Methanol gel electrophoresis: Separation of human fast and slow myosin light chain 1 and other myofibrillar protein isoforms on a single gel format.

This report describes a novel sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) resolving gel format that consistently yields the electrophoretic separation of the fast and slow isoforms of human sarcomeric myosin light chain 1 (MLC1). The inclusion of methanol as a constituent of the resolving gel impacted the electrophoretic mobility of proteins across a broad range of molecular masses. There was greater separation of the fast and slow isoforms of human MLC1, as well as separation and high resolution of fast and slow isoforms of the three myosin heavy chain isoforms that are expressed in human skeletal muscle on the same gel format. Furthermore, the same resolving gel format substantially altered the electrophoretic mobility of at least one isoform of tropomyosin in human striated muscle. It is possible that the inclusion of methanol in SDS-PAGE resolving gels could improve the separation of other proteins that are expressed in muscle and in other tissues and cell types.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscles and Structure-Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium-binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering the systolic calcium concentration, thereby strengthening the cardiac function. Here, we examined the effect of our previously identified calcium-sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR-derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This integrated structural-biochemical-physiological approach led to the identification of three novel low-affinity binders, which had similar binding affinities to the known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM.

Targeting Troponin C with Small Molecules Containing Diphenyl Moieties: Calcium Sensitivity Effects on Striated Muscle and Structure Activity Relationship.

Despite large investments from academia and industry, heart failure, which results from a disruption of the contractile apparatus, remains a leading cause of death. Cardiac muscle contraction is a calcium-dependent mechanism, which is regulated by the troponin protein complex (cTn) and specifically by the N-terminal domain of its calcium binding subunit (cNTnC). There is an increasing need for the development of small molecules that increase calcium sensitivity without altering systolic calcium concentration, thereby strengthening cardiac function. Here, we examined the effect of our previously identified calcium sensitizing small molecule, ChemBridge compound 7930079, in the context of several homologous muscle systems. The effect of this molecule on force generation in isolated cardiac trabeculae and slow skeletal muscle fibers was measured. Furthermore, we explored the use of Gaussian accelerated molecular dynamics in sampling highly predictive receptor conformations based on NMR derived starting structures. Additionally, we took a rational computational approach for lead optimization based on lipophilic diphenyl moieties. This led to the identification of three novel low affinity binders, which had similar binding affinities to known positive inotrope trifluoperazine. The most potent identified calcium sensitizer was compound 16 with an apparent affinity of 117 ± 17 µM .

Small Molecule RPI-194 Stabilizes Activated Troponin to Increase the Calcium Sensitivity of Striated Muscle Contraction.

Small molecule cardiac troponin activators could potentially enhance cardiac muscle contraction in the treatment of systolic heart failure. We designed a small molecule, RPI-194, to bind cardiac/slow skeletal muscle troponin (Cardiac muscle and slow skeletal muscle share a common isoform of the troponin C subunit.) Using solution NMR and stopped flow fluorescence spectroscopy, we determined that RPI-194 binds to cardiac troponin with a dissociation constant KD of 6-24 µM, stabilizing the activated complex between troponin C and the switch region of troponin I. The interaction between RPI-194 and troponin C is weak (KD 311 µM) in the absence of the switch region. RPI-194 acts as a calcium sensitizer, shifting the pCa50 of isometric contraction from 6.28 to 6.99 in mouse slow skeletal muscle fibers and from 5.68 to 5.96 in skinned cardiac trabeculae at 100 µM concentration. There is also some cross-reactivity with fast skeletal muscle fibers (pCa50 increases from 6.27 to 6.52). In the slack test performed on the same skinned skeletal muscle fibers, RPI-194 slowed the velocity of unloaded shortening at saturating calcium concentrations, suggesting that it slows the rate of actin-myosin cross-bridge cycling under these conditions. However, RPI-194 had no effect on the ATPase activity of purified actin-myosin. In isolated unloaded mouse cardiomyocytes, RPI-194 markedly decreased the velocity and amplitude of contractions. In contrast, cardiac function was preserved in mouse isolated perfused working hearts. In summary, the novel troponin activator RPI-194 acts as a calcium sensitizer in all striated muscle types. Surprisingly, it also slows the velocity of unloaded contraction, but the cause and significance of this is uncertain at this time. RPI-194 represents a new class of non-specific troponin activator that could potentially be used either to enhance cardiac muscle contractility in the setting of systolic heart failure or to enhance skeletal muscle contraction in neuromuscular disorders.

3-Chlorodiphenylamine activates cardiac troponin by a mechanism distinct from bepridil or TFP.

Despite extensive efforts spanning multiple decades, the development of highly effective Ca2+ sensitizers for the heart remains an elusive goal. Existing Ca2+ sensitizers have other targets in addition to cardiac troponin (cTn), which can lead to adverse side effects, such as hypotension or arrhythmias. Thus, there is a need to design Ca2+-sensitizing drugs with higher affinity and selectivity for cTn. Previously, we determined that many compounds based on diphenylamine (DPA) were able to bind to a cTnC-cTnI chimera with moderate affinity (Kd ∼10-120 µM). Of these compounds, 3-chlorodiphenylamine (3-Cl-DPA) bound most tightly (Kd of 10 µM). Here, we investigate 3-Cl-DPA further and find that it increases the Ca2+ sensitivity of force development in skinned cardiac muscle. Using NMR, we show that, like the known Ca2+ sensitizers, trifluoperazine (TFP) and bepridil, 3-Cl-DPA is able to bind to the isolated N-terminal domain (N-domain) of cTnC (Kd of 6 µM). However, while the bulky molecules of TFP and bepridil stabilize the open state of the N-domain of cTnC, the small and flexible 3-Cl-DPA molecule is able to bind without stabilizing this open state. Thus, unlike TFP, which drastically slows the rate of Ca2+ dissociation from the N-domain of isolated cTnC in a dose-dependent manner, 3-Cl-DPA has no effect on the rate of Ca2+ dissociation. On the other hand, the affinity of 3-Cl-DPA for a cTnC-TnI chimera is at least an order of magnitude higher than that of TFP or bepridil, likely because 3-Cl-DPA is less disruptive of cTnI binding to cTnC. Therefore, 3-Cl-DPA has a bigger effect on the rate of Ca2+ dissociation from the entire cTn complex than TFP and bepridil. Our data suggest that 3-Cl-DPA activates the cTn complex via a unique mechanism and could be a suitable scaffold for the development of novel treatments for systolic heart failure.

Desensitizing mouse cardiac troponin C to calcium converts slow muscle towards a fast muscle phenotype.

KEY POINTS: The Ca2+ -desensitizing D73N mutation in slow skeletal/cardiac troponin C caused dilatated cardiomyopathy in mice, but the consequences of this mutation in skeletal muscle were not known. The D73N mutation led to a rightward shift in the force versus pCa (-log [Ca]) relationship in slow-twitch mouse fibres. The D73N mutation led to a rightward shift in the force-stimulation frequency relationship and reduced fatigue resistance of mouse soleus muscle. The D73N mutation led to reduced cross-sectional area of slow-twitch fibres in mouse soleus muscle without affecting fibre type composition of the muscle. The D73N mutation resulted in significantly shorter times to peak force and to relaxation during isometric twitches and tetani in mouse soleus muscle. The D73N mutation led to major changes in physiological properties of mouse soleus muscle, converting slow muscle toward a fast muscle phenotype. ABSTRACT: The missense mutation, D73N, in mouse cardiac troponin C has a profound impact on cardiac function, mediated by a decreased myofilament Ca2+ sensitivity. Mammalian cardiac muscle and slow skeletal muscle normally share expression of the same troponin C isoform. Therefore, the objective of this study was to determine the consequences of the D73N mutation in skeletal muscle, as a potential mechanism that contributes to the morbidity associated with heart failure or other conditions in which Ca2+ sensitivity might be altered. Effects of the D73N mutation on physiological properties of mouse soleus muscle, in which slow-twitch fibres are prevalent, were examined. The mutation resulted in a rightward shift of the force-stimulation frequency relationship, and significantly faster kinetics of isometric twitches and tetani in isolated soleus muscle. Furthermore, soleus muscles from D73N mice underwent a significantly greater reduction in force during a fatigue test. The mutation significantly reduced slow fibre mean cross-sectional area without affecting soleus fibre type composition. The effects of the mutation on Ca2+ sensitivity of force development in soleus skinned slow and fast fibres were also examined. As expected, the D73N mutation did not affect the Ca2+ sensitivity of force development in fast fibres but resulted in substantially decreased Ca2+ sensitivity in slow fibres. The results demonstrate that a point mutation in a single constituent of myofilaments (slow/cardiac troponin C) led to major changes in physiological properties of skeletal muscle and converted slow muscle toward a fast muscle phenotype with reduced fatigue resistance and Ca2+ sensitivity of force generation.

Euflammation attenuates peripheral inflammation-induced neuroinflammation and mitigates immune-to-brain signaling.

Peripheral inflammation can trigger a number of neuroinflammatory events in the CNS, such as activation of microglia and increases of proinflammatory cytokines. We have previously identified an interesting phenomenon, termed "euflammation", which can be induced by repeated subthreshold infectious challenges. Euflammation causes innate immune alterations without overt neuroimmune activation. In the current study, we examined the protective effect of euflammation against peripheral inflammation-induced neuroinflammation and the underlying mechanisms. When Escherichia coli or lipopolysaccharide (LPS) was injected inside or outside the euflammation induction locus (EIL), sickness behavior, global microglial activation, proinflammatory cytokine production in the brain, expression of endothelial cyclooxygenase II and induction of c-fos expression in the paraventricular nucleus of the hypothalamus were all attenuated in the euflammatory mice compared with those in the control unprimed mice. Euflammation also modulated innate immunity outside the EIL by upregulating receptors for pathogen-associated molecular patterns in spleen cells. In addition, euflammation attenuated CNS activation in response to an intra-airpouch (outside the EIL) injection of LPS without suppressing the cytokine expression in the airpouch. Collectively, our study demonstrates that signaling of peripheral inflammation to the CNS is modulated dynamically by peripheral inflammatory kinetics. Specifically, euflammation can offer effective protection against both bacterial infection and endotoxin induced neuroinflammation.

Interleukin 1 type 1 receptor restore: a genetic mouse model for studying interleukin 1 receptor-mediated effects in specific cell types.

Interleukin-1 (IL-1) mediates diverse neurophysiological and neuropathological effects in the CNS through type I IL-1 receptor (IL-1R1). However, identification of IL-1R1-expressing cell types and cell-type-specific functions of IL-1R1 remains challenging. In this study, we created a novel genetic mouse model in which IL-1R1 gene expression is disrupted by an intronic insertion of a loxP flanked disruptive sequence that can be deleted by Cre recombinase, resulting in restored IL-1R1 gene expression under its endogenous promoters. A second mutation was introduced at stop codon of the IL-1R1 gene to allow tracking of the restored IL-1R1 protein by a 3HA tag and IL-1R1 mRNA by tdTomato fluorescence. These animals were designated as IL-1R1(r/r) and exhibited an IL-1R1 knock-out phenotype. We used IL-1R1 globally restored mice (IL-1R1(GR/GR)) as an IL-1R1 reporter and observed concordant labeling of IL-1R1 mRNA and protein in brain endothelial cells. Two cell-type-specific IL-1R1 restore lines were generated: Tie2Cre-IL-1R1(r/r) and LysMCre-IL-1R1(r/r). Brain endothelial COX-2 expression, CNS leukocyte infiltration, and global microglia activation induced by intracerebroventricular injection of IL-1ß were not observed in IL-1R1(r/r) or LysMCre-IL-1R1(r/r) mice, but were restored in Tie2Cre-IL-1R1(r/r) mice. These results reveal IL-1R1 expression in endothelial cells alone is sufficient to mediate these central IL-1-induced responses. In addition, ex vivo IL-1ß stimulation increased IL-1ß expression in bone marrow cells in wild-type, Tie2Cre-IL-1R1(r/r), and LysMCre-IL-1R1(r/r), but not IL-1R1(r/r) mice. These results demonstrate this IL-1R1 restore model is a valuable tool for studying cell-type-specific functions of IL-1R1.

Prostacyclin mediates endothelial COX-2-dependent neuroprotective effects during excitotoxic brain injury.

In a previous study, we found that intracerebral administration of excitotoxin (RS)-(tetrazole-5yl) glycine caused increased neural damage in the brain in an endothelial COX-2 deleted mouse line (Tie2Cre COX-2(flox/flox) ). In this study, we investigated whether prostacyclin might mediate this endothelial COX-2-dependent neuroprotection. Administration of excitotoxin into the striatum induced the production of prostacyclin (PGI2) in wild type, but not in endothelial COX-2 deleted mice. Inhibition of PGI2 synthase exacerbated brain lesions induced by the excitotoxin in wild type, but not in endothelial COX-2 deleted mice. Administration of a PGI2 agonist reduced neural damage in both wild type and endothelial COX-2 deleted mice. Increased PGI2 synthase expression was found in infiltrating neutrophils. In an ex vivo assay, PGI2 reduced the excitotoxin-induced calcium influx into neurons, suggesting a cellular mechanism for PGI2 mediated neuroprotection. These results reveal that PGI2 mediates endothelial COX-2 dependent neuroprotection.

Neuronal and nonneuronal COX-2 expression confers neurotoxic and neuroprotective phenotypes in response to excitotoxin challenge.

Treating acute brain injuries with COX-2 inhibitors can produce both neuroprotective and neurotoxic effects. This study investigated the role of COX-2 in modulating acute brain injury induced by excitotoxic neural damage. Intrastriatal injection of excitotoxin (RS)-(tetrazole-5yl) glycine elicited COX-2 expression in two distinct groups of cells. cortical neurons surrounding the lesion and vascular cells in the lesion core. The vascular COX-2 was expressed in two cell types, endothelial cells and monocytes. Selective deletion of COX-2 in vascular cells in Tie2Cre Cox-2(flox/flox) mice did not affect the induction of COX-2 in neurons after the excitotoxin injection but resulted in increased lesion volume, indicating a neuroprotective role for the COX-2 expressed in the vascular cells. Selective deletion of monocyte COX-2 in LysMCre Cox-2(flox/flox) mice did not reduce COX-2-dependent neuroprotection, suggesting that endothelial COX-2 is sufficient to confer neuroprotection. Pharmacological inhibition of COX-2 activity in Tie2Cre Cox-2(flox/flox) mice reduced lesion volume, indicating a neurotoxic role for the COX-2 expressed in neurons. Furthermore, COX-2-dependent neurotoxicity was mediated, at least in part, via the activation of the EP1 receptor. These results show that Cox-2 expression induced in different cell types can confer opposite effects.

Interleukin-1R3 mediates interleukin-1-induced potassium current increase through fast activation of Akt kinase.

Inflammatory cytokine interleukin-1 (IL-1) performs multiple functions in the central nervous system. The type 1 IL-1 receptor (IL-1R1) and the IL-1 receptor accessory protein (IL-1RAcP) form a functional IL-1 receptor complex that is thought to mediate most, if not all, IL-1-induced effects. Several recent studies, however, suggest the existence of a heretofore-unidentified receptor for IL-1. In this study, we report that the IL-1R1 gene contains an internal promoter that drives the transcription of a shortened IL-1R1 mRNA. This mRNA is the template for a unique IL-1R protein that is identical to IL-1R1 at the C terminus, but with a shorter extracellular domain at the N terminus. We have termed this molecule IL-1R3. The mRNA and protein for IL-1R3 are expressed in normal and two strains of commercially available IL-1R1 knockout mice. Western blot analysis shows IL-1R3 is preferentially expressed in neural tissues. Furthermore, IL-1ß binds specifically to IL-1R3 when it is complexed with the newly discovered alternative IL-1 receptor accessory protein, IL-1RAcPb. Stimulation of neurons expressing both IL-1R3 and IL-1RAcPb with IL-1ß causes fast activation of the Akt kinase, which leads to an increase in voltage-gated potassium current. These results demonstrate that IL-1R3/IL-1RAcPb complex mediates a unique subset of IL-1 activity that accounts for many previously unexplained IL-1 effects in the central nervous system.

Endothelial IL-1R1 is a critical mediator of EAE pathogenesis.

Interleukin-1 (IL-1) has been implicated in the disease progression of multiple sclerosis (MS). In the animal model of MS, experimental autoimmune encephalomyelitis (EAE), the induction of disease is significantly attenuated in mice lacking the type I IL-1 receptor (IL-1R1). In this study, we created a transgenic mouse (eIL-1R1 kd) in which IL-1R1 expression is knocked down specifically in endothelial cells. Induction of EAE in eIL-1R1 kd mice results in a decrease in incidence, severity and delayed onset of EAE. In addition, eIL-1R1 kd mice show significant decrease in VCAM-1 expression and diminished CD45(+) and CD3(+) infiltrating leukocytes in the spinal cord in animals challenged with EAE. Further, IL-1 and IL-23 stimulate IL-17 production by splenocytes from both wild type and the eIL-1R1 kd animals. Similarly, IL-1 and IL-23 synergistically stimulate splenocytes proliferation in these two strains of animals. After immunization with MOG(79-96), although eIL-1R1 kd mice displayed greatly reduced clinical scores, their splenocytes produced IL-17 and proliferated in response to a second MOG challenge, similar to wild type animals. These findings indicate a critical role for endothelial IL-1R1 in mediating the pathogenesis of EAE, and describe a new model that can be used to study endothelial IL-1R1.

Location-specific activation of the paraventricular nucleus of the hypothalamus by localized inflammation.

The existence of an immunological homunculus has been proposed, but evidence for location-specific response of the central nervous system to immunological stimulation is lacking. In this study, we show that inflammation induced by injection of casein into one of the causes c-fos expression in the paraventricular nucleus of the hypothalamus (PVN) in an asymmetrical manner: much stronger activation is always induced in the contralateral PVN. Unilateral sciatic nerve transection abolished the casein-induced PVN activation if casein was injected into the hindlimb with the nerve transection, but had no effect if casein was injected into the hindlimb with intact nerve innervation. Injection of casein into one the forelimbs also caused contralateral PNV activation. Further, stronger PVN activation was found in the anterior PVN after the forelimb injection, but in the posterior PVN after the hindlimb injection. Casein-induced PVN activation is absent in IL-1R1 KO, IL-6 KO, TNFα KO, and in C3H/HeJ (TLR4 mutant) animals. In comparison, injection of LPS, a systemic inflammagen, into one hindlimb induced bilateral PVN activation but injection of live Escherichia coli into one hindlimb induced contralateral PVN activation. These results support the notion that local inflammation may activate the PVN by neural routes in a location-specific manner.

Endothelial-specific knockdown of interleukin-1 (IL-1) type 1 receptor differentially alters CNS responses to IL-1 depending on its route of administration.

Interleukin-1 (IL-1) has been implicated as a critical mediator of neuroimmune communication. In the brain, the functional receptor for IL-1, type 1 IL-1 receptor (IL-1R1), is localized primarily to the endothelial cells. In this study, we created an endothelial-specific IL-1R1 knockdown model to test the role of endothelial IL-1R1 in mediating the effects of IL-1. Neuronal activation in the hypothalamus was measured by c-fos expression in the paraventricular nucleus and the ventromedial preoptic area. In addition, two specific sickness symptoms, febrile response and reduction of locomotor activity, were studied. Intracerebroventricular injection of IL-1 induced leukocyte infiltration into the CNS, activation of hypothalamic neurons, fever, and reduced locomotor activity in normal mice. Endothelial-specific knockdown of IL-1R1 abrogated all these responses. Intraperitoneal injection of IL-1 also induced neuronal activation in the hypothalamus, fever, and reduced locomotor activity, without inducing leukocyte infiltration into the brain. Endothelial-specific knockdown of IL-1R1 suppressed intraperitoneal IL-1-induced fever, but not the induction of c-fos in hypothalamus. When IL-1 was given intravenously, endothelial knockdown of IL-1R1 abolished intravenous IL-1-induced CNS activation and the two monitored sickness symptoms. In addition, endothelial-specific knockdown of IL-1R1 blocked the induction of cyclooxygenase-2 expression induced by all three routes of IL-1 administration. These results show that the effects of intravenous and intracerebroventricular IL-1 are mediated by endothelial IL-1R1, whereas the effects of intraperitoneal IL-1 are partially dependent on endothelial IL-1R1.