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 signaling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU)3–5 are not known. The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is activated in ischemia reperfusion (I/R), myocardial infarction (MI) and neurohumoral injury, common causes of myocardial death and heart failure, suggesting 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 (CsA), an mPTP antagonist with clinical efficacy in I/R injury6, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to I/R injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition are resistant to I/R injury, MI and neurohumoral injury, suggesting pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry and suggest mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure dysfunction in response to common experimental forms of pathophysiological stress.

Ion channel function is fundamental to the existence of life. In metazoans, the coordinate activities of voltagegated Na + channels underlie cellular excitability and control neuronal communication, cardiac excitationcontraction coupling, and skeletal muscle function. However, despite decades of research and linkage of Na + channel dysfunction with arrhythmia, epilepsy, and myotonia, little progress has been made toward understanding the fundamental processes that regulate this family of proteins. Here, we have identified β IV -spectrin as a multifunctional regulatory platform for Na + channels in mice. We found that β IV -spectrin targeted critical structural and regulatory proteins to excitable membranes in the heart and brain. Animal models harboring mutant β IV -spectrin alleles displayed aberrant cellular excitability and whole animal physiology. Moreover, we identified a regulatory mechanism for Na + channels, via direct phosphorylation by β IV -spectrin-targeted calcium/calmodulin-dependent kinase II (CaMKII). Collectively, our data define an unexpected but indispensable molecular platform that determines membrane excitability in the mouse heart and brain.

During the classic "fight-or-flight" stress response, sympathetic nervous system activation leads to catecholamine release, which increases heart rate and contractility, resulting in enhanced cardiac output. Catecholamines bind to β-adrenergic receptors, causing cAMP generation and activation of PKA, which phosphorylates multiple targets in cardiac muscle, including the cardiac ryanodine receptor/calcium release channel (RyR2) required for muscle contraction. PKA phosphorylation of RyR2 enhances channel activity by sensitizing the channel to cytosolic calcium (Ca 2+ ). Here, we found that mice harboring RyR2 channels that cannot be PKA phosphorylated (referred to herein as RyR2-S2808A +/+ mice) exhibited blunted heart rate and cardiac contractile responses to catecholamines (isoproterenol). The isoproterenol-induced enhancement of ventricular myocyte Ca 2+ transients and fractional shortening (contraction) and the spontaneous beating rate of sinoatrial nodal cells were all blunted in RyR2-S2808A +/+ mice. The blunted cardiac response to catecholamines in RyR2-S2808A +/+ mice resulted in impaired exercise capacity. RyR2-S2808A +/+ mice were protected against chronic catecholaminergic-induced cardiac dysfunction. These studies identify what we believe to be new roles for PKA phosphorylation of RyR2 in both the heart rate and contractile responses to acute catecholaminergic stimulation. IntroductionDuring exercise, heart rate (chronotropy) and cardiac contractility (inotropy) increase to meet the metabolic demands of the organs. Stress-induced activation of the sympathetic nervous system (SNS) results in catecholamine release, stimulation of β-adrenergic receptors (β-ARs), generation of cAMP, and activation of cAMP-dependent protein kinase (PKA) in cardiac myocytes. Catecholaminergic stimulation of the heart increases both heart rate and contractility (1). The essential role of β-ARs in the stress-induced enhancement of cardiac function has been demonstrated using β 1 -AR knockout mice that are unable to develop normal responses to stress (2). However, the complexity of the β-AR signaling cascade has made it difficult to elucidate specific contributions of downstream targets to the physiologic responses to stress.β-AR stimulation and downstream activation of PKA enhances calcium (Ca 2+ ) signaling in myocytes (3). During excitation-contraction (EC) coupling in the heart, depolarization of the sarcolemmal membrane activates the voltage-gated calcium channel (Ca V 1.2), causing a small Ca 2+ influx into the cell. This in turn triggers the opening of ryanodine receptor/calcium release channel (RyR2) and the release of Ca 2+ from the sarcoplasmic reticu-

Expression of the mTOR pathway components, which are related with the transferability and invasive capacity of HCC cells, may be used as prognostic indicators in HCC.