Morphological and Immunohistochemical Features of the Heart under the Effect of Hydrogen Sulfide Balneotherapy.

The effects of hydrogen sulfide baths and sulfide-silt mud applications on the abdominal wall were studied in outbred white rats. Heart preparations stained with hematoxylin and eosin were analyzed and the mean numbers of cells expressing Ki-67, Nkx-2.5, and mesenchymal stem cell markers were determined. Hydrogen sulfide balneotherapy promoted the development of blood capillaries in rat hearts and an increased the expression of CD34. A decrease in the regenerative potential of the myocardium was found due to a decrease in the content of proliferating cells and cells expressing CD73, CD90, and CD105, and also due to the absence of cells stained for cardiomyogenic differentiation marker Nkx-2.5.

Study of the cardioprotective effects of crocin on Human Cardiac Myocyte cells and reduction of oxidative stress produced by aluminum phosphide poisoning.

OBJECTIVES: The effects of Crocin as a cardioprotective material against Aluminum phosphide poisoning by reducing the oxidative stress is investigated. METHODS: The level of biomarkers of oxidative stress (Catalase, Superoxide dismutase, Malondialdehyde and Protein carbonyl) were measured in the cell culture model on Human Cardiac Myocyte cells to detect the protective effect of crocin. Initially, to define the pure impact of aluminum phosphide poison and crocin on the heart cells, their effects on the biomarkers quantity in cell line were measured, separately, using the standard related kits. Later the effect of crocin with different concentration as a treatment on the oxidative stress biomarkers of the poisoned heart cells were monitored. Note that in pre-treatment case, the crocin was initially added to the cells before poisoning them. Data were analyzed using the analysis of variance method. KEY FINDINGS: Results showed that crocin treatment reduced the aluminum phosphide (AlP) poisoning effect significantly. The treatment resulted in substantial deviation in the biomarkers of oxidative stress at the pre- and post-treatment phases for all groups. The oxidative markers values of the poisoned cells were recovered by crocin treatment. CONCLUSIONS: Crocin is proposed as a potentially powerful antioxidant to treat the cardiotoxicity caused by aluminum phosphide poisoning.

In silico design and pharmacological evaluation of conjugates of atenolol with modified saccharide for cardiovascular targeting.

Amongst a wide range of biological macromolecules, saccharides exhibit the potential to be specifically recognized by cell-surface receptors and hence can be utilized as ligands in targeted drug delivery. The current study aims to use saccharides viz. Galactose, Pectin and Chitosan to improve targeting of Atenolol by oxalyl chloride mediated grafting. Conjugates were engineered by grafting Atenolol, a cardiovascular agent with the modified saccharide units. The conjugates were characterized by FTIR, DSC and 1H NMR study. Drug release analysis and cellular uptake study was carried out using H9c2 cell lines which represent that concentration of drug in cells treated with all atenolol-saccharide conjugates is enhanced by almost two-folds in comparison with cells treated with atenolol solution. Thus cell line study confers the evidence of selective cardiac delivery. No significant cytotoxicity was observed in case of all synthesized conjugates in the Brine shrimp lethality bioassay. Possible binding of the developed conjugates with the GLUT-4 receptors was assessed by in silico analysis using homology model developed by Swiss Model server. Hence it was concluded that the application of these conjugates with saccharides in selective cardiovascular drug delivery can be a promising approach to increase bioavailability, minimize drug loss by degradation and prevent harmful side effects by increasing specific cell targeting.

Apigenin suppresses TGF-ß1-induced cardiac fibroblast differentiation and collagen synthesis through the downregulation of HIF-1α expression by miR-122-5p.

BACKGROUND: Apigenin can reduce cardiomyocyte hypertrophy by downregulating hypoxia inducible factor-1 alpha (HIF-1α) expression. However, its effects on cardiac fibroblasts (CFs) and its exact inhibitory molecular mechanisms on HIF-1α remain unclear. PURPOSE: This study aims to examine the effects of apigenin on cell proliferation and differentiation, microRNA-122-5p (miR-122-5p) expression, and HIF-1α-mediated Smad signaling pathway in transforming growth factor beta 1 (TGF-ß1)-stimulated CFs and cardiac fibrosis and to investigate the relationship between miR-122-5p and HIF-1α. METHODS: The TGF-ß1-stimulated CFs, the combination of TGF-ß1-stimulated and miR-122-5p mimic-transfected CFs, the combination of TGF-ß1-stimulated and miR-122-5p inhibitor-transfected CFs, and the isoproterenol-induced cardiac fibrotic mice were used and treated with or without apigenin. The recombinant lentiviruses overexpressing HIF-1α vector and miR-122-5p mimic were co-transfected to observe their interaction. Related mRNA and protein expressions and myocardial collagen were determined. The luciferase reporter gene that contains HIF-1α wild type or mutant type 3'-UTR was used, and the luciferase activity was determined to verify the direct link between miR-122-5p and HIF-1α. RESULTS: In the TGF-ß1-stimulated CFs, apigenin treatment increased the miR-122-5p and Smad7 expressions and decreased the HIF-1α, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 expressions. Similar and inverse results were observed in the miR-122-5p mimic- and inhibitor-transfected CFs, respectively. Moreover, the miR-122-5p mimic could antagonize the effects of TGF-ß1 in the TGF-ß1 and miR-122-5p mimic-combined CFs, and the miR-122-5p inhibitor could enhance the effects of TGF-ß1 in the TGF-ß1 and miR-122-5p inhibitor-combined CFs. In the two aforementioned cell models, the addition of apigenin could further enhance the effects of miR-122-5p mimic and partially reverse the effects of miR-122-5p inhibitor. After treatment of HIF-1α-transfected CFs with miR-122-5p mimic, the HIF-1α expression decreased. Further study confirmed that HIF-1α was a direct target of miR-122-5p. Apigenin also decreased the myocardial collagen accumulation in cardiac fibrotic mice. CONCLUSION: Apigenin could suppress the differentiation and collagen synthesis of TGF-ß1-stimulated CFs and mouse cardiac fibrosis, and its mechanisms were related to the increment of miR-122-5p expression and subsequent downregulation of HIF-1α expression via direct interaction, which might finally result in the decrements of Smad2/3 and p-Smad2/3 expressions and increment of Smad7 expression.

Pathophysiological Basis for Nutraceutical Supplementation in Heart Failure: A Comprehensive Review.

There is evidence demonstrating that heart failure (HF) occurs in 1-2% of the global population and is often accompanied by comorbidities which contribute to increasing the prevalence of the disease, the rate of hospitalization and the mortality. Although recent advances in both pharmacological and non-pharmacological approaches have led to a significant improvement in clinical outcomes in patients affected by HF, residual unmet needs remain, mostly related to the occurrence of poorly defined strategies in the early stages of myocardial dysfunction. Nutritional support in patients developing HF and nutraceutical supplementation have recently been shown to possibly contribute to protection of the failing myocardium, although their place in the treatment of HF requires further assessment, in order to find better therapeutic solutions. In this context, the Optimal Nutraceutical Supplementation in Heart Failure (ONUS-HF) working group aimed to assess the optimal nutraceutical approach to HF in the early phases of the disease, in order to counteract selected pathways that are imbalanced in the failing myocardium. In particular, we reviewed several of the most relevant pathophysiological and molecular changes occurring during the early stages of myocardial dysfunction. These include mitochondrial and sarcoplasmic reticulum stress, insufficient nitric oxide (NO) release, impaired cardiac stem cell mobilization and an imbalanced regulation of metalloproteinases. Moreover, we reviewed the potential of the nutraceutical supplementation of several natural products, such as coenzyme Q10 (CoQ10), a grape seed extract, Olea Europea L.-related antioxidants, a sodium-glucose cotransporter (SGLT2) inhibitor-rich apple extract and a bergamot polyphenolic fraction, in addition to their support in cardiomyocyte protection, in HF. Such an approach should contribute to optimising the use of nutraceuticals in HF, and the effect needs to be confirmed by means of more targeted clinical trials exploring the efficacy and safety of these compounds.

Apigenin attenuates TGF-ß1-stimulated cardiac fibroblast differentiation and extracellular matrix production by targeting miR-155-5p/c-Ski/Smad pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin is a natural flavonoid compound present in chamomile (Matricaia chamomilla L.) from the Asteraceae family, which is used in the treatment of cardiovascular diseases by traditional healers, but its effects on differentiation and extracellular matrix (ECM) production of cardiac fibroblasts (CFs) induced by transforming growth factor beta 1 (TGF-ß1) are poorly understood. AIM OF THE STUDY: This study aimed to examine these effects and potential molecular mechanisms and to provide a new application of apigenin in the prevention and treatment of cardiac fibrosis. MATERIALS AND METHODS: The TGF-ß1-stimulated CFs or the combination of TGF-ß1-stimulated and microRNA-155-5p (miR-155-5p) inhibitor- or mimic-transfected CFs were treated with or without apigenin. The expression levels of intracellular related mRNA and proteins were detected by real-time polymerase chain reaction and Western blot methods, respectively. The luciferase reporter gene containing cellular Sloan-Kettering Institute (c-Ski) wild or mutant type 3'-UTR was used and the luciferase activity was examined to verify the direct link of miR-155-5p and c-Ski. RESULTS: After treatment of TGF-ß1-stimulated CFs with 6-24 µM apigenin, the expression of c-Ski was increased, while levels of miR-155-5p, α-smooth muscle actin, collagen Ⅰ/Ⅲ, Smad2/3, and p-Smad2/3 were decreased. After transfection of CFs with the miR-155-5p inhibitor or mimic, the similar or inverse results were respectively observed as well. The combination of TGF-ß1 and miR-155-5p inhibitor or mimic might cause an antagonistical or synergistic effect, respectively, and apigenin addition could enhance the effects of the inhibitor and antagonize the effects of the mimic. Luciferase reporter gene assay demonstrated that c-Ski was a direct target of miR-155-5p. CONCLUSION: These findings suggested that apigenin could inhibit the differentiation and ECM production in TGF-ß1-stimulated CFs, and its mechanisms might partly be attributable to the reduction of miR-155-5p expression and subsequent increment of c-Ski expression, which might result in the inhibition of Smad2/3 and p-Smad2/3 expressions.

Metabolic environment in vivo as a blueprint for differentiation and maturation of human stem cell-derived cardiomyocytes.

Patient-derived human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are increasingly being used for disease modeling, drug screening and regenerative medicine. However, to date, an immature, fetal-like, phenotype of hPSC-CMs restrains their full potential. Increasing evidence suggests that the metabolic state, particularly important for provision of sufficient energy in highly active contractile CMs and anabolic and regulatory processes, plays an important role in CM maturation, which affects crucial functional aspects of CMs, such as contractility and electrophysiology. During embryonic development the heart is subjected to metabolite concentrations that differ substantially from that of hPSC-derived cardiac cell cultures. A deeper understanding of the environmental and metabolic cues during embryonic heart development and how these change postnatally, will provide a framework for optimizing cell culture conditions and maturation of hPSC-CMs. Maturation of hPSC-CMs will improve the predictability of disease modeling, drug screening and drug safety assessment and broadens their applicability for personalized and regenerative medicine.

Screening ginseng saponins in progenitor cells identifies 20(R)-ginsenoside Rh2 as an enhancer of skeletal and cardiac muscle regeneration.

Aging is associated with increased prevalence of skeletal and cardiac muscle disorders, such as sarcopenia and cardiac infarction. In this study, we constructed a compendium of purified ginsenoside compounds from Panax ginseng C.A. Meyer, which is a traditional Korean medicinal plant used to treat for muscle weakness. Skeletal muscle progenitor cell-based screening identified three compounds that enhance cell viability, of which 20(R)-ginsenoside Rh2 showed the most robust response. 20(R)-ginsenoside Rh2 increased viability in myoblasts and cardiomyocytes, but not fibroblasts or disease-related cells. The cellular mechanism was identified as downregulation of cyclin-dependent kinase inhibitor 1B (p27Kip1) via upregulation of Akt1/PKB phosphorylation at serine 473, with the orientation of the 20 carbon epimer being crucially important for biological activity. In zebrafish and mammalian models, 20(R)-ginsenoside Rh2 enhanced muscle cell proliferation and accelerated recovery from degeneration. Thus, we have identified 20(R)-ginsenoside Rh2 as a p27Kip1 inhibitor that may be developed as a natural therapeutic for muscle degeneration.

Cotreatments with Dex and Na2SeO3 further improved antioxidant and anti-inflammatory protection of myocardial cells from I/R injury compared to their individual treatments.

Dexmedetomidine (Dex), a sedative and analgesic agent, is known to have a cardioprotective effect against ischaemia/reperfusion (I/R) injury via regulation of antioxidant and anti-inflammatory signals. In contrast, Se shows a cardioprotective effect against I/R injury, because it is a key component of selenoproteins, most of which are antioxidant enzymes such as GPxs and TrxRs. This study aimed to determine whether the protective effects on myocardial cells against I/R injury were further improved when treatment with Dex and Se in combination. H9C2 cells were treated with Dex and Na2SeO3, alone or in combination, before oxygen glucose deprivation/reoxygenation (OGD/R). OGD/R-induced myocardial cell injury was evaluated using cell viability, apoptosis rate, the release of LDH, and intracellular ROS levels. Both Dex and Na2SeO3 improved cell viability and reduced the apoptosis rate, LDH release, and intracellular ROS. This cytoprotection was higher with Dex and Na2SeO3 cotreatment than their individual treatments. Treatment with Dex increased the SOD1, SOD2, GPx1, and GPx2 expression in H9C2 cells in OGD/R, while Na2SeO3 increased the GPx1-4 and TrxR1-3 mRNA levels. Notably, cotreatment with Dex and Na2SeO3 increased the mRNA expression of all these antioxidant enzymes. Dex treatment attenuated the activation of JNK, p65 (NF-κB), Camk1, and NLRP3 signals. Na2SeO3 enhanced the inhibitory effect of Dex on phosphorylated (p)-p65, p65, and NLRP3 in OGD/R. However, TrxR1 knockdown attenuated the positive effect of Na2SeO3 on Dex-mediated anti-inflammatory effects. In summary, cotreatments with Dex and Na2SeO3 further improved antioxidant and anti-inflammatory protection of myocardial cells from I/R injury compared to their individual treatments.

Dissection of mechanisms of Chinese medicinal formula Si-Miao-Yong-an decoction protects against cardiac hypertrophy and fibrosis in isoprenaline-induced heart failure.

ETHNOPHARMACOLOGICAL RELEVANCE: Si-Miao-Yong-An decoction (SMYAD) is a traditional Chinese herbal formulation. SMYAD first appeared in the Eastern Han Dynasty according to the "Shen Yi Mi Zhuan". Then the formula was recorded in the "Yan Fang Xin Bian" edited by medical scientist Bao Xiangao in the Qing Dynasty. This well-known prescription has been traditionally used for gangrene and vascular vasculitis. It is mainly used for cardiovascular and endocrine diseases in current clinical applications and research. AIM OF STUDY: In this study, the potential mechanisms of SMYAD against cardiac fibrosis and hypertrophy in the ß-adrenoceptor agonist isoprenaline induced heart failure model were investigated. MATERIALS AND METHODS: The heart failure animal model was established via injected isoprenaline in rats. Echocardiography was used to detect the structure and function of the heart. HE staining and Masson's trichrome staining was performed to assess myocardial tissue morphology. The serum biochemical indexes were detected by dedicated biochemical kit. BNP was tested by ELISA kit. The levels of mRNA were detected by RT-qPCR. Cardiomyocyte morphology was assessed by immunofluorescence. Phosphorylated and total p38, Akt were analyzed by Western blot. The production of reactive oxygen species (ROS) was tested by CM-H2DCFDA probe. Formula identification of chemical constituents of SMYAD in plasma was disclosed through ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS). RESULTS: SMYAD was able to improve the heart function in ISO induced heart failure rat model via protecting rat from developing cardiac hypertrophy and fibrosis. SMYAD also decreased plasma expression of these biochemical indexes. It was found that SMYAD could regulate cardiac hypertrophy and fibrosis makers' mRNA levels in vitro and vivo. In addition, SMYAD inhibited the phosphorylation of p38 and Akt, which are key mediators in the pathological process of ISO-induced cardiac hypertrophy and myocardial fibrosis. It also showed that the components of SMYAD in rat plasma exerted myocardial cell protective activity. CONCLUSION: In summary, SMYAD may comprise more than one active ingredient to the pursuit of combination therapies instead of specifically target a single disease-causing molecule. These experimental results suggest that SMYAD may be a potential drug candidate in diseases of cardiac hypertrophy and myocardial fibrosis caused by ß-adrenoceptor abnormalities.

Astragaloside IV inhibits cardiac fibrosis via miR-135a-TRPM7-TGF-ß/Smads pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Cardiac fibrosis is a common characteristic of many cardiac diseases. Our previous results showed that TRPM7 channel played an important role in the fibrosis process. MicroRNA-135a was reported to get involved in the fibrotic process. Astragalus membranaceus (Fisch.) Bunge was widely used in Chinese traditional medicine and showed cardiac protective effects in previous researches. Astragaloside IV(ASG), which is regarded as the most important ingredient of Astragalus, has been showed the effect of cardiac protection via various mechanisms, while no data suggested its action related to miRNAs regulation. AIM OF THE STUDY: The objective of this article is to investigate the inhibition effect of ASG on cardiac fibrosis through the miR-135a-TRPM7-TGF-ß/Smads pathway. MATERIALS AND METHODS: We extracted the active components from herb according to the paper and measured the content of ASG from the mixture via HPLC. The inhibition potency of cardiac hypertrophy between total extract of Astragalus and ASG was compared. SD rats were treated with ISO (5 mg/kg/day) subcutaneously (s.c.) for 14 days, ASG (10 mg/kg/d) and Astragalus extract (AE) (4.35 g/kg/d, which contained about ASG 10 mg) were given p.o. from the 6th day of the modeling. Cardiac fibroblasts (CFs) of neonatal rats were incubated with ISO (10 µM) and treated with ASG (10 µM) simultaneously for 24 h. RESULTS: The results showed that both AE and ASG treatment reduced the TRPM7 expression from the gene level and inhibited cardiac fibrosis. ASG group showed similar potency as the AE mixture. ASG treatment significantly decreased the current, mRNA and protein expression of TRPM7 which was one of targets of miR-135a. The activation of TGF-ß/Smads pathway was suppressed and the expression of α-SMA and Collagen I were also decreased obviously. In addition, our results showed that there was a positive feedback between the activation of TGF-ß/Smads pathway and the elevation of TRPM7, both of which could promote the development of myocardial fibrosis. CONCLUSIONS: AE had the effect of cardiac fibrosis inhibition and decreased the mRNA expression of TRPM7. ASG, as one of the effective ingredients of AE, showed the same potency when given the same dose. ASG inhibited cardiac fibrosis by targeting the miR-135a-TRPM7-TGF-ß/Smads pathway.

Review: Contemporary biological therapies for cardiovascular diseases.

Cardiovascular diseases are top cause of mortality in the world. Current interventional therapy and pharmacotherapy may alleviate symptoms or slow disease progression but are unable to cure or treat them. Molecular and pathophysiological advances have paved the way for contemporary biological therapies to be tested and standardized for the treatment of these diseases. Stem cells therapy and gene therapy has shown promise in the treatment of CVDs. Various types of stem cells used in cardiac conditions like myocardial infarction with the aim of regenerating the damaged myocardium have had variable success rates in clinical and preclinical trials. Improvements in methods and routes of cell delivery have improved clinical outcomes. Gene therapy employs therapeutic genes to treat diseases. Advances in vectors have improved transfection efficiencies and transgene expression and enhanced role in Heart failure, ischemic disease as well as arrhythmias. Clinical trials have shown improved cardiac function upon treatment with genes which promote angiogenesis. The current review looks at the role of these biological therapies in cardiovascular diseases.

Spatiotemporal Characterizations of Spontaneously Beating Cardiomyocytes with Adaptive Reference Digital Image Correlation.

We developed an Adaptive Reference-Digital Image Correlation (AR-DIC) method that enables unbiased and accurate mechanics measurements of moving biological tissue samples. We applied the AR-DIC analysis to a spontaneously beating cardiomyocyte (CM) tissue, and could provide correct quantifications of tissue displacement and strain for the beating CMs utilizing physiologically-relevant, sarcomere displacement length-based contraction criteria. The data were further synthesized into novel spatiotemporal parameters of CM contraction to account for the CM beating homogeneity, synchronicity, and propagation as holistic measures of functional myocardial tissue development. Our AR-DIC analyses may thus provide advanced non-invasive characterization tools for assessing the development of spontaneously contracting CMs, suggesting an applicability in myocardial regenerative medicine.

Zn(ii)-Curcumin supplementation alleviates gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity in rats.

Doxorubicin is a powerful anticancer agent used to treat a variety of human neoplasms. However, the clinical use of doxorubicin is hampered by cardiotoxicity and effective cardioprotective adjuvants do not exist. Dietary zinc, an essential nutrient, is required to maintain steady-state tissue zinc levels and intestinal homeostasis and may yield therapeutic benefits in diseases associated with zinc dysregulation or gut dysbiosis. Here, we investigated the effects of dietary Zn(ii)-curcumin (ZnCM) solid dispersions on gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity in rats. Rats were injected with multiple low doses of doxorubicin and orally administered ZnCM daily over four weeks. Daily administration of ZnCM not only alleviated Dox-induced gut dysbiosis-as indicated by the increased Firmicutes-to-Bacteroidetes ratio and the maintenance of the relative abundances of major beneficial bacteria including Clostridium_XIVa, Clostridium_IV, Roseburia, Butyricicoccus and Akkermansia-but also maintained intestinal barrier integrity and decreased the lipopolysaccharide (LPS) contents of feces and plasma. ZnCM also significantly attenuated doxorubicin-induced zinc dyshomeostasis, which was mirrored by preservation of zinc levels and expression of zinc-related transporters. Furthermore, ZnCM significantly improved heart function and reduced cardiomyocyte apoptosis and myocardial injury in doxorubicin-treated rats. Notably, the regulation of zinc homeostasis and cardioprotective and microbiota-modulating effects of ZnCM were transmissible through horizontal feces transfer from ZnCM-treated rats to normal rats. Thus, ZnCM supplementation has potential as an effective therapeutic strategy to alleviate gut dysbiosis and zinc dyshomeostasis during doxorubicin-induced cardiotoxicity.

Rare ginsenosides ameliorate lipid overload-induced myocardial insulin resistance via modulating metabolic flexibility.

BACKGROUND: Rare ginsenosides are found in ginseng and notoginseng, two medicinal plants widely used in China for treatment of cardiovascular diseases and type 2 diabetes. However, their pharmacological studies regarding myocardial fuel metabolism and insulin signaling are not clear. PURPOSE: To explore the effect of a rare ginsenoside-standardized extract (RGSE), derived from steamed notoginseng, on cardiac fuel metabolism and insulin signaling. STUDY DESIGN: We used palmitic acid (PA) to treat H9c2 cells in vitro and high fat diet (HFD) to mice to induce insulin resistance in vivo. METHODS: In vitro, differentiated H9c2 cells were pretreated with RGSE, metformin, mildronate or dichloroacetate (DCA) and stimulated with PA. In vivo, mice were fed with HFD and received RGSE, metformin or DCA for 6 weeks. Protein expression was determined by Western blotting. Mitochondrial membrane potential (Δψm), glucose uptake and reactive oxygen species (ROS) production were measured by fluorescence labeling. Other assessments including oxygen consumption rate (OCR) were also performed. RESULTS: RGSE prevented PA-induced decrease in pyruvate dehydrogenase (PDH) activity and increase in carnitine palmitoyltransferase 1 (CPT1) expression, and ameliorated insulin-mediated glucose uptake and utilization in H9c2 cells. Metformin and mildronate exhibited similar effects. In vivo, RGSE counteracted HFD-induced increase in myocardial expression of p-PDH and CPT1 and ameliorated cardiac insulin signaling. Metformin and DCA also showed beneficial effects. Further study showed that RGSE decreased OCR and mitochondrial complex I activity in PA-treated H9c2 cells, reduced ROS production and relieved mitochondrial oxidative stress, thus decreased serine phosphorylation in IRS-1. CONCLUSION: RGSE ameliorated myocardial insulin sensitivity under conditions of lipid overload, which was tightly associated with the decrease in mitochondrial oxidative stress via modulating glucose and fatty acid oxidation.

Drug Screening in Human PSC-Cardiac Organoids Identifies Pro-proliferative Compounds Acting via the Mevalonate Pathway.

We have previously developed a high-throughput bioengineered human cardiac organoid (hCO) platform, which provides functional contractile tissue with biological properties similar to native heart tissue, including mature, cell-cycle-arrested cardiomyocytes. In this study, we perform functional screening of 105 small molecules with pro-regenerative potential. Our findings reveal surprising discordance between our hCO system and traditional 2D assays. In addition, functional analyses uncovered detrimental effects of many hit compounds. Two pro-proliferative small molecules without detrimental impacts on cardiac function were identified. High-throughput proteomics in hCO revealed synergistic activation of the mevalonate pathway and a cell-cycle network by the pro-proliferative compounds. Cell-cycle reentry in hCO and in vivo required the mevalonate pathway as inhibition of the mevalonate pathway with a statin attenuated pro-proliferative effects. This study highlights the utility of human cardiac organoids for pro-regenerative drug development, including identification of underlying biological mechanisms and minimization of adverse side effects.

Iron-deficiency anemia reduces cardiac contraction by downregulating RyR2 channels and suppressing SERCA pump activity.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation.

Endothelial cells of different organs exhibit heterogeneity in von Willebrand factor expression in response to hypoxia.

BACKGROUND AND AIMS: We have previously demonstrated that in response to hypoxia, von Willebrand factor (VWF) expression is upregulated in lung and heart endothelial cells both in vitro and in vivo, but not in kidney endothelial cells. The aim of our current study was to determine whether endothelial cells of different organs employ distinct molecular mechanisms to mediate VWF response to hypoxia. METHODS: We used cultured human primary lung, heart and kidney endothelial cells to determine the activation of endogenous VWF as well as exogenously expressed VWF promoter in response to hypoxia. Chromatin immunoprecipitation and siRNA knockdown analyses were used to determine the roles of VWF promoter associated transacting factors in mediating its hypoxia response. Platelet aggregates formations in vascular beds of mice were used as a marker for potential functional consequences of hypoxia-induced VWF upregulation in vivo. RESULTS: Our analyses demonstrated that while Yin Yang 1 (YY1) and specificity protein 1 (Sp1) participate in the hypoxia-induced upregulation of VWF specifically in lung endothelial cells, GATA6 mediates this process specifically in heart endothelial cells. In both cell types, the response to hypoxia involves the decreased association of the NFIB repressor with the VWF promoter, and the increased acetylation of the promoter-associated histone H4. In mice exposed to hypoxia, the upregulation of VWF expression was concomitant with the presence of thrombi in heart and lung, but not kidney vascular beds. CONCLUSIONS: Heart and lung endothelial cells demonstrated VWF upregulation in response to hypoxia, using distinct mechanisms, while this response was lacking in kidney endothelial cells.

Cardiotoxicity of Consolida rugulosa, a poisonous weed in Western China.

Poisonous weeds are a global problem since they not only hinder local economic development, but also cause ecological harm. Consolida rugulosa (family Ranunculaceae) is a weed that is widespread in Northwestern China and causes severe poisoning when ingested by livestock. In the present study, we purified the toxins in this plant and investigated their mechanism of action. Five natural diterpene alkaloids (compounds 1-5)-including two new compounds (1 and 2)-were isolated, and five semi-synthetic derivatives (6-10) were synthesised based on 4 or 5 for structure-activity analysis. The toxicity of the compounds was evaluated in vitro with lactate dehydrogenase (LDH) assay. All of the compounds-especially 1-stimulated LDH release in primary cultured rat myocardial cells, an effect that was blocked by the Na+ channel blocker lidocaine. Electrocardiography revealed that rats treated with 1 had severe arrhythmia, while heart Doppler echocardiography and analysis of serum biomarkers levels revealed that administration of 1 for 15 days induced changes in cardiac structure and myocardial enzyme levels. These effects were antagonised by lidocaine treatment. Thus, diterpene alkaloids are the main compounds responsible for the cardiotoxicity of C. rugulosa, which can be mitigated by co-administration of lidocaine.

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 (25 mM), 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.