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.

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.

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.

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.

Glenzocimab: A GPVI (Glycoprotein VI)-Targeted Potential Antiplatelet Agent for the Treatment of Acute Ischemic Stroke.

It has previously been shown in several animal experiments that platelet GPVI (glycoprotein VI) contributes to thrombosis, particularly in ischemic stroke. Moreover, GPVI levels are upregulated in stroke patients. This review describes the therapeutic roles of anti-GPVI antibody in preclinical models of ischemic stroke and provides the current evidence for potential benefits of glenzocimab, a Fab fragment of humanized anti-GPVI monoclonal antibody, in stroke patients. Anti-GPVI antibody, JAQ1, significantly decreased infarct volume and improved neurological function in mice with transient middle cerebral artery occlusion, a model of ischemic stroke, with no or minor bleeding tendency. Intravenous injection of glenzocimab in nonhuman primates produced rapid inhibition of ex vivo platelet aggregation induced by collagen (a GPVI ligand). Complete platelet inhibition is observed at 30 minutes following administration without increasing the risk of bleeding. In humans, glenzocimab is well tolerated and produces dose-dependent antiplatelet activity. More importantly, glenzocimab (125-1000 mg) was safe when administered as soon as possible (<3 hours) following reperfusion with the r-tPA (recombinant tissue-type plasminogen activator) in patients with acute ischemic stroke. Although glenzocimab 1000 mg (a selected dose) did not demonstrate a significant improvement in overall clinical outcomes, it appeared to provide benefits in severe cases and in patients who required thrombectomy. This promising efficacy together with a good safety profile of glenzocimab warrant further investigation in phase III (ACTISAVE [Adaptive Efficacy and Safety Study of Glenzocimab Used as an Add-On Therapy on Top of Standard of Care in the 4.5 Hours Following an Acute Ischemic Stroke]) clinical study.

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.

Cardioprotective Effects of Glucagon-like Peptide-1 (9-36) Against Oxidative Injury in H9c2 Cardiomyoblasts: Potential Role of the PI3K/Akt/NOS Pathway.

ABSTRACT: Glucagon-like peptide (GLP)-1(7-36), a major active form of GLP-1 hormone, is rapidly cleaved by dipeptidyl peptidase-4 to generate a truncated metabolite, GLP-1(9-36) which has a low affinity for GLP-1 receptor (GLP-1R). GLP-1(7-36) has been shown to have protective effects on cardiovascular system through GLP-1R-dependent pathway. Nevertheless, the cardioprotective effects of GLP-1(9-36) have not fully understood. The present study investigated the effects of GLP-1(9-36), including its underlying mechanisms against oxidative stress and apoptosis in H9c2 cells. Here, we reported that GLP-1(9-36) protects H9c2 cardiomyoblasts from hydrogen peroxide (H2O2)-induced oxidative stress by promoting the synthesis of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1. In addition, treatment with GLP-1(9-36) suppressed H2O2-induced apoptosis by attenuating caspase-3 activity and upregulating antiapoptotic proteins, Bcl-2 and Bcl-xL. These protective effects of GLP-1(9-36) are attenuated by blockade of PI3K-mediated Akt phosphorylation and prevention of nitric oxide synthase-induced nitric oxide production. Thus, GLP-1(9-36) represents the potential therapeutic target for prevention of oxidative stress and apoptosis in the heart via PI3K/Akt/nitric oxide synthase signaling pathway.

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.

Andrographolide Exhibits Anticancer Activity against Breast Cancer Cells (MCF-7 and MDA-MB-231 Cells) through Suppressing Cell Proliferation and Inducing Cell Apoptosis via Inactivation of ER-α Receptor and PI3K/AKT/mTOR Signaling.

Breast cancer is the most common cancer among women worldwide. Chemotherapy followed by endocrine therapy is the standard treatment strategy after surgery or radiotherapy. However, breast cancer is highly resistant to the treatments leading to the recurrence of breast cancer. As a result, the development of alternative medicines derived from natural plants with fewer side effects is being emphasized. Andrographolide isolated from Andrographis paniculata is one of the potential substances with anti-cancer properties in a variety of cell types, including breast cancer cells. This study aims to investigate the anti-cancer effects of andrographolide in breast cancer cells by evaluating cell viability and apoptosis as well as its underlying mechanisms through estrogen receptor (ER)-dependent and PI3K/AKT/mTOR signaling pathways. Cell viability, cell apoptosis, mRNA or miRNA, and protein expression were examined by MTT assay, Annexin V-FITC, qRT-PCR, and Western blot analysis, respectively. MCF-7 and MDA-MB-231 cell viability was reduced in a concentration- and time-dependent manner after andrographolide treatment. Moreover, andrographolide induced cell apoptosis in both MCF-7 and MDA-MB-231 cells by inhibiting Bcl-2 and enhancing Bax expression at both mRNA and protein levels. In MCF-7 cells, the ER-positive breast cancer, andrographolide showed an inhibitory effect on cell proliferation through downregulation of ERα, PI3K, and mTOR expression levels. Andrographolide also inhibited MDA-MB-231 breast cancer cell proliferation via induction of cell apoptosis. However, the inhibition of MCF-7 and MDA-MB-231 cell proliferation of andrographolide treatment did not disrupt miR-21. Our findings showed that andrographolide possesses an anti-estrogenic effect by suppressing cell proliferation in MCF-7 cells. The effects were comparable to those of the anticancer drug fulvestrant in MCF-7 cells. This study provides new insights into the anti-cancer effect of andrographolide on breast cancer and suggests andrographolide as a potential alternative from the natural plant for treating breast cancer types that are resistant to tamoxifen and fulvestrant.

Plumbagin Suppresses Breast Cancer Progression by Downregulating HIF-1α Expression via a PI3K/Akt/mTOR Independent Pathway under Hypoxic Condition.

Hypoxia-inducible factor-1α (HIF-1α) is a major transcriptional regulator that plays a crucial role in the hypoxic response of rapidly growing tumors. Overexpression of HIF-1α has been associated with breast cancer metastasis and poor clinical prognosis. Plumbagin, the main phytochemical from Plumbago indica, exerts anticancer effects via multiple mechanisms. However, its precise mechanisms on breast cancer cells under hypoxic conditions has never been investigated. This study aims to examine the anticancer effect of plumbagin on MCF-7 cell viability, transcriptional activity, and protein expression of HIF-1α under normoxia and hypoxia-mimicking conditions, as well as reveal the underlying signaling pathways. The results demonstrate that plumbagin decreased MCF-7 cell viability under normoxic conditions, and a greater extent of reduction was observed upon exposure to hypoxic conditions induced by cobalt chloride (CoCl2). Mechanistically, MCF-7 cells upregulated the expression of HIF-1α protein, mRNA, and the VEGF target gene under CoCl2-induced hypoxia, which were abolished by plumbagin treatment. In addition, inhibition of HIF-1α and its downstream targets did not affect the signaling transduction of the PI3K/Akt/mTOR pathway under hypoxic state. This study provides mechanistic insight into the anticancer activity of plumbagin in breast cancer cells under hypoxic conditions by abolishing HIF-1α at transcription and post-translational modifications.

Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation.

Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A2 receptors (A2Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A2Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A2B receptor (A2BR) subtype. Stimulation of A2BR exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A2BR-mediated antifibrotic effects. Thus, A2BR is one of the potential therapeutic targets against cardiac fibrosis.

Prolonged stimulation of ß2-adrenergic receptor with ß2-agonists impairs insulin actions in H9c2 cells.

Insulin resistance is a condition in which there is a defect in insulin actions to induce glucose uptake into the cells. Overstimulation of ß2-adrenergic receptors (ß2ARs) is associated with the pathogenesis of insulin resistance in the heart. However, the mechanisms by which ß2-agonists affect insulin resistance in the heart are incompletely understood. The ß2-agonists are used for treatment of asthma due to bronchodilating effects. We also investigated the effects of ß2-agonists in human bronchial smooth muscle (HBSM) cells. In this study, we demonstrate that chronic treatment with salbutamol, salmeterol, and formoterol inhibited insulin-induced glucose uptake and GLUT4 synthesis in H9c2 myoblast cells. Sustained ß2AR stimulation also attenuated GLUT4 translocation to the plasma membrane, whereas short-term stimulation had no effect. In HBSM cells, prolonged treatment with ß2-agonists had no effect on insulin-induced glucose uptake and did not alter insulin-induced expressions of GLUT1, GLUT4, and GLUT10. In addition, genetic polymorphisms at amino acid positions 16 and 27 of ß2AR are linked to insulin resistance by significant suppression of GLUT4 translocation compared to wild-type. Thus, prolonged ß2AR stimulation by ß2-agonists impairs insulin actions through suppression of GLUT synthesis and translocation only in H9c2 cells.

Resveratrol Specifically Kills Cancer Cells by a Devastating Increase in the Ca2+ Coupling Between the Greatly Tethered Endoplasmic Reticulum and Mitochondria.

BACKGROUND/AIMS: Resveratrol and its derivate piceatannol are known to induce cancer cell-specific cell death. While multiple mechanisms of actions have been described including the inhibition of ATP synthase, changes in mitochondrial membrane potential and ROS levels, the exact mechanisms of cancer specificity of these polyphenols remain unclear. This paper is designed to reveal the molecular basis of the cancer-specific initiation of cell death by resveratrol and piceatannol. METHODS: The two cancer cell lines EA.hy926 and HeLa, and somatic short-term cultured HUVEC were used. Cell viability and caspase 3/7 activity were tested. Mitochondrial, cytosolic and endoplasmic reticulum Ca2+ as well as cytosolic and mitochondrial ATP levels were measured using single cell fluorescence microscopy and respective genetically-encoded sensors. Mitochondria-ER junctions were analyzed applying super-resolution SIM and ImageJ-based image analysis. RESULTS: Resveratrol and piceatannol selectively trigger death in cancer but not somatic cells. Hence, these polyphenols strongly enhanced mitochondrial Ca2+ uptake in cancer exclusively. Resveratrol and piceatannol predominantly affect mitochondrial but not cytosolic ATP content that yields in a reduced SERCA activity. Decreased SERCA activity and the strongly enriched tethering of the ER and mitochondria in cancer cells result in an enhanced MCU/Letm1-dependent mitochondrial Ca2+ uptake upon intracellular Ca2+ release exclusively in cancer cells. Accordingly, resveratrol/piceatannol-induced cancer cell death could be prevented by siRNA-mediated knock-down of MCU and Letm1. CONCLUSIONS: Because their greatly enriched ER-mitochondria tethering, cancer cells are highly susceptible for resveratrol/piceatannol-induced reduction of SERCA activity to yield mitochondrial Ca2+ overload and subsequent cancer cell death.

UCP2 modulates single-channel properties of a MCU-dependent Ca(2+) inward current in mitochondria.

The mitochondrial Ca(2+) uniporter is a highly Ca(2+)-selective protein complex that consists of the pore-forming mitochondrial Ca(2+) uniporter protein (MCU), the scaffolding essential MCU regulator (EMRE), and mitochondrial calcium uptake 1 and 2 (MICU1/2), which negatively regulate mitochondrial Ca(2+) uptake. We have previously reported that uncoupling proteins 2 and 3 (UCP2/3) are also engaged in the activity of mitochondrial Ca(2+) uptake under certain conditions, while the mechanism by which UCP2/3 facilitates mitochondrial Ca(2+) uniport remains elusive. This work was designed to investigate the impact of UCP2 on the three distinct mitochondrial Ca(2+) currents found in mitoplasts isolated from HeLa cells, the intermediate- (i-), burst- (b-) and extra-large (xl-) mitochondrial/mitoplast Ca(2+) currents (MCC). Using the patch clamp technique on mitoplasts from cells with reduced MCU and EMRE unveiled a very high affinity of MCU for xl-MCC that succeeds that for i-MCC, indicating the coexistence of at least two MCU/EMRE-dependent Ca(2+) currents. The manipulation of the expression level of UCP2 by either siRNA-mediated knockdown or overexpression changed exclusively the open probability (NPo) of xl-MCC by approx. 38% decrease or nearly a 3-fold increase, respectively. These findings confirm a regulatory role of UCP2 in mitochondrial Ca(2+) uptake and identify UCP2 as a selective modulator of just one distinct MCU/EMRE-dependent mitochondrial Ca(2+) inward current.

Mitochondrial Ca(2+) uniporter (MCU)-dependent and MCU-independent Ca(2+) channels coexist in the inner mitochondrial membrane.

A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

Mitochondrial Ca2+ uptake 1 (MICU1) and mitochondrial ca2+ uniporter (MCU) contribute to metabolism-secretion coupling in clonal pancreatic ß-cells.

In pancreatic ß-cells, uptake of Ca(2+) into mitochondria facilitates metabolism-secretion coupling by activation of various matrix enzymes, thus facilitating ATP generation by oxidative phosphorylation and, in turn, augmenting insulin release. We employed an siRNA-based approach to evaluate the individual contribution of four proteins that were recently described to be engaged in mitochondrial Ca(2+) sequestration in clonal INS-1 832/13 pancreatic ß-cells: the mitochondrial Ca(2+) uptake 1 (MICU1), mitochondrial Ca(2+) uniporter (MCU), uncoupling protein 2 (UCP2), and leucine zipper EF-hand-containing transmembrane protein 1 (LETM1). Using a FRET-based genetically encoded Ca(2+) sensor targeted to mitochondria, we show that a transient knockdown of MICU1 or MCU diminished mitochondrial Ca(2+) uptake upon both intracellular Ca(2+) release and Ca(2+) entry via L-type channels. In contrast, knockdown of UCP2 and LETM1 exclusively reduced mitochondrial Ca(2+) uptake in response to either intracellular Ca(2+) release or Ca(2+) entry, respectively. Therefore, we further investigated the role of MICU1 and MCU in metabolism-secretion coupling. Diminution of MICU1 or MCU reduced mitochondrial Ca(2+) uptake in response to d-glucose, whereas d-glucose-triggered cytosolic Ca(2+) oscillations remained unaffected. Moreover, d-glucose-evoked increases in cytosolic ATP and d-glucose-stimulated insulin secretion were diminished in MICU1- or MCU-silenced cells. Our data highlight the crucial role of MICU1 and MCU in mitochondrial Ca(2+) uptake in pancreatic ß-cells and their involvement in the positive feedback required for sustained insulin secretion.

Targeting collagen homeostasis for the treatment of liver fibrosis: Opportunities and challenges.

Liver fibrosis is an excessive production, aberrant deposition, and deficit degradation of extracellular matrix (ECM). Patients with unresolved fibrosis ultimately undergo end-stage liver diseases. To date, the effective and safe strategy to cease fibrosis progression remains an unmet clinical need. Since collagens are the most abundant ECM protein which play an essential role in fibrogenesis, the suitable regulation of collagen homeostasis could be an effective strategy for the treatment of liver fibrosis. Therefore, this review provides a brief overview on the dysregulation of ECM homeostasis, focusing on collagens, in the pathogenesis of liver fibrosis. Most importantly, promising therapeutic mechanisms related to biosynthesis, deposition and extracellular interactions, and degradation of collagens, together with preclinical and clinical antifibrotic evidence of drugs affecting each target are orderly criticized. In addition, challenges for targeting collagen homeostasis in the treatment of liver fibrosis are discussed.

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.

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.

Determination of the Potential Clinical Benefits of Small Molecule Factor XIa Inhibitors in Arterial Thrombosis.

Anticoagulants are the mainstay for the prevention and treatment of thrombosis. However, bleeding complications remain a primary concern. Recent advances in understanding the contribution of activated factor XI (FXIa) in arterial thrombosis with a limited impact on hemostasis have led to the development of several FXIa-targeting modalities. Injectable agents including monoclonal antibodies and antisense oligonucleotides against FXIa have been primarily studied in venous thrombosis. The orally active small molecules that specifically inhibit the active site of FXIa are currently being investigated for their antithrombotic activity in both arteries and veins. This review focuses on a discussion of the potential clinical benefits of small molecule FXIa inhibitors, mainly asundexian and milvexian, in arterial thrombosis based on their pharmacological profiles and the compelling results of phase 2 clinical studies. The preclinical and epidemiological basis for the impact of FXIa in hemostasis and arterial thrombosis is also addressed. In recent clinical study results, asundexian appears to reduce ischemic events in patients with myocardial infarction and minor-to-moderate stroke, whereas milvexian possibly provides benefits in patients with minor stroke or high-risk transient ischemic attack (TIA). In addition, asundexian and milvexian had a minor impact on hemostasis even in combination with dual-antiplatelet therapy. Other orally active FXIa inhibitors also produce antithrombotic activity in vivo with low bleeding risk. Therefore, FXIa inhibitors might represent a new class of direct-acting oral anticoagulants (DOACs) for the treatment of thrombosis, although the explicit clinical positions of asundexian and milvexian in patients with ischemic stroke, high-risk TIA, and coronary artery disease require confirmation from the outcomes of ongoing phase 3 trials.