Sperm-egg interaction at fertilization results in a rise in the intracellular concentration of Ca2+ that triggers a series of events known as egg activation. Several calcium-dependent proteins have been postulated to mediate the different events. One of them is calcium/calmodulin-dependent protein kinase II (CaMKII). In Xenopus eggs, the involvement of CaMKII in metaphase II exit has been recently demonstrated. However, whether the same signaling cascade operates in mice is unknown. Moreover, CaMKII's role in triggering other events of egg activation and the identity of the isoform/s present in mouse eggs are still unclear. Here we show that CaMKIIγ is the only isoform present in mouse eggs. We generated CaMKIIγ-deficient mice and report that they are viable, but CaMKIIγ-/- females are infertile. While CaMKIIγ-/- eggs exhibit a normal pattern of calcium oscillations, they fail to get fertilized or parthenogenetically activated. Instead, they remain arrested at metaphase II with high mitogen-activated protein kinase (MAPK) and maturation-promoting factor (MPF) activities. Maternal mRNA recruitment does not occur in CaMKIIγ-deficient eggs. On the other hand, null eggs are able to mount a post-fertilization block to polyspermy, as revealed by their ability to proteolytically cleave the zona pellucida protein ZP2. This demonstrates that while cell cycle resumption and mRNA recruitment are CaMKII-dependent, the block to polyspermy is not. Additionally, we report that the egg activation defects observed in CaMKIIγ-/- eggs can be reversed by introduction of either CaMKIIγ or CaMKIIδ mRNA into mouse oocytes, suggesting that CaMKII's role during egg activation is not isoform-specific.

Fertilization triggers a rise in intracellular Ca 2+ concentration ([Ca 2+ ] i ) in the egg that initiates a series of events known as egg activation. These events include cortical granule exocytosis that establishes a block to polyspermy, resumption of meiosis, and recruitment of maternal mRNAs into polysomes for translation. Several calcium-dependent proteins, including calcium/calmodulin-dependent protein kinase II (CaMKII), have been implicated in egg activation. However, the precise role of CaMKII in mediating specific events of egg activation and the identity of the isoform(s) present in mouse eggs have not been unequivocally established. Through targeted deletion of the γ isoform of CaMKII, we find that CaMKIIγ is the predominant CaMKII isoform in mouse eggs and that it is essential for egg activation. Although CaMKIIγ −/− eggs exhibit a normal pattern of Ca 2+ oscillations after insemination and undergo cortical granule exocytosis, they fail to resume meiosis or to recruit maternal mRNAs. Surprisingly, we find that the recruitment of maternal mRNAs does not directly depend on CaMKII, but requires elevated [Ca 2+ ] i and metaphase II exit. We conclude that CaMKIIγ specifically controls mouse egg activation by regulating cell cycle resumption.

ER stress-induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress-mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress-induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIgamma (CaMKIIgamma) and JNK. Remarkably, CaMKIIgamma was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress-induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIgamma-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress-induced apoptosis.

Acute and chronic injuries to the heart result in perturbation of intracellular calcium signaling, which leads to pathological cardiac hypertrophy and remodeling. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been implicated in the transduction of calcium signals in the heart, but the specific isoforms of CaMKII that mediate pathological cardiac signaling have not been fully defined. To investigate the potential involvement in heart disease of CaMKII delta, the major CaMKII isoform expressed in the heart, we generated CaMKII delta-null mice. These mice are viable and display no overt abnormalities in cardiac structure or function in the absence of stress. However, pathological cardiac hypertrophy and remodeling are attenuated in response to pressure overload in these animals. Cardiac extracts from CaMKII delta-null mice showed diminished kinase activity toward histone deacetylase 4 ( HDAC4), a substrate of stress-responsive protein kinases and suppressor of stress-dependent cardiac remodeling. In contrast, phosphorylation of the closely related HDAC5 was unaffected in hearts of CaMKII delta-null mice, underscoring the specificity of the CaMKII delta signaling pathway for HDAC4 phosphorylation. We conclude that CaMKII delta functions as an important transducer of stress stimuli involved in pathological cardiac remodeling in vivo, which is mediated, at least in part, by the phosphorylation of HDAC4. These findings point to CaMKII delta as a potential therapeutic target for the maintenance of cardiac function in the setting of pressure overload.

Calcium/calmodulin-dependent protein kinase II (CaMKII) phosphorylates histone deacetylase 4 (HDAC4), a class IIa HDAC, resulting in the cytosolic accumulation of HDAC4 and the derepression of the transcription factor myocyte enhancer factor 2. Phosphorylation by CaMKII requires docking of the kinase to a specific domain of HDAC4 not present in other HDACs. Paradoxically, however, CaMKII signaling can also promote the nuclear export of other class IIa HDACs, such as HDAC5. Here, we show that HDAC4 and HDAC5 form homo- and hetero-oligomers via a conserved coiled-coil domain near their amino termini. Whereas HDAC5 alone is unresponsive to CaMKII, it becomes responsive to CaMKII in the presence of HDAC4. The acquisition of CaMKH responsiveness by HDAC5 is mediated by HDAC5's direct association with HDAC4 and can occur by phosphorylation of HDAC4 or by transphosphorylation by CaMKII bound to HDAC4. Thus, HDAC4 integrates upstream Ca2+-dependent signals via its association with CaMKH and transmits these signals to HDAC5 by protein-protein interactions. We conclude that HDAC4 represents a point of convergence for CaMKII signaling to downstream HDAC-regulated genes, and we suggest that modulation of the interaction of CaMKII and HDAC4 represents a means of regulating CaMKII-dependent gene programs.

Abstract 281 Johannes Backs, David Patrick, Thea Backs, Svetlana Bezprozvannaya, Xiaoxia Qi, Joseph A Hill, Eric N Olson, UT Southwestern Medical Center at Dallas, Dallas, TX Johannes Backs, 2007 Finalist and Presenting Author