In vitro to in vivo extrapolation from three-dimensional hiPSC-derived cardiac microtissues and physiologically based pharmacokinetic modeling to inform next-generation arrhythmia risk assessment.

Proarrhythmic cardiotoxicity remains a substantial barrier to drug development as well as a major global health challenge. In vitro human pluripotent stem cell-based new approach methodologies have been increasingly proposed and employed as alternatives to existing in vitro and in vivo models that do not accurately recapitulate human cardiac electrophysiology or cardiotoxicity risk. In this study, we expanded the capacity of our previously established three-dimensional human cardiac microtissue model to perform quantitative risk assessment by combining it with a physiologically based pharmacokinetic model, allowing a direct comparison of potentially harmful concentrations predicted in vitro to in vivo therapeutic levels. This approach enabled the measurement of concentration responses and margins of exposure for two physiologically relevant metrics of proarrhythmic risk (ie, action potential duration and triangulation assessed by optical mapping) across concentrations spanning three orders of magnitude. The combination of both metrics enabled accurate proarrhythmic risk assessment of four compounds with a range of known proarrhythmic risk profiles (ie, quinidine, cisapride, ranolazine, and verapamil) and demonstrated close agreement with their known clinical effects. Action potential triangulation was found to be a more sensitive metric for predicting proarrhythmic risk associated with the primary mechanism of concern for pharmaceutical-induced fatal ventricular arrhythmias, delayed cardiac repolarization due to inhibition of the rapid delayed rectifier potassium channel, or hERG channel. This study advances human induced pluripotent stem cell-based three-dimensional cardiac tissue models as new approach methodologies that enable in vitro proarrhythmic risk assessment with high precision of quantitative metrics for understanding clinically relevant cardiotoxicity.

Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo.

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Molecular chaperones and the regulation of neurotransmitter exocytosis.

Regulated neurotransmitter release depends on a precise sequence of events that lead to repeated cycles of exocytosis and endocytosis. These events are mediated by a series of molecular interactions among vesicular, plasma membrane, and cytosolic proteins. An emerging theme has been that molecular chaperones may guide the sequential restructuring of stable or transient protein complexes to promote a temporal and spatial regulation of the endo- and exocytotic machinery and to ensure a vectorial passage through the vesicle cycle. Chaperones, specialized for a few substrates, are ideally suited to participate in regulatory processes that require some molecular dexterity to rearrange conformational or oligomeric protein structures. This article emphasizes the significance of three molecular chaperone systems in regulated neurotransmitter release: the regulation of soluble NSF attachment protein receptor (SNARE) complexes by N-ethylmaleimide-sensitive factor (NSF) and the soluble NSF attachment protein (SNAP), the uncoating of clathrin-coated vesicles by the 70 kDa heat-shock cognate protein (Hsc70), and the regulation of SNARE complex-associated protein interactions by cysteine-string protein and Hsc70.

Cysteine-string protein increases the calcium sensitivity of neurotransmitter exocytosis in Drosophila.

Previous studies suggest that the vesicular cysteine-string protein (CSP) may modulate presynaptic Ca(2+) channel activity in fast neurotransmitter release. To test this hypothesis, we analyzed the dynamics of presynaptic Ca(2+) ion influx with the Ca(2+) indicator fluo-4 AM at csp mutant neuromuscular junctions of Drosophila. From 24 to 30 degrees C, stimulus-evoked, relative presynaptic Ca(2+) signals were increasingly larger in csp mutant boutons than in controls. Above 30 degrees C, Ca(2+) signals declined and were similar to controls at 34 degrees C. A prolonged decay of Ca(2+) signals in mutant boutons at high temperatures indicated abnormally slow Ca(2+) clearance. Cytosolic Ca(2+) at rest was determined with the ratiometric Ca(2+) indicator fura-2 AM and was similar in mutant and control boutons at 24 degrees C but higher in mutant boutons at 34 degrees C. Despite larger Ca(2+) signals in mutant boutons, evoked neurotransmitter release was always reduced in csp mutants and exhibited pronounced facilitation. Thus, a lack of Ca(2+) entry cannot explain the reduction of neurotransmitter release in csp mutants. At all temperatures tested, raising extracellular Ca(2+) increased transmitter release elicited by single stimuli in csp mutants. Collectively, these data suggest multiple functions for CSP at synaptic terminals. Increased Ca(2+) signals coupled with reduced release suggest a direct function of CSP in exocytosis downstream from Ca(2+) entry. Because the reduction of evoked release in csp mutants is counteracted by increased Ca(2+) levels, we suggest that CSP primarily increases the Ca(2+) sensitivity of the exocytotic machinery.

Multiple local contact sites are induced by GPI-linked influenza hemagglutinin during hemifusion and flickering pore formation.

Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapid freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed numerous hourglass shaped points of membrane contact (approximately 10-130 nm waist) when viewed by electron microscopy. Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, but wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores, a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA, lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pull membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing.

Overexpression of cysteine-string proteins in Drosophila reveals interactions with syntaxin.

Cysteine-string proteins (CSPs) are associated with secretory vesicles and critical for regulated neurotransmitter release and peptide exocytosis. At nerve terminals, CSPs have been implicated in the mediation of neurotransmitter exocytosis by modulating presynaptic calcium channels; however, studies of CSPs in peptidergic secretion suggest a direct role in exocytosis independent of calcium transmembrane fluxes. Here we show that the individual expression of various CSP isoforms in Drosophila similarly rescues the loss of evoked neurotransmitter release at csp null mutant motor nerve terminals, suggesting widely overlapping functions for each isoform. Thus, the structural difference of CSP variants may not explain the opposing putative functions of CSP in neurotransmitter and peptide exocytosis. Consistently, the individual overexpression of each CSP isoform in wild-type Drosophila shows similar effects such as impaired viability and interference with wing and eye development. The dominant effects caused by the overexpression of CSP are suppressed by the simultaneous overexpression of syntaxin-1A but not by the coexpression of SNAP-25. Although overexpression of CSP itself has no apparent effect on the synaptic physiology of larval motor nerve terminals, it fully suppresses the decrease of evoked release induced by the overexpression of syntaxin-1A. A direct protein-protein interaction of CSP with syntaxin is further supported by coimmunoprecipitations of syntaxin with CSP and by protein binding assays using recombinant fusion proteins. Together, the genetic and biochemical interactions of CSP and syntaxin-1A suggest that CSP may chaperone or modulate protein-protein interactions of syntaxin-1A with either calcium channels or other components of the regulatory machinery mediating depolarization-dependent neurotransmitter exocytosis.

The pathway of membrane fusion catalyzed by influenza hemagglutinin: restriction of lipids, hemifusion, and lipidic fusion pore formation.

The mechanism of bilayer unification in biological fusion is unclear. We reversibly arrested hemagglutinin (HA)-mediated cell-cell fusion right before fusion pore opening. A low-pH conformation of HA was required to form this intermediate and to ensure fusion beyond it. We present evidence indicating that outer monolayers of the fusing membranes were merged and continuous in this intermediate, but HA restricted lipid mixing. Depending on the surface density of HA and the membrane lipid composition, this restricted hemifusion intermediate either transformed into a fusion pore or expanded into an unrestricted hemifusion, without pores but with unrestricted lipid mixing. Our results suggest that restriction of lipid flux by a ring of activated HA is necessary for successful fusion, during which a lipidic fusion pore develops in a local and transient hemifusion diaphragm.

Cysteine string protein is required for calcium secretion coupling of evoked neurotransmission in drosophila but not for vesicle recycling.

The entire deletion of the cysteine string protein (CSP) gene causes a temperature-sensitive (ts) block of evoked neurotransmission in Drosophila. CSP has been found to interact in vitro with the clathrin-uncoating ATPase HSC70, suggesting a potential role of CSP in vesicle recycling. Using FM1-43 imaging, we analyzed whether the ts block of neurotransmission in csp mutants is caused by a defect in vesicle exocytosis or vesicle recycling. We determined that FM1-43-labeled synaptic boutons of csp mutant neuromuscular junctions fail to destain at 32 degrees C after K+ depolarization, and that FM1-43 dye uptake cannot be evoked by K+ stimulation at 32 degrees C. However, when we stimulated dye uptake independent of depolarization by using black widow spider venom (BWSV), we observed endocytotic uptake of FM1-43. This suggests that endocytosis exhibits no primary ts defect. In addition, we found no ts defect of vesicle recycling at 32 degrees C that would correlate with the ts block of neurotransmission. We also discovered that BWSV and the calcium ionophore calcimycin stimulate FM1-43 destaining and quantal release in csp mutants at 32 degrees C when depolarization fails to evoke any response. The wild-type-like, calcimycin-induced response in csp null mutants indicates that some aspect of the depolarization-dependent calcium signaling pathway must be impaired, either calcium entry, calcium action, or both. Collectively, our results indicate that the csp mutation affects calcium secretion coupling of evoked exocytosis but not vesicle recycling. This supports the hypothesis that CSP links synaptic vesicles to calcium secretion coupling.

An early stage of membrane fusion mediated by the low pH conformation of influenza hemagglutinin depends upon membrane lipids.

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus-cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell-cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH-induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone-shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.