Inactivation of Malic Enzyme 1 in Endothelial Cells Alleviates Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive cardiopulmonary disease with a high mortality rate. Although growing evidence has revealed the importance of dysregulated energetic metabolism in the pathogenesis of PH, the underlying cellular and molecular mechanisms are not fully understood. In this study, we focused on ME1 (malic enzyme 1), a key enzyme linking glycolysis to the tricarboxylic acid cycle. We aimed to determine the role and mechanistic action of ME1 in PH. METHODS: Global and endothelial-specific ME1 knockout mice were used to investigate the role of ME1 in hypoxia- and SU5416/hypoxia (SuHx)-induced PH. Small hairpin RNA and ME1 enzymatic inhibitor (ME1*) were used to study the mechanism of ME1 in pulmonary artery endothelial cells. Downstream key metabolic pathways and mediators of ME1 were identified by metabolomics analysis in vivo and ME1-mediated energetic alterations were examined by Seahorse metabolic analysis in vitro. The pharmacological effect of ME1* on PH treatment was evaluated in PH animal models induced by SuHx. RESULTS: We found that ME1 protein level and enzymatic activity were highly elevated in lung tissues of patients and mice with PH, primarily in vascular endothelial cells. Global knockout of ME1 protected mice from developing hypoxia- or SuHx-induced PH. Endothelial-specific ME1 deletion similarly attenuated pulmonary vascular remodeling and PH development in mice, suggesting a critical role of endothelial ME1 in PH. Mechanistic studies revealed that ME1 inhibition promoted downstream adenosine production and activated A2AR-mediated adenosine signaling, which leads to an increase in nitric oxide generation and a decrease in proinflammatory molecule expression in endothelial cells. ME1 inhibition activated adenosine production in an ATP-dependent manner through regulating malate-aspartate NADH (nicotinamide adenine dinucleotide plus hydrogen) shuttle and thereby balancing oxidative phosphorylation and glycolysis. Pharmacological inactivation of ME1 attenuated the progression of PH in both preventive and therapeutic settings by promoting adenosine production in vivo. CONCLUSIONS: Our findings indicate that ME1 upregulation in endothelial cells plays a causative role in PH development by negatively regulating adenosine production and subsequently dysregulating endothelial functions. Our findings also suggest that ME1 may represent as a novel pharmacological target for upregulating protective adenosine signaling in PH therapy.

Adenovirus-mediated Overexpression of FcγRIIB Attenuates Pulmonary Inflammation and Fibrosis.

Progressive fibrosing interstitial lung diseases (PF-ILDs) result in high mortality and lack effective therapies. The pathogenesis of PF-ILDs involves macrophages driving inflammation and irreversible fibrosis. Fc-γ receptors (FcγRs) regulate macrophages and inflammation, but their roles in PF-ILDs remain unclear. We characterized the expression of FcγRs and found upregulated FcγRIIB in human and mouse lungs after exposure to silica. FcγRIIB deficiency aggravated lung dysfunction, inflammation, and fibrosis in silica-exposed mice. Using single-cell transcriptomics and in vitro experiments, FcγRIIB was found in alveolar macrophages, where it regulated the expression of fibrosis-related genes Spp1 and Ctss. In mice with macrophage-specific overexpression of FcγRIIB and in mice treated with adenovirus by intratracheal instillation to upregulate FcγRIIB, silica-induced functional and histological changes were ameliorated. Our data from three genetic models and a therapeutic model suggest that FcγRIIB plays a protective role that can be enhanced by adenoviral overexpression, representing a potential therapeutic strategy for PF-ILDs.

Strong, Tough, and Anti-Swelling Supramolecular Conductive Hydrogels for Amphibious Motion Sensors.

Conductive polymer hydrogels (CPHs) are widely employed in emerging flexible electronic devices because they possess both the electrical conductivity of conductors and the mechanical properties of hydrogels. However, the poor compatibility between conductive polymers and the hydrogel matrix, as well as the swelling behavior in humid environments, greatly compromises the mechanical and electrical properties of CPHs, limiting their applications in wearable electronic devices. Herein, a supramolecular strategy to develop a strong and tough CPH with excellent anti-swelling properties by incorporating hydrogen, coordination bonds, and cation-π interactions between a rigid conducting polymer and a soft hydrogel matrix is reported. Benefiting from the effective interactions between the polymer networks, the obtained supramolecular hydrogel has homogeneous structural integrity, exhibiting remarkable tensile strength (1.63 MPa), superior elongation at break (453%), and remarkable toughness (5.5 MJ m-3 ). As a strain sensor, the hydrogel possesses high electrical conductivity (2.16 S m-1 ), a wide strain linear detection range (0-400%), and excellent sensitivity (gauge factor = 4.1), sufficient to monitor human activities with different strain windows. Furthermore, this hydrogel with high swelling resistance has been successfully applied to underwater sensors for monitoring frog swimming and underwater communication. These results reveal new possibilities for amphibious applications of wearable sensors.

Ahf-Caltide, a Novel Polypeptide Derived from Calpastatin, Protects against Oxidative Stress Injury by Stabilizing the Expression of CaV1.2 Calcium Channel.

Reperfusion after ischemia would cause massive myocardial injury, which leads to oxidative stress (OS). Calcium homeostasis imbalance plays an essential role in myocardial OS injury. CaV1.2 calcium channel mediates calcium influx into cardiomyocytes, and its activity is modulated by a region of calpastatin (CAST) domain L, CSL54-64. In this study, the effect of Ahf-caltide, derived from CSL54-64, on myocardial OS injury was investigated. Ahf-caltide decreased the levels of LDH, MDA and ROS and increased heart rate, coronary flow, cell survival and SOD activity during OS. In addition, Ahf-caltide permeated into H9c2 cells and increased CaV1.2, CaVß2 and CAST levels by inhibiting protein degradation. At different Ca2+ concentrations (25 nM, 10 µM, 1 mM), the binding of CSL to the IQ motif in the C terminus of the CaV1.2 channel was increased in a H2O2 concentration-dependent manner. CSL54-64 was predicted to be responsible for the binding of CSL to CaV1.2. In conclusion, Ahf-caltide exerted a cardioprotective effect on myocardial OS injury by stabilizing CaV1.2 protein expression. Our study, for the first time, proposed that restoring calcium homeostasis by targeting the CaV1.2 calcium channel and its regulating factor CAST could be a novel treatment for myocardial OS injury.

Binding characteristics of calpastatin domain L to NaV1.5 sodium channel and its IQ motif mutants.

NaV1.5 channel is an integral membrane protein involved in the initiation and conduction of action potentials. IQ motif is located in the C-terminal domain of NaV1.5 sodium channel, which is highly conserved in human sodium channel subtypes. IQ motif is involved in the Ca2+-dependent regulation through interaction with the regulatory proteins such as calpastatin domain L (CSL). Mutations in SCN5A, the gene encoding NaV1.5 channel, have been linked to many cardiac arrhythmias, such as Long QT syndrome type 3 (LQT3) and Brugada syndrome (BRS). LQT3-associated mutations in NaV1.5 IQ motif, IQQ1909R and IQR1913H, have been reported to affect the late INa. A BRS-associated mutation in NaV1.5 IQ motif, IQA1924T, has been reported to affect the peak INa. But the detailed pathogenic mechanisms of LQT3 and BRS remains unclear. To explore the binding properties of CSL to IQ motif and its muants associated with LQT3/BRS, molecular docking and GST pull down assay were performed in this study. As a result, S58 and E59 in CSL activating channel effect region L54-64 were involved in the conformation of the CSL/IQWT complex by protein-protein docking. IQ motif could bind to CSL in a [CSL]-dependent and [Ca2+]-dependent manner by pull down assay. However, the binding affinities of IQQ1909R and IQR1913H to CSL were decreased and its reaction rates with CSL were slower. The binding characteristics of IQA1924T to CSL was opposite in a [Ca2+]-dependent manner and its binding efficacy became smaller. The changes of the binding characteristics of IQmutants to CSL would affect the regulation of NaV1.5 channel, which may be related to LQT3 and BRS.

FcεRI deficiency alleviates silica-induced pulmonary inflammation and fibrosis.

Silicosis is one of the most important occupational diseases worldwide, caused by inhalation of silica particles or free crystalline silicon dioxide. As a disease with high mortality, it has no effective treatment and new therapeutic targets are urgently needed. Recent studies have identified FCER1A, encoding α-subunit of the immunoglobulin E (IgE) receptor FcεRI, as a candidate gene involved in the biological pathways leading to respiratory symptoms. FcεRI is known to be important in allergic asthma, but its role in silicosis remains unclear. In this study, serum IgE concentrations and FcεRI expression were assessed in pneumoconiosis patients and silica-exposed mice. The role of FcεRI was explored in a silica-induced mouse model using wild-type and FcεRI-deficient mice. The results showed that serum IgE concentrations were significantly elevated in both pneumoconiosis patients and mice exposed to silica compared with controls. The mRNA and protein expression of FcεRI were also significantly increased in the lung tissue of patients and silica-exposed mice. FcεRI deficiency significantly attenuated the changes in lung function caused by silica exposure. Silica-induced elevations of IL-1ß, IL-6, and TNF-α were significantly attenuated in the lung tissue and bronchoalveolar lavage fluid (BALF) of FcεRI-deficient mice compared with wild-type controls. Additionally, FcεRI-deficient mice showed a significantly lower score of pulmonary fibrosis than wild-type mice following exposure to silica, with significantly lower hydroxyproline content and expression of fibrotic genes Col1a1 and Fn1. Immunofluorescent staining suggested FcεRI mainly on mast cells. Mast cell degranulation took place after silica exposure, as shown by increased serum histamine levels and ß-hexosaminidase activity, which were significantly reduced in FcεRI-deficient mice compared with wild-type controls. Together, these data showed that FcεRI deficiency had a significant protective effect against silica-induced pulmonary inflammation and fibrosis. Our findings provide new insights into the pathophysiological mechanisms of silica-induced pulmonary fibrosis and a potential target for the treatment of silicosis.

Distinct roles of calmodulin and Ca2+/calmodulin-dependent protein kinase II in isopreterenol-induced cardiac hypertrophy.

Intracellular calcium is related to cardiac hypertrophy. The CaV1.2 channel and Ca2+/calmodulin-dependent protein kinase II (CaMKII) and CaM regulate the intracellular calcium content. However, the differences in CaMKII and CaM in cardiac hypertrophy are still conflicting and are worthy of studying as drug targets. Therefore, in this study, we aim to investigate the roles and mechanism of CaM and CaMKII on CaV1.2 in pathological myocardial hypertrophy. The results showed that ISO stimulation caused SD rat heart and cardiomyocyte hypertrophy. In vivo, the HW/BW, LVW/BW, cross-sectional area, fibrosis ratio and ANP expression were all increased. There were no differences in CaV1.2 channel expression in the in vivo model or the in vitro model, but the ISO stimulation induced channel activity, and the [Ca2+]i increased. The protein expression levels of CaMKII and p-CaMKII were all increased in the ISO group, but the CaM expression level decreased. AIP inhibited ANP, CaMKII and p-CaMKII expression, and ISO-induced [Ca2+]i increased. AIP also reduced HDAC4, p-HDAC and MEF2C expression. However, CMZ did not play a cardiac hypertrophy reversal role in vitro. In conclusion, we considered that compared with CaM, CaMKII may be a much more important drug target in cardiac hypertrophy reversal.

A potent antiarrhythmic drug N-methyl berbamine extends the action potential through inhibiting both calcium and potassium currents.

N-methyl berbamine (N-MB) is a berberine derivative. Its analogue berbamine has been reported to have remarkable antiarrhythmic and ischemic protective effects. However, the pharmacological effects of N-MB are ill-defined. In this study, molecular docking was used to evaluate the binding of N-MB to CaV1.2 Ca2+ and KV11.1 K+ channels, and the effects of N-MB on action potential and ionic currents were observed in the ventricular myocytes of rabbits, HEK293 cells stably transfected with the hCaV1.2 gene and CHO cells stably transfected with hERG (human ether-a-go-go related gene). The results showed that N-MB was able to bind to both CaV1.2 and KV11.1 channels. Following a perfusion with N-MB, the durations of action potentials (APD20, APD50 and APD90) were extended, and the outward tail current, Itail, as well as the hERG current, IhERG, were inhibited, while the amplitude of action potential (APA) was only slightly reduced. N-MB also decreased the peak amplitude of the L-type Ca2+ channel current, ICaL, as well as the CaV1.2 current, ICaV1.2; this may limit the prolongation of APD. In conclusion, N-MB is a potent and natural antiarrhythmic multitarget drug that may elicit its antiarrhythmic effect through blocking both Ca2+ and K+ channel currents.

Sustained increased CaMKII phosphorylation is involved in the impaired regression of isoproterenol-induced cardiac hypertrophy in rats.

To understand the mechanism underlying the regression of cardiac hypertrophy, we investigated the pathological changes after isoproterenol (ISO) withdrawal in ISO-induced cardiomyopathy models in rats and neonatal cardiomyocytes. Cardiac hypertrophy was induced in rats by two weeks of ISO administration; however, the hypertrophy did not regress after three weeks of natural maintenance after ISO administration was withdrawn (ISO-wdr group). The remaining hypertrophy in the ISO-wdr group was accompanied by a sustained increase in the level of phosphorylated Ca2+/calmodulin-dependent protein kinase II (p-CaMKII). Additionally, the increased expression levels of histone deacetylase 4 (HDAC4) and the CaV1.2 channel and amounts of CaMKII bound with HDAC4 and CaV1.2 were not recovered in the ISO-wdr group. The results in cardiomyocyte models were similar to those seen in rat models. Losartan, metoprolol or amlodipine neither ameliorated the increase in atrial natriuretic peptide nor inhibited the increase in p-CaMKII and bound CaMKII. In contrast, autocamtide-2-related inhibitor peptide, a CaMKII inhibitor, reduced these increases. This study investigated the phosphorylation status of CaMKII after hypertrophic stimulus was withdrawn for the first time and proposed that CaMKII as well as its complexes with CaV1.2 could be potential targets to achieve effective regression of cardiac hypertrophy.

The Effect of Ca2+, Lobe-Specificity, and CaMKII on CaM Binding to NaV1.1.

Calmodulin (CaM) is well known as an activator of calcium/calmodulin-dependent protein kinase II (CaMKII). Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are crucial molecular targets for nervous system agents. However, the way in which Ca2+/CaM/CaMKII cascade modulates NaV1.1 IQ (isoleucine and glutamine) domain of VGSCs remains obscure. In this study, the binding of CaM, its mutants at calcium binding sites (CaM12, CaM34, and CaM1234), and truncated proteins (N-lobe and C-lobe) to NaV1.1 IQ domain were detected by pull-down assay. Our data showed that the binding of Ca2+/CaM to the NaV1.1 IQ was concentration-dependent. ApoCaM (Ca2+-free form of calmodulin) bound to NaV1.1 IQ domain preferentially more than Ca2+/CaM. Additionally, the C-lobe of CaM was the predominant domain involved in apoCaM binding to NaV1.1 IQ domain. By contrast, the N-lobe of CaM was predominant in the binding of Ca2+/CaM to NaV1.1 IQ domain. Moreover, CaMKII-mediated phosphorylation increased the binding of Ca2+/CaM to NaV1.1 IQ domain due to one or several phosphorylation sites in T1909, S1918, and T1934 of NaV1.1 IQ domain. This study provides novel mechanisms for the modulation of NaV1.1 by the Ca2+/CaM/CaMKII axis. For the first time, we uncover the effect of Ca2+, lobe-specificity and CaMKII on CaM binding to NaV1.1.