Domain zipping and unzipping modulates TRPM4's properties in human cardiac conduction disease.

The transient receptor potential melastatin 4 (TRPM4) is a Ca2+ -activated nonselective cation channel linked to human cardiac diseases. The human mutation K914R within TRPM4's S4-S5 linker was identified in patients with atrioventricular block. During UV-flash-mediated Ca2+ transients, TRPM4K914R  generated a threefold augmented membrane current concomitant with 2 to 3-fold slowed down activation and deactivation kinetics resulting in excessive membrane currents during human cardiac action potentials. Mutagenesis of K914 paired with molecular modeling suggested the importance of the nanoscopic interface between the S4-S5 linker, the MHR4-, and TRP-domain as a major determinant for TRPM4's behavior. Rational mutagenesis of an interacting amino acid (R1062Q) in the TRP domain was able to offset K914R`s gain-of-function by zipping and unzipping of this nanoscopic interface. In conclusion, repulsion and attraction between the amino acids at positions 914 and 1062 alters the flexibility of the nanoscopic interface suggesting a zipping and unzipping mechanism that modulates TRPM4's functions. Pharmacological modulation of this intramolecular mechanism might represent a novel therapeutic strategy for the management of TRPM4-mediated cardiac diseases.

Genetically Encoded Voltage Indicators in Circulation Research.

Membrane potentials display the cellular status of non-excitable cells and mediate communication between excitable cells via action potentials. The use of genetically encoded biosensors employing fluorescent proteins allows a non-invasive biocompatible way to read out the membrane potential in cardiac myocytes and other cells of the circulation system. Although the approaches to design such biosensors date back to the time when the first fluorescent-protein based Förster Resonance Energy Transfer (FRET) sensors were constructed, it took 15 years before reliable sensors became readily available. Here, we review different developments of genetically encoded membrane potential sensors. Furthermore, it is shown how such sensors can be used in pharmacological screening applications as well as in circulation related basic biomedical research. Potentials and limitations will be discussed and perspectives of possible future developments will be provided.

Design, synthesis and evaluation of the antibacterial enhancement activities of amino dihydroartemisinin derivatives.

Artemisinin (ART) and its derivatives artesunate (AS), dihydroartemisinin (DHA) are a group of drugs containing a sesquiterpene lactone used to treat malaria. Previously, AS was shown to not have antibacterial activity but to significantly increase the antibacterial activities of ß-lactam antibiotics against E. coli. Herein, molecular docking experiments showed that ART, AS and DHA could dock into AcrB very well, especially DHA and AS; both DHA and AS had the same docking pose. The affinity between AS and AcrB seemed weaker than that of DHA, while the succinate tail of AS, which was like a "bug", could extend in the binding pocket very well. Imitating the parent nucleus of DHA and the succinate tail of AS, twenty-one DHA derivatives 4a-u were designed and synthesized. Among them, seventeen were new compounds. The synergistic effects against E. coli AG100A/pET28a-AcrB showed among the new structures 4k, 4l, 4m, 4n, and 4r exhibited significant synergism with ß-lactam antibiotics although they had no direct antibacterial activities themselves. The bacterial growth assay showed that only 4k in combination with ampicillin or cefuroxime could totally inhibit bacterial growth from 0 to 12 h, demonstrating that 4k had the best antibacterial enhancement effect. In conclusion, our results provided a new idea and several candidate compounds for antibacterial activity enhancers against multidrug resistant E. coli.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

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 five 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 CaCLEAN analysis to identify functional EC-coupling sites (couplons) 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-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. CONCLUSION: BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.

DREADD technology reveals major impact of Gq signalling on cardiac electrophysiology.

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.

Aberrant Deactivation-Induced Gain of Function in TRPM4 Mutant Is Associated with Human Cardiac Conduction Block.

A gain-of-function mutation in the Ca2+-activated transient receptor potential melastatin member 4 (TRPM4A432T) is linked to life-threatening cardiac conduction disturbance, but the underlying mechanism is unclear. For deeper insights, we used photolysis of caged Ca2+, quantitative Ca2+, and electrophysiological measurements. TRPM4A432T's 2-fold larger membrane current was associated with 50% decreased plasma membrane expression. Kinetic analysis unveiled 4-fold slower deactivation that was responsible for the augmented membrane current progressively rising during repetitive human cardiac action potentials. Rational mutagenesis of TRPM4 at position 432 revealed that the bulkiness of the amino acid was key to TRPM4A432T's aberrant gating. Charged amino acids rendered the channel non-functional. The slow deactivation caused by an amino acid substitution at position 432 from alanine to the bulkier threonine represents a key contributor to the gain of function in TRPM4A432T. Thus, our results add a mechanism in the etiology of TRP channel-linked human cardiac channelopathies.