Calmodulin-dependent Protein Kinase II Signaling Research Articles

TRPM2 and CaMKII Signaling Drives Excessive GABAergic Synaptic Inhibition Following Ischemia.

Excitotoxicity and the concurrent loss of inhibition are well-defined mechanisms driving acute elevation in excitatory/inhibitory (E/I) balance and neuronal cell death following an ischemic insult to the brain. Despite the high prevalence of long-term disability in survivors of global cerebral ischemia (GCI) as a consequence of cardiac arrest, it remains unclear whether E/I imbalance persists beyond the acute phase and negatively affects functional recovery. We previously demonstrated sustained impairment of long-term potentiation (LTP) in hippocampal CA1 neurons correlating with deficits in learning and memory tasks in a murine model of cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Here, we use CA/CPR and an in vitro ischemia model to elucidate mechanisms by which E/I imbalance contributes to ongoing hippocampal dysfunction in male mice. We reveal increased postsynaptic GABAA receptor (GABAAR) clustering and function in the CA1 region of the hippocampus that reduces the E/I ratio. Importantly, reduced GABAAR clustering observed in the first 24 h rebounds to an elevation of GABAergic clustering by 3 d postischemia. This increase in GABAergic inhibition required activation of the Ca2+-permeable ion channel transient receptor potential melastatin-2 (TRPM2), previously implicated in persistent LTP and memory deficits following CA/CPR. Furthermore, we find Ca2+-signaling, likely downstream of TRPM2 activation, upregulates Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity, thereby driving the elevation of postsynaptic inhibitory function. Thus, we propose a novel mechanism by which inhibitory synaptic strength is upregulated in the context of ischemia and identify TRPM2 and CaMKII as potential pharmacological targets to restore perturbed synaptic plasticity and ameliorate cognitive function.

Ca2+/Calmodulin-Dependent Protein Kinase II Enhances Retinal Ganglion Cell Survival But Suppresses Axon Regeneration after Optic Nerve Injury.

Neuroprotection after injury or in neurodegenerative disease remains a major goal for basic and translational neuroscience. Retinal ganglion cells (RGCs), the projection neurons of the eye, degenerate in optic neuropathies after axon injury, and there are no clinical therapies to prevent their loss or restore their connectivity to targets in the brain. Here we demonstrate a profound neuroprotective effect of the exogenous expression of various Ca2+/calmodulin-dependent protein kinase II (CaMKII) isoforms in mice. A dramatic increase in RGC survival following the optic nerve trauma was elicited by the expression of constitutively active variants of multiple CaMKII isoforms in RGCs using adeno-associated viral (AAV) vectors across a 100-fold range of AAV dosing in vivo. Despite this neuroprotection, however, short-distance RGC axon sprouting was suppressed by CaMKII, and long-distance axon regeneration elicited by several pro-axon growth treatments was likewise inhibited even as CaMKII further enhanced RGC survival. Notably, in a dose-escalation study, AAV-expressed CaMKII was more potent for axon growth suppression than the promotion of survival. That diffuse overexpression of constitutively active CaMKII strongly promotes RGC survival after axon injury may be clinically valuable for neuroprotection per se. However, the associated strong suppression of the optic nerve axon regeneration demonstrates the need for understanding the intracellular domain- and target-specific CaMKII activities to the development of CaMKII signaling pathway-directed strategies for the treatment of optic neuropathies.

MFG-E8 facilitates heart repair through M1/M2 polarization after myocardial infarction by inhibiting CaMKII

BackgroundM1/M2 macrophage polarization affects patient outcomes after myocardial infarction (MI). The relationship between milk fat globule-epidermal growth factor 8 (MFG-E8) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) on macrophage polarization after MI is unknown. To investigate the functional role of MFG-E8 in modulating cardiac M1/M2 macrophage polarization after MI, especially its influence on CaMKII signaling. MethodsHuman ventricular tissue and blood were obtained from patients with MI and controls. MFG-E8-KO mice were constructed (C57BL/6). The mice were randomized to WT-sham, sham-MFG-E8-KO, WT-PBS, rmMFG-E8 (WT injected with rmMFG-E8 10 min after MI), and MFG-E8-KO. The mouse macrophage cell line RAW264.7 was obtained. CaMKII, p-CaMKII, Akt, and NF-κB p65 were determined by qRT-PCR, western blot, and immunofluorescence. ResultsThe MFG-E8 levels were significantly enhanced after MI in the hearts and plasma of patients with MI compared with controls. The MFG-E8 levels were significantly increased in the hearts and plasma of mice after MI. MFG-E8 was derived from cardiac fibroblasts. The administration of rmMFG-E8 improved ventricular remodeling and cardiac function after MI. rmMFG-E8 did not suppress infiltrating monocyte/macrophages into the peri-infarct area. rmMFG-E8 suppressed the polarization of macrophages to the M1 phenotype and promoted the polarization of macrophages to the M2 phenotype. rmMFG-E8 suppressed CaMKII-dependent signaling in macrophages. ConclusionsMFG-E8 and CaMKII appear to collaboratively regulate myocardial remodeling and M1/M2 macrophage polarization after MI. These observations suggest new roles for MFG-E8 in inhibiting M1 but promoting M2 macrophage polarization.

Short-term CaMKII inhibition with tatCN19o does not erase pre-formed memory in mice and is neuroprotective in pigs

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a central regulator of learning and memory, which poses a problem for targeting it therapeutically. Indeed, our study supports prior conclusions that long-term interference with CaMKII signaling can erase pre-formed memories. By contrast, short-term pharmacological CaMKII inhibition with the neuroprotective peptide tatCN19o interfered with learning in mice only mildly and transiently (for less than 1h) and did not at all reverse pre-formed memories. These results were obtained with ≥500-fold of the dose that protected hippocampal neurons from cell death after a highly clinically relevant pig model of transient global cerebral ischemia: ventricular fibrillation followed by advanced life support and electrical defibrillation to induce the return of spontaneous circulation. Of additional importance for therapy development, our preliminary cardiovascular safety studies in mice and pig did not indicate any concerns with acute tatCN19o injection. Taken together, although prolonged interference with CaMKII signaling can erase memory, acute short-term CaMKII inhibition with tatCN19o did not cause such retrograde amnesia that would pose a contraindication for therapy.

CaMKIIα as a Promising Drug Target for Ischemic Grey Matter.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a major mediator of Ca2+-dependent signaling pathways in various cell types throughout the body. Its neuronal isoform CaMKIIα (alpha) centrally integrates physiological but also pathological glutamate signals directly downstream of glutamate receptors and has thus emerged as a target for ischemic stroke. Previous studies provided evidence for the involvement of CaMKII activity in ischemic cell death by showing that CaMKII inhibition affords substantial neuroprotection. However, broad inhibition of this central kinase is challenging because various essential physiological processes like synaptic plasticity rely on intact CaMKII regulation. Thus, specific strategies for targeting CaMKII after ischemia are warranted which would ideally only interfere with pathological activity of CaMKII. This review highlights recent advances in the understanding of how ischemia affects CaMKII and how pathospecific pharmacological targeting of CaMKII signaling could be achieved. Specifically, we discuss direct targeting of CaMKII kinase activity with peptide inhibitors versus indirect targeting of the association (hub) domain of CaMKIIα with analogues of γ-hydroxybutyrate (GHB) as a potential way to achieve more specific pharmacological modulation of CaMKII activity after ischemia.

Xiaoyu Jiangzhi capsule protects against heart failure via Ca2+/CaMKII signaling pathways in mice

Heart failure (HF), a worldwide health condition, is the result of many cardiovascular diseases. The traditional Chinese medicine (TCM) Xiaoyu Jiangzhi capsule (XYC) has long been in use in China to treat hyperlipidemia and inhibit platelet aggregation. This study explores the effects of XYC on heart failure (HF) and its detailed mechanisms. Isoproterenol (ISO, 30 mg/kg) was injected intraperitoneally for 7 days to copy a HF model of 10–12 weeks old, 20–30 g male mice. We then compared the CON (control) group, ISO (HF model) group, MET (metoprolol) group, and XYC group. Cardiac systolic function and left wall thickness were evaluated by echocardiograph. Using western blot analysis, we detected the proteins of calmodulin dependent protein kinase II (CaMKII) and sarco/endoplasmic reticulum Ca 2+ -ATPase (Serca). Furthermore, tsA201 cells were cultured and the human CaV1.2 calcium channel current (hCaV1.2) were detected by patch clamp experiments. XYC reduced HF, inhibiting the protein expression of CaMKII, but Serca did not change significantly. Moreover, XYC inhibited the peak amplitude of the hCaV1.2 current, depolarizing shifted the activation curve 27.6 mV, and shifted the inactivation curve toward a positive potential 17.6 mV. The fraction recovered from inaction was reduced in XYC group compared with that in CON group. XYC could inhibit ISO-induced HF by reducing the Ca 2+ /CaMKII signaling pathway in mice.

Task-Dependent Effects of SKF83959 on Operant Behaviors Associated With Distinct Changes of CaMKII Signaling in Striatal Subareas.

BackgroundSKF83959, an atypical dopamine (DA) D1 receptor agonist, has been used to test the functions of DA-related receptor complexes in vitro, but little is known about its impact on conditioned behavior. The present study examined the effects of SKF83959 on operant behaviors and assayed the neurochemical mechanisms involved.MethodsMale rats were trained and maintained on either a fixed-interval 30-second (FI30) schedule or a differential reinforcement of low-rate response 10-second (DRL10) schedule of reinforcement. After drug treatment tests, western blotting assayed the protein expressions of the calcium-/calmodulin-dependent protein kinase II (CaMKII) and the transcription factor cyclic AMP response element binding protein (CREB) in tissues collected from 4 selected DA-related areas.ResultsSKF83959 disrupted the performance of FI30 and DRL10 behaviors in a dose-dependent manner by reducing the total number of responses in varying magnitudes. Moreover, the distinct profiles of the behavior altered by the drug were manifested by analyzing qualitative and quantitative measures on both tasks. Western-blot results showed that phospho-CaMKII levels decreased in the nucleus accumbens and the dorsal striatum of the drug-treated FI30 and DRL10 subjects, respectively, compared with their vehicle controls. The phospho-CREB levels decreased in the nucleus accumbens and the hippocampus of drug-treated FI30 subjects but increased in the nucleus accumbens of drug-treated DRL10 subjects.ConclusionsOur results provide important insight into the neuropsychopharmacology of SKF83959, indicating that the drug-altered operant behavior is task dependent and related to regional-dependent changes of CaMKII-CREB signaling in the mesocorticolimbic DA systems.

Dysregulation of cardiac CaMKII pathway is increased in aging and chronic inflammation

Chronic elevation of inflammatory mediators is common in frail older adults and predicts a host of adverse cardiovascular outcomes, including heart failure and sudden death. To date, few specific molecular mechanisms have been identified that connect chronic inflammation to age-related cardiomyopathies. Dysregulation in Ca2+and calmodulin-dependent protein kinase II (CaMKII) signaling has been implicated in promoting inflammatory responses in cardiomyopathies but is understudied in the context of aging. We compared myocardial CaMKII signaling in young (32 weeks) and old (104 weeks) wild type (WT) and interleukin 10 null mice (IL-10tm/tm), a model of chronic inflammation and frailty. Aged cohorts were treated with 0.6 mg of losartan. Hearts from mouse cohorts were assayed for total and autophosphorylated CaMKII (P-CaMKII) and the phosphorylation status of phospholamban (PLN) (downstream signaling pathway) using western blot techniques. We observed significant increases in total and activated, autophosphorylated, CaMKII (P-CaMKII) (2.4 ± 0.38 vs 1.2 ± 0.12 p< 0.02; 9.0 ± 1.9 vs 0.03 ± 0.01 p< 0.002), but decreases in the phosphorylation of PLN (0.6 ±0.3 vs 1.7 ± 0.3 p<0.03) at a validated CaMKII target site (T17) in old compared to young WT hearts. Similarly, we found increased P-CaMKII (6.9 ± 1.5 vs 0.7 ± 0.4 p< 0.004) and diminished PLN T17 phosphorylation (0.8 ± 0.2 vs 1.5 ± 0.2 p<0.02) in old compared to young IL-10tm/tm mice. Old IL-10tm/tm mouse hearts had less total CaMKII compared to old WT mouse hearts (1.1 ± 0.3 vs 2.9 ±0.2 p< 0.002). Four weeks treatment of old WT mice with Losartan was associated with reversal of observed age-related changes in the CaMKII signaling pathway. We observed decreases in total CaMKII, P-CaMKII (2.9 ± 0.2 vs.1.5 ± 0.2 p<0.001; 2.2 ± 0.9 vs 0.47 ± 0.1 p <0.03), and an increase in PLN T17 phosphorylation (0.55±0.3 vs 2.23 ± 0.4 p< 0.007). The effects of Losartan on the animals aging with chronic inflammation (old IL-10tm/tm) was less clear. We observed a losartan associated increase in total CaMKII (1.41 ± 0.02 vs 1.3 ± 0.02 p<0.02) but no impact on P-CaMKII or PLN T17 phosphorylation. We interpret our findings to suggest that changes in myocardial CaMKII signaling are a feature of aging that is affected by chronic inflammation.

The Role of CaMKII and ERK Signaling in Addiction.

Nicotine is the predominant addictive compound of tobacco and causes the acquisition of dependence through its interactions with nicotinic acetylcholine receptors and various neurotransmitter releases in the central nervous system. The Ca2+/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) play a pivotal role in synaptic plasticity in the hippocampus. CaMKII is involved in long-term potentiation induction, which underlies the consolidation of learning and memory; however, the roles of CaMKII in nicotine and other psychostimulant-induced addiction still require further investigation. This article reviews the molecular mechanisms and crucial roles of CaMKII and ERK in nicotine and other stimulant drug-induced addiction. We also discuss dopamine (DA) receptor signaling involved in nicotine-induced addiction in the brain reward circuitry. In the last section, we introduce the association of polyunsaturated fatty acids and cellular chaperones of fatty acid-binding protein 3 in the context of nicotine-induced addiction in the mouse nucleus accumbens and provide a novel target for the treatment of drug abuse affecting dopaminergic systems.

Pka and CaMKII Signaling Synergistically Promotes Atrial Arrhythmia in Populations of Human Atrial Tissues

The β-adrenergic receptor (βAR)/cAMP/PKA and multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) signaling pathways are key mediators of cardiac excitation-contraction coupling (ECC) and share multiple downstream targets. Hyperactivation of both signaling pathways contributes to the initiation and maintenance of atrial fibrillation (AF), the world's most common arrhythmia. Using novel populations of computational models coupling atrial electrophysiology and Ca2+ handling with detailed descriptions of βAR-PKA and CaMKII pathways, we have previously shown that PKA and CaMKII effects on atrial ECC proteins synergistically promote a vicious cycle of Ca2+and membrane potential instabilities. Since both kinases are known to affect tissue-level modifiers of atrial electrophysiology, here we sought to test whether PKA and CaMKII signaling also synergize to mediate arrhythmias in tissue, and to determine the mechanisms underlying the increased vulnerability. Populations of 1D strand models revealed increased vulnerability to unidirectional block with hyperactive CaMKII, mediated by Na+ channel loss of function and cell-to-cell uncoupling, which was exacerbated by activation of βARs. Simulations of 2D heterogeneous tissues demonstrated synergistic enhancement of arrhythmia vulnerability by βAR stimulation and CaMKII hyperactivation through dysregulating conduction and modifying source-sink mismatch, as well as promoting tissue repolarization dispersion and invoking spontaneous Ca2+-overload-mediated action potentials. CaMKII inhibition substantially reduced the vulnerability. Collectively, our simulations reveal synergy in PKA and CaMKII effects on tissue-level outcomes, and depict a novel paradigm for Ca2+-CaMKII-dependent involvement in both enhanced triggered activity and conduction disturbances in AF. These findings suggest that interrupting the vicious cycle of ever-increasing CaMKII and Ca2+ (e.g., via CaMKII inhibition) may be a valuable pharmacotherapy approach to counteract both AF triggers and functional substrate.

Interactive Roles of CaMKII/Ryanodine Receptor Signaling and Inflammation in Lung Diseases.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.

Feeding habit transition induced by social learning through CaMKII signaling in Chinese perch (Siniperca chuatsi)

Social learning plays pivotal roles in gaining new foraging skills and food preferences. However, the molecular mechanism underlying the effects of social learning on feeding habits is unclear. Chinese perch, as a demersal piscivore, has a unique feeding habit. Once the fry of Chinese perch start feeding, they only accept live prey fish in the wild and refuse dead prey fish or artificial diets. In the present study, we used the inhibitor of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) signaling pathway KN-93 in vivo and in vitro, to explore how social learning can affect the transition from live prey fish to dead prey fish in Chinese perch. Analysis of gene expression, protein phosphorylation, success rate, learning times, and food intake allowed us to gain insights into the molecular mechanism of social learning in acquiring new feeding habits in Chinese perch. Our results showed that the p-CaMKII, phospho-cAMP-response element binding (p-CREB) and proto-oncogenes c-fos (c-fos), and melanin-concentrating hormone (mch) expression levels were significantly lower with KN-93 treatment than levels in the control condition, whereas the expression of the anorexigenic gene proopiomelanocortin (pomc) was significantly higher in vitro. The increased success rate and food intake of Chinese perch induced by social learning, were diminished after the injection of the CaMKII inhibitor KN-93 in vivo. Inhibited signaling of CaMKII was observed in Chinese perch after KN-93 treatment. We also found that the expression levels of the c-fos, mch or pomc genes were increased or decreased in the learning group and then decreased or increased in the learning group with KN-93 injection, respectively. These results indicate that the higher expression of the orexigenic mch gene and the decreased expression of the anorexigenic pomc gene might contribute to the learning of eating dead fish prey in Chinese perch. Altogether, our results showed that social learning could promote the feeding habit transition from live prey fish to dead prey fish through the CaMKII - c-fos - pomc/mch signaling pathway in Chinese perch.

Cardiac CaMKIIδ and Wenxin Keli Prevents Ang II-Induced Cardiomyocyte Hypertrophy by Modulating CnA-NFATc4 and Inflammatory Signaling Pathways in H9c2 Cells.

Previous studies have demonstrated that calcium-/calmodulin-dependent protein kinase II (CaMKII) and calcineurin A-nuclear factor of activated T-cell (CnA-NFAT) signaling pathways play key roles in cardiac hypertrophy (CH). However, the interaction between CaMKII and CnA-NFAT signaling remains unclear. H9c2 cells were cultured and treated with angiotensin II (Ang II) with or without silenced CaMKIIδ (siCaMKII) and cyclosporine A (CsA, a calcineurin inhibitor) and subsequently treated with Wenxin Keli (WXKL). Patch clamp recording was conducted to assess L-type Ca2+ current (ICa-L), and the expression of proteins involved in signaling pathways was measured by western blotting. Myocardial cytoskeletal protein and nuclear translocation of target proteins were assessed by immunofluorescence. The results indicated that siCaMKII suppressed Ang II-induced CH, as evidenced by reduced cell surface area and ICa-L. Notably, siCaMKII inhibited Ang II-induced activation of CnA and NFATc4 nuclear transfer. Inflammatory signaling was inhibited by siCaMKII and WXKL. Interestingly, CsA inhibited CnA-NFAT pathway expression but activated CaMKII signaling. In conclusion, siCaMKII may improve CH, possibly by blocking CnA-NFAT and MyD88 signaling, and WXKL has a similar effect. These data suggest that inhibiting CaMKII, but not CnA, may be a promising approach to attenuate CH and arrhythmia progression.

Ethanol-activated CaMKII signaling induces neuronal apoptosis through Drp1-mediated excessive mitochondrial fission and JNK1-dependent NLRP3 inflammasome activation

BackgroundNeurodegeneration is a representative phenotype of patients with chronic alcoholism. Ethanol-induced calcium overload causes NOD-like receptor protein 3 (NLRP3) inflammasome formation and an imbalance in mitochondrial dynamics, closely associated with the pathogenesis of neurodegeneration. However, how calcium regulates this process in neuronal cells is poorly understood. Therefore, the present study investigated the detailed mechanism of calcium-regulated mitochondrial dynamics and NLRP3 inflammasome formation in neuronal cells by ethanol.MethodsIn this study, we used the SK-N-MC human neuroblastoma cell line. To confirm the expression level of the mRNA and protein, real time quantitative PCR and western blot were performed. Co-immunoprecipitation and Immunofluorescence staining were conducted to confirm the complex formation or interaction of the proteins. Flow cytometry was used to analyze intracellular calcium, mitochondrial dysfunction and neuronal apoptosis.ResultsEthanol increased cleaved caspase-3 levels and mitochondrial reactive oxygen species (ROS) generation associated with neuronal apoptosis. In addition, ethanol increased protein kinase A (PKA) activation and cAMP-response-element-binding protein (CREB) phosphorylation, which increased N-methyl-D-aspartate receptor (NMDAR) expression. Ethanol-increased NMDAR induced intracellular calcium overload and calmodulin-dependent protein kinase II (CaMKII) activation leading to phosphorylation of dynamin-related protein 1 (Drp1) and c-Jun N-terminal protein kinase 1 (JNK1). Drp1 phosphorylation promoted Drp1 translocation to the mitochondria, resulting in excessive mitochondrial fission, mitochondrial ROS accumulation, and loss of mitochondrial membrane potential, which was recovered by Drp1 inhibitor pretreatment. Ethanol-induced JNK1 phosphorylation activated the NLRP3 inflammasome that induced caspase-1 dependent mitophagy inhibition, thereby exacerbating ROS accumulation and causing cell death. Suppressing caspase-1 induced mitophagy and reversed the ethanol-induced apoptosis in neuronal cells.ConclusionsOur results demonstrated that ethanol upregulated NMDAR-dependent CaMKII phosphorylation which is essential for Drp1-mediated excessive mitochondrial fission and the JNK1-induced NLRP3 inflammasome activation resulting in neuronal apoptosis.EBGddboEPhESjQm19dbAFrVideo abstractGraphical abstract

Challenges and Opportunities for Therapeutic Targeting of Calmodulin Kinase II in Heart.

Heart failure remains a major health burden around the world. Despite great progress in delineation of molecular mechanisms underlying development of disease, standard therapy has not advanced at the same pace. The multifunctional signaling molecule Ca2+/calmodulin-dependent protein kinase II (CaMKII) has received considerable attention over recent years for its central role in maladaptive remodeling and arrhythmias in the setting of chronic disease. However, these basic science discoveries have yet to translate into new therapies for human patients. This review addresses both the promise and barriers to developing translational therapies that target CaMKII signaling to abrogate pathologic remodeling in the setting of chronic disease. Efforts in small molecule design are discussed, as well as alternative targeting approaches that exploit novel avenues for compound delivery and/or genetic approaches to affect cardiac CaMKII signaling. These alternative strategies provide hope for overcoming some of the challenges that have limited the development of new therapies.

Interplay Between β-Adrenergic Stimulation and CaMKII Signaling Favors Human Atrial Arrhythmogenesis: Insights from Populations of Models

Excessive β-adrenergic receptor (βAR) stimulation and hyperactivation of the multifunctional Ca2+-calmodulin-dependent protein kinase II (CaMKII) play important roles in the initiation and maintenance of atrial fibrillation (AF), the world's most common cardiac arrhythmia. It has been postulated that the interplay of these two pathways with intracellular Ca2+ signaling drives a vicious cycle that promotes arrhythmogenesis. Here, we coupled our well-established computational model of human atrial cellular electrophysiology (extended to incorporate two recently-characterized atrial-predominant K+ currents) and Ca2+ handling (Grandi et al., 2011) with detailed descriptions of βAR/cAMP/PKA and CaMKII pathways (Soltis and Saucerman 2010; Morotti et al., 2014) to investigate their respective roles and potential synergy in promoting AF.

Regulation of intracellular Ca2+/CaMKII signaling by TRPV4 membrane translocation during osteoblastic differentiation

Bone constantly remodels between resorption by osteoclasts and formation by osteoblasts; therefore the functions of osteoblasts are pivotal for maintaining homeostasis of bone mass. Transient receptor potential vanilloid 4 (TRPV4), a type of mechanosensitive channel, has been reported to be a key regulator in bone remodeling. However, the relationship between TRPV4 and osteoblast function remains largely elusive. Only little is known about the spatial distribution change of TRPV4 during osteoblastic differentiation and related signal events. Based on three-dimensional super-resolution microscopy, our results clearly showed a different distribution of TRPV4 in undifferentiated and differentiated osteoblasts, which reflected the plasma membrane translocation of TRPV4 along with prolonged differentiation. GSK1016790A (GSK101), the most potent agonist of TRPV4, triggered rapid calcium entry and calmodulin-dependent protein kinase II (CaMKII) phosphorylation via TRPV4 activation in a differentiation-dependent manner, indicating that the abundance of TRPV4 at the cell surface resulting from differentiation may be related to the modulation of Ca2+ response and CaMKII activity. These data provide compelling evidences for the plasma membrane translocation of TRPV4 during osteoblastic differentiation as well as demonstrate the regulation of downstream Ca2+/CaMKII signaling.

CREB activity is required for epidermal growth factor-induced mouse cumulus expansion.

The release of a fertilizable oocyte from the ovary is dependent upon the expansion of the cumulus cells. The expansion requires cooperation between epidermal growth factor (EGF) family peptide-activated mitogen-activated protein kinase (MAPK)3/1 and oocyte paracrine factor-activated-Sma- and Mad-related protein (SMAD)2/3 signaling in cumulus cells. However, the mechanism underlying (MAPK)3/1 signaling is unclear. In the present study, the EGF-activation of EGF receptor (EGFR) induced cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB) phosphorylation in cumulus cells, and the interruption of CREB functional complex formation by naphthol AS-E phosphate (KG-501) completely blocked the EGF-stimulated expansion-related gene expression. EGF-stimulated phosphorylation of CREB was completely inhibited by MAPK3/1 inhibitor U0126, suggesting that EGF-activated MAPK3/1 results in the activation of CREB for cumulus expansion. Also, the role of EGF-stimulated calcium signaling was studied. Calcium-elevating reagents ionomycin and sphingosine-1-phosphate mimicked, but calcium chelators bis-(o'aminophenoxy)-ethane-N,N,N,N-tetraacetic acid, tetra(acetoxymethyl)-ester, and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate abolished the activity of EGF on CREB phosphorylation, cumulus expansion, and expansion-related gene expression. Furthermore, EGF-induced cumulus expansion was inhibited by calmodulin (CaM)-dependent protein kinase II (CaMKII) inhibitors, KN-93 and autocamtide-2-related inhibitory peptide. However, the inhibition of SMAD2/3 activity by removal of oocyte from cumulus-oocyte complexes did not affect the EGF-induced CREB phosphorylation, indicating that EGF-activated CREB is independent of oocyte-activated SMAD2/3 signaling. Therefore, EGF-induced CREB activity by MAPK3/1 and Ca2+ /CaMKII signaling pathways promotes the expansion-related gene expression and consequent cumulus expansion.

Social Learning of Acquiring Novel Feeding Habit in Mandarin Fish (Siniperca chuatsi).

Social learning plays important roles in gaining new foraging skills and food preferences. However, the potential role and molecular mechanism of social learning in acquiring new feeding habits is less clear in fish. In the present study, we examined the success rate of feeding habit domestication from live prey fish to dead prey fish, as well as the food intake of dead prey fish in mandarin fish with or without feeders of dead prey fish as demonstrators. Here, we found that mandarin fish can learn from each other how to solve novel foraging tasks, feeding on dead prey fish. In addition, the analysis of gene expressions and signaling pathways of learning through Western blotting and transcriptome sequencing shows that the expression of the c-fos, fra2, zif268, c/ebpd and sytIV genes were significantly increased, and the anorexigenic pomc and leptin a expressions were decreased in fish of the learning group. The phosphorylation levels of protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) in the learning group were significantly higher than those of the control group, while the phosphorylation level of S6 ribosomal protein (S6) was lower. With the inhibitors of PKA and CaMKII signaling and the chromatin immunoprecipitation (ChIP) assay, we further found that the social learning of new feeding habits in mandarin fish could be attributed to the activation of the CaMKII signaling pathway and then the stimulation of the expression of the c-fos gene, which might be an important transcriptional factor to inhibit the expression of the anorexigenic gene pomc, resulting in the food intake of dead prey fish in mandarin fish. Altogether, our results support the hypothesis that social learning could facilitate the acquisition of novel feeding habits in fish, and it considerably increases the rate of subsequent individual food intake and domestication through the interaction between the learning gene c-fos and the appetite control gene pomc.

β-adrenergic regulation of late Na+ current during cardiac action potential is mediated by both PKA and CaMKII

Late Na+ current (INaL) significantly contributes to shaping cardiac action potentials (APs) and increased INaL is associated with cardiac arrhythmias. β-adrenergic receptor (βAR) stimulation and its downstream signaling via protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) pathways are known to regulate INaL. However, it remains unclear how each of these pathways regulates INaL during the AP under physiological conditions. Here we performed AP-clamp experiments in rabbit ventricular myocytes to delineate the impact of each signaling pathway on INaL at different AP phases to understand the arrhythmogenic potential. During the physiological AP (2 Hz, 37 °C) we found that INaL had a basal level current independent of PKA, but partially dependent on CaMKII. βAR activation (10 nM isoproterenol, ISO) further enhanced INaL via both PKA and CaMKII pathways. However, PKA predominantly increased INaL early during the AP plateau, whereas CaMKII mainly increased INaL later in the plateau and during rapid repolarization. We also tested the role of key signaling pathways through exchange protein activated by cAMP (Epac), nitric oxide synthase (NOS) and reactive oxygen species (ROS). Direct Epac stimulation enhanced INaL similar to the βAR-induced CaMKII effect, while NOS inhibition prevented the βAR-induced CaMKII-dependent INaL enhancement. ROS generated by NADPH oxidase 2 (NOX2) also contributed to the ISO-induced INaL activation early in the AP. Taken together, our data reveal differential modulations of INaL by PKA and CaMKII signaling pathways at different AP phases. This nuanced and comprehensive view on the changes in INaL during AP deepens our understanding of the important role of INaL in reshaping the cardiac AP and arrhythmogenic potential under elevated sympathetic stimulation, which is relevant for designing therapeutic treatment of arrhythmias under pathological conditions.