On the interdependence of ketone body oxidation, glycogen content, glycolysis and energy metabolism in the heart.

In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-ß-hydroxybutyrate (ßHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on ßHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11 mM [5-3 H]glucose and/or 4 mM [14 C]ßHB to measure glycolytic rates or ßHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+ ]/[NADH] and mitochondrial [Q+ ]/[QH2 ] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi ) concentrations were measured using 31 P-nuclear magnetic resonance spectroscopy. Rates of ßHB oxidation in LG hearts were half that in HG hearts, with ßHB oxidation directly proportional to glycogen content. ßHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with ßHB alone was twice that of hearts perfused with ßHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+ ]/[NADH]. ßHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+ ]/[QH2 ], and increased the calculated free energy of ATP hydrolysis (∆GATP ). Although ßHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. ßHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP . We conclude that glycogen facilitates cardiac ßHB oxidation by anaplerosis. KEY POINTS: Ketone bodies (d-ß-hydroxybutyrate, acetoacetate) are increasingly recognised as important cardiac energetic substrates, in both healthy and diseased hearts. As 2-carbon equivalents they are cataplerotic, causing depletion of Krebs cycle intermediates; therefore their utilisation requires anaplerotic supplementation, and intra-myocardial glycogen has been suggested as a potential anaplerotic source during ketone oxidation. It is demonstrated here that cardiac glycogen does indeed provide anaplerotic substrate to facilitate ß-hydroxybutyrate oxidation in isolated perfused rat heart, and this contribution was quantified using a novel pulse-chase metabolic approach. Further, using metabolomics and 31 P-MR, it was shown that glycolytic flux from myocardial glycogen increased the heart's ability to oxidise ßHB, and ßHB oxidation increased the mitochondrial redox potential, ultimately increasing the free energy of ATP hydrolysis.

Modelling Diabetic Cardiomyopathy: Using Human Stem Cell-Derived Cardiomyocytes to Complement Animal Models.

Diabetes is a global epidemic, with cardiovascular disease being the leading cause of death in diabetic patients. There is a pressing need for an in vitro model to aid understanding of the mechanisms driving diabetic heart disease, and to provide an accurate, reliable tool for drug testing. Human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have potential as a disease modelling tool. There are several factors that drive molecular changes inside cardiomyocytes contributing to diabetic cardiomyopathy, including hyperglycaemia, lipotoxicity and hyperinsulinemia. Here we discuss these factors and how they can be seen in animal models and utilised in cell culture to mimic the diabetic heart. The use of human iPSC-CMs will allow for a greater understanding of disease pathogenesis and open up new avenues for drug testing.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart.

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.

Acute intermittent hypoxia drives hepatic de novo lipogenesis in humans and rodents.

Background and aims: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver condition. It is tightly associated with an adverse metabolic phenotype (including obesity and type 2 diabetes) as well as with obstructive sleep apnoea (OSA) of which intermittent hypoxia is a critical component. Hepatic de novo lipogenesis (DNL) is a significant contributor to hepatic lipid content and the pathogenesis of NAFLD and has been proposed as a key pathway to target in the development of pharmacotherapies to treat NAFLD. Our aim is to use experimental models to investigate the impact of hypoxia on hepatic lipid metabolism independent of obesity and metabolic disease. Methods: Human and rodent studies incorporating stable isotopes and hyperinsulinaemic euglycaemic clamp studies were performed to assess the regulation of DNL and broader metabolic phenotype by intermittent hypoxia. Cell-based studies, including pharmacological and genetic manipulation of hypoxia-inducible factors (HIF), were used to examine the underlying mechanisms. Results: Hepatic DNL increased in response to acute intermittent hypoxia in humans, without alteration in glucose production or disposal. These observations were endorsed in a prolonged model of intermittent hypoxia in rodents using stable isotopic assessment of lipid metabolism. Changes in DNL were paralleled by increases in hepatic gene expression of acetyl CoA carboxylase 1 and fatty acid synthase. In human hepatoma cell lines, hypoxia increased both DNL and fatty acid uptake through HIF-1α and -2α dependent mechanisms. Conclusions: These studies provide robust evidence linking intermittent hypoxia and the regulation of DNL in both acute and sustained in vivo models of intermittent hypoxia, providing an important mechanistic link between hypoxia and NAFLD.

Cardiac regeneration following myocardial infarction: the need for regeneration and a review of cardiac stromal cell populations used for transplantation.

Myocardial infarction is a leading cause of death globally due to the inability of the adult human heart to regenerate after injury. Cell therapy using cardiac-derived progenitor populations emerged about two decades ago with the aim of replacing cells lost after ischaemic injury. Despite early promise from rodent studies, administration of these populations has not translated to the clinic. We will discuss the need for cardiac regeneration and review the debate surrounding how cardiac progenitor populations exert a therapeutic effect following transplantation into the heart, including their ability to form de novo cardiomyocytes and the release of paracrine factors. We will also discuss limitations hindering the cell therapy field, which include the challenges of performing cell-based clinical trials and the low retention of administered cells, and how future research may overcome them.

Acidic environments trigger intracellular H+-sensing FAK proteins to re-balance sarcolemmal acid-base transporters and auto-regulate cardiomyocyte pH.

AIMS: In cardiomyocytes, acute disturbances to intracellular pH (pHi) are promptly corrected by a system of finely tuned sarcolemmal acid-base transporters. However, these fluxes become thermodynamically re-balanced in acidic environments, which inadvertently causes their set-point pHi to fall outside the physiological range. It is unclear whether an adaptive mechanism exists to correct this thermodynamic challenge, and return pHi to normal. METHODS AND RESULTS: Following left ventricle cryo-damage, a diffuse pattern of low extracellular pH (pHe) was detected by acid-sensing pHLIP. Despite this, pHi measured in the beating heart (13C NMR) was normal. Myocytes had adapted to their acidic environment by reducing Cl-/HCO3- exchange (CBE)-dependent acid-loading and increasing Na+/H+ exchange (NHE1)-dependent acid-extrusion, as measured by fluorescence (cSNARF1). The outcome of this adaptation on pHi is revealed as a cytoplasmic alkalinization when cells are superfused at physiological pHe. Conversely, mice given oral bicarbonate (to improve systemic buffering) had reduced myocardial NHE1 expression, consistent with a needs-dependent expression of pHi-regulatory transporters. The response to sustained acidity could be replicated in vitro using neonatal ventricular myocytes incubated at low pHe for 48 h. The adaptive increase in NHE1 and decrease in CBE activities was linked to Slc9a1 (NHE1) up-regulation and Slc4a2 (AE2) down-regulation. This response was triggered by intracellular H+ ions because it persisted in the absence of CO2/HCO3- and became ablated when acidic incubation media had lower chloride, a solution manoeuvre that reduces the extent of pHi-decrease. Pharmacological inhibition of FAK-family non-receptor kinases, previously characterized as pH-sensors, ablated this pHi autoregulation. In support of a pHi-sensing role, FAK protein Pyk2 (auto)phosphorylation was reduced within minutes of exposure to acidity, ahead of adaptive changes to pHi control. CONCLUSIONS: Cardiomyocytes fine-tune the expression of pHi-regulators so that pHi is at least 7.0. This autoregulatory feedback mechanism defines physiological pHi and protects it during pHe vulnerabilities.

Activation of HIF1α Rescues the Hypoxic Response and Reverses Metabolic Dysfunction in the Diabetic Heart.

Type 2 diabetes (T2D) impairs hypoxia-inducible factor (HIF)1α activation, a master transcription factor that drives cellular adaptation to hypoxia. Reduced activation of HIF1α contributes to the impaired post-ischemic remodeling observed following myocardial infarction in T2D. Molidustat is an HIF stabilizer currently undergoing clinical trials for the treatment of renal anemia associated with chronic kidney disease; however, it may provide a route to pharmacologically activate HIF1α in the T2D heart. In human cardiomyocytes, molidustat stabilized HIF1α and downstream HIF target genes, promoting anaerobic glucose metabolism. In hypoxia, insulin resistance blunted HIF1α activation and downstream signaling, but this was reversed by molidustat. In T2D rats, oral treatment with molidustat rescued the cardiac metabolic dysfunction caused by T2D, promoting glucose metabolism and mitochondrial function, while suppressing fatty acid oxidation and lipid accumulation. This resulted in beneficial effects on post-ischemic cardiac function, with the impaired contractile recovery in T2D heart reversed by molidustat treatment. In conclusion, pharmacological HIF1α stabilization can overcome the blunted hypoxic response induced by insulin resistance. In vivo this corrected the abnormal metabolic phenotype and impaired post-ischemic recovery of the diabetic heart. Therefore, molidustat may be an effective compound to further explore the clinical translatability of HIF1α activation in the diabetic heart.

Metabolic maturation of differentiating cardiosphere-derived cells.

Cardiosphere-derived cells (CDCs) can be expanded in vitro and induced to differentiate along the cardiac lineage. To recapitulate the phenotype of an adult cardiomyocyte, differentiating progenitors need to upregulate mitochondrial glucose and fatty acid oxidation. Here we cultured and differentiated CDCs using protocols aimed to maintain stemness or to promote differentiation, including triggering fatty acid oxidation using an agonist of peroxisome proliferator-activated receptor alpha (PPARα). Metabolic changes were characterised in undifferentiated CDCs and during differentiation towards a cardiac phenotype. CDCs from rat atria were expanded on fibronectin or collagen IV via cardiosphere formation. Differentiation was assessed using flow cytometry and qPCR and substrate metabolism was quantified using radiolabelled substrates. Collagen IV promoted proliferation of CDCs whereas fibronectin primed cells for differentiation towards a cardiac phenotype. In both populations, treatment with 5-Azacytidine induced a switch towards oxidative metabolism, as shown by changes in gene expression, decreased glycolytic flux and increased oxidation of glucose and palmitate. Addition of a PPARα agonist during differentiation increased both glucose and fatty acid oxidation and expression of cardiac genes. We conclude that oxidative metabolism and cell differentiation act in partnership with increases in one driving an increase in the other.

Mechanical and Degradation Properties of Hybrid Scaffolds for Tissue Engineered Heart Valve (TEHV).

In addition to biocompatibility, an ideal scaffold for the regeneration of valvular tissue should also replicate the natural heart valve extracellular matrix (ECM) in terms of biomechanical properties and structural stability. In our previous paper, we demonstrated the development of collagen type I and hyaluronic acid (HA)-based scaffolds with interlaced microstructure. Such hybrid scaffolds were found to be compatible with cardiosphere-derived cells (CDCs) to potentially regenerate the diseased aortic heart valve. This paper focused on the quantification of the effect of crosslinking density on the mechanical properties under dry and wet conditions as well as degradation resistance. Elastic moduli increased with increasing crosslinking densities, in the dry and wet state, for parent networks, whereas those of interlaced scaffolds were higher than either network alone. Compressive and storage moduli ranged from 35 ± 5 to 95 ± 5 kPa and 16 ± 2 kPa to 113 ± 6 kPa, respectively, in the dry state. Storage moduli, in the dry state, matched and exceeded those of human aortic valve leaflets (HAVL). Similarly, degradation resistance increased with increasing the crosslinking densities for collagen-only and HA-only scaffolds. Interlaced scaffolds showed partial degradation in the presence of either collagenase or hyaluronidase as compared to when exposed to both enzymes together. These results agree with our previous findings that interlaced scaffolds were composed of independent collagen and HA networks without crosslinking between them. Thus, collagen/HA interlaced scaffolds have the potential to fill in the niche for designing an ideal tissue engineered heart valve (TEHV).

Physiological and pharmacological stimulation for in vitro maturation of substrate metabolism in human induced pluripotent stem cell-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) enable human cardiac cells to be studied in vitro, although they use glucose as their primary metabolic substrate and do not recapitulate the properties of adult cardiomyocytes. Here, we have explored the interplay between maturation by stimulation of fatty acid oxidation and by culture in 3D. We have investigated substrate metabolism in hiPSC-CMs grown as a monolayer and in 3D, in porous collagen-derived scaffolds and in engineered heart tissue (EHT), by measuring rates of glycolysis and glucose and fatty acid oxidation (FAO), and changes in gene expression and mitochondrial oxygen consumption. FAO was stimulated by activation of peroxisome proliferator-activated receptor alpha (PPARα), using oleate and the agonist WY-14643, which induced an increase in FAO in monolayer hiPSC-CMs. hiPSC-CMs grown in 3D on collagen-derived scaffolds showed reduced glycolysis and increased FAO compared with monolayer cells. Activation of PPARα further increased FAO in cells on collagen/elastin scaffolds but not collagen or collagen/chondroitin-4-sulphate scaffolds. In EHT, FAO was significantly higher than in monolayer cells or those on static scaffolds and could be further increased by culture with oleate and WY-14643. In conclusion, a more mature metabolic phenotype can be induced by culture in 3D and FAO can be incremented by pharmacological stimulation.

Investigation of the Cellular Pharmacological Mechanism and Clinical Evidence of the Multi-Herbal Antiarrhythmic Chinese Medicine Xin Su Ning.

Xin Su Ning (XSN), a China patented and certified multi-herbal medicine, has been available in China since 2005 for treating cardiac ventricular arrhythmia including arrhythmia induced by ischemic heart diseases and viral myocarditis, without adverse reactions being reported. It is vitally important to discover pharmacologically how XSN as a multicomponent medicine exerts its clinical efficacy, and whether the therapeutic effect of XSN can be verified by standard clinical trial studies. In this paper we report our discoveries in a cellular electrophysiological study and in a three-armed, randomized, double-blind, placebo-controlled, parallel-group, multicenter trial. Conventional electrophysiological techniques were used to study the cellular antiarrhythmic mechanism of XSN. Data was then modeled with computational simulation of human action potential (AP) of the cardiac ventricular myocytes. The clinical trial was conducted with a total of 861 eligible participants randomly assigned in a ratio of 2:2:1 to receive XSN, mexiletine, or the placebo for 4 weeks. The primary and secondary endpoint was the change of premature ventricular contraction (PVC) counts and PVC-related symptoms, respectively. This trial was registered in the Chinese Clinical Trial Register Center (ChiCTR-TRC-14004180). We found that XSN prolonged AP duration of the ventricular myocytes in a dose-dependent, reversible manner and blocked potassium channels. Patients in XSN group exhibited significant total effective responses in the reduction of PVCs compared to those in the placebo group (65.85% vs. 27.27%, P < 0.0001). No severe adverse effects attributable to XSN were observed. In conclusion, XSN is an effective multicomponent antiarrhythmic medicine to treat PVC without adverse effect in patients, which is convincingly supported by its class I & III pharmacological antiarrhythmic mechanism of blocking hERG potassium channels and hNaV1.5 sodium channel reported in our earlier publication and prolongs AP duration both in ventricular myocytes and with computational simulation of human AP presented in this report.

Context-dependent regulation of endothelial cell metabolism: differential effects of the PPARß/δ agonist GW0742 and VEGF-A.

Peroxisome proliferator activated receptor ß/δ (PPARß/δ) has pro-angiogenic functions, but whether PPARß/δ modulates endothelial cell metabolism to support the dynamic phenotype remains to be established. This study characterised the metabolic response of HUVEC to the PPARß/δ agonist, GW0742, and compared these effects with those induced by VEGF-A. In HUVEC monolayers, flux analysis revealed that VEGF-A promoted glycolysis at the expense of fatty acid oxidation (FAO), whereas GW0742 reduced both glycolysis and FAO. Only VEGF-A stimulated HUVEC migration and proliferation whereas both GW0742 and VEGF-A promoted tubulogenesis. Studies using inhibitors of PPARß/δ or sirtuin-1 showed that the tubulogenic effect of GW0742, but not VEGF-A, was PPARß/δ- and sirtuin-1-dependent. HUVEC were reliant on glycolysis and FAO, and inhibition of either pathway disrupted cell growth and proliferation. VEGF-A was a potent inducer of glycolysis in tubulogenic HUVEC, while FAO was maintained. In contrast, GW0742-induced tubulogenesis was associated with enhanced FAO and a modest increase in glycolysis. These novel data reveal a context-dependent regulation of endothelial metabolism by GW0742, where metabolic activity is reduced in monolayers but enhanced during tubulogenesis. These findings expand our understanding of PPARß/δ in the endothelium and support the targeting of PPARß/δ in regulating EC behaviour and boosting tissue maintenance and repair.

Complexation of the nickel and cobalt transcriptional regulator RcnR with DNA.

RcnR is a transcription factor that regulates the homeostasis of cobalt and nickel in bacterial cells. Escherichia coli RcnR was crystallized with DNA that encompasses the DNA-binding site. X-ray diffraction data were collected to 2.9 Šresolution. The crystal belonged to space group P6122 or P6522, with unit-cell parameters a = b = 73.59, c = 157.66 Å, α = ß = 90, γ = 120°.

Trilayer scaffold with cardiosphere-derived cells for heart valve tissue engineering.

Natural polymers collagen, glycosaminoglycans, and elastin are promising candidate materials for heart valve tissue engineering scaffolds. This work produced trilayer scaffolds that resembled the layered structures of the extracellular matrices of native heart valves. The scaffolds showed anisotropic bending moduli (in both dry and hydrated statuses) depending on the loading directions (lower in the With Curvature direction than in the Against Curvature direction), which mimicked the characteristic behavior of the native heart valves. The interactions between cardiosphere-derived cells and the scaffolds were characterized by multiphoton microscopy, and relatively similar cell distributions were observed on different layers (a cell density of 3,000-4,000 mm-3 and a migration depth of 0.3-0.4 mm). The trilayer scaffold has represented a forwarding step from the previous studies, in attempting to better replicate a native heart valve structurally, mechanically, and biologically.

Correction: Fast, quantitative, murine cardiac 19F MRI/MRS of PFCE-labeled progenitor stem cells and macrophages at 9.4T.

[This corrects the article DOI: 10.1371/journal.pone.0190558.].

A Network Pharmacology Study of the Multi-Targeting Profile of an Antiarrhythmic Chinese Medicine Xin Su Ning.

Xin Su Ning (XSN) is a China patented and certified traditional Chinese herbal medicine used to treat premature ventricular contractions (PVCs) since 2005. XSN is formulated with 11 herbs, designed to treat arrhythmia with phlegm-heat heart-disturbed syndrome (PHHD) according to Chinese medicine theory. The rational compatibility of the 11 herbs decides the therapeutic outcome of XSN. Due to the multicomponent nature of traditional Chinese medicine, it is difficult to use conventional pharmacology to interpret the therapeutic mechanism of XSN in terms of clear-cut drug molecule and target interactions. Network pharmacology/systematic pharmacology usually consider all the components in a formula with the same weight; therefore, the proportion of the weight of the components has been ignored. In the present study, we introduced a novel coefficient to mimic the relative amount of all the components in relation with the weight of the corresponding herb in the formula. The coefficient is also used to weigh the pharmacological effect of XSN on all relative biological pathways. We also used the cellular electrophysiological data generated in our lab, such as the effect of liensinine and isoliquiritigenin on NaV1.5 channels; we therefore set sodium channel as one of the targets of these two components, which would support the clinical efficacy of XSN in treating tachyarrhythmia. Combining the collected data and our discovery, a panoramagram of the pharmacological mechanism of XSN was established. Pathway enrichment and analysis showed that XSN treated PHHD arrhythmia through multiple ion channels regulation, protecting the heart from I/R injury, inhibiting the apoptosis of cardiomyocyte, and improving glucose and lipid metabolism.

Intracellular iron deficiency in pulmonary arterial smooth muscle cells induces pulmonary arterial hypertension in mice.

Iron deficiency augments hypoxic pulmonary arterial pressure in healthy individuals and exacerbates pulmonary arterial hypertension (PAH) in patients, even without anemia. Conversely, iron supplementation has been shown to be beneficial in both settings. The mechanisms underlying the effects of iron availability are not known, due to lack of understanding of how cells of the pulmonary vasculature respond to changes in iron levels. The iron export protein ferroportin (FPN) and its antagonist peptide hepcidin control systemic iron levels by regulating release from the gut and spleen, the sites of absorption and recycling, respectively. We found FPN to be present in pulmonary arterial smooth muscle cells (PASMCs) and regulated by hepcidin cell autonomously. To interrogate the importance of this regulation, we generated mice with smooth muscle-specific knock in of the hepcidin-resistant isoform fpn C326Y. While retaining normal systemic iron levels, this model developed PAH and right heart failure as a consequence of intracellular iron deficiency and increased expression of the vasoconstrictor endothelin-1 (ET-1) within PASMCs. PAH was prevented and reversed by i.v. iron and by the ET receptor antagonist BQ-123. The regulation of ET-1 by iron was also demonstrated in healthy humans exposed to hypoxia and in PASMCs from PAH patients with mutations in bone morphogenetic protein receptor type II. Such mutations were further associated with dysregulation of the HAMP/FPN axis in PASMCs. This study presents evidence that intracellular iron deficiency specifically within PASMCs alters pulmonary vascular function. It offers a mechanistic underpinning for the known effects of iron availability in humans.

Metabolic flux analyses to assess the differentiation of adult cardiac progenitors after fatty acid supplementation.

Myocardial infarction is the most prevalent of cardiovascular diseases and pharmacological interventions do not lead to restoration of the lost cardiomyocytes. Despite extensive stem cell therapy studies, clinical trials using cardiac progenitor cells have shown moderate results. Furthermore, differentiation of endogenous progenitors to mature cardiomyocytes is rarely reported. A metabolic switch from glucose to fatty acid oxidation occurs during cardiac development and cardiomyocyte maturation, however in vitro differentiation protocols do not consider the lack of fatty acids in cell culture media. The aim of this study was to assess the effect of this metabolic switch on control and differentiated adult cardiac progenitors, by fatty acid supplementation. Addition of oleic acid stimulated the peroxisome proliferator-activated receptor alpha pathway and led to maturation of the cardiac progenitors, both before and after transforming growth factor-beta 1 differentiation. Addition of oleic acid following differentiation increased expression of myosin heavy chain 7 and connexin 43. Also, total glycolytic metabolism increased, as did mitochondrial membrane potential and glucose and fatty acid transporter expression. This work provides new insights into the importance of fatty acids, and of peroxisome proliferator-activated receptor alpha, in cardiac progenitor differentiation. Harnessing the oxidative metabolic switch induced maturation of differentiated endogenous stem cells. (200 words).

Collagen type I and hyaluronic acid based hybrid scaffolds for heart valve tissue engineering.

Tissue engineers have achieved limited success so far in designing an ideal scaffold for aortic valve; scaffolds lack in mechanical compatibility, appropriate degradation rate, and microstructural similarity. This paper, therefore, has demonstrated a carbodiimide-based sequential crosslinking technique to prepare aortic valve extracellular matrix mimicking (ECM) hybrid scaffolds from collagen type I and hyaluronic acid (HA), the building blocks of heart valve ECM, with tailorable crosslinking densities. Swelling studies revealed that crosslinking densities of parent networks increased with increasing the concentration of the crosslinking agents whereas crosslinking densities of hybrid scaffolds averaged from those of parent collagen and HA networks. Hybrid scaffolds also offered a wide range of pore size (66-126 µm) which fulfilled the criteria for valvular tissue regeneration. Scanning electron microscopy and images of Alcian blue-Periodic acid Schiff stained samples suggested that our crosslinking technique yielded an ECM mimicking microstructure with interlaced bands of collagen and HA in the hybrid scaffolds. The mutually reinforcing networks of collagen and HA also resulted in increased bending moduli up to 1660 kPa which spanned the range of natural aortic valves. Cardio sphere-derived cells (CDCs) from rat hearts showed that crosslinking density affected the available cell attachment sites on the surface of the scaffold. Increased bending moduli of CDCs seeded scaffolds up to two folds (2-6 kPa) as compared to the non-seeded scaffolds (1 kPa) suggested that an increase in crosslinking density of the scaffolds could not only increase the in vitro bending modulus but also prevented its disintegration in the cell culture medium.

ß-Adrenergic Receptor Stimulation and Alternans in the Border Zone of a Healed Infarct: An ex vivo Study and Computational Investigation of Arrhythmogenesis.

Background: Following myocardial infarction (MI), the myocardium is prone to calcium-driven alternans, which typically precedes ventricular tachycardia and fibrillation. MI is also associated with remodeling of the sympathetic innervation in the infarct border zone, although how this influences arrhythmogenesis is controversial. We hypothesize that the border zone is most vulnerable to alternans, that ß-adrenergic receptor stimulation can suppresses this, and investigate the consequences in terms of arrhythmogenic mechanisms. Methods and Results: Anterior MI was induced in Sprague-Dawley rats (n = 8) and allowed to heal over 2 months. This resulted in scar formation, significant (p < 0.05) dilation of the left ventricle, and reduction in ejection fraction compared to sham operated rats (n = 4) on 7 T cardiac magnetic resonance imaging. Dual voltage/calcium optical mapping of post-MI Langendorff perfused hearts (using RH-237 and Rhod2) demonstrated that the border zone was significantly more prone to alternans than the surrounding myocardium at longer cycle lengths, predisposing to spatially heterogeneous alternans. ß-Adrenergic receptor stimulation with norepinephrine (1 µmol/L) attenuated alternans by 60 [52-65]% [interquartile range] and this was reversed with metoprolol (10 µmol/L, p = 0.008). These results could be reproduced by computer modeling of the border zone based on our knowledge of ß-adrenergic receptor signaling pathways and their influence on intracellular calcium handling and ion channels. Simulations also demonstrated that ß-adrenergic receptor stimulation in this specific region reduced the formation of conduction block and the probability of premature ventricular activation propagation. Conclusion: While high levels of overall cardiac sympathetic drive are a negative prognostic indicator of mortality following MI and during heart failure, ß-adrenergic receptor stimulation in the infarct border zone reduced spatially heterogeneous alternans, and prevented conduction block and propagation of extrasystoles. This may help explain recent clinical imaging studies using meta-iodobenzylguanidine (MIBG) and 11C-meta-hydroxyephedrine positron emission tomography (PET) which demonstrate that border zone denervation is strongly associated with a high risk of future arrhythmia.