Cardiac Cell Growth Research Articles

An Alginate/Gelatin Injectable Hydrogel Containing Au Nanoparticles for Transplantation of Embryonic Mouse Cardiomyocytes in Myocardial Repair.

In advancing cardiac tissue engineering (CTE), the development of injectable hydrogels mirroring myocardial properties is pivotal. The designed hydrogels must not only support cardiac cell growth but also have to be conductive to properly promote the functionalities of cardiac cells. Here, a facile approach is developed to incorporate gold nanoparticles (AuNPs) into an injectable hydrogel composed of Alginate (Alg) and Gelatin (Gel). The resultant nanocomposite hydrogel boasts a porous interconnected network and superior conductivity (2.04 × 10-4 Scm-1) compared to the base Alg/Gel hydrogel. Hydrogel hydration and in vitro degradation profiles affirm their suitability as carriers for cardiac cells. Importantly, Alg/Gel+AuNPs hydrogels exhibit no toxicity to mouse Embryonic Cardiac Cells (mECCs) over 7 days, elevating connexin 43 (Cx43) and cardiac troponin T (CTnT) gene expression compared to controls. Then, the Alg/Gel+AuNPs hydrogel is used as a carrier for intramyocardial delivery of mECCs in rats with myocardial infarction. The significant increase in α-Smooth Muscle Actin (α-SMA) and cardiac troponin T (CTnT) expression along with the increase in ejection fraction (EF), smaller infarction size, less fibrosis area confirmed that the hydrogel efficiently promoted the transmission of mechanical and electrical signals between transplanted cells and surrounding tissue.

Cardiac organ chip: advances in construction and application.

Cardiovascular diseases are a leading cause of death worldwide, and effective treatment for cardiac disease has been a research focal point. Although the development of new drugs and strategies has never ceased, the existing drug development process relies primarily on rodent models such as mice, which have significant shortcomings in predicting human responses. Therefore, human-based in vitro cardiac tissue models are considered to simulate physiological and functional characteristics more effectively, advancing disease treatment and drug development. The microfluidic device simulates the physiological functions and pathological states of the human heart by culture, thereby reducing the need for animal experimentation and enhancing the efficiency and accuracy of the research. The basic framework of cardiac chips typically includes multiple functional units, effectively simulating different parts of the heart and allowing the observation of cardiac cell growth and responses under various drug treatments and disease conditions. To date, cardiac chips have demonstrated significant application value in drug development, toxicology testing, and the construction of cardiac disease models; they not only accelerate drug screening but also provide a new research platform for understanding cardiac diseases. In the future, with advancements in functionality, integration, and personalised medicine, cardiac chips will further simulate multiorgan systems, becoming vital tools for disease modelling and precision medicine. Here, we emphasised the development history of cardiac organ chips, highlighted the material selection and construction strategy of cardiac organ chip electrodes and hydrogels, introduced the current application scenarios of cardiac organ chips, and discussed the development opportunities and prospects for their of biomedical applications.

Imageable AuNP-ECM Hydrogel Tissue Implants for Regenerative Medicine.

In myocardial infarction, a blockage in one of the coronary arteries leads to ischemic conditions in the left ventricle of the myocardium and, therefore, to significant death of contractile cardiac cells. This process leads to the formation of scar tissue, which reduces heart functionality. Cardiac tissue engineering is an interdisciplinary technology that treats the injured myocardium and improves its functionality. However, in many cases, mainly when employing injectable hydrogels, the treatment may be partial because it does not fully cover the diseased area and, therefore, may not be effective and even cause conduction disorders. Here, we report a hybrid nanocomposite material composed of gold nanoparticles and an extracellular matrix-based hydrogel. Such a hybrid hydrogel could support cardiac cell growth and promote cardiac tissue assembly. After injection of the hybrid material into the diseased area of the heart, it could be efficiently imaged by magnetic resonance imaging (MRI). Furthermore, as the scar tissue could also be detected by MRI, a distinction between the diseased area and the treatment could be made, providing information about the ability of the hydrogel to cover the scar. We envision that such a nanocomposite hydrogel may improve the accuracy of tissue engineering treatment.

3D Graphene Oxide-Polyethylenimine Scaffolds for Cardiac Tissue Engineering.

The development of novel three-dimensional (3D) nanomaterials combining high biocompatibility, precise mechanical characteristics, electrical conductivity, and controlled pore size to enable cell and nutrient permeation is highly sought after for cardiac tissue engineering applications including repair of damaged heart tissues following myocardial infarction and heart failure. Such unique characteristics can collectively be found in hybrid, highly porous tridimensional scaffolds based on chemically functionalized graphene oxide (GO). By exploiting the rich reactivity of the GO's basal epoxydic and edge carboxylate moieties when interacting, respectively, with NH2 and NH3+ groups of linear polyethylenimines (PEIs), 3D architectures with variable thickness and porosity can be manufactured, making use of the layer-by-layer technique through the subsequent dipping in GO and PEI aqueous solutions, thereby attaining enhanced compositional and structural control. The elasticity modulus of the hybrid material is found to depend on scaffold's thickness, with the lowest value of 13 GPa obtained in samples containing the highest number of alternating layers. Thanks to the amino-rich composition of the hybrid and the established biocompatibility of GO, the scaffolds do not exhibit cytotoxicity; they promote cardiac muscle HL-1 cell adhesion and growth without interfering with the cell morphology and increasing cardiac markers such as Connexin-43 and Nkx 2.5. Our novel strategy for scaffold preparation thus overcomes the drawbacks associated with the limited processability of pristine graphene and low GO conductivity, and it enables the production of biocompatible 3D GO scaffolds covalently functionalized with amino-based spacers, which is advantageous for cardiac tissue engineering applications. In particular, they displayed a significant increase in the number of gap junctions compared to HL-1 cultured on CTRL substrates, which render them key components for repairing damaged heart tissues as well as being used for 3D in vitro cardiac modeling investigations.

Melatonin inhibits embryonic rat H9c2 cells growth through induction of apoptosis and cell cycle arrest via PI3K‐AKT signaling pathway

Our recent epidemiological study revealed that maternal sleep during the periconceptional period should be involved in the risk of congenital heart disease (CHD) in offspring. Melatonin, a sleep related hormone, has been suggested to play a crucial role in embryonic development based on the emerging evidence. In this study, we set out to assess the effect of melatonin on the embryonic cardiac cell growth and to explore the underlying mechanisms. We observed the effect of different gradient doses of melatonin as 10, 100, or 1,000 μM on cell proliferation in H9c2 embryonic rat cardiac cells. Furthermore, flow cytometry was applied to evaluate the impact on apoptosis and cell cycle. RNA-seq was conducted to screen the changes in expression of mRNA and signaling pathways. Quantitative Real-Time-PCR (qRT-PCR) was then conducted to validate the results. It was observed that melatonin could inhibit H9c2 cell growth, at the doses of 100 and 1,000 μM, but not at 10μM. Moreover, melatonin ranged from 100 to 1,000 μM could instigate cell cycle arrest at G1 phase and simulate apoptosis, in a dose-dependent manner. In addition, melatonin was found to down-regulate the expression of a number of genes, which are related to heart development (SPARC, IFITM3, TNNT2, LOX), and PI3K-Akt signaling pathway activation (FN1, HSP90B1, THBS1, MFGE8, and CLU). Our findings suggested that high level of melatonin could be capable of inhibiting growth through the induction of apoptosis and cell cycle arrest via PI3K-AKT signaling pathway, thereby interfering with embryonic heart development. Considering this study is based on H9c2 embryonic rat cardiac cells, future additional studies using human embryonic cardiac cell are warranted.

MicroRNA 21 Emerging Role in Diabetic Complications: A Critical Update.

Diabetes Mellitus is a multifactorial disease encompassing various pathogenic pathways. To avoid morbidity and mortality related to diabetic complications, early detection of disease complications as well as targeted therapeutic strategies are essential. MicroRNAs (miRs) are short non-coding RNA molecules that regulate eukaryotic posttranscriptional gene expression. MicroRNA-21 has diverse gene regulatory functions and plays a significant role in various complications of Type 2 diabetes mellitus (T2DM). The study included electronic database searches on Pubmed, Embase, and Web of Science with the search items MicroRNA21 and each of the diabetic complications. The search was carried out up to November, 2019. MicroRNA-21 modulates diabetic cardiomyopathy by affecting vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions. At the renal tubules, miR-21 can regulate the mesangial expansion, interstitial fibrosis, macrophage infiltration, podocyte loss, albuminuria and fibrotic and inflammatory gene expression related to diabetic nephropathy. Overexpression of miR-21 has been seen to play a pivotal role in the pathogenesis of diabetic retinopathy by contributing to diabetes-induced endothelial dysfunction as well as low-grade inflammation. Considering the raised levels of miR-21 in various diabetic complications, it may prove to be a candidate biomarker for diabetic complications. Further, miR-21 antagonists have shown great potential in the treatment of diabetic cardiomyopathy, diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy related complications in the future. The current review is the first of its kind encompassing the roles miR-21 plays in various diabetic complications, with a critical discussion of its future potential role as a biomarker and therapeutic target.

Dietary Fat and Sugar Differentially Affect Beta‐Adrenergic Stimulation of Cardiac ERK and AKT Pathways in Mice Subjected to High‐Calorie Feeding

Introduction. High dietary fat and sugar promote cardiac hypertrophy independently from an increase in blood pressure. The respective contribution that each macronutrient exerts on cardiac growth signaling pathways remains unclear. The goal of this study was to investigate the mechanisms by which high dietary fat and sugar levels affect cardiac growth regulatory pathways in the mouse heart.Methods. Nine‐week‐old male C57BL/6 mice (n = 20/group) were fed a standard rodent diet (STD; 3.0 kcal/g), a high‐fat diet (HFD; 5.24 kcal/g), a high‐fat and high‐sugar Western diet (WD; 4.73 kcal/g), a high‐sugar diet with mixed carbohydrates (HCD; 3.85 kcal/g), or a high‐sucrose diet (HSD; 3.85 kcal/g). Body composition was assessed weekly by EchoMRI. Whole‐body glucose utilization was assessed with an intraperitoneal glucose tolerance test. After six weeks on diets, mice were treated with saline or 20 mg/kg isoproterenol (ISO) and the activity of cardiac growth regulatory pathways was analyzed by immunoblotting. One‐ or two‐way ANOVA was used for statistical comparisons.Results. Compared with STD, HFD increased body fat mass 4.5‐fold (P < 0.0001), induced glucose intolerance (P < 0.0001), and increased insulin levels 3.6‐fold (P < 0.0001), thereby enhancing basal and ISO‐stimulated AKT phosphorylation at both threonine 308 and serine 473 residues (+37–100%; P < 0.05). Both HCD and HSD potentiated ISO‐mediated stimulation of the glucose‐sensitive kinases PYK2 (>50%; P < 0.05) and ERK (>30%; P < 0.05), thereby leading to increased phosphorylation of protein synthesis regulator S6K1 at threonine 389 residue (≥70%; P < 0.05). All these anthropometric, metabolic and cardiac growth signaling alterations occurred simultaneously in WD‐fed mice.Conclusions. The results indicate that dietary fat and sugar affect cardiac cell growth signaling pathways in C57BL/6 mice through distinct and additive mechanisms. The findings may provide new insights into the role of overnutrition in pathological remodeling of the heart.Support or Funding InformationThis work was supported by grants R01HL136438, P20GM104357 and P01HL051971 from the National Institutes of Health.

MicroRNA and Cardiovascular Diseases

Cardiovascular diseases are one of the most common causes of death in both developing and developed countries worldwide. Even though there have been improvements in primary prevention, the prevalence of cardiovascular diseases continues to increase in recent years. Hence, it is crucial to both investigate the molecular pathophysiology of cardiovascular diseases in-depth and find novel biomarkers regarding the early and proper prevention and diagnosis of these diseases. MicroRNAs, or miRNAs, are endogenous, conserved, single-stranded non-coding RNAs of 21-25 nucleotides in length. miRNAs have important roles in various cellular events such as embryogenesis, proliferation, vasculogenesis, apoptosis, cell growth, differentiation, and tumorigenesis. They also have potential roles in the cardiovascular system, including angiogenesis, cardiac cell contractility, control of lipid metabolism, plaque formation, the arrangement of cardiac rhythm, and cardiac cell growth. Circulating miRNAs are promising novel biomarkers for purposes of the diagnosis and prognosis of cardiovascular diseases. Cell or tissue specificity, stability in serum or plasma, resistance to degradative factors such as freeze-thaw cycles or enzymes in the blood, and fast-release kinetics, provide the potential for miRNAs to be surrogate markers for the early and accurate diagnosis of disease and for predicting middle- or long-term prognosis. Moreover, it may be a logical approach to combine miRNAs with traditional biomarkers to improve risk stratification and long-term prognosis. In addition to their efficacy in both diagnosis and prognosis, miRNA-based therapeutics may be beneficial for treating cardiovascular diseases using novel platforms and computational tools and in combination with traditional methods of analysis. microRNAs are promising, novel therapeutic agents, which can affect multiple genes using different signaling pathways. miRNAs therapeutic modulation techniques have been used in the settings of atherosclerosis, acute myocardial infarction, restenosis, vascular remodeling, arrhythmias, hypertrophy and fibrosis, angiogenesis and cardiogenesis, aortic aneurysm, pulmonary hypertension, and ischemic injury. This review presents detailed information about miRNAs regarding structure and biogenesis, stages of synthesis and functions, expression profiles in serum/plasma of living organisms, diagnostic and prognostic potential as novel biomarkers, and therapeutic applications in various diseases.

Differential expression of novel MicroRNAs from developing fetal heart of Gallus gallus domesticus implies a role in cardiac development.

Heart development is a complex process regulated by multi-layered genetic as well epigenetic regulators many of which are still unknown. Besides their critical role during cardiac development, these molecular regulators emerge as key modulators of cardiovascular pathologies, where fetal cardiac genes' re-expression is witnessed. MicroRNAs have recently emerged as a crucial part of signalling cascade in both development and diseases. We aimed to identify, validate, and perform functional annotation of putative novel miRNAs using chicken as a cardiac development model system. Novel miRNAs were obtained through deep sequencing of small RNAs extracted from chicken embryonic cardiac tissue of different developmental stages. After filtering out real pre-miRNAs, their expression analysis, potential target gene's prediction and functional annotations were performed. Expression analysis revealed that miRNAs were differentially expressed during different developmental stages of chicken heart. The expression of selected putative novel miRNAs was further validated by real-time PCR. Our analysis indicated the presence of novel cardiac miRNAs that might be regulating critical cardiac development events such as cardiac cell growth, differentiation, cardiac action potential generation and signal transduction.

Abstract 423: Cardiomyocyte Maturation and Multinucleation in Postnatal Swine

Objectives: Cardiomyocyte (CM) cell cycle arrest and decline of mononucleated diploid CMs have been implicated in loss of regenerative potential in postnatal mouse hearts. Sarcomeric and extracellular matrix (ECM) maturation also occur concurrently in mice, influencing CM proliferative arrest. Recent studies show a 3-day neonatal period of cardiac regeneration in pigs similar to mice, but the dynamics of postnatal pig CM growth are unknown. Our objective is to explore cardiac cell cycling, growth and maturation in postnatal pigs to understand the events guiding loss of cardiac regenerative capacity in large mammals. Methods & Results: Left-ventricular tissue from farm pigs (White Yorkshire-Landrace) at Postnatal day (P)0, P7, P15, P30, 2 months (2mo) and 6mo were utilized. CM dissociations revealed predominant CM mononucleation at birth in swine, with persistence of ~50% (1186/2537 cells, n=5) mononucleated CMs at P15, and ~10% (227/1785 cells, n=4) at 2mo. By 6mo, pig CMs are entirely multinucleated, exhibiting 4-16 nuclei per cell. Assessing hypertrophic growth in dissociated pig CMs revealed longitudinal CM growth relative to increased nucleation at all ages. However, onset of diametric hypertrophy only occurs beyond 2mo. When nuclear pHH3 and mRNA expression of cell cycle genes was assessed, pig hearts show robust cell-cycling up to 2mo. Also, fetal TNNI1 and MYH6 are active up to 2mo in pig hearts. Ongoing studies on collagen remodeling indicate ECM remodeling in swine occurs beyond P7. Future studies are designed to identify nuclear ploidy in postnatal pig CMs and measure cytokinesis. Conclusions: Cardiac maturational events are staggered over a 2-6 month postnatal window in pigs, with older pig hearts exhibiting extensive CM multinucleation and differences in longitudinal versus diametric CM growth. These fundamental variations in CM growth characteristics are important to consider when designing preclinical trials for cardiac regenerative strategies in pigs. Also, despite a similar period of regenerative capacity as mice, pig hearts do not undergo loss of CM cell cycling and mononucleation until 2mo after birth. Utilizing pigs may thus offer unique opportunities to study aspects of heart regeneration unavailable in other animal models.

ASSESSMENT OF MICRORNA-21 GENE EXPRESSION LEVELS IN RELATION WITH THE DETERIORATION OF LEFT VENTRICULAR STRAIN IN HYPERTENSIVE PATIENTS

Objective: MicroRNAs (miRs) modulate cardiovascular development and disease by post-transcriptional gene expression regulation and thus they are emerging as potential biomarkers and promising therapeutic targets in cardiovascular disease. miR-21 is found to play important roles in vascular smooth muscle cell proliferation and apoptosis, cardiac cell growth and death, and cardiac fibroblast functions.. We evaluated miR-21 gene expression levels in peripheral blood mononuclear cells in patients with essential hypertension in relation to left ventricular global longitudinal peak strain (GLPS). Design and method: We included 50 patients with newly diagnosed essential hypertension stage 1 and 2 (31 men, mean age 69.5 ± 11.8 years, mean systolic blood pressure (SBP) 167 ± 19 mmHg and mean diastolic blood pressure (DBP) 107 ± 11 mmHg). All patients underwent a serial assessment with standard conventional transthoracic and a two-dimensional speckle tracking echocardiography at baseline and at 24 months follow-up. miR-21 gene expression levels in peripheral blood mononuclear cells were quantified by real-time reverse transcription polymerase chain reaction. Results: Systolic and diastolic blood pressure was significantly reduced in the 12 months follow up under antihypertensive medication (SBP: from 167 ± 19 mmHg to 147 ± 15 mmHg and DBP: from 107 ± 11 mmHg to 95 ± 15 mmHg, p < 0.05 for both). GLPS showed a significant reduction during the 24 months follow-up (from −18.3 ± −4.9 % at baseline to −16.5 ± −6.9%, p = 0.04). miR-21 gene expression levels at baseline revealed a significant positive correlation with the reduction of GLPS (r = 0.52, p < 0.001). This correlation was independent of the patients’ clinical parameters Conclusions: Our data reveal that miR-21 might have a prognostic value in the deterioration of left ventricular GLPS in essential hypertension. In addition, it may be involved in the pathophysiology of in hypertensive heart disease and may be a promising therapeutic target.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR

The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Flexible and Stretchable PEDOT-Embedded Hybrid Substrates for Bioengineering and Sensory Applications.

Herein, we introduce a flexible, biocompatible, robust and conductive electrospun fiber mat as a substrate for flexible and stretchable electronic devices for various biomedical applications. To impart the electrospun fiber mats with electrical conductivity, poly(3,4-ethylenedioxythiophene) (PEDOT), a conductive polymer, was interpenetrated into nitrile butadiene rubber (NBR) and poly(ethylene glycol) dimethacrylate (PEGDM) crosslinked electrospun fiber mats. The mats were fabricated with tunable fiber orientation, random and aligned, and displayed elastomeric mechanical properties and high conductivity. In addition, bending the mats caused a reversible change in their resistance. The cytotoxicity studies confirmed that the elastomeric and conductive electrospun fiber mats support cardiac cell growth, and thus are adaptable to a wide range of applications, including tissue engineering, implantable sensors and wearable bioelectronics.

Gold Nanoparticle-Functionalized Reverse Thermal Gel for Tissue Engineering Applications.

Utilizing polymers in cardiac tissue engineering holds promise for restoring function to the heart following myocardial infarction, which is associated with grave morbidity and mortality. To properly mimic native cardiac tissue, materials must not only support cardiac cell growth but also have inherent conductive properties. Here, we present an injectable reverse thermal gel (RTG)-based cardiac cell scaffold system that is both biocompatible and conductive. Following the synthesis of a highly functionalizable, biomimetic RTG backbone, gold nanoparticles (AuNPs) were chemically conjugated to the backbone to enhance the system's conductivity. The resulting RTG-AuNP hydrogel supported targeted survival of neonatal rat ventricular myocytes (NRVMs) for up to 21 days when cocultured with cardiac fibroblasts, leading to an increase in connexin 43 (Cx43) relative to control cultures (NRVMs cultured on traditional gelatin-coated dishes and RTG hydrogel without AuNPs). This biomimetic and conductive RTG-AuNP hydrogel holds promise for future cardiac tissue engineering applications.

Cardiac manifestations of inherited metabolic disease linked to cellular vitamin B12 (cobalamin) uptake: Study in murine model of invalidation of Mtr gene in the heart

Heart failure is one of the most common cause of death in Western societies. Deficiency in folates and vitamin B12 during gestation and lactation causes foetal programming effect with metabolic cardiomyopathy related to decreased synthesis of methionine by methionine synthase encoded by MTR Gene. Methionine is the precursor for S-adenosyl methionine (SAM) the universal methyl donor. Mutations in MTR gene causes cardiac decompensation in new-borns by unknown mechanisms. To investigate cardiac metabolic and functional consequences of inhibition of methionine synthesis in conditional knock out of Mtr gene in the heart of C57BL/6 mice. Systolic Blood Pressure in conscious mice was measured using tail-cuff plethysmography. Left ventricular (LV) functions were assessed by Mini-PET and Echocardiography. Transcriptomics analysis was achieved by RNA deep sequencing and proteomic study by LC-MS/MS. Systolic hypertension, decreased LV ejection fraction, increased LV mass and heart/body weight ratio were observed in Mtr KO compared to control. RT-qPCR confirmed upregulated expression of natriuretic peptides (ANP and BNP) and decreased alpha MHC/beta MHC ratio in Mtr KO. Biochemical studies revealed decreased SAM/SAH ratio, elevated plasma acylcarnitine and cardiac fibrosis in Mtr KO. The whole heart transcriptome consisted of 16540 expressed genes which were clustered by K-means into three signatures, these signatures were correlated to proteomic results. Dysregulated genes and proteins in Mtr KO were implicated in cardiac contraction, cell growth and energy metabolism. Our results suggest that silencing of Methionine synthase in the heart induces hypertension, hypertrophic cardiomyopathy with LV systolic dysfunction. The related mechanisms were dysregulation of energy metabolism and fibrosis. This data provides a novel insight on physiopathological mechanisms of cardiomyopathies of inherited disorders of cobalamin metabolism.

Guided evaluation and standardisation of mesenchymal stem cell culture conditions to generate conditioned medium favourable to cardiac c-kit cell growth.

Mesenchymal stem cells (MSCs) are known to secrete cardioprotective paracrine factors that can potentially activate endogenous cardiac c-kit cells (CCs). This study aims to optimise MSC growth conditions and medium formulation for generating the conditioned medium (CdM) to facilitate CC growth and expansion in vitro. The quality of MSC-CdM after optimisation of seeding density during MSC stabilisation and medium formulation used during MSC stimulation including glucose, ascorbic acid, serum and oxygen levels and the effects of treatment concentration and repeated CdM harvesting were assessed based on CC viability in vitro under growth factor- and serum-deprived condition. Our data showed that functional CdM can be produced from MSCs with a density of 20,000 cells/cm2, which were stimulated using high glucose (25mM), ascorbic acid supplemented, serum-free medium under normoxic condition. The generated CdM, when applied to growth factor- and serum-deprived medium at 1:1 ratio, improved CC viability, migration and proliferation in vitro. Such an effect could further be augmented by generating CdM concentrates without compromising CC gene and protein expressions, while retaining its capability to undergo differentiation to form endothelial, smooth muscle and cardiomyocytes. Nevertheless, CdM could not be repeatedly harvested from the same MSC culture, as the protein content and its effect on CC viability deteriorated after the first harvest. In conclusion, this study provides a proof-of-concept strategy to standardise the production of CdM from MSCs based on rapid, stepwise assessment of CC viability, thus enabling production of CdM favourable to CC growth for in vitro or clinical applications.

Influence of injectable microparticle size on cardiac progenitor cell response.

Injectable scaffolds are emerging as a promising strategy in the field of myocardial tissue engineering. Among injectable scaffolds, microparticles have been poorly investigated. The goal of this study was the development of novel gelatin/gellan microparticles that could be used as an injectable scaffold to repair the infarcted myocardium. In particular, the effect of particle size on cardiac progenitor cell response was investigated. Particles were produced by a water-in-oil emulsion method. Phosphatidylcholine was used as a surfactant. Particles with different diameter ranges (125-300 µm and 350-450 µm) were fabricated using two different surfactant concentrations. Morphological, physicochemical, and functional characterizations were carried out. Cardiac progenitor cell adhesion and growth on microparticles were tested both in static and dynamic suspension culture conditions. Morphological analysis of the produced particles showed a spherical shape and porous surface. The hydrophilicity of particle matrix and the presence of intermolecular interactions between gellan and gelatin were pointed out by the physicochemical characterization. A weight loss of 75 ± 5 % after 90 days of hydrolytic degradation was observed. Injectability through a narrow needle (26 G) and persistence of the microparticles at the injection site were preliminarily verified by ex vivo test. In vitro cell culture tests showed a preservation of rat cardiac progenitor biologic properties and indicated a preferential cell adherence to microparticles with a smaller size. Overall, the obtained results indicate that the produced gelatin/gellan microparticles could be potentially employed as injectable scaffolds for myocardial regeneration.

Attenuation of type-1 diabetes-induced cardiovascular dysfunctions by direct thrombin inhibitor in rats: a mechanistic study.

Chronic diabetes is associated with ventricular dysfunctions in the absence of hypertension and coronary artery diseases. This condition is termed as diabetic cardiomyopathy (DCM). There is no favourable treatment available for the management of diabetic cardiomyopathy. Recent studies have reported increase in circulating thrombin level among diabetic patients which is responsible for hypercoagulability of blood. Thrombin induces inflammation and fibrosis, and enhances cardiac cell growth and contractility in vitro. In this study, we have investigated the effects of argatroban; a direct thrombin inhibitor against DCM in streptozotocin-induced type-1 diabetes. Diabetes was induced by single dose of streptozotocin (STZ; 50mg/kg, i.p.) in male Sprague-Dawley rats. After 4 weeks of diabetes induction, the animals were treated with argatroban (0.3 and 1mg/kg, i.p. daily) for the next 4 weeks. The effect of argatroban was evaluated against diabetes-associated cardiac dysfunction, structural alteration and protein expression. STZ-induced diabetic rats exhibited significant decline in left ventricular functions. Four weeks of treatments with argatroban significantly improved ventricular functions without affecting heart rate. Further, it also protected heart against structural changes induced by diabetes as shown by reduction in fibrosis, hypertrophy and apoptosis. The improvement in cardiac functions and structural changes was associated with significant reduction in left ventricular expression of thrombin receptor also termed as protease-activated receptor-1 or PAR1, p-AKT (ser-473), p-50 NFκB and caspase-3 proteins. This study demonstrates beneficial effects of argatroban via improvement in cardiac functions and structural changes in STZ-induced DCM. These effects may be attributed through reduction in cardiac inflammation, fibrosis and apoptosis.

Abstract 107: RNA Seq Transcriptome Analysis Reveals Genes and Pathways Involved in the Cardiac Protection of VCP Against Pressure Overload

Aims: We previously showed that the valosin containing protein (VCP), a member of ATPases associated protein, protects the heart against pressure overload induced cardiac hypertrophy and dysfunction in transgenic (TG) mice. The mechanisms remain unknown. We hypothesize that VCP regulated alteration in transcriptome contributes to its cardio-protection by targeting cardiac cell growth and survival. Methods and results: Cardiac-specific VCPTG mice and their litter-matched wild type (WT) mice were subjected to transverse aortic constriction (TAC) for 2 and 5 weeks and compared to the sham mice. By using echocardiography and hemodynamic analysis, cardiac hypertrophy and dysfunction were confirmed in 2 weeks- and 5 weeks- TAC models respectively in WT but not in VCPTG mice. Total RNA was extracted from left ventricular tissues and gene expression was determined by RNA-Seq transcriptome analysis. Upon 2 weeks TAC, 690 differentially expressed genes were identified between VCPTG and WT (Fold Change ≥ 1.5, P value ≤ 0.05). Among these genes, VCPTG TAC mice, compared to WT TAC mice, showed significant activation of the genes linked to transcriptional factors, Protein Kinase CGMP-Dependent Type I ( Prkg1 ) and Kruppel Like factor 15 ( Klf15 ), the known repressors of cardiac hypertrophy under stress. On the contrary, there is significant increase in cardiac hypertrophy associated genes in WT TAC mice, such as myosin light chain 7 ( Myl7 ), periostin ( Postn ) and tropomyosin beta chain ( Tpm2) , and in fetal gene natriuretic peptide A ( Nppa) , but these alterations were not observed in VCPTG TAC mice, which may contribute to repression of cardiac hypertrophy in VCPTG mice. In addition, pro-apoptosis genes, such as platelet factor 4 ( Pf4), Pleckstrin homology like domain A1 ( Phlda1 ) and Radical S-Adenosyl Methionine Domain Containing 2 ( Rsad2 ), are significantly downregulated continually in 2 weeks through 5 weeks TAC in VCPTG mice, but not in WT TAC, which may contribute to the protection of cell survival in VCPTG mice. Conclusion: Significant difference of gene regulation exists between VCPTG and WT in the heart under the pressure overload which may be the mechanism of VCP mediated cardiac protection.

Role of angiotensin II in stem cell therapy of cardiac disease.

The renin angiotensin system (RAS) is closely related to the cardiovascular system, body fluid regulation and homeostasis. Despite common therapeutic methods, stem cell/progenitor cell therapy is daily increasing as a term of regenerative medicine. RAS and its pharmacological inhibitors are not only involved in physiological and pathological aspects of the cardiovascular system, but also affect the different stages of stem cell proliferation, differentiation and function, via interfering cell signaling pathways. This study reviews the new role of RAS, in particular Ang II distinct from other common roles, by considering its regulating impact on the different signaling pathways involved in the cardiac and endothelial tissue, as well as in stem cell transplantation. This review focuses on the impact of stem cell therapy on the cardiovascular system, the role of RAS in stem cell differentiation, and the role of RAS inhibition in cardiac stem cell growth and development.