The molecular carcinogenesis of lung cancer has yet to be clearly elucidated. We investigated the possible oncogenic function of SEC62 in lung cancer, which was predicted based on our previous findings that lung and thyroid cancer tissue samples exhibited increased Sec62 protein levels. The SEC62 gene locus is at 3q26.2, and 3q amplification is reportedly the most common genomic alteration in non-small cell lung cancer. We analyzed SEC62 mRNA and protein levels in tissue samples from lung cancer patients by real-time quantitative PCR, Western blot, and IHC and found significantly increased SEC62 mRNA and protein levels in tumors compared with tumor-free tissue samples from the same patients. Correlation analyses revealed significantly higher Sec62 levels in tumors with lymph node metastases compared with nonmetastatic tumors, as well as in poorly compared with moderately differentiated tumors. On the basis of these promising results, we examined the role of Sec62 in cancer cell biology in vitro. Cell migration assays with lung and thyroid cancer cells showed distinct stimulation of migration in SEC62-overexpressing cells and inhibition of migration in Sec62-depleted cells. Moreover, we found that SEC62 silencing sensitized the cells to thapsigargin-induced endoplasmic reticulum stress. Thus, our results indicate that SEC62 represents a potential candidate oncogene in the amplified 3q region in cases of non-small cell lung cancer and harbors various functions in cancer cell biology.

Aims Signalling via Gq-coupled receptors is of profound importance in many cardiac diseases such as hypertrophy and arrhythmia. Nevertheless, owing to their widespread expression and the inability to selectively stimulate such receptors in vivo, their relevance for cardiac function is not well understood. We here use DREADD technology to understand the role of Gq-coupled signalling in vivo in cardiac function. Methods and results We generated a novel transgenic mouse line that expresses a Gq-coupled DREADD (Dq) in striated muscle under the control of the muscle creatine kinase promotor. In vivo injection of the DREADD agonist clozapine-N-oxide (CNO) resulted in a dose-dependent, rapid mortality of the animals. In vivo electrocardiogram data revealed severe cardiac arrhythmias including lack of P waves, atrioventricular block, and ventricular tachycardia. Following Dq activation, electrophysiological malfunction of the heart could be recapitulated in the isolated heart ex vivo. Individual ventricular and atrial myocytes displayed a positive inotropic response and arrhythmogenic events in the absence of altered action potentials. Ventricular tissue sections revealed a strong co-localization of Dq with the principal cardiac connexin CX43. Western blot analysis with phosphor-specific antibodies revealed strong phosphorylation of a PKC-dependent CX43 phosphorylation site following CNO application in vivo. Conclusion Activation of Gq-coupled signalling has a major impact on impulse generation, impulse propagation, and coordinated impulse delivery in the heart. Thus, Gq-coupled signalling does not only modulate the myocytes’ Ca2+ handling but also directly alters the heart’s electrophysiological properties such as intercellular communication. This study greatly advances our understanding of the plethora of modulatory influences of Gq signalling on the heart in vivo.

Aims In ventricular myocytes, Transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC) coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). Methods and Results In heart samples from healthy human donors expression patterns of 5 BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray we employed high-speed confocal calcium imaging and Ca-CLEAN analysis to identify functional EC-coupling sites and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI motiv induced de-novo generation of T-tubules, functional EC-coupling sites and enhanced calcium handling. Conclusion(s) BIN1 is essential for the maintenance, regeneration, and de-novo generation of functional T-tubules, especially isoforms with PI motifs. These T-tubules trigger the development of functional EC couplons resulting in enhanced calcium handling. Translational Perspective Cardiomyopathy and heart failure are among the most frequent causes of death in modern societies. Gene therapies and hiPSC technology are becoming increasingly promising, both for treatment and therapy development. On the cellular level, one of the common denominators of cardiac diseases is the concurrent loss of T-tubules essential for efficient EC-coupling. While initial approaches in animal models employing gene therapy with BIN1 have depicted encouraging improvements the expression pattern of BIN1 isoforms in the human heart is still elusive. The present study identifies a unique set of five distinct BIN1 isoforms in healthy human hearts and demonstrates their potency in both, T-tubule maintenance and re-generation after loss resulting in efficient EC-coupling. Noteworthy, PI-motif containing isoforms were potent trigger of de-novo generation of T-tubules and establishment of efficient EC-coupling in hiPSC-CMs. Therefore, the expression of BIN1 might be novel and promising for pharmaceutical treatment and gene therapy.

CaMKII is a central regulator of cardiac Ca handling, electrophysiology and transcription. Its persistent over-activation has been implicated in arrhythmias, heart failure, and other diseases. Typically, CaMKII activates upon Ca-CaM binding during elevated [Ca] free and inactivates upon Ca-CaM dissociation when [Ca] free drops. Several post-translational-modifications (PTMs) also trap CaMKII in an autonomously activated state even after [Ca] i declines. The PTMs include autophosphorylation, oxidation, S-nitrosylation, and O-GlcNAcylation. Exactly how these PTMs regulate CaM binding to CaM-KIId-the predominant cardiac isoform-is unknown. Schulman's group showed that autophosphorylation of CaMKIIa (neuronal isoform) increases its CaM affinity by 1,000-fold in lysates. CaMKII activity that persists during low [Ca] free creates a form of molecular memory. Here we perform novel tests of changes in CaM affinity (or trapping) for CaMKIId by known PTMs. Importantly, these are measured in adult rabbit ventricular myocytes, CaMKIId's native environment. Briefly, we express CaMKIId-GFP in isolated ventricular myocytes and load fluorescent-CaM after saponin-permeabilization of the myocytes. We use FRET as a readout for CaM dissociation upon the addition of unlabeled CaM or upon rapidly dropping [Ca] free with BAPTA. In myocytes, expressing the phosphomimetic CaMKIId (T287D) caused a dramatic slowing of CaM dissociation (>5 fold) upon abrupt reduction of [Ca] i (<50 nM) when compared to the non-phosphorylatable mutant (T287A). These differences provide a quantitative, mechanistic basis for PTM-dependent regulation of CaM-KIId in cardiac myocytes and its potential role in disease. We extended this analysis with CaMKIId-GFP mutants and corresponding agonists for other PTMs: e.g. 100uM H 2 O 2 -WT vs. M281/282V and analogous for the S-nitrosylation sites (C273S, C290A) and the O-GlcNAcylation site (S280A). This allows us to test which of these PTMs creates the longest CaM-binding state which will influence its relative impact on cardiac CaMKII memory and contributions to disease.