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Patchouli oil has exhibited remarkable efficacy in the treatment of colitis. However, its volatility and potential irritancy are often drawbacks when extensively used in clinical applications. Oil gel is a semisolid and thermoreversible system that has received extensive interest for its solubility enhancement, inhibition of bioactive component recrystallization, and the facilitation of controlled bioactive release. Therefore, we present a strategy to develop an oil gel formulation that addresses this multifaceted problem. Notably, a patchouli oil gel formulation was designed to solidify and trap patchouli oil into a spatially stable crystal-particle structure and colonic released delivery, which has an advantage of the stable structure and viscosity. The patchouli oil gel treatment of zebrafish with colitis improved goblet cells and decreased macrophages. Additionally, patchouli oil gel showed superior advantages for restoring the tissue barrier. Furthermore, our investigative efforts unveiled patchouli oil’s influence on TRP channels, providing evidence for its potential role in mechanisms of anti-inflammatory action. While the journey continues, these preliminary revelations provide a robust foundation for considering the adoption of patchouli oil gel as a pragmatic intervention for managing colitis.

N-methyl-D-aspartate receptor (NMDAR)-mediated glutamate excitotoxicity significantly contributes to ischemic neuronal death and post-recanalization infarction expansion. Despite tremendous efforts, targeting NMDARs has proven unsuccessful in clinical trials for mitigating brain injury. Here, we show the discovery of an interaction motif for transient receptor potential melastatin 2 (TRPM2) and protein kinase Cγ (PKCγ) association and demonstrate that TRPM2-PKCγ uncoupling is an effective therapeutic strategy for attenuating NMDAR-mediated excitotoxicity in ischemic stroke. We demonstrate that the TRPM2-PKCγ interaction allows TRPM2-mediated Ca2+influx to promote PKCγ activation, which subsequently enhances TRPM2-induced potentiation of extrasynaptic NMDAR (esNMDAR) activity. By identifying the PKCγ binding motif on TRPM2 (M2PBM), which directly associates with the C2 domain of PKCγ, an interfering peptide (TAT-M2PBM) is developed to disrupt TRPM2-PKCγ interaction without compromising PKCγ function. M2PBM deletion or TRPM2-PKCγ dissociation abolishes both TRPM2-PKCγ and TRPM2-esNMDAR couplings, resulting in reduced excitotoxic neuronal death and attenuated ischemic brain injury.

Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However, applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here, we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines, we found that mutations in CDKAL1, KCNQ1, and KCNJ11 led to impaired glucose secretion in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform, which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects, paves the way for precision therapy of metabolic diseases.

Introduction:Ischemic heart disease is the leading cause of death in the USA. Despite the progress of interventional coronary reperfusion strategies, myocardial ischemia reperfusion (I/R) injury remains a substantial challenge for treatment. TRPM7 is a Ca2+-permeable nonselective channel which plays an important role in cardiogenesis and atrial fibrillation. However the role of TRPM7 in pathogenesis of myocardial I/R is unknown.Hypothesis:TRPM7 contributes to myocardial I/R injury.Methods:TRPM7 knockout mice (M7KO) and Cre-control littermates (WT) mice were exposed to 45 min ischemia or sham procedure followed by 24 hrs of reperfusion (n=8 to 12/group). Heart function was evaluated by echocardiograph, and in vitro assays were used for mechanistic investigation.Results:We found that TRPM7 expression was upregulated in I/R injury WT mouse hearts. Global M7KO (gM7KO) improved heart performance via increasing the ejection fraction and fraction shortening by 29.1 ± 4.2 % and 10.8 ± 3.0 % respectively (p<0.05). Trpm7deletion also reduced the infarct size by 31.1 ± 1.2 % in comparison with WT mice (P<0.05). Myocytes specific knockout (mM7KO) exhibited similar protective effect against I/R injury. Moreover, deletion of Trpm7significantly reduced tunnel positive cells, suggesting that upregulated TRPM7 in WT mice contributed to myocyte apoptosis in I/R injury. Interestingly, deletion of Trpm7also significantly reduced the protein level of NLRP3, cleaved-caspase-1 and mature IL-1β after I/R injury (p<0.05), indicating that TRPM7 contributed to pyroptosis during I/R injury. Consistent with the increased IL-1β level induced by I/R injury in WT mice, the cleaved N-terminal Gasdermin D (N-GSDMD) level was also increased in WT I/R injury hearts versus sham control hearts. Deletion of Trpm7significantly attenuated the increase of the cleaved N-GSDMD level induced by I/R injury (p<0.05).Conclusions:Trpm7deletion in cardiac myocytes protects mice against myocardial ischemic injury. This is the first study to demonstrate the important role of TRPM7 in I/R injury through the mechanisms of activation of both apoptosis and pyroptosis. Our results suggest that TRPM7 may serve as a novel therapeutic target for myocardial ischemia/reperfusion injury.

The vast potential of human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs) in preclinical models of cardiac pathologies, precision medicine, and drug screening remains to be fully realized because hiPSC-CMs are immature without adult-like characteristics. Here, we present a method to accelerate hiPSC-CM maturation on a substrate, cardiac mimetic matrix (CMM), mimicking adult human heart matrix ligand chemistry, rigidity, and submicron ultrastructure, which synergistically mature hiPSC-CMs rapidly within 30 days. hiPSC-CMs matured on CMM exhibit systemic transcriptomic maturation toward an adult heart state, are aligned with high strain energy, metabolically rely on oxidative phosphorylation and fatty acid oxidation, and display enhanced redox handling capability, efficient calcium handling, and electrophysiological features of ventricular myocytes. Endothelin-1-induced pathological hypertrophy is mitigated on CMM, highlighting the role of a native cardiac microenvironment in withstanding hypertrophy progression. CMM is a convenient model for accelerated development of ventricular myocytes manifesting highly specialized cardiac-specific functions.

Introduction:Atherosclerosis and its major complications, ischemic heart diseases and stroke, are the leading causes of mortality worldwide. The prominent feature of atherosclerosis is the formation of foam cells, the infiltrated macrophages overloaded with lipids. Lipid uptake by macrophages is mainly mediated by CD36.Hypothesis:Previously we reported that: (1) global Trpm2deletion attenuates atherosclerosis; (2), TRPM2 and CD36 enhance the activation of each other in macrophages, thereby promoting foam cell formation. Thus, we hypothesized that macrophage specific Trpm2deletion protects mice against atherosclerosis.Methods:Macrophage-specific Trpm2deletion in was achieved by crossing Apoe-/-/ Trpm2fl/flmice with Cd11b-cremice. High-fat diet (HFD) was used to induce atherosclerosis, and total serum cholesterol level was measured. Oil Red O (ORO) staining was used to evaluate atherosclerotic lesion size. Immunofluorescence staining of Mac-1, F4/80 and CD80 was used to examine the macrophage content in the plaques, which was further confirmed by flow cytometry analysis of the immune cell populations in digested aortas. Inflammation of the aortas was examined by WB analysis of expression of NLRP3, ASC, Caspase-1, IL-1β, macrophage chemoattractant protein-1 (MCP1) and migration-inhibitory factor (MIF).Results:Macrophage-specific Trpm2deletion: (1) did not influence the serum total cholesterol level; (2) decreased plaque lesion ratio as shown by en-face aorta ORO staining and reduced plaque size as shown by aortic root ORO staining; (3) reduced macrophage content and immune cell infiltration in the plaques; (4) inhibited NLRP3 inflammasome activation and inflammatory cytokines expression in the atherosclerotic aortas.Conclusion:Macrophage specific Trpm2deletion attenuates HFD-induced atherosclerosis in Apoe-/-mice.

Cardiac fibrosis is associated with most cardiac diseases. Fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) synthesized by cardiac fibroblasts and myofibroblasts. Fibroblasts are the most prevalent cell type in the heart, comprising 75 of cardiac cells. Myofibroblasts are hardly present in healthy normal heart tissue, but appear abundantly in diseased hearts. Cardiac fibroblasts are activated by a variety of pathological stimuli, such as myocardial injury, oxidative stress, mechanical stretch, and elevated autocrine-paracrine mediators, thereby undergoing proliferation, differentiation to myofibroblasts, and production of various cytokines and ECM proteins. A number of signaling pathways and bioactive molecules are involved and work in concert to activate fibroblasts and myofibroblasts in the fibrogenesis cascade. Fibroblasts and myofibroblasts are not only principal ECM producers, but also play a central role in fibrogenesis and myocardial remodeling in fibrotic heart disease. Thus, understanding the biological processes of cardiac fibroblasts will provide novel insights into the underlying mechanisms of fibrosis and provide potential targets for developing antifibrotic drugs. Recent studies demonstrate that Ca2 signal is essential for fibroblast proliferation, differentiation, and ECM-protein production. This review focuses on the recent advances in understanding molecular mechanisms of Ca2 signaling in cardiac fibrogenesis, and potential role of Ca2-permeable channels, in particular, the transient potential (TRP) channels in fibrotic heart disease. TRP channels are highly expressed in cardiac fibroblasts. TRPM7 has been shown to be essential in TGF1 mediated fibrogenesis, and TRPC3 has been demonstrated to play an essential role in regulating fibroblast function. Thus, the Ca2-permeable TRP channels may serve as potential novel targets for developing anti-fibrotic drugs.

Supravalvular aortic stenosis (SVAS) is caused by mutations in the elastin (ELN) gene and is characterized by abnormal proliferation of vascular smooth muscle cells (SMCs) that can lead to narrowing or blockage of the ascending aorta and other arterial vessels. Having patient-specific SMCs available may facilitate the study of disease mechanisms and development of novel therapeutic interventions.

Introduction:Atherosclerosis and its complications are the leading cause of death. The central pathological features of atherosclerosis are macrophage infiltration and foam cell formation, in which CD36 is crucial in activating macrophages and mediating the uptake of cholesterol. However, mechanisms regulating the activation of CD36 remain unclear. TRPM2 is an Ca2+permeable channel activated by oxidative stress, which occurs in the activation of macrophages.Hypothesis:We hypothesized that during atherosclerosis, TRPM2 is critical for the activation of CD36 in macrophages. Thus, Trpm2deletion inhibits macrophage activation and foam cell formation.Methods:ApoE-/-mice and ApoE-/-mice with Trpm2deletion were fed with high-fat food to induce atherosclerosis. Oil red O and immunofluorescence staining were performed to evaluate lipid deposition and macrophage load in aorta. Transwell assay was used to test macrophage infiltration and emigration. Oxidized LDL (oxLDL) or thrombospondin-1 (TSP1) was used to activate CD36. Western blot was used to examine the activation of CD36 signaling. ROS and Ca2+imaging were performed to examine the activation of macrophages by CD36.Results:Trpm2deletion: (1) reduced lesion ratio (0.13 ± 0.02 vs 0.36 ± 0.02) and macrophage load (20.93 ± 4.82 / mm2vs 60.20 ± 8.36 / mm2) in aorta; (2) inhibited oxLDL uptake (32.24 ± 7.65 % vs 100 %) by macrophages; (3) suppressed macrophage infiltration (15.83 ± 2.44 / mm2vs 46.83 ± 4.62 / mm2) and preserved macrophage emigration (15.5 ± 1.73 / mm2vs 5.0 ± 0.73 / mm2); (4) inhibited ROS production (5.32 ± 0.61 % vs 28.82 ± 1.48 %) and increase of intracellular Ca2+(2.92 ± 0.86 % vs 11.95 ± 1.60 %) in macrophages induced by oxLDL (n= 6 ~ 8 for all data). Also, oxLDL and TSP1 activated TRPM2 via CD36, and TRPM2 was required for the activation of CD36 signaling in macrophages by oxLDL or TSP1.Conclusions:In summary, TRPM2 and CD36 activated each other in macrophages, which promoted the development of atherosclerosis.

1We previously described an ultrarapid delayed rectifier current in dog atrial myocytes (IKur,d) with properties resembling currents reported for Kv3.1 channels in neural tissue; however, there was no direct molecular evidence for Shawsubfamily (Kv3) subunit expression in the heart. To identify the molecular basis of IKur,d, we cloned a full‐length cDNA (dKv3.1) from canine atrium with homology‐based reverse transcription (RT)‐ polymerase chain reaction (PCR) cloning techniques.2A 1755 bp full‐length cDNA (dKv3.1) was obtained, with 94.2 % homology to rat brain Kv3.1 (rbKv3.1). The deduced amino acid sequence had 99.3 % homology with rbKv3.1.3Heterologous expression of dKv3.1 in Xenopusoocytes produced currents with activation voltage dependence, rectification, and activation and deactivation kinetics that strongly resemble native IKur,d. Like IKur,d, dKv3.1 was found to be highly sensitive to extracellular 4‐aminopyridine (4‐AP) and tetraethylammonium (TEA).4RNase protection assays, Western blots and immunohistochemical studies demonstrated the presence of dKv3.1 transcripts and proteins in dog atrial preparations and isolated canine atrial myocytes. Protein corresponding to the Kv1.5 subunit, which can also carry ultrarapid delayed rectifier current, was absent. Unlike neural tissues, which express two splice variants (Kv3.1a and Kv3.1b), canine atrium showed only Kv3.1b transcripts.5Whole‐cell patch‐clamp studies showed that IKur,dis absent in canine ventricular myocytes, and immunohistochemical and Western blot analysis demonstrated the absence of dKv3.1 protein in canine ventricle.6We conclude that the Shaw‐type channel dKv3.1 is present in dog atrium, but not ventricle, and is the likely molecular basis of canine atrial IKur,d.

1The effects of adrenergic stimulation on the ultrarapid delayed rectifier K+current (IKur,d) of dog atrial myocytes was studied with patch‐clamp methods.2Isoproterenol (isoprenaline) increased IKur,din a concentration‐dependent fashion with an EC50of 7·3 ± 0·8 nM. The effect of isoproterenol was blocked by propranolol, mimicked by forskolin and 8‐bromo‐cAMP, and prevented by inhibition of protein kinase A.3Phenylephrine (in the presence of propranolol) increased IKur,dwith an EC50of 0·49 ± 0·06 μM. The effect of phenylephrine was blocked by prazosin, prevented by inhibition of protein kinase C, and mimicked by activation of protein kinase C with phorbol ester.4Phenylephrine significantly abbreviated canine atrial action potential duration in the absence of tetraethylammonium (TEA). When TEA was present under both control conditions and in the presence of phenylephrine, phenylephrine failed to alter canine atrial repolarization.5We conclude that β‐ and α‐adrenergic stimulation increase IKur,dvia protein kinase A and C, respectively, and that the induced changes in IKur,dmay play a role in adrenergic control of canine atrial repolarization.

Classical cardiac delayed rectifier currents activate at least two orders of magnitude slower than delayed rectifier currents in nerve and skeletal muscle tissue. It has recently become evident that many cardiac tissues express delayed rectifier currents with kinetics similar to those of nerve and muscle. These currents have been designated IKur (for ‘ultrarapid’ delayed rectifier), in contrast to the classical cardiac rapid (IKr) and slow (IKs) delayed rectifier components. Although the kinetics of IKur in different species and tissues are similar, their pharmacological properties vary greatly. It now appears that the differences among various IKurs are due to differences in the molecular basis. A variety of Shaker-related clones (Kv1.2, 1.5, 2.1 and 3.1) that form IKur channels upon heterologous expression have been identified with specific IKurs (e.g. Kv1.2, rat atrium; Kv1.5, mouse ventricle and human atrium; Kv3.1, dog atrium). The present article reviews the distribution, biophysical and pharmacological properties, molecular basis and functional role of IKur, as well as the potential value of IKur as a target for new antiarrhythmic drug development.

Objective: A dog atrial ultra-rapid delayed rectifier current (IKur.d) is involved in canine atrial repolarization and shares similarities with the human atrial ultra-rapid delayed rectifier (IKur). Almost no information is available about the actions of antiarrhythmic drugs on IKur.d. This study evaluated effects of ambasilide, quinidine, flecainide and verapamil on IKur.d in isolated canine atrial myocytes. Methods: Standard whole-cell patch clamp techniques were used to study the effects of multiple concentrations of each drug. Results: All drugs produced reversible concentration-, voltage- and time-dependent IKur.d inhibition. Significant effects of quinidine, flecainide and ambasilide were noted at atrial-effective antiarrhythmic concentrations in the dog. Upon the onset of a depolarizing pulse, block developed exponentially in relation to time, with the blocking rate-constant increasing with drug concentration, consistent with open-channel blockade and permitting the calculation of forward and reverse rate-constants. For all drugs, the 50% blocking concentration (EC50) showed significant voltage-dependence, decreasing at more positive potentials. The magnitude of voltage-dependent block was directly related to the degree of drug-induced shift in the voltage dependence of activation (r=0.97), pointing to open-channel block as a mechanism for voltage-dependent action. An additional component of voltage-dependence suggested that blocking sites were subjected to 17–21% of the transmembrane voltage field. Conclusions: Ambasilide, quinidine, flecainide and verapamil inhibit IKur.d, with preferential action on the open state. IKur.d inhibition may play a role in antiarrhythmic effects in canine atrial arrhythmia models. Comparisons between the effects of these drugs on IKur.d and previously studied effects on IKur suggest potential opportunities for investigating the molecular structural determinants of drug-blocking action on atrial-specific ultrarapid delayed rectifiers.

Background: We have shown that rapid atrial activation, as occurs during atrial fibrillation (AF), reduces L-type Ca2+ current (ICa) and that this is the principal mechanism of the action potential duration and refractoriness changes that characterize tachycardia-induced atrial remodeling. The present study was designed to determine whether atrial tachycardia alters biochemical indices of the number of L-type Ca2+ channels and/or of the number and binding affinity of β-adrenergic receptors. Methods: In canine atrial sarcolemmal preparations, the number and binding affinity of dihydropyridine receptors were determined with the use of 3H-nitrendipine and that of β-adrenergic receptors with 125I-iodocyanopindolol. Results were obtained with preparations from dogs paced at 400/min for 1 (P1, n=20), 7 (P7, n=9), and 42 (P42, n=9) days, and compared with observations in sham-operated controls (P0, n=14). Results: Pacing reduced the Bmax of dihydropyridine receptors, from 157±18 fmol/mg (P0) to 116±9 fmol/mg (P1, P <0.05), 100±14 fmol/mg (P7, P <0.05) and 94±9 fmol/mg (P42, P <0 .01). The affinity of dihydropyridine receptors was unchanged, with the Kd averaging 711±102 pM, 656±74 pM, 633±155 pM and 585±92 pM in P0, P1, P7 and P42 dogs. Neither Bmax nor Kd of β-adrenergic receptors was altered by rapid pacing. Values of Bmax of dihydropyridine receptors correlated with atrial ICa current density (r2=0.95) and ERP (r2=0.99). Conclusions: Rapid atrial activation results in downregulation in the number of dihydropyridine receptors without altering the number or affinity of β-adrenergic receptors. The reductions in ICa that play an important role in the atrial electrical remodeling by which ‘AF begets AF’ appear to be due at least in part to a decrease in the number of L-type Ca2+ channels in cardiac cell membranes.