Calmodulin-dependent Kinase Research Articles

Immunofluorescent Localization of Ca²⁺-Sensor Proteins in The Somatic Motor Muscles of The Earthworm <i>Lumbricus terrestris</i>

The method of immunofluorescent staining of earthworm somatic muscle samples showed the presence of calmodulin, Ca²⁺-calmodulin-dependent protein kinases type 1 and type 2, synaptotagmin type 2 and type 7 and calcineurin A. These proteins are detected in both synaptic and extrasynaptic regions of the motor muscle. However, for synaptotagmin type 2 and type 7, calcineurin A, their predominant localization in the area of neuromuscular synapses has been established. Besides, synaptic localization for synaptotagmin 7 and calcineurin A is most clearly expressed.

Releasing a sugar brake generates sweeter tomato without yield penalty.

In tomato, sugar content is highly correlated with consumer preferences, with most consumers preferring sweeter fruit1-4. However, the sugar content of commercial varieties is generally low, as it is inversely correlated with fruit size, and growers prioritize yield over flavour quality5-7. Here we identified two genes,tomato(Solanum lycopersicum) calcium-dependent protein kinase 27 (SlCDPK27; also known as SlCPK27) and its paralogue SlCDPK26, that control fruit sugar content. They act as sugar brakes by phosphorylating a sucrose synthase, which promotes degradationof the sucrose synthase. Gene-edited SlCDPK27 and SlCDPK26 knockouts increased glucose and fructose contents by up to 30%, enhancing perceived sweetness without fruit weight or yield penalty. Although there are fewer, lighter seeds in the mutants, they exhibit normal germination. Together, these findings provide insight into the regulatory mechanisms controlling fruit sugar accumulation in tomato and offer opportunities to increase sugar content in large-fruited cultivars without sacrificing size and yield.

CSIG-22. PHARMACOLOGIC INHIBITION AND KNOCKDOWN OF CAMK2 IMPAIR GROWTH OF PATIENT-DERIVED GLIOBLASTOMA CULTURES

Abstract INTRODUCTION Glioblastoma (GBM), a primary brain malignancy with a median survival of 15-18 months, presents a pressing challenge in neuro-oncology. Patient-derived GBM cultures (PDGCs) exhibit spontaneous calcium (Ca2+) waves, which are thought to drive tumor growth and represent a therapeutic target. The mechanisms underlying the generation and the effectors of these Ca2+ waves remain elusive. We hypothesize CAMK2, a Ca2+/calmodulin-dependent protein kinase expressed as four distinct isoforms, is a key effector of such Ca2+ waves by phosphorylating substrates that promote tumor growth. METHODS Using multiple PDGCs, CAMK2 isoform-specific mRNA levels were assessed via qRT-PCR both at baseline and after shRNA-mediated knockdown (KD). Tumorsphere formation and WST-8 assays were performed to assess PDGC clonogenic potential after either KD or pharmacologic inhibition with the cell-permeable CAMK2 inhibitor KN93. RESULTS The relative mRNA levels of CAMK2 isoforms across three PDGC lines were, in decreasing order: CAMK2D, CAMK2G, CAMK2B, and CAMK2A. Expression of CAMK2D- and CAMK2B-specific shRNAs significantly reduced tumorsphere growth compared to control shRNA. CAMK2D KD reduced sphere formation by 55.13 ± 3.54% (p=0.0001) and 47.72 ± 9.55% (p=0.008) in separate PDGC lines, while CAMK2B KD reduced sphere formation by 41.15 ± 11.51% (p=0.021) and sphere size by 41.78% ± 10.50% (p=0.049) (n=3/experiment). KN93 treatment reduced tumorsphere formation by 59.23 ± 1.40% (p=0.0006) and 42.94 ± 13.0% (p=0.032) in two PDGC lines (n=3) and PDGC viability, while CAMK2-inactive analogue KN92 and DMSO minimally impacted viability (p<0.0001, two-way ANOVA). CONCLUSION CAMK2D and CAMK2B KD, as well as KN93 treatment, reduce tumorsphere formation, suggesting CAMK2 has a tumorigenic role in GBM. Further investigation of CAMK2 isoforms and in vivo validation are underway. Future aims include directly testing the link between spontaneous Ca2+ waves and CAMK2 activation and identifying CAMK2 phosphorylation targets that mediate tumor growth.

Establishment of potential lncRNA-related hub genes involved competitive endogenous RNA in lung adenocarcinoma

Long non-coding RNAs (lncRNAs) have a notable role in the diagnosis and prognosis of cancer. However, the associations between lncRNA-related hub genes (LRHGs) expression and the corresponding outcomes have not been fully understood in lung adenocarcinoma (LUAD). Here, a total of 71 patients diagnosed with LUAD and 60 healthy volunteers at The First Affiliated Hospital of Huzhou University from April, 2023 to December, 2023 were enrolled in the present study. A LRHGs model was established using least absolute shrinkage and selection operator analyses of The Cancer Genome Atlas-LUAD datasets. The underlying mechanisms of the LRHGs were investigated via Gene Set Enrichment Analysis and Gene Set Variation Analysis. Additionally, the diagnostic role of serum HOXD cluster antisense RNA 2 (HOXD-AS2) was assessed by receiver operating characteristic (ROC) curve analysis. Lastly, TCGA-LUAD samples were divided into high- and low-HOXD-AS2 expression groups based on the median expression. The associations between HOXD-AS2 expression and miR-4538 as well as Calmodulin-Dependent Protein Kinase Type II subunit Beta (CAMK2B) levels were conducted through Pearson correlation analysis. A comprehensive analysis identified 141 differentially expressed lncRNAs between 539 LUAD tissues and 59 normal samples. A prognostic marker for overall survival was established by constructing a predictive signature consisting of 9 LRHGs. Subsequently, 474 LUAD samples were categorized into a high or low-risk group based on the median of the risk score. An independent prognostic model was constructed to confirm the validity of this categorization. Further comparisons of the clinicopathological features and LRHG-related pathways were performed between the two groups. Examinations of LRHG expression in two LUAD clusters and of the association between LRHG expression and immune infiltration were also conducted. HOXD-AS2 expression was shown to be elevated in LUAD tissues compared with matched normal tissues, and the serum HOXD-AS2 level was also notably increased in LUAD samples compared with healthy controls. The results of the ROC analysis indicated that the sensitivity and specificity of HOXD-AS2 were higher than that of cytokeratin-19 fragment (CYFRA21-1), which is a serum marker for LUAD. Pearson analyses indicated that miR-4538 level was negatively associated with HOXD-AS2 expression, but CAMK2B level showed positive correlation in LUAD. The results of the present study therefore indicated that the constructed LRHG model, particularly HOXD-AS2, could independently diagnose and predict the prognosis of LUAD, which suggested the underlying mechanism of the HOXD-AS2/miR-4538/CAMK2B, and might offer efficient strategies for LUAD treatment.

NMDA receptor blockade attenuates Japanese encephalitis virus infection-induced microglia activation

Neurodegeneration and neuroinflammation are key components in the pathogenesis of Japanese Encephalitis caused by Japanese Encephalitis Virus (JEV) infection. The N-methyl-D-aspartate (NMDA)-type glutamate receptor displays excitatory neurotoxic and pro-inflammatory properties in a cell context-dependent manner. Herein, potential roles of the NMDA receptor in excitatory neurotoxicity and neuroinflammation and effects of NMDA receptor blockade against JEV pathogenesis were investigated in rat microglia, neuron/glia, neuron cultures, and C57BL/6 mice. In microglia, JEV infection induced glutamate release and activated post-receptor NMDA signaling, leading to activation of Ca2+ mobilization and Calcium/Calmodulin-dependent Protein Kinase II (CaMKII), accompanied by pro-inflammatory NF-κB and AP-1 activation and cytokine expression. Additionally, increased Dynamin-Related Protein-1 protein phosphorylation, NAPDH Oxidase-2/4 expression, free radical generation, and Endoplasmic Reticulum stress paralleled with the reactive changes of microglia after JEV infection. JEV infection-induced biochemical and molecular changes contributed to microglia reactivity and pro-inflammatory cytokine expression. NMDA receptor antagonists MK801 and memantine alleviated intracellular signaling and pro-inflammatory cytokine expression in JEV-infected microglia. JEV infection induced neuronal cell death in neuron/glia culture associated with the concurrent production of pro-inflammatory cytokines. Conditioned media of JEV-infected microglia compromised neuron viability in neuron culture. JEV infection-associated neuronal cell death was alleviated by MK801 and memantine. Activation of NMDA receptor-related inflammatory changes, microglia activation, and neurodegeneration as well as reversal effects of memantine were revealed in the brains of JEV-infected mice. The current findings highlight a crucial role of the glutamate/NMDA receptor axis in linking excitotoxicity and neuroinflammation during the course of JEV pathogenesis, and proposes the anti-inflammatory and neuroprotective potential of NMDA receptor blockade.

Curcumin Mitigates Muscle Atrophy Potentially by Attenuating Calcium Signaling and Inflammation in a Spinal Nerve Ligation Model

Denervation-induced calcium/calmodulin-dependent protein kinase II (CaMKII) activation and inflammation can result in muscle atrophy. Curcumin and bisdemethoxycurcumin are well known to exhibit an anti-inflammatory effect. In addition, curcumin has been shown to attenuate CaMKII activation in neuronal cells. This study aimed to examine the effect of curcumin or bisdemethoxycurcumin on CaMKII activation, inflammation, and muscle cross-sectional area (CSA) in spinal nerve ligated rats. Sixteen female rats were assigned to sham (CON), spinal nerve ligation (SNL), SNL+ curcumin 100 mg/kg BW (100CUR), and SNL+ bisdemethoxycurcumin 50 mg/kg BW (50CMO) for 4 weeks. Ipsilateral (surgical) soleus and tibialis anterior (TA) muscles was stained for dystrophin to measure CSA. Ipsilateral and contralateral (non-surgical) plantaris muscles were analyzed for protein content for acetylcholine receptor (AChR), CaMKII, CaMKIIThr286, nuclear factor-κB (NF-κB), NF-κBSer536, and interleukin-1β (IL-1β) and normalized to α-tubulin and then CON. A significant (p < 0.050) group effect was observed for TA CSA where CON (11,082.25 ± 1617.68 μm2; p < 0.001) and 100CUR (9931.04 ± 2060.87 μm2; p = 0.018) were larger than SNL (4062.25 ± 151.86 μm2). In the ipsilateral plantaris, the SNL (4.49 ± 0.69) group had greater CaMKII activation compared to CON (1.00 ± 0.25; p = 0.010), 100CUR (1.12 ± 0.45; p = 0.017), and 50CMO (0.78 ± 0.19; p = 0.009). The ipsilateral plantaris (2.11 ± 0.66) had greater IL-1β protein content than the contralateral leg (0.65 ± 0.14; p = 0.041) in the SNL group. In plantaris, the SNL (1.65 ± 0.51) group had greater NF-κB activation compared to CON (1.00 ± 0.29; p = 0.021), 100CUR (0.61 ± 0.10; p = 0.003), 50CMO (0.77 ± 0.25; p = 0.009) groups. The observed reduction in Ca2+ signaling and inflammation in type II plantaris muscle fibers might reflect the changes within the type II TA muscle fibers which may contribute to the mitigation of TA mass loss with curcumin supplementation.

The ZmCPK39-ZmDi19-ZmPR10 immune module regulates quantitative resistance to multiple foliar diseases in maize.

Gray leaf spot, northern leaf blight and southern leaf blight are three of the most destructive foliar diseases affecting maize (Zea mays L.). Here we identified a gene, ZmCPK39, that encodes a calcium-dependent protein kinase and negatively regulates quantitative resistance to these three diseases. The ZmCPK39 allele in the resistant line displayed significantly lower pathogen-induced gene expression than that in the susceptible line. A marked decrease in ZmCPK39 abundance mitigated the phosphorylation and degradation of the transcription factor ZmDi19. This led to elevated expression of ZmPR10, a gene known to encode an antimicrobial protein, thereby enhancing maize resistance to foliar diseases. Moreover, the F1 hybrid with reduced ZmCPK39 expression favored disease resistance, thereby increasing yield. Hence, the discovery of the ZmCPK39-ZmDi19-ZmPR10 immune module provides insight into the mechanisms underlying broad-spectrum quantitative disease resistance and also offers a new avenue for the genetic control of maize foliar diseases.

CPK28-mediated phosphorylation enhances nitrate transport activity of NRT2.1 during nitrogen deprivation.

Nitrate (NO3 -) serves as the primary inorganic nitrogen source assimilated by most terrestrial plants. The acquisition of nitrate from the soil is facilitated by NITRATE TRANSPORTERS (NRTs), with NRT2.1 being the key high-affinity nitrate transporter. The activity of NRT2.1, which has multiple potential phosphorylation sites, is intricately regulated under various physiological conditions. Here, we discovered that CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) positively regulates nitrate uptake under nitrogen deprivation conditions. We found CPK28 as the kinase targeted by immunoprecipitation followed by mass spectrometry and examined the in-planta phosphorylation status of NRT2.1 in cpk28 mutant plants by employing quantitative MS-based phosphoproteomics. Through a combination of in vitro phosphorylation experiment and immunoblotting using phospho-specific antibody, we successfully demonstrated that CPK28 specifically phosphorylates NRT2.1 at Ser21. Functional analysis conducted in Xenopus oocytes revealed that co-expression of CPK28 significantly enhanced high-affinity nitrate uptake of NRT2.1. Further investigation using transgenic plants showed that the phosphomimic variant NRT2.1S21E, but not the nonphosphorylatable variant NRT2.1S21A, fully restored high-affinity 15NO3 - uptake ability in both nrt2.1 and cpk28 mutant backgrounds. This study clarifies that the kinase activity of CPK28 is promoted during nitrogen deprivation conditions. These significant findings provide valuable insights into the intricate regulatory mechanisms that govern nitrate-demand adaptation.

CaMKII Exacerbates Doxorubicin-Induced Cardiotoxicity by Promoting Ubiquitination Through USP10 Inhibition.

Doxorubicin (DOX) is an effective anticancer drug, but it has a problem of cardiotoxicity that cannot be ignored. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is tightly associated with the pathological progression of DOX-induced cardiotoxicity. Ubiquitin-specific protease 10 (USP10) plays an important role in many biological processes and cancers. However, its association with DOX-induced cardiotoxicity and CaMKII remains unclear. H9C2 cells, HL-1 cells and C57BL/6 mice were used to establish the DOX-induced cardiotoxicity model, and the CaMKII-specific inhibitor KN-93 and USP10 specific inhibitor Spautin-1 were used to observe the CaMKII and USP10 effect. In cell experiments, CCK-8 method was used to assess cell viability, LDH kit was used to assess lactate dehydrogenase expression, DCFH-DA staining was used to observe changes in active oxygen content, TUNEL staining was used to observe cell apoptosis, and Western blotting method was used to detect relevant protein markers. The expression of p-CaMKII and USP10 was assessed by immunofluorescence staining. In animal experiments, mouse echocardiograph was used were used to evaluate cardiac function, and HE staining and Masson staining were used to evaluate myocardial injury. Cardiomyocyte apoptosis was detected by TUNEL staining. Western blotting method was used to detect relevant protein markers. Our results demonstrated that activation of CaMKII and inhibition of USP10 pathway related to DOX-induced cardiotoxicity. Inhibition of CaMKII with KN-93 ameliorated DOX-induced cardiac dysfunction and cytotoxicity. In addition, CaMKII inhibition prevented DOX-induced apoptosis and ubiquitination. Furthermore, CaMKII inhibition increased USP10 expression in DOX-treated mouse hearts, H9C2 cells and HL-1 cells. At last, the USP10 inhibitor, Spautin-1, blocked the regulatory effect of CaMKII inhibition on apoptosis and ubiquitination in DOX-induced cardiotoxicity. Our findings revealed that DOX-induced myocardial apoptosis and activated CaMKII through cellular and animal levels, while providing a novel probe into the mechanism of CaMKII action: promoting ubiquitination by inhibiting USP10 aggravated apoptosis.

Hyperfunction of AMPA receptors in the amygdala and hippocampus contributes to enhanced fear memory in diabetic mice

Patients with diabetes mellitus show an elevated prevalence of psychiatric disorders such as anxiety. We have reported that fear memory, a model related to anxiety as reflected in the freezing response, is enhanced in diabetic mice and was ameliorated by an AMPA receptor antagonist. The present study investigated whether functions of AMPA receptors in the amygdala and hippocampus are altered in streptozotocin (STZ)-induced diabetic mice. While protein levels of the GluA1 subunit of AMPA receptors were not altered in the amygdala and hippocampus, protein levels of GluA1 phosphorylated at serine 845 in the amygdala and hippocampus and of GluA1 phosphorylated at serine 831 in the hippocampus were increased in STZ-induced diabetic mice. L-lactate, which is increased in the amygdala and hippocampus of STZ-induced diabetic mice, did not alter these protein levels in either brain area. In contrast, protein levels of phosphorylated protein kinase A (PKA) catalytic subunit and phosphorylated calcium calmodulin kinase II (CaMKII), which are known to phosphorylate serine 845 and serine 831 of GluA1, respectively, were increased in the amygdala and hippocampus of STZ-induced diabetic mice. In the fear memory test, the PKA inhibitor H-89 injected before test sessions and the CaMKII inhibitor KN-62 injected before conditioning or test sessions each reduced the increase in freezing in STZ-induced diabetic mice. These results indicate that the functions of AMPA receptors in the amygdala and hippocampus are enhanced due to increased phosphorylation by PKA and CaMKII, which enhances fear memory in diabetic mice.

Beta-Adrenergic Activation of the Inward Rectifier K+ Current Is Mediated by the CaMKII Pathway in Canine Ventricular Cardiomyocytes.

Several ion currents in the mammalian ventricular myocardium are substantially regulated by the sympathetic nervous system via β-adrenergic receptor activation, including the slow delayed rectifier K+ current and the L-type calcium current. This study investigated the downstream mechanisms of β-adrenergic receptor stimulation by isoproterenol (ISO) on the inward rectifier (IK1) and the rapid delayed rectifier (IKr) K+ currents using action potential voltage clamp (APVC) and conventional voltage clamp techniques in isolated canine left ventricular cardiomyocytes. IK1 and IKr were dissected by 50 µM BaCl2 and 1 µM E-4031, respectively. Acute application of 10 nM ISO significantly increased IK1 under the plateau phase of the action potential (0-+20 mV) using APVC, and similar results were obtained with conventional voltage clamp. However, β-adrenergic receptor stimulation did not affect the peak current density flowing during terminal repolarization or the overall IK1 integral. The ISO-induced enhancement of IK1 was blocked by the calcium/calmodulin kinase II (CaMKII) inhibitor KN-93 (1 µM) but not by the protein kinase A inhibitor H-89 (3 µM). Neither KN-93 nor H-89 affected the IK1 density under baseline conditions (in the absence of ISO). In contrast, parameters of the IKr current were not affected by β-adrenergic receptor stimulation with ISO. These findings suggest that sympathetic activation enhances IK1 in canine left ventricular cells through the CaMKII pathway, while IKr remains unaffected under the experimental conditions used.

Carvedilol suppresses coronary artery spasm in A-kinase anchoring protein 150 knockout mouse

Abstract Backgrounds A-kinase anchoring proteins (AKAPs) act as scaffold to regulate activation of protein kinases and subsequent downstream signaling. AKAP 150 interacts with protein kinase A and protein kinase C, and has a critical role in regulating L-type calcium channel activity. We previously reported enhanced phospholipase C activity as an important mechanism of coronary spastic angina (CSA), known as Prinzmetal’s angina. Coronary artery spasm was induced by intravenous ergometrine injection in mouse model of vascular smooth muscle cell (VSMC)-specific enhanced phospholipase C activity concomitantly with enhanced intracellular Ca2+ concentration ([Ca2+]i). Then, we hypothesized that coronary artery spasm would be inhibited by AKAP 150 knockout, however, coronary artery spasm was unexpectedly induced in AKAP 150 knockout (AKAP-KO) mice without affecting [Ca2+]i, suggesting that AKAP-KO mouse is an unique model of CSA independent of [Ca2+]i. Previous report showed that AKAP-KO mice have a high Ca2+/calmodulin-dependent protein kinase (CaMK) II activity, and carvedilol, a non-selective beta-adrenergic receptor antagonist, inhibits CaMK II activity. Aim We tested the hypothesis that carvedilol could prevent coronary artery spasm via inhibiting CaMK II activity, which is a key molecule to regulate VSMC contraction, in AKAP-KO mice. Methods and Results We pretreated 14-18-weeks-old AKAP-KO mice with intraperitoneal injection of carvedilol (2.5mg/kg), propranolol (20mg/kg), or vehicle (control). Coronary artery spasm, evaluated by ST-segment elevation on lead II of body surface electrocardiogram, was induced by intravenous ergometrine injection (30mg/kg) in all controls (5/5, 100%) and all propranolol-treated mice (5/5, 100%), whereas it tended to be inhibited in carvedilol-treated mice (3/6, 50%) (p=0.08 vs control). In isolated VSMCs obtained from the aorta of AKAO-KO mice, the changes in [Ca2+]i from baseline in response to acetylcholine (ACh, 100μM) did not differ among the groups of pretreatment with carvedilol (10μM), propranolol (10μM), or vehicle, indicating that coronary artery spasm in AKAP-KO mice was induced by Ca2+-independent mechanism. In response to ACh, the phosphorylation of CaMK II was tended to be decreased (p=0.07), and the phosphorylation of myosin light chain was decreased (p<0.05) in VSMCs treated with carvedilol compared with control. Conclusions Carvedilol may inhibit coronary artery spasm probably via inhibiting CaMK II activity in AKAP-KO mice. Our results may provide a new paradigm into the pathogenesis of CSA as a model of enhanced Ca2+ sensitivity and important clinical implications for the mechanism of calcium channel antagonist-refractory coronary artery spasm.

The CNP analogue vosoritide reduces arrhythmia in diabetic cardiomyopathy via cGMP-dependent PDE2 stimulation on cellular and organ level

Abstract Background Detrimental arrhythmia occurs frequently in patients with diabetic cardiomyopathy (DiabCM), but safe and effective therapies are limited. Diabetes-associated neuropathological alterations increase the sympathetic tone, leading to hyperactivation of β-adrenoceptor-mediated cAMP signalling in cardiomyocytes (CM). Increased activity of deleterious cAMP-dependent kinases causes pro-arrhythmic dysregulation of intracellular Ca2+ homeostasis. Phosphodiesterases (PDE) can reduce cAMP levels. Exceptionally, the cAMP-hydrolysing activity of PDE2 can be cGMP-dependently increased by C-type natriuretic peptide (CNP). Thus, the CNP analogue vosoritide (approved for the treatment of achondroplasia) might mediate this cGMP/cAMP crosstalk, reducing arrhythmia. Purpose Here, we aim to study whether the cGMP-dependent activation of PDE2 by vosoritide (VO) is sufficient to reduce arrhythmia and arrhythmogenic triggers in DiabCM. Methods Diabetes was induced by 5 consecutive injections of streptozotocin (50 mg/kg) in control (WT) mice and mice with a CM-specific PDE2 knockout (KO). All experiments were performed 5 weeks later. Cardiac function was characterised by echocardiography. Primary ventricular CM were isolated to assess protein expression and phosphorylation levels by Western blot (WB) and cAMP levels by ELISA. Patch-clamp and Ca2+ imaging experiments were used to investigate pro-arrhythmic triggers. Arrhythmic events were quantified in ex vivo perfused hearts after ischaemia-reperfusion (I/R) injury. Results Compared to healthy controls, diabetic animals showed impaired systolic function with a ~9 % reduction in ejection fraction. The significant increase in E/E’ ratio by ~34 % indicated a diastolic dysfunction. Interestingly, the cardiac function was further worsened in diabetic PDE2 KO. WB analysis revealed upregulation of the CNP receptor NPR-B and PDE2 in diabetic CM and altered regulation of proteins involved in cAMP-dependent Ca2+ cycling such as protein kinase A, Ca2+/calmodulin-dependent kinase II and phospholamban. Interestingly, VO significantly reduced isoprenaline (Iso)-induced cAMP levels in diabetic CM. The effect of VO was prevented by specific PDE2 inhibition with BAY 60-7550 (BAY). Furthermore, VO was able to reduce the Iso-induced increase in L-type Ca2+ current, whereas BAY or genetic PDE2 deletion counteracted this effect. Accordingly, VO significantly decreased the Iso-triggered Ca2+ spark frequency by ~40 %. Concomitant PDE2 inhibition or PDE2 KO prevented this anti-arrhythmic effect. Finally, ECG analysis revealed that VO clearly reduced arrhythmia development after I/R in ex vivo perfused diabetic hearts via PDE2 activation. Conclusion Our data indicate that vosoritide-induced PDE2 stimulation may attenuate abnormal cardiac Ca2+ cycling and thereby reduce arrhythmia in diabetic hearts. Thus, vosoritide may be repurposed as a novel anti-arrhythmic drug in DiabCM.

Secretoneurin plasma levels are decreased after catheter ablation for atrial fibrillation, patients with AF produce lower SN levels than healthy individuals

Abstract Background Secretoneurin (SN) is a novel biomarker with potential use in cardiovascular medicine. The main effect of SN is mediated through its inhibition of calmodulin-dependent kinase II (CaMKII), which influences calcium handling[1], plasma SN levels differ in ischemic vs. dilated cardiomyopathy [2]. We aim to associate the levels of SN pre- and postprocedurally and according to the type of atrial fibrillation (AF). Methods We prospectively enrolled patients indicated for catheter ablation (CA) for AF, sequentially sampled patients for SN before, during and after CA for AF. We also sampled healthy individuals as controls. SN was analysed from venous, left atrial and coronary sinus (CS) blood by the use of the ELISA method. Values are expressed as means±SD, variables are compared by standard statistical methods. Results A total of 104 patients (59.4% male) with AF aged 64±10.4, with BMI 30.1±5 and 34 healthy individuals (38% male) were included in the analysis. SN plasma levels were significantly lower 32.3±15.1 after the procedure (SN2) as compared to levels before (SN1) 38.4±17.5 pmol/L (P=0.02, Fig.1). SN was not associated with any marker of cardiac injury or overload, echocardiographic parameters, or ablation characteristics (P=NS), even though Troponin I was significantly increased after the procedure (P<0.0001, Fig.2). Left atrial SN was numerically higher 40.9±19.1 than SN sampled from CS 32.9±13 (P=NS).The SN levels in our patients with AF were generally lower than in healthy individuals (50.7±15.3 pmol/L) before (P=0.0004) as well as after the procedure (P=0.0001). Conclusion Catheter ablation of AF leads to a decrease in SN plasma levels even though markers of cardiac injury are increased. Patients with AF irrespective of actual rhythm produce lower levels of SN than healthy individuals.

Functions and mechanisms of CDPKs in plant responses to abiotic stress

Calcium-dependent protein kinases (CDPKs/CPKs) are members of the Ca2+-sensitive Ser/Thr protein kinase family and play a crucial role in plant growth and development and responses to abiotic stress. CDPKs are capable of rapidly sensing changes in intracellular Ca2+ signals and recognizing and phosphorylating specific substrates, thereby transmitting and amplifying Ca2+ signal cascades downstream. They are involved in plant responses to stress conditions such as drought, saline-alkali stress, and injuries and regulate plant growth and development, gene expression, ion channel activity, and stomatal movement. The autophosphorylation of CDPKs can affect their activities and substrate specificity. CDPKs have the ability to bind to and phosphorylate multiple substrates. In addition to participating in respiratory burst oxidase homolog (RBOH), mitogen-activated protein kinase (MAPK), and plant hormone signaling pathways, CDPKs can also bind to 14-3-3 proteins, which enables the regulation of plant responses to stress and promotes plant growth and development. This paper summarized the research findings on the discovery, structure, classification, and roles of CDPKs in plant responses to stress and proposed the future research directions, aiming to provide the genetic resources and a theoretical basis for improving the stress tolerance of crops.

Subfamily C7 Raf-like kinases MRK1, RAF26, and RAF39 regulate immune homeostasis and stomatal opening in Arabidopsis thaliana.

The calcium-dependent protein kinase CPK28 regulates several stress pathways in multiple plant species. Here, we aimed to discover CPK28-associated proteins in Arabidopsis thaliana. We used affinity-based proteomics and identified several potential CPK28 binding partners, including the C7 Raf-like kinases MRK1, RAF26, and RAF39. We used biochemistry, genetics, and physiological assays to gain insight into their function. We define redundant roles for these kinases in stomatal opening, immune-triggered reactive oxygen species (ROS) production, and resistance to a bacterial pathogen. We report that CPK28 associates with and trans-phosphorylates RAF26 and RAF39, and that MRK1, RAF26, and RAF39 are active kinases that localize to endomembranes. Although Raf-like kinases share some features with mitogen-activated protein kinase kinase kinases (MKKKs), we found that MRK1, RAF26, and RAF39 are unable to trans-phosphorylate any of the 10 Arabidopsis mitogen-activated protein kinase kinases (MKKs). Overall, our study suggests that C7 Raf-like kinases associate with and are phosphorylated by CPK28, function redundantly in stomatal opening and immunity, and possess substrate specificities distinct from canonical MKKKs.

Heat stress causes chromatin accessibility and related gene expression changes in crown tissues of barley (Hordeum vulgare).

Plant responses to stress caused by high temperatures involve changes occurring at the molecular, metabolic, and physiological levels. Understanding the mechanisms by which plants recognize signals to activate this response is a prerequisite for identifying key genes and signaling pathways and for obtaining heat-tolerant plants. We demonstrated the first implementation of an assay for transposase-accessible chromatin to identify open chromatin regions (OCRs) in crown tissues of barley using three genotypes carrying different allelic forms of the sdw1 gene encoding gibberellin 20-oxidase subjected to elevated temperatures. In parallel, we performed gene expression analysis, which allowed us to relate changes in chromatin state to changes in transcriptional activity. The obtained data revealed that the hypersensitive chromatin regions within the genes were more repeatable than those outside the gene intervals. We observed that prolonged exposure to high temperatures increased chromatin accessibility. Genes with OCRs in their regulatory regions were involved in stress signaling and tolerance, including calcium-dependent protein kinase, mitogen-activated protein kinase (MAPK3), receptor-like cytoplasmic kinase (RLK), TIFY domain-containing transcriptional regulator, bZIP transcription factor, and regulatory protein NPR1. The effect of genotype on gene expression was not as pronounced as that of temperature. By combining results from the differential analysis of chromatin accessibility and expression profiles, we identified genes with high temperature-induced changes in chromatin accessibility associated with expression alterations. Importantly, our data revealed a relationship between the loss of chromatin accessibility in response to heat and the downregulation of genes related to gibberellin signaling.

Baicalein Ameliorates Insulin Resistance of HFD/STZ Mice Through Activating PI3K/AKT Signal Pathway of Liver and Skeletal Muscle in a GLP-1R-Dependent Manner.

Insulin resistance (IR) is the principal pathophysiological change occurring in diabetes mellitus (DM). Baicalein, a bioactive flavonoid primarily extracted from the medicinal plant Scutellaria baicalensis Georgi, has been shown in our previous research to be a potential natural glucagon-like peptide-1 receptor (GLP-1R) agonist. However, the exact therapeutic effect of baicalein on DM and its underlying mechanisms remain elusive. In this study, we investigated the therapeutic effects of baicalein on diabetes and sought to clarify its underlying molecular mechanisms. Our results demonstrated that baicalein improves hyperglycemic, hyperinsulinemic, and glucometabolic disorders in mice with induced diabetes via GLP-1R. This was confirmed by the finding that baicalein's effects on improving IR were largely diminished in mice with whole-body Glp1r ablation. Complementarily, network pharmacology analysis highlighted the pivotal involvement of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) insulin signaling pathway in the therapeutic actions of baicalein on IR. Our mechanism research significantly confirmed that baicalein mitigates hepatic and muscular IR through the PI3K/AKT signal pathway, both in vitro and in vivo. Furthermore, we demonstrated that baicalein enhances glucose uptake in skeletal muscle cells under IR conditions through the Ca2+/calmodulin-dependent protein kinase II (CaMKII)-adenosine 5'-monophosphate-activated protein kinase (AMPK)-glucose transporter 4 (GLUT4) signaling pathway in a GLP-1R-dependent manner. In conclusion, our findings confirm the therapeutic effects of baicalein on IR and reveal that it improves IR in liver and muscle tissues through the PI3K/AKT insulin signaling pathway in a GLP-1R dependent manner. Moreover, we clarified that baicalein enhances the glucose uptake in skeletal muscle tissue through the Ca2+/CaMKII-AMPK-GLUT4 signal pathway.

Transcriptome sequencing analysis of overexpressed SikCDPK1 in tobacco reveals mechanisms of cold stress response

BackgroundCold is a significant limiting factor in productivity, particularly in northwestern and eastern China. Calcium-Dependent Protein Kinases (CDPKs), a primary calcium signaling sensor in plants, play an important role in their response to cold. Snow lotus (Sasussured involucrata Kar L) is a plant that thrives in harsh climates and grows in northwest China. However, there were no reports on the transcriptome of OE-SikCDPK1 transgenic tobacco in response to cold.ResultsWhen exposed to cold stress, OE-SikCDPK1 plants displayed a cold-tolerant phenotype compared to non-transgenic tobacco. Under cold conditions, relative water content reduced, relative conductivity increased, malondialdehyde levels rose, and cold-responsive gene expression increased. The OE-SikCDPK1 gene and non-transgenic tobacco were employed for research purposes. The transcriptome of leaves was sequenced using the HISAT2 sequencing platform, and the data were used to examine gene function annotation and differentially expressed genes (DEGs). 53,022 DEGs in tobacco leaves under cold treatment were obtained. The GO enrichment results revealed that it was enriched for biological-process, defense response and other processes under cold stress. The KEGG pathway enrichment analysis revealed that the metabolic pathways of significant enrichment of DEGs under cold stress mainly involved MAPK signaling pathway transduction. The transcription factor identification results showed that the transcription factors with the largest number of differential expressions under cold treatment were mainly from WRKY, AP2, MYB, bHLH, NAC and other transcription factor families.ConclusionThe cold tolerance mechanism of snow lotus SikCDPK1 was comprehensively analyzed at the transcriptional level for the first time using RNA-seq technology. This study demonstrates that SikCDPK1 can respond to cold by participating in the MAPK signaling pathway and regulating the expression levels of transcription factors, including WRKY, AP2, MYB, bHLH, and NAC. These results offer valuable insights for further exploration of the cold tolerance mechanism associated with SikCDPK1.

Extracellular RIPK3 Acts as a Danger-Associated Molecular Pattern to Exaggerate Cardiac Ischemia/Reperfusion Injury.

Cardiac ischemia/reperfusion (I/R) injury has emerged as an important therapeutic target for ischemic heart disease. Currently, there is no effective therapy for reducing cardiac I/R injury. Damage-associated molecular patterns are endogenous molecules released after cellular damage to exaggerate tissue inflammation and injury. RIPK3 (receptor-interacting protein kinase 3), a well-established intracellular mediator of cell necroptosis and inflammation, serves as a circulating biomarker of multiple diseases. However, whether extracellular RIPK3 also exerts biological functions in cardiac I/R injury remains totally unknown. Patients with acute myocardial infarction receiving percutaneous coronary intervention (PCI) were recruited independently in the discovery cohort (103 patients) and validation cohort (334 patients), and major adverse cardiovascular events were recorded. Plasma samples were collected before and after PCI (6 and 24 h) for RIPK3 concentration measurement. Cultured neonatal rat ventricular myocytes, macrophages and endothelial cells, and in vivo mouse models with myocardial injury induced by I/R (or hypoxia/reoxygenation) were used to investigate the role and mechanisms of extracellular RIPK3. Another cohort including patients with acute myocardial infarction receiving PCI and healthy volunteers was recruited to further explore the mechanisms of extracellular RIPK3. In the discovery cohort, elevated plasma RIPK3 levels after PCI are associated with poorer short- and long-term outcomes in patients with acute myocardial infarction, as confirmed in the validation cohort. In both cultured cells and in vivo mouse models, recombinant RIPK3 protein exaggerated myocardial I/R (or hypoxia/reoxygenation) injury, which was alleviated by the RIPK3 antibody. Mechanistically, RIPK3 acted as a damage-associated molecular pattern and bound with RAGE (receptor of advanced glycation end-products), subsequently activating CaMKII (Ca2+/calmodulin-dependent kinase II) to elicit the detrimental effects. The positive correlation between plasma RIPK3 concentrations and CaMKII phosphorylation in human peripheral blood mononuclear cells was confirmed. We identified the positive relationship between plasma RIPK3 concentrations and the risk of major adverse cardiovascular events in patients with acute myocardial infarction receiving PCI. As a damage-associated molecular pattern, extracellular RIPK3 plays a causal role in multiple pathological conditions during cardiac I/R injury through RAGE/CaMKII signaling. These findings expand our understanding of the physiological and pathological roles of RIPK3, and also provide a promising therapeutic target for myocardial I/R injury and the associated complications.