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BackgroundIncreased small-conductance Ca2+-activated K+ current (SK), abnormal intracellular Ca2+ handling, and enhanced expression and activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been found in clinical and/or experimental models of atrial fibrillation (AF), but the cumulative effect of these phenomena and their mechanisms in AF are still unclear. This study aimed to test the hypothesis that CaMKII increases SK current in human chronic AF.Material/MethodsRight atrial appendage tissues from patients with either sinus rhythm (SR) or AF and neonatal rat atrial myocytes were used. Patch clamp, qRT-PCR, and Western blotting techniques were used to perform the study.ResultsCompared to SR, the apamin-sensitive SK current (IKAS) was significantly increased, but the mRNA and protein levels of SK1, SK2, and SK3 were significantly decreased. In AF, the steady-state Ca2+ response curve of IKAS was shifted leftward and the [Ca2+]i level was significantly increased. CaMKII inhibitors (KN-93 or autocamtide-2-related inhibitory peptide (AIP)) reduced the IKAS in both AF and SR. The inhibitory effect of KN-93 or AIP on IKAS was greater in AF than in SR. The expression levels of calmodulin, CaMKII, and autophosphorylated CaMKII at Thr287 (but not at Thr286) were significantly increased in AF. Furthermore, KN-93 inhibited the expression of (Thr287)p-CaMKII and SK2 in neonatal rat atrial myocytes.ConclusionsSK current is increased via the enhanced activation of CaMKII in patients with AF. This finding may explain the difference between SK current and channels expression in AF, and thus may provide a therapeutic target for AF.

Previous studies have shown that propofol, an intravenous anesthetic commonly used in clinical practice, protects the myocardium from injury. Mitochondria- and endoplasmic reticulum (ER)-mediated oxidative stress and apoptosis are two important signaling pathways involved in myocardial injury and protection. The present study aimed to test the hypothesis that propofol could exert a cardio-protective effect via the above two pathways. Cultured neonatal rat cardiomyocytes were treated with culture medium (control group), HO at 500 μM (HO group), propofol at 50 μM (propofol group), and HO plus propofol (HO + propofol group), respectively. The oxidative stress, mitochondrial membrane potential (ΔΨm) and apoptosis of the cardiomyocytes were evaluated by a series of assays including ELISA, flow cytometry, immunofluorescence microscopy and Western blotting. Propofol significantly suppressed the HO-induced elevations in the activities of caspases 3, 8, 9 and 12, the ratio of Bax/Bcl-2, and cell apoptosis. Propofol also inhibited the HO-induced reactive oxygen species (ROS) generation, lactic dehydrogenase (LDH) release and mitochondrial transmembrane potential (ΔΨm) depolarization, and restored the HO-induced reductions of glutathione (GSH) and superoxide dismutase (SOD). In addition, propofol decreased the expressions of glucose-regulated protein 78 kDa (Grp78) and inositol-requiring enzyme 1α (IRE1α), two important signaling molecules in the ER-mediated apoptosis pathway. Propofol protects cardiomyocytes from HO-induced injury by inhibiting the mitochondria- and ER-mediated apoptosis signaling pathways.

Dexmedetomidine (DEX), an α2 adrenoceptor agonist, has sedative and analgesic properties and myocardial protective effects. However, the mechanism underlying the protective effects of DEX on the myocardium remain unclear. The present study aimed to determine whether DEX serves an important role on cardioprotection through the endoplasmic reticulum (ER)- and mitochondria-mediated apoptosis signaling pathways. Neonatal rat cardiomyocytes (NRCMs) were cultured and divided four groups: i) Normal culture medium with 10% fetal bovine serum (control group); ii) H2O2 at 500 µM (H2O2 group); iii) DEX at 5 µM (DEX group); and iv) H2O2 plus DEX (H2O2 + DEX group). The levels of apoptosis and oxidative stress of NRCMs were investigated by ELISA, western blotting, flow cytometry and cell immunofluorescence. DEX significantly suppressed H2O2-induced apoptosis, and increased activity of caspases 3, 8 and 9 of NRCMs. DEX inhibited mitochondria-mediated oxidative stress and apoptosis, as evidenced by decreased levels of reactive oxygen species and lactic dehydrogenase, alleviated mitochondrial membrane potential depolarization, and increased Bcl-2-associated X protein/B-cell lymphoma 2 ratio. In addition, DEX decreased the activity of caspase 12, and the expression levels of glucose-regulated protein 78 kDa and serine/threonine-protein kinase/endoribonuclease IRE1, three major signaling molecules involved in the ER stress-mediated apoptosis pathway. Preventive treatment with DEX alleviates cardiomyocyte against H2O2-induced oxidative stress injury through attenuating the mitochondria- and ER-mediated apoptosis pathways.

Resveratrol (RES) is a naturally occurring antioxidant compound found in red wine. Although it has been demonstrated to have a cardioprotective effect, the mechanism underlying this effect remains to be fully elucidated. The aim of the present study was to determine whether RES exerts a protective effect against mitochondrial oxidative stress and apoptosis in neonatal rat cardiomyocytes (NRCMs) induced by hypoxia/reoxygenation (H/R) injury. Primary cultured NRCMs were used as a model system and were divided into four experimental groups: Control, H/R, H/R + DMSO (H/R with 0.2% DMSO) and H/R + RES (H/R with 100 µM RES) groups. Mitochondrial oxidative stress was determined by measuring the alteration in the mitochondrial membrane potential (ΔΨm) of NRCMs, the release of lactate dehydrogenase (LDH) and the ratio of B-cell lymphoma 2 (Bcl-2)/Bcl-2-associated X protein (Bax) from NRCMs. Cell apoptosis was assessed by measuring cell apoptotic rates and the activity of caspase 3. In the H/R+RES group, RES significantly alleviated structural impairment, including disordered α-actin and F-actin, in the NRCMs induced by H/R injury. RES attenuated H/R injury-induced mitochondria oxidative stress. RES also attenuated H/R injury-induced cell apoptosis; it decreased the NRCM apoptotic rate from 84.25±7.41% (H/R) to 46.39±5.43% (H/R+RES) (P<0.05, n=4), rescued the decrease in the Bcl2/Bax ratio induced by H/R from 0.53±0.08-fold (H/R) to 0.86±0.06-fold (H/R+RES) (P<0.05, n=5) and alleviated the increased activity of caspase 3 induced by H/R from 1.32±0.06-fold to 1.02±0.04-fold (P<0.05, n=5). Furthermore, RES significantly attenuated the increment of LDH release induced by H/R injury in NRCMs from 1.41±0.03-fold (H/R) to 1.02±0.06-fold (H/R+RES) (P<0.01, n=4) and alleviated the depolarization of ΔΨm induced by H/R, shifting the ratio of JC-1 monomer from 62.39±1.82% (H/R) to 35.31±8.63% (H/R+RES) (P<0.05, n=4). RES alleviated the decrease in sirtuin 1 induced by H/R injury from 0.61±0.06-fold (H/R) to 1.01±0.05-fold (H/R+RES) (P<0.05, n=5). In conclusion, the present study is the first, to the best of our knowledge, to demonstrate that RES provides cardioprotection against H/R injury through decreasing mitochondria-mediated oxidative stress injury and structural impairment in NRCMs. These results provide scientific evidence for the clinical application of RES in the treatment of cardiac conditions.

Action potential (AP) induces presynaptic membrane depolarization and subsequent opening of Ca2+ channels, and then triggers neurotransmitter release at the active zone of presynaptic terminal. Presynaptic Ca2+ channels and SNARE proteins (SNAREs) interactions form a large signal transfer complex, which are core components for exocytosis. Ca2+ channels serve to regulate the activity of Ca2+ channels through direct binding and indirect activation of active zone proteins and SNAREs. The activation of Ca2+ channels promotes synaptic vesicle recruitment, docking, priming, fusion and neurotransmission release. Intracellular calcium increase is a key step for the initiation of vesicle fusion. Various voltage-gated calcium channel (VGCC) subtypes exert different physiological functions. Until now, it has not been clear how different subtypes of calcium channels integrally regulate the release of neurotransmitters within 200 μs of the AP arriving at the active zone of synaptic terminal. In this mini review, we provide a brief overview of the structure and physiological function of Ca2+ channel subtypes, interactions of Ca2+ channels and SNAREs in neurotransmitter release, and dynamic fine-tune Ca2+ channel activities by G proteins (Gβγ), multiple protein kinases and Ca2+ sensor (CaS) proteins.

Fibroblast proliferation and migration are central in atrial fibrillation (AF) promoting structure remodeling, which is strongly associated with aging and hypertension. Transient receptor potential canonical-3 channel (TRPC3) is a key mediator of cardiac fibrosis and the pathogenesis of AF. Here, we have observed the increased TRPC3 expression that induced atrial fibrosis which possibly is either mediated by the aging process or related to hypertensive progression. In this study, we measured the pathological structure remodeling by H&E staining, Masson staining, and transmission electron microscope (TEM). The protein expression levels of fibrotic biomarkers and TRPC3 were measured by Western blotting with atrial tissues from normotensive Wistar Kyoto rats (WKY 4m-o (4 months old)), old WKY (WKY 24m-o (24 months old)), spontaneously hypertensive rat (SHR 4m-o (4 months old)), and old SHR (SHR 24m-o (24 months old)). To illuminate the molecular mechanism of TRPC3 in atrial fibrosis of aging rats and SHR, we detected the inhibited role of TRPC3 selective blocker ethyl-1-(4-(2,3,3-trichloroacrylamide) phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate,pyrazole-3 (Pyr3) on angiotensin II (Ang II) induced fibrosis in neonatal rat atrial fibroblasts. The pathological examination showed that the extracellular matrix (ECM) and collagen fibrils were markedly increased in atrial tissues from aged and hypertensive rats. The protein expressions of fibrotic biomarkers (collagen I, collagen III, and transforming growth factor-β1 (TGF-β1)) were significantly upregulated in atrial tissues from the WKY 24m-o group, SHR 4m-o group, and SHR 24m-o group compared with the WKY 4m-o group. Meanwhile, the expression level of TRPC3 was significantly upregulated in WKY 24m-o and SHR 4m-o atrial tissues compared to WKY 4m-o rats. In isolated and cultured neonatal rat atrial fibroblasts, Ang II induced the atrial fibroblast migration and proliferation and upregulated the expression levels of TRPC3 and fibrotic biomarkers. TRPC3 selected blocker Pyr3 attenuated the migration and proliferation in neonatal rat atrial fibroblasts. Furthermore, Pyr3 significantly alleviated Ang II-induced upregulation of TRPC3, collagen I, collagen III, and TGF-β1 through the molecular mechanism of the TGF-β/Smad2/3 signaling pathway. Similarly, knocking down TRPC3 using short hairpin RNA (shTRPC3) also attenuated Ang II-induced upregulation of TGF-β1. Pyr3 preconditioning decreased Ang II-induced intracellular Ca2+ transient amplitude elevation. Furthermore, AT1 receptor was involved in Ang II-induced TRPC3 upregulation. Hence, upregulation of TRPC3 in aging and hypertension is involved in an atrial fibrosis process. Inhibition of TRPC3 contributes to reverse Ang II-induced fibrosis. TRPC3 may be a potential therapeutic target for preventing fibrosis in aging and hypertension.

The limited sensitivity of Förster Resonance Energy Transfer (FRET) biosensors hinders their broader applications. Here, we develop an approach integrating high-throughput FRET sorting and next-generation sequencing (FRET-Seq) to identify sensitive biosensors with varying substrate sequences from large-scale libraries directly in mammalian cells, utilizing the design of self-activating FRET (saFRET) biosensor. The resulting biosensors of Fyn and ZAP70 kinases exhibit enhanced performance and enable the dynamic imaging of T-cell activation mediated by T cell receptor (TCR) or chimeric antigen receptor (CAR), revealing a highly organized ZAP70 subcellular activity pattern upon TCR but not CAR engagement. The ZAP70 biosensor elucidates the role of immunoreceptor tyrosine-based activation motif (ITAM) in affecting ZAP70 activation to regulate CAR functions. A saFRET biosensor-based high-throughput drug screening (saFRET-HTDS) assay further enables the identification of an FDA-approved cancer drug, Sunitinib, that can be repurposed to inhibit ZAP70 activity and autoimmune-disease-related T-cell activation.