The Ca 2+ release channel ryanodine receptor 2 (RyR2) is required for excitation-contraction coupling in the heart and is also present in the brain. Mutations in RyR2 have been linked to exercise-induced sudden cardiac death (catecholaminergic polymorphic ventricular tachycardia [CPVT]). CPVT-associated RyR2 mutations result in "leaky" RyR2 channels due to the decreased binding of the calstabin2 (FKBP12.6) subunit, which stabilizes the closed state of the channel. We found that mice heterozygous for the R2474S mutation in Ryr2 (Ryr2-R2474S mice) exhibited spontaneous generalized tonic-clonic seizures (which occurred in the absence of cardiac arrhythmias), exercise-induced ventricular arrhythmias, and sudden cardiac death. Treatment with a novel RyR2-specific compound (S107) that enhances the binding of calstabin2 to the mutant Ryr2-R2474S channel inhibited the channel leak and prevented cardiac arrhythmias and raised the seizure threshold. Thus, CPVT-associated mutant leaky Ryr2-R2474S channels in the brain can cause seizures in mice, independent of cardiac arrhythmias. Based on these data, we propose that CPVT is a combined neurocardiac disorder in which leaky RyR2 channels in the brain cause epilepsy, and the same leaky channels in the heart cause exerciseinduced sudden cardiac death. IntroductionPharmacological seizure models have implicated abnormalities in intracellular Ca 2+ cycling of inhibitory interneurons and/or astrocytes as a mechanism of seizure generation (1, 2), and the inositol 1,4,5-trisphosphate receptor (IP3R), an intracellular calcium release channel on the ER, has been associated with seizures in mice (3). However, a causal relationship between defective intracellular calcium release channels and seizures has not been reported. Calcium stored within the ER contributes to neuronal signaling and is controlled by intracellular Ca 2+ release channels, in particular ryanodine receptors (RyRs) (4-6) and IP3Rs (7,8). To explore the underlying mechanism for seizures in CPVT we generated mice that harbor a missense mutation (RyR2-R2474S) that has been linked to exercise-induced cardiac arrhythmias in humans (9-12).More than 50 distinct RYR2 mutations have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT), an arrhythmogenic cardiomyopathy (13-15). CPVT patients experience syncope and sudden cardiac death (SCD) from the toddler to adult ages, and by 35 years age the mortality is up to 50% (13,16,17).

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

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-

See Appendix for individual names.Background: Pertuzumab combined with trastuzumab and docetaxel is the standard first-line therapy for HER2-positive metastatic breast cancer, based on results from the phase III CLEOPATRA trial. PERUSE was designed to assess the safety and efficacy of investigator-selected taxane with pertuzumab and trastuzumab in this setting. Patients and methods:In the ongoing multicentre single-arm phase IIIb PERUSE study, patients with inoperable HER2-positive advanced breast cancer (locally recurrent/metastatic) (LR/MBC) and no prior systemic therapy for LR/MBC (except endocrine therapy) received docetaxel, paclitaxel or nab-paclitaxel with trastuzumab [8 mg/kg loading dose, then 6 mg/kg every 3 weeks (q3w)] and pertuzumab (840 mg loading dose, then 420 mg q3w) until disease progression or unacceptable toxicity. The primary end point was safety. Secondary end points included overall response rate (ORR) and progression-free survival (PFS). Results:Overall, 1436 patients received at least one treatment dose (initially docetaxel in 775 patients, paclitaxel in 589, nabpaclitaxel in 65; 7 discontinued before starting taxane). Median age was 54 years; 29% had received prior trastuzumab. Median treatment duration was 16 months for pertuzumab and trastuzumab and 4 months for taxane. Compared with docetaxel-containing therapy, paclitaxel-containing therapy was associated with more neuropathy (all-grade peripheral neuropathy 31% versus 16%) but less febrile neutropenia (1% versus 11%) and mucositis (14% versus 25%). At this preliminary analysis (52 months' median follow-up), median PFS was 20.6 [95% confidence interval (CI) 18.9-22.7] months overall (19.6, 23.0 and 18.1 months with docetaxel, paclitaxel and nab-paclitaxel, respectively). ORR was 80% (95% CI 78%-82%) overall (docetaxel 79%, paclitaxel 83%, nab-paclitaxel 77%).Conclusions: Preliminary findings from PERUSE suggest that the safety and efficacy of first-line pertuzumab, trastuzumab and taxane for HER2-positive LR/MBC are consistent with results from CLEOPATRA. Paclitaxel appears to be a valid alternative taxane backbone to docetaxel, offering similar PFS and ORR with a predictable safety profile.ClinicalTrials.gov: NCT01572038.

Background: Many patients with metastatic human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) are candidates for trastuzumab emtansine (T-DM1) treatment sometime in their disease history. KAMILLA evaluated safety of T-DM1 in patients with previously treated HER2-positive locally advanced or metastatic BC (advanced BC).