Opposing mitochondrial fission and fusion reactions determine the shape and interconnectivity of mitochondria. Dynamin‐related protein 1 (Drp1) is an ancient mechanoenzyme that uses GTP hydrolysis to power the constriction and division of mitochondria. Although Drp1‐mediated mitochondrial fragmentation is recognized as an early event in the apoptotic programme, acute regulation of Drp1 activity is poorly understood. Here, we identify a crucial phosphorylation site that is conserved in all metazoan Drp1 orthologues. Ser 656 is phosphorylated by cyclic AMP‐dependent protein kinase and dephosphorylated by calcineurin, and its phosphorylation state is controlled by sympathetic tone, calcium levels and cell viability. Pseudophosphorylation of Drp1 by mutation of Ser 656 to aspartic acid leads to the elongation of mitochondria and confers resistance to various pro‐apoptotic insults. Conversely, the constitutively dephosphorylated Ser656Ala mutant Drp1 promotes mitochondrial fragmentation and increases cell vulnerability. Thus, Drp1 phosphorylation at Ser 656 provides a mechanism for the integration of cAMP and calcium signals in the control of mitochondrial shape, apoptosis and other aspects of mitochondrial function.

Increased mitochondrial calcium entry has been implicated in myocardial death, heart failure and related conditions. Here, the authors show that inhibition of the multifunctional Ca2+- and calmodulin-dependent protein kinase II (CaMKII) in a mouse model of ischaemia reperfusion injury reduces infarct size and mitochondrial-triggered cell death and dysfunction. This is due to reduced mitochondrial Ca2+ entry through the mitochondrial calcium uniporter and enhanced Ca2+ tolerance of the mitochondrial permeability transition pore. This suggests that CaMKII inhibition could reduce adverse responses to common forms of pathological myocardial stress. Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm)1,2. However, the signalling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3,4,5 are not known. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced IMCU and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

Activation and Thr²⁸⁶autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca²⁺ influx viaN-methyl-d-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII·NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca²⁺influx, and Thr²⁸⁶ autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca²⁺channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Drp1 oligomers also translocate directionally along mitochondria. Ionomycin, a calcium ionophore, causes rapid mitochondrial accumulation of actin filaments followed by Drp1 accumulation at the fission site, and increases fission rate. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites.