s2fieldsofstudy: Biology Medicine

text:
AID-induced remodeling of immunoglobulin genes and B cell fate.

Survival and phenotype of normal and malignant B lymphocytes are critically dependent on constitutive signals by the B cell receptor (BCR) for antigen. In addition, either antigen ligation of the BCR or various mitogenic stimuli result in B cell activation and induction of activation-induced deaminase (AID). AID activity can in turn mediate somatic hypermutation (SHM) of immunoglobulin (Ig) V regions and also deeply remodel the Ig heavy chain locus through class switch recombination (CSR) or locus suicide recombination (LSR). In addition to changes linked to affinity for antigen, modifying the class/isotype (i.e. the structure and function) of the BCR or suddenly deleting BCR expression also modulates the fate of antigen-experienced B cells.

AID FUNCTION AND SITES OF ACTION
Activation-induced deaminase (AID), an ancestral AID/APOBEC family member, deaminates DNA cytidines into uridines within immunoglobulin (Ig) variable (V) regions in all vertebrate species carrying B cells, thus supporting their Ag-driven diversification through gene conversion (GCV) and/or somatic hypermutation (SHM) [1]. It also diversifies expression of Ig heavy chain (IgH) constant (CH) regions in frogs, birds and mammals, who have developed class switch recombination (CSR) of CH genes.
AID was first identified as specifically expressed during the antigen-driven B cell maturation that mostly occurs in germinal centers (GC) of peripheral lymphoid organs [2]. It is mandatory for SHM and CSR [3] while its defect in patients results in hyper-IgM immune deficiency [4]. Its random mutagenic activity alters V domain complementarity determining regions, and thus modulates BCR (and later on antibody) binding affinity in a selection process where SHM is coordinated with cell competition for optimal intra-GC interactions with antigen-presenting cells [5]. In some mammals, especially in cattle, AIDmediated SHM can also begin in fetal gut associated lymphoid tissues prior to any contact with exogenous antigens [6].
Biochemically, G:U mismatches created through AID deamination can be processed in several ways, preferentially leading to mutations rather than repair within Ig genes. In « phase 1 » mutations, direct replication across a G:U mismatch can generate transitions from G:C to A:T base pairs. Base excision repair (BER) and uracil removal by uracil N-glycosylase (UNG) instead generate abasic sites, which subsequently undergo DNA nicking by apurinic/apyrimidinic endonuclease, and are repaired during replication by error-prone DNA polymerases as both transitions and transversions. G:U mismatches can also be processed by the mismatch repair (MMR) pathway involving MSH2/MSH6, with associated error-prone DNA polymerases and then result in patches of « phase 2 » mutations at both G:C and (preferentially) A:T base pairs around targeted cytosines.
Primary regulation of AID activity in B cells relies on its strictly controlled tissue-specific and stage-specific expression upon cell activation, due to control of the level of AID transcripts by both ubiquitous and lymphoidspecific transcription factors (Pax-5, STAT6, SP1, C/EBP) and miRNAs (miR155 and miR181b). This ensures high AID expression only in activated B cells with appropriate signals, as occurring within GCs upon interaction with follicular dendritic cells and T follicular helper cells. In addition, AID can appear at low levels in some bone marrow developing B cells upon stimulation of toll like receptors (TLR) [7,8].
AID requires transcription of target regions and also preferentially deaminates cytosine into uracil within WRC motifs (W = A/T, R = A/G) [9]. Besides potential constraints concerning the "accessibility" of target DNA, another major link between AID targeting and transcription is that AID loading onto Ig genes requires physical interaction with stalled RNAPII and bound Spt5 that occurs immediately downstream from transcription start sites [10]. The RNAPII associated polymerase associated factor (PAF) complex also helps recruit AID [11]. CH regions are protected from AID attack due to the absence of RNAPII pausing. Switch (S)-region transcription before AID recruitment is under the control of cytokine-dependent germline promoters preceding CH regions and a series of B cell activationdependent transcriptional enhancers located in the 3' regulatory region (3'RR) of the IgH locus [12][13][14][15]. While AID generates mutations in V regions, it initiates DNA breaks (DSBs) in S regions, thereby promoting large deletions [16,17]. DSBs activate the ubiquitous DNA damage response, which is then resolved through classical (C-) or alternative non-homologous end joining (A-NHEJ). Recruitment of 53BP1 and Rif1 [18] to broken DNA ends (and subsequent formation of γH2AX foci) is required for protection of DNA ends from resection before repair and ligation by C-NHEJ rather than A-NHEJ [19,20]. AID recruitment to both V and S regions (and S-S region synapses, likely favored by IgH locus DNA loops) requires IgH 3'RR enhancer activity elements [13] [15] [14] [21] [22]. Multiple 3'RR genetic alterations affected transcription of AID targeted regions [12][13][14][15]. However, transcription was often partially reduced while being associated with complete CSR and/or SHM blockades. In addition to boosting transcription, the 3'RR thus likely promotes AID activity through epigenetic changes of targets, or by attracting and recruiting AID and/or AID partners. Figure 1 resumes the different targets of AID in the IgH locus.

Structure and function of the B Cell Receptor
The B cell receptor is composed of a membranebound immunoglobulin (mIg) associated with the

IgM/IgD BCR
While all mIg associate with the Igɑ/ß complex to constitute the BCR, signaling from this complex has mostly been studied for IgM [24][25]. The membrane µ HC has multiple roles by first providing differentiation signals during early development, survival signals in resting B cells and activation signals during peripheral antigen-dependent maturation ( Figure 2). IgM and IgD have an identical intracellular positively charged short tail (including residues KVK). Replacement of µ by δ HC expression resulted in a modest phenotype and suggested that IgM and IgD can signal in a roughly similar manner [26]. This replacement completely deleted both mIgM expression and secretion of soluble IgM. The main feature of the resulting phenotype was delayed affinity maturation of immune humoral responses, i.e. a phenotype similar to mice simply lacking the secreted form of IgM [26] [27]. It was also shown later that, despite their roughly normal peripheral B cell compartments, mice in which µ HC was replaced by δ HC expression had a partial blockage of pro-B to pre-B transition due to lack of autonomous pre-BCR signaling normally provided by a glycosylation site within the µ chain CH1 domain [28].
IgD-deficient mice have also been generated through gene targeting and only showed slightly reduced B-cell spleen and lymph node compartments with higher mIgM expression than wt B cells. Early and late B cell differentiation, GC formation and humoral responses to immunization were unaffected [29]. However, a later study showed autoantibodies accumulating in IgD-deficient/lpr mice, suggesting that mIgD plays a role in B cell tolerance and limits auto-reactive B cell differentiation into plasma cells [30].

mIgG BCR
There are clear qualitative differences in BCR signaling and responses to antigen that are conferred by IgG vs IgM HC tails [31] (Figure 2). IgG carry longer recently emerged B cells circulate as transitional cells and can be committed to various compartments depending upon BCR signalling (Notch2 expression and weak BCR tonic signaling commit B cells to a marginal zone fate). After maturation into IgM+, IgD+ cells, naïve B cells encounter T-dependent Ag and undergo cognate interactions with pre-TFH cells. B cells then initially activate in extra-follicular foci and differentiate into short-lived plasma cells, or participate with TFH in the formation of GCs where their BCRs will be deeply remodeled by SHM (mostly in the proliferating dark zone) or CSR (mostly in the GC light zone), or eventually LSR. The GC reaction will yield both memory cells and plasma cells, some being long-lived and surviving after their migration to several protective niches (spleen red pulp, bone marrow, MALT…).

Ag-activated B cells CSR Long-lived plasma cells Niches in spleen red pulp, bone marrow, MALT…
T-dependent Ag

Marginal zone commitment Ag-independent maturation (in human)
T-independent Ag

Dark zone proliferation
Light zone selection TFH apoptosis LSR AID expression SHM and highly conserved cytoplasmic tails. Deletion or replacement of the IgG tail with an IgM tail in mice resulted, for unexplained reasons, in lower levels of membrane expression and also strikingly disturbed IgG1 memory, showing that the IgG tail is necessary for high-titer IgG secretion and IgG1-switched memory cell survival [32]. In contrast, studies in transgenic mice carrying IgM, IgG or IgM/G (IgM with an IgG tail) BCRs of identical antigen specificity for hen-egg lysozyme, showed that the IgG tail increased production of plasma cells during extra-follicular T cell−dependent antibody reactions [33]. It was also observed in B cell transfectants that signaling through the mIgG BCR, but not through mIgM nor mIgD, was resistant to CD22-mediated signal inhibition [34]. However, this CD22-independence was not confirmed by other detailed studies in primary cells from transgenic or knock-in mice [31] [35]. Rather, it was then suggested that IgG enhancement of intracellular calcium responses did not reflect independence from CD22 inhibition or changes in tyrosine phosphorylation, ERK signaling, or global gene induction, but increased differentiation into marginal zone cells, extrafollicular proliferation, and plasma cell differentiation. Despite increased Ca2+ responses, several BCR ligation response genes showed lower induction by the IgG tail, in agreement with the observation that marginal zone B cell differentiation is favored by conditions that decrease BCR signaling [36]. This includes poor CCR7 induction by antigen ligation of the IgG BCR, while before entry into follicles, CCR7 is known to attract B cells to CCL19/ CCL-21-rich T-zones rather than towards the marginal zone.
Premature expression of IgG rather than IgM in B cell progenitors also resulted in abnormal pre-BCR signaling and partial blockage of pro-B to pre-B cell transition, emphasizing that mIgM is optimal for early B cell maturation. In contrast, maintenance of mIgG+ cells might be less dependent on Igα/β than mIgM+ cells [35]. These observations led Goodnow's group to propose a "less-is-more" hypothesis where decreased induction of some BCR response genes might increase plasma cell formation, and to globally conclude that signaling differences between IgG and IgM BCR classes were qualitative rather than quantitative [31].
In comparison to IgM, another specific signaling feature of mIgG (shared with mIgE), is recruitment of the adaptor Grb2, which can bind to a tyrosine of the mIgG intracellular tail upon phosphorylation, and then modulate signaling and increase cell proliferation [37]. Interestingly, Grb2 is a modulator of cell signaling by tyrosine kinase receptors, with the dual function, in some instances, to decrease basal signaling while increasing signals emanating from receptor ligation [38].
Another feature of the mIgG1 cytoplasmic tail was demonstrated: enhancing early BCR oligomerization that immediately follows ligation of membrane bound antigen leading to increased Syk recruitment and calcium mobilization [39]. Importantly, a recent study has shown that the strong tendency of mIgG1 cells to differentiate into plasma cells is restricted to those Ag-experienced cells and then translated into Bach2 repression, thus clearly involving more than just mIgG intracellular tail structure [40].

mIgA BCR
Few studies have explored a potential role for mIgA in conferring specific properties to memory mIgA+ B cells in comparison to naive mIgM+ cells ( Figure 2). mIgA cross-linking raises intracellular calcium concentrations so that mIgA+ B cells residing in mucosa associated lymphoid tissues (MALT) can mediate IgA responses to local immunization [41] [42]. As for IgG, it was also shown by targeted deletion in the mouse, that the Cα membrane anchoring region allowing mIgA expression, is necessary for differentiation of IgA secreting cells in vivo [43]. The μMT mutation removing Cμ membrane exons and IgM/IgD expression did not result in a complete B cell defect but resulted in accumulation of IgA+ plasma cells in MALT, also suggesting that B cell progenitors can undergo CSR to mIgA, survive under expression of this BCR and then differentiate into ASCs [44]. That early B cell development can be ensured by expression of membrane α HC has been shown in the α1KI model where the Sµ region of the mouse IgH locus was replaced with an Ig HC ɑ1 gene [45].
In α1KI mice, a partial defect in pre-B cells was, however, noticed, indicating that the α-class pre-BCR was less efficient than μ-class pre-BCR for signaling completion of V(D)J rearrangement upon assembly of a functional pre-BCR or BCR complex. This was reminiscent of data in mice with premature expression of membrane γ HC instead of µ/δ [35].
Despite this developmental defect, B cells accumulated in lymphoid organs of ɑ1KI mice including spleen follicles, marginal zone, lymph nodes, Peyer's patches and the peritoneal B1 compartment. Splenic marginal zone and follicular B cell numbers were affected in the same proportion, in contrast to the increased marginal zone differentiation reported in mice expressing an IgG BCR [31,35]. As mentioned in a previous study [32], BCR density appeared lower in ɑ1KI than in wt mice, despite normal association of mIgA1 with Igɑ/ß. Another feature of mIgA-driven B cell differentiation in ɑ1KI mice was the abundance of plasma cells, affecting to the same extent long-lived (accumulated) and shortlived (recently differentiated) plasma cells. Rather than just compensatory plasma cell accumulation in response to B cell lymphopenia, the α1KI intrinsic commitment to plasma cell differentiation was also manifested in vitro by higher differentiation of B cells activated by BCR crosslinking or mitogens into CD138+ cells.
Interestingly, normal human blood mIgA+ cells also include a high proportion of CD38 expressing cells, a marker of engagement in plasma cell differentiation [46]. It should also be noticed that the IgA BCR has been shown to be sensitive to CD22 inhibition as for IgM [47]. In addition, a recent study demonstrated a specific transcriptional program for mIgA+ memory cells, notably depending upon high RORɑ expression, while mIgG2a+ cells were intrinsically dependent upon high expression of T-bet [48].

mIgE BCR
Secreted IgE is the least prevalent Ig class in blood, with only nanograms per milliliter compared to micrograms for IgD and milligrams for other classes in healthy populations. Maintaining low IgE levels is controlled by stringent mechanisms in order to restrict the very potent effects and side-effects of IgE immune responses. The IgE molecule is evolutionarily conserved and present in all mammalian species studied. In contrast to its active role in immune responses against parasites, IgE can also be deleterious and mediate atopic diseases of various intensities (eventually lethal in the case of anaphylactic shock).
Production of the double-sworded IgE antibody thus requires tight regulation, for which multiple mechanisms might be at work. IgE expression was first postulated to be restrained at the CSR level. Whereas IgG1 and IgE class switching can be both induced by IL4 in mice, in vitro experiments most often show lower switching frequency to IgE than to IgG1 [49]. Preferential CSR to Cγ1 might thus reflect intrinsic differences between the 10kb-long Sγ1 and the much shorter SƐ region. Furthermore, IgE class switching is often a sequential process with an IgG1 intermediate [50]. Interestingly, direct CSR to IgE (without prior IgG1 CSR) occurs preferentially in immature B cells, and this feature may reflect epigenetic marks modulating accessibility of the IgH locus during B cell differentiation [51].
Beyond IgE CSR, it is clear that expression of mIgE BCR is mandatory for further differentiation into IgE secreting plasma cells (Figure 2). Mice deleted for IgE membrane exons have reduced (by more than 95 percent) serum IgE levels and fail to develop antigen specific IgE responses [52]. To a milder extent, replacement of the IgE tail with the much shorter IgM tail also decreased (~50%) IgE secretion [52]. It was also shown that treatment of mice with an antibody specifically targeting a mIgEspecific epitope on mIgE+ cells efficiently inhibited secreted IgE production [53] [54], further suggesting that mIgE+ B cells are a necessary step for differentiation of IgE secreting plasma cells.
In this regard, a structural feature of the Cε gene was postulated to restrain the production of membranetype Cε transcripts which drive mIgE expression. Whereas all documented Ig genes have classical polyA sites (AATAAA) after both their secretory (CH3 or CH4) and membrane (M1/M2) exons, human and mouse Cε genes carry an atypical polyA site downstream from the M2 exon [55]. By comparing the sequence of CƐ genes in the IMGT data base (http://www.imgt.org), we observed that a canonical AATAAA sequence downstream from CƐ M2 was absent in all published mammalian IgH loci. This conserved suboptimal polyA site may down-regulate the ratio of membrane-vs secreted-form Cε mRNA transcripts during alternative splicing of murine (and human) Cε primary transcripts [56] and thus limit mIgE BCR expression . In an utmost form, CƐ gene membrane exons are lacking in one lineage of primates, the tarsiers [55]. Despite these limitations, mIgE expression can be characterized in vitro in human or mouse stimulated B cells and has also been characterized in a B lymphoma cell line [57]. Exploring mIgE expression requires rigorous staining protocols eliminating passive binding of soluble IgE to FcεRII receptors (whose expression is strongly induced in the presence of IL4) [58].
While present in a single (short) form in most mammalian species, mIgE is additionally present in humans and apes as a long isoform generated through alternate splicing of the M1 exon, and including either a long (66 amino acids) or a short (14 amino acids) extracellular membrane proximal domain (EMPD) between CH4 and the transmembrane domain. Both human mIgE isoforms have been expressed as functional BCRs in transfected B cells, showing that the short isoform was more efficiently transported to the cell surface and that its ligation resulted in growth inhibition of the WEHI immature B cell line, similar to mIgM BCR [59]. The EMPD domain was shown to be necessary for B cell activation through mIgE ligation in transfectants from the mature B cell line A20, while its deletion yielded a form of mIgE with proapoptotic activity upon ligation [60].
Another particularity of the IgE BCR is the intracellular tail structure, and phage display experiments identified 2 proteins interacting with the IgE tail. HS1associated protein X-1 (HAX-1) notably seems to be associated with the IgE specific YANIL motif (where Y is a potentially phosphorylated tyrosine) of the intra-cellular tail and may influence BCR mediated Ag-internalization and cognate Ag-presentation to T cells [61]. Hematopoietic progenitor kinase 1 (HPK1) was also shown to interact with mIgE and mIgG (but not mIgM) tails and could differentially affect signaling from these receptors [62]. IgE committed B cells were also shown to be exquisitely sensitive to inhibition through ligation of LXR receptors, potentially due to increased CD23 expression and in turn down-regulating IgE production [63].
In order to study IgE responses in vivo, different mouse models have been developed and characterized.
Xiong et al used T and B monoclonal mice (with antichicken ovalbumin (OVA) T cell receptor transgenes and anti-influenza hemagglutinin (HA) knock-in B cell receptor genes on a RAG1-deficient background), immunized with OVA-HA and OVA-PEP1 (a variant of HA) and a classical model of helminth infection in wt mice. They showed that IgE+ cells were found outside the GC (in contrast to IgG1 cells) and displayed a plasma cell transcriptional program. They also showed that IgEs accumulate hypermutation and affinity selection (and are able to bind the PEP1 Ag) and proposed a sequential maturation program: a pre-IgE phase with SHM and affinity maturation in IgG1+ cells and a post-IgEswitching phase where IgE+ rapidly differentiate into plasma cells [64]. In another study this team used the same T/B monoclonal model and immunized wt or hMT mice (which are deficient for IgG1 populations) with NP19KLH Ag. They demonstrated that high affinity IgE Abs responsible for anaphylactic reactions were generated through the sequential µ/γ1/Ɛ CSR pathway (and were absent in hMT mice), with IgG1 intermediate cells supporting SHM and affinity selection. In contrast, low affinity IgE producing cells were generated through direct µ/Ɛ CSR, were less mutated, and may play a beneficial role during anaphylaxis, by competing with high affinity Ab for FcƐR1 binding [65].
Two transgenic models were created to study rare IgE+ cells in vivo. The first model used a GFP reporter system by inserting a bi-cistronic GFP reporter gene downstream from the mouse IgE M2 exon. The long human EMPD domain was introduced upstream to the IgE M1 exon, resulting in a chimeric IgE BCR with an additional EMPD domain (allowing specific detection of these cells but denaturing the natural architecture of this receptor). This model was subjected to helminth infection by N. brasiliensis and TNP-OVA immunization. In contrast to previous models, this strategy demonstrated IgE+ cells in GC and suggested the existence of IgE+ memory and plasma cells [66]. In another model, the T2A strategy tagged IgE+ cells by linking the Cε M2 exon with Venus (a derivative of yellow fluorescent protein) and used a viral 2A sequence enabling expression of 2 proteins from a single ORF. Translation of Venus was thus linked to translation of a nearly normal mIgE (except for an additional 17 amino acid peptide). These mice and wt mice were immunized with NP-KLH and infected with N. brasiliensis. This model suggested that IgE+ cells can differentiate into GC B cells during primary immune responses and confirmed a strong propensity of these cells to differentiate rapidly into plasma cells which were mostly short-lived [67]. In agreement with this conclusion, another model where membrane IgE exons were replaced by membrane IgG1 exons suggested that IgE+ plasma cells have an intrinsically lower chance than IgG1+ cells to migrate towards the chemokine CXCL12 and thus to contribute to the long-lived plasma cell pool [68].

LSR ELIMINATION OF B CELLS
B cell fate can also be more dramatically modified by AID after CSR-like events resulting in complete deletion of the IgH CH gene cluster and thus inducing B cell death through locus suicide recombination (LSR) (Figure 1) [16].
During AID-mediated competition that occurs among B cells within germinal centers, a few with high affinity are selected (Figure 3). Besides these winners, many cells are losers or undesired responders deserving elimination. Although some unfavorable mutations of V regions can promote apoptosis [69], abundant survival signals from the GC microenvironment might also activate or maintain bystander cells with useless or eventually harmful BCRs (the latter having, for example, acquired specificity for self or environmental antigens after random remodeling of V sequences). Since class-switched antibodies are potent actors of auto-immunity and/or hypersensitivity, means for restricting CSR and reentry of class-switched cells into SHM would help ensure the specificity of immune responses. Yet, how the post-GC repertoire is controlled remains poorly understood.
3'RR elements regulate AID-mediated SHM and CSR by physically interacting with AID-targeted IgH promoters [70,74,79,80,89]. Since 3'RR enhancer elements and LS regions are themselves transcribed, it was logically found that they also undergo SHM and DNA breaks. AID-initiated junctions then occur in activated B cells between Sµ and 3'RR LS regions and delete the complete C gene cluster, thereby eliminating BCR expression (or Ig secretion for cells engaged in plasma cell differentiation). Given that BCR expression is mandatory for B cell survival, this process termed "locus suicide recombination" (LSR) necessarily leads to B cell death.
How the CSR/LSR balance is finely regulated in vivo remains to be determined. Beyond IgH locus accessibility, intra-GC interactions govern the selection of high affinity class-switched cells. In this context, LSR might contribute to B cell homeostasis and lead to death of suboptimally stimulated cells, while optimal stimulation would promote CSR and survival of selected antigen-specific B cell clones. LSR could then help shape the peripheral B cell repertoire and ensure BCR antigen specificity.

DEVELOPMENT AND FEATURES OF MEMORY B CELLS
B cell responses to T-dependent antigens involve germinal center reactions featuring B cell proliferation and expression of AID for SHM and CSR in parallel interactions with follicular dendritic cells and T follicular helper cells that will help select those B cells with the highest affinity for Ag. GC reactions peak after 7 days and wane after a few weeks, once having generated long-lived plasma cells and memory B cells with an affinity matured BCR. Besides intra-GC competition between B cells for limited antigen, soluble antibody feedback also ensures inter-GC communication concerning access to antigen [90]. Secreted antibodies were thus shown to inhibit or even terminate GC proliferation with an efficiency directly correlated with their affinity for antigen [90]. Interestingly in the absence of T cell help, soluble antigen might also inhibit GC development [91].
Regarding the prolonged maintenance (eventually lifelong) of GC-born memory B cells, it was shown that Ag-persistence within lymphoid organs is unnecessary [92]. Rather, further reintroduction of Ag into the organism will trigger memory cell differentiation into plasma cells, with or without parallel formation of new GC [93]. A recent study by Reynaud and colleagues followed Ag-specific memory B cells for up to 1 year in mice and showed that while GCs waned a few weeks after immunization by a soluble antigen, they persisted up to 10 months after immunization with a particulate Ag [94]. Several types of memory cells were demonstrated in the latter model: either IgM+IgD+, or IgM-only, or classswitched (and then mostly mIgG1+). Ag-experienced cells were located in the spleen T cell zone, red pulp, and follicles, with very few in the marginal zone. Upon transfer into pre-immunized recipient animals, the mIgM+ subset gave rise to centroblasts, part of them switching to IgG1, and to a small proportion of Ag-specific IgM+ plasma cells. The mIgG1 subset yielded Ag-specific IgG1 plasma cells and maintenance of a mIgG1+ memory pool. While the average number of mutations was stable in persisting memory B cells, it was increased upon differentiation of mIgM+ memory cells into centroblasts, showing that such cells can reenter new cycles of AID-mediated affinity maturation.
In another model, two layers of Ag-specific B memory cells were demonstrated, either mIgM+ or having undergone class switching (swIg+ memory cells). The mIgM+ memory cells were very stable and long-lived, able to form GC when transferred into a naïve animal but inhibited upon the presence of soluble Ag-specific Ig (then inhibiting GC formation). The swIg+ memory cells persisted for a shorter time, and mostly differentiated into plasmablasts upon activation but kept the ability to be activated even in the presence of preexisting antibodies. This led to the proposition that memory 1) initially relies on swIg+ cells, able to be rapidly activated and yield antibodies even in the presence of preexisting serum antibodies, and 2) later relies on longlived mIgM+ memory cells, able to form new GC once initial antibodies have vanished [93]. If this model can be generalized, "early" memory responses would classically overwhelmingly involve class-switched antibodies, while "late" memory responses would be expected to recapitulate the kinetics of the primary response, first with IgM (albeit with low affinity) then followed with high affinity classswitched antibodies. Interestingly, it was recently shown in a model of bacterial infection that in vivo depletion of the mIgM memory B cells abrogated the IgG recall responses to specific Ag challenge, demonstrating that mIgM cells were then required for humoral memory, and underwent CSR and plasma cell differentiation upon Ag re-challenge [95].
Upstream of GC reactions, some specific functions have been reported during immune responses for B cells with low affinity BCRs. For example, mouse B1 cells show polyreactivity and bind multiple exogenous antigens or auto-antigens with low affinity, being responsible for generation of natural antibodies that preexist prior to any antigenic challenge and play an innate function. Some tissue B cells binding antigens with low affinity (notably in lung, with intra-nasally administered antigens) have also been demonstrated to shuttle and carry antigens towards spleen follicles, then initiating GC formation by resident B cells with higher affinities [96]. BCR independent Ag-binding by B cells was also reported for marginal zone B cells recognizing particulate antigens within immune complexes through complement or Fc receptors, which were also postulated to carry antigens towards FDCs within follicles [97]. Human and mouse MZ B cells might somehow differ although they both respond to T-independent antigens. While mouse MZ B cells have mostly unmutated BCRs and do not recirculate, human IgM+IgD+CD21highCD27+ B cells recirculate in blood and carry mutated Ig and Bcl6 genes. Their V(D) J repertoire is very large without clonal restrictions and hallmarks of antigen-induced activation, similar to naive cells and in contrast to GC mIgM+ cells, suggesting that MZ B cells provide a GC-independent IgM memory layer [98]. MZ B cells likely differentiate through an innate extracellular TLR-dependent pathway allowing AID induction outside GC, as demonstrated in both mouse and humans for immature B cells then undergoing CSR and SHM [99] [7]. A helper role for neutrophils in the induction of MZ B cell CSR, SHM and plasma cell differentiation was also demonstrated during T-independent responses to bacterial antigens [100]. www.impactjournals.com/oncotarget

LYMPHOMAGENESIS AND BCR SIGNALS
AID also induces mutations and breaks in numerous non-Ig genes, many of which (but not all) are transcribed in B cells, and many of which overlap with translocation or recombination sites found in B cell lymphomas or correspond to repetitive DNA sequences. [101,102]. Studies of B cell malignancies since the early 1980s also identified a number of translocations linking oncogenes such as c-MYC, c-MAF, CYCLIN D1, and BCL2 to breakpoints located within Ig loci. Some of these events were recurrently identified as the clonal driving forces of lymphomagenesis. This pointed to a major role of errors in the processes of antibody gene diversification in tumorigenesis. Recently, it was also shown that breakpoints located outside Ig loci constitute frequent side targets of AID attacks in activated B cells. It is thus now clear that in B cell malignancies as in normal B cells, AIDinduced mutations accumulate on multiple loci including those of Ig (with the higher level of error-prone repair after AID deamination), BCL6 and a large list of oncogenes and tumor suppressors. [101] Precise characterization of translocation breakpoints and development of whole genome sequencing showed that mutations and recombinations detected in activated B cells and in tumors with mature B cell phenotypes all carry hallmarks of WRC sequence-specific AID on-target and off-target attacks. [103,104] In addition, more than half of translocations seen in immature B cell malignancies also show breakpoints at sites of AID deamination corresponding to mCG dinucleotides and might imply the combined action of AID, RAG and ARTEMIS. [105,106] However, this issue can still be debated and it is noteworthy that lymphoma breakpoints targeting CG dinucleotides were also recently identified by A. Nussenzweig and colleagues, as early replicating fragile sites susceptible to AIDindependent genomic instability [107].
Besides the role of translocations, it is important to note that BCR signals most likely play a crucial role in lymphomagenesis. These signals might vary with SHM of V regions, which probably modulate interactions of malignant cells with various endogenous ligands. Indeed, N-glycosylation sites carrying high-mannose glycans and created by SHM within V domains are a frequent feature of follicular lymphoma cells, and may promote B cell interactions with lectins such as DC-SIGN [108]. The BCR Ig class might also somehow control the phenotype of B cell malignant proliferations. Indeed, it is well known that plasmacytic lymphomas overwhelmingly express mIgM (in striking contrast with myelomas, where the secreted Ig is always a non-IgM class). In diffuse large B cell lymphomas, it was recently shown that ABC cases mostly express mIgM, while GC cases overwhelmingly express IgG or IgA [109].
Autonomous BCR signaling in chronic lymphocytic leukemia cases was also recently demonstrated, but appeared independent from the mutated or unmutated BCR status [110]. Finally a number of AID-mediated mutations affecting proteins of BCR-or TLR-signaling cascades and resulting in constitutive B cell activation have been observed in many different types of lymphoproliferation [111,112].

CONCLUSIONS
Early B cell differentiation is optimally driven through expression of germinally encoded and diversified IgM molecules. This generates transitional B cells that are affected to the various mature B cell compartments and then express variable levels of IgM and IgD. Such cells can then undergo either Ag-dependent (within B cell follicles) or Ag-independent (for splenic marginal zone B cells), AID remodeling of their Ig V regions through SHM or GCV. Cell survival, fate and phenotype are all highly dependent at that time on constitutive signals provided by the BCR. Either antigen ligation of the BCR and/or stimulation by various TLR ligands can additionally result in B cell activation and rapid AID induction. By improving BCR affinity for antigen by SHM, AID will help select follicular cells having the strongest interactions with antigen exposed at the surface of FDC along the process of SHM, as winners of the competition for activation and survival signals provided by FDC and TFH cells within the GC. In parallel, these interactions further stimulate expression and accessibility of the IgH locus then resulting in CSR for expression of a new BCR with conserved Ag-specificity but new signaling functions. Finally in some instances, another dramatic IgH remodeling event implying complete deletion of the IgH locus C region through a "sterile CSR" (also defined as "locus suicide recombination" or LSR), can occur and result in loss of BCR expression and B cell death.


s2fieldsofstudy: Biology Chemistry

text:
Autophagy in Muscle of Glucose-Infusion Hyperglycemia Rats and Streptozotocin-Induced Hyperglycemia Rats via Selective Activation of m-TOR or FoxO3

Autophagy is a conserved process in eukaryotes required for metabolism and is involved in diverse diseases. To investigate autophagy in skeletal muscle under hyperglycemia status, we established two hyperglycemia-rat models that differ in their circulating insulin levels, by glucose infusion and singe high-dose streptozotocin injection. We then detected expression of autophagy related genes with real-time PCR and western blot. We found that under hyperglycemia status induced by glucose-infusion, autophagy was inhibited in rat skeletal muscle, whereas under streptozotocin-induced hyperglycemia status autophagy was enhanced. Meanwhile, hyperglycemic gastrocnemius muscle was more prone to autophagy than soleus muscle. Furthermore, inhibition of autophagy in skeletal muscle in glucose-infusion hyperglycemia rats was mediated by the m-TOR pathway while m-TOR and FoxO3 both contributed to enhancement of autophagy in gastrocnemius muscle in streptozotocin-induced hyperglycemia rats. These data shows that insulin plays a relatively more important role than hyperglycemia in regulating autophagy in hyperglycemia rat muscle through selectively activating the m-TOR or FoxO3 pathway in a fiber-selective manner.

Introduction
Hyperglycemia impairs cell function and biological processes, leading to severe physical injury and increased mortality [1,2,3]. Recent findings have demonstrated that autophagy is involved in diabetes and diabetes complications such as diabetic nephropathy [4,5,6,7], suggesting that autophagy is closely related to hyperglycemia.
Autophagy is an evolutionarily conserved process in eukaryotes that recycles cellular components [8]. It involves numerous autophagy related genes (ATGs) such as Beclin1, Atg5, Atg7, Atg12. The most typical trigger of autophagy is nutrient starvation [7]. Starvation-induced autophagy was observed in many organisms including liver, muscle, heart, pancreas etc. Compared with most other organs, autophagy in skeletal muscle is more sensitive for an obvious longer time of autophagy activation [9]. Autophagy controls muscle mass during catabolic conditions and required for basal myofiber homeostasis [10,11]. Recently, He C et al. [12] found that autophagy plays a crucial role in glucose homeostasis in mice muscle during excise, suggesting that autophagy is closely to metabolism in skeletal muscle. However, whether loss of homeostasis, such as hyperglycemia, in turn affected autophagy in muscle got less attention. Thus, our interest was aroused to focus on autophagy in skeletal muscle under hyperglycemia status.
Skeletal muscle contains slow as well as fast twitch muscle fibers exhibiting different proteomics and transcriptomics [13]. Gastrocnemius muscle is composed mainly by fast-twitch muscle fibers and soleus muscle is composed mainly of slow-twitch muscle fibers [14,15]. A variety of recent reports have indicated that gastrocnemius and soleus differ in many properties [16,17,18], and diversity of responses of different skeletal muscles to starvationinduced autophagy has been observed [9]. The AKT signaling pathway plays a crucial role in autophagy in rat skeletal muscle. Downstream of AKT, m-TOR and FoxO3 regulate autophagy under certain circumstances [19]. Activation of the FoxO3 pathway enhances autophagy [20] while the m-TOR pathway plays a negative role in autophagy [21].
In our study, we focus on autophagy in skeletal muscle (gastrocnemius and soleus) under hyperglycemia induced by different mechanisms. Comparing glucose-infusion hyperglycemia rats (GLU-rats) with streptozotocin-induced hyperglycemia rats (STZ-rats), we found that effect on autophagy in these two hyperglycemia rats was opposite. We attribute this to the different insulin levels and provide evidence that the effect is mediated by the mTOR pathway and FoxO3 pathway. In addition, the response differs in the two fiber types studied.

Autophagy is Inhibited in Gastrocnemius of GLU-rats
To investigate autophagy under hyperglycemia status in skeletal muscle, we first studied the gastrocnemius of GLU-rats. GLU-rats showed a significant increase in blood glucose (1864.0 mmol/L) and insulin (11.061.5 mmol/L) levels compared with control rats (6.361.1 mmol/L, 0.5760.50 mmol/L) ( Figure 1A), implying that the GLU-rats exhibited hyperglycemia with a high insulin level.
Expression of the autophagy-related genes LC3, Atg5, Atg7, Atg12, and BECN1 was analyzed by RT-PCR ( Figure 1B). Compared with control rats, mRNA levels of LC3, Atg7, BECN1 significantly decreased while Atg5, Atg12 showed a weak decrease that was not statistically significant. To examine whether translation levels were affected, we used Western blot to detect LC3, Beclin1 and ATG7 ( Figure 1C). LC3-II, which is one marker for autophagy [22], was showed a dramatic decrease in GLU-rats. Compared with control rats, Beclin1, an essential autophagy related protein in skeletal muscle, was also significantly reduced. Since the ratio of LC3-II to LC3-I may be more correlated with autophagy than the absolute levels [22], we compared the ratio LC3-II/LC3-I between GLU-rats and control rats ( Figure 1C) and also found a significant decrease. Therefore, autophagy was suppressed in gastrocnemius of GLU-rats.

Autophagy is Enhanced in Gastrocnemius of STZ-rats
Next, we investigated autophagy in gastrocnemius of the other hyperglycemia rat model, STZ-rats, which was established by a single high-dose of streptozotocin and characterized by damage tobcells. In order to eliminate the potential possibility that STZ may directly induce autophagy, we performed western blot in C2C12 muscle fibers and got a negative result ( Figure S1).
STZ-rats showed a significant increase in blood glucose level (2563.0 mmol/L) compared with control rats (6.161.5 mmol/L). However, the insulin level in STZ-rats (0.1560.020 mmol/L) exhibited a dramatic decrease compared with control rats (1.260.11 mmol/L), which differed from GLU-rats ( Figure 2A).
RT-PCR and western blot showed that the mRNA levels of the autophagy related genes LC3, Atg5 and BECN1 significantly increased, while Atg5 and Atg12 showed a slight increase ( Figure 2B). The changes in the levels of the autophagy related proteins mirrored the changes in the mRNAs. The translation levels of LC3-II, Beclin1 and LC3-II/LC3-I showed a statistically significant increase, with ATG7 showing a marginal increase ( Figure 2C). Thus, autophagy was enhanced in the gastrocnemius of STZ-rats.

Autophagy in the Soleus in the Two Hyperglycemia Rat Models
To investigate whether soleus differs from gastrocnemius in skeletal muscle autophagy under hyperglycemia, the autophagy related proteins LC3, Beclin1, ATG7 were analyzed by western blot in soleus of the two hyperglycemia rat models. In GLU-rats, LC3-II/LC3-I and Beclin1 showed a statistically significant decrease, while ATG7 exhibited a slight decrease ( Figure 3A). Although autophagy in the gastrocnemius and soleus of GLU-rats were both reduced, the soleus showed a lesser decrease compared with the gastrocnemius. However, in STZ-rats, expression of the autophagy related proteins LC3, Beclin1 and ATG7 in the soleus remained unchanged compared with control rats ( Figure 3B). Figure 1. Autophagy in gastrocnemius is inhibited in Glucose-infusion hyperglycemia rats (GLU-rats). A: GLU-rats were treated with 50% glucose via carotid artery infusion for 24 h (2 mg/kg/h). Control rats were treated with an equivalent volume of 0.9% sodium chloride for 24 h. Blood glucose and insulin levels were measured before the rats were killed. B: mRNA levels of LC3, Atg5, Atg7, Atg12, BECN1 were detected by RT-PCR in gastrocnemius. C: Gastrocnemius lysates were analyzed by Western blot using anti-LC3, anti-Beclin 1, anti-ATG7 and anti-Actin. (*P,0.05, **P,0.01, ***P,0.001). doi:10.1371/journal.pone.0087254.g001 From the above results, the two hyperglycemia rat models have nearly opposite effects on autophagy in skeletal muscles. We investigated whether m-TOR and FoxO3 [19] are involved in mediating this process.
P-S6 and p-FoxO3a are commonly used as indicators for activation of the m-TOR pathway and FoxO3 pathway, respectively. In GLU-rats, we observed a noticeable activation of  m-TOR in gastrocnemius ( Figure 4A) and soleus ( Figure 4B), as determined by an increase of p-S6. Meanwhile, the FoxO3 pathway showed no significant alteration in skeletal muscle of GLU-rats. These results indicate that activation of the m-TOR pathway led to the repression of autophagy. In STZ-rats, the alteration in the m-TOR pathway in gastrocnemius was the reverse of that in GLU-rats; p-S6 decreased and p-FoxO3a significantly increased, indicating repression of m-TOR together with activation in FoxO3 led to enhancement in autophagy in the gastrocnemius. In soleus of STZ-rats, no significant alteration in the m-TOR or FoxO3 pathway was detected, in which correlates with no change in autophagy ( Figure 4D). The above results suggest that m-TOR is mainly involved in autophagy in hyperglycemia rat skeletal muscle. In addition, FoxO3 is involved in the enhancement of autophagy in the gastrocnemius in STZrats. The M-TOR and FoxO3 pathways showed no alteration in the soleus of STZ-rats which may may due to the non-sensitive property to lack of insulin of the soleus fiber in STZ-rats [23]. Alteration in p-mTOR was also detected as shown in supplement materials ( Figure S2).

Discussion
In our study, we found that autophagy was altered in hyperglycemia rat skeletal muscles, and the effects were opposite in the two hyperglycemia models. Autophagy was inhibited in both gastrocnemius and soleus and mediated by the m-TOR pathway in GLU-rats. In STZ-rats, autophagy was enhanced in the gastrocnemius and mediated by the m-TOR and FoxO3 pathway, while autophagy in the soleus was unchanged. In addition, the gastrocnemius was more sensitive to autophagy than the soleus in hyperglycemia rats.
The glucose-infusion hyperglycemia model and the streptozotocin-induced hyperglycemia model are both classic hyperglycemia models that are used widely in research. In the former model, pancreatic b cell proliferation is activated, leading to a relatively higher insulin level [24]. In the latter model, the single high dose of streptozotocin induces damage in rat b cells [25], causing a lower insulin level. The difference in autophagy observed in our study suggested that insulin may be a crucial regulator to autophagy under hyperglycemia conditions. Actually, high fat diet induced hyperinsulinemia was reported to suppress autophagy in mice liver [26]. However, more specific researched are needed to certificate the relationship between insulin and autophagy in skeletal muscle under hyperglycemia.
The gastrocnemius was found to be more sensitive to autophagy than the soleus. The diversity of autophagy responses in skeletal muscle may due to different regulation of protein synthesis and degradation in fast-twitch and slow-twitch muscle fibers [27]. As previously reported, the gastrocnemius is more prone to be affected by external changes such as food deprivation, resulting in protein turnover [27,28]. Since autophagy is a process of degradation, our observations seems to be consistent with previous studies. However, we didn't detect specific markers for protein synthesis or degradation in this study. Therefore, although we found that gastrocnemius and soleus differed in responses to autophagy, underlying mechanism needs further evidence. What's more, different oxidative capacity of fast-switching muscle and slow-switching muscle may be another cause. Fast-switching muscle, which possesses a low oxidative capacity, showed a greater extent in basal and starvation-induced autophagy [29]. In STZ-rats,results were more complicate since we did not detect an alteration in autophagy in the soleus. Different properties of Figure 4. Signaling pathway involved in autophagy in muscle of GLU-rats and STZ-rats. Gastrocnemius lysates (A) and soleus lysates (B) from GLU-rats were analyzed by Western blot using anti-pS6, anti-pFoxO3 and anti-Actin and compared with control rats. In STZ-rats, western blots were performed with these same antibodies in gastrocnemius (C) and soleus (D) and compared with control rats. (***P,0.001). doi:10.1371/journal.pone.0087254.g004 muscle fibers in STZ-rats aroused researchers' attention ever since 1975. Armstrong R et al pointed out that soleus was not sensitive to lack of insulin induced by STZ, exhibiting a least alteration in histochemical properties. Maybe our results could be a supplement to their research that soleus also appeared to be least affected related to autophagy under hyperglycemia status [23].
The M-TOR pathway showed detectable changes in the two skeletal muscle in both GLU-rats and STZ-rats, while FoxO3 changed only in gastrocnemius in STZ-rats. Maybe the roles of mTOR and FoxO3 playing in protein turnover was a potent cause. mTOR regulates cell function mainly via regulation of protein synthesis [30] while FoxO3 mainly controls protein degradation [31]. Autophagy is a main pathway of degradation [32]. FoxO3 acted together with m-TOR when autophagy was activated, whereas m-TOR played an important role alone when autophagy was inhibited.
The alteration in autophagy in hyperglycemia rats is shown in Figure 5. In GLU-rats, hyperglycemia associated with a high insulin level activated the m-TOR pathway and inhibited autophagy in the gastrocnemius and soleus. The gastrocnemius is more sensitive to autophagy, so the change in autophagy in the gastrocnemius was more significant than in the soleus. In STZrats, hyperglycemia associated with lack of insulin inhibited the m-TOR pathway and activated FoxO3 pathway, triggering autophagy in the gastrocnemius. Our data suggests that insulin may play a relatively more important role than hyperglycemia in autophagy in hyperglycemia rat muscle in a fiber-selective manner, with both mTOR and FoxO3 involved. These findings might provide a new understanding of autophagy under hyperglycemia status in skeletal muscle. However, more research will be needed to specify the underlying mechanism.

Animals
Four-week-old female SD rats (weight = 6200 g) were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). All animals were housed at 18uC-25uC under 12 h light and dark cycles and allowed access to food and water ad libitum. The National Institutes of Health principles of laboratory animal use and care were strictly followed with institutional approval of Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences.

Development of GLU-rats and STZ-rats
GLU-rats were treated with 50% glucose injection via carotid artery infusion for 24 h (5 g/kg/h, n = 6). Control animals received an equivalent volume of 0.45% sodium chloride (n = 6); STZ-rats were treated with streptozotocin via intraperitoneal injection (65 mg/kg, n = 6). Control animals received an equivalent volume of citric acid buffer (n = 6). Glucose level and insulin level were detected from the second day after streptozotocin injection until rats were killed in the forth day, then gastrocnemius and soleus muscle were prepared for western blot and RT-PCR.

Western Blotting
Soluble lysates (20 mg per lane) were subjected to 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were then transferred to nitrocellulose membranes and blocked with 5% nonfat milk/TBST for 1 h at room temperature. Membranes were incubated with primary antibodies (diluted by 1:1000) for 18 h at 4uC. After washing membranes with TBST three times, membranes were incubated with horseradish-peroxidase-conjugated antibody for 1 h at room temperature followed by three washings. Western blots were developed using Pierce ECL Western blotting substrate and quantified by Quantity One software.

RT-PCR
RNA was collected with trizol from all rats. Two micrograms of total RNA was used for first-strand cDNA synthesis by using SuperScript II Reverse Transcription Kit. Primers were designed using Primer 5 software. Duplicate PCR was carried out with the intercalating dye SybrGreen. PCR cycles were programmed on an ABI Prism 7000 Sequence Detection System (Applied Biosystems). Figure 5. Autophagy in hyperglycemia rat skeletal muscle. In GLU-rats, the m-TOR pathway is activated in gastrocnemius and soleus, inhibiting autophagy. In STZ-rats, the m-TOR pathway is repressed and the FoxO3 pathway is activated in gastrocnemius, enhancing autophagy. doi:10.1371/journal.pone.0087254.g005 Statistics All values are expressed as means 6 S.D. Significances of differences among groups were evaluated using unpaired t test. A P value of less than 0.05 was considered statistically significant. Figure S1 STZ has no effect on autophagy in C2C12 skeletal muscle cells. Mature C2C12 skeletal muscle cells were exposed to 2.5/5 mM STZ for 0 h, 3 h, 6 h, 9 h, 12 h and 16 h, then western blot was performed to detect autophagy using anti-LC3 and anti-GAPDH. (TIF) Figure S2 Alteration in p-mTOR in muscles of GLU-rats and STZ-rats. Gastrocnemius lysates and soleus lysates from GLU-rats and STZ-rats were analyzed by Western blot using antip-mTOR and anti-Actin. (TIF) Author Contributions


extfieldsofstudy: Biology Medicine

text:
CaMKIIdelta subtypes: localization and function

In this review we discuss the localization and function of the known subtypes of calcium/calmodulin dependent protein kinase IIδ (CaMKIIδ) and their role in cardiac physiology and pathophysiology. The CaMKII holoenzyme is comprised of multiple subunits that are encoded by four different genes called CaMKIIα, β, γ, and δ. While these four genes have a high degree of sequence homology, they are expressed in different tissues. CaMKIIα and β are expressed in neuronal tissue while γ and δ are present throughout the body, including in the heart. Both CaMKIIγ and δ are alternatively spliced in the heart to generate multiple subtypes. CaMKIIδ is the predominant cardiac isoform and is alternatively spliced in the heart to generate the CaMKIIδB subtype or the slightly less abundant δC subtype. The CaMKIIδB mRNA sequence contains a 33bp insert not present in δC that codes for an 11-amino acid nuclear localization sequence. This review focuses on the localization and function of the CaMKIIδ subtypes δB and δC and the role of these subtypes in arrhythmias, contractile dysfunction, gene transcription, and the regulation of Ca2+ handling.

EXPRESSION AND LOCALIZATION
Calcium/calmodulin dependent protein kinase II (CaMKII) is a multimeric enzyme consisting of distinct subunits encoded by four different genes known as CaMKIIα, β, γ, and δ. These genes have a high degree of sequence homology but show differential tissue expression. CaMKIIα and β are predominantly expressed in neuronal tissue while γ and δ are present throughout the body, including the heart (Bennett et al., 1983;Tobimatsu and Fujisawa, 1989). CaMKIIδ is the predominant cardiac isoform and is alternatively spliced to generate multiple subtypes . Schworer et al. (1993) were the first to demonstrate that there are different subtypes of CaMKIIδ expressed in various tissues. The authors reported four distinct proteins with differential expression patterns and named them CaMKIIδ 1−4 . CaMKIIδ 2 , and CaMKIIδ 3 were shown to be identical except for the insertion of an 11-amino acid sequence in the variable domain of CaMKIIδ 3 , the more abundant of the two subtypes in the heart (Schworer et al., 1993). Around the same time, Edman and Schulman (1994) identified these same CaMKIIδ subtypes in rat heart and characterized their catalytic activity and regulation by calcium-liganded calmodulin (Ca 2+ /CaM). They refer to the predominant cardiac subtypes as CaMKIIδ B and CaMKIIδ C (the convention that will be used in this review), which correspond to the δ 3 and δ 2 subtypes, respectively. The structure of these proteins is shown in Figure 1. CaMKIIδ B and δ C possess similar catalytic activity and sensitivity to Ca 2+ /CaM. Furthermore, both subtypes can undergo autophosphorylation and acquire a similar degree of Ca 2+ -independent or autonomous activity . In the years that followed, seven additional splice variants of the CaMKIIδ gene, referred to as CaMKIIδ 5−11 , were identified. Only one of these, CaMKIIδ 9 , is expressed in the adult heart (Figure 1; Mayer et al., 1994Mayer et al., , 1995Hoch et al., 1998Hoch et al., , 1999. The 11-amino acid insert in CaMKIIδ B ( 328 KKRKSSSSVQMM) is also present in some splice variants of CaMKIIα and γ; this conservation suggests an important function (Schworer et al., 1993). Srinivasan et al. (1994) showed that when constructs of CaMKIIδ B are transfected into fibroblasts the expressed protein is localized to the nucleus. This is not the case for constructs of CaMKIIδ C , implying that that the additional amino acid sequence present in CaMKIIδ B is responsible for nuclear localization (Srinivasan et al., 1994). A similar differential localization pattern was also observed when CaMKIIδ subtypes were expressed neonatal rat ventricular myocytes (NRVMs; Ramirez et al., 1997). Further studies showed that the 11-amino acid insert in CaMKIIδ B can confer nuclear localization when inserted into the variable domain of CaMKIIα and that mutagenesis of the first two lysines in the insert abrogates the nuclear localization of these constructs. Thus it is widely accepted that the CaMKIIδ B variable domain contains a nuclear localization signal (NLS).
CaMKII heteromultimerization is permissive in that the CaMKII holoenzyme can include subunits from multiple CaMKII genes and multiple splice variants of those genes (Bennett et al., 1983;Yamauchi et al., 1989). It seems likely that more than a single CaMKIIδ subtype is present in a single CaMKIIδ multimer and accordingly the ratio of δ B to δ C in a multimer could regulate the localization of the holoenzyme. This has been demonstrated experimentally. When CaMKIIδ B and δ C are cotransfected into fibroblasts or NRVMs, the localization of the expressed protein can be shifted in accordance with the ratio of the expressed CaMKIIδ subtypes, i.e., highly expressed δ C sequesters δ B in the cytosol and blocks its nuclear localization (Srinivasan et al., 1994;Ramirez et al., 1997). The opposite is also true: high relative expression FIGURE 1 | Domain scheme of CaMKIIδ monomers. The four subtypes shown are distinguished by the amino acid sequence of a variable region between the regulatory and association domains. CaMKIIδ 2/C lacks this variable domain while CaMKIIδ 3/B contains an 11-amino acid insertion that contains the canonical nuclear localization signal (NLS) sequence KKRK. An asterisk denotes a phosphorylation site (Ser 332 ) in this domain that affects NLS accessibility. CaMKIIδ 9 contains a different variable domain sequence that is also present in the longer variable region of CaMKIIδ 1/A . All of these subtypes can be detected in adult murine heart with the exception of CaMKIIδ 1/A , which is normally only present in neonatal mouse heart. of δ B can localize δ C to the nucleus. While not well appreciated, CaMKIIδ B localization can also be regulated by phosphorylation. A serine (Ser 332 ) immediately adjacent to the NLS of CaMKIIδ B was shown to be a site of phosphorylation by CaMKI and CaMKIV in vitro. Phosphorylation prevents association of δ B with the NLS receptor m-pendulin and thus limits localization of CaMKIIδ B to the nucleus (Heist et al., 1998). Remarkably this mode of regulation is also seen when the NLS is moved from the middle of the protein to the N-terminus, suggesting that conformational changes are not required for phosphorylation to block the NLS.
Relative expression of CaMKIIδ subtypes is altered during cardiomyocyte differentiation and maturation and in association with the development of heart failure and ischemia/reperfusion (I/R) injury (Hoch et al., 1998(Hoch et al., , 1999Colomer et al., 2003;Peng et al., 2010). In humans CaMKIIδ B mRNA is selectively upregulated during heart failure (Hoch et al., 1999). The altered expression of particular subtypes suggests the possibility of a regulated process governing CaMKIIδ mRNA splicing because transcriptional regulation would not be expected to alter the ratio of CaMKIIδ subtypes. Alternative splicing factor/pre-mRNA-splicing factor SF2 (ASF/SF2) was initially described by Krainer and Maniatis (1985) and subsequently mice lacking ASF/SF2 expression were demonstrated to have incomplete processing of CaMKIIδ mRNA (Krainer and Maniatis, 1985;Xu et al., 2005). Specifically, enhanced expression of the δ A subtype [δ 1 in the nomenclature of Schworer et al. (1993)] was observed while expression of CaMKIIδ B and δ C was diminished. Figure 1 also depicts the structure of the δ A subtype, which is expressed in the fetal heart. ASF/SF2 can be regulated by phosphorylation. Protein kinase A (PKA)-mediated ASF/SF2 phosphorylation has been correlated with alternative splicing of CaMKIIδ in heart and brain (Gu et al., 2011). Additionally, regulation of ASF/SF2 by Protein phosphatase 1 γ (PP1γ) has been demonstrated to affect CaMKIIδ splicing (Huang et al., 2013). CaMKIIδ A expression is increased in models of isoproterenol-induced cardiac hypertrophy and thus regulation of CaMKIIδ splicing by PKA and PP1γ may be relevant in the context of chronic β-adrenergic stimulation . The RNA binding proteins Fox 1 (RBFOX1) and 2 (RBFOX2) collaborate with ASF/SF2 to induce proper CaMKIIδ splicing (Han et al., 2011) and factors that regulate these proteins could also influence the expression of CaMKIIδ subtypes. Thus, CaMKIIδ splicing is a dynamic and regulated process. The role of this system in the heart has not been extensively explored but could be of major importance since regulation of CaMKIIδ splicing may account for altered subtype expression and CaMKIIδ signaling in physiological and pathophysiological settings.

CaMKIIδ B TRANSGENIC MICE
The differential localization and function of CaMKIIδ subtypes could be of considerable importance to understanding the role of this enzyme in normal physiology and disease states. Early studies demonstrated that expression of CaMKIIδ B in NRVMs induced atrial natriuretic factor (ANF) expression and led to increased myofilament organization, both hallmarks of cardiac hypertrophy, while expression of CaMKIIδ C did not (Ramirez et al., 1997). This finding suggested that nuclear CaMKIIδ localization is required to regulate gene expression. Consistent with this notion are data indicating that CaMKIIδ B signaling activates several transcription factors including myocyte enhancer factor 2 (MEF2), GATA4, and heat shock factor 1 (HSF1; Little et al., 2009;Lu et al., 2010;Peng et al., 2010). The significance of the hypertrophic responses elicited by δ B in vitro was explored further Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies by generation of CaMKIIδ B transgenic (TG) mice . These animals, which overexpress δ B under the control of the cardiac-specific α-myosin heavy chain (α-MHC) promoter, demonstrate the enhanced expression of hypertrophic markers observed in NRVMs expressing CaMKIIδ B . CaMKIIδ B TG animals develop hypertrophy and moderate cardiac dysfunction by 4 months of age. Thus, CaMKIIδ B expression appears to be sufficient to induce cardiac hypertrophy. Surprisingly, despite the increased CaMKII activity in the CaMKIIδ B TG mouse heart, phosphorylation of the canonical cardiac CaMKII substrate phospholamban (PLN) at its CaMKII site (Thr 17 ) was not increased but rather was decreased relative to WT mice. PLN phosphorylation at the PKA site (Ser 16 ) was similarly reduced. These data were related to increases in phosphatase activity , but also implied that CaMKIIδ B did not lead to robust phosphorylation of PLN. A subsequent paper that examined CaMKIIδ B TG animals at a younger age to avoid changes in phosphatase activity confirmed that phosphorylation of PLN and another cardiac CaMKII substrate, the cardiac ryanodine receptor (RyR2), was not increased by cardiac CaMKIIδ B expression (Zhang et al., 2007). This finding is consistent with a predominantly nuclear localization and function of the δ B subtype.
CaMKIIδ B has also been suggested to regulate expression of the Na + /Ca 2+ exchanger (NCX1) during the development of cardiac dysfunction following trans-aortic constriction (TAC; Lu et al., 2011). The conclusion that δ B was the subtype involved in NCX1 regulation relied on the use of a constitutively active construct of CaMKIIδ B in which a Thr to Asp mutation (T287D) simulates autophosphorylation. Interestingly, the authors found that this construct was excluded from the nucleus (Lu et al., 2010). This differs from the localization pattern described above (Srinivasan et al., 1994;Ramirez et al., 1997) and can be explained as a result of phosphorylation of Ser 332 in the 11-amino acid insert of δ B (Figure 1). The observation that mutation of Ser 332 to Ala restores nuclear localization of constitutively active CaMKIIδ B (Backs et al., 2006) confirms the role of this site in the cytosolic localization of the active construct. The possibility that phosphorylation of Ser 332 might regulate CaMKIIδ B localization in the intact heart has not been evaluated, but such a mechanism could contribute to the observation that CaMKIIδ B is found outside the nucleus even in the absence of multimerization with δ C .

CaMKIIδ C TRANSGENIC MICE
CaMKIIδ C transgenic mice have also been generated and demonstrate a strikingly different phenotype from mice that express CaMKIIδ B . While cardiac dysfunction is relatively moderate and takes months to develop in CaMKIIδ B TG animals, mice expressing δ C rapidly progress to heart failure and premature death . By 6 weeks of age CaMKIIδ C TG animals display marked changes in cardiac morphology and by 12 weeks these animals display severe cardiac dysfunction and upregulation of hypertrophic genes.

Ca 2+ HANDING AND ARRHYTHMIA
Expression of the cardiac sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) is diminished in δ C TG mice as occurs in other models of heart failure. Since SERCA regulates Ca 2+ reuptake into the sarcoplasmic reticulum (SR), this decrease would diminish SR Ca 2+ loading. On the other hand, the CaMKIIδ C TG mice show hyperphosphorylation of PLN at Thr 17 , which should improve SERCA function. In addition δ C TG animals display marked increases in phosphorylation of the RyR2, the channel through which Ca 2+ exits the SR. Taken together, these changes would predict dysregulation of SR Ca 2+ cycling and excitationcontraction coupling. This was substantiated in an accompanying paper that systematically analyzed and demonstrated dysregulation of cardiac Ca 2+ handling in mice expressing δ C . Specifically it was shown that SR Ca 2+ stores were depleted in myocytes from these animals, explaining the observation that isolated myocytes displayed diminished twitch shortening amplitude. Furthermore, Maier et al. (2003) showed that the frequency and duration of Ca 2+ sparks, or spontaneous intracellular Ca 2+release events, was markedly increased in myocytes from animals expressing δ C . Hyperphosphorylation of RyR2 by CaMKIIδ C was hypothesized to underly the enhanced leak of Ca 2+ from the SR, and this was verified by the demonstration that acute inhibition of CaMKII in δ C TG myocytes rescues the altered Ca 2+ handling . In other experiments, acute expression of δ C in rabbit cardiomyocytes was shown to be sufficient to induce SR Ca 2+ sparks and diminished SR Ca 2+ loading (Kohlhaas et al., 2006). These findings imply that direct regulation of Ca 2+ handling targets including RyR2 by CaMKIIδ C can account for the dysregulation of Ca 2+ and contractile function seen in myocytes from δ C TG animals (Figure 2).
Dysregulation of excitation-contraction coupling by CaMKII is thought to contribute to arrythmogenesis in a variety of contexts, as supported by the increased incidence of arrhythmogenic events in CaMKIIδ C TG mice (Anderson et al., 1998;Wu et al., 2002;Wagner et al., 2006). Overexpression of CaMKIIδ C not only induces more spontaneous arrhythmias but also enhances the susceptibility of mice to arrhythmogenic challenge by β-adrenergic stimulation. Sag et al. (2009) found that much of the proarrhythmogenic effects of β-adrenergic stimulation on SR Ca 2+ leak were significantly inhibited by treatment of myocytes with KN-93, an inhibitor of CaMKII. Furthermore the SR Ca 2+ leak induced by isoproterenol did not occur in myocytes from mice lacking CaMKIIδ. These findings collectively implicate SR Ca 2+ leak as one of the key mechanisms in δ C -mediated arrhythmias (Sag et al., 2009). The notion that hyperphosphorylation of RyR2 at the CaMKII site (Ser 2814 ) contributes to arrhythmias and SR Ca 2+ leak is supported by the finding that mutation of Ser 2814 to Ala (S2814A) blocks the ability of CaMKII to induce Ca 2+ sparks (van Oort et al., 2010). The autosomal dominant form of catecholaminergic polymorphic ventricular tachycardia (CPVT) can be caused by the RyR2 mutation R4496C and mice carrying this mutation are predisposed to arrhythmia and ventricular fibrillation. Enhanced CaMKIIδ C expression and activity are implicated in the etiology of premature death in CPVT as expression of CaMKIIδ C exacerbates the effects of the R4496C mutation (Dybkova et al., 2011). As mentioned earlier RyR2 Ser 2814 phosphorylation is increased by expression of CaMKIIδ C (but not by δ B ) in vivo (Zhang et al., 2007) and the effects of mutating this site (van Oort et al., 2010) emphasize the importance of RyR2 phosphorylation by CaMKII in SR Ca 2+ leak and arrhythmia. www.frontiersin.org FIGURE 2 | Localization and function of CaMKIIδ subtypes in the adult cardiomyocyte. The circles labeled δ C and δ B represent CaMKIIδ multimers that are composed primarily of δ C and δ B subunits, respectively. Documented phosphorylation events are indicated by dashed lines. CaMKIIδ C regulates Ca 2+ homeostasis and currents involved in arrhythmogenesis through phosphorylation of Ca 2+ handling proteins and channels. CaMKIIδ C can also affect gene transcription through direct and indirect mechanisms including phosphorylation of NFAT and HDAC (sequestering them in the cytosol) , increases in p53, and increased nuclear import of NF-κB. The CaMKIIδ B subtype has little effect on phosphorylation of Ca 2+ handling proteins but increases gene expression through HDAC phosphorylation and nuclear export and activation of HSF1 and GATA4. A putative mechanism for δ B redistribution is depicted, showing δ B exiting or being excluded from the nucleus due to phosphorylation at a site (Ser 332 ) adjacent to its NLS.
Other targets besides those at the SR may contribute to the arrhythmogenic phenotype of CaMKIIδ C mice. The cardiac sodium channel Na V 1.5 is physically associated with CaMKIIδ C based on coimmunoprecipitation of these proteins from CaMKIIδ C TG animals and Na V 1.5 is phosphorylated in mice expressing δ C (Wagner et al., 2006). CaMKIIδ C phosphorylates Na V 1.5 at multiple sites and phosphorylation appears to elicit the loss-of-function changes in Na V 1.5 gating that are observed in the context of CaMKIIδ C expression in vitro (Ashpole et al., 2012;Koval et al., 2012). Incomplete inactivation of Na V 1.5 generates a late Na + current (I Na ), which can prolong the duration of the action potential and contribute to arrhythmias. Additionally, increased I Na can lead to Na + -overloading of the cardiomyocyte, which contributes to diminished diastolic contractile performance (Maltsev et al., 1998). Late I Na is observed in CaMKIIδ C TG mice and inhibition of this current ameliorates arrhythmia and diastolic dysfunction in these animals (Sossalla et al., 2011). Modulation of I Na therefore appears to contribute to the phenotype of CaMKIIδ C mice with respect to arrhythmia development; additionally the CaMKIIδ C subtype likely regulates the L-type Ca 2+ channel (LTCC) and repolarizing potassium currents (I to and I K1 ; McCarron et al., 1992;Wagner et al., 2009). Thus, a multitude of mechanisms link CaMKIIδ C to arrhythmogenesis.

CONTRACTILE DYSFUNCTION
Arrhythmias may contribute to the premature death of CaMKIIδ C TG mice but there are also marked decreases in contractile function in these animals. Since alterations to cardiomyocyte Ca 2+ handling are seen in relatively young CaMKIIδ C TG mice and precede the development of heart failure, it is possible that dysregulated Ca 2+ homeostasis (specifically SR Ca 2+ leak) is an initiating event in δ C -induced heart failure. Specifically, as a consequence of SR Ca 2+ leak and SERCA downregulation, the SR Ca 2+ load is diminished which would compromise contractile function. To determine whether diminished SR Ca 2+ load is the primary causal event leading to contractile dysfunction and premature death in response to δ C overexpression, we crossed the Frontiers in Pharmacology | Pharmacology of Ion Channels and Channelopathies δ C TG mice with mice in which the SERCA regulatory protein PLN was deleted (PLN-KO). Deletion of PLN in the context of δ C overexpression normalized SR Ca 2+ levels and the contractile function of isolated myocytes was restored . Remarkably the development of cardiac dysfunction in vivo was not rescued but instead was accelerated in the δ C TG/PLN-KO mice. In addition SR Ca 2+ leak was enhanced. It was hypothesized that the increased SR Ca 2+ load, in the context of RyR2 hyperphosphorylation, precipitated greater Ca 2+ leak and further suggested that the accelerated development of cardiac dysfunction was due to mitochondrial Ca 2+ overloading . These observations and their interpretation places central importance on the Ca 2+ leak elicited by δ C -mediated phosphorylation of RyR2 in the development of heart failure. Further support for this hypothesis comes from the finding that CaMKIIδ knockout mice have attenuated contractile dysfunction in response to pressure overload induced by TAC and myocytes from these animals show diminished SR Ca 2+ leak in response to TAC (Ling et al., 2009). Additionally, mice expressing the RyR2 S2814A mutation are protected from the development of heart failure in response to pressure overload (Respress et al., 2012) consistent with a critical role for CaMKII-mediated RyR2 phosphorylation. We recently crossed CaMKIIδ C mice with those expressing RyR2 S2814A; if the hypothesis is correct these mice will show diminished SR Ca 2+ leak and improved contractile function when compared to CaMKIIδ C TG mice.
Another approach used to determine the role of RyR2 phosphorylation and SR Ca 2+ leak in the phenotype of CaMKIIδ C TG mice was to cross the CaMKIIδ C TG mice with mice expressing SRtargeted autocamtide-2-related inhibitory peptide (SR-AIP; Huke et al., 2011). AIP simulates the regulatory domain of CaMKII and inhibits the kinase, and SR-AIP mice have been shown to display diminished phosphorylation of CaMKII substrates at the SR (Ji et al., 2003). A reduction in the extent of PLN and RyR2 hyperphosphorylation observed in CaMKIIδ C TG mice was conferred by SR-AIP. There were associated changes in Ca 2+ handling that indicated a modest improvement in SR Ca 2+ leak. Despite the salutary effects of SR-AIP in cells from δ C TG mice, in vivo cardiac function was not improved. One possible explanation for these findings is that the degree of inhibition of RyR2 phosphorylation conferred by SR-AIP was insufficient to prevent the effects of CaMKIIδ C overexpression. Alternatively, while δ C -mediated phosphorylation of targets at the SR including RyR2 and PLN is of considerable consequence, targets of CaMKII elsewhere in the cell may also contribute to the pathogenesis of cardiac dysfunction induced by CaMKIIδ C .
Mitochondrial Ca 2+ is elevated in mice overexpressing δ C in the context of intact SR Ca 2+ load  and increases in mitochondrial Ca 2+ are known to induce opening of the mitochondrial permeability transition pore (MPTP) and cell death (Halestrap and Davidson, 1990). Considering the central importance of mitochondria in the regulation of cell death and of cell death in the development of heart failure (Wencker et al., 2003), any pathway by which CaMKIIδ C induces mitochondrial Ca 2+ overloading and subsequent loss of mitochondrial integrity would be predicted to contribute to the development of contractile dysfunction and heart failure. To test the role of mitochondrial dysregulation in the cardiomyopathy that develops in δ C TG animals, CaMKIIδ C TG mice were crossed with mice lacking expression of cyclophilin D, a mitochondrial protein required for the formation of the MPTP. The ability of high Ca 2+ to induce swelling of isolated mitochondria, an index of MPTP opening, was impaired in the CaMKIIδ C TG mice lacking cyclophilin D, but development of dilated cardiomyopathy and premature death of these mice was not diminished. Indeed these responses were exacerbated when compared to δ C TG mice with intact cyclophilin D expression. The authors suggest that cyclophilin D may actually play a beneficial role in stress responses, as they observed that TACinduced heart failure development was also made more severe by genetic deletion of cyclophilin D (Elrod et al., 2010). However, CaMKIIδ C is found at mitochondria and a recent seminal study by Joiner et al. (2012) identified the mitochondrial Ca 2+ uniporter (MCU) as a potential target of CaMKII Joiner et al., 2012). While phosphorylation of the MCU by CaMKII was not shown to occur in vivo, a CaMKII-dependent change in the function of the MCU was evidenced by data demonstrating that a CaMKII inhibitory peptide targeted to the mitochondria diminished mitochondrial Ca 2+ uptake and inhibited apoptosis in mice subjected to myocardial infarction and I/R injury.

CaMKIIδ SUBTYPES IN GENE TRANSCRIPTION
The discussion above, and indeed most of the literature, considers the role of CaMKIIδ-mediated phosphorylation and regulation of Ca 2+ handling proteins and ion channels. Chronic elevations in CaMKIIδ expression and activity are observed in humans with heart failure (Hoch et al., 1999) and these long-term changes are likely to elicit altered gene expression. As discussed earlier, CaMKIIδ B induces the expression of hypertrophic genes in myocytes and transgenic mice, consistent with its primarily nuclear localization (Ramirez et al., 1997;Zhang et al., 2002). Other work showed that the CaMKIIδ B subtype is required for GATA-4 binding to the B cell lymphoma 2 (Bcl-2) promoter and subsequent gene expression (Little et al., 2009). Furthermore, CaMKIIδ B was shown to phosphorylate the transcription factor HSF1 thereby increasing its transcriptional activity (Peng et al., 2010). Taken together, these observations imply that it is the CaMKIIδ B subtype that regulates gene expression as a result of its actions in the nucleus.
It is not necessarily the case, however, that gene regulation requires CaMKIIδ to be localized to the nuclear compartment. Despite its primarily cytosolic localization, CaMKIIδ C overexpressed in mouse heart increased phosphorylation of histone deacetylase 4 (HDAC4), resulting in activation of the transcription factor MEF2 (Zhang et al., 2007). CaMKIIδ C has also been demonstrated to regulate nuclear localization of nuclear factor of activated T cells (NFATs) in NRVM. The ability of CaMKIIδ C to decrease nuclear NFAT was blocked by coexpression of a dominant-negative construct of CaMKIIδ C and was shown to be elicited by phosphorylation and inhibition of the Ca 2+ /CaM dependent phosphatase calcineurin (Cn;MacDonnell et al., 2009), presumably in the cytosol. Alteration of Ca 2+ homeostasis by cytosolic CaMKIIδ C expression may indirectly affect gene expression and additionally the constitutively active CaMKIIδ B utilized in the studies discussed www.frontiersin.org above (Lu et al., 2011) is cytosolic and yet regulates expression of NCX1.
Regulation of gene expression by CaMKIIδ B has been demonstrated to promote cardiomyocyte survival while the opposite is true for CaMKIIδ C . CaMKIIδ B was shown to protect cardiomyocytes from doxorubicin-induced cell death via transcriptional upregulation of Bcl-2 (Little et al., 2009). Along similar lines, CaMKIIδ B contributes to cardioprotection from H 2 O 2 by increasing inducible heat shock protein 70 (iHSP70) expression (Peng et al., 2010). Conversely, CaMKIIδ C activation is implicated in cell death elicited by a variety of stimuli (Zhu et al., 2007). It has been suggested that CaMKIIδ C (but not δ B ) upregulates the proapoptotic transcription factor p53 (Toko et al., 2010), and recent work from our laboratory demonstrates that CaMKIIδ C expression in NRVMs activates the proinflammatory transcription factor nuclear factor κB (NF-κB; Ling et al., 2013). We demonstrated that CaMKIIδ C increased phosphorylation of IκB Kinase (IKK) and since IKK activation can also upregulate p53 (Jia et al., 2013), this pathway may contribute to the proapoptotic response reported by Toko et al. (2010).

FUTURE DIRECTIONS
There is compelling evidence that the CaMKIIδ B and δ C subtypes differentially regulate cardiomyocyte Ca 2+ handling and survival in vitro. Whether this occurs in vivo under physiological or pathophysiological conditions, and whether δ B and δ C subserve different functions based on their localization or selective activation, remains to be determined.
It seems likely that the relative levels of endogenous δ B and δ C determine localization and could therefore impact CaMKIIδ signaling. Hypothetically, a selective increase in CaMKIIδ C would result in accumulation of cytosolic CaMKIIδ and depletion of nuclear CaMKIIδ while a selective increase in CaMKIIδ B would have the opposite effect. CaMKIIδ redistribution in this manner may contribute to the phenotype of mice that overexpress δ B and δ C and importantly there are changes in the relative expression of δ B and δ C in models of heart failure and I/R injury. In both models δ C expression is enhanced relative to that of δ B Peng et al., 2010). It is not known how this occurs but it is of interest to postulate that in heart failure and during I/R regulation of CaMKIIδ splicing is altered. ASF/SF2 and RBFOX1/2 regulate the splicing of the CaMKIIδ gene and thus expression of δ B and δ C , but whether changes in splicing occur in and contribute to the development of heart failure or I/R injury remains to be determined. It is likely that the increased δ C expression observed in these models is pathogenic.
While CaMKIIδ B contains an NLS, this subtype is not completely sequestered in the nucleus . As mentioned previously the NLS within the variable domain of δ B can be regulated by phosphorylation, which prevents nuclear localization. This type of regulation could be of considerable importance since the nuclear localization of δ B appears to correlate with enhanced expression of protective genes and cell survival while cytosolic localization does not (Little et al., 2009;Peng et al., 2010;Lu et al., 2011).
Of additional interest is the neglected CaMKIIδ 9 . The pioneering work of (Hoch et al., 1998;Mayer et al., 1995) identified δ 9 as one of the three subtypes of CaMKIIδ in the adult heart and showed that it is expressed at similar levels to those of CaMKIIδ B . δ 9 contains a sequence ( 328 EPQTTVIHNPDGNK) not present in δ B or δ C and thus may possess unique properties that merit further investigation, as the function and localization of δ 9 in vivo has not been explored. Along similar lines, CaMKIIδ A expression is increased in a model of cardiac hypertrophy , but the possibility that this splice variant is upregulated in and contributes to cardiovascular disease has not been investigated.