Alignment Of Myofibrils Research Articles

Effect of stepwise cooking on the water-retention capacity and protein denaturation degree of chicken breast

This study investigated the impact of different center temperatures (60 °C, 65 °C, 70 °C) combined with low temperature long time cooking (cooking at 60 °C for 30 min or 60 min) on the water-retention capacity of chicken breasts. The effects of different heat treatment methods on microstructure, texture parameters, water content, cooking loss, color, pH value, protein denaturation degree and secondary structure content of chicken breast were analyzed. The results revealed that both center temperature and low temperature long times cooking (LTLT cooking) time significantly influenced the water-retention capacity of chicken breasts (P < 0.05). With increasing heat exposure levels, there was a corresponding rise in pH value as well as protein solubility and hydrophobicity in meat. This led to increased alignment of myofibrils along with gradual structural disruption and connective tissue efflux resulting in significant increases in meat hardness, chewiness and cooking loss (P < 0.05). However, when the center temperature reached 70 °C with an LTLT cooking time of 60 min, the muscle fiber structure is almost completely destroyed, causing the connective tissue between the muscle bundles or fibers to refill, resulting in a random coil content increase of about 2%, increased the water content by around 12% and decreased hardness by about 31%. These findings demonstrate that the group subjected to a combination treatment involving a center temperature of 70 °C and an LTLT cooking time of 60 min was effective in enhancing the water content while reducing the hardness of meat.

Abstract Mo088: RBFOX2 haploinsufficiency impairs cardiomyocyte adhesion and contractility through faulty RNA metabolism

Background: Hypoplastic left heart syndrome (HLHS) is a severe form of congenital heart disease that is uniformly lethal if left untreated. Protein damaging mutations in one allele of RBFOX2, a highly conserved RNA binding protein, are enriched in HLHS patients, suggesting they are causal. However, it remains unclear how RBFOX2 haploinsufficiency contributes to disease pathogenesis as heterozygous animal models appear healthy. Methods: We generated and characterized RBFOX2 heterozygous and null human induced pluripotent stem cells (hiPSCs) and assessed their phenotypes in differentiated cardiomyocytes (iPSC-CMs) in 2D culture. To learn if altered function might be revealed in a more native microenvironment, we also generated 3D engineered heart tissues (EHTs) from RBFOX2 heterozygous hiPSC-CMs. To delineate the underlying molecular mechanisms, we performed bulk RNA-seq to reveal differential gene expression (DGE) and alternative splicing events (ASEs) between the different genetic cohorts and eCLIP-seq to detect RNAs directly bound by RBFOX2 in the CM lineage. Results: We observed dose-dependent reductions in cell adhesion, size, maturation, respiration, calcium handling, and action potential duration with no significant effect on proliferation in 2D culture. Although myofibril alignment and contractility were completely abolished in null hiPSC-CMs, these features were surprisingly preserved in heterozygous cells. However, in EHTs, we found that RBFOX2 is haploinsufficient for tissue maturation and contractility, suggesting that myocardial-intrinsic defects contribute to RBFOX2 -mediated HLHS. Integration of DGE, ASEs and eCLIP-seq datasets uncovered significant enrichment of DGE and/or ASEs of RBFOX2-bound and unbound transcripts involved in cell adhesion between the extracellular matrix (ECM) and the actin cytoskeleton/sarcomere network. Included in the direct targets with altered expression and alternative splicing was the ECM ligand FIBRONECTIN 1 ( FN1 ), which when downregulated was previously associated with HLHS. Conclusions: Our data suggest that RBFOX2 sits atop an expression and splicing hierarchy that promotes optimal CM cell growth, adhesion, and contractility that protects the heart from HLHS pathogenesis.

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

MYH7 mutations induce changes in single cell mechanobiology.

Hypertrophic Cardiomyopathy (HCM) is characterized by thickening of the left ventricular wall and hypercontractility and has been linked to mutations in the sarcomere motor protein β-myosin (MYH7). Using single cell mechanobiology studies, we examined how the effects of single point mutations propagate to change the contractile dynamics and cellular morphology (sarcomere spacing, spread area, myofibril alignment) of human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). We micropattern islands of adhesive protein to constraining the spreading and alignment of hiPSC-CM on hydrogel substrates containing fluorescent microbeads as fiducial markers for traction force microscopy (TFM). We deployed substrate stiffnesses ranging from physiological (10 kPa) to heavily diseased/fibrotic (100 kPa) to test the role of increased “afterload” in functional phenotypes. We use image and video analysis to assess the contractile dynamics of the hiPSC-CM in terms of force, power, and velocities of relaxation and contraction. For example, we assessed multiple MYH7 mutations edited into the WTC line along with isogenic controls. Some lines carried an endogenously labeled alpha-actinin GFP reporter of sarcomere structure to enable visualization of sarcomere structure and dynamics. We assessed the magnitude and dynamics of contractile force output from TFM video analysis and observed increased the contractile force when compared to the control hiPSC-CMs. We also measured significantly different dynamics in the relaxation or contraction velocities compared to control hiPSC-CMs. Interestingly, not all HCM mutant lines presented a significant increase in cell spread area, a proxy for hypertrophy, and this correlated with culture conditions, such as the size of the protein pattern constraining the cells or stiffness of the substrate. Taken together, these results suggest a role for MYH7 mutations driving remodeling of structure and function at a cell-intrinsic level via changes in mechanosignaling.

Abstract 15136: Matrix Mechanics Regulate Engineered Myocardial Microtissue Organization and Contractility

Introduction: The mechanical function of the myocardium is dictated by cardiomyocytes (CMs) and the surrounding fibrous extracellular matrix (ECM). Due to limitations in existing approaches for engineering myocardial tissue, how CMs sense and respond to mechanical microenvironmental changes remains understudied. Here, we established a method for creating arrays of cardiac microtissues with highly tunable mechanical features by integrating synthetic, fibrous matrices and iPSC-CMs. Through real-time contractility readouts enabled, we explored how biomechanical cues influence engineered cardiac tissue assembly and function. Methods: Arrayed cardiac microtissues were generated by selectively photo-crosslinking electrospun dextran vinyl sulfone fiber matrices onto pairs of microfabricated PDMS cantilevers. Subsequently, iPSC-CMs were patterned onto matrices using a microfabricated seeding mask. Matrix and cantilever stiffnesses were tuned by adjusting photoinitiator concentrations and cantilever geometry, respectively. Results: In tissues contracting against soft cantilevers (0.41 N/m), tissue contractile stress decreased with increasing matrix stiffness (E = 0.68 to 17.3 kPa). We also observed an increase in contractile stress on soft and aligned matrices, compared to non-aligned matrices. However, tissues contracting against stiffer cantilevers (1.2 N/m) exerted similar contractile stresses independent of matrix alignment. This may be explained by the enhanced alignment of myofibrils in non-aligned matrices contracting against stiffer posts. The size of vinculin-rich cell-ECM adhesion decreased on stiff matrices. Vinculin also localized to z-discs forming organized costameres on soft matrices. Tissue response to mavacamten suggests that β-cardiac myosins are required for costamere formation and myofibril maturation. Conclusions: We developed a new microfabrication strategy to create tunable cardiac microtissues and examined how biomechanical cues affect tissue assembly and function. We found that matrix stiffness, matrix alignment, and tissue constraint distinctly affect iPSC-CM function and structure, potentially due to differential influences on costamere formation and myofibrillar assembly.

Are human induced pluripotent stem cell derived cardiomyocytes a good cellular model for studying incomplete penetrance in arrhythmogenic cardiomyopathy?

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Regional Development Fund and Interreg V-A Italy-Austria 2014-2020 and Department of Innovation, Research and University of the Autonomous Province of Bolzano-South Tyrol (Italy). Background Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) were previously used to model several inherited heart diseases (1). Among these, arrhythmogenic cardiomyopathy (ACM) is characterized by a fibrofatty myocardial replacement and severe ventricular arrhythmias (2). ACM is mainly caused by mutations affecting proteins of intercalated discs (3) and shows incomplete penetrance and variable expressivity (4). Purpose To evaluate whether hiPSC-CMs provide a good in vitro model to study incomplete penetrance in ACM. Material and methods hiPSCs were generated from buffy coats of 6 close relatives. Three of them were ACM patients carrying a deletion of exon 4 in the PKP2 gene causing a premature stop codon, 2 were asymptomatic (ASY) PKP2 mutation carriers and 1 was a healthy control (CTR). Whole exome sequencing of the 6 individuals was performed. The purified hiPSC-CMs were cultured in basal and adipogenic medium and examined by ddPCR, western blot, Wes™ immunoassay system, patch clamp and immunofluorescence. Results All family members tested negative for additional mutations in ACM genes. As expected, half the amount of wild type (wt) PKP2 mRNA was found in ACM and ASY than in CTR hiPSC-CMs (2.25±0.27vs4.29±0.70 wtPKP2/HPRT1; p=0.028) (2.08±0.38vs4.29±0.70 wtPKP2/HPRT1; p=0.052). Of note, the mutated (mut) PKP2 mRNA was detected and significantly more expressed in ACM than in ASY hiPSC-CMs (0.33±0.04vs0.11±0.06 mutPKP2/HPRT1; p=0.029). While we confirmed a reduced amount of wt plakophillin-2 protein in ACM and ASY compared to CTR hiPSC-CMs (0.19±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.016), (0.25±0.07vs0.81±0.28 wtPKP2/GAPDH; p=0.11), the truncated protein was not detected. Moreover, a significantly lower expression of the active form of β-catenin (ABC) has been shown in ACM and ASY than in CTR hiPSC-CMs (0.93±0.18vs1.30±0.09 ABC/GAPDH; p=0.057), (0.76±0.10vs1.30±0.09 ABC/GAPDH; p=0.0057). In basal medium, ACM hiPSC-CMs had a significant lower overall sodium current density compared to CTR (-49.82±2.8 vs -69.38±4.8 pA/pF; p&amp;lt;0.0001) and to ASY (-49.82±2.8 vs -68.92±4.3 pA/pF; p&amp;lt;0.0001) hiPSC-CMs. In adipogenic medium, ACM hiPSC-CMs showed a higher lipid accumulation and a higher sarcomere disorganization than CTR (1580±219vs615±199 intensity/nuclei; p=0.0351) (0.019±0.001vs0.028±0.003 myofibril alignment index; p=0.039) and ASY (1580±219vs689±146 intensity/nuclei; p=0.0059) (0.019±0.001vs0.027±0.001 myofibril alignment index; p=0.010) hiPSC-CMs. Conclusions Here we report that ACM express a significantly higher amount of mutated PKP2 mRNA than ASY iPSC-CMs. Even though no differences were detected for plakophilin-2 and active β-catenin proteins between ACM and ASY iPSCM-CMs, the ACM showed a fatty phenotype and altered electrical activity that differed significantly from the CTR as well as from the ASY hiPSC-CMs, demonstrating that these cells provide a valid model to study incomplete penetrance in ACM.

The larval scaffold controls fascicle number but is not required for formation of the dorsolongitudinal flight muscles in Manduca sexta

During metamorphosis, the dorsolongitudinal flight muscles (DLMs) of both the moth Manduca sexta and the fly Drosophila melanogaster develop from the remnants of larval muscles called larval scaffolds. Although this developmental program has been conserved across highly disparate taxa, the role of the larval scaffold remains unclear. Ablation experiments have demonstrated that the Drosophila DLM does not require the scaffold, but the resulting de novo muscles vary highly in fiber number, and their functional characteristics were not examined. To address this question in Manduca, we have surgically ablated the DLM precursors in Manduca sexta larvae and assayed the resulting DLMs in pharate adults using X-ray micro-CT and phalloidin histology. Following ablation, animals were able to form de novo DLMs with normal myofibril alignment, but these muscles had an altered shape and highly variable number of fascicles. Our results suggest that the larval scaffold is not required for DLM development in Manduca sexta, but appears to define the number of fascicles in the adult muscle, as previously found in Drosophila. Additionally, our ablated animals were able to generate flight, further suggesting that the use of a larval scaffold is a modification on the more ancestral myogenesis program.

Glabrol impurity exacerbates glabridin toxicity in zebrafish embryos by increasing myofibril disorganization

Ethnopharmacological relevanceGlabridin, extracted from Glycyrrhiza glabra L., is widely used for the treatment of hyperpigmentation because of its anti-inflammatory and antioxidant activities and its ability to inhibit melanin synthesis. This led to the strict regulation of its quality and safety. However, traditional quality control methods used for plant extracts cannot reflect the product quality owing to multiple unknown impurities, which necessitates the further analysis of impurities. Aim of the studyThe study identified the toxic impurities of glabridin and their toxicological mechanism. Materials and methodsIn total, 10 glabridin samples from different sources were quantified using high-performance liquid chromatography. Sample toxicities were evaluated using zebrafish and cell models. To identify impurities, samples with different toxicity were analyzed by ultra-high-performance liquid chromatography coupled with quadrupole-Orbitrap mass spectrometry. The toxicity of related impurities was verified in the zebrafish model. Phalloidin stain was used to evaluate subtle changes in myofibril alignment. ResultsAlthough glabridin content in the samples was similar, there were significant differences in toxicity. The results were verified using four different mammalian cell lines. Higher contents of glabrone and glabrol were identified in the sample with the highest toxicity. In the zebrafish model, the addition of glabrol reduced the LC50 of glabridin to 9.224, 6.229, and 5.370 μM at 48, 72, and 96 h post-fertilization, respectively, whereas glabrone did not have any toxic effect. Phalloidin staining indicated that a glabrol impurity exacerbates the myotoxicity of glabridin in zebrafish embryos. ConclusionGlabrol, but not glabrone, was identified as a key impurity that increased glabridin toxicity. This finding indicates that controlling glabrol content is necessary during glabridin product production.

Aligned human cardiac syncytium for in vitro analysis of electrical, structural, and mechanical readouts.

Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC-CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC-CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two-dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC-CMs.

HIGHLIGHTS ACROSS MYOLOGY: O.13 Altered calcium handling in a zebrafish model of SELENON congenital muscular dystrophy

SELENON congenital muscular dystrophy (previously designated SEPN1 congenital muscular dystrophy) causes muscle weakness, scoliosis, and respiratory insufficiency in infants and children. Targeted treatments for this devastating disease are currently lacking. SELENON encodes the protein Selenoprotein N, a member of the selenoprotein family. Selenoprotein N is expressed at high levels in developing and immature tissues and adopts a ubiquitous, low-level expression pattern in mature tissues. This raises questions about how loss of Selenoprotein N leads to muscle-specific disease. Overlap in clinical and histopathologic characteristics between patients with SELENON congenital muscular dystrophy and certain types of RYR1 myopathy raise the possibility of interactions between Selenoprotein N and the RYR1 calcium channel. This possibility is supported by altered calcium release in SELENON-deficient myotubes in vitro but has not been tested in vivo. Here, calcium release was measured from Selenoprotein N-deficient zebrafish muscle fibers in vivo. Selenoprotein N-deficient zebrafish show decreased amplitude but increased frequency of calcium spikes during myogenesis with subsequent abnormal myofibril alignment in slow-twitch muscle fibers. Further, mature muscle fibers of Selenoprotein N-deficient zebrafish show decreased amplitude and duration of calcium release in response to depolarization compared to wildtype and control muscle fibers. The characteristics of calcium release in Selenoprotein N-deficient zebrafish muscle is suggestive of a leaky RYR1 calcium channel, as is typically seen in the setting of oxidative stress. These data support a role for Selenoprotein N in modulating RYR1 function in vivo, both during muscle development and during excitation-contraction coupling in mature muscle fibers.

Direct 3D bioprinting of cardiac micro-tissues mimicking native myocardium

The heart possesses a complex three-dimensional (3D) laminar myofiber organization; however, because engineering physiologically relevant 3D tissues remains a technical challenge, the effects of cardiomyocyte alignment on excitation-contraction coupling, shortening and force development have not been systematically studied. Cellular shape and orientations in 3D can be controlled by engineering scaffold microstructures and encapsulating cells near these geometric cues. Here, we show that a novel method of cell encapsulation in 3D methacrylated gelatin (GelMA) scaffolds patterned via Microscale Continuous Optical Printing (μCOP) can rapidly micropattern neonatal mouse ventricular cardiomyocytes (NMVCMs) in photocrosslinkable hydrogels. Encapsulated cardiomyocytes preferentially align with the engineered microarchitecture and can display morphology and myofibril alignment phenotypic of myocardium in vivo. Utilizing the μCOP system, an asymmetric, multi-material, cantilever-based scaffold was directly printed, so that the force produced by the microtissue was transmitted onto a single deformable pillar. Aligned 3D encapsulated NMVCM scaffolds produced nearly 2 times the force compared to aligned 2D seeded samples. To further highlight the flexibility of μCOP, NMVCMs were encapsulated in several patterns to compare the effects of varying degrees of alignment on tissue displacement and synchronicity. Well aligned myofiber cultured patterns generated 4–10 times the contractile force of less anisotropically patterned constructs. Finally, normalized fluo-4 fluorescence of NMVCM-encapsulated structures showed characteristic calcium transient waveforms that increased in magnitude and rate of decline during treatment with 100 nM isoproterenol. This novel instrumented 3D cardiac microtissue serves as a physiologically relevant in vitro model system with great potential for use in cardiac disease modeling and drug screening.

Enhancing Stem Cell‐based Toxicity Assays by Engineering the Culture Niche in a High‐throughput, Assay‐agnostic Manner

Human induced pluripotent stem cell (hiPSC)‐based assays hold great promise for ameliorating human disease and improving the translation of new therapies, but they have shortcomings that hamper their ability to accurately model human physiology. For example, hiPSC‐Cardiomyocytes (CMs) mis‐classify several compounds of known cardiotoxicity. One likely source of this is the maturity of the cells; most culture techniques result in hiPSC‐CMs that express fetal phenotypes, leading to inaccurate or difficult‐to‐translate results. While engineering the extracellular matrix (ECM) has demonstrated improvements to both structural and functional phenotypes, it is often hampered by challenges with integration into industry‐standard assays or instrumentation. Further, the ability to engineer increasingly intricate and multi‐faceted artificial culture environments often drives decisions that trade off biological complexity with experimental throughput. Here, we developed bioengineering techniques to create biomimetic patterns that mimic the shape and structure of the ECM on a transparent polymer‐coated glass layer. The resultant structure directs focal adhesion assembly mechanisms in individual cells to drive higher‐ordered structuring of 2D tissues. This forms 2D anisotropic syncytia from iPSC‐CMs in industry‐standard microplate formats that are compatible with transmitted light microscopy. The method is extended to micro‐electrode arrays (MEA) in a manner that maintains both the quality of MEA recordings and baseline electrophysiological properties. iPSC‐CMs grown this way exhibit several structural and biochemical phenotypes including adult protein isoform expression, myofibril alignment, and sarcomere spacing, length, and width. We show that biomimetic cues enhance the electrophysiological response of cardiomyocytes to various drugs of known effect. Since ECM structuring recapitulates the polarized nature of cells and their concomitant gap junctions, we used the gap junction blocker carbenoxolone for validation studies. Polarized tissues enable differential conduction block measurements between longitudinal and transverse directions. Our MEA data show that patterned tissues exhibit increased sensitivity to carbenoxolone over unpatterned constructs (IC50’s of 14.71 nM versus 398.1 nM, respectively) comparing favorably to clinically relevant unbound concentrations reported in the low nM range. Our biomimetic engineering approach is instrument‐ and assay agnostic, effective on a variety of cell types and lines (including neuronal cells; examples to be presented), and compatible with high‐throughput approaches.Support or Funding InformationNational Institutes of Health Small Business Innovation Research Grant.(A) decellularized ex‐vivo cardiac tissue slices demonstrate a polarized meshwork of extracellular matrix (ECM) fibers; (B) our biomimetic patterning technique recapitulates the nanoscale structure and orientation of the ECM; (C) the technique can be expanded to the surface of high‐throughput MEAs without degradation to electrical signal detection while maintaining the nanoscale pattern (inset); (D) FCDI iCell2 Cardiomyocytes cultured on traditional plastic surfaces show unstructured morphology; (E) iCell2 Cardiomyocytes cultured on our biomimetic surfaces recapitulate the anisotropic structure of adult heart tissue.Figure 1(A) sarcomere lengths (B) and widths of cells on biomimetic nanosurfaces (green) compared to conventional surfaces (gray) are increased; (C) adult isoforms of structural proteins are enhanced with biomimetic nanopatterning; (D) patterned cells exhibit higher oxygen consumption rate; (E) patch clamp data demonstrate increased action potential duration on patterned tissues; (F) asymmetric conduction on patterned tissues enhances the sensitivity of MEA assays to conduction blockers;Figure 2

Embryonic exposure to 4-methylimidazole leads to zebrafish myofibril misalignment.

4-methylimidazole (4-MI) is an imidazole-derived organic chemical compound that can be used as a raw material in the manufacture of diverse chemicals and has been identified as an ingredient of caramel color in soybean sauce, beers, and other soft drinks. The aim of the present study was to investigate the teratogenic effects of 4-MI during zebrafish embryogenesis. Zebrafish embryos were treated with different dosages of 4-MI (0-120 mM) for different exposure durations (12-60 hours). The percentages of embryos with malformed phenotypes increased as the exposure dosages and duration time of 4-MI increased. We also used immunofluorescence and transmission microscopy to evaluate the subtle changes in the myofibril alignment and ultrastructure of muscle organization. Our data showed that 4-MI treatment disturbs muscle fiber alignment. Electron microscopy data indicated that Z-lines were undetectable in the 4-MI-treated embryos. Although the thick and thin filaments were visible, they were all disorganized. In addition, zebrafish embryos treated by 4-MI exhibited aberrant expression of 2 muscle-specific genes, myod and myogenin. Taken together, we concluded that early exposure to 4-MI affects zebrafish myogenesis, especially in myofibril alignment.

Mechanobiology of Myosin Mutations and Myofibril Remodeling in iPSC-Cardiomyocytes

Hypertrophic cardiomyopathies are the most prevalent form of heritable cardiovascular disease and are caused by a host of mutations that result in hypercontractility, though the largest number of known mutations are found in beta cardiac myosin heavy chain (βMYH). Myosin is a key protein responsible for muscle contraction and is arranged into a dynamic, hierarchical structure of sarcomeres and myofibrils within cardiomyocytes. We have previously measured differences in the kinetics of isolated myosin proteins with different hypertrophic cardiomyopathy mutations in βMYH, but it has been difficult to determine how these alterations manifest at the cellular level. Induced pluripotent stem cell-derived cardiomyocytes provide a powerful tool for studying specific features of human cardiomyocyte biology including contractility, hypertrophic growth, and intracellular organization. We have developed a micropatterned hydrogel platform that promotes myofibril alignment, cardiomyocyte force generation, and other metrics of maturation. Using CRISPR-Cas9 gene-editing, we have created cell lines with specific βMYH mutations, including D239N and P710R. We used traction force microscopy in a preliminary study and measured increased forces in the mutant cells compared to isogenic controls, and we have observed larger cell size in one of the mutant lines in both patterned and unpatterned contexts. Infection with LifeAct adenovirus also allows for the visualization of actin structures including myofibrils and sarcomeres during cell spreading and the recovery of beating in cells recently transferred to patterned substrates. Immunostaining for βMYH revealed higher expression and more regular organization of sarcomeres in patterned cells, and we plan to use these techniques to quantify differences in myofibril and sarcomere organization in cells with βMYH mutations. This work provides valuable insight into the etiology of hypertrophic cardiomyopathy and the utility of induced pluripotent stem cell-derived cardiomyocytes as a model for human cardiac biology and disease.

Efficacy of gold nanoparticles against isoproterenol induced acute myocardial infarction in adult male albino rats

ABSTRACTThis study was undertaken to investigate the role of gold nanoparticles (GNPs) of 50 nm diameter on isoproterenol (ISO) induced acute myocardial infarction in adult male albino rats. Forty five adult Wistar male albino rats were equally divided into three groups. Control (group I) was further subdivided into three subgroups. In group II, the rats received ISO subcutaneously at a dose of 100 mg/kg for three days. In group III, rats received ISO as group II and then GNPs (400 μg/kg/day) intravenously for 14 consecutive days. Echocardiography was performed. Left ventricular specimens were prepared for H&E, van Gieson staining, immunohistochemical analysis for (eNOs and Bcl-2), and Electron microscope examination. Energy dispersive X-ray microanalysis was also performed. Cardiac markers such as creatine Kinase-MB (CK-MB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and cardiac troponin T (cTnT) were measured. Group II revealed cardiomyocytes with deeply stained acidophilic cytoplasm, small dark nuclei, intracellular vacuolations, wide intercellular spaces, and extravasated red blood cells. Increased collagen fibers were observed. Electron microscope examination showed cardiomyocyte with small and irregular outlined nuclei, mitochondria with irregular cristae and others with ruptured mitochondrial membrane, abnormal alignment of myofibrils, dilated cisternae of smooth endoplasmic reticulum, and disorganized intercalated discs. Group III showed that most cardiomyocytes preserved the normal architecture. Increased expression of eNOs immunoreaction and decreased Bcl-2 immunoreaction were detected in group II as compared to the control and GNP-treated groups. These findings suggested that GNPs of 50 nm diameter improved myocardial injury after ISO-induced myocardial infarction in rats.Abbreviations: Myocardial infarction (MI), Isoproterenol (ISO), Nitric oxide (NO), Neuronal NOS (nNOs), Endothelial NOs (eNOs), Gold nanoparticle (GNPs), Diamiobenzidine (DAB), Serum Creatine Kinase-MB (CK-MB), Alanine aminotransferase (ALT), Cardiac troponin T (cTnT), Electrochemiluminiscence (ECLIA), Cardiomyocytes (CMC), Peroxisomal proliferator activated receptor (PPARs), Reactive oxygen species (ROS)

Human Stem Cell-Derived Cardiac Model of Chronic Drug Exposure.

Animal models have been instrumental in providing insight into the molecular basis of disease. While such information has been successfully applied to the study of human disease, this translation would be significantly strengthened by the availability of models based on human cells. This would be particularly important for cardiovascular disease, as the physiology of human cardiomyocytes (CMs) differs significantly from rodents. Here, we have generated a three-dimensional human engineered cardiac tissue, termed biowire, from human embryonic stem cell-derived CMs to investigate the effects of chronic (7 day) treatment with isoproterenol, endothelin-1, or angiotensin II. We show that biowires chronically treated with either isoproterenol, endothelin-1, or angiotensin II have disrupted myofibril alignment and significantly reduced force of contraction. Isoproterenol-treated biowires have upregulated brain natriuretic peptide and atrial natriuretic peptide gene expression. Endothelin-1 and angiotensin II-treated biowires demonstrated a significantly increased cell size. Endothelin-1-treated biowires exhibited increased cardiac troponin secretion into the culture media. This demonstrates that human biowires treated for 7 days with isoproterenol, angiotensin II, or endothelin-1 exhibit some changes compatible with hypertrophic cardiomyopathy.

A Tuned Tension Regulates the Contractility of Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells

The ability to differentiate human cardiomyocytes (heart muscle cells) from induced pluripotent stem cells (iPSCs) presents high potential to model heart contractility. Shortening of sarcomeres in series along intracellular myofibrils enables the beating of cardiomyocytes. We developed a platform with patterned single iPSC-cardiomyocytes in arrays and tested the contractility of these cells as a function of their shape and of substrate stiffness. We fluorescently labelled sarcomeres with LifeAct to analyze their shortening and organization. We measured the mechanical output of contractile cycles with traction force microscopy and adapted cross-correlation algorithms to characterize movement of sarcomeres. We assayed single cells on patterns of 2000 μm2 with aspect ratios (length:width) ranging from 1:1 to 7:1 and on substrates with varied stiffnesses: 6 kPa, 10 kPa and 35 kPa. Preliminary studies suggested that cell morphology and substrate stiffness affect the organization of sarcomeres with a potential impact in cardiomyocyte contractility. We aimed to understand how the contractility of iPSC-cardiomyocytes is affected by the improved organization of sarcomeres induced by these cues. For a substrate stiffness of 10 kPa, we observed that cells with a 7:1 aspect ratio produced higher contractile forces per μm of sarcomere shortening. Improved sarcomere alignment seemed to drive increased contractility of cells with this shape. Substrate stiffness also affected the contractility of 7:1 iPSC-cardiomyocytes. 35 kPa substrates induced sarcomere ruptures and lower contractile forces. Cells on 6 kPa substrates developed sarcomeres that buckled. These results suggested a role of intracellular tension in contractility. We further tested how tension affects contractility and sarcomere organization in 7:1 cells through cell stretching, culture in high calcium and by inhibiting non-muscle myosin. Results showed evidence that a tuned intracellular tension mechanism drives myofibril alignment and improved contractility in iPSC-cardiomyocytes.

Contractility of single cardiomyocytes differentiated from pluripotent stem cells depends on physiological shape and substrate stiffness

Single cardiomyocytes contain myofibrils that harbor the sarcomere-based contractile machinery of the myocardium. Cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs) have potential as an in vitro model of heart activity. However, their fetal-like misalignment of myofibrils limits their usefulness for modeling contractile activity. We analyzed the effects of cell shape and substrate stiffness on the shortening and movement of labeled sarcomeres and the translation of sarcomere activity to mechanical output (contractility) in live engineered hPSC-CMs. Single hPSC-CMs were cultured on polyacrylamide substrates of physiological stiffness (10 kPa), and Matrigel micropatterns were used to generate physiological shapes (2,000-µm(2) rectangles with length:width aspect ratios of 5:1-7:1) and a mature alignment of myofibrils. Translation of sarcomere shortening to mechanical output was highest in 7:1 hPSC-CMs. Increased substrate stiffness and applied overstretch induced myofibril defects in 7:1 hPSC-CMs and decreased mechanical output. Inhibitors of nonmuscle myosin activity repressed the assembly of myofibrils, showing that subcellular tension drives the improved contractile activity in these engineered hPSC-CMs. Other factors associated with improved contractility were axially directed calcium flow, systematic mitochondrial distribution, more mature electrophysiology, and evidence of transverse-tubule formation. These findings support the potential of these engineered hPSC-CMs as powerful models for studying myocardial contractility at the cellular level.

Development of a Cyclic Strain Bioreactor for Mechanical Enhancement and Assessment of Bioengineered Myocardial Constructs.

The purpose of this study was to develop enabling bioreactor technologies using a novel voice coil actuator system for investigating the effects of periodic strain on cardiac patches fabricated with rat cardiomyocytes. The bioengineered muscle constructs used in this study were formed by culturing rat neonatal primary cardiac cells on a fibrin gel. The physical design of the bioreactor was initially conceived using Solidworks to test clearances and perform structural strain analysis. Once the software design phase was completed the bioreactor was assembled using a combination of commercially available, custom machined, and 3-D printed parts. We utilized the bioreactor to evaluate the effect of a 4-h stretch protocol on the contractile properties of the tissue after which immunohistological assessment of the tissue was also performed. An increase in contractile force was observed after the strain protocol of 10% stretch at 1 Hz, with no significant increase observed in the control group. Additionally, an increase in cardiac myofibril alignment, connexin 43 expression, and collagen type I distribution were noted. In this study we demonstrated the effectiveness of a new bioreactor design to improve contractility of engineered cardiac muscle tissue.

Perturbation of cytosolic calcium by 2-aminoethoxydiphenyl borate and caffeine affects zebrafish myofibril alignment.

The objective of the current study was to investigate the effects of Ca(2+) levels on myofibril alignment during zebrafish embryogenesis. To investigate how altered cytoplasmic Ca(2+) levels affect myofibril alignment, we exposed zebrafish embryos to 2-aminothoxyldiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor inhibitor that reduces cytosolic Ca(2+) levels) and caffeine (a ryanodine receptor activator that enhances cytosolic Ca(2+) levels). The results demonstrated that the most evident changes in zebrafish embryos treated with 2-APB were shorter body length, curved trunk and malformed somite boundary. In contrast, such malformed phenotypes were evident neither in untreated controls nor in caffeine-treated embryos. Subtle morphological changes, including changes in muscle fibers, F-actin and ultrastructures were easily observed by staining with specific monoclonal antibodies (F59 and α-laminin), fluorescent probes (phalloidin) and by transmission electron microscopy. Our data suggested that: (1) the exposure to 2-APB and/or caffeine led to myofibril misalignment; (2) 2-APB-treated embryos displayed split and short myofibril phenotypes, whereas muscle fibers from caffeine-treated embryos were twisted and wavy; and (3) zebrafish embryos co-exposed to 2-APB and caffeine resulted in normal myofibril alignment. In conclusion, we proposed that cytosolic Ca(2+) is important for myogenesis, particularly for myofibril alignment.