Journal of General Physiology

Contraction force in muscle is produced by the interaction of myosin motors in the thick filaments and actin in the thin filaments and is fine-tuned by other proteins such as myosin-binding protein C (MyBP-C). One form of control is through the regulation of myosin heads between an ON and OFF state in passive sarcomeres, which leads to their ability or inability to interact with the thin filaments during contraction, respectively. MyBP-C is a flexible and long protein that is tightly bound to the thick filament at its C-terminal end but may be loosely bound at its middle- and N-terminal end (MyBP-CC1C7). Under considerable debate is whether the MyBP-CC1C7 domains directly regulate myosin head ON/OFF states, and/or link thin filaments ("C-links"). Here, we used a combination of mechanics and small-angle X-ray diffraction to study the immediate and selective removal of the MyBP-CC1C7 domains of fast MyBP-C in permeabilized skeletal muscle. After cleavage, the thin filaments were significantly shorter, a result consistent with direct interactions of MyBP-C with thin filaments thus confirming C-links. Ca2+ sensitivity was reduced at shorter sarcomere lengths, and crossbridge kinetics were increased across sarcomere lengths at submaximal activation levels, demonstrating a role in crossbridge kinetics. Structural signatures of the thick filaments suggest that cleavage also shifted myosin heads towards the ON state - a marker that typically indicates increased Ca2+ sensitivity but that may account for increased crossbridge kinetics at submaximal Ca2+ and/or a change in the force transmission pathway. Taken together, we conclude that MyBP-CC1C7 domains play an important role in contractile performance which helps explain why mutations in these domains often lead to debilitating diseases.

Using both optical and electrical methods, we document that solute diffusion in the cytoplasm of BL6 murine cardiac myocytes becomes restricted >30-fold as molecular weight increases from 30 to 2000, roughly as expected for pores with dimensions of cardiac porin channels. The Bodipy-FL ATP analogue diffuses [~]50-fold slower in BL6 cardiac cytoplasm than in free water. From several fluorophores analyzed, our estimates of bound fluorophore fractions range from 0.1 for a 2 kD FITC-labeled polyethylene glycol to 0.93 for sulforhodamine. We estimate that diffusion coefficients of unbound fluorophores range from 0.5 to 8 x 10-7 cm2/s. Analysis of Na/K pump and veratridine-modified Na channel currents confirms that Na diffusion is nearly unrestricted (time constant for equilibration with the pipette tip, [~]20 s). Using three different approaches, we estimate that ATP diffuses 8 to 10-times slower in the cytoplasm of BL6 myocytes than in free water. To address whether restrictions are caused more by cytoplasmic protein or membrane networks, we verified first that a protein gel, 10 gram% gelatin, restricts solute diffusion with strong dependence on molecular weight. Solute diffusion in membrane-extracted cardiac myofilaments, confined laterally by suction into large-diameter pipette tips, is however less restricted than in intact myocytes. Notably, myofilaments from equivalently extracted skeletal (diaphragm) myocytes restrict diffusion less than cardiac myofilaments. Solute diffusion in myocytes with sarcolemma permeabilized by {beta}-escin (80 {micro}M) is similarly restricted as in intact myocytes. Diffusion restriction in cardiac myocytes is strain-dependent, being about two-fold greater in BL6 myocytes than in myocytes with a CD1/J6/129svJ background. Furthermore, diffusion is 2.5-fold more restricted in CD1/J6/129svJ myocytes lacking the mitochondrial porin, Vdac1, than in WT CD1/J6/129svJ myocytes. We conclude that both myofilaments and mitochondria networks restrict diffusion in cardiac myocytes. As a result, long-range solute diffusion may preferentially occur via passage through porin channels and intramembrane mitochondrial spaces, where diffusion is less restricted than in myofilament spaces.

SCN5A-encoded NaV1.5 is a voltage-gated Na+ channel expressed in cardiac myocytes and human gastrointestinal (GI) smooth muscle cells (SMCs). NaV1.5 contributes to electrical excitability in the heart and slow waves in the gut. NaV1.5 is also mechanosensitive, and mechanical force modulates several modes of NaV1.5s voltage-dependent function. NaV1.5 mutations in patients with cardiac arrhythmias and gastrointestinal diseases lead to abnormal mechano- and voltage-sensitivity. Membrane permeable amphipathic drugs that target NaV1.5 in the heart and GI tract alter NaV1.5 mechanosensitivity (MS), suggesting that amphipaths may be a viable therapeutic option for modulating NaV1.5 function. We therefore searched for membrane-permeable amphipathic agents that would modulate NaV1.5 MS with minimal effect on NaV1.5 voltage-gating intact to more selectively target mechanosensitivity. We used two methods to assess NaV1.5 MS: (1) membrane suction in cell-attached macroscopic patches and (2) fluid shear stress on whole cells. We tested the effect of capsaicin on NaV1.5 MS by examining macropatch and whole-cell Na+ current parameters with and without force. The pressure- and shear-mediated peak current increase and acceleration were effectively abolished by capsaicin. Capsaicin abolished the mechanosensitive shifts in the voltage-dependence of activation (shear) and inactivation (pressure and shear). Exploring the recovery from inactivation and use-dependent entry into inactivation, we found divergent stimulus-dependent effects that could potentiate or mitigate the effect of capsaicin, suggesting that mechanical stimuli may differentially modulate NaV1.5 MS. We conclude that selective modulation of MS makes capsaicin is a novel modulator of NaV1.5 MS and a promising therapeutic candidate.

Myosin binding protein-C (MyBP-C) consists of a family of regulatory proteins expressed in sarcomeres of cardiac, fast and slow twitch skeletal muscles. The 3 MyBP-C paralogs expressed in each muscle type are encoded by separate genes but maintain a similar structure. Given the overall similarity in structure and localization of each of paralog, it is assumed that MyBP-C expressed in different muscles have similar functional effects. Here we directly tested this assumption by making use of our cut and paste approach to remove and replace N-terminal regions of MyBP-C in sarcomeres of different muscle types. We found that the different MyBP-C paralogs similarly slowed cross-bridge cycling kinetics, increased Ca2+ sensitivity of tension, and damped force oscillations. However, responses to a rapid stretch in actively contracting fibers, taken as indices of cross-bridge detachment and attachment kinetics, differed in each muscle type and responses depended on the presence or absence of a given paralog of MyBP-C. Altered responses to stretch were most evident for fast MyBP-C where loss of MyBP-C in psoas muscle resulted in transient responses to stretch that resembled those found in cardiomyocytes. Replacement of cardiac MyBP-C with fast MyBP-C in cardiomyocytes led to responses similar to psoas muscle. In separate X-ray diffraction experiments we also found that loss of MyBP-C in Ca2+-activated psoas muscle increased lattice disorder, reduced the ordering of myosin heads, and decreased thin filament length. Taken together, these results indicate that the different MyBP-C paralogs exert both common and unique effects on myosin cross-bridge kinetics. Significance StatementMyBP-C is a family of regulatory proteins found in muscle sarcomeres, where they regulate contraction and relaxation. Mutations in all MyBP-C paralogs cause disease in skeletal and cardiac muscles. We used a powerful "cut and paste" strategy to selectively remove MyBP-C from slow-twitch, fast-twitch, and cardiac muscle to show that each MyBP-C effects cross-bridge behavior similarly, though to varying degrees. Each MyBP-C had a notable effect on transient responses to rapid stretch, where MyBP-C was found to limit strain-induced cross-bridge detachment, especially in fast-twitch muscles. Strain-induced cross-bridge detachment is critical for rapid filling of the left ventricle in diastole and for sustained contraction in skeletal muscle. MyBP-C paralogs appear adapted to meet the mechanical demands of each muscle type.

There is a growing awareness that both thick filament and classical thin filament regulation play central roles in modulating muscle contraction. Myosin ATPase assays have demonstrated that under relaxed conditions, myosin may reside in either a high energy-consuming disordered-relaxed (DRX) state available for binding actin to generate force, or in an energy-sparing super-relaxed (SRX) state unavailable for actin binding. X-ray diffraction studies have shown the majority of myosin heads are in a quasi-helically ordered OFF state in a resting muscle and that this helical ordering is lost when myosin heads are turned ON for contraction. It has been assumed that myosin heads in SRX and DRX states are equivalent to the OFF and ON state respectively and the terms have been used interchangeably. Here, we use X-ray diffraction and ATP turnover assays to track the structural and biochemical transitions of myosin heads respectively induced with either omecamtiv mecarbil (OM) or piperine in relaxed porcine myocardium. We find that while OM and piperine induce dramatic shifts of myosin heads from the OFF to ON states, there are no appreciable changes in the population of myosin heads in the SRX and DRX states in both unloaded and loaded preparations. Our results show that biochemically defined SRX and DRX can be decoupled from structurally-defined OFF and ON states. In summary, while SRX/DRX and OFF/ON transitions can be correlated in some cases, these two phenomena are measured using different approaches, do not necessarily reflect the same properties of the thick filament and should be investigated and interpreted separately. SignificanceMyosin based thick filament regulation is now known to be critical for muscle contraction with myosin dysregulation found in hypertrophic and dilated cardiomyopathies. While previously thought to be synonymous, this study finds that biochemical and structural thick filament disengagement are distinct properties and should be investigated as independent phenomena. Understanding the details of thick filament regulation will be of great relevance to defining sarcomere-level dysfunction in myopathies and understanding and better designing and testing sarcomere therapies aimed at reversing them for treatment of cardiomyopathy.

Voltage-gated Na+ (Nav) channels, including Nav1.5, are responsible for the initiation of cardiac and neuronal action potentials. Regulation of Nav1.5 inactivation is linked to multiple accessory proteins that bind its C-terminal domain (CTD) including calmodulin (CaM) and intracellular fibroblast growth factors (iFGF). Previous results demonstrate that Ca2+-bound CaM preferentially binds to iFGF12A. The role of intracellular Ca2+ ([Ca2+]i) in regulating Nav1.5 gating, either directly or via auxiliary proteins like CaM, is controversial. We hypothesize that CaM binding to the Nav1.5 CTD and iFGF12A synergistically alters channel inactivation in a previously unobserved calcium-dependent manner. We performed Fluorescence Resonance Energy Transfer (FRET) imaging in live cells to observe the interaction between the Nav1.5 alpha subunit, CaM and iFGF12A. At resting [Ca2+]i, a 2-fold difference between acceptor and donor FRET efficiency was observed, implying that a single CaM acceptor is present on the Nav1.5 CTD even in the presence of FGF12A. After increasing [Ca2+]i, the donor and acceptor FRET efficiencies equalize, suggesting a 2:1:1 ratio between CaM, FGF12A, and the Nav1.5 CTD. We then compared the voltage-dependent gating kinetics of Nav1.5 with FGF12A in the presence/absence of calcium. With low [Ca2+]i, the steady-state inactivation of Nav1.5 with FGF12A was significantly shifted toward hyperpolarized potential compared to resting [Ca2+]i. Thus, the FGF12A:CaM complex confers a Ca2+-dependent mechanism enabling FGF12A modulates the Nav1.5 steady-state inactivation. Additionally, the ability of multiple subunits to bring CaM to the Nav1.5 CTD implies biological redundancy to prevent major alteration to Nav1.5 inactivation in the absence of CaM.

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming 1S subunit and the auxiliary 2{delta}-1 and {gamma}1 subunits. Particularly, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage-dependence of activation to positive potentials outside the physiological range. We generated a new HEK293-cell line stably expressing 2{delta}-1, {beta}3, and STAC3. When the adult (CaV1.1a) and the embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage-dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further co-expressed the {gamma}1 subunit, the current density of CaV1.1a, but not of CaV1.1e, was reduced by >50 %. In addition, {gamma}1 caused a shift of the voltage-dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of {gamma}1, but not its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between oppositely charged residues in the IVS3-S4 loop and the {gamma}1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the {gamma}1-dependent reduction of current density, suggesting that structural rearrangements of CaV1.1a induced by inclusion of exon 29 allosterically empower the {gamma}1 subunit to exert its inhibitory action on CaV1.1 calcium currents. SummaryEl Ghaleb et al. analyzed the effects of the {gamma}1 subunit on current properties and expression of the adult (CaV1.1a) and embryonic (CaV1.1e) calcium channel splice variants, demonstrating that {gamma}1 reduces the current amplitude in a splicing-dependent manner.

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain EM and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin. Significance StatementDilated cardiomyopathy can be caused by single point mutations in cardiac muscle myosin, the motor protein that powers contraction of the myocardium. We found that the E525K DCM mutation in the cardiac myosin heavy chain stabilizes the auto-inhibited, super-relaxed state, suggesting a mechanism by which this mutation reduces muscle force and power. The E525K mutation also highlights critical electrostatic interactions important for forming the conserved, auto-inhibited conformational state of striated muscle myosins.

Mutations in the gene encoding the sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy (SMLM) and modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, groove, and T-tubules in wild-type, dystrophin-deficient (mdx) mice, and mice expressing C-terminally truncated Nav1.5 ({Delta}SIV). We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density and distribution partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and {Delta}SIV cells results in a non-uniform redistribution with Nav1.5 being specifically reduced at the groove of {Delta}SIV and increased in T-tubules of mdx cardiomyocytes. Nav1.5 mutations may therefore site-specifically affect Nav1.5 localization and distribution depending on site-specific interacting proteins.

Two-pore domain potassium (K2P) channels play a central role in modulating cellular excitability and neuronal function. The unique structure of the selectivity filter in K2P and other potassium channels determines their ability to allow the selective passage of potassium ions across cell membranes. The nematode C. elegans has one of the largest K2P families, with 47 subunit-coding genes. This remarkable expansion has been accompanied by the evolution of atypical selectivity filter sequences that diverge from the canonical TxGYG motif. Whether and how this sequence variation may impact the function of K2P channels has not been investigated so far. Here we show that the UNC-58 K2P channel is constitutively permeable to sodium ions and that a cysteine residue in its selectivity filter is responsible for this atypical behavior. Indeed, by performing in vivo electrophysiological recordings and Ca2+ imaging experiments, we demonstrate that UNC-58 has a depolarizing effect in muscles and sensory neurons. Consistently, unc-58 gain-of-function mutants are hypercontracted, unlike the relaxed phenotype observed in hyperactive mutants of many neuromuscular K2P channels. Finally, by combining molecular dynamics simulations with functional studies in Xenopus laevis oocytes, we show that the atypical cysteine residue plays a key role in the unconventional sodium permeability of UNC-58. As predicting the consequences of selectivity filter sequence variations in silico remains a major challenge, our study illustrates how functional experiments are essential to determine the contribution of such unusual potassium channels to the electrical profile of excitable cells. SIGNIFICANCEPotassium channels play a central role in modulating cellular excitability, particularly of neuronal cells. Their unique structure determines their ability to let ions pass selectively through cell membranes. The impact of pathological or evolutionary variations in this selectivity filter remains difficult to predict. Here, we reveal that UNC-58, a member of the two-pore domain potassium channel family of C. elegans, exhibits an unusual sodium permeability due to a unique cysteine residue in its selectivity filter. Our findings underscore the importance of functional studies to determine how sequence variation in potassium channel selectivity filters can shape the electrical profiles of excitable cells.

The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2s Ca2+ permeability and regulation remain ill-defined. In this study, we assessed Ca2+ transport by PC2iv, in a lipid bilayer reconstitution system in the presence of a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans). PC2iv channel reconstitution was conducted in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline (POPC) and ethanolamine (POPE) lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents, in both lipid mixtures with a maximum conductance of 63 {+/-} 13 pS (n = 19) and 105 pS {+/-} 9.8 (n = 9), respectively. In both cases, experimental data were best fitted with the Goldman-Hodgkin-Katz equation, showing a reversal potential (Vrev ~ -27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, spontaneous Ca2+ current oscillations were observed whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed data provide new evidence to support a high Ca2+ permeability by PC2, and a novel regulatory feedback mechanism dependent on the presence of Ca2+ and phospholipids on its function. Statement of SignificanceThe regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. The Transient Potential Receptor channel Polycystin-2 (TRPP2, PC2), is a Ca2+ permeable non-selective cation channel. Ca2+ transport by PC2 has largely been inferred by changes in reversal potential. This study provides experimental evidence on the Ca2+-transporting capabilities of PC2 in high Ca2+ that is modulated by lipids and generates a novel phenomenon of oscillatory currents by channel clustering and multiple subconductance behavior. PC2 can be self-regulated by feedback mechanisms, which are independent of external regulatory proteins. This oscillatory behavior, previously unknown for a single channel species, depend on the presence of Ca2+ interaction sites as have been postulated for the channel protein.

The first-in-its-class cardiac drug mavacamten reduces the proportion of so-called ON-state myosin heads in relaxed sarcomeres, altering contraction performance. However, mavacamten is not completely specific to cardiac myosin and can also affect skeletal muscle myosin, an important consideration since mavacamten is administered orally and so will also be present in skeletal tissue. Here, we studied the effect of mavacamten on skeletal muscle structure using small-angle X-ray diffraction. Mavacamten treatment reduced the proportion of ON myosin heads but did not eliminate the molecular underpinnings of length-dependent activation, demonstrating similar effects to those observed in cardiac muscle. These findings provide valuable insights for the potential use of mavacamten as a tool to study muscle contraction across striated muscle.

In striated muscle, some sarcomere proteins regulate crossbridge cycling by varying the propensity of myosin heads to interact with actin. Myosin-binding protein C (MyBP-C) is bound to the myosin thick filament and is predicted to interact and stabilize myosin heads in a docked position against the thick filament and limit crossbridge formation, the so-called OFF state. Via an unknown mechanism, MyBP-C is thought to release heads into the so-called ON state, where they are more likely to form crossbridges. To study this proposed mechanism, we used the C2-/- mouse line to knock down fast-isoform MyBP-C completely and total MyBP-C by [~]24%, and conducted mechanical functional studies in parallel with small-angle X-ray diffraction to evaluate the myofilament structure. We report that C2-/- fibers presented deficits in force production and reduced calcium sensitivity. Structurally, passive C2-/- fibers presented altered SL-independent and SL-dependent regulation of myosin head ON/OFF states, with a shift of myosin heads towards the ON state. Unexpectedly, at shorter sarcomere lengths, the thin filament was axially extended in C2-/- vs. non-transgenic controls, which we postulate is due to increased low-level crossbridge formation arising from relatively more ON myosins in the passive muscle that elongates the thin filament. The downstream effect of increasing crossbridge formation in a passive muscle on contraction performance is not known. Such widespread structural changes to sarcomere proteins provide testable mechanisms to explain the etiology of debilitating MyBP-C-associated diseases.

Human ERG is a voltage-activated, K-selective channel whose physiological role is to drive action potential repolarization in cardiac myocytes. To carry out its role in the heart, hERG has specialized gating (opening and closing) transitions that are regulated by the internal N-terminal PAS and C-terminal CNBH domains. The PAS and CNBHD domains interact directly and this interaction is required for the characteristic slow deactivation (closing) of hERG channels. But it is unclear whether PAS remains globally attached or dislodges from the CNBHD during gating. Interestingly the direct PAS-CNBHD interaction can be formed in trans by co-expression of the PAS domain and hERG channels with a deleted PAS domain (hERG {Delta}PAS) in which the PAS domain is not attached to the channel with a peptide bond. In trans expression allows us to probe the biophysical mechanism for PAS domain attachment to the rest of the channel and in a broader sense allows us to test the mechanism for intracellular domain function in an ion channel, and test whether the PAS domain detaches or remains attached to the channel during gating. We report here that in excised patches from cells containing the hERG PAS domain fused to CFP and hERG {Delta}PAS channels fused to Citrine that 1) regulation of deactivation (slow deactivation conveyed by the PAS domain) was similar in on-cell and excised, inside-out patch configurations, 2) that regulation of deactivation persists for the lifetime of the patch (up to 30 minutes) in excised, inside-out mode, 3) that channel activity measured by activation of the channel with voltage pulses did not alter channel deactivation and 4) dual fluorescence and ionic current measurements using patch-clamp fluorometry (PCF) showed that only membrane patches containing PAS-CFP + hERG {Delta}PAS-Citrine had CFP and Citrine fluorescence and slow (regulated) deactivation, whereas control patches with hERG {Delta}PAS -Citrine had fast (unregulated) deactivation and Citrine fluorescence (but not CFP fluorescence) and control patches from hERG PAS-CFP - injected cells had neither currents nor CFP or Citrine fluorescence. Moreover, in PCF mode, we detected FRET from PAS-CFP + hERG {Delta}PAS-Citrine channels. Taken together, these results suggested that PAS - CFP remained associated with hERG {Delta}PAS-Citrine channels after membrane excision. We interpret these results to mean that the PAS domain was not dislodged from the channel despite mechanical (excised patch) and conformational (voltage) challenges and suggests that the PAS domain remained firmly attached to the hERG channel during gating.

Mutations in myosin alter its motor functions in diverse ways by affecting different structural and chemo-mechanical events. Multidisciplinary strategies can be used to understand how varying alterations in motor function converge to common phenotypes like hypercontractility and hypertrophic cardiomyopathy (HCM). Here, we combined molecular dynamics (MD) simulations with protein biochemical and myofibril mechanical analyses to study the HCM-causing myosin variant G256E. MD simulations demonstrated that G256E induces structural changes that increase the work required to displace ADP.Mg2+ from actomysoin complex. Stopped-flow biochemical analysis demonstrated increased ADP affinity for actomyosin and single myofibril mechanics analysis demonstrated increased force generation and reduced ADP sensitivity of the early, slow phase of relaxation. Together, these results demonstrate that slower ADP release from myosin during contraction is a significant contributor to pathological contractile nature of the G256E mutation. This study highlights the importance of detailed chemo-mechanical analysis of mutations associated with hereditary cardiac diseases.

The myosin-containing thick filament has recently been shown to alter its resting activation level in response to multiple diseases and therapeutics. Changes in thick filament resting activation level are caused by myosin heads transitioning between OFF and ON conformational states. Functionally, this modulation of thick filament activation level is a key regulatory step in muscle contraction and a promising therapeutic target. The availability of resting ON-state myosin heads governs dynamic contractility, which is critical to physical function and well-being. At present, there is a lack of compounds favouring this ON-state in resting skeletal muscle. Piperine is a molecule known to bind to myosin and increase submaximal isometric contractility in fast and slow skeletal muscle. Yet, effects on dynamic contractility and the underlying mechanism responsible for the observed effects in skeletal muscles remain unclear. Here, we used fibre small-angle X-ray diffraction and intact-muscle ex vivo contractility experiments to determine the effects of piperine on resting myosin structure and dynamic contractility in fast and slow rat muscles. X-ray diffraction data suggest that piperine promotes an OFF-to-ON transition of myosin in resting skeletal muscle, increasing the availability of myosin heads for force generation. Functionally, piperine substantially enhanced dynamic contractility in both muscle types, with greater improvements in slow muscle during maximal activation. These findings establish piperine as a tool to probe thick-filament activation in skeletal muscle, highlighting fibre-type-specific effects of thick-filament activation on the recruitment of the contractile reserve capacity. Key Points- Piperine is a compound known to bind to skeletal muscle myosin and enhance isometric contractility in fast and slow muscles, but the underlying molecular mechanisms and effects on dynamic contractile function remain unknown. - We show that piperine increases the activation level of the myosin-containing thick filament in resting fast and slow skeletal muscle, which may explain the effect of piperine on contractile function. - Piperine substantially increases the maximal contractile power of both fast and slow skeletal muscles at low-frequency activation; however, it only enhances the maximal power in slow skeletal muscle at high-frequency activation. - Our data reveal potentiation of dynamic contractility with fibre-type-dependent magnitudes in response to piperine-induced activation of the resting thick filament, a phenomenon that requires further investigation and may ultimately be exploited in the treatment of diseases characterised by muscle weakness. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/689918v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1ff3672org.highwire.dtl.DTLVardef@4f809forg.highwire.dtl.DTLVardef@1857404org.highwire.dtl.DTLVardef@83d958_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO Abstract figure legend: We investigated the effects of piperine on 1) the activation level of the resting thick filament and 2) dynamic contractility in fibres and intact slow (soleus) and fast (extensor digitorum longus, EDL) rat muscles, respectively. The activation level of the resting thick filament was assessed pre- and post-piperine incubation using small-angle X-ray diffraction. Dynamic contractility was assessed at submaximal and maximal activation levels by constructing low- and high-frequency force-velocity curves and corresponding power curves using an ex vivo contraction setup. The setup allows for simultaneous experiments on the effects of piperine and vehicle treatment in contralateral muscles. We found that piperine increased the activation level of the resting thick filament by favouring the ON-myosin state in fibres from both muscle types. In whole muscle preparations, piperine also induced substantial increases in dynamic contractility, especially in slow soleus muscle. C_FIG

The block of voltage-dependent sodium channels by saxitoxin (STX) and tetrodotoxin (TTX) was investigated in voltage-clamped squid giant axons internally perfused with a variety of permeant monovalent cations. Substitution of internal Na+ by either NH4+ or N2H5+ resulted in a reduction of outward current through sodium channels under control conditions. In contrast, anomalous increases in both inward and outward currents were seen for the same ions if some of the channels were blocked by STX or TTX, suggesting a relief of block by these internal cations. External NH4+ was without effect on the apparent magnitude of toxin block. Likewise, internal inorganic monovalent cations were without effect, suggesting that proton donation by NH4+ might be involved in reducing toxin block. Consistent with this hypothesis, decreases in internal pH mimicked internal perfusion with NH4+ in reducing toxin block. The interaction between internally applied protons and externally applied toxin molecules appears to be competitive, as transient increases in sodium channel current were observed during step increases in intracellular pH in the presence of a fixed STX concentration. In addition to these effects on toxin block, low internal pH produced a voltage-dependent block of sodium channels and enhanced steady-state inactivation. Elevation of external buffer capacity only marginally diminished the modulation of STX block by internal NH4+, suggesting that alkalinization of the periaxonal space and a resultant decrease in the cationic STX concentration during NH4+ perfusion may play only a minor role in the effect. These observations indicate that internal monovalent cations can exert trans-channel influences on external toxin binding sites on sodium channels.

In genetic cardiomyopathies, a frequently described phenomenon is how similar mutations in one protein can lead to discrete clinical phenotypes. One example is illustrated by two mutations in beta myosin heavy chain ({beta}-MHC) that are linked to hypertrophic cardiomyopathy (HCM) (Ile467Val, I467V) and left ventricular non-compaction (LVNC) (Ile467Thr, I467T). To investigate how these missense mutations lead to independent diseases, we studied the molecular effects of each mutation using recombinant human {beta}-MHC Subfragment 1 (S1) in vitro assays. Both HCM-I467V and LVNC-I467T S1 mutations exhibited similar mechanochemical functions, including unchanged ATPase and enhanced actin velocity but had distinct effects on the basal activity of myosin. HCM-I467V S1 showed no change in basal ATPase activity of myosin while LVNC-I467T reduced the basal ATPase activity by 50%. Molecular dynamics simulations reveal that I467T allosterically disrupts nucleotide binding of myosin, which may contribute to the uncoupled reduced basal activity and enhanced actin velocity observed in this mutation. These contrasting molecular effects may lead to contractile dysregulation that initiates LVNC-associated signaling pathways that progress the phenotype. Together, analysis of these mutations provides evidence that phenotypic complexity originates at the molecular level and is critical to understanding disease progression and developing therapies.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in {beta}-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus- pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human {beta}, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins, with the most dramatic in perinatal, but minimal effects in {beta} myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing ATPase cycle rate. In contrast, the only measured effect of R671C in {beta} myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not {beta}, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents the first direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are yet another testament to myosins highly allosteric nature.

In muscle, titin proteins connect myofilaments together and are thought to be critical for contraction, especially during residual force enhancement (RFE) when force is elevated after an active stretch. We investigated titins function during contraction using small-angle X-ray diffraction to track structural changes before and after 50% titin cleavage and in the RFE-deficient, mdm titin mutant. We report that the RFE state is structurally distinct from pure isometric contractions, with increased thick filament strain and decreased lattice spacing, most likely caused by elevated titin-based forces. Furthermore, no RFE structural state was detected in mdm muscle. We posit that decreased lattice spacing, increased thick filament stiffness, and increased non-crossbridge forces are the major contributors to RFE. We conclude that titin directly contributes to RFE. One-Sentence SummaryTitin contributes to active force production and residual force enhancement in skeletal muscles.