Regulation of ß-Adrenergic Receptors in the Heart: A Review on Emerging Therapeutic Strategies for Heart Failure.

The prolonged overstimulation of ß-adrenergic receptors (ß-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with ß-AR antagonists (ß-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of ß-ARs involved in the pathogenesis of HF. As ß-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), ß-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of ß-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for ß-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of ß-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of ß-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF.

Antihyperglycemic activity of a novel polyherbal formula (HF344), a mixture of fifteen herb extracts, for the management of type 2 diabetes: Evidence from in vitro, ex vivo, and in vivo studies.

Antihyperglycemic effects of a novel polyherbal formula (HF344), comprising fifteen Thai herbal extracts, were elucidated for pharmacological mechanisms and potential for managing type 2 diabetes mellitus, by employing in vitro, ex vivo, and in vivo approaches. LC/MS analysis of HF344 extract revealed several phytoconstituents, with piperine identified as the major active compound. HF344 extract significantly enhanced insulin secretion in RINm5F cells in vitro and inhibited glucose uptake into the everted sacs of the mouse small intestine ex vivo in a concentration-dependent manner compared to the control (p < 0.05). It exhibited potent α-glucosidase inhibition in vitro, with an IC50 of 96.74 µg/mL. Moreover, HF344 extract upregulated mRNA levels of GLUT1 in L6 skeletal myoblasts, suggesting increased glucose uptake into skeletal muscle. In addition, in vivo antihyperglycemic effects were assessed in streptozotocin (STZ)-nicotinamide (NA)-induced diabetic mice. Acute oral toxicity testing confirmed the HF344 extract's safety, with an LD50 exceeding 2000 mg/kg. Oral administration of HF344 extract (500 and 1000 mg/kg) in STZ-NA-induced diabetic mice significantly reduced the area under the fasting blood glucose (FBG)-time curve (AUC) in the oral glucose tolerance test (OGTT) model and treatment for 28-day reduced the FBG levels as compared with control (p < 0.05). This was accompanied by increased serum insulin levels and improved insulin resistance. HF344 extract also demonstrated a concentration-dependent inhibitory effect on malondialdehyde (MDA) production in vitro, with an IC50 of 7.24 µg/mL. Oral treatment with HF344 extract decreased MDA production in the homogenized muscle ex vivo collected from STZ-NA-induced mice. Furthermore, pretreatment with HF344 extract effectively restored the survival of RINm5F cells from STZ-induced damage. These findings suggest that HF344 is a promising polyherbal formula for managing blood glucose levels, enhancing insulin production, and providing antioxidant benefits in T2DM. Further research is required to evaluate the clinical efficacy and safety profiles of HF344.

Harnessing Cannabis sativa Oil for Enhanced Skin Wound Healing: The Role of Reactive Oxygen Species Regulation.

Cannabis sativa emerges as a noteworthy candidate for its medicinal potential, particularly in wound healing. This review article explores the efficacy of cannabis oil in reducing reactive oxygen species (ROS) during the healing of acute and chronic wounds, comparing it to the standard treatments. ROS, produced from various internal and external sources, play a crucial role in wound development by causing cell and tissue damage. Understanding the role of ROS on skin wounds is essential, as they act both as signaling molecules and contributors to oxidative damage. Cannabis oil, recognized for its antioxidant properties, may help mitigate oxidative damage by scavenging ROS and upregulating antioxidative mechanisms, potentially enhancing wound healing. This review emphasizes ongoing research and the future potential of cannabis oil in dermatological treatments, highlighted through clinical studies and patent updates. Despite its promising benefits, optimizing cannabis oil formulations for therapeutic applications remains a challenge, underscoring the need for further research to realize its medicinal capabilities in wounds.

Exendin-4 exhibits cardioprotective effects against high glucose-induced mitochondrial abnormalities: Potential role of GLP-1 receptor and mTOR signaling.

Mitochondrial dysfunction is associated with hyperglycemic conditions and insulin resistance leading to cellular damage and apoptosis of cardiomyocytes in diabetic cardiomyopathy. The dysregulation of glucagon-like peptide-1 (GLP-1) receptor and mammalian target of rapamycin (mTOR) is linked to cardiomyopathies and myocardial dysfunctions mediated by hyperglycemia. However, the involvements of mTOR for GLP-1 receptor-mediated cardioprotection against high glucose (HG)-induced mitochondrial disturbances are not clearly identified. The present study demonstrated that HG-induced cellular stress and mitochondrial damage resulted in impaired ATP production and oxidative defense markers such as catalase and SOD2, along with a reduction in survival markers such as Bcl-2 and p-Akt, while an increased expression of pro-apoptotic marker Bax was observed in H9c2 cardiomyoblasts. In addition, the autophagic marker LC3-II was considerably reduced, together with the disruption of autophagy regulators (p-mTOR and p-AMPKα) under the hyperglycemic state. Furthermore, there was a dysregulated expression of several indicators related to mitochondrial homeostasis, including MFN2, p-DRP1, FIS1, MCU, UCP3, and Parkin. Remarkably, treatment with either exendin-4 (GLP-1 receptor agonist) or rapamycin (mTOR inhibitor) significantly inhibited HG-induced mitochondrial damage while co-treatment of exendin-4 and rapamycin completely reversed all mitochondrial abnormalities. Antagonism of GLP-1 receptors using exendin-(9-39) abolished these cardioprotective effects of exendin-4 and rapamycin under HG conditions. In addition, exendin-4 attenuated HG-induced phosphorylation of mTOR, and this inhibitory effect was antagonized by exendin-(9-39), indicating the regulation of mTOR by GLP-1 receptor. Therefore, improvement of mitochondrial dysfunction by stimulating the GLP-1 receptor/AMPK/Akt pathway and inhibiting mTOR signaling could ameliorate cardiac abnormalities caused by hyperglycemic conditions.

Anti-Inflammatory Activities of Yataprasen Thai Traditional Formulary and Its Active Compounds, Beta-Amyrin and Stigmasterol, in RAW264.7 and THP-1 Cells.

Yataprasen (YTPS) remedy formulary, a national Thai traditional medicine formulary, comprises 13 herbal plants. It has been extensively prescribed to relieve osteoarthritis and musculoskeletal pain in the Thai traditional medicine healthcare system. The aim of this study was to investigate the antioxidant and anti-inflammatory properties of the bioactive compounds (ß-amyrin and stigmasterol) of YTPS remedy formulary ethanolic extract, along with its composition. The YTPS formulary extract contains 70.30 nM of ß-amyrin and 605.76 nM of stigmasterol. The YTPS formulary extract exhibited ABTS and DPPH free radical scavenging activity, with IC50 values of 144.50 ± 2.82 and 31.85 ± 0.18 µg/mL, respectively. The ethanolic extract of YTPS at a concentration of 1000 µg/mL showed a significant (p < 0.01) anti-inflammatory effect, mainly by reducing IL-6 and TNF-α release in response to LPS. NO production was prominently lowered by 50% at 24.76 ± 1.48 µg/mL, 55.52 ± 24.40 µM, and more than 570 µM of YTPS formulary extract, ß-amyrin, and stigmasterol, respectively. Major components of YTPS, ß-amyrin, and stigmasterol exerted significant anti-inflammatory effects by inhibiting LPS-induced IL-1ß, IL-6, TNF-α secretion in THP-1 cells. Our findings suggest that the ethanolic extract from YTPS holds promise as an alternative topical treatment for osteoarthritis and inflammatory disorders, potentially with fewer side effects than non-steroidal anti-inflammatory medications (NSAIDs).

Adenosine A3 Receptor: From Molecular Signaling to Therapeutic Strategies for Heart Diseases.

Cardiovascular diseases (CVDs), particularly heart failure, are major contributors to early mortality globally. Heart failure poses a significant public health problem, with persistently poor long-term outcomes and an overall unsatisfactory prognosis for patients. Conventionally, treatments for heart failure have focused on lowering blood pressure; however, the development of more potent therapies targeting hemodynamic parameters presents challenges, including tolerability and safety risks, which could potentially restrict their clinical effectiveness. Adenosine has emerged as a key mediator in CVDs, acting as a retaliatory metabolite produced during cellular stress via ATP metabolism, and works as a signaling molecule regulating various physiological processes. Adenosine functions by interacting with different adenosine receptor (AR) subtypes expressed in cardiac cells, including A1AR, A2AAR, A2BAR, and A3AR. In addition to A1AR, A3AR has a multifaceted role in the cardiovascular system, since its activation contributes to reducing the damage to the heart in various pathological states, particularly ischemic heart disease, heart failure, and hypertension, although its role is not as well documented compared to other AR subtypes. Research on A3AR signaling has focused on identifying the intricate molecular mechanisms involved in CVDs through various pathways, including Gi or Gq protein-dependent signaling, ATP-sensitive potassium channels, MAPKs, and G protein-independent signaling. Several A3AR-specific agonists, such as piclidenoson and namodenoson, exert cardioprotective impacts during ischemia in the diverse animal models of heart disease. Thus, modulating A3ARs serves as a potential therapeutic approach, fueling considerable interest in developing compounds that target A3ARs as potential treatments for heart diseases.

Anti-inflammatory effects of the Thai herbal remedy Yataprasen and biflavonoids isolated from Putranjiva roxburghii in RAW264.7 macrophages.

ETHNOPHARMACOLOGICAL RELEVANCE: Yataprasen is a topical Thai herbal remedy for the treatment of musculoskeletal pain and is included in Kumpe Thart Phra Narai, the first Thai textbook of traditional medicine. The herbal preparation is made from a hydroethanolic extract of a mixture of 13 medicinal plants, of which Putranjiva roxburghii Wall. leaves are the major ingredient. AIM OF THE STUDY: In this study, we investigated the underlying mechanism of action for the anti-inflammatory effects of the Yataprasen remedy, its main ingredients, and the phytochemicals isolated from P. roxburghii leaves. MATERIALS AND METHODS: The anti-inflammatory effects of the Yataprasen remedy, along with its main ingredients, including the leaves of Baliospermum solanifolium (Burm.) Suresh, Melia azedarach L., P. roxburghii, Senna siamea (Lam.) Irwin & Barneby, and Tamarindus indica L. were determined by measuring prostaglandin E2 (PGE2) secretion, nitric oxide (NO) production, and the synthesis of inflammatory biomarkers in lipopolysaccharide (LPS)-treated RAW264.7 macrophage cells. The active ingredients of the P. roxburghii leaves were separated by chromatography and spectroscopic measurements were used to identify their chemical structures. RESULTS: Ethanol extracts of the Yataprasen remedy and some of its ingredients significantly suppressed LPS-induced PGE2 secretion and NO production in a dose-dependent manner. Treatment of RAW264.7 cells with ethanolic extracts of the Yataprasen remedy (50 µg/mL) significantly inhibited LPS-induced mRNA expression of TNF-α, COX-2, iNOS, and NF-κB. Among the plant ingredient extracts, P. roxburghii leaf extract exhibited the highest inhibitory effects on LPS-induced TNF-α and iNOS expression. Moreover, T. indica leaf extract showed the highest activity on the inhibition of LPS-induced COX-2 and NF-κB expression. Putraflavone, podocarpusflavone A, and amentoflavone were isolated biflavonoids from P. roxburghii leaf extract and showed the inhibitory effects on LPS-induced PGE2 secretion and NO synthesis in RAW264.7 cells. Of the isolated biflavonoids, amentoflavone exhibited the strongest anti-inflammatory activity by inhibiting the expression of TNF-α, COX-2, and iNOS. CONCLUSION: The results support reported the anti-inflammatory effects of the Yataprasen remedy, which are associated with the downregulation of proinflammatory mediators. P. roxburghii, along with its biflavonoids, are the impact components that contribute to the anti-inflammatory effects of the herbal remedy.

New Therapeutics for Heart Failure: Focusing on cGMP Signaling.

Current drugs for treating heart failure (HF), for example, angiotensin II receptor blockers and ß-blockers, possess specific target molecules involved in the regulation of the cardiac circulatory system. However, most clinically approved drugs are effective in the treatment of HF with reduced ejection fraction (HFrEF). Novel drug classes, including angiotensin receptor blocker/neprilysin inhibitor (ARNI), sodium-glucose co-transporter-2 (SGLT2) inhibitor, hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, soluble guanylyl cyclase (sGC) stimulator/activator, and cardiac myosin activator, have recently been introduced for HF intervention based on their proposed novel mechanisms. SGLT2 inhibitors have been shown to be effective not only for HFrEF but also for HF with preserved ejection fraction (HFpEF). In the myocardium, excess cyclic adenosine monophosphate (cAMP) stimulation has detrimental effects on HFrEF, whereas cyclic guanosine monophosphate (cGMP) signaling inhibits cAMP-mediated responses. Thus, molecules participating in cGMP signaling are promising targets of novel drugs for HF. In this review, we summarize molecular pathways of cGMP signaling and clinical trials of emerging drug classes targeting cGMP signaling in the treatment of HF.

Pyridylnidulin exerts anti-diabetic properties and improves non-alcoholic fatty liver disease in diet-induced obesity mice.

Introduction: Non-alcoholic fatty liver disease (NAFLD) is one of the metabolic disorders related to the pathophysiology of type 2 diabetes mellitus (T2DM). Therapeutic strategies are focused on the improvement of energy balance and lifestyle modification. Additionally, the derivative of the bioactive fungal metabolite is of interest to provide health benefits, especially in obese and pre-diabetic conditions. In our screening of anti-diabetic compounds from fungal metabolites and semisynthetic derivatives, a depsidone derivative, namely pyridylnidulin (PN), showed potent glucose uptake-inducing activity. The present study aimed to investigate the liver lipid metabolism and anti-diabetic properties of PN in diet-induced obesity mice. Methods: Male C57BL/6 mice were induced obesity and pre-diabetic conditions by dietary intervention with a high-fat diet (HFD) for 6 weeks. These obese mice were orally administered with PN (40 or 120 mg/kg), metformin (150 mg/kg), or vehicle for 4 weeks. Glucose tolerance, plasma adipocytokines, hepatic gene and protein expressions were assessed after treatment. Results: Improved glucose tolerance and reduced fasting blood glucose levels were found in the PN and metformin-treated mice. Additionally, hepatic triglyceride levels were consistent with the histopathological steatosis score regarding hepatocellular hypertrophy in the PN and metformin groups. The levels of plasma adipocytokines such as tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were reduced in the PN (120 mg/kg) and metformin-treated mice. In addition, hepatic gene expression involved in lipid metabolism, including lipogenic enzymes was significantly reduced in the PN (120 mg/kg) and metformin-treated mice. The increased protein expression levels of phosphorylated AMP-activated protein kinase (p-AMPK) was also found in PN and metformin-treated mice. Discussion: Considering the increased p-AMPK protein expression levels in PN and metformin-treated mice were revealed as the underlying mechanisms to improve metabolic parameters. These results suggested that PN provided the health benefit to slow the progression of NAFLD and T2DM in obese and pre-diabetic conditions.

Exendin-4 protects against high glucose-induced mitochondrial dysfunction and oxidative stress in SH-SY5Y neuroblastoma cells through GLP-1 receptor/Epac/Akt signaling.

Mitochondrial dysfunction under diabetic condition leads to the development and progression of neurodegenerative complications. Recently, the beneficial effects of glucagon-like peptide-1 (GLP-1) receptor agonists on diabetic neuropathies have been widely recognized. However, molecular mechanisms underlying the neuroprotective effects of GLP-1 receptor agonists against high glucose (HG)-induced neuronal damages is not completely elucidated. Here, we investigated the underlying mechanisms of GLP-1 receptor agonist treatment against oxidative stress, mitochondrial dysfunction, and neuronal damages under HG conditions mimicking a diabetic hyperglycemic state in SH-SY5Y neuroblastoma cells. We revealed that treatment with exendin-4, a GLP-1 receptor agonist, not only increased the expression of survival markers, phospho-Akt/Akt and Bcl-2, but also decreased the expression of pro-apoptotic marker, Bax, and reduced the levels of reactive oxygen species (ROS) defense markers (catalase, SOD-2, and HO-1) under HG conditions. The expressions of mitochondrial function associated genes, MCU and UCP3, and mitochondrial fission genes, DRP1 and FIS1, were decreased by exendin-4 compared to non-treated levels, while the protein expression levels of mitochondrial homeostasis regulators, Parkin and PINK1, were enhanced. In addition, blockade of Epac and Akt activities was able to antagonize these neuroprotective effects of exendin-4. Collectively, we demonstrated that stimulation of GLP-1 receptor propagates a neuroprotective cascade against the oxidative stress and mitochondrial dysfunction as well as augments survival through the Epac/Akt-dependent pathway. Therefore, the revealed mechanisms underlying GLP-1 receptor pathway by preserving mitochondrial homeostasis would be a therapeutic candidate to alleviate neuronal dysfunctions and delay the progression of diabetic neuropathies.

Multifaceted Roles of GLP-1 and Its Analogs: A Review on Molecular Mechanisms with a Cardiotherapeutic Perspective.

Diabetes is one of the chronic metabolic disorders which poses a multitude of life-debilitating challenges, including cardiac muscle impairment, which eventually results in heart failure. The incretin hormone glucagon-like peptide-1 (GLP-1) has gained distinct recognition in reinstating glucose homeostasis in diabetes, while it is now largely accepted that it has an array of biological effects in the body. Several lines of evidence have revealed that GLP-1 and its analogs possess cardioprotective effects by various mechanisms related to cardiac contractility, myocardial glucose uptake, cardiac oxidative stress and ischemia/reperfusion injury, and mitochondrial homeostasis. Upon binding to GLP-1 receptor (GLP-1R), GLP-1 and its analogs exert their effects via adenylyl cyclase-mediated cAMP elevation and subsequent activation of cAMP-dependent protein kinase(s) which stimulates the insulin release in conjunction with enhanced Ca2+ and ATP levels. Recent findings have suggested additional downstream molecular pathways stirred by long-term exposure of GLP-1 analogs, which pave the way for the development of potential therapeutic molecules with longer lasting beneficial effects against diabetic cardiomyopathies. This review provides a comprehensive overview of the recent advances in the understanding of the GLP-1R-dependent and -independent actions of GLP-1 and its analogs in the protection against cardiomyopathies.

Short-Term Growth Hormone Administration Mediates Hepatic Fatty Acid Uptake and De Novo Lipogenesis Gene Expression in Obese Rats.

Obesity has been linked to metabolic syndrome, type 2 diabetes, and non-alcoholic fatty liver disease (NAFLD). Obesity causes a decrease in growth hormone (GH) levels and an increase in insulin levels. Long-term GH treatment increased lipolytic activity as opposed to decreasing insulin sensitivity. Nonetheless, it is possible that short-term GH administration had no impact on insulin sensitivity. In this study, the effect of short-term GH administration on liver lipid metabolism and the effector molecules of GH and insulin receptors were investigated in diet-induced obesity (DIO) rats. Recombinant human GH (1 mg/kg) was then administered for 3 days. Livers were collected to determine the hepatic mRNA expression and protein levels involved in lipid metabolism. The expression of GH and insulin receptor effector proteins was investigated. In DIO rats, short-term GH administration significantly reduced hepatic fatty acid synthase (FASN) and cluster of differentiation 36 (CD36) mRNA expression while increasing carnitine palmitoyltransferase 1A (CPT1A) mRNA expression. Short-term GH administration reduced hepatic FAS protein levels and downregulated gene transcription of hepatic fatty acid uptake and lipogenesis, while increasing fatty acid oxidation in DIO rats. DIO rats had lower hepatic JAK2 protein levels but higher IRS-1 levels than control rats due to hyperinsulinemia. Our findings suggest that short-term GH supplementation improves liver lipid metabolism and may slow the progression of NAFLD, where GH acts as the transcriptional regulator of related genes.

Gαq protein-biased ligand of angiotensin II type 1 receptor mediates myofibroblast differentiation through TGF-ß1/ERK axis in human cardiac fibroblasts.

Angiotensin II receptors are members of G protein-coupled receptor superfamily that manifest biased signals toward G protein- and ß-arrestin-dependent pathways. However, the role of angiotensin II receptor-biased ligands and the mechanisms underlying myofibroblast differentiation in human cardiac fibroblasts have not been fully elucidated. Our results demonstrated that antagonism of angiotensin II type 1 receptor (AT1 receptor) and blockade of Gαq protein suppressed angiotensin II (Ang II)-induced fibroblast proliferation, overexpression of collagen I and α-smooth muscle actin (α-SMA), and stress fibre formation, indicating the AT1 receptor/Gαq axis is necessary for fibrogenic effects of Ang II. Stimulation of AT1 receptors by their Gαq-biased ligand (TRV120055), but not ß-arrestin-biased ligand (TRV120027), substantially exerted fibrogenic effects at a level similar to that of Ang II, suggesting that AT1 receptor induced cardiac fibrosis in a Gαq-dependent and ß-arrestin-independent manner. Valsartan prevented TRV120055-mediated fibroblast activation. TRV120055 mediated the upregulation of transforming growth factor-beta1 (TGF-ß1) through the AT1 receptor/Gαq cascade. In addition, Gαq protein and TGF-ß1 were necessary for ERK1/2 activation induced by Ang II and TRV120055. Collectively, TGF-ß1 and ERK1/2 are downstream effectors of the Gαq-biased ligand of AT1 receptor for the induction of cardiac fibrosis.

Kombucha Beverages Produced from Fruits, Vegetables, and Plants: A Review on Their Pharmacological Activities and Health Benefits.

Kombucha is a traditional health beverage produced by fermenting sweetened tea with a symbiotic culture of bacteria and yeasts. Consumption of kombucha beverages has been growing and there is kombucha commercially available worldwide as one of the most famous low-alcohol beverages. Kombucha beverages have been claimed to have beneficial effects on human health because they contain a variety of bioactive compounds that possess various functional properties. At present, several kinds of raw material (e.g., milk, fruit, vegetables, and herbs) have been fermented with kombucha consortium and consumed as kombucha beverages. Although several studies have been written regarding the biological activities of kombucha and raw materials, there is however little information available on the characterization of their components as well as the biological activities of fermented kombucha from many raw material mixtures. Several pharmacological activities were reviewed in the scientific literature, describing their potential implications for human health. In addition, the adverse effects and toxicity of kombucha consumption were also reviewed. In this study, we focused on the main and latest studies of the pharmacological effects of kombucha beverages produced from various kinds of raw materials, including antioxidant, anti-inflammatory, immunomodulatory, antimicrobial, anticancer, antidiabetic, antihypertensive, and antihyperlipidemic effects in in vitro and in vivo studies.

Dual Blockade of TGF-ß Receptor and Endothelin Receptor Synergistically Inhibits Angiotensin II-Induced Myofibroblast Differentiation: Role of AT1R/Gαq-Mediated TGF-ß1 and ET-1 Signaling.

Angiotensin II (Ang II) upregulates transforming growth factor-beta1 (TGF-ß1) and endothelin-1 (ET-1) in various types of cells, and all of them act as profibrotic mediators. However, the signal transduction of angiotensin II receptor (ATR) for upregulation of TGF-ß1 and ET-1, and their effectors that play an essential role in myofibroblast differentiation, are not fully understood. Therefore, we investigated the ATR networking with TGF-ß1 and ET-1 and identified the signal transduction of these mediators by measuring the mRNA expression of alpha-smooth muscle actin (α-SMA) and collagen I using qRT-PCR. Myofibroblast phenotypes were monitored by α-SMA and stress fiber formation with fluorescence microscopy. Our findings suggested that Ang II induced collagen I and α-SMA synthesis and stress fiber formation through the AT1R/Gαq axis in adult human cardiac fibroblasts (HCFs). Following AT1R stimulation, Gαq protein, not Gßγ subunit, was required for upregulation of TGF-ß1 and ET-1. Moreover, dual inhibition of TGF-ß and ET-1 signaling completely inhibited Ang II-induced myofibroblast differentiation. The AT1R/Gαq cascade transduced signals to TGF-ß1, which in turn upregulated ET-1 via the Smad- and ERK1/2-dependent pathways. ET-1 consecutively bound to and activated endothelin receptor type A (ETAR), leading to increases in collagen I and α-SMA synthesis and stress fiber formation. Remarkably, dual blockade of TGF-ß receptor and ETR exhibited the restorative effects to reverse the myofibroblast phenotype induced by Ang II. Collectively, TGF-ß1 and ET-1 are major effectors of AT1R/Gαq cascade, and therefore, negative regulation of TGF-ß and ET-1 signaling represents a targeted therapeutic strategy for the prevention and restoration of cardiac fibrosis.

Endothelin-1 Induces Cell Proliferation and Myofibroblast Differentiation through the ETAR/Gαq/ERK Signaling Pathway in Human Cardiac Fibroblasts.

Endothelin-1 (ET-1) has been implicated in the pathogenesis of cardiac fibrosis. Stimulation of endothelin receptors (ETR) with ET-1 leads to fibroblast activation and myofibroblast differentiation, which is mainly characterized by an overexpression of α-smooth muscle actin (α-SMA) and collagens. Although ET-1 is a potent profibrotic mediator, the signal transductions and subtype specificity of ETR contributing to cell proliferation, as well as α-SMA and collagen I synthesis in human cardiac fibroblasts are not well clarified. This study aimed to evaluate the subtype specificity and signal transduction of ETR on fibroblast activation and myofibroblast differentiation. Treatment with ET-1 induced fibroblast proliferation, and synthesis of myofibroblast markers, α-SMA, and collagen I through the ETAR subtype. Inhibition of Gαq protein, not Gαi or Gßγ, inhibited these effects of ET-1, indicating the essential role of Gαq protein-mediated ETAR signaling. In addition, ERK1/2 was required for ETAR/Gαq axis-induced proliferative capacity and overexpression of these myofibroblast markers. Antagonism of ETR with ETR antagonists (ERAs), ambrisentan and bosentan, inhibited ET-1-induced cell proliferation and synthesis of α-SMA and collagen I. Furthermore, ambrisentan and bosentan promoted the reversal of myofibroblasts after day 3 of treatment, with loss of proliferative ability and a reduction in α-SMA synthesis, confirming the restorative effects of ERAs. This novel work reports on the ETAR/Gαq/ERK signaling pathway for ET-1 actions and blockade of ETR signaling with ERAs, representing a promising therapeutic strategy for prevention and restoration of ET-1-induced cardiac fibrosis.

High Glucose-Induced Cardiomyocyte Damage Involves Interplay between Endothelin ET-1/ETA/ETB Receptor and mTOR Pathway.

Patients with type two diabetes mellitus (T2DM) are at increased risk for cardiovascular diseases. Impairments of endothelin-1 (ET-1) signaling and mTOR pathway have been implicated in diabetic cardiomyopathies. However, the molecular interplay between the ET-1 and mTOR pathway under high glucose (HG) conditions in H9c2 cardiomyoblasts has not been investigated. We employed MTT assay, qPCR, western blotting, fluorescence assays, and confocal microscopy to assess the oxidative stress and mitochondrial damage under hyperglycemic conditions in H9c2 cells. Our results showed that HG-induced cellular stress leads to a significant decline in cell survival and an impairment in the activation of ETA-R/ETB-R and the mTOR main components, Raptor and Rictor. These changes induced by HG were accompanied by a reactive oxygen species (ROS) level increase and mitochondrial membrane potential (MMP) loss. In addition, the fragmentation of mitochondria and a decrease in mitochondrial size were observed. However, the inhibition of either ETA-R alone by ambrisentan or ETA-R/ETB-R by bosentan or the partial blockage of the mTOR function by silencing Raptor or Rictor counteracted those adverse effects on the cellular function. Altogether, our findings prove that ET-1 signaling under HG conditions leads to a significant mitochondrial dysfunction involving contributions from the mTOR pathway.

Sustained AT1R stimulation induces upregulation of growth factors in human cardiac fibroblasts via Gαq/TGF-ß/ERK signaling that influences myocyte hypertrophy.

Stimulation of angiotensin II receptor (ATR) with angiotensin II (Ang II) accelerates cardiac fibroblast activation, resulting in upregulation of cytokines and growth factors. Growth factors were strongly upregulated in animal models of myocardial fibrosis and hypertrophy as well as patients with heart failure. Nevertheless, the signal transduction of ATR for upregulation of growth factors in human cardiac fibroblasts contributing to myocyte hypertrophy have not fully understood. Long-term Ang II treatment of human cardiac fibroblasts provokes the synthesis and secretion of connective tissue growth factor (CTGF), transforming growth factor beta1 (TGF-ß1), and vascular endothelial growth factor (VEGF) through the AT1R subtype. Blockade of Gαq, not Gαi or Gα12/13, protein signaling inhibited AT1R-mediated upregulation of CTGF, TGF-ß1, and VEGF. In addition, AT1R overstimulation induced upregulation of growth factors via the TGF-ß-dependent and ERK1/2-dependent pathways. Growth factors secreted from cardiac fibroblasts are necessary for the induction of hypertrophic markers, atrial natriuretic peptide (ANP) and ß-myosin heavy chain (ß-MHC), resulting in myocyte hypertrophy. Candesartan, irbesartan, and valsartan had greater effects than losartan for blockade of fibrotic and hypertrophic effects of Ang II. Our data support the concept whereby sustained AT1R stimulation contributes to the development of myocardial fibrosis and hypertrophy, and advances understanding of this complex AT1R signaling, including fibroblasts-myocytes communication during pathological conditions.

Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through ß-adrenoceptors.

Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates ß-adrenoceptor (ßAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated ßAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting ß-arrestin-mediated ßAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.

Stimulation of adenosine A1 receptor prevents oxidative injury in H9c2 cardiomyoblasts: Role of Gßγ-mediated Akt and ERK1/2 signaling.

Oxidative stress causes cellular injury and damage in the heart primarily through apoptosis resulting in cardiac abnormalities such as heart failure and cardiomyopathy. During oxidative stress, stimulation of adenosine receptor (AR) has been shown to protect against oxidative damage due to their cytoprotective properties. However, the subtype specificity and signal transductions of adenosine A1 receptor (A1R) on cardiac protection during oxidative stress have remained elusive. In this study, we found that stimulation of A1Rs with N6-cyclopentyladenosine (CPA), a specific A1R agonist, attenuated the H2O2-induced intracellular and mitochondrial reactive oxygen species (ROS) production and apoptosis. In addition, A1R stimulation upregulated the synthesis of antioxidant enzymes (catalase and GPx-1), antiapoptotic proteins (Bcl-2 and Bcl-xL), and mitochondria-related markers (UCP2 and UCP3). Blockades of Gßγ subunit of heterotrimeric Gαi protein antagonized A1R-mediated antioxidant and antiapoptotic effects, confirming the potential role of Gßγ subunit-mediated A1R signaling. Additionally, cardioprotective effects of CPA mediated through PI3K/Akt- and ERK1/2-dependent signaling pathways. Thus, we propose that A1R represents a promising therapeutic target for prevention of oxidative injury in the heart.