The results showed that matrine inhibited cell viability and induced cell apoptosis in AML cell lines as well as primary AML cells from patients with AML in a dose- and time-dependent manner. Matrine induced apoptosis by collapsing the mitochondrial membrane potential, inducing cytochrome c release from mitochondria, reducing the ratio of Bcl-2/Bax, increasing activation of caspase-3, and decreasing the levels of p-Akt and p-ERK1/2. The apoptotic effects of matrine on AML cells were partially blocked by a caspase-3 inhibitor Z-DEVD-FMK and a PI3K/Akt activator IGF-1, respectively. Matrine potently inhibited in vivo tumor growth following subcutaneous inoculation of HL-60 cells in SCID mice. These findings indicate that matrine can inhibit cell proliferation and induce apoptosis of AML cells and may be a novel effective candidate as chemotherapeutic agent against AML.Acute myeloid leukemia (AML) is a hematological malignancy characterized by a rapid increase in the number of immature myeloid cells in bone marrow. Despite recent advances in the treatment, AML remains an incurable disease. Matrine, a major component extracted from Sophora flavescens Ait, has been demonstrated to exert anticancer effects on various cancer cell lines. However, the effects of matrine on AML remain largely unknown. Here we investigated its anticancer effects and underlying mechanisms on human AML cells  Therefore, alternative treatments still need to be found for patients with AML.Acute myeloid leukemia (AML) is a group of clonal hematopoietic stem cell disorders in which both failure to differentiate and overproliferation in the stem cell compartment lead to accumulation of non-functional cells termed myeloblasts 15H24N20. Matrine has been found to exhibit many biological activities, such as anti-inflammation, anti-virus, anti-fibrosis, anti-arrhythmia, and immunosuppression, leading to wide clinical use in the treatment of viral hepatitis, liver fibrosis, heart arrhythmia and skin diseases in China et alSome natural products have been used as alternative treatments for cancers including AML because of their extensive biological activities and comparatively low toxicities in vitro and in vivo. We found that matrine could induce apoptosis of AML cells by collapse of mitochondrial membrane potential (Δψm), release of cytochrome c (cyt c) from mitochondria to cytosol, reduction of the ratio of Bcl-2/Bax, activation of caspase-3, and down-regulation of p-Akt and p-ERK1/2.In the present study, we investigated the cytotoxic effects of matrine on AML cell lines HL-60, NB4, and U937 as well as primary AML cells obtained from patients with AML and its underlying mechanisms +lin− cells were sorted from BMMNCs of patients with AML using the Diamond CD34 Isolation kit  according to the manufacturer’s protocols. Normal CD34+lin− cells were sorted from BMMNCs of patients with non-Hodgkin’s lymphoma (NHL) without lymphoma involvement in BM. All protocols and experiments were approved by the First Affiliated Hospital of Wenzhou Medical College institutional review board for clinical experiments and use of human samples; written consents were obtained from all subjects participated in this study in accordance with the Declaration of Helsinki protocol.Human AML cell lines HL-60, U937 , and NB4  of matrine  for 12 h, 24 h, and 48 h, and cell viabilities were assessed using the 3--2,5-diphenyltetrazolium bromide (MTT)  assay. The absorbance (A) was read at 490 nm using an ELISA reader . Cell viability rate (%) = A490, matrine/A490, control×100%.AML cells , leukemic CD345 cells/well in 12-well plates. The cells were treated with various concentrations of matrine for 24 h or 1.5 g/L matrine for three different time points . In addition, leukemic CD34+lin− cells and normal CD34+lin− cells were plated and treated with 1.5 g/L matrine for 24 h. Apoptosis was measured using FITC Annexin V Apoptosis Detection Kit II  according to the manufacturer’s protocols. Data acquisition and analysis were performed using CellQuest software on a flow cytometry . In addition, AML cells were treated with or without Z-Asp(O-Me)-Glu(O-Me)-Val-Asp(O-Me) fluoromethyl ketone , a caspase-3 inhibitor for 1 h before treatment with matrine for 24 h, then, the apoptosis analysis was performed as described above.AML cells were plated at a density of 2.0×10Rhodamine 123 is a yellow-green fluorescent probe that stains mitochondria in living cells in a membrane potential-dependent fashion. Cells were plated into 12-well plates and treated with matrine for 24 h. Then, cells were collected, washed twice with cold phosphate-buffered saline (PBS), and incubated with 5 µM Rhodamine 123 (Sigma-Aldrich) at 37°C in the dark for 30 min before FCM analysis. The cells were subsequently washed twice and analyzed by FCM. As described previously AML cells were plated into 12-well plates and treated with various concentrations  of matrine for 24 h. Then, the cells were collected, fixed, permeabilized, stained with Bcl-2 PE, Bax PE , and activated caspase-3 PE (BD Pharmingen™), respectively. The expression levels were subsequently detected and analyzed by FCM.After treatment with matrine, the related changes of protein expression in cell lysates were detected using western blot. Briefly, collected cells were lysed immediately in M-PER® Mammalian protein extraction reagent  supplemented with Halt protease and phosphatase inhibitor cocktail (Pierce). Protein concentration was assessed using BCA protein assay kit . Then, equal amounts of protein (30 µg) were boiled for 5 min, separated by SDS-PAGE, and electroblotted onto a PVDF membrane. After blocking, membranes were incubated with respective antibodies including Akt, p-Akt (Ser473), ERK1/2, and p-ERK1/2 (Thr202/Tyr204)  at appropriate dilutions overnight at 4°C. Membranes were then washed and incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology) for 2 h at room temperature. Membranes were then washed again and developed using enhanced chemiluminescence. The optical densities of the bands were measured using Scion image analysis .6) were suspended in 100 µl PBS and subcutaneously injected into the right hind leg of recipient mice. On day 5, these mice were randomly divided into 4 groups, with 8 animals in each group. Then, the two treatment groups were injected intraperitoneally at two doses of matrine (50 mg/kg and 100 mg/kg) on alternative days, respectively. Positive and negative control group animals were given CTX (20 mg/kg) and 0.9% sodium chloride, respectively. All mice were killed on day 22 after these drugs had been administered seven times and tumor masses were carefully dissected out and weighted. Animal procedures were carried out in accordance with institutional guidelines after Wenzhou Medical College and Fudan University Animal Care and Use Committee approved the study protocol.The severe combined immunodeficiency (SCID) mice , bred in-house, were maintained throughout in specific pathogen-free (SPF) environment. Exponentially growing HL-60  was used to examine differences among the matrine groups with different doses. Differences were considered significant when 50 at 24 h of 0.91, 0.66, and 0.59 g/L, respectively . The positive expression rate of cytoplasmic myeloperoxidase was (82.1±6.2) % in BMMNCs from 9 patients with AML. Matrine inhibited the proliferation of AML cell lines HL-60, NB4, and U937 in a dose- and time-dependent manner, with ICectively . Further0.99 g/L . 1.5 andn− cells . Howevern− cells . The res+lin− cells treated with 1.5 g/L matrine for 24 h was 5.1%  and primary AML cells after treatment with high concentrations of matrine (1.5 and 2.0 g/L) for 24 h . In addiTo investigate whether the Akt pathway and the ERK pathway involved in matrine-induced apoptosis, AML cells were treated with 1.5 g/L matrine for 24 h. Then, the expression levels of p-Akt and p-ERK1/2 were analyzed using western blot. As shown in in vivo. The heterogeneous AML model was established in SCID mice following subcutaneous transplantation of HL-60 cells. As shown in P>0.05). The 20 mg/kg CTX-treated mice had the smallest tumor weight among 4 groups. These results indicated that matrine has a significant anti-leukemic effect in vivo.We further studied the effect of matrine-mediated AML cell growth inhibition in vitro and subcutaneous xenograft HL-60 cells in mice. Furthermore, we also exhibited a mechanism of action by which matrine induced apoptosis of AML cells via the mitochondrial-mediated pathway. In addition, it has been demonstrated that only a slight effect was observed on normal CD34+lin− cells and peripheral blood mononuclear cells treated by matrine less than 2 g/L in our present and previous study in vitro and in vivo.The present study has shown that matrine induces marked growth inhibition and apoptosis in AML cell lines as well as primary AML cells Uncontrolled proliferation is a key aspect of tumorigenesis, and inhibiting cell proliferation can achieve growth arrest in tumor cells. The proliferation of AML cell lines HL-60, NB4, and U937 was significantly inhibited by matrine. We also showed that matrine had a marked cytotoxicity in BMMNCs from 9 patients with AML, in which included approximately 80% primary AML cells. We and others have reported that apoptosis is a key mechanism by which matrine kills tumor cells Δψm loss and cyt c leak out.It has been widely recognized that apoptosis is initiated by two principal pathways: the mitochondria-mediated intrinsic pathway and the death-receptor-induced extrinsic pathway The release of cyt c from the mitochondrion plays a crucial role for the execution of the apoptotic pathway because it activates caspase-9, which subsequently activates caspase-3. The activated caspase-3 leads to the final destruction of the target cell. We have shown that the levels of activated caspase-3 of AML cells induced by matrine increased in a dose-dependent manner. The activation of caspase-3 occurred concomitantly with apoptosis in AML cells, suggesting that the activation of caspase-3 might lead to apoptosis. This viewpoint was further verified by the addition of Z-DEVD-FMK. Combined with with 1.5 g/L matrine and 5 µM Z-DEVD-FMK, apoptosis was partially blocked. These results indicated that matrine-induced apoptosis in AML cells was partially dependent on the activation of caspase-3.et alSome signaling pathways, such as PI3K/Akt and ERK/MAPK, play a critical role in the pathogenesis and progression of AML in vitro and in vivo. The anticancer activity of matrine could be attributed to its inhibition of proliferation and apoptosis induction of AML cells through mitochondria-mediated pathway and inhibition of the Akt and ERK1/2 pathways. Given our findings that matrine inhibits proliferation and induces apoptosis in AML cell lines and primary AML cells, matrine may be a useful candidate as chemotherapeutic agent against AML.In conclusion, matrine exerts a significant anti-leukemic effect on AML cells


While the effect of β-adrenergic related PKA phosphorylation of troponin I at Ser23/24 is well established, the effects of α-adrenergic induced PKC phosphorylation on multiple sites of TnI  and TnT  are much less clear. By utilizing an IAANS labeled fluorescent troponin C, 2+ binding properties in the Tn complex and reconstituted thin filament. The majority of the phosphomemetics had little effect on the Ca2+ binding properties of the isolated Tn complex. However, when incorporated into the thin filament, the phosphomimetics typically altered thin filament Ca2+ sensitivity in a way consistent with their respective effects on Ca2+ sensitivity of skinned muscle preparations. The altered Ca2+ sensitivity could be generally explained by a change in Ca2+ dissociation rates. Within TnI, phosphomimetic Asp and Glu did not always behave similar, nor were Ala mutations (used to mimic non-phosphorylatable states) benign to Ca2+ binding. Our results suggest that Troponin may act as a hub on the thin filament, sensing physiological stimuli to modulate the contractile performance of the heart.Adrenergic stimulation modulates cardiac function by altering the phosphorylation status of several cardiac proteins. The Troponin complex, which is the Ca Instead, it may act as a central hub that integrates information from the myofilament  to adjust its Ca2+ binding properties and regulate cardiac muscle mechanics Troponin (Tn) is the CaTnC, troponin I (TnI), and troponin T (TnT) form the trimeric Tn complex that regulates myofilament activation. Both TnI and TnT are important targets of adrenergic stimulation induced phosphorylation 2+ binding properties. We studied not only the effects of these phosphomimetic mutants on Tn’s Ca2+ sensitivity, but also their effects on Tn’s Ca2+ exchange kinetics, which may be even more significant to how the heart performs since the heart is dynamic and does not function in a static steady-state. Additionally, we examined the effects of different substitution residues that mimic different phosphorylation states (Asp or Glu for phosphorylation and Ala for non-phosphorylatable) on Tn’s Ca2+ binding at select sites. Our results show that all the phosphorylation related protein modifications of TnI or TnT alter Tn’s Ca2+ binding in a way that can be related to previous physiological studies. These results are consistent with the notion that Tn acts as a central hub on the thin filament by sensing physiological stimuli to alter cardiac contractile properties.In this study, we generated a series of phosphomimetic mutants of TnI and TnT at multiple PKC phosphorylation sites to systematically examine the site-specific effects on Tn’s CaPhenyl-Sepharose CL-4B, Tween-20, and EGTA were purchased from Sigma Chemical Co. . IAANS and phalloidin were purchased from Invitrogen . Affi-Gel 15 affinity media was purchased from Bio-Rad .The pET3a plasmid encoding human cardiac TnC was a generous gift from Dr. Lawrence Smillie . The pET3a plasmids encoding human cardiac TnI and TnT were graciously provided by Dr. James Potter . TnC, TnI and TnT mutants were constructed from their respective pET3a plasmids using the primer-based QuikChange Site-Directed Mutagenesis Kit  as previously described E. coli BL21(DE3)pLysS cells , while the TnI and TnT plasmids were transformed into Rosetta™(DE3)pLysS cells . The proteins were expressed and purified as previously described The plasmid encoding human cardiac TnC was transformed into Rabbit skeletal actin and bovine ventricular tropomyosin (Tm) were purified from acetone powders as previously described C35S,T53C,C84S (herein denoted as TnCT53C) was labeled with the environmentally sensitive thiol-reactive fluorescent probe IAANS as previously described TnCThe reconstituted Tn complexes and regulated thin filaments were prepared as previously described 2 were added to 2 ml of each labeled Tn complex (0.15 µM) in a titration buffer (200 mM MOPS (to prevent pH changes upon addition of Ca2+), 150 mM KCl, 2 mM EGTA, 1 mM DTT, 3 mM MgCl2, 0.02% Tween-20, pH 7.0) with constant stirring. Reconstituted thin filaments were titrated in an identical buffer composition (excluding Tween-20). The [Ca2+]free was calculated using the computer program EGCA02 developed by Robertson and Potter 2+ sensitivities were reported as a dissociation constant Kd, representing a mean of three to four separate titrations ± S.E.M. The data were fit with a logistic sigmoid function , as previously described All steady-state fluorescence measurements were performed using a Perkin-Elmer LS55 spectrofluorimeter at 15°C. IAANS fluorescence was excited at 330 nm and monitored at 450 nm as microliter amounts of CaCl2+ exchange rates were characterized using an Applied Photophysics model SX.20 stopped-flow instrument with a dead time of 1.4 ms at 15°C. IAANS fluorescence was excited at 330 nm. The IAANS emission was monitored through either a 420–470 nm band-pass interference filter for 2, 0.02% Tween-20 (excluded for thin filament kinetic measurements), at pH 7.0. 10 mM EGTA was utilized to remove 200 µM Ca2+ from the Tn complexes or thin filaments.CaStatistical significance was determined by ANOVA followed by a Dunnett’s post-hoc t-test using the statistical analysis software Minitab . Two means were considered to be significantly different when the P value was < 0.05. The data is shown as a mean value ± S.E.M.2+ sensitivity of force development as well as delayed and accelerated relaxation kinetics 2+ binding properties of the Tn complex were examined first since Tn is the simplest biochemical system to test the effects of TnI and TnT modifications on TnC. The Ca2+ sensitivity of TnC within the various Tn complexes was measured by following the Ca2+ dependent decrease in IAANS fluorescence. Similar to previous studies 2+ induced half-maximal fluorescence decrease at 0.89±0.02 µM . As shown in ectively . While T1.8 fold , Table 12+ dissociation from unlabeled wild type Tn and a series of rationally engineered TnC mutants with high fidelity 2+ dissociation from the phosphomimetic 2+ dissociation from control Previously, we demonstrated that the fluorescence of .3±0.2/s .2+ sensitivity of the Tn complex, PKC phosphorylation mimicking TnI T144E had no effect on the rate of Ca2+ dissociation from the Tn complex. However, TnI T144D and T144A slightly increased the rate of Ca2+ dissociation from the Tn complex  did not alter the rate of Ca2+ dissociation from the Tn complex . Intere2.2 fold . Additio binding . As show3.3 fold . On the 1.5 fold . TnT S191.8 fold , while T binding .2+ dissociation from thin filament bound 2+ dissociation from thin filaments reconstituted with control Fluorescence stopped-flow measurements were conducted to determine the rate of Ca.5±0.8/s .2+ sensitizing effect on steady state Ca2+ sensitivity among the three mutations   . Additio4.7 fold . As show3.7 fold . The othilaments .2+ sensitivity of TnC is determined by the kinetics of Ca2+ association and dissociation. Based on the experimentally measured steady-state Ca2+ sensitivity and Ca2+ dissociation rates, we calculated the rates of Ca2+ association to thin filaments for each of the phosphomimetic mutants  or potentially slow the rate of Ca2+ dissociation (TnI T144D), which may contribute to the observed acceleration and slowing of relaxation during dynamic regulation of PKC pathways The thin filament kinetic studies are generally in line with the steady state studies. While the physiological significance of the Ca2+ sensitivity of TnC is determined by the kinetics of Ca2+ association and dissociation. It is generally assumed that alterations in the steady-state Ca2+ sensitivity of TnC operate exclusively through changes in the rate of Ca2+ dissociation, since Ca2+ association to TnC has been traditionally thought to be diffusion controlled  cross-talk to alter the Ca2+ sensitivity of the thin filament 2+ binding properties of the thin filament.The next logical step to these studies is to delve into how the various PKC sites may work together to tune the Ca2+ binding sensitivity and slowed the rate of Ca2+ dissociation. Thus, slight side chain differences between Glu and Asp could potentially cause significant differences. On the other hand, when Ser43/45 were replaced by Glu, Asp, or Ala, all three mutants desensitized thin filament Ca2+ binding, with the biggest effect seen for S43/45E. Interestingly, S43/45A had a bigger Ca2+ desensitizing effect than S43/45D, which might question the validity of trusting the Ala substitution for mimicking a non-phosphorylatable state. These results are consistent with a recent report that an Ala mutation in polo-like kinase 1 behaved similarly to both Asp and Glu pseudo-phosphorylation mutations Within the Tn literature, both Asp and Glu have been utilized to mimic phosphporylated residues 2+ binding properties of TnC, with the largest influence being the binding of TnI to TnC 2+ sensitivity, whereas just the opposite will occur when TnI binding is hindered in any way 2+ sensitivity of TnC, we speculate that this is due to a decreased ability of TnC to bind TnI. Since the majority of the mutations had little effect on the isolated Tn complex, we do not think there are any major deficits in the intrinsic ability of TnC to bind TnI, but that TnI is in some way restrained from binding to TnC. This could occur more readily on the thin filament where enhanced actin-Tm binding of TnI can more effectively outcompete the binding of TnI to TnC. Thus, we predict that PKC phosphorylation of TnI and TnT increases the affinity of TnI for actin-Tm. Alternatively, phosphorylation may affect the flexibility of Tn, which can alter both the apparent Ca2+ sensitivity and cooperativity of the thin filament There are potentially multiple mechanisms that exist to alter the Ca2+ binding properties of TnC in a way consistent with their previously reported effects in skinned cardiac muscle or myocyte preparations. Therefore, TnC may act as a central hub that converges these physiological stimuli to affect cardiac contractile properties.In conclusion, the phosphomimetics studied in this work altered the steady state and kinetic Ca


Mentoring is important for the recruitment and retention of qualified nurse faculty, their ongoing career development, and leadership development. However, what are current best practices of mentoring? The purpose of this paper is to provide an overview of a model for excellence in establishing a formal mentoring program for academic nurse educators. Six themes for establishing a formal mentoring program are presented, highlighting best practices in mentoring as culled from experience and the literature. Themes reflect aims to achieve appropriately matched dyads, establish clear mentorship purpose and goals, solidify the dyad relationship, advocate for and guide the protégé, integrate the protégé into the academic culture, and mobilize institutional resources for mentoring support. Attending to the six themes will help mentors achieve important protégé outcomes, such as orientation to the educator role, integration into the academic community, development of teaching, scholarship, and service skills, as well as leadership development. The model is intended to be generalizable for faculty teaching in a variety of academic nursing institution types and sizes. Mentoring that integrates the six themes assists faculty members to better navigate the academic environment and more easily transition to new roles and responsibilities. Mentoring is important for the recruitment and retention of qualified nurse faculty, their ongoing career development, and leadership development. The functional outcomes of mentoring encompass orientation to the educator role, integration into the academic community, development of teaching, scholarship, and service skills, as well as leadership development . Given tThe above questions are examples of some of the inquiries that motivated the authors to conduct an integrated review to design a template for excellence in mentoring in the context of nursing education. The authors aimed to create a template addressing the “what” and “how” of mentoring that would serve as a standardized best-practice model targeting faculty across the career span. This paper reflects the yearlong work of the group project from the fourth cohort and Project Director of the National League for Nursing (NLN)/Johnson & Johnson (J&J) Faculty Leadership and Mentoring Program. The NLN, with generous support from J&J, established a faculty leadership and mentoring program in 2007 with the overall goal to prepare leaders to transform the future of nursing education . The word “mentor” derives from Greek mythology when Odysseus entrusted the care of his son to his friend “Mentor,” to serve as guide and teacher while he went to fight the Trojan War . Since tResearch indicates many positive outcomes as a result of mentorship. For example, when a novice educator is formally mentored by a more experienced and accomplished academician, the novice educator more quickly assumes the full scope of the academic role and is more productive . Across Often, nurse educators enter the academic role without a clear idea of the full scope of their responsibilities, or how they can actually achieve them at a level sufficient to become productive academicians. Others labor under the misconception that teaching is the academicians' primary responsibility. Mentoring relationships can help educators understand the multifaceted roles of an academician, which facilitates achieving success in a timely manner in the areas of teaching, scholarship. and service. Research demonstrates that careers did not progress as satisfactorily when faculty did not have mentors, compared to those who did , 9, 12. The purpose of this paper is to provide an overview of a model for excellence in establishing a formal mentoring program for academic nurse educators. The model is intended to be generalizable for faculty teaching in a variety of academic nursing institution types and sizes.The authors participated in a formal distance mentoring program and determined to engage in a heuristic inquiry to study mentoring. Initially, the authors used an inductive process to identify mentoring themes. At a face-to-face meeting, each participant shared lived experiences (good or bad) of either being mentored or mentoring someone and described the significance of the experience. The group discussed practices of mentoring revealed in each story; a recording secretary listed practices that group members agreed upon. Through reflection and dialogue, the group clustered 25 original practices into six categories based on similar thematic content. The six categories served as the basis for six in-depth reviews of the literature; refinement of the categories occurred over several months during exploration of research literature. Databases searched included Academic Search Elite, CINAHL, ERIC, PUBMED, Google, and Google Scholar. Search terms included mentor, protégé, mentee, mentoring, faculty mentoring programs, mentorship, mentorship advocacy, collegiality, academic networking, academic socialization, matching, resources, and workload release. Only articles in English were considered for the integrated review.Six categories reflecting aims for establishing a formal mentoring program are presented, highlighting six best practices in mentoring. These practices support the four pillars of excellence . The disAppropriate fit is an important aspect for creating a successful mentor/mentee (protégé) relationship; therefore, matching the right mentor to the right protégé is the first best practice theme. Pairing can be accomplished by a variety of methods described below. There is, however, no clear evidence as to which method is best. Fortunately, the literature does give guidance that seeking individual input from both the mentor and protégé will result in the best fit.The mentor and protégé are often referred to as a dyad or pair. There are five basic ways that mentors and protégés can come together to form a mentoring dyad: (a) paired administratively based on arbitrary criteria, (b) paired administratively based on specified criteria, (c) paired based on protégé selection of mentor, (d) paired based on mentor selection of a protégé based on recognized potential and a desire to “take under wing,” and (e) paired based on finding each other and creating their own dyad relationship. Formal mentoring programs tend to use one of the first three approaches to pair mentor and protégé  which meClearly, administrators often assign mentors based on availability: whose turn it is, who is most friendly, or who is coteaching a course. While this approach is common practice in contemporary nursing education, it may not produce the best fit. Mentor-protégé mismatch has been identified as a common problem in formal mentoring programs from both perspectives . DiffereThe significance of obtaining dyad input during the matching process has been noted by many researchers . Allen eParise and Forret  studied One strategy to elicit input into a matching process is to match the dyad using criteria—in this way, the potential for compatibility increases. Headlam-Wells et al.  tested cAnother strategy that encourages input of both mentor and protégé is a variation on speed dating. Berk  recommenBozeman and Feeney  offered endowments ;mentoring content ;preferences . The notion of goodness-to-fit generates questions for future research on how preferences affect endowments and endowments affect preferences. The fit will shape mentorship outcomes. For example, the dyad fit is good when “the mentor has the knowledge preferred by the protégé, and the ability to transmit that knowledge effectively…and the protégé has the ability and skill to fully [grasp] the knowledge being transmitted” [Regardless of the strategy employed, the recommended best practice to achieve appropriately matched dyads is to obtain input in the matching process. Once dyads have been matched using input by both parties, the next best practice is to establish clear purposes and goals for the mentoring relationship. The following section provides direction as to what mentors should aim for when starting out in a new mentoring relationship.Once paired, the dyad must clearly articulate the purpose of the mentorship relationship and set initial goals early on to give direction and clarity of future responsibilities. In addition to helping the protégé establish personal career goals, Chao  recommenThe concept of reciprocity occurred frequently in the mentoring literature. Sorcinelli and Yun  called t Formal mentoring programs are characterized by a clearly stated timeframe for the dyad relationship that focuses on the protégé's development . A typic Ideally, activities should be spread over time. While dyads have freedom to negotiate how they will communicate with one another, the key point is that both the mentor and the protégé are committed to and engaged in the mentoring relationship and participate in multiple activities over time , 25. RegThe formation of a relationship between mentor and protégé is crucial to effective mentoring. As the relationship unfolds and expectations are clarified, the dyad must strive to deepen the relationship. The relationship may be further developed through four strategies, namely, (a) creating collegiality, (b) establishing regular communication, (c) exchanging regular feedback from mentor and protégé, and (d) building a supportive environment.Once the dyad has been matched appropriately and goals set, the next step in creating successful mentoring is for the pair to establish a collegial relationship. Collegial and reciprocal relationships are the basis for effective mentoring which ultimately fosters the protégé's academic success . CollegiOrganizations have used multiple strategies for recruitment and retention of faculty. Whereas, financial incentives and promotional campaigns are effective in recruiting faculty in the short term, they do not necessarily retain faculty in the long term. Emerging evidence suggests that an environment of incivility in the workplace is often the reason for employee attrition , 30. In  To solidify a relationship, regular “connectedness” cannot be understated. The more formalized and set the pattern of communication is the better the connection occurs between the pair. Smith and Zsohar  and Whit Mentors can be successful resources when dyads prepare for relationships by not only reflecting on and defining goals, but also identifying challenges and asking for feedback to evaluate relationship effectiveness , 36–39.  Creating an environment where the protégé feels supported cannot be overemphasized; this action has a direct effect on solidifying the dyad relationship . When prWhat does a supportive environment look like? Mentors who show positive regard and genuine caring are willing to listen, display empathy and trustworthiness, give encouragement, provide authentic feedback, and create supportive environments , 25, 40.The fourth theme of best practices is advocating for and guiding the protégé. Although much of the research in this area is less recent, it is relevant to review. Advocacy provides tangible benefits for the protégé throughout the mentoring process. An advocate is someone who supports another and acts for another's benefit or one who speaks for another's behalf. The literature provides many areas for mentor advocacy/guidance. This paper, however, focuses on three primary strategies: (a) providing psychosocial support, (b) achieving life balance, and (c) advising career progression.motivating protégés as one of the top activities, followed by 91% providing moral support. Protégés who were queried about mentor activities ranked being acknowledged as a person and as a professional very highly (4.8 of 5), while having mentors who listened to their expressed concerns ranked a perfect 5 of 5 [ With multifaceted academic responsibilities, faculty may easily feel pulled in many directions and become discouraged. Earlier research indicated that a major aspect of advocacy is for mentors to attend to psychosocial undertakings , 41, 42. Achievement of life balance between personal and professional work is critical to the success of a new faculty member. Straus et al.  saw the  An obvious area for protégé advocacy/guidance is advising career progression while helping the protégé achieve balance between personal and professional responsibilities. Mentors often promote progress by helping protégés set professional goals, mapping a career plan, and establishing clear career milestones , 43. MenThe fifth theme of best practices offers two separate but critical activities that support the program aim of integrating protégés into the academic culture: (a) teaching networking skills and (b) facilitating socialization to the academic culture. The value of integrating protégés into the academic culture is that it allows mentors to share intellectual capital and emotional intelligence. The mentor should give due diligence to this theme, since these qualities affect the protégé's ability to become a productive member of academe. Mentors can facilitate protégé integration into the culture of academe by helping protégés learn to network and establish professional contacts. Coleman et al. identified networking as extremely important for getting to know professional contacts in the clinical area . NetworkA second way mentors can facilitate protégé integration into the culture of academe is by helping protégés navigate the social structure and culture of academics. Faculty new to academia are rarely prepared educationally or experientially for the multiple roles and expectations as well as the isolation that may present itself with academe—the reason being, academia is an unfamiliar culture to them . There iA smooth transition into the academic culture by a novice educator may not be realized without the assistance of a mentor since the social norms and expectations are most often not written—or easily understood . SocialiThe sixth theme of best practices, mobilizing institutional resources, requires institutional responsibility in order for the intended mentoring program to thrive. Four strategies are identified here that reflect administrative, collegial, or financial investments.resources, they often think of money. Although money is a key component, the primary resource requirement for an effective formal mentoring program is administrative support and commitment. Therefore, a critical strategy to assist mobilizing resources in mentoring is to gain administrative support at both the departmental and college level prior to initiating any formal mentoring program [ When people think of the term  program , 48, 49.Another action that can be readily accomplished is the inclusion of mentoring activities into faculty expectations; these can be demonstrated in the criteria for promotion and tenure, salary merit, and workload calculation documents. Including expectations that experienced faculty mentor junior faculty in these documents sends a strong message of acceptance by both administration and faculty. Plus, faculty receive acknowledgement and credit for time spent mentoring other faculty.The time is ripe for this action; a practice analysis conducted in 2005 by the National League for Nursing showed that, nationally, faculty and administrators identified mentoring as part of the role of nurse faculty . It must Because effective mentorship skills do not always occur naturally, a third strategy is to train mentors. Effective mentorship extends beyond simply sharing one's knowledge or expertise; mentors can also be taught how to be effective mentors . This be This best practice requires some financial expense in the form of release time for mentor and protégé participants . ReleaseThe model see  builds o“Best practice” is operationally defined as those actions that produce the most desirable faculty outcomes, based on evidence and real life experiences. An underlying assumption is that relationships play a key role in any successful mentorship experience, as evidenced by the focus of the best-practice themes as it pertains to ways to initiate, build, solidify, advocate, or integrate the relationship.Mentoring programs have many benefits and contribute to improved faculty morale, higher career satisfaction, and increased self-confidence in professional development. Mentored faculty publish more, obtain more grants, and are promoted more quickly. Institutions providing mentoring programs experience increased retention and improved sense of community and professional identity.Best Practices in Academic Mentoring: A Model for Excellence,” provides a schema that can be used to create programs of mentoring and functions as a thematic basis for evaluation of program effectiveness. Mentoring assists faculty members to better navigate the academic environment and more easily transition to new roles and responsibilities. A work environment where collaborative and reciprocal peer and co-mentoring are present results in a rich, satisfying, and rewarding career experience for both mentor and protégé. It ultimately moves the profession forward.The model, “Imagine the impact on faculty career attainment, institutional culture, the science of nursing, and leadership development in nursing education, if all faculty were mentored. Mentoring programs are especially important at a time when academia is experiencing a shortage of nurse faculty members. A trait of a true leader then is being an excellent mentor and developing future leaders.


Chronic overexpression of HRC that may disrupt intracellular Ca2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively.Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca2+ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay.AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca2+ leak and cytosolic Ca2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca2+ cycling.Increased Ca However, the precise mechanism of HRC mediated inhibition of Ca2+ cycling and the long term cardiac remodeling has remained to be clarified.We have previously reported that HRC overexpression increased SR Ca2+ cycling and cardiac function through the increased activity of SERCA2 and RyR2. Thus, we used synthetic siRNA oligonucleotides and adeno-associated virus (AAV) to knock-down HRC expression in vitro (for short term effect) and in vivo (for chronic effect), respectively. HRC-KD in neonatal rat ventricular cells (NRVCs) or HL-1 cells showed enhanced Ca2+ cycling, but the resting Ca2+ concentration was increased due possibly to Ca2+ leak through the activated RyR2. HRC-KD using AAV9-shHRC resulted in more decreased cardiac function, and increased cardiac fibrosis and apoptosis causing more severe heart failure in mice under pressure-overload by transverse aortic constriction (TAC). Our concomitant biochemical study showed that the increased Ca2+-leak and elevated cytosolic Ca2+ due to HRC-KD could enhance phosphorylation of CaMKII - p38 MAPK pathway resulting in the increased apoptosis and heart failure. Collectively, the present study suggests that HRC is an important regulator of Ca2+ homeostasis which is essential for normal functions of the heart.The present study was designed on the basis of the hypothesis that HRC knock-down (KD) enhances Ca2+ cycling without possible change in protein expression related to Ca2+ cycling. The siRNA oligonucleotides were successfully transfected to NRVCs  (The chronic HRC overexpression or KO studies presented higher expression of TRN protein han 99%)  and the han 99%) . We alsohan 99%) .2+ transients in siNC and siHRC oligo transfected NRVCs to investigate whether HRC-KD affected Ca2+ transients in these cells under basal and isoproterenol (ISO) treated conditions (2+ concentration (baseline) was significantly increased in HRC-KD NRVCs (n = 33) , and the peak amplitude of the Ca2+ transients was also increased in HRC-KD cells , suggesting increased Ca2+ release through RyR2. The time to reach 50% baseline (T50 baseline), on the other hand, was significantly reduced , suggesting faster Ca2+ uptake through SERCA2 (2+ concentration (baseline) was again significantly increased in HRC-KD NRVCs (n = 25) , and the peak amplitude of Ca2+ transients was also increased in HRC-KD cells , suggesting increased Ca2+ release through RyR2. The time to reach 50% baseline (T50 baseline), on the other hand, was significantly reduced , suggesting faster Ca2+ uptake through SERCA2   2+ was released from the SR upon electrical stimulation in HRC-KD cells   could be due to fact that Ca2+ release was already activated under ISO condition in siNC cells. HL-1 cells showed no significant change of caffeine-induced Ca2+ transients, but significantly enhanced fractional Ca2+ release under basal condition, similar to NRVCs   and 3C. 2±7.57%)  and 3C. to NRVCs .2+ cycling in HRC-KD NRVCs  was significantly increased in siHRC cells (n = 27), compared with siNC cells (n = 17)  without a significant change in dissociation constant of [3H]-ryanodine binding (Kd) (siNC: 2.786±0.664 nM vs. siHRC: 3.052±0.702)  . In prev2+ uptake rate was highly increased in siHRC cells (n = 7) compared with siNC cells (n = 5)  (2+ cycling in HRC-KD cells is caused by the increased activities of RyR2 and SERCA2.The SR Ca P<0.05) , suggest P<0.05) . These r2+ cycling  and further increase in dilation of the heart at 11 weeks post operation in the failure group . HRC-KD It has been reported that apoptosis is the critical mechanism present in hypertensive heart disease 2+ concentration in NRVCs and HL-1 cells  Tpathways  and 8. C2+ transient amplitude and enhanced activities of RyR2 and SERCA2, suggestive of an enhanced Ca2+ cycling phenotype .2 and 95% air in an incubator.HL-1 cells obtained as a kind gift from Dr. W. Claycomb  were maintained as described previously HRC gene. siRNA transfection was performed as described previously Primary cultures of neonatal rat ventricular cells (NRVCs) from 2-day-old Sprague-Dawley rats were prepared as described siRNA-transfected NRVCs or HL-1 cells were washed in cold PBS and lysed in lysis buffer containing 1% SDS, protease inhibitor cocktail (Roche Applied Science), 10 mM Tris-HCl (pH 7.4) and frozen hearts of shNC sham, shNC failure, shHRC sham and shHRC failure mice were grounded in a mortar and lysed in the same buffer. Cell lysates (60 µg) and heart homogenates (60 µg) were separated by electrophoresis on 6–15% gels and transferred to 0.2 µm nitrocellulose or 0.45 µm PVDF membranes. After blocking with 5% skim milk in 1× TBST, the membranes were incubated with respective antibodies: HRC, CSQ, TRN ; DHPR, NCX, CaMKII (Abcam); RyR2, SERCA2a, phospho-CaMKII and PLB (ABR); phospho-RyR2 and phospho-PLB-T17 (Badrilla); phospho-p38MAPK ; α-tubulin (Santa Cruz Biotech); phospho-PLB-S16 (Upstate). After primary antibody incubation, membranes were washed with 1× TBST and further incubated with proper peroxidase-conjugated secondary antibody. Western blot signal was detected by ImageQuant LAS 4000 mini  with a SuperSignal West Pico chemiluminescence kit . Western blot band intensities were measured by using ImageJ software.2+ transients in NRVCs or HL-1 cells were measured as described previously 2, 1.8 mM CaCl2, and 10 mM glucose for 30 min and washed in dye-free Tyrode solution. The cells were placed in a circulating bath with Tyrode solution held at 37°C under an inverted microscope. A dual-beam excitation spectrofluorometer setup (IONOPTIX) was used to record fluorescence emissions (505 nm) elicited from exciting wavelengths of 340 and 380 nm. Ca2+ transient amplitude measured as fluorescence ratio (340∶380 nm), cytosolic free Ca2+ concentration (baseline), time required to reach 50% of baseline (T50), and time to peak of Ca2+ transients were acquired. SR Ca2+ content was estimated by rapid application of 20 mM caffeine in Ca2+-free Tyrode solution. Data were analyzed by using Ion Wizard software (IONOPTIX). In the experiment shown in CaMicrosome preparations were carried out as described previously 3H]ryanodine binding assay in NRVCs or HL-1 cells was performed as described previously µl of reaction mixture containing 1 M KCl, 20 mM Mops (pH 7.4), various concentration of [3H]ryanodine and 50 µM free Ca2+ for 2 hours at 37°C. PEG (poly-ethylene glycol) solution  was added to each vial and further incubated for 5 min at room temperature. Precipitated protein was segregated for 5 min at 12000 g in an Eppendorf microcentrifuge, and the pellets were rinsed twice with 0.4 ml of the washing buffer containing 1 M KCl, 20 mM Mops (pH 7.4) and 50 µM free Ca2+. The pellets were then solubilized in 200 µl of Soluene 350  at 70°C for 45 min and then radioactivity was counted with 4 ml of Picofluor (Packard) using a liquid scintillation counter . For nonspecific binding, a 1000-fold amount of non-radioactive ryanodine  was included. For the PEG solution, BSA and γ -globulin (5 mg of each per ml) were used as carrier proteins. [3H]ryanodine binding for NRVCs, whole homogenates were used.-ryanodine binding and oxalate-supported Ca2+ uptake assays in HL-1 cells.-ryanodine binding and oxalate-supported Ca2+ uptake assays. A: [3H]-ryanodine binding assay results using siNC and siHRC transfected HL-1 cells. The significantly different Bmax values of [3H]-ryanodine binding were 0.157±0.008 and 0.209±0.015 pmol/mg for siNC (○) and siHRC (•) samples, respectively. The Kd values are not significantly different between the 2 samples (siNC: 6.015±0.644 nM vs. siHRC: 5.314±0.654). B: Oxalate-supported Ca2+ uptake assay results. Oxalate-supported SR-based Ca2+ uptake in siNC (○) and siHRC (•) transfected HL-1 cells were determined at 1, 2, and 3 min. The rates of Ca2+ uptake were 19.29±2.62 and 26.14±1.42 nmol/mg/min for siNC and siHRC samples, respectively. Five sets of siHRC and 4 sets of siNC HL-1 cells were used for statistical analyses .(TIF)Click here for additional data file.Figure S4AAV transduction efficacy in mouse heart. DsRed was removed to enhance AAV-mediated knock-down (KD) efficiency of HRC in mouse heart. GFP-expressing AAV (AAV-GFP) was used to examine the transduction efficiency of our AAV systems. A: Western blot result showing AAV-mediated GFP expression in mouse heart. B: Fluorescence microscopic result for GFP in AAV-GFP transduced heart sample.(TIF)Click here for additional data file.


This study investigated the effects of alterations in Ca2+ homeostasis and sarcoplasmic reticulum Ca2+-associated proteins on cardiac function in the development of diabetic cardiomyopathy.Cardiac dysfunction in diabetic cardiomyopathy may be associated with abnormal Ca2+ spark images were formed by introducing fluorescent dye Fluo-4 and obtained with confocal scanning microscopy. Expressions of Ca2+-associated proteins were assessed by Western blotting.Sprague–Dawley rats were divided into 4 groups : a control group, and streptozotocin-induced rat models of diabetes groups, examined after 4, 8, or 12 weeks. Evaluations on cardiac structure and function were performed by echocardiography and hemodynamic examinations, respectively. Cardiomyocytes were isolated and spontaneous Ca2+ spark frequency . However, Ca2+ spark decay time constants increased significantly, relative to the control group. Expressions of ryanodine receptor 2 (RyR2), sarcoplasmic reticulum Ca2+-2ATPase (SERCA) and Na+/Ca2+ exchanger (NCX1) were decreased, together with quantitative alterations in Ca2+regulatory proteins, FKBP12.6 and phospholamban progressively and respectively in the diabetic rats.Echocardiography and hemodynamic measurements revealed that cardiac dysfunction is associated with the progression of diabetes, which also correlated with a gradual but significant decline in Ca2+ sparks exhibited a time-dependent decay with progression of diabetic cardiomyopathy, which may partly contribute to cardiac dysfunction. This abnormality may be attributable to alterations in the expressions of some Ca2+-associated proteins.Ca A small amount of Ca2+ influx through L-type Ca2+ channels can activate a further discharge of Ca2+ from the sarcoplasmic reticulum (SR) via ryanodine receptor 2 (RyR2), resulting in an increase in global intracellular Ca2+ which activates contraction [2+ release events occur spontaneously as clusters, termed Ca2+ sparks [2+ into the SR via Ca2+-ATPase (SERCA) on the SR, and concurrently Ca2+ is pumped out of the cardiomyocytes via the Na+/Ca2+ exchanger (NCX). An imbalance of Ca2+ flux may induce pathological conditions such as arrhythmia and decreased contractility of the cardiomyocytes. It has been reported that a certain portion of the contractile deficit in heart failure is due to an impairment of Ca2+ homeostasis [Catraction . Elementeostasis ,4.2+ homeostasis. As shown in a previous study, the ability of Ca2+ current to trigger SR Ca2+ release is decreased significantly in diabetic cardiomyocytes. Spontaneous Ca2+ spark frequency and peak amplitude were also both significantly reduced (P < 0.05) in diabetic myocytes compared with the control myocytes [2+ release rate and Ca2+ stores were depressed in the cardiomyocytes of type 1 diabetic rats. These changes may account for cardiac dysfunction in diabetic models. However, inconsistent results have been reported. For instance, according to Yaras et al. [2+ sparks was not significantly changed, but the spontaneous Ca2+ spark frequency was elevated in diabetic cardiomyocytes, compared with control cells.Diabetic cardiomyopathy was initially recognized by Rubler et al.  in diabemyocytes . In addimyocytes  found ths et al.  the maxi2+ involvement in diabetic cardiomyopathy is still not fully known and requires further investigation. As animal models in different diabetic stages have been used in various studies, we hypothesized that the inconsistent results regarding Ca2+ homeostasis were due to different severities and stages of diabetes. Therefore, this study investigated the progressive alterations in Ca2+ homeostasis and expressions of SR Ca2+- associated proteins in the development of diabetes, and the association between Ca2+ homeostasis and cardiac dysfunction in diabetic cardiomyopathy.It is obvious that the role of CaThe Beijing Friendship Hospital Animal Care Committee approved all animal handling protocols. The diabetic rat model was induced with a single intraperitoneal injection of streptozotocin  in male rats . Rats in the control group were given an injection of a matched volume of citrate buffer (0.1 M). Subsequently on day three and day five after the injection, random blood glucose concentrations were measured. Only rats with blood glucose levels ≥16.7 mmol/L on both days were defined as diabetic and used in the study.Four experimental groups  consisted of the control group (group A) and the diabetic model rats, examined at 4, 8, or 12 weeks after injection . All the rats were housed (three per cage) in a controlled environment at 20 ± 2°C, 30-70% humidity, and a 12:12 h light–dark cycle, and given standard chow and water ad libitum. In accordance with the protocol, all diabetic model rats were euthanized prior to the experiments at the assigned time points. Control rats were euthanized at the twelfth week after an injection with citrate buffer.in vivo imaging system . The transthoracic echocardiography images were obtained via long- and short-axis views using standard echocardiography techniques [Echocardiography was performed to evaluate cardiac structure and function in all animals involved in the study . The rats were weighed and anesthetized with 10% chloral hydrate at 0.3 mL/100 g body weight . Two-dimmax). Myocardial relaxation was evaluated according to the left ventricular end-diastolic pressure (LVEDP) and the maximum descending rate of left ventricular pressure (−dP/dtmax) [To assess the hemodynamic condition, an ultra-miniature catheter connected to a polygraph instrument  was inserted into the carotid artery, and then to the left ventricle of the anesthetized animals . After a 5-min period of stabilization, the parameters were acquired and recorded. The myocardial contractility was assessed according to the left ventricular systolic peak pressure (LVPSP) and the maximum rate of ascending pressure change in the left ventricle (+dP/dtP/dtmax) . The hea2+-free buffer, containing 145 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 1.4 mM Na2HPO4, 0.4 mM NaH2PO4, 5 mM HEPES, and 10 mM glucose; pH 7.4, adjusted with NaOH. Then the hearts were perfused with an enzyme solution, containing 0.6 mg/mL collagenase type II  for about 15 min. The ventricular muscle tissue was cut into small pieces, and the ventricular myocytes were released by agitating the cell/tissue suspension. After filtration through a nylon mesh, Ca2+ was gradually reintroduced up to 1.8 mM in the cell suspension. The ventricular myocytes were then ready to be used in further studies.Ventricular myocytes were isolated from the rats  as described previously . In brie2+ spark imaging, cardiomyocytes were incubated with 10 μM Fluo-4 AM  in a normal Tyrode’s solution for 5 min at 37°C, and transferred into a recording chamber. Cells were perfused with the Tyrode’s solution for 20 min for de-esterification. SR Ca2+ load was estimated by a rapid application of 10 mM caffeine via a nearby pipette. To control the rest potentiation, experiments were performed after about 30 minutes, upon reintroduction of Ca2+ to a concentration of 1.8 mM in each group. Confocal line-scan imaging was performed using a Leica SP5 confocal microscope  with an excitation at 488 nm. Line-scan images were acquired at a sampling rate of 1.43 ms per line, along the longitudinal axis of the cell. Digital image processing was performed using MATLAB 7.1 (Math Works). For a minimal detection of Ca2+ sparks, the criteria was set at greater than 3.8× the standard deviation (SD) of the background noise over the mean background noise, and Ca2+ sparks were automatically counted with the Sparkmaster Plug-in for Image [For the Caor Image .2+-associated proteins RyR2, SERCA, NCX1, FKBP12.6, and phospholamban (PLB), and the phosphorylation status of PLB at serine-16 (PLB-Ser16) and threonine-17 (PLB-Thr17). In brief, the heart tissue was homogenized in a cold Tris–HCl buffer . Total protein was quantified using a BCA protein assay kit as required . Equal amounts of protein (40 μg) were separated by SDS-PAGE. After electrophoresis, proteins were transferred to a polyvinylidene fluoride membrane and blocked with Tris buffered saline containing 5% skim milk powder and 0.05% Tween 20. Then the corresponding primary antibodies, RyR2 , SERCA , NCX1 , PLB , PLB-Thr17 , PLB-Ser16 , and FKBP12.6  were added in series. The membranes were incubated with the diluted antibody preparations overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) and goat anti-mouse IgG (H + L) antibodies  for 40 min at room temperature. The blots were visualized using an enhanced chemiluminescence detection kit . Target proteins were quantified and normalized relative to β-actin .Western blot analyses  were conducted as previously described  to assesP-value < 0.05 was considered significant.Data were evaluated by one-way ANOVA expressed as the mean ± SD. When a significant difference was identified by ANOVA, a post-hoc analysis was performed using the Student-Newman-Keuls test. Data were analyzed using SPSS 13.0 software, and a P = 0.00), but were not significantly different among the diabetic groups . Heart weights also decreased during the 12-week development of diabetes, although the difference was statistically significant only in group D compared with the control group (P = 0.013).Blood glucose levels were significantly higher in all diabetic rats (22.62 ± 5.07 mmol/L) compared with the control rats . With regard to ejection fraction (EF) and fractional shortening (FS), both showed a trend toward gradual decline in the diabetic rats over the 12 weeks, with significant reductions (P < 0.05) appearing at the twelfth week after diabetes was induced . In group D, there was a slight but substantial decline in E/A ratio compared with that in control group (P = 0.027).Similar heart rates were observed in all groups (P = 0.009), which was consistent with the marked drops in EF and FS. Concurrently, the left ventricular end-diastolic pressure (LVEDP) increased gradually. Relative to the control rats, a significant increase in LVEDP first appeared in group C  to group D , and was sustained in groups C (8 weeks) and D  was unchanged in group B, but significantly increased from group C to group D  increased significantly and progressively from group B to group D  were significantly different in the different stages of diabetes from that of the control rats  could indicate a decline of Ca2+ efflux rate, which might partly account for the reduction of –dP/dtmax, as well as the impairment of diastolic function in the diabetic rats. The SR Ca2+ load gradually declined over the experimental period, and might be partly responsible for the drop in Ca2+ spark frequency in the diabetic rats. It was also noticed that cardiac dysfunction occurred after alterations in Ca2+ handling. This implies that the depression of cardiac function may result, at least in part, from progressive Ca2+ mishandling.Our data showed that Ca2+ channel proteins, RyR2, SERCA, and NCX1 declined in a time-dependent manner with the progression of diabetes. Since these declines appeared earlier than Ca2+ mishandling, Ca2+ mishandling could partly result from the changes in channel protein levels. Those of RyR2, the major Ca2+ release channel protein on the SR, exhibited a downward trend in the diabetic rats, which was primarily responsible for the decrease in Ca2+ diffusion away from the SR and Ca2+ spark decay.We also showed that levels of the major Ca2+ leak [2+ leak results in a reduction of SR Ca2+ load and Ca2+ spark frequency, and an increase in the Tau, thereby contributing to the impairment of left ventricular systolic and diastolic functions.Simultaneously, the levels of FKBP12.6, the regulatory protein of RyR2, also declined in a time-dependent manner in the diabetic rats. FKBP12.6 is a small cytosolic protein which binds to the RyR2 protein. The binding stabilizes the RyR2 in the channel’s closed state during diastole . A reduc2+ leak . Diastol2+ channel protein on the SR, and is responsible for Ca2+ reuptake (70-92%) into the SR [2+ channel protein on sarcolemma, and is responsible for Ca2+ efflux from cardiomyocyte during diastole. Reuptake or efflux of Ca2+ via SERCA or NCX1 decreases the Ca2+ concentration in cardiomyocytes, which facilitates myocardial relaxation and maintains the Ca2+ content in the SR. Decreases in SERCA and NCX1 in this study were primarily the result of intracellular accumulation of Ca2+ during diastole and a prolonged Tau, which corresponded to the impairment of left ventricular diastolic function in the development of diabetes. Moreover, the reduction in SERCA protein was also related to a decrease in the Ca2+ reuptake rate, leading to a decline of SR Ca2+ load.SERCA is another major Cao the SR . NCX1 is2+ flux via SERCA [2+ reuptake decreases. In the present study, the PLB protein levels progressively increased over the experimental period, and phosphorylation of PLB experienced a gradual decline in the diabetic rats. Both these changes in PLB and its phosphorylation status attenuated the Ca2+ flux by SERCA during diastole in diabetic rats, which was also related to the impairment of left ventricular diastolic function.The regulation of SERCA is dependent on interaction with PLB. It has been confirmed that PLB acts as an inhibitor on SERCA, whereas phosphorylation of PLB can attenuate the inhibition and induce a substantial increase in Caia SERCA -20. Upon2+ sparks decay with progression of diabetes, accompanied by a successive impairment of cardiac function. Alterations in Ca2+ -associated protein levels were detected in the development of diabetes, which partly accounted for the Ca2+ spark decay and subsequent cardiac dysfunction. These results may indicate a possible molecular mechanism underlying cardiac dysfunction in the diabetic rat model, and thereby contribute to the search for a possible intervention. Further studies are warranted to investigate the signal pathways responsible for alterations in the levels of Ca2+-associated proteins.The present study showed that Ca+/Ca2+ exchanger; PLB: Phospholamban; RyR2: Ryanodine receptor 2; SERCA: Sarcoplasmic reticulum Ca2+-2ATPase; SR: Sarcoplasmic reticulum; Tau: Ca2+ spark decay time constant.EF: Ejection fraction; FS: Fractional shortening; FWHM: Full width at half-maximum amplitude; LVEDD: Left ventricular end-diastolic diameters; LVEDP: Left ventricular end-diastolic pressure; LVESD: Left ventricular end systolic diameters; LVPSP: Left ventricular systolic peak pressure; NCX: NaThe authors declare that they have no competing interests.2+ homeostasis in cardiomyocytes, participated in the sequence alignment, and drafted the manuscript. Y-LW carried out the Western blot analysis. C-YG performed echocardiographic and hemodynamic measurements. J-LC carried out the animal feeding and performed the statistical analysis. Y-QW conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.S-MZ carried out the examination of Ca


We specifically blocked the Ca2+ pumps in the endoplasmic reticulum and assessed the rate at which the cells reduced an elevated cytosolic Ca2+ concentration after a period of enhanced Ca2+ entry. Because external Ca2+ was removed and endoplasmic reticulum Ca2+ pumps were blocked, Ca2+ extrusion was the only process responsible for recovery. Cells lacking Bcl-2 restored the basal cytosolic Ca2+ level much faster than control cells. The enhanced Ca2+ extrusion in cells from Bcl-2 knockout (Bcl-2 KO) mice was not due to increased Na+/Ca2+ exchange activity, because removal of external Na+ did not influence the Ca2+ extrusion rate. Overexpression of Bcl-2 in the pancreatic acinar cell line AR42J decreased Ca2+ extrusion, whereas silencing Bcl-2 expression (siRNA) had the opposite effect. Loss of Bcl-2, while increasing Ca2+ extrusion, dramatically decreased necrosis and promoted apoptosis induced by oxidative stress, whereas specific inhibition of Ca2+ pumps in the plasma membrane (PMCA) with caloxin 3A1 reduced Ca2+ extrusion and increased necrosis. Bcl-2 regulates PMCA function in pancreatic acinar cells and thereby influences cell fate.The antiapoptotic protein Bcl-2  plays imignaling  by influ release . Here we 2+ extrusion through PMCA in control cells as compared to Bcl-2 KO ► Bcl-2 reduces Ca2+ extrusion though PMCA in AR42J cells overexpressing Bcl-2 ► Loss of Bcl-2 reduces Ca2+ overload, decreases necrosis, and promotes apoptosis► Bcl-2 reduces Ca We used a protocol allowing us to monitor the rate of reducing the cytosolic Ca2+ concentration ([Ca2+]i) following a maximal elevation of [Ca2+]i during complete blockade of Ca2+ uptake into the endoplasmic reticulum (ER) (2+-free solution were treated with thapsigargin (Tg), a specific inhibitor of Ca2+ pumps in the ER, in order to empty the ER Ca2+ stores. The extracellular Ca2+ concentration ([Ca2+]o) was then increased to 1 mM, 5 mM, or 10 mM, which induced rapid influx of Ca2+ to the cytosol. After a stable [Ca2+]i plateau had been attained, extracellular Ca2+ was removed and [Ca2+]i declined until the baseline level had been reestablished  than WT cells (100.3 ± 5.6 nM SE)  A and 1B.ablished A and 1B.6 nM SE) C. This s2+ uptake into the ER was blocked by Tg, the rate of decline of [Ca2+]i—after removal of external Ca2+—must reflect the rate of Ca2+ extrusion. Calcium extrusion rates (d[Ca2+]i/dt) were calculated and then plotted as a function of the [Ca2+]i values obtained from the exponential fit. Initial d[Ca2+]i/dt values (for the highest [Ca2+]i) from each trace were plotted together on a graph and fitted by linear regression (2+]i levels in the experiments shown in Because Cagression D. Similagression C and S1D2+ pumps (sarcoendoplasmic reticulum Ca2+-activated ATPase [SERCA]). In such conditions, Ca2+ removal from the cytosol is only dependent on Ca2+ export across the plasma membrane. The two main proteins responsible for this process are the plasma membrane calcium-activated ATPase (PMCA) i after an imposed period of elevated [Ca2+]i using our standard protocol  of AR42J cells ER , whereas the opposite effect was obtained by knocking down Bcl-2 with specific siRNA . Our data are in line with the majority of previous studies [2+ leak channel in the ER [2+]ER directly, because indirect cytosolic measurements can be misleading with regard to information about ER Ca2+ loading.It was shown previously  that Bclratories , but notratories . We meas2J cells  with ER-2J cells  in untra studies  suggestin the ER  and possn the ER  are regu2+ extrusion, we performed cell death assays on freshly isolated WT and Bcl-2 KO pancreatic acinar cells treated with 30 μM menadione [2+] (5 mM) for 30 min. Control groups were incubated for 30 min without any treatment. Our results (2+] reduced the number of live WT cells in comparison to WT controls (p = 0.019). The same phenomenon was not observed in Bcl-2 KO cells (p = 0.05). Menadione (30 μM) caused a substantial reduction in live WT cells in comparison to WT controls (p < 0.001), increasing both apoptosis (p < 0.001) and necrosis (p = 0.01). Treatment of WT cells with both menadione and high Ca2+ increased necrosis even more than treatment with menadione alone (p = 0.018). Bcl-2 KO cells treated with 30 μM menadione had decreased numbers of live cells (p = 0.001) and markedly increased numbers of apoptotic cells (p = 0.003) in comparison to the untreated Bcl-2 KO cells. However, necrosis levels were not significantly different when compared to the Bcl-2 KO control (p = 0.138). Combined treatment with menadione and 5 mM Ca2+ did not substantially increase necrosis levels above those induced by menadione alone (p = 0.282). Importantly, Bcl-2 KO cells treated with menadione had increased apoptosis levels and decreased necrosis levels as compared to WT cells treated in the same way . Necrosis inhibition in Bcl-2 KO versus WT cells was even more pronounced between the groups incubated with menadione and high Ca2+ (p = 0.008).In order to investigate the pathophysiological importance of Bcl-2 and in particular its regulation of Caenadione  and/or h results A and 3B 2+]i in Fura-2-loaded WT cells and Bcl-2 KO cells in response to menadione (30 μM) or to raising the external [Ca2+] from 1 to 5 mM or to both raising [Ca2+]o to 5 mM and simultaneously adding menadione. Typical traces are shown in 2+]o from 1 mM to 5 mM resulted in a small, slowly developing elevation of [Ca2+]i in WT cells but had almost no effect on Bcl-2 KO cells . Menadione applied in the presence of both 1 mM and 5 mM extracellular Ca2+ induced much larger responses in WT cells as compared to Bcl-2 KO cells . In WT cells, the presence of 5 mM Ca2+ together with menadione caused much larger responses than menadione alone (p = 0.017), whereas in Bcl-2 KO cells, the presence of 5 mM Ca2+ did not affect menadione-dependent responses (p = 0.38). These data demonstrate that removal of Bcl-2 affords a remarkable degree of protection against attempts to raise [Ca2+]i by elevation of the external Ca2+ and/or stimulation with menadione.We also compared the rise in [Ca2+ extrusion by using the specific PMCA inhibitor peptide caloxin 3A1 [2+]i in response to Tg and ACh in normal control pancreatic acinar cells as compared to cells preincubated for 300 s in the presence of caloxin 3A1. Caloxin 3A1 increased the basal resting [Ca2+]i and significantly inhibited Ca2+ extrusion  generating reactive oxygen species.Our results reveal a new and unexpected role for Bcl-2 in the regulation of cellular Caignaling , we now 2+ measurements were performed with Fluo-4 or Fura-2 [2+]i was calculated (Fura-2) and recovery phases were fitted with an exponential decay function ifunction . Immunobfunction .


These Ca2+ signals, which emerge within seconds after iron addition, arise mostly from Ca2+ release through the redox-sensitive ryanodine receptor (RyR) channels present in the endoplasmic reticulum. We have reported also that addition of synaptotoxic amyloid-β oligomers to primary hippocampal neurons stimulates RyR-mediated Ca2+ release, generating long-lasting Ca2+ signals that activate Ca2+-sensitive cellular effectors and promote the disruption of the mitochondrial network. Here, we describe that 24 h incubation of primary hippocampal neurons with iron enhanced agonist-induced RyR-mediated Ca2+ release and promoted mitochondrial network fragmentation in 43% of neurons, a response significantly prevented by RyR inhibition and by the antioxidant agent N-acetyl-L-cysteine. Stimulation of RyR-mediated Ca2+ release by a RyR agonist promoted mitochondrial Ca2+ uptake in control neurons and in iron-treated neurons that displayed non-fragmented mitochondria, but not in neurons with fragmented mitochondria. Yet, the global cytoplasmic Ca2+ increase induced by the Ca2+ ionophore ionomycin prompted significant mitochondrial Ca2+ uptake in neurons with fragmented mitochondria, indicating that fragmentation did not prevent mitochondrial Ca2+ uptake but presumably decreased the functional coupling between RyR-mediated Ca2+ release and the mitochondrial Ca2+ uniporter. Taken together, our results indicate that stimulation of redox-sensitive RyR-mediated Ca2+ release by iron causes significant neuronal mitochondrial fragmentation, which presumably contributes to the impairment of neuronal function produced by iron accumulation.Mounting evidence indicates that iron accumulation impairs brain function. We have reported previously that addition of sub-lethal concentrations of iron to primary hippocampal neurons produces Ca Under physiological conditions, mitochondria undergo constant structural changes, forming interconnected networks or punctiform organelles depending on cell type . Cells f2+ buffers participate in intracellular Ca2+ homeostasis. Due to the low Ca2+ affinity of the mitochondrial Ca2+ uniporter, effective mitochondrial Ca2+ uptake requires the proximity of mitochondria to endoplasmic reticulum (ER) or plasma membrane Ca2+ channels, since their opening generates transient microdomains of high Ca2+ concentration, a requisite feature of mitochondrial Ca2+ uptake  recepto  and iron accumulation are characteristic hallmarks of Alzheimer’s disease, a highly prevalent neurodegenerative disease , a requisite step of long-lasting synaptic plasticity . We also show that RyR-mediated Ca2+ release stimulated mitochondrial Ca2+ uptake only in iron-treated neurons that displayed a non-fragmented mitochondrial network structure.Here, we investigated in primary hippocampal neurons the effects of iron treatment on mitochondrial network dynamics and function. We found that 24 h exposure of primary hippocampal cultures to iron promoted significant mitochondrial network fragmentation in a sizable fraction of neurons; this fragmentation process required functional RyR channels and was prevented by ®488 anti-mouse were from Molecular Probes . Neurobasal medium, B27 supplement, and horse serum were from Gibco . Dulbeco’s modified essential medium (DMEM) and lipofectamine 2000 were from Invitrogen . Ryanodine was from Alexis . The mito-pericam plasmid was a kind gift from Dr. Eisner. Protease inhibitors leupeptin and pepstatin A were from Calbiochem , and 3--2,5-diphenyl tetrazolium bromide (MTT) was from Sigma Chemical . Bicinchoninic acid assay kit, horseradish peroxidase (HRP)-conjugated secondary antibodies and m-Hsp-70 antibody were from Pierce Biotecnology , 4-cloro-methyl-cresol was from Merck  and polyvinylidene difluoride (PDVF) membranes from Millipore . The antibody anti-Cox IV was from Cell Signaling , anti-Drp-1 was from Thermo Scientific , anti m-Hsp-70 was from Pierce Biotecnology .Fluo4-AM, calcein-AM, MitoTracker Orange CMTMRos and Alexa Fluorin vitro (DIV) at 37oC in a humidified 5% CO2 atmosphere prior to experimental manipulations. The resulting cultures were highly enriched in neuronal cells . Cells were homogenized using a homogenizer with a tight fitting Teflon pestle. The homogenates were maintained on ice for 5 min and then centrifuged  to remove nuclei and unbroken cells. The supernatants were centrifuged  to obtain a pellet enriched in mitochondria, which was re-suspended in buffer A supplemented with 1% Triton X-100 and centrifuged . The protein content in the resultant purified pellet, highly enriched with mitochondria, was determined by using the Bicinchoninic acid assay.Mitochondrial fractions were obtained by differential centrifugation of hippocampal homogenates as previously described . Cells w® 488 anti-mouse secondary antibody  for 1 h at room temperature. Cells were rinsed three times with PBS, and the coverslips were mounted in DAKO mounting medium on glass slides. To control for the specificity of Hsp-70 as a mitochondrial label, cultures were stained with 50 nM MitoTracker Orange for 20 min at 37°C.Hippocampal cultures were fixed by adding equal volumes of 4% formaldehyde and 4% sucrose fixation solution, in phosphate buffered saline (PBS) to the culture medium for 5 min. The fixation solution was replaced by fresh solution in the absence of culture medium, and incubation was continued for 10 min. Cells were rinsed three times with PBS and were incubated with 0.1% Triton X-100 in PBS (permeabilization solution) for 30 min and blocked with 5% BSA in PBS (blocking solution) for 1 h. Cells were immunostained with anti-Hsp-70 primary antibody  at 4°C overnight. After this incubation period, cells were rinsed three times with PBS and were incubated with Alexa Fluor(a) Fluorescence: Confocal image stacks were captured with a Zeiss LSM-5, Pascal 5 Axiovert 200 microscope, using LSM 5 3.2 image capture, analysis software and a Plan-Apochromat 40x, 1.4 oil differential interference contrast objective. One-channel Alexa Fluor 488 (λex/λem = 488/505–530 nm) and image stacks  were acquired. We made sure that I did not saturate and that image background was slightly above zero by adjusting the laser power, the detector gain, and the detector offset. Image stacks were deconvoluted with Huygens Scripting . All image-processing routines were developed in our laboratory based on IDL . Mitochondria were identified by staining fixed cultures with Hsp-70, as described in our earlier work (b)Determination of mitochondrial protein Hsp-70 in soma and neurite volumes by 3D reconstruction of the segmented objects: 3D models were reconstructed from successive xy-images along the z-axis. Based on their volumes, we defined four different clusters to characterize mitochondrial connectivity. First, we determined the mean volume of single mitochondria, yielding 0.15 ± 0.04 μm3 . The mean volume of single mitochondria was used to define connected clusters: (i) 1–3 mitochondria  4–10 mitochondria  11–50 mitochondria ; (iv) over 50 mitochondria .ier work . The speImage deconvolution was performed with the Image J software and z-stacks from images were projected in one image, which was analyzed. In accordance to mitochondrial morphology, neurons were classified as exhibiting fragmented or continuous mitochondrial network, by visual examination in ten optical fields for each condition, counting 15 neurons approximately in each field. The percentage of cells with a fragmented pattern was determined respect to the total number of cells analyzed.Fractions enriched in mitochondrial proteins were resolved by 12% SDS-PAGE and then transferred to PDVF membranes. Blots were blocked for 1 h at room temperature in Tris-buffered saline (TBS) containing 0.2% Tween-20 and 5% fat-free milk. Overnight incubation with primary antibody against Drp-1 (1:1000) was performed at 4°C. After incubation for 1.5 h with HRP-conjugated secondary antibodies, membranes were developed by enhanced chemiluminescence . To correct for loading, membranes were stripped and blotted against Cox-IV (1:1000) or mHsp-70 (1:1000). The films were scanned and the Image J program was employed for densitometric analysis of the bands.3- sodium nitrilotriacetate complex (2.2:1 molar ratio)] or vehicle in Neurobasal medium supplemented with B-27 were placed in modified Tyrode solution  prior to fluorescence measurements. This medium change was performed in order to avoid color interference from the Neurobasal medium. Cultures were loaded next with 5 μM Fluo4-AM for 30 min at 37°C. After washing three times with modified Tyrode solution, cultures were maintained in this solution during the experiment. Neurons were stimulated with 4-cloro-methyl-cresol (4-CMC) at the microscope stage (which was not removed from the medium). Fluorescence images of intracellular Ca2+ signals in primary hippocampal neurons were recorded every 5 s in a Carl Zeiss LSM Pascal 5 confocal microscope system using 63x Oil DIC objective, excitation 488 nm, argon laser beam. Frame scans were averaged using the equipment data acquisition program. Ca2+ signals are presented as F/Fo values, where F corresponds to the experimental fluorescence and Fo to the basal fluorescence.Cultures treated for 24 h with 30 μM Fe-NTA  (We propose that fragmented mitochondria loose their physical interactions with the ER, resulting in decreased functional coupling between RyR channels and the mitochondrial Cadiseases , would pg [Ca2+] , which m2+ in response to RyR-mediated Ca2+ release. The loss of mitochondrial Ca2+ uptake exhibited by fragmented mitochondria, which is likely to disrupt cellular Ca2+ homeostasis, presumably contributes to the increased fragility observed in neurons subjected to a high iron oxidative load, as observed in Parkinson’s and Alzheimer’s disease. The present results add to previous reports showing significant contributions of RyR-mediated Ca2+ release from the ER to neuronal function, including synaptic plasticity (In conclusion, we found that iron treatment promoted mitochondrial fission through a process that required functional RyR channels, and that fragmented mitochondria did not take up Caasticity  and hippasticity .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Primary hippocampal neurons were cultured from suckling neonatal SD rats. Cells were cultured for 7–9 days and the Ca2+ oscillations in response to perfusion with Ade were detected using confocal laser scanning microscopy in combination with Fluo-3/AM labeling. This study found that Ade inhibits the spontaneous synchronized Ca2+ oscillation frequency and amplitude in mature hippocampal neurons and such inhibition depends on the Ade dosage level to a certain extent. Ade also had a significant inhibitory effect on high potassium-induced Ca2+ oscillation frequency and amplitude. Ade had a significant inhibitory effect on high-voltage-activated Ca2+ channel-mediated Ca2+ influx and Ca2+ oscillations in neurons. This may be one of the mechanisms for Ade to exert antiepileptic effects as an endogenous substance.Adenosine (Ade) is an antiepileptic agent. In order to investigate the possible mechanism of action of Ade, its effect on calcium (Ca The electrophysiological characteristics of an epileptic attack mainly include hypersynchronous discharge of neurons of the local or whole brain; therefore Ca2+ oscillations have always been considered as a simple mode of electrophysiological movement of the epileptic nerves. At present, the biggest problem in epilepsy treatment is the insensitivity to existing exogenous substances  rats (no more than 48 h old) provided by the Experimental Animal Center of Binzhou Medical University were randomly selected without restriction of gender. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Hospital of Binzhou Medical University.The primary hippocampal neurons were cultured referring to related studies ,3. The s2+ oscillations, the hippocampal neurons cultured with Krebs-Ringer solution as a basal medium were divided into 4 groups: control group, high potassium treament group, Ade treament group, high potassium and Ade co-treatment group. High potassium group: neurons were treated with 60 mmol/l KCl. Ade treament group: neurons were treated with Ade at 0.1 μmol/l and 50 μmol/l, seperately. High potassium and Ade co-treatment group: neurons were treated with 50 μmol/l Ade and 60 mmol/l KCl. Control group: neurons were just treated with Krebs-Ringer solution.In order to record Ca2+ concentration. Therefore, the hippocampal neurons were carried in Krebs-Ringer solution  of Fluo-3/AM in an incubator with 5% CO2 at 37°C for ∼30 min, followed by quick flushing three times and then suspending in the recording solution for delipidation with Fluo-3/AM for ∼15 min  is proportional to the intracellular free Ca ∼15 min . When th0, the relative fluorescence intensity change of Fluo-3 shows the intracellular Ca2+ concentration change, and is used to reflect the amplitude of Ca2+ oscillation. ΔF is the Ca2+ concentration at the moment of t and F0 is the average baseline value obtained within the unit time of t±10 sec. Ca2+ oscillation is defined when ΔF/F0 is dramatically increased by more than 20%. Ca2+ oscillation frequency and amplitude are obtained from calculating the frequency and average amplitude within the fixed unit time of 2 min, and are used as the statistical record of the experimental data.Data were obtained from fluorescence images by analyzing the average fluorescence intensity in the pixel region of ∼3×3 in the center of the hippocampal neuronal cell bodies which had pyramidal shapes, well developed branches and good adherence. ΔF/FData were analyzed using SPSS 11.0  statistical software. Measured data were expressed as the means ± SEM using the paired t-test. P<0.05 was considered to indicate a statistically significant result.Hippocampal neurons were observed with a high power microscope after 7–9 days using indirect immunofluorescence staining with Tubulin. Green represents neuronal matter and blue represents the nerve cell nucleus .2+ oscillations were observed in primary cultured hippocampal neurons using confocal laser technology. Ca2+ oscillation frequency and amplitude were 1.2±0.12/min (0.02±0.002 Hz) and 1.87±0.17, respectively . Its frequency was reduced from 1.2±0.2/min (0.02±0.003 Hz) before addition of Ade to 0.3±0.05/min (0.005±0.001 Hz) and its amplitude was decreased from 1.87±0.17 before addition of Ade to 1.1±0.07  was prepared in a dish and observed for ∼3 min, which was taken as the control Ade intervention. The solution was then replaced by 1 ml freshly prepared Ade solution for continual recording and neurons maintained in 1 ml of Krebs-Ringer solution were used as a control. Following the addition of Ade, spontaneous Ca1.1±0.07 .2+ oscillations were significantly enhanced in respect of frequency and amplitude. After recording for 3 min, the Ade (50 μmol/l) intervention began and Ca2+ oscillation frequency and amplitude were inhibited again . The frequency was reduced from 2.39±0.22/min (0.04±0.003 Hz) before addition of Ade to 0.44±0.13/min (0.01±0.002 Hz) and its amplitude was decreased from 2.45± 0.27 before addition of Ade to 1.12±0.08 . During an epileptic attack, however, the Ade concentration is increased by 6–31 times. Some researchers have reported that Ade secretion and Ade receptor expression were significantly decreased in the refractory epilepsy model  ,6. In thsy model . Some stt of Ade , so a pr2+ oscillation is the temporal and spatial undulation of Ca2+. The formation of Ca2+ oscillations within the neuron network is modified through extracellular Ca2+ influx in combination with the release and assimilation of intracellular Ca2+ stores. Ca2+ oscillation is an ubiquitous phenomenon in the nervous system tissues and Ca2+ can rapidly diffuse in several ways, including gap junctions. Its frequency and amplitude changes code neural network information and play an important role in synaptic plasticity and neuronal transfer (2+ oscillations exist in the hippocampal neurons of rats; such Ca2+ oscillation is closely related to the synapse stimuli and can trigger excitation. Some experiments proved that the synchronization of neuronal electrical activity marked by the Ca2+ oscillation is crucial to the propagation of epileptiform discharges (2+ passing through the Ca2+ channels and intracellular Ca2+ store release plays an important role. At the same time, Ca2+ overload phenomenon exists in neurons during an epileptic attack. Calcium is known as the basic promoter of the excitatory toxic action of epilepsy. In the central nervous system, temporal and spatial undulation of Ca2+ at a certain frequency and amplitude is known as the Ca2+ oscillation. In primary cultured hippocampal neurons, synchronous primary Ca2+ oscillation is associated with dynamic changes of the membrane potential. The synergistic effect of NMDA receptors and voltage-dependent L-type Ca2+ channel activation allows the Ca2+ to move from the intracellular to the extracellular area, thereby modifying the shape of Ca2+ oscillation every time (2+ oscillation by regulating the Ca2+ channel current in neurons and decrease Ca2+ oscillation frequency and amplitude, so as to further play a role in inhibiting the pathological processes such as synchronized electrical activity between neurons and peripheral excitatory neurotransmitter release during epileptic attack, finally reduce the excitability of neural networks in the central nervous system, and inhibit epileptic attack. As a result, spontaneous synchronized Ca2+ oscillations are considered the electro-physiological basis of epileptic activity (Catransfer . Koizumitransfer  reportedscharges . The relery time . High-leactivity ,15.2+ oscillations in primary cultured hippocampal neurons using confocal laser scanning microscopy. Based on this, the effect of Ade on Ca2+ oscillations was further investigated. Low-level Ade does not have a significant effect on Ca2+ oscillations in neurons but Ade at a concentration of 50 μmol/l causes the spontaneous Ca2+ oscillation frequency and amplitude in hippocampal neurons to be decreased. Ade may influence intra-cellular Ca2+ oscillations by regulating the balance between the release and assimilation of the spontaneous intracellular Ca2+ stores in neurons. This effect shows some dependence on the Ade concentration. The high extracellular potassium environment allows the intracellular and extracellular potassium concentration gradient to be decreased, the negative value of the resting potential to be reduced, the threshold potential gap to be shortened and finally results in abnormal rise of the cell excitability. Potassium and Ca2+ are mutually antagonistic, so the influx of potassium is reduced and the Ca2+ influx is increased correspondingly, which can further trigger the electrical imbalance. Therefore, a high potassium solution was used in this study as the depolarization stimuli to activate voltage-gated Ca2+ channels. This was to facilitate the extracellular Ca2+ influx, accelerate Ca2+ circulation in the cytosol and Ca2+ stores and simulate the epileptic discharge model to observe the effect of Ade. The results showed that Ade at certain concentrations plays a role in inhibiting the high potassium-induced Ca2+ oscillations. When the Ade is above the physiological level, it will decrease the Ca2+ oscillation frequency and amplitude and is likely to further inhibit the pathological processes such as synchronized electrical activity between neurons and peripheral excitatory neurotransmitter release during epileptic attack, thereby finally reducing the excitability of neural network in the central nervous system, which is also consistent with the research findings of Brundege and Dunwiddie (This study first observed spontaneous synchronized Caunwiddie  and Philunwiddie .2+ transport within the sarcoplasmic reticulum of myocardial cells and studies on the protective effect of the A3 receptor on the central nervous system, have also been reported (2+ oscillations by acting on A3 receptors, thereby eventually antagonizing the synchronized electrical activity between neurons during an epileptic attack.Ade has several receptors including A1, A2a, A2b and A3, which are all distributed throughout the central nervous system, as indicated by autoradiography. Research has shown that activated A3 receptors stabilize Careported . Ade at 2+ influx to be reduced, thereby inhibiting the excitability of neurons. Many studies have shown that Ade can inhibit the release of glutamate. Flint et al (2+ oscillations mediated by metabotropic glutamate receptors in embryonic cortical slices. Sohn et al (2+ waves are mediated by metabotropic glutamate receptor 5. It was further hypothesized that Ade may open Ca2+ channels by acting on A1 receptors at certain concentrations. This results in hyperpolarization, inhibition of the excitatory effect of glutamate and regulation of the spatiotemporal changes of intracellular Ca2+ concentration. This eventually antagonizes the synchronized excitatory electrical activity between neurons during an epileptic attack. Proteomic mass spectrometry analysis in this study showed that when epilepsy is induced by pentrazole, there is no expression of glutamine synthetase in the cerebral cortex of Ade Al receptor gene knockout mice, compared with wild-type controls. Glutamine synthetase is the key enzyme in the glutamate/glutamine pathway. The Ade A1 receptor is likely to have important relevance with the activation of glutamine synthetase and acceleration of the metabolic hydrolysis of excitatory neurotransmitter glutamate in the brain, which also corroborates the results in this study to a certain extent.As the most important member of the Ade receptor family, Ade receptor A1 with high affinity is highly expressed in the cerebral cortex, hippocampus and other sites. Pék and Lutz  proved tnt et al  recordedhn et al  further To understand the action mechanism of Ade, it is necessary to further study the neuronal membrane surface receptor characteristics and intracellular and extracellular complex signaling pathways. This research provides a basis and lays preliminary research foundation for new clinical therapy and preventive approaches against epilepsy.


The second law [of thermodynamics] is one of the all-time great laws of science, for it illuminates why anything—anything from the cooling of hot matter to the formulation of a thought—happens at all.Peter Atkins.Four laws that drive the universeIt seems already like a long time ago that we used to think about heart failure as a problem in which mitochondria and energy were not involved. Energy has been for so long granted in the mind of mainstream electrophysiologists, but not anymore. Although energy can be taken for granted under normal conditions, this is not the case in heart failure where dysfunctional mitochondria, lack of substrate, and downstream effects become dominant factors. In the last decade, this unrealistic assumption has dramatically changed. Increasing experimental evidence supports the role of mitochondrial malfunction in the origin of arrhythmias  is dissipated or ATP synthesis inhibited. Alternans—a well-established risk factor for ventricular and atrial dysrhythmias—occur in cardiac failure and during myocardial ischemia. Hindering either of the two main components of the energy transducing machinery of mitochondria: the energy flow (ATP) and its main driving force (ΔΨm), trigger ailleurs Ca2+ alternans, or the beat-to-beat variations in Ca2+ transient amplitude at constant stimulation frequency. Further enhancement of alternans also occurred after inhibition of respiratory complexes or the Ca2+-sensitive dehydrogenases from the tricarboxylic acid cycle. Together, these data suggested that factors diminishing mitochondrial Ca2+ sequestration generate favorable conditions for Ca2+ alternans. Affecting mitochondrial Ca2+ handling by decreasing either uptake through the uniporter or extrusion through the Na+/Ca2+ exchanger also increased alternans. Then, Florea and Blatter made the following intriguing observation: alternans may happen in the absence of effects on the sarcoplasmic reticulum (SR) Ca2+ replenishment, and despite impaired mitochondrial energetics and Ca2+ dynamics. They suggested that under these conditions cytosolic ATP levels are still sufficiently high to allow for normal sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) activity and/or ATP for SERCA is supplied by glycolysis.A push forward of this idea came in 2010, when 2+ alternans even in the presence of selective inhibition of either glycolytic or mitochondrial ATP supply, both enhancers of alternans occurrence. Only severe deficit of energy supply given by simultaneous inhibition of both catabolic pathways prevented β-adrenergic stimulation-mediated abrogation of Ca2+ alternans. The authors suggest that enhanced Ca2+ sequestration together with β-adrenergic-mediated effects on the ryanodine receptor appear to act in tandem to protect against pacing-induced Ca2+ alternans. In fact, all cellular and transport processes involving subcellular organelles that contribute to clear cytosolic Ca2+ protect against alternans, and that conditions that impair cytosolic Ca2+ sequestration promote alternans. Impairment of mitochondrial Ca2+ uptake during excitation-contraction (EC) coupling can potentially have an impact on alternans incidence, although its quantitative importance is still a matter of debate. In this scenario, beta adrenergic receptor stimulation triggers a tug of war between protective and enhancing effects on alternans, the former through increase in Ca2+ sequestration and the latter by augmenting SR load and fractional release.In more recent work, Florea and Blatter  investigm depolarization that under critical conditions can scale up to the whole heart, thereby producing fatal arrhythmias (Aon et al., 2+ ATPase to sequester cytosolic Ca2+ back into the SR or its efflux from the cell (Aggarwal and Makielski, Mitochondrial redox and energetic functions are inextricably linked, and both are liable in heart dysfunction in chronic diseases such as obesity and diabetes (Bugger and Abel,