training time for many mouse strains. With these limitations in mind, we designed a novel, high-intensity interval training modality (HIIT) for mice that is carried out on a rotarod. Abbreviated as RotaHIIT, this protocol establishes interval workload intensities that are not time or resource intensive, maintains excellent training compliance over time, and results in improved exercise capacity independent of sex when measured by treadmill graded exercise testing (GXT) and rotarod specific acceleration and endurance testing. This protocol may therefore be useful and easily implemented for a broad range of research investigations. As RotaHIIT training was not associated cardiac structural or functional changes, or changes in oxidative capacity in cardiac or skeletal muscle tissue, further studies will be needed to define the physiological adaptations and molecular transducers that are driving the training effect of this exercise modality.

Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-Nitroso- N -Pivaloyl- d -Penicillamine: Implications for Improved Diastolic Function and Cardiac Performance.

We previously reported a novel compound called S-nitroso- N -pivaloyl- d -penicillamine (SNPiP), which was screened from a group of nitric oxide donor compounds with a basic chemical structure of S-nitroso- N -acetylpenicillamine, to activate the nonneuronal acetylcholine system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The nonneuronal acetylcholine-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 hours after SNPiP administration) revealed that SNPiP initially induced Wnt and cyclic guanosine monophosphate-protein kinase G signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in response to SNPiP treatment, resulting in a metabolic shift from oxidative phosphorylation to glycolysis, which was suggested by reduced cardiac glucose contents while maintaining adenosine tri-phosphate levels. In addition, SNPiP significantly upregulated atrial natriuretic peptide and sarcolipin, which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

Effect of moxibustion combined with intraperitoneal injection of benazepril on endoplasmic reticulum stress-related protein phosphorylation in rats with chronic heart failure.

To observe the effect of moxibustion at "Xinshu"(BL15) and "Feishu"(BL13) combined with intraperitoneal injection of benazepril on cardiac function and phosphorylation of protein kinase R-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (elF2α) proteins in myocardium of rats with chronic heart failure (CHF), so as to explore its potential mechanism underlying improvement of CHF. A total of 42 male SD rats were randomly assigned to blank control (n=10), CHF model (n=7), medication (benazepril, n=8), moxibustion (n=8) and moxibustion+benazepril (n=9) groups, after cardiac ultrasound model identification and elimination of the dead. The CHF model was established by intraperitoneal injection of doxorubicin hydrochloride (DOX), once every week for 6 weeks. Mild moxibustion was applied to bilateral BL15 and BL13 regions for 20 min, once daily for 3 weeks. The rats of the medication group and moxibustion+benazepril group (benazepril was given first, followed by moxibustion) received intraperitoneal injection of benazepril (0.86 mg/kg) solution once daily for 3 weeks . The cardiac ejection fraction (EF) and left ventricular fractional shortening (FS) were measured using echocardiography. Histopathological changes of the cardiac muscle tissue were observed under light microscope after hematoxylin-eosin (H.E.) staining. Serum contents of B-type brain natriuretic peptide (BNP) and angiotensin Ⅱ (AngⅡ) were measured by enzyme-linked immunosorbent assay (ELISA). The expressions of phospho-PERK (p-PERK) and phospho-elF2α (p-elF2α) in the myocardium were detected by Western blot. Compared with the blank control group, the EF and FS of the left cardiac ventricle were significantly decreased (P<0.01), while the contents of serum BNP and AngⅡ, and expression levels of p-PERK and p-eIF2α significantly increased in the model group (P<0.01). In comparison with the model group, both the decreased EF and FS and the increased BNP and AngⅡ contents as well as p-PERK and p-elF2α expression levels were reversed by moxibustion, medication and moxibustion+benazepril (P<0.01). The effects of moxibustion+benazepril were markedly superior to those of simple moxibustion and simple medication in raising the levels of EF and FS rate and in down-regulating the contents of BNP, Ang Ⅱ, levels of p-PERK and p-elF2α (P<0.01, P<0.05). Outcomes of H.E. staining showed irregular arrangement of cardiomyocytes, cell swelling, vacuole and inflammatory infiltration in the model group, which was relatively milder in the 3 treatment groups. The effects of moxibustion+benazepril were superior to those of moxibustion or benazepril. Moxibustion combined with Benazepril can improve the cardiac function in CHF rats, which may be related to its functions in down-regulating the expression levels of myocardial p-PERK and p-elF2α to inhibit endoplasmic reticulum stress response.

Bioactive Glass and Silica Particles for Skeletal and Cardiac Muscle Tissue Regeneration.

When skeletal and cardiac tissues are damaged, surgical approaches are not always successful and tissue regeneration approaches are investigated. Reports in the literature indicate that silica nanoparticles and bioactive glasses (BGs), including silicate bioactive glasses (e.g., 45S5 BG), phosphate glass fibers, boron-doped mesoporous BGs, borosilicate glasses, and aluminoborates, are promising for repairing skeletal muscle tissue. Silica nanoparticles and BGs have been combined with polymers to obtain aligned nanofibers and to maintain controlled delivery of nanoparticles for skeletal muscle repair. The literature indicates that cardiac muscle regeneration can be also triggered by the ionic products of BGs. This was observed to be due to the release of vascular endothelial growth factor and other growth factors from cardiomyocytes, which regulate endothelial cells to form capillary structures (angiogenesis). Specific studies, including both in vitro and in vivo approaches, are reviewed in this article. The analysis of the literature indicates that although the research field is still very limited, BGs are showing great promise for muscle tissue engineering and further research in the field should be carried out to expand our basic knowledge on the application of BGs in muscle (skeletal and cardiac) tissue regeneration. Impact statement This review highlights the potential of silica particles and bioactive glasses (BGs) for skeletal and cardiac tissue regeneration. These biomaterials create scaffolds triggering muscle cell differentiation. Ionic products from BGs stimulate growth factors, supporting angiogenesis in cardiac tissue repair. Further research is required to expand our know-how on silica particles and BGs in muscle tissue engineering.

ATR-FTIR Spectroscopic Analysis of Novel Fixatives and their Histological Properties On Sheep Heart

Objective: In this study, new fixative solutions that are odorless and non-toxic were tested on the hearts of sheep to preserve the cellular structure and tissue architecture in a life-like manner. Material and Method: The changes of new fixatives before and after contact with tissue were detected for the first time by the ATR-FTIR spectroscopic method. After a preliminary inspection, hearts were fixed with six different solutions. It was periodically evaluated for weight, size, color, and hardness. Samples taken from the left vertical of each heart and stained with Hematoxylin eosin were observed histologically and alterations at the cellular level were evaluated. Results: As a result of the measurements of heart weights, reduction in Solution 1 (Standard Formaldehyde Fixative), Solution 4 and Solution 5 were detected at 5.64%, 23.70% and 14.38%, respectively. In Solution 4 and Solution 5, it was observed that the integrity of the myocardium was protected in terms of morphology, the stiffness was suited for sectioning and the coronary vessels were well preserved according to Solution 1. Further, compared to solution 1, it was determined that the endocardium and myocardium layers were well preserved in the tissues fixed in Solution 4 and Solution 5. Typical transversal streaking appearance in cardiac muscle tissue existed in all three Solution 1, Solution 4 and Solution 5. Conclusion: Solution 4 and Solution 5 were found to have superior fixative properties than Solution 1 (Formaldehyde). As a result of ATR-FTIR studies, it was determined that formaldehyde was converted into toxic formic acid in Standard Solution 1, while very few and harmless changes were detected in Solution 4 and Solution 5.

Calcium Channel Blocker Overdose.

Emergency medicine residents and medical students on emergency medicine rotation. Calcium channel blocker (CCB) overdoses can be severe with potentially serious adverse outcomes. CCBs work by blocking the calcium channels on smooth and cardiac muscle tissue. At low dose ranges, dihydropyridine CCBs (such as nifedipine, amlodipine, and nicardipine) block the L-type calcium receptors in the peripheral vasculature, whereas non-dihydropyridine CCBs (such as: verapamil and diltiazem) affect the L-type calcium receptors in the myocardium.1 Because of this distinction, dihydropyridine CCB toxicity manifests as arterial vasodilation and non-dihydropyridine CCB toxicity is associated with cardiac manifestations such as bradycardia and negative inotropy.2 It is important to note that in high concentrations (such as in overdoses), CCBs lose specificity for their specific receptors and can show all the manifestations of toxicity such as bradycardia, peripheral vasodilation, and hypotension. Patients can develop both vasoplegic shock from peripheral vasodilation and cardiogenic shock. This is a high acuity low occurrence case with infrequently used but specific treatments, and thus this case provides educational value. At the end of this oral board session, examinees will: (1) demonstrate ability to evaluate a patient with undifferentiated shock with bradycardia and discuss the differential diagnosis, (2) recognize the signs and symptoms of calcium channel blocker overdose, (3) demonstrate ability to manage treatment of a patient with calcium channel overdose. This oral board case followed the standard American Board of Emergency Medicine-style case in a tertiary care hospital with access to all specialists and resources needed. This case was tested using 12 resident volunteers ranging from PGY 1-2 in an ACGME (Accreditation Council for Graduate Medical Education) accredited emergency medicine residency program. Immediate feedback was solicited both from the learners and from the evaluators following the debriefing session. Residents were asked to evaluate the educational value of the case using a 1-5 Likert scale (5 being excellent). Evaluators were asked to score the residents using the ACGME core competencies with a scale of 1-8, 1-4 being unacceptable and 5-8 being acceptable. Seven PGY1 residents and five PGY2 residents, thus twelve residents in total, completed the case. The average score was 5.10/8. Three residents missed zero critical actions. The most common critical action missed was consulting cardiology or cardiothoracic surgery for circulatory support options. Many residents failed to recognize that the patient did not have a perfusing blood pressure at the beginning of the case and did not start CPR. Although most residents recognized the patient's hemodynamic collapse was from a calcium channel blocker overdose, most did not know the treatment for this beyond atropine and intravenous fluids.The learners rated the educational value of the case as 4.9/5. Seven residents reported that the case definitely increased their medical knowledge; five residents reported that it somewhat increased their medical knowledge. All residents rated the case as helpful in preparing to manage this medical condition. The educational content from this case was effective. This is a high acuity low occurrence case that has unique treatments that are not commonly used. This makes this case excellent for practice and discussion. We learned during implementation that this case has a high degree of difficulty compared to other cases, and junior learners will need more prompting. It is also important for the proctor to keep the case moving because there is a lot to cover in the allotted amount of time. Calcium channel blocker overdose, toxicology.

Essential Role of Anisotropy in Bioengineered Cardiac Tissue Models.

As regulatory bodies encourage alternatives to animal testing, there is renewed interest in engineering disease models, particularly for cardiac tissues. The aligned organization of cells in the mammalian heart controls the electrical and ionic currents and its ability to efficiently circulate blood to the body. Althoughthe development of engineered cardiac systems is rising, insights into the topographical aspects, in particular, the necessity to design in vitro cardiac models incorporating cues for unidirectional cell growth, is lacking. This review first summarizes the widely used methods to organize cardiomyocytes (CMs) unidirectionally and the ways to quantify the resulting cellular alignment. The behavior of CMs in response to alignment is described, with emphasis on their functions and underlying mechanisms. Lastly, the limitations of state-of-the-art techniques to modulate CM alignment in vitro and opportunities for further development in the future to improve the cardiac tissue models that more faithfully mimic the pathophysiological hallmarks are outlined. This review serves as a call to action for bioengineers to delve deeper into the in vivo role of cellular organization in cardiac muscle tissue and draw inspiration to effectively mimic in vitro for engineering reliable disease models.

Abstract 16049: Hydrogen Sulfide Attenuates Duchenne Cardiomyopathy via Suppression of Sterile Inflammation

Background: Duchenne muscular dystrophy (DMD) is a lethal, X-linked recessive disease caused by mutations in dystrophin. There is currently no cure for DMD, and the main cause of death is dilated cardiomyopathy. Hydrogen sulfide (H 2 S) is a gaseous molecule produced endogenously, and disruptions in basal H 2 S levels have been associated with various pathophysiological conditions, including heart failure. In this study, we investigated whether H 2 S could prevent cardiac dysfunction in a mouse model of DMD. Methods: Male mdx 4cv /mTR KO mice received early treatment (ET) with the H 2 S donor, SG1002 (40 mg/kg/day in chow starting at the time of weaning) or no treatment (NT), and wildtype C57BL (WT) mice were used as control. Left ventricular ejection fraction (LVEF) was assessed by transthoracic echocardiography. Skeletal and cardiac fibrosis was determined by picrosirius red staining. NLRP3 inflammasome activity was measured through apoptosis speck like protein (ASC) staining in cardiac muscle tissue. Results: NT mice exhibited significant LVEF decline by 4 months compared to ET and WT mice and declined further by 12 months (n=15-17, NT: 45.2% ± 7.9 vs ET: 57.1% ± 8.9% and WT: 56.5% ± 7.0%, P&lt;0.001). In addition, ET mice showed significantly reduced fibrosis compared to NT mice in the myocardium (n=5-8, 2.62% ± 0.87% vs 11.32% ± 4.70%, respectively, P=0.002), diaphragm (n=11-14, 15.84% ± 9.0% vs 31.14% ± 7.1%, respectively, P&lt;0.001), and gastrocnemius (n=12-17, 7.60% ± 3.14% vs 13.48% ± 5.97%, respectively, P=0.004) at 12 months. ASC aggregation score was significantly higher in cardiac tissue samples (Fig 1) from NT mice at 12 months compared with WT mice. ET attenuated the increase in cardiac ASC aggregation at 12 months compared with NT mice (P&lt;0.001). Conclusions: Our results demonstrate that early treatment with SG1002 preserves cardiac function in a mouse model of DMD and suggest a potential therapeutic role of NLRP3 inflammasome suppression as a protective strategy in DMD.

296 Comparison of Dna Methylation Patterns in Sperm Collected from Brahman Bulls with Differences in Embryo Development

Abstract The objective was to determine if methylation patterns in sperm differed between bulls with good vs poor embryo development. Semen collected from sexually mature Brahman bulls was purified from frozen-thawed straws using a gradient [50% (vol/vol) and 90% (vol/vol)] of Isolate (Irvine Scientific, Santa Ana, CA), and used to fertilize abattoir-derived cumulus oocyte complexes (n = 81-100/bull in 2 wells). Number of blastocysts were recorded on d 8, and bulls were classified as Good (GD; &amp;gt;35% blastocyst rate; n = 5) or Poor (PD; &amp;lt; 35% blastocyst rate; n = 4) embryo development. Methylation of sperm DNA was assessed by reduced representation sodium bisulfite sequencing. Differences in methylation were determined with the Dispersion Shrinkage for Sequencing method in conjunction with Wald test. Differentially methylated cytosines and regions located in promoters of genes and gene bodies were analyzed to reveal gene ontology terms associated with them. Proportion of sperm cells that were viable (before and after a three-hour stress test), with inceased reactive oxygen species (ROS), and with damaged chromatin was determined. Statistical analysis (PROC GLIMMIX; SAS 9.4) included the fixed effect of embryo development classification which was considered significant at P &amp;lt; 0.05. Mean separation was performed using LSmeans. The proportion of viable (P &amp;gt; 0.21; GD = 46 ± 5%; PD = 36 ± 5%) sperm cells did not differ before the stress test. Post-stress test, PD bulls had more viable sperm cells (P &amp;lt; 0.02; 37 ± 3%) than GD (25 ± 2%) bulls. There was no difference between GD and PD for ROS (H2O2; P &amp;gt; 0.16 and •O2; P &amp;gt; 0.59). Proportion of sperm cells with damaged chromatin was greater (P &amp;lt; 0.01) in PD (3 ± 0.3%) than GD bulls (1 ± 0.01%). There were 1,742 differentially methylated cytosines and 343 differentially methylated regions when GD bulls were compared with PD bulls. Genes whose promoters were differentially methylated included DDC, GTSF1L, PPP1R27, NTM, HCK, GPR173, and TNFRSF1A. Functionally enriched pathways for differentially methylated promoters included apoptosis and amyotrophic lateral sclerosis while some of the significant biological processes were regulation of apoptotic process involved in morphogenesis, negative regulation of neuron migration, pyrimidine nucleoside catabolic process, and regulation of cardiac muscle tissue development. Differentially methylated gene bodies were significantly related to cellular components such as cell surface, endomembrane system, synapse, and cell periphery while molecular functions included binding, protein binding, and transmembrane receptor protein tyrosine kinase activity. Biological processes impacted by differentially methylated gene bodies included system development, neurogenesis, animal organ development, anatomical structure development, generation of neurons, nervous system development, and multicellular organism development. In summary, differences in embryo development are likely due to differences in methylation patterns impacting developmental processes, as semen characteristics were mainly similar between bulls with good versus poor embryo development.

Real‐time and Multichannel Measurement of Contractility of hiPSC‐Derived 3D Skeletal Muscle using Fiber Optics‐Based Sensing

AbstractAs the field of cardiac and skeletal muscle tissue engineering expands, so does the need for accurate and reliable systems to generate in vitro 3D tissues and analyze their functional properties. In this study, the Cuore is introduced, a system that integrates sensors based on optical fibers and uses the principle of light interferometry to detect the contraction of 3D Tissue Engineered Skeletal Muscles (3D‐TESMs). The technology employed in the Cuore allows for reproducible and multichannel force measurements down to a nano‐Newtons resolution while maintaining sterility and permitting continuous non‐invasive recording within and outside standard tissue culture incubators. Thanks to the integrated electrodes for electrical pulse stimulation (EPS), 3D‐TESMs generated from three independent hiPSC‐derived myogenic progenitors (MPs) lines are stimulated and the contractility is recorded over the course of a week. Through the modulation of different EPS parameters, the optimal combination to induce the 3D‐TESMs in producing fully fused tetani without causing damage is determined. Furthermore, 3D‐TESMs from different lines exhibit characteristic signatures of spontaneous contractility and response to caffeine, verapamil, and the β‐agonist clenbuterol. The ease of use, high sensitivity, and the integrated electrodes and sensors make the Cuore an ideal technology to investigate the biology of contractile tissues and their response to drugs.