Search

Keratin (KRT), a natural fibrous structural protein, can be classified into two categories: "soft" cytosolic KRT that is primarily found in the epithelia tissues (e.g., skin, the inner lining of digestive tract) and "hard" KRT that is mainly found in the protective tissues (e.g., hair, horn). The latter is the predominant form of KRT widely used in biomedical research. The oxidized form of extracted KRT is exclusively denoted as keratose (KOS) while the reduced form of KRT is termed as kerateine (KRTN). KOS can be processed into various forms (e.g., hydrogel, films, fibers, and coatings) for different biomedical applications. KRT/KOS offers numerous advantages over other types of biomaterials, such as bioactivity, biocompatibility, degradability, immune/inflammatory privileges, mechanical resilience, chemical manipulability, and easy accessibility. As a result, KRT/KOS has attracted considerable attention and led to a large number of publications associated with this biomaterial over the past few decades; however, most (if not all) of the published review articles focus on KRT regarding its molecular structure, biochemical/biophysical properties, bioactivity, biocompatibility, drug/cell delivery, and in vivo transplantation, as well as its applications in biotechnical products and medical devices. Current progress that is directly associated with KOS applications in tissue regeneration and drug delivery appears an important topic that merits a commentary. To this end, the present review aims to summarize the current progress of KOS-associated biomedical applications, especially focusing on the in vitro and in vivo effects of KOS hydrogel on cultured cells and tissue regeneration following skin injury, skeletal muscle loss, peripheral nerve injury, and cardiac infarction.

Trimethylamine N-oxide (TMAO) is a gut microbiota metabolite derived from trimethylamine- containing nutrient precursors such as choline, L-carnitine, and betaine, which are rich in many vegetables, fruits, nuts, dairy products, and meats. An increasing number of clinical studies have demonstrated a strong relationship between elevated plasma TMAO levels and adverse cardiovascular events. It is commonly agreed that TMAO acts as an independent risk factor and a prognostic index for patients with cardiovascular disease. Although most animal (mainly rodent) data support the clinical findings, the mechanisms by which TMAO modulates the cardiovascular system are still not well understood. In this context, we provide an overview of the potential mechanisms underlying TMAO-induced cardiovascular diseases at the cellular and molecular levels, with a focus on atherosclerosis. We also address the direct effects of TMAO on cardiomyocytes (a new and under-researched area) and finally propose TMAO as a potential biomarker and/or therapeutic target for diagnosis and treatment of patients with cardiovascular disease.

Current medicinal treatments for diseases comprise largely of two categories: small molecular (chemical) (e.g., aspirin) and larger molecular (peptides/proteins, e.g., insulin) drugs. Whilst both types of therapeutics can effectively treat different diseases, ranging from well-understood (in view of pathogenesis and treatment) examples (e.g., flu), to less-understood chronic diseases (e.g., diabetes), classical small molecule drugs often possess significant side-effects (a major cause of drug withdrawal from market) due to their low- or non-specific targeting. By contrast, therapeutic peptides, which comprise short sequences from naturally occurring peptides/proteins, commonly demonstrate high target specificity, well-characterized modes-of-action, and low or non-toxicity in vivo. Unfortunately, due to their small size, linear permutation, and lack of tertiary structure, peptidic drugs are easily subject to rapid degradation or loss in vivo through chemical and physical routines, thus resulting in a short half-life and reduced therapeutic efficacy, a major drawback that can reduce therapeutic efficiency. However, recent studies demonstrate that the short half-life of peptidic drugs can be significantly extended by various means, including use of enantiomeric or non-natural amino acids (AAs) (e.g., L-AAs replacement with D-AAs), chemical conjugation [e.g., with polyethylene glycol], and encapsulation (e.g., in exosomes). In this context, we provide an overview of the major in vivo degradation forms of small therapeutic peptides in the plasma and anti-degradation strategies. We also update on the progress of small peptide therapeutics that are either currently in clinical trials or are being successfully used in clinical therapies for patients with non-infectious diseases, such as diabetes, multiple sclerosis, and cancer.

Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) directly interact with intracellular interleukin receptor associated kinase (IRAK) family members to initialize innate immune and inflammatory responses following activation by pathogen-associated or hostderived elements. Although four IRAK family members [IRAK1, 2, 3 (i.e., IRAK-M), and 4] are involved in TLR and IL-1R signaling pathways, IL-1R > IRAK1 signaling appears to be the most studied pathway, with sufficient evidence to support its central role linking the innate immune response to the pathogenesis of various diseases, including cancers, metabolic disorders, and non-infectious immune disorders. However, IRAK1's involvement in cardiovascular diseases was only recently revealed and the detailed mechanism underling the pathogenesis of cardiovascular diseases, such as atherosclerosis, myocardial infarction, and heart failure (all non-infectious disorders), remains largely unknown with very limited publications to date. This review aims to summarize the overall roles of the IRAK family, especially IRAK1, in mediating the development of cardiovascular diseases. NIH [1R15HL140528-01]; College of Veterinary Medicine at Virginia Tech; Edward Via College of Osteopathic Medicine, Interdisciplinary Graduate Education Program of Regenerative Medicine; Internal Research Competition (IRC) [178391] Published version This work was supported by the NIH grant (1R15HL140528-01 for JQH) , One-Health seed grant (PJ6SPVHJ for JQH) by the College of Veterinary Medicine at Virginia Tech and the Edward Via College of Osteopathic Medicine, Interdisciplinary Graduate Education Program of Regenerative Medicine (IGEP-RM for YJZ) , and Internal Research Competition (IRC) Seed Grant (#178391 for JQH) by the College of Veterinary Medicine at Virginia Tech. The funders had no role in the study design, data collection and analysis, decision to publish, and preparation of the manuscript.

Stem cell-based therapies have demonstrated great potential for the treatment of cardiac diseases, for example, myocardial infarction; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation are still major limitations, which lead to low therapeutic efficiency. Biomaterials provide a promising solution to overcome these issues due to their biocompatibility, biodegradability, low/nonimmunogenicity, and low/noncytotoxicity. The present study aimed to investigate the impacts of keratose (KOS) hydrogel biomaterial on cellular viability, proliferation, and differentiation of c-kit+ human cardiac stem cells (hCSCs). Briefly, hCSCs were cultured on both KOS hydrogel-coated dishes and regular tissue culture dishes (Blank control). Cell viability, stemness, proliferation, cellular morphology, and cardiac lineage differentiation were compared between KOS hydrogel and the Blank control at different time points. We found that KOS hydrogel is effective in maintaining hCSCs without any observable toxic effects, although cell size and proliferation rate appeared smaller on the KOS hydrogel compared to the Blank control. To our surprise, KOS hydrogel significantly promoted vascular smooth muscle cell (VSMC) differentiation (∼72%), while on the Blank control dishes, most of the hCSCs (∼78%) became cardiomyocytes. Furthermore, we also observed "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. To the best of our knowledge, this is the first report to discover the preferred differentiation of hCSCs toward VSMCs on KOS hydrogel. The underlying mechanism remains unknown. This innovative methodology may offer a new approach to generate a robust number of VSMCs simply by culturing hCSCs on KOS hydrogel, and the resulting VSMCs may be used in animal studies and clinical trials in combination with an injectable KOS hydrogel to treat cardiovascular diseases.

Background and aim Although femoral artery ligation-induced ischemia is commonly used in C57BL/6 or Balb/c mice, direct comparisons between femoral artery/vein (FAV) versus femoral artery (FA) excisions have not been reported. The goal of the present study is to investigate the effects of FAV versus FA excisions on hindlimb models using adult CD-1 mice. Methods Two groups (n = 10/group) of adult, mixed gender CD-1 mice were used to generate hindlimb ischemic models by excising either the FAV or FA. Laser Doppler Imaging was used to evaluate blood flow before surgery, immediately after surgery (Day 0), and then on Days 14 and 28. Toe necrosis was checked every 14 days while skeletal muscle cellular remodeling and vascular networks were analyzed at the end of the experiment using pathohistological, Dil-vessel painting, and immunohistochemical approaches. Results During the 4-week period, no statistical differences were found between FAV and FA excision-induced ischemia in terms of reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell sizes. However, significant increases in centrally-located nuclei cells, adipose cells, diameters of Dil-stained arterioles, and CD31+ capillary densities, but decreases in arteriole densities/lengths were observed in ischemic limbs of both FAV and FA groups compared to control limbs. Conclusion We conclude that FAV and FA excision in CD-1 mice generate a comparable degree of hindlimb ischemia, suggesting that, as expected, FAV is no more severe than FA. These findings may provide important information for researchers when selecting ligation methods for their hindlimb models.

Regeneration of cardiomyocytes, endothelial cells and vascular smooth muscle cells (three major lineages of cardiac tissues) following myocardial infarction is the critical step to recover the function of the damaged heart. Myeloid ecotropic viral integration site 1 (Meis1) was first discovered in leukemic mice in 1995 and its biological function has been extensively studied in leukemia, hematopoiesis, the embryonic pattering of body axis, eye development and various genetic diseases, such as restless leg syndrome. It was found that Meis1 is highly associated with Hox genes and their cofactors to exert its regulatory effects on multiple intracellular signaling pathways. Recently with the advent of bioinformatics, biochemical methods and advanced genetic engineering tools, new function of Meis1 has been found to be involved in the cell cycle regulation of cardiomyocytes and endothelial cells. For example, inhibition of Meis1 expression increases the proliferative capacity of neonatal mouse cardiomyocytes, whereas overexpression of Meis1 results in the reduction in the length of cardiomyocyte proliferative window. Interestingly, downregulation of one of the circular RNAs, which acts downstream of Meis1 in the cardiomyocytes, promotes angiogenesis and restores the myocardial blood supply, thus reinforcing better regeneration of the damaged heart. It appears that Meis1 may play double roles in modulating proliferation and regeneration of cardiomyocytes and endothelial cells post-myocardial infarction. In this review, we propose to summarize the major findings of Meis1 in modulating fetal development and adult abnormalities, especially focusing on the recent discoveries of Meis1 in controlling the fate of cardiomyocytes and endothelial cells.

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit(+) human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5µm) elastomeric meshes consisting of aligned 1.7µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases.

Correct selection of an appropriate animal mode to closely mimic human extremity diseases or to exhibit desirable phenotypes of limb regeneration is the first critical step for all scientists in biomedical and regenerative researches. The commonly-used animals in limb regeneration and repairing studies, such as axolotl, mice, and rats, are discussed in the review and other models including cockroaches, dogs, and horses are also mentioned. The review weighs the general advantages, disadvantages, and precedent uses of each model in the context of limb and peripheral injury and subsequent regeneration. We hope that this review can provide the reader an overview of each model, from which to select one for their specific purpose.

Previous studies indicated that the fast-healer strain of MRL/MpJ-Fas(lpr)/J (MRL) mice demonstrated superior regenerative capabilities for digit wound healing and/or regeneration compared with the non-healer strain of C57BL/6 (C57) mice. These reports, however, mainly focused on morphological observations and analysis of gene expression with little attention on the role of angiogenesis in the amputated digits. By taking advantage of Laser Doppler Imaging and histological analysis, we examined the potential role(s) of angiogenesis in facilitating tissue regrowth/regeneration by comparing two strains of mice (MRL versus C57). The three middle digits on the mouse's right foot (RF) were amputated at the middle level of phalanx 2 (P2) on postnatal day 2 (Day 0), while the left foot (LF) remained intact and served as a control. Laser Doppler images and digital photographs were taken of both feet before, immediately after surgery, and on Day 7, 14, 21, and 28 to evaluate blood flow and overall length of digit regrowth. All measurements from the amputated digits of the RF were divided by those of the control LF to obtain normalized ratios for statistical comparisons between groups. It was found that MRL mice demonstrated an approximately 220% increase in regrowth ratios over that of C57 mice from Day 21-28 (p

3D bioprinting is an emerging biofabrication process for the production of adherent cell-based products, including engineered tissues and foods. While process innovations are rapidly occurring in the area of process monitoring, which can improve fundamental understanding of process-structure-property relations as well as product quality by closed-loop control techniques, in-line sensing of the bioink composition remains a challenge. Here, we report that hollow multifunctional fibers enable in-line impedimetric sensing of bioink composition and exhibit selectivity for real-time classification of cell type, viability, and state of differentiation during bioprinting. Continuous monitoring of the fiber impedance magnitude and phase angle response from 102 to 106 Hz during microextrusion 3D bioprinting enabled compositional and quality analysis of alginate bioinks that contained fibroblasts, neurons, or mouse embryonic stem cells (mESCs). Fiber impedimetric responses associated with the bioinks that contained differentiated mESCs were consistent with differentiation marker expression characterized by immunocytochemistry. 3D bioprinting through hollow multifunctional fiber impedimetric sensors enabled classification of stem cells as stable or randomly differentiated populations. This work reports an advance in monitoring 3D bioprinting processes in terms of in-line sensor-based bioink compositional analysis using fiber technology and provides a non-invasive sensing platform for achieving future quality-controlled bioprinted tissues and injectable stem-cell therapies.

Introduction: Meis1 belongs to the TALE (three amino-acid loop extension) subclass of the homeobox gene families. It is a highly conserved transcription factor in all eukaryotes and plays a crucial role in anatomical development during embryogenesis and maintaining homeostasis after birth. However, little is known about the role of Meis1 gene on regulating functions of endothelial cells (ECs). Our preliminary study found that EC-specific knockout (KO) of Meis1 showed protective benefits in response to hindlimb ischemia, such as increased blood flow and decreased toe apoptosis. We hypothesize that deletion of Meis1 may enhance EC viability or resistance to harsh environment. The present study aims to explore the potential underlying mechanisms using an in vitro apoptotic condition induced by doxorubicin (Dox). Methods: EC-specific deletion of Meis1 gene was generated by crossbreeding Meis1 flox/flox mice with Tie2-Cre mice. Following confirmation of genotyping, ECs were isolated from the lungs of 3-5 WT and KO newborn pups using CD31 microbeads. Cells were then plated and treated with 0, 0.5, and 1 μM Dox for 24 hours prior to various assays. Results: Our qPCR data indicated that KDR expression was ~100-times more in CD31 + fraction than in CD31 - fraction both in WT and KO groups (n=4); while in KO group, Meis1 expression was significantly decreased to 20% of WT control, suggesting the successful isolation of Meis1-KO ECs. Further analysis with immunocytochemistry indicated that 83.1±2.3% CD31 + cells were also KDR + , compared to 8.3±1.2% KDR + cells in CD31 - fraction (n=7-8). Using EC-specific dye uptake assay, we found that 84.6±2.4% CD31 + cells were positive for Dil-Ac-LDL, compared to 7.9±3.2% in CD31 - cells (n=6), which appears to correspond to more tube formation observed in KO than WT groups. As we expected, Meis1-KO ECs demonstrated greater cell viability (81.2±6.1%) than WT cells (58.0±1.6%) under 0.5 μM Dox treatment (n=5-6). The increased viability in KO ECs may be due to the decreased expression of Bax , a pro-apoptotic factor. Conclusions: We conclude that activation of Meis1 gene may facilitate EC death under ischemic condition while deletion of Mei1 gene promote protective effects on ECs.

Alginate-based microencapsulation of cells has made a significant impact on the fields of regenerative medicine and tissue engineering mainly because of its ability to provide immunoisolation for the encapsulated material. This characteristic has allowed for the successful transplantation of non-autologous cells in several clinical trials for life threatening conditions, such as diabetes, myocardial infarction, and neurodegenerative disorders. Methods for alginate hydrogel microencapsulation have been well developed for various types of cells and can generate microcapsules of different diameters, degradation time, and composition. It appears the most prominent and successful method in clinical applications is the coaxial electrospray method, which can be used to generate both homogenous and non-homogeneous microcapsules with uniform size on the order of 100 μm. The present review aims to discuss why alginate hydrogel is an ideal biomaterial for the encapsulation of cells, how alginate-based microcapsules are generated, and methods of modifying the microcapsules for specific clinical treatments. This review will also discuss clinical applications that have utilized alginate-based microencapsulation in the treatment of diabetes, ischemic heart disease, and neurodegenerative diseases.

Due to the biological complexity of the cardiovascular system, the animal model is an urgent pre-clinical need to advance our knowledge of cardiovascular disease and to explore new drugs to repair the damaged heart. Ideally, a model system should be inexpensive, easily manipulated, reproducible, a biological representative of human disease, and ethically sound. Although a larger animal model is more expensive and difficult to manipulate, its genetic, structural, functional, and even disease similarities to humans make it an ideal model to first consider. This review presents the commonly-used large animals—dog, sheep, pig, and non-human primates—while the less-used other large animals—cows, horses—are excluded. The review attempts to introduce unique points for each species regarding its biological property, degrees of susceptibility to develop certain types of heart diseases, and methodology of induced conditions. For example, dogs barely develop myocardial infarction, while dilated cardiomyopathy is developed quite often. Based on the similarities of each species to the human, the model selection may first consider non-human primates—pig, sheep, then dog—but it also depends on other factors, for example, purposes, funding, ethics, and policy. We hope this review can serve as a basic outline of large animal models for cardiovascular researchers and clinicians. Published version

Background The relatively high radiation dose from micro-CT is a cause for concern in preclinical research involving animal subjects. Interior region-of-interest (ROI) imaging was proposed for dose reduction, but has not been experimentally applied in micro-CT. Objective Our aim is to implement interior ROI imaging in a carbon nanotube (CNT) x-ray source based micro-CT, and present the ROI image quality and radiation dose reduction for interior cardiac micro-CT imaging of a mouse heart in situ. Methods An aperture collimator was mounted at the source-side to induce a small-sized cone beam (10 mm width) at the isocenter. Interior in situ micro-CT scans were conducted on a mouse carcass and several micro-CT phantoms. A GPU-accelerated hybrid iterative reconstruction algorithm was employed for volumetric image reconstruction. Radiation dose was measured for the same system operated at the interior and global micro-CT modes. Results Visual inspection demonstrated comparable image quality between two scan modes. Quantitative evaluation demonstrated high structural similarity index (up to 0.9614) with improved contrast-noise-ratio (CNR) on interior micro-CT mode. Interior micro-CT mode yielded significant reduction (up to 83.9%) for dose length product (DLP). Conclusions This work demonstrates the applicability of using CNT x-ray source based interior micro-CT for preclinical imaging with significantly reduced radiation dose.

There is an urgent clinical need to develop new therapeutic approaches for treating cardiovascular disease, but the biology of cardiovascular regeneration is complex. Model systems are required to advance our understanding of the pathogenesis, progression, and mechanisms underlying cardiovascular disease as well as to test therapeutic approaches to regenerate tissue and restore cardiac function following injury. An ideal model system should be inexpensive, easily manipulated, reproducible, physiologically representative of human disease, and ethically sound. The choice of animal model needs to be considered carefully since it affects experimental outcomes and whether findings of the study can be reasonably translated to humans. This review presents a guideline for the commonly used small animal models (mice, rats, rabbits, and cats) used in cardiac research as an effort to standardize the most relevant procedures and obtain translatable and reproducible results.

Uniquely identifying research animals is a widespread and essential procedure. Potential disadvantages of commonly used identification methods such as toe clipping, ear punching, and ear tagging include tissue loss and adverse effects in physiologic homeostasis and animal behavior. In addition, the labels produced by using these methods can become unreadable, potentially leading to misidentification. In this study, we proposed a combined approach involving ear, tail, and toe tattooing that can be used to permanently identify mice regardless of their age. Four groups (neonatal and adult C57BL/6J [black] and CD1 [white] mice) were used. Single- or 2-color tattooing (ear, tail, or toe or combinations thereof) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than 3 wk. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. We conclude that tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals.

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H2O2, concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

The effect of environmental temperature on the damping capacity of Cu-7.66Al-9.52Mn (mass fraction, %) alloy was studied. The result shows that with increasing the environmental temperature, the logarithmic decrement increases firstly and reaches the maximal value of 0.118. The reason is that more phase interfaces and twinning boundaries can move at a higher temperature, leading to higher consumption of energy, in despite of the decreasing of the amount of martensite. When the environmental temperature is above M s , with further increase in the environmental temperature, the logarithmic decrement decreases sharply mainly because there is little martensite remaining in the alloy.

Human embryonic stem cells (hESCs) can provide potentially unlimited quantities of a wide range of human cell types that can be used in drug discovery and development, basic research and regenerative medicine. In this review, the authors describe the differentiation of hESCs into cardiomyocytes and outline the properties of hESC-derived cardiomyocytes (hESC-CMs), including their cardiac-type action potentials and contractile characteristics. In vitro cellular assays using hESC-CMs, which can be genetically engineered to create target-specific reporters as well as human disease models, will have applications at multiple stages of the drug discovery process. Furthermore, cardiac safety pharmacology assays evaluating the risk of proarrhythmic side effects associated with QT prolongation may be enhanced in their predictive value with the use of hESC-CMs.

The mechanisms underlying the blunted contractile response to β-adrenergic receptor (β-AR) stimulation in heart failure (HF) are incompletely understood, especially with regard to β-AR subtype–specific regulation of L-type Ca 2+ channels. We evaluated the impact of HF induced by pacing tachycardia on β-AR regulation of L-type Ca 2+ channels in a canine model. To evaluate changes in the relative subcellular distribution of β-AR subtypes, left ventricular membranes enriched in surface sarcolemma and T-tubular sarcolemma were prepared. Radioligand binding using [ 125 I]cyanopindolol revealed that HF resulted in a comparable decrease in the density of β 1 -ARs in both surface and T-tubule sarcolemma (55±4%, n=7, P P 2 -AR density was observed. Whole-cell patch clamp studies demonstrated a markedly blunted increase in I Ca,L in response to saturating concentrations of the nonselective β-AR agonist isoproterenol (0.1 μmol/L) in failing myocytes compared with control (129±20%, n=11, versus 332±35%, n=7; P 1 -AR– and β 2 -AR–selective stimulation showed that the major component of the blunted response to nonselective β-AR stimulation in HF was caused by β 2 -AR activation, resulting in a pertussis toxin–sensitive, G i -mediated inhibition of the β 1 -AR–induced increase in I Ca,L . In conclusion, canine HF results in the following: (1) a uniform reduction in β 1 -AR density in surface and T-tubule membrane fractions without a change in β 2 -AR density; and (2) the emergence of distinct G i -coupling to β 2 -ARs resulting in accentuated antagonism of β 1 -AR–mediated stimulation of I Ca,L . These results have implications for optimizing the use of β-AR drugs in HF.

Cardiovascular Diseases (CVD) constitute the single leading cause of death in the United States. According to the heart disease and stroke statistics released by American Heart Association in 2010, 1 out of every 2.9 deaths is due to cardiovascular disease. Even with early diagnosis and timely reperfusion of ischemic myocardium, up to 30% patients develop Left Ventricular (LV) remodeling and loss of cardiac function [1,2]. Current treatment modality of cardiac failure is mainly focused to relieve symptoms. Although cardiac transplantation has offered some hope, organ shortage, donor-recipient organ mismatch, and transplant rejection are the major challenges limiting its widespread use.

L-type Ca2+ channels are essential for the initiation and regulation of excitation-contraction (EC) coupling in adult cardiac muscle.1 The rapid influx of Ca2+ through these channels triggers release of intracellular Ca2+ from the sarcoplasmic reticulum (SR) stores, and the resulting Ca2+ transient activates the myofilaments and thus contraction. Given that failing ventricular myocytes exhibit impaired contractility and abnormal Ca2+ transients, the L-type Ca2+ channel makes for a good suspect as a contributor to the derangement of EC coupling. However, most initial studies of whole-cell currents through L-type Ca2+ channels ( I Ca) in both human heart failure and animal models did not show a significant difference between nonfailing and failing myocytes.2 In the meantime, important changes in the abundance and/or function of other Ca2+ cycling proteins including the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), Na+-Ca2+ exchanger (NCX), and ryanodine receptors (RyRs) took the spotlight as the bad actors in heart failure.3 The L-type Ca2+ channel was left behind as a boring, obligatory participant in EC coupling. Times may be changing for the status of the L-type Ca2+ channel in the world of failing hearts. The article by Chen et al,4 in this issue of Circulation Research , unmasks important changes in the density as well as regulation of L-type Ca2+ channels in failing human ventricular myocytes. And, perhaps most encouragingly, the authors show that these changes can be partly reversed in patients with left ventricular assist devices (LVADs). This article adds more evidence to recent publications suggesting significant changes in L-type Ca2+ channel abundance and function in heart failure.5,6⇓ To appreciate the story, one needs to step back from the macroscopic or whole-cell currents through L-type Ca2+ channels and …

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC 50 . Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr , prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.

Cardiac I Kr is a critical repolarizing current in the heart and a target for inherited and acquired long-QT syndrome (LQTS). Biochemical and functional studies have demonstrated that I Kr channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I Kr functional properties derives primarily from studies of homooligomers of the original hERG 1a isolate. Here, we examine currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We find that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a 2-fold increase in the apparent rates of activation and recovery from inactivation, thus reducing rectification and facilitating current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the 2 channel types. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding 4-fold shift in the IC 50 . The importance of hERG 1b in vivo is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients that was absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells dramatically reduced 1b protein levels. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I Kr and enhance drug sensitivity, and represent a potential mechanism underlying inherited or acquired LQTS.

Current procedures for the maintenance of cardiomyocytes from human embryonic stem (hES) cells rely on either co-culture with mouse cells or medium containing fetal bovine serum (FBS). Due to exposure to animal products, these methods carry the risk of potential pathogen contamination and increased immunogenicity. Additionally, FBS introduces inherent variability in the cultures due to the inevitable differences in serum lots. Here we investigated whether a defined serum-free medium containing creatine, carnitine, taurine, and insulin (CCTI) could maintain hES cell-derived cardiomyocytes. We show that hES cell-derived cardiomyocytes maintained in the CCTI medium in the absence of any feeders exhibit similar phenotypes to those maintained in serum, as indicated by the following observations: (1) comparable levels of cardiac gene transcription were found in cells grown in serum-containing medium versus those in the CCTI medium; (2) cardiomyocyte-associated proteins were expressed in cells cultured in the CCTI medium; (3) beating cells in the CCTI medium responded to pharmacological agents in a dose-dependent manner; and (4) the vast majority of the beating embryoid bodies displayed ventricular-like action potentials (APs), and the ventricular cells in serum-containing medium and the CCTI medium had indistinguishable AP properties. Therefore, culturing hES cell-derived cardiomyocytes in serum-free medium as described here should facilitate the use of the cells for in vitro and in vivo applications.

Human embryonic stem (hES) cells can differentiate in vitro, forming embryoid bodies (EBs) composed of derivatives of all three embryonic germ layers. Spontaneously contracting outgrowths from these EBs contain cardiomyocytes (CMs); however, the types of human CMs and their functional properties are unknown. This study characterizes the contractions and action potentials (APs) from beating EB outgrowths cultured for 40 to 95 days. Spontaneous and electrical field-stimulated contractions were measured with video edge-detection microscopy. β-Adrenergic stimulation with 1.0 μmol/L isoproterenol resulted in a significant increase in contraction magnitude. Intracellular electrical recordings using sharp KCl microelectrodes in beating EB outgrowths revealed three distinct classes of APs: nodal-like, embryonic atrial-like, and embryonic ventricular-like. The APs were described as embryonic based on the relatively depolarized resting membrane potential and slow AP upstroke. Repeated impalements of an individual beating outgrowth revealed a reproducible AP morphology recorded from different cells, suggesting that each outgrowth is composed of a predominant cell type. Complex functional properties typical of cardiac muscle were observed in the hES cell-derived CMs including rate adaptation of AP duration and provoked early and delayed afterdepolarizations. Repolarization of the AP showed a significant role for I Kr based on E-4031 induced prolongation of AP duration as anticipated for human CMs. In conclusion, hES cells can differentiate into multiple types of CMs displaying functional properties characteristic of embryonic human cardiac muscle. Thus, hES provide a renewable source of distinct types of human cardiac myocytes for basic research, pharmacological testing, and potentially therapeutic applications.

Objective: Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation–contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca2+ through L-type Ca2+ channels ( I Ca) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca2+ channels change in canine tachycardia pacing-induced cardiomyopathy. Methods: Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I Ca and intramembrane charge movement. Results: A complex staining pattern of interconnected tubules including prominent transverse components spaced every ∼1.6 μm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5±0.4 in control to 8.7±0.4% in failing cells ( P

The amphotericin B-perforated whole-cell patch clamp technique was used to determine the modulation of L-type Ca2+ channels by protein kinase C (PKC)-mediated pathways in adult rat ventricular myocytes. Application of 10 nM endothelin-1 (ET-1) increased peak Ca2+ current (ICa) by 28.2 +/- 2.5 % (n = 13) and slowed current decay. These effects were prevented by the endothelin receptor antagonist PD145065 (10 microM) and by the PKC inhibitor chelerythrine (8 microM). To establish if direct activation of PKC mimicked the ET-1 effect, the active and inactive phorbol esters (phorbol-12-myristate-13-acetate and 4alpha-phorbol-12, 13-didecanoate) were tested. Both phorbol esters (100 nM) resulted in a small (approximately 10%) increase in ICa, suggesting PKC-independent effects. Bath application of dioctanoylglycerol (diC8), a diacylglycerol (DAG) analogue which is capable of directly activating PKC, caused a gradual decline in peak ICa (50.4 +/- 6.2 %, n = 5) and increased the rate of current decay. These effects were unaffected by the PKC inhibitor chelerythrine (8 microM). Intracellular photorelease of caged diC8 with 3 or 10 s exposure to UV light produced a concentration-dependent increase in peak ICa (20. 7 +/- 8.5 % (n = 8) for 3 s UV and 60.8 +/- 11.4 % (n = 13) for 10 s UV), which could be inhibited by chelerythrine. Our results demonstrate that both ET-1 and intracellularly photoreleased diC8 increase ICa by a PKC-mediated pathway, which is in direct contrast to the PKC-independent inhibition of ICa produced by bath-applied diC8. We conclude that specific cellular pools of DAG are crucially important in the regulation of ICa by PKC.