1 2 3 4 5 Effect of hypernatremia on injury caused by energy deficiency : role of T - type Ca 2 + 6 channel 7 8

27 Hypernatremia exerts multiple cellular effects, many of which could influence the 28 outcome of an ischemic event. To further evaluate these effects of hypernatremia, 29 isolated neonatal cardiomyocytes were chronically incubated with medium containing 30 either normal (142 mM) or elevated sodium (167 mM) and then transferred to medium 31 containing deoxyglucose and the electron transport chain inhibitor, amobarbital. 32 Chronic hypernatremia diminished the degree of calcium accumulation and reactive 33 oxygen species (ROS) generation during the period of metabolic inhibition. The 34 improvement in calcium homeostasis was traced in part to the downregulation of the 35 CaV3.1 T-type calcium channel, as deficiency in the CaV3.1 subtype using shRNA or 36 treatment with an inhibitor of the CaV3.1 variant of the T-type calcium channel (i.e. 37 diphenylhydantoin) attenuated energy deficiency-mediated calcium accumulation and 38 cell death. Although hyperosmotically stressed cells (exposed to 50 mM mannitol) had 39 no effect on T-type calcium channel activity, they were also resistant to death during 40 metabolic inhibition. Both hyperosmotic stress and hypernatremia activated Akt, 41 suggesting that they initiate the PI 3-kinase/Akt cytoprotective pathway, which protects 42 the cell against calcium overload and oxidative stress. Thus, hypernatremia appears to 43 protect the cell against metabolic inhibition by promoting the downregulation of the T44 type calcium channel and stimulating cytoprotective protein kinase pathways. 45 46