Organization Of Cardiomyocytes Research Articles

WDR1-regulated actin dynamics is required for outflow tract and right ventricle development

Outflow tract (OFT) anomalies account for about 30% of human congenital heart defects detected at birth. The second heart field (SHF) progenitors contribute to OFT and right ventricle (RV) development, but the process largely remains unknown. WDR1 (WD-repeat domain 1) is a major co-factor of actin depolymerizing factor (ADF)/cofilin that actively disassembles ADF/cofilin-bound actin filaments. Its function in embryonic heart development has been unknown. Using Wdr1 floxed mice and Nkx2.5-Cre, we deleted Wdr1 in embryonic heart (Wdr1F/F;Nkx2.5-Cre) and found that these mice exhibited embryonic lethality, and hypoplasia of OFT and RV. To investigate the role of WDR1 in OFT and RV development, we generated SHF progenitors-specific Wdr1 deletion mice (shfKO). shfKO mice began to die at embryonic day 11.5 (E11.5), and displayed decreased size of the proximal OFT and RV at E10.5. In shfKO embryos, neither the number of SHF cells deployment to OFT nor cell proliferation and the cell number were changed, whereas the cellular organization and myofibrillar assembly of cardiomyocytes were severely disrupted. In the proximal OFT and RV of both shfKO and Wdr1F/F;Nkx2.5-Cre embryos, cardiomyocytes were dissociated from the outer compact myocardial layer and loosely and disorderly arranged into multilayered myocardium. Our results demonstrate that WDR1 is indispensable for normal OFT and RV development, and suggest that WDR1-mediated actin dynamics functions in controlling the size of OFT and RV, which might through regulating the spatial arrangement of cardiomyocytes.

Cardiac cell modelling of myofibrillar myopathy by AAV-mediated transduction of desmin mutations in human iPSC-derived and neonatal rat cardiomyocytes

Myofibrillar myopathy (MFM) is a group of severe cytokeletal proteins known to be caused by mutations in genes encoding. Among the myofibrillar proteins that are accumulated within the muscle fibers of affected patients, they appear to be responsible for the structural integrity of striated muscle. Mutations in the gene lead not only to skeletal muscle myopathies but also to cardiomyopathies, however the cardiac pathological consequences caused by them remain poorly understood. The objective of this work is to examine the effects of MFM-induced mutations in the structural organization of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) as identified by troponinT immunostaining, and rat neonatal cardiomyocytes. We propose an in vitro cardiac disease-based cellular model based on adeno-associated virus (AAV)-mediated transduction of wild-type (WT) and mutated desmin in fusion with a c-myc tag to distinguish between endogenous and transgenic desmin proteins. Our preliminary results demonstrated that cardiomyocytes were successfully transduced by AAV vectors and expressed exogenous WT desmin as revealed by c-myc immunostaining. For better recapitulating the in vivo anisotropic cardiomyocyte organization, we have developed micro-patterned culture plates made by microcontact printing where thin strips of gelatin have been deposited on top of glass. Culture of both hiPSC-derived and rat neonatal cardiomyocytes on this system improved their sarcomere alignment. The analysis based on immunostaining of protein-markers to examine structural organization in AAV-transduced cardiomyocytes is in progress. Our results indicate that the proposed in vitro model is suitable for the study of the structural defects of desmin-mutated cardiomyocytes.

Geranylgeranyl pyrophosphate synthase facilitates the organization of cardiomyocytes during mid-gestation through modulating protein geranylgeranylation in mouse heart.

With the maturation of placenta, ventricular chamber maturation enhances cardiac contractile performance to adapt to the metabolic demand of growing embryo. The organization of cardiomyocytes is required for the morphological remodelling in ventricular chamber maturation. However, the mechanism governing the establishment of cardiac cytoarchitecture during ventricular chamber maturation is still poorly studied. Here, we found that the expression of geranylgeranyl pyrophosphate synthase (Ggpps), which mediates protein geranylgeranylation, increased in the mouse heart after the onset of placental function. By using different Cre lines, we found that the cardiac inactivation of Ggpps by the Nkx2.5Cre/+ line disrupted protein geranylgeranylation as early as E9.5, which affected ventricular chamber maturation and resulted in mid-gestational embryonic lethality. In contrast, α-SMA-Cre line mediated the disruption of protein geranylgeranylation from E13.5 did not affect embryonic heart development. Further analysis of Nkx2.5Cre/+; Ggppsfl/fl mutants showed that the loss of Ggpps caused disorganized cardiac cytoarchitecture as early as E11.5 by disturbing cell-cell junctions. Ggpps inactivation decreased Rho GTPase geranylgeranylation and their activity, which might account for the disruption of cell-cell junctions. Moreover, elevating the protein geranylgeranylation by supplement of geranylgeranyl pyrophosphate (GGPP) could recover the Ggpps deficient induced defects of cytoarchitecture and cell-cell junctions in vitro and in vivo. Our present study demonstrates that GGPPS-mediated protein geranylgeranylation plays an indispensable role in the ventricular chamber maturation and acts as a stage-specific signal to regulate the establishment of cardiac cytoarchitecture during mid-gestation.

Human Embryonic Stem Cell-Derived Cardiomyocytes Self-Arrange with Areas of Different Subtypes During Differentiation.

The derivation of functional cardiomyocytes (CMs) from human embryonic stem cells (hESCs) represents a unique way of studying human cardiogenesis, including the development of CM subtypes. In this study, we investigated the development and organization of hESC-derived cardiomyocytes (hESC-CMs) and examined how the expression levels of CM subtypes correspond to human in vivo cardiogenesis. Beating clusters were used to determine cardiac differentiation, which was evaluated by the expression of cardiac genes GATA4 and TNNT2 and subcellular localization of GATA4 and NKX2.5. Sharp electrode recordings to determine action potentials (APs) further revealed spatial organization of intracluster CM subtypes (ie, complex clusters). Nodal-, atrial-, and ventricular-like AP morphologies were detected within distinct regions of complex clusters. The ability of different CM subtypes to self-organize was documented by immunohistochemical analyses and a differential spatial expression of β-III tubulin, myosin light chain 2v (MLC-2V), and α-smooth muscle actin (α-SMA). Furthermore, all hESC-CM subtypes formed expressed primary cilia, which are known to coordinate cellular signaling pathways during cardiomyogenesis and heart development. This study expands the foundation for studying regulatory pathways for spatial and temporal CM differentiation during human cardiogenesis.

Abstract 9: Cardiac BIN1 Improves Dyad Organization and Calcium Transient in Cardiomyocytes

BIN1 (bridging integrator 1) is a membrane scaffolding protein that forms microfolds at t-tubules and organizes dyad microdomains essential for a normal calcium transient. Reduction of cBIN1 has emerged as a new hallmark in heart failure and is associated with impaired calcium transient and increased risk of arrhythmias. We hypothesized that gene transfer of cardiac BIN1 (cBIN1) in vivo can improve calcium transients in disease models. We introduced V5-tagged cBIN1 or GFP through AAV9-mediated expression in adult mice. Confocal imaging of isolated cardiomyocytes was used to assess organization of dyad microdomain. Calcium transients in response to acute administration of isoproterenol (ISO) was measured as functional readout of excitation-contraction (EC) coupling. Here we report that cBIN1 significantly enhanced both basal and ISO-induced increase in calcium transient compared to cells expressing GFP. Immunofluorescent labeling revealed that cBIN1 expression increased both LTCC and RyR at t-tubule. In addition, in a mouse model of ischemic cardiomyopathy, permanent LAD ligation resulted in a marked reduction in ISO-induced increment in calcium transient, indicating blunted beta-adrenergic responsiveness and impaired regulation of EC coupling machinery. In comparison, an ISO-induced increment in calcium transient was largely maintained in cBIN1-expressing cells. These results demonstrate that cBIN1 increases calcium transients by upregulating and organizing dyad proteins at t-tubule microfolds. More importantly, cBIN1 rescues diminished beta-adrenergic responsiveness in ischemic cardiomyopathy and improves EC coupling of viable cardiomyocytes. This work suggests that cBIN1 provides a therapeutic opportunity to improve cardiac function in ischemic cardiomyopathy and heart failure.

Decreased Rac1 Cardiac Expression in Nitrofen-Induced Diaphragmatic Hernia.

The high incidence of cardiac malformations in humans and animal models with congenital diaphragmatic hernia (CDH) is well known. The hypoplasia of left heart is common among fetuses with CDH and has been identified as a poor prognostic factor. However, the precise mechanisms underlying cardiac maldevelopment in CDH are not fully understood. Ras-related C3 botulinum toxin substrate 1 (Rac1) plays a key role in cardiomyocyte polarity and embryonic heart development. Deficiency of Rac1 is reported to impair elongation and cytoskeletal organization of cardiomyocytes, resulting in congenital cardiac defects. We designed this study to test the hypothesis that Rac1 expression is downregulated in the developing hearts of rats with nitrofen-induced CDH. Following ethical approval (REC1103), time-pregnant Sprague Dawley rats received nitrofen or vehicle on gestational day 9 (D9). Fetuses were sacrificed on D18 and D21 and divided into CDH and control (CTRL) (n = 6 for each group and time point). Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, and confocal-immunofluorescence microscopy were performed to detect cardiac gene and protein expression of Rac1. qRT-PCR and Western blot analysis revealed that Rac1 expression was significantly decreased in the CDH group compared with controls (p < 0.05). Confocal-immunofluorescence microscopy revealed that Rac1 cardiac expression was markedly decreased in the CDH group compared with controls. Decreased cardiac Rac1 expression in the nitrofen-induced CDH suggests that Rac1 deficiency during morphogenesis may impair structural cardiac remodeling, resulting in congenital cardiac defects.

Sept7b is required for the subcellular organization of cardiomyocytes and cardiac function in zebrafish.

Myofibrils made up of actin, myosin, and associated proteins generate the contractile force in muscle, and, consequently, mutations in these proteins may lead to heart failure. Septins are a conserved family of small GTPases that associate with actin filaments, microtubules, and cellular membranes. Despite the importance of septins in cytoskeleton organization, their role in cardiomyocyte organization and function is poorly characterized. Here, we show that septin 7 is expressed in both embryonic and adult zebrafish hearts and elucidate the physiological significance of sept7b, the zebrafish ortholog of human septin 7, in the heart in embryonic and larval zebrafish. Knockdown of sept7b reduced F-actin and α-cardiac actin expression in the heart and caused disorganization of actin filaments. Electron microscopy of sept7b-depleted larvae showed disorganization of heart myofibrils and partial detachment from Z-disks. Functional studies revealed that knockdown of sept7b leads to reduced ventricular dimensions, contractility, and cardiac output. Furthermore, we found that depletion of sept7b diminished the expression of retinaldehyde dehydrogenase 2, which catalyzes the synthesis of retinoic acid necessary for heart morphogenesis. We further observed that the sept7b and retinoic acid signaling pathways converge to regulate cardiac function. Together, these results specify an essential role for sept7b in the contractile function of the heart.NEW & NOTEWORTHY Knockdown of the zebrafish ortholog of human septin 7 (sept7b) destabilizes cardiac actin and reduces ventricular dimensions, contractility, and cardiac output in larval zebrafish, indicating that sept7b is essential for cardiac function. We further found that sept7b and retinoic acid signaling pathways converge to regulate cardiac function. These data prompt further studies defining the role of sept7b in cardiomyopathies.

Myofibril Organization in Cardiomyocytes is Guided by the Maximal Tensile Stress Direction

The mechanisms underlying the spatial organization of self-assembled myofibrils in cardiac tissues remain incompletely understood. By modeling cells as elastic solids under active cytoskeletal contraction, we found a remarkable correlation between the predicted maximal principal stress directions and the in vitro myofibril orientations in individual cardiomyocytes. This implies that actomyosin fibers tend to assemble along the maximal tensile stress directions. By considering the dynamics of focal adhesion and myofibril formation in the model, we revealed that the different myofibril organizations in mature versus immature cardiomyocytes are due to the different levels of force-dependent remodeling of focal adhesions. Further we found that the myofibril organizations in microtissues are regulated by a combination of multiple factors including cell/tissue shape, cell-substrate adhesions, and cell-cell adhesions. These findings can guide the rational design in cardiac tissue engineering where recapitulating in vivo myofibril organizations is crucial to the contractile function of the heart.

Melatonin-Induced Protective Effects on Cardiomyocytes Against Reperfusion Injury Partly Through Modulation of IP3R and SERCA2a Via Activation of ERK1.

BackgroundMelatonin is a neuroendocrine hormone synthesized primarily by the pineal gland that is indicated to effectively prevent myocardial reperfusion injury. It is unclear whether melatonin protects cardiac function from reperfusion injury by modulating intracellular calcium homeostasis.ObjectiveDemonstrate that melatonin protect against myocardial reperfusion injury through modulating IP3R and SERCA2a to maintain calcium homeostasis via activation of ERK1 in cardiomyocytes.MethodsIn vitro experiments were performed using H9C2 cells undergoing simulative hypoxia/reoxygenation (H/R) induction. Expression level of ERK1, IP3R and SERCA2a were assessed by Western Blots. Cardiomyocytes apoptosis was detected by TUNEL. Phalloidin-staining was used to assess alteration of actin filament organization of cardiomyocytes. Fura-2 /AM was used to measure intracellular Ca2+ concentration. Performing in vivo experiments, myocardial expression of IP3R and SERCA2a were detected by immunofluorescence staining using myocardial ischemia/ reperfusion (I/R) model in rats.ResultsIn vitro results showed that melatonin induces ERK1 activation in cardiomyocytes against H/R which was inhibited by PD98059 (ERK1 inhibitor). The results showed melatonin inhibit apoptosis of cardiomyocytes and improve actin filament organization in cardiomyocytes against H/R, because both could be reversed by PD98059. Melatonin was showed to reduce calcium overload, further to inhibit IP3R expression and promote SERCA2a expression via ERK1 pathway in cardiomyocytes against H/R. Melatonin induced lower IP3R and higher SERCA2a expression in myocardium that were reversed by PD98059.Conclusionmelatonin-induced cardioprotection against reperfusion injury is at least partly through modulation of IP3R and SERCA2a to maintain intracellular calcium homeostasis via activation of ERK1.

Anisotropic microfibrous scaffolds enhance the organization and function of cardiomyocytes derived from induced pluripotent stem cells

Engineering of myocardial tissue constructs is a promising approach for treatment of coronary heart disease. To engineer myocardial tissues that better mimic the highly ordered physiological arrangement and function of native cardiomyocytes, we generated electrospun microfibrous polycaprolactone scaffolds with either randomly oriented (14 μm fiber diameter) or parallel-aligned (7 μm fiber diameter) microfiber arrangement and co-seeded the scaffolds with human induced pluripotent stem cell-derived cardiomyocytes (iCMs) and endothelial cells (iECs) for up to 12 days after iCM seeding. Here we demonstrated that aligned microfibrous scaffolds induced iCM alignment along the direction of the aligned microfibers after 2 days of iCM seeding, as well as promoted greater iCM maturation by increasing the sarcomeric length and gene expression of myosin heavy chain adult isoform (MYH7), in comparison to randomly oriented scaffolds. Furthermore, the benefit of scaffold anisotropy was evident in the significantly higher maximum contraction velocity of iCMs on the aligned scaffolds, compared to randomly oriented scaffolds, at 12 days of culture. Co-seeding of iCMs with iECs led to reduced contractility, compared to when iCMs were seeded alone. These findings demonstrate a dominant role of scaffold anisotropy in engineering cardiovascular tissues that maintain iCM organization and contractile function.

TGF-β receptor type II costameric localization in cardiomyocytes and host cell TGF-β response is disrupted by Trypanosoma cruzi infection.

Transforming growth factor beta (TGF-β) cytokine is involved in Chagas disease establishment and progression. Since Trypanosoma cruzi can modulate host cell receptors, we analysed the TGF-β receptor type II (TβRII) expression and distribution during T. cruzi - cardiomyocyte interaction. TβRII immunofluorescent staining revealed a striated organization in cardiomyocytes, which was co-localized with vinculin costameres and enhanced (38%) after TGF-β treatment. Cytochalasin D induced a decrease of 45·3% in the ratio of cardiomyocytes presenting TβRII striations, demonstrating an association of TβRII with the cytoskeleton. Western blot analysis showed that cytochalasin D significantly inhibited Smad 2 phosphorylation and fibronectin stimulation after TGF-β treatment in cardiomyocytes. Trypanosoma cruzi infection elicited a decrease of 79·8% in the frequency of cardiomyocytes presenting TβRII striations, but did not interfere significantly in its expression. In addition, T. cruzi-infected cardiomyocytes present a lower response to exogenous TGF-β, showing no enhancement of TβRII striations and a reduction of phosphorylated Smad 2, with no significant difference in TβRII expression when compared to uninfected cells. Together, these results suggest that the co-localization of TβRII with costameres is important in activating the TGF-β signalling cascade, and that T. cruzi-derived cytoskeleton disorganization could result in altered or low TGF-β response in infected cardiomyocytes.

Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells.

Understanding the phenotypic development of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is a prerequisite to advancing regenerative cardiac therapy, disease modeling, and drug screening applications. Lack of consistent hiPSC-CM in vitro data can be largely attributed to the inability of conventional culture methods to mimic the structural, biochemical, and mechanical aspects of the myocardial niche accurately. Here, we present a nanogrid culture array comprised of nanogrooved topographies, with groove widths ranging from 350 to 2000 nm, to study the effect of different nanoscale structures on the structural development of hiPSC-CMs in vitro. Nanotopographies were designed to have a biomimetic interface, based on observations of the oriented myocardial extracellular matrix (ECM) fibers found in vivo. Nanotopographic substrates were integrated with a self-assembling chimeric peptide containing the Arg-Gly-Asp (RGD) cell adhesion motif. Using this platform, cell adhesion to peptide-coated substrates was found to be comparable to that of conventional fibronectin-coated surfaces. Cardiomyocyte organization and structural development were found to be dependent on the nanotopographical feature size in a biphasic manner, with improved development achieved on grooves in the 700-1000 nm range. These findings highlight the capability of surface-functionalized, bioinspired substrates to influence cardiomyocyte development, and the capacity for such platforms to serve as a versatile assay for investigating the role of topographical guidance cues on cell behavior. Such substrates could potentially create more physiologically relevant in vitro cardiac tissues for future drug screening and disease modeling studies.

Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart

Invaginations of the plasma membrane within cardiomyocytes form the transverse tubule network (t-tubules), and at “dyadic junctions” these come into close association with regions of the sarcoplasmic reticulum (SR). In addition to many of the key excitation-contraction (EC) coupling proteins, junctions contain junctophilin-2 (JPH2), which is implicated in both the formation and maintenance of junctions. In human heart failure there is a loss of cardiac function which is generally associated with impairment of EC coupling processes, as well as changes to t-tubule structure and protein distribution. The aim of this study was to investigate the link between structural re-organization of cardiomyocytes and the loss of contractile function in trabeculae from failing human hearts.We obtained trabeculae from explanted human hearts in end-stage heart failure and performed force measurements followed by immuno-labelling to determine correlations between protein distribution and functional performance in the failing human heart. Cardiac trabeculae were processed along with samples taken from the free wall to investigate the variability of structure and protein distribution, including t-tubules, JPH2 and ryanodine receptors (the SR calcium release channel) using confocal imaging. Findings from these experiments revealed that there is a high degree of variability of trabecular structure and function, both within and between hearts. Trabeculae exhibiting poor contractile function often had low cardiomyocyte content and remaining myocytes showed disorganization of t-tubules and EC coupling proteins.These findings demonstrate a clear link between trabecula structure and function in the diseased human heart and support the idea that cardiomyocyte organization at cell and tissue levels is correlated with cardiac function. Furthermore, while trabeculae are useful for studying cardiac function, trabecula and cardiomyocyte structure must be considered when interpreting functional results.

Multi-facets of Serum Response Factor in the Cardiac Pathophysiology

Heart is a vital organ that pumps blood to all body parts of an organism through synchronous contraction and relaxation movements of the cardiac muscle. Homeostasis of cardiac muscle is thus necessary for normal heart functioning. Fine tuning of the cardiac muscle is maintained by numerous signal transduction pathways which are not only responsible for physiological functions, but pathological lesions as well. Many intrinsic or extrinsic stimuli like physical stress, pressure overload, hypertension, etc. disturbs cardiac homeostasis and affects signal transduction pathways causing disease phenotype such as hypertrophy, cardiomyopathy, and consequent heart failure. The most unique characteristic of cardiomyocytes is that they are terminally differentiated and do not grow in number postnatally, they only grow in size by lateral/longitudinal hypertrophy. Hypertrophy is also induced in cardiomyocytes to overcome acute wall tension caused by various biochemical stresses. At molecular level, several signalling pathways and interacting networks have been implicated with the heart’s molecular response to physiological and/or pathological biomechanical stress, reviewed in detail elsewhere [1‐4]. Calcineurin, a Ca2+-calmodulin activation dependent serine‐threonine phosphatase is one of the key signaling molecules strongly linked to cardiac hypertrophy [5‐8]. Its prohypertrophic effects are well established through series of in vitro and in vivo studies and is highly correlated with human patients of cardiac hypertrophy and cardiomyopathy [6,9]. Moreover, Calcineurin signaling is found to be interconnected with many other important hypertrophic pathways, such as those controlled by glycogen synthase kinase (GSK) 3β and mitogenactivated protein (MAP) kinase signaling [3,10]. Interestingly, Rho family of small GTPase proteins, consisting of Rho, Rac, and Cdc42 subfamilies regulate the sarcomere organization in cardiomyocytes, one of the hallmarks of hypertrophy [11]. These GTPase effectors are also upstream of a very important cardiac signaling molecule, serum response factor (SRF) [12,13].

Micro-patterning directional fibronectin cues onto hydrogel scaffolds for cardiac tissue engineering

Event Abstract Back to Event Micro-patterning directional fibronectin cues onto hydrogel scaffolds for cardiac tissue engineering Quentin Jallerat1, Vinit S. Palayekar1, 2, Madeline N. Monroe1, 2, Ivan Batalov2, Gaurav Begur1, 2 and Adam W. Feinberg1, 2 1 Carnegie Mellon University, Biomedical Engineering, United States 2 Carnegie Mellon University, Materials Science And Engineering, United States Introduction: Myocardial infarction kills one in six people in the US, due to an inability to regenerate cardiac muscle lost after infarct. Cardiac muscle engineered in vitro is a potential approach to create new myocardium for infarct repair, however multiple challenges exist. Specifically, while spatially well-defined micropatterns of ECM proteins such as fibronectin (FN) have been shown to direct cardiomyocyte (CM) organization on 2D surfaces, extending this micropatterning to 3D scaffolds has been difficult. To address this, we have developed a technique to micropattern directional FN cues into 3D hydrogel scaffolds to direct CM organization. We compared CM alignment in response to FN lines on collagen type I (COL1) or fibrin hydrogels, with or without Matrigel and identified a specific scaffold composition that maximized CM uniaxial alignment. We then used this technique to build aligned 2D human CM sheets and multilayered 3D tissues. Methods: To engineer micropatterned hydrogel scaffolds, we modified a previously reported surface-initiated assembly technique[1]. Briefly, 20 μm wide FN lines were microcontact printed onto a sacrificial poly(N-isopropylacrylamide) (PIPAAm) substrate. PIPAAm was then dissolved to transfer the FN lines, first on gelatin, then onto hydrogels consisting of COL1 or fibrin with or without Matrigel. Embryonic chick CMs were cultured for 4 days onto patterned hydrogels, then fixed and stained for nuclei, actin, and α-actinin, with CMs seeded on micropatterned polydimethylsiloxane used as controls. Similarly, human embryonic stem cell derived CMs were seeded on COL1 patterned with FN lines. Orientational order parameter (OOP) of the actin cytoskeleton was analyzed to determine the hydrogel composition that maximized alignment of both CM types. To create multilayered constructs, engineered cardiac tissues were cultured for 2 days, stacked, and maintained in contact for 3 days before fixing. Results: FN patterns were well defined on the COL1 and fibrin, with <10% variation in line width. Chick CM alignment on fibrin was low for all concentrations of fibrin and Matrigel (OOP<0.5). In contrast, CMs aligned well on COL1 without Matrigel (OOP = 0.66 vs. 0.38 with Matrigel), and reached maximum anisotropy on high concentration COL1 (6 mg/mL) without Matrigel (OOP=0.75±0.1). Human CMs seeded on the high COL1 concentration substrate formed anisotropic contractile monolayers with distinct sarcomeres (OOP = 0.65±0.1). To build 3D tissues, we stacked cardiac sheets 3 layer high with a single layer thickness of 17 ± 1.8 µm. Conclusions: We engineered directional, microscale ECM cues into hydrogel scaffolds and successfully guided the alignment of cardiac cell sheets, including human CMs. The high concentration COL1 with integrated FN micropatterns maximized both chick and human CM alignment. Future work will focus on engineering multi-layered and aligned human cardiac tissues and assessing electrophysiology and contractility. Financial support from the Dowd-ICES fellowship to Q. Jallerat and from National Institutes of Health 1DP2HL117750 and American Heart Association 12SDG11800036 to A. W. Feinberg.

In vitro maturation of large-scale cardiac patches based on a perfusable starter matrix by cyclic mechanical stimulation

The ultimate goal of tissue engineering is the generation of implants similar to native tissue. Thus, it is essential to utilize physiological stimuli to improve the quality of engineered constructs. Numerous publications reported that mechanical stimulation of small-sized, non-perfusable, tissue engineered cardiac constructs leads to a maturation of immature cardiomyocytes like neonatal rat cardiomyocytes or induced pluripotent stem cells/embryonic stem cells derived self-contracting cells. The aim of this study was to investigate the impact of mechanical stimulation and perfusion on the maturation process of large-scale (2.5×4.5cm), implantable cardiac patches based on decellularized porcine small intestinal submucosa (SIS) or Biological Vascularized Matrix (BioVaM) and a 3-dimensional construct containing neonatal rat heart cells. Application of cyclic mechanical stretch improved contractile function, cardiomyocyte alignment along the stretch axis and gene expression of cardiomyocyte markers. The development of a complex network formed by endothelial cells within the cardiac construct was enhanced by cyclic stretch. Finally, the utilization of BioVaM enabled the perfusion of the matrix during stimulation, augmenting the beneficial influence of cyclic stretch. Thus, this study demonstrates the maturation of cardiac constructs with clinically relevant dimensions by the application of cyclic mechanical stretch and perfusion of the starter matrix. Statement of significanceConsidering the poor endogenous regeneration of the heart, engineering of bioartificial cardiac tissue for the replacement of infarcted myocardium is an exciting strategy. Most techniques for the generation of cardiac tissue result in relative small-sized constructs insufficient for clinical applications. Another issue is to achieve cardiomyocytes and tissue maturation in culture. Here we report, for the first time, the effect of mechanical stimulation and simultaneous perfusion on the maturation of cardiac constructs of clinical relevant dimensions, which are based on a perfusable starter matrix derived from porcine small intestine. In response to these stimuli superior organization of cardiomyocytes and vascular networks was observed in contrast to untreated controls. The study provides substantial progress towards the generation of implantable cardiac patches.

Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes

Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also down-regulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway.

Evaluation of sarcomeric organization in human pluripotent stem cell-derived cardiomyocytes.

High-resolution optical imaging provides valuable window in examining transitional and systemic changes in cellular processes. The relative spatial relationship of structural, transport, and signaling proteins, surface antigens, and transcription factors may reveal developmental state of the cellular system. Here, we describe the use of confocal microscopy to evaluate the organization of sarcomeric structural proteins, sarcoplasmic channel proteins, and cardiac transcription factors in human pluripotent stem cell (PSC)-derived cardiomyocytes.

Facile One-step Micropatterning Using Photodegradable Methacrylated Gelatin Hydrogels for Improved Cardiomyocyte Organization and Alignment.

Hydrogels are often employed as temporary platforms for cell proliferation and tissue organization in vitro. Researchers have incorporated photodegradable moieties into synthetic polymeric hydrogels as a means of achieving spatiotemporal control over material properties. In this study protein-based photodegradable hydrogels composed of methacrylated gelatin (GelMA) and a crosslinker containing o-nitrobenzyl ester groups have been developed. The hydrogels are able to degrade rapidly and specifically in response to UV light and can be photopatterned to a variety of shapes and dimensions in a one-step process. Micropatterned photodegradable hydrogels are shown to improve cell distribution, alignment and beating regularity of cultured neonatal rat cardiomyocytes. Overall this work introduces a new class of photodegradable hydrogel based on natural and biofunctional polymers as cell culture substrates for improving cellular organization and function.

NEONATAL RAT MYOCARDIUM SURVIVAL IN TERMS OF CHRONIC HEMIC HYPOXIA

Hypoxia problem and its consequences are still relevant problems in perinatal medicine. The aim of the research was to investigate the neonatal rat’s myocardium structural adaptation in terms of chronic hemic hypoxia. In the experiment there were involved 15 female three-months old Wistar rats which were daily administered sodium nitrite solution at a dose of 50 mg/1 kg body weight throughout the pregnancy and for three days after giving birth to pups. Myocardium was taken from the pups that were fed the by above females and reached 3 days of age. Preparation of material for electron microscopy studies was conducted according to the standard methods. Additionally we applied La20H3 solution (lanthanum hydroxide) to determine the permeability of cell membranes. Content of malondialdehyde (MDA) was detected by the method suggested by M. Mihara. Cytochemical research included identification of the succinate dehydrogenase enzyme (SDH) by Nartsissov quantitative method. It was found out that changes in the ultrastructural organization of cardiomyocytes and endothelial cells of capillaries in the myocardium of newborn rats with chronic hemic hypoxia are accompanied by a very vivid mitochondrial dysfunction. It is structurally expressed by the outer mitochondrial membrane damage and lysis of crists confirmed by increased levels of MDA in serum and decreased activity of SDH in lymphocytes. The most pronounced damage occurs in the mitochondrial membranes accompanied by destructive structural vascular changes and focal lysis of myofibrils. Thus, hemic hypoxia of mother has a strong toxic effect on the myocardium of the neonates.