The role of action potential changes in depolarization-induced failure of excitation contraction coupling in mouse skeletal muscle

Excitation-contraction coupling (ECC) is the process by which electrical excitation of muscle is converted into force generation. Depolarization of skeletal muscle resting potential contributes to failure of ECC in diseases such as periodic paralysis, intensive care unit acquired weakness and possibly fatigue of muscle during vigorous exercise. When extracellular K+ is raised to depolarize the resting potential, failure of ECC occurs suddenly, over a narrow range of resting potentials. Simultaneous imaging of Ca2+ transients and recording of action potentials (APs) demonstrated failure to generate Ca2+ transients when APs peaked at potentials more negative than –30mV. An AP property that closely correlated with failure of the Ca2+ transient was the integral of AP voltage with respect to time. Simultaneous recording of Ca2+ transients and APs with electrodes separated by 1.6mm revealed AP conduction fails when APs peak below –21mV. We hypothesize propagation of APs and generation of Ca2+ transients are governed by distinct AP properties: AP conduction is governed by AP peak, whereas Ca2+ release from the sarcoplasmic reticulum is governed by AP integral. The reason distinct AP properties may govern distinct steps of ECC is the kinetics of the ion channels involved. Na channels, which govern propagation, have rapid kinetics and are insensitive to AP width (and thus AP integral) whereas Ca2+ release is governed by gating charge movement of Cav1.1 channels, which have slower kinetics such that Ca2+ release is sensitive to AP integral. The quantitative relationships established between resting potential, AP properties, AP conduction and Ca2+ transients provide the foundation for future studies of failure of ECC induced by depolarization of the resting potential.

SUMO-specific Isopeptidases Tuning Cardiac SUMOylation in Health and Disease

SUMOylation is a transient posttranslational modification with small-ubiquitin like modifiers (SUMO1, SUMO2 and SUMO3) covalently attached to their target-proteins via a multi-step enzymatic cascade. SUMOylation modifies protein-protein interactions, enzymatic-activity or chromatin binding in a multitude of key cellular processes, acting as a highly dynamic molecular switch. To guarantee the rapid kinetics, SUMO target-proteins are kept in a tightly controlled equilibrium of SUMOylation and deSUMOylation. DeSUMOylation is maintained by the SUMO-specific proteases, predominantly of the SENP family. SENP1 and SENP2 represent family members tuning SUMOylation status of all three SUMO isoforms, while SENP3 and SENP5 are dedicated to detach mainly SUMO2/3 from its substrates. SENP6 and SENP7 cleave polySUMO2/3 chains thereby countering the SUMO-targeted-Ubiquitin-Ligase (StUbL) pathway. Several biochemical studies pinpoint towards the SENPs as critical enzymes to control balanced SUMOylation/deSUMOylation in cardiovascular health and disease. This study aims to review the current knowledge about the SUMO-specific proteases in the heart and provides an integrated view of cardiac functions of the deSUMOylating enzymes under physiological and pathological conditions.

Bioluminescent Sensors for Ca++ Flux Imaging and the Introduction of a New Intensity-Based Ca++ Sensor

Sensitive detection of biological events is a goal for the design and characterization of sensors that can be used in vitro and in vivo. One important second messenger is Ca++ which has been a focus of using genetically encoded Ca++ indicators (GECIs) within living cells or intact organisms in vivo. An ideal GECI would exhibit high signal intensity, excellent signal-to-noise ratio (SNR), rapid kinetics, a large dynamic range within relevant physiological conditions, and red-shifted emission. Most available GECIs are based on fluorescence, but bioluminescent GECIs have potential advantages in terms of avoiding tissue autofluorescence, phototoxicity, photobleaching, and spectral overlap, as well as enhancing SNR. Here, we summarize current progress in the development of bioluminescent GECIs and introduce a new and previously unpublished biosensor. Because these biosensors require a substrate, we also describe the pros and cons of various substrates used with these sensors. The novel GECI that is introduced here is called CalBiT, and it is a Ca++ indicator based on the functional complementation of NanoBiT which shows a high dynamic change in response to Ca++ fluxes. Here, we use CalBiT for the detection of Ca++ fluctuations in cultured cells, including its ability for real-time imaging in living cells.

The M2 Gene Is a Determinant of Reovirus-Induced Myocarditis

Although a broad range of viruses cause myocarditis, the mechanisms that underlie viral myocarditis are poorly understood. Here, we report that the M2 gene is a determinant of reovirus myocarditis. The reovirus M2 gene encodes outer capsid protein µ1, which influences both cell entry and cell death. We infected newborn mice with reovirus strain type 1 Lang (T1L) or a reassortant reovirus in which the M2 gene from strain type 3 Dearing (T3D) was substituted into the T1L background (T1L/T3DM2). T1L was non-lethal in wild-type mice, whereas ~ 90% of mice succumbed to T1L/T3DM2. T1L/T3DM2 produced higher viral loads than T1L at the site of inoculation. In secondary organs, T1L/T3DM2 was detected with more rapid kinetics and reached higher peak titers than T1L. We found that hearts from T1L/T3DM2-infected mice were grossly abnormal, with large lesions corresponding to substantial cardiac injury with inflammatory infiltrates. Lesions in T1L/T3DM2-infected mice contained aggregates of necrotic cardiomyocytes with pyknotic debris, and prominent lymphocyte and histiocyte infiltration. In contrast, T1L induced the formation of smaller lesions in a subset of animals, consistent with T1L being mildly myocarditic. Finally, more activated caspase-3-positive cells were observed in hearts from animals infected with T1L/T3DM2 compared to T1L. Together, our findings indicate that substitution of the T3D M2 allele into an otherwise T1L genetic background is sufficient to change a non-lethal infection into a lethal infection. Our results further indicate that T3D M2 enhances T1L replication and dissemination in vivo, which potentiates the capacity of reovirus to cause myocarditis.

Hexacyanometallate aqueous flow battery

Aqueous redox flow batteries (RFBs) have attracted significant attention as energy storage systems by virtue of their inexpensive nature and long-lasting features. Although all-vanadium RFBs exhibit long lifetimes, the cost of vanadium resources fluctuates considerably, and is generally expensive. Iron–chromium RFBs take advantage of utilizing a low-cost and large abundance of iron and chromite ore; however, the redox chemistry of CrII/III generally involves strong Jahn–Teller effects. Herein, we introduce a new Cr-based negolyte coordinated with strong-field ligands capable of mitigating strong Jahn–Teller effects, thereby facilitating low redox potential, high stability, and rapid kinetics. Density functional theory (DFT) calculations reveal that the complex of [Cr(CN)6]4− prefers low-spin states, facilitating a stable and fast redox reaction. The prototype full-cell configuration features a high-energy density of 11.4 Wh L− 1 and a stable lifetime of 250 cycles. Consequently, our proposed system opens new avenues for the development of high-performance RFBs.

Guiding Stem Cell Differentiation and Proliferation Activities Based on Nanometer-Thick Functionalized Poly-p-xylylene Coatings

Modifications of biomaterials based on the combination of physical, chemical, and biological cues for manipulating stem cell growth are needed for modern regenerative medicine. The exploitation of these sophisticated modifications remains a challenge, including substrate limitation, biocompatibility, and versatile and general cues for stem cell activities. In this report, a vapor-phase coating technique based on the functionalization of poly-p-xylylene (PPX) was used to generate a surface modification for use with stem cells in culture. The coating provided the ability for covalent conjugation that immobilized bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2), and the modified coating surfaces enabled direct stem cell differentiation and controlled proliferation because of the specific activities. The ligations were realized between the growth factors and the maleimide-modified surface, and the conjugation reactions proceeded with high specificity and rapid kinetics under mild conditions. The conjugation densities were approximately 140 ng·cm−2 for BMP-2 and 155 ng·cm−2 for FGF-2. Guiding the activities of the human adipose-derived stem cells (hADSCs) was achieved by modifying surfaces to promote the hADSC differentiation capacity and proliferation rate. The reported coating system demonstrated biocompatibility, substrate-independent conformity, and stability, and it could provide an effective and versatile interface platform for further use in biomedical applications.

Flexible pivoting of dynamin PH-domain catalyzes fission: Insights into molecular degrees of freedom

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) through the centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission (in model membranes) can be managed, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, the absence of the PHD renders a dramatic dampening of the rate of fission. These observations suggest that the PHD-PIP2 containing membrane interaction could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches to explore PHD-membrane interactions. Our results reveal that (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, and (b) PHD associates with membrane in multiple orientations using variable loops as pivots. We identify a new loop (VL4), which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane — a mechanism we believe may be important for high fidelity dynamin collar assembly. Together, these insights provide a molecular-level understanding of the catalytic role of PHD in dynamin-mediated membrane fission. [Media: see text] [Media: see text] [Media: see text] [Media: see text]