Orientation Of Cardiomyocytes Research Articles

Use of the intrusion angle to describe the radial orientation of local cardiomyocytes in the left ventricle.

The projected transverse angle and the nonprojected intrusion angle can be used to describe the radial orientation of local cardiomyocytes in the left ventricle wall, although to date their descriptive relevance has not been demonstrated. This paper compares the evolution of the transverse angle and the intrusion angle in five left ventricle wall samples, and investigates in more detail their respective behaviors when the nonprojected helical angle varies. We show that the intrusion angle avoids the "projection" effect, and contrary to the transverse angle, it remains stable whatever the values taken by the nonprojected helical angle, even when this approaches 90°. The intrusion angle is the better choice, rather than the transverse angle, in describing the radial orientation of local cardiomyocytes. Furthermore, the oscillation of the intrusion angle in the samples is assessed, whose results indicate that the intrusion angle's oscillation amplitude and period are regional and related to the local tissue architecture.

Cardiac-Adaptive Conductive Hydrogel Patch Enabling Construction of Mechanical-Electrical Anisotropic Microenvironment for Heart Repair.

The biomimetic construction of a microstructural-mechanical-electrical anisotropic microenvironment adaptive to the native cardiac tissue is essential to repair myocardial infarction (MI). Inspired by the 3D anisotropic characteristic of the natural fish swim bladder (FSB), a novel flexible, anisotropic, and conductive hydrogel was developed for tissue-specific adaptation to the anisotropic structural, conductive, and mechanical features of the native cardiac extracellular matrix. The results revealed that the originally stiff, homogeneous FSB film was tailored to a highly flexible anisotropic hydrogel, enabling its potential as a functional engineered cardiac patch (ECP). Invitro and invivo experiments demonstrated the enhanced electrophysiological activity, maturation, elongation, and orientation of cardiomyocytes (CMs), and marked MI repair performance with reduced CM apoptosis and myocardial fibrosis, thereby promoting cell retention, myogenesis, and vascularization, as well as improving electrical integration. Our findings offer a potential strategy for functional ECP and provides a novel strategy to bionically simulate the complex cardiac repair environment.

Human pluripotent stem cell-derived cardiomyocytes align under cyclic strain when guided by cardiac fibroblasts.

The myocardium is a mechanically active tissue typified by anisotropy of the resident cells [cardiomyocytes (CMs) and cardiac fibroblasts (cFBs)] and the extracellular matrix (ECM). Upon ischemic injury, the anisotropic tissue is replaced by disorganized scar tissue, resulting in loss of coordinated contraction. Efforts to re-establish tissue anisotropy in the injured myocardium are hampered by a lack of understanding of how CM and/or cFB structural organization is affected by the two major physical cues inherent in the myocardium: ECM organization and cyclic mechanical strain. Herein, we investigate the singular and combined effect of ECM (dis)organization and cyclic strain in a two-dimensional human in vitro co-culture model of the myocardial microenvironment. We show that (an)isotropic ECM protein patterning can guide the orientation of CMs and cFBs, both in mono- and co-culture. Subsequent application of uniaxial cyclic strain-mimicking the local anisotropic deformation of beating myocardium-causes no effect when applied parallel to the anisotropic ECM. However, when cultured on isotropic substrates, cFBs, but not CMs, orient away from the direction of cyclic uniaxial strain (strain avoidance). In contrast, CMs show strain avoidance via active remodeling of their sarcomeres only when co-cultured with at least 30% cFBs. Paracrine signaling or N-cadherin-mediated communication between CMs and cFBs was no contributing factor. Our findings suggest that the mechanoresponsive cFBs provide structural guidance for CM orientation and elongation. Our study, therefore, highlights a synergistic mechanobiological interplay between CMs and cFBs in shaping tissue organization, which is of relevance for regenerating functionally organized myocardium.

Age-related changes of myofibrils in white laboratory rats of different ages

The study of isolated preparations of experimental white rats of 14 months and older reveals that the longitudinal orientation of cardiomyocytes is replaced by an angular orientation in older mice. The number and structural integrity of myofibrils occupy one of the leading places in the regulation of the contraction-relaxation cycle and the implementation of the adaptive capabilities of the myocardium in the normal functioning of cardiomyocytes. Our experimental studies aim at describing the quantitative and morphological characteristics of myofibrils in rats of two age groups and analyzing the morphological and functional results. Isolated heart preparations were examined in two age groups of white rats (middle - 8-14 years; elderly - 14 months and more. The number of rats in each group amounted to six) using different histological staining methods. The results obtained show that, compared with middle-aged rats, the development of degenerative changes in myofibrils is observed in old rats, which is manifested in structural inferiority and quantitative reduction. Degenerative changes revealed with age at the intracellular level can be considered an important morphological basis for the formation of functional heart failure and the depletion of adaptive capabilities.

Myoarchitectural disarray of hypertrophic cardiomyopathy begins pre-birth.

Myoarchitectural disarray – the multiscalar disorganisation of myocytes, is a recognised histopathological hallmark of adult human hypertrophic cardiomyopathy (HCM). It occurs before the establishment of left ventricular hypertrophy (LVH) but its early origins and evolution around the time of birth are unknown. Our aim is to investigate whether myoarchitectural abnormalities in HCM are present in the fetal heart. We used wild‐type, heterozygous and homozygous hearts (n = 56) from a Mybpc3‐targeted knock‐out HCM mouse model and imaged the 3D micro‐structure by high‐resolution episcopic microscopy. We developed a novel structure tensor approach to extract, display and quantify myocyte orientation and its local angular uniformity by helical angle, angle of intrusion and myoarchitectural disarray index, respectively, immediately before and after birth. In wild‐type, we demonstrate uniformity of orientation of cardiomyocytes with smooth transitions of helical angle transmurally both before and after birth but with traces of disarray at the septal insertion points of the right ventricle. In comparison, heterozygous mice free of LVH, and homozygous mice showed not only loss of the normal linear helical angulation transmural profiles observed in wild‐type but also fewer circumferentially arranged myocytes at birth. Heterozygous and homozygous showed more disarray with a wider distribution than in wild‐type before birth. In heterozygous mice, disarray was seen in the anterior, septal and inferior walls irrespective of stage, whereas in homozygous mice it extended to the whole LV circumference including the lateral wall. In conclusion, myoarchitectural disarray is detectable in the fetal heart of an HCM mouse model before the development of LVH.

Dependences of the geometrical parameters of cell community on stimulation voltage and frequency in chick embryonic cardiomyocytes

To investigate the optimal conditions for electrical stimulation, communities of lined-up chick embryonic cardiomyocytes were evaluated in terms of their threshold voltage for pacing (PVMin) and the half-maximum paced frequency (PF50), with a focus on the following factors: (1) the orientation of the major axis of cell communities to the electric field (EF) direction as the external factor; (2) the number of cells in a cell community, the length of the cell community, and the mean length of cells comprising the community as the internal factors. Firstly, PVMin decreased with increasing length of the cell network oriented parallel to the EF. PVMin was approximately 0.041 ± 0.025 V/mm when the community was sufficiently long. On the other hand, PVMin in the orthogonal orientation was constant at 1.7 ± 0.047 V/mm with no dependence on the length of the cell network. Secondly, we found that PF50 increased with increasing length of the cell network or the number of cells in the network; the PF50 values were 2.03 ± 0.05 and 3.39 ± 0.05 Hz when the respective cell network lengths were 100 µm (n = 43) and more than 300 µm (n = 6) and the cells were oriented parallel to the EF. These findings indicate that it is important to suppress ventricular fibrillation with minimal efficient stimulation by considering the EF direction with respect to the orientation of cardiomyocytes. Furthermore, expanded cells showed the loss of ability to respond to stimulation at higher frequencies. Cardiomyocytes combined with seeded fibroblasts as a cell network at a low density are a possible model of a ventricular remodeling heart.

Alignment of human cardiomyocytes on laser patterned biphasic core/shell nanowire assemblies

The management of end stage heart failure patients is only possible by heart transplantation or by the implantation of artificial hearts as a bridge for later transplantation. However, these therapeutic strategies are limited by a lack of donor hearts and by the associated complications, such as coagulation and infection, due to the used artificial mechanical circulatory assist devices. Therefore, new strategies for myocardial regenerative approaches are under extensive research to produce contractile myocardial tissue in the future to replace non-contractile myocardial ischemic and scarred tissue. Different approaches, such as cell transplantation, have been studied intensively. Although successful approaches have been observed, there are still limitations to the application. It is envisaged that myocardial tissue engineering can be used to help replace infarcted non-contractile tissue. The developed tissue should later mimic the aligned fibrillar structure of the extracellular matrix and provide important guidance cues for the survival, function and the needed orientation of cardiomyocytes. Nanostructured surfaces have been tested to provide a guided direction that cells can follow. In the present study, the cellular adhesion/alignment of human cardiomyocytes and the biocompatibility have been investigated after cultivation on different laser-patterned nanowires compared with unmodified nanowires. As a result, the nanostructured surfaces possessed good biocompatibility before and after laser modification. The laser-induced scalability of the pattern enabled the growth and orientation of the adhered myocardial tissue. Such approaches may be used to modify the surface of potential scaffolds to develop myocardial contractile tissue in the future.

Guided orientation of human cardiomyocytes on laser patterned biphasic core/shell nanowires

Guided orientation of human cardiomyocytes on laser patterned biphasic core/shell nanowires

Guided orientation of cardiomyocytes on electrospun aligned nanofibers for cardiac tissue engineering

Cardiac tissue engineering (TE) is one of the most promising strategies to reconstruct the infarct myocardium and the major challenge involves producing a bioactive scaffold with anisotropic properties that assist in cell guidance to mimic the heart tissue. In this study, random and aligned poly(ε-caprolactone)/gelatin (PG) composite nanofibrous scaffolds were electrospun to structurally mimic the oriented extracellular matrix (ECM). Morphological, chemical and mechanical properties of the electrospun PG nanofibers were evaluated by scanning electron microscopy (SEM), water contact angle, attenuated total reflectance Fourier transform infrared spectroscopy and tensile measurements. Results indicated that PG nanofibrous scaffolds possessed smaller fiber diameters (239 ± 37 nm for random fibers and 269 ± 33 nm for aligned fibers), increased hydrophilicity, and lower stiffness compared to electrospun PCL nanofibers. The aligned PG nanofibers showed anisotropic wetting characteristics and mechanical properties, which closely match the requirements of native cardiac anisotropy. Rabbit cardiomyocytes were cultured on electrospun random and aligned nanofibers to assess the biocompatibility of scaffolds, together with its potential for cell guidance. The SEM and immunocytochemical analysis showed that the aligned PG scaffold greatly promoted cell attachment and alignment because of the biological components and ordered topography of the scaffolds. Moreover, we concluded that the aligned PG nanofibrous scaffolds could be more promising substrates suitable for the regeneration of infarct myocardium and other cardiac defects.

Modulation of alignment, elongation and contraction of cardiomyocytes through a combination of nanotopography and rigidity of substrates

The topographic and mechanical characteristics of engineered tissue constructs, simulating native tissues, should benefit tissue engineering. Previous studies reported that surface topography and substrate rigidity provide biomechanical cues to modulate cellular responses such as alignment, migration and differentiation. To fully address this issue, the present study aimed to examine the influence of nanogrooved substrates with different stiffnesses on the responses of rat cardiomyocytes. Nanogrooved substrates (450 nm in groove/ridge width; 100 or 350 nm in depth) made of polystyrene and polyurethane were prepared by imprinting from polydimethylsiloxane molds. The morphology and orientation of cardiomyocytes attached to the substrates were found to be influenced mainly by the nanogrooved structures, while the contractile function of the cells was regulated by the coupled effect of surface topography and substrate stiffness. The distribution of intracellular structural proteins such as vinculin and F-actin showed that the surface topography and substrate stiffness regulated the organization of the actin cytoskeleton and focal adhesion complexes, and consequently the contractile behavior of the cardiomyocytes. The beating rates of the cultured cardiomyocytes were dependent on both the surface topography and the substrate stiffness. The study provides insights into the interaction between cardiomyocytes and biomaterials, and benefits cardiac tissue engineering.

Contractile tension and beating rates of self-exciting monolayers and 3D-tissue constructs of neonatal rat cardiomyocytes

The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.

Interactive effects of surface topography and pulsatile electrical field stimulation on orientation and elongation of fibroblasts and cardiomyocytes

In contractile tissues such as myocardium, functional properties are directly related to the cellular orientation and elongation. Thus, tissue engineering of functional cardiac patches critically depends on our understanding of the interaction between multiple guidance cues such as topographical, adhesive or electrical. The main objective of this study was to determine the interactive effects of contact guidance and electrical field stimulation on elongation and orientation of fibroblasts and cardiomyocytes, major cell populations of the myocardium. Polyvinyl surfaces were abraded using lapping paper with grain size 1–80 μm, resulting in V-shaped abrasions with the average abrasion peak-to-peak width in the range from 3 to 13 μm, and the average depth in the range from 140 to 700 nm (AFM). The surfaces with abrasions 13 μm wide and 700 nm deep, exhibited the strongest effect on neonatal rat cardiomyocyte elongation and orientation as well as statistically significant effect on orientation of fibroblasts, thus they were utilized for electrical field stimulation. Electrical field stimulation was performed using a regime of relevance for heart tissue in vivo as well as for cardiac tissue engineering. Stimulation (square pulses, 1 ms duration, 1 Hz, 2.3 or 4.6 V/cm) was initiated 24 h after cell seeding and maintained for additional 72 h. The cover slips were positioned between the carbon rod electrodes such that the abrasions were either parallel or perpendicular to the field lines. Non-abraded surfaces were utilized as controls. Field stimulation did not affect cell viability. The presence of a well-developed contractile apparatus in neonatal rat cardiomyocytes (staining for cardiac Troponin I and actin filaments) was identified in the groups cultivated on abraded surfaces in the presence of field stimulation. Overall we observed that (i) fibroblast and cardiomyocyte elongation on non-abraded surfaces was significantly enhanced by electrical field stimulation, (ii) electrical field stimulation promoted orientation of fibroblasts in the direction perpendicular to the field lines when the abrasions were also placed perpendicular to the field lines and (iii) topographical cues were a significantly stronger determinant of cardiomyocyte orientation than the electrical field stimulation. The orientation and elongation response of cardiomyocytes was completely abolished by inhibition of actin polymerization (Cytochalasin D) and only partially by inhibition of phosphatidyl-inositol 3 kinase (PI3K) pathway (LY294002).

N-cadherin-mediated cell adhesion determines the plasticity for cell alignment in response to mechanical stretch in cultured cardiomyocytes

Mechanical stretch has been implicated as the growth stimuli in the heart. Physiologically, mechanical stretch is reported to contribute to the orientation of cardiomyocytes, though the molecular mechanism remains to be elucidated. This study was designed to make clear functional significances of N-cadherin in plasticity of cell alignment in response to mechanical stretch. Neonatal rat cardiomyocytes, cultured on silicone dishes, were subjected to artificial uniaxial cyclic stretch. Mechanical stretch was started at certain times (3–75 h) after seeding and continued for 24 h. Stretch stimulation in 3 h after cultivation promoted cell orientation running parallel to tension direction. In contrast, cardiac myocytes fail to align when exposed to stretch 24–75 h after cultivation. To address the importance of N-cadherin in the responsiveness to stretch, the expression and distribution of N-cadherin were analyzed. Immediately after seeding, N-cadherin showed dispersed distributions. During cultivation, N-cadherin localized to cell–cell contacts accompanied by the upregulation of its protein. Next, to investigate influence of cell–cell adhesion, cardiomyocytes cultured for 72 h were replated by trypsin treatment and exposed to stretch 3 h after replating. The cardiomyocytes replated by trypsinization were oriented in parallel to tension direction by mechanical stretch. Finally, adenoviral transfection of dominant-negative N-cadherin recovered the ability to exhibit cell orientation in response to stretch. Our results suggested that N-cadherin was involved in the oriented responses of cardiomyocytes induced by mechanical stretch.

A Titin mutation defines roles for circulation in endothelial morphogenesis

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell–cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.

A Titin mutation defines roles for circulation in endothelial morphogenesis

Morphogenesis of the developing vascular network requires coordinated regulation of an extensive array of endothelial cell behaviors. Precisely regulated signaling molecules such as vascular endothelial growth factor (VEGF) direct some of these endothelial behaviors. Newly forming blood vessels also become subjected to novel biomechanical forces upon initiation of cardiac contractions. We report here the identification of a recessive mouse mutation termed shrunken-head (shru) that disrupts function of the Titin gene. Titin was found to be required for the initiation of proper heart contractions as well as for maintaining the correct overall shape and orientation of individual cardiomyocytes. Cardiac dysfunction in shrunken-head mutant embryos provided an opportunity to study the effects of lack of blood circulation on the morphogenesis of endothelial cells. Without blood flow, differentiating endothelial cells display defects in their shapes and patterns of cell–cell contact. These endothelial cells, without exposure to blood circulation, have an abnormal distribution within vasculogenic vessels. Further effects of absent blood flow include abnormal spatial regulation of angiogenesis and elevated VEGF signaling. The shrunken-head mutation has provided an in vivo model to precisely define the roles of circulation on cellular and network aspects of vascular morphogenesis.