Magnesium increases numbers of Foxp3+ Treg cells and reduces arthritis severity and joint damage in an IL-10-dependent manner mediated by the intestinal microbiome.

BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. METHODS: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). FINDINGS: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1ß, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. INTERPRETATION: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. FUNDING: None.

Adeno-Associated virus 8 delivers an immunomodulatory peptide to mouse liver more efficiently than to rat liver.

Targeting the Kv1.3 potassium channel has proven effective in reducing obesity and the severity of animal models of autoimmune disease. Stichodactyla toxin (ShK), isolated from the sea anemone Stichodactyla helianthus, is a potent blocker of Kv1.3. Several of its analogs are some of the most potent and selective blockers of this channel. However, like most biologics, ShK and its analogs require injections for their delivery, and repeated injections reduce patient compliance during the treatment of chronic diseases. We hypothesized that inducing the expression of an ShK analog by hepatocytes would remove the requirement for frequent injections and lead to a sustained level of Kv1.3 blocker in the circulation. To this goal, we tested the ability of Adeno-Associated Virus (AAV)8 vectors to target hepatocytes for expressing the ShK analog, ShK-235 (AAV-ShK-235) in rodents. We designed AAV8 vectors expressing the target transgene, ShK-235, or Enhanced Green fluorescent protein (EGFP). Transduction of mouse livers led to the production of sufficient levels of functional ShK-235 in the serum from AAV-ShK-235 single-injected mice to block Kv1.3 channels. However, AAV-ShK-235 therapy was not effective in reducing high-fat diet-induced obesity in mice. In addition, injection of even high doses of AAV8-ShK-235 to rats resulted in a very low liver transduction efficiency and failed to reduce inflammation in a well-established rat model of delayed-type hypersensitivity. In conclusion, the AAV8-based delivery of ShK-235 was highly effective in inducing the secretion of functional Kv1.3-blocking peptide in mouse, but not rat, hepatocytes yet did not reduce obesity in mice fed a high-fat diet.

A bioengineered probiotic for the oral delivery of a peptide Kv1.3 channel blocker to treat rheumatoid arthritis.

Engineered microbes for the delivery of biologics are a promising avenue for the treatment of various conditions such as chronic inflammatory disorders and metabolic disease. In this study, we developed a genetically engineered probiotic delivery system that delivers a peptide to the intestinal tract with high efficacy. We constructed an inducible system in the probiotic Lactobacillus reuteri to secrete the Kv1.3 potassium blocker ShK-235 (LrS235). We show that LrS235 culture supernatants block Kv1.3 currents and preferentially inhibit human T effector memory (TEM) lymphocyte proliferation in vitro. A single oral gavage of healthy rats with LrS235 resulted in sufficient functional ShK-235 in the circulation to reduce inflammation in a delayed-type hypersensitivity model of atopic dermatitis mediated by TEM cells. Furthermore, the daily oral gavage of LrS235 dramatically reduced clinical signs of disease and joint inflammation in rats with a model of rheumatoid arthritis without eliciting immunogenicity against ShK-235. This work demonstrates the efficacy of using the probiotic L. reuteri as a novel oral delivery platform for the peptide ShK-235 and provides an efficacious strategy to deliver other biologics with great translational potential.

Syndecan-2 regulates PAD2 to exert antifibrotic effects on RA-ILD fibroblasts.

Rheumatoid arthritis (RA)-associated interstitial lung disease (RA-ILD) is the most common pulmonary complication of RA, increasing morbidity and mortality. Anti-citrullinated protein antibodies have been associated with the development and progression of both RA and fibrotic lung disease; however, the role of protein citrullination in RA-ILD remains unclear. Here, we demonstrate that the expression of peptidylarginine deiminase 2 (PAD2), an enzyme that catalyzes protein citrullination, is increased in lung homogenates from subjects with RA-ILD and their lung fibroblasts. Chemical inhibition or genetic knockdown of PAD2 in RA-ILD fibroblasts attenuated their activation, marked by decreased myofibroblast differentiation, gel contraction, and extracellular matrix gene expression. Treatment of RA-ILD fibroblasts with the proteoglycan syndecan-2 (SDC2) yielded similar antifibrotic effects through regulation of PAD2 expression, phosphoinositide 3-kinase/Akt signaling, and Sp1 activation in a CD148-dependent manner. Furthermore, SDC2-transgenic mice exposed to bleomycin-induced lung injury in an inflammatory arthritis model expressed lower levels of PAD2 and were protected from the development of pulmonary fibrosis. Together, our results support a SDC2-sensitive profibrotic role for PAD2 in RA-ILD fibroblasts and identify PAD2 as a promising therapeutic target of RA-ILD.

Kv1.3 Channel Up-Regulation in Peripheral Blood T Lymphocytes of Patients With Multiple Sclerosis.

Voltage-gated Kv1.3 potassium channels are key regulators of T lymphocyte activation, proliferation and cytokine production, by providing the necessary membrane hyper-polarization for calcium influx following immune stimulation. It is noteworthy that an accumulating body of in vivo and in vitro evidence links these channels to multiple sclerosis pathophysiology. Here we studied the electrophysiological properties and the transcriptional and translational expression of T lymphocyte Kv1.3 channels in multiple sclerosis, by combining patch clamp recordings, reverse transcription polymerase chain reaction and flow cytometry on freshly isolated peripheral blood T lymphocytes from two patient cohorts with multiple sclerosis, as well as from healthy and disease controls. Our data demonstrate that T lymphocytes in MS, manifest a significant up-regulation of Kv1.3 mRNA, Kv1.3 membrane protein and Kv1.3 current density and therefore of functional membrane channel protein, compared to control groups (p < 0.001). Interestingly, patient sub-grouping shows that Kv1.3 channel density is significantly higher in secondary progressive, compared to relapsing-remitting multiple sclerosis (p < 0.001). Taking into account the tight connection between Kv1.3 channel activity and calcium-dependent processes, our data predict and could partly explain the reported alterations of T lymphocyte function in multiple sclerosis, while they highlight Kv1.3 channels as potential therapeutic targets and peripheral biomarkers for the disease.

Antioxidant Carbon Nanoparticles Inhibit Fibroblast-Like Synoviocyte Invasiveness and Reduce Disease Severity in a Rat Model of Rheumatoid Arthritis.

Reactive oxygen species have been involved in the pathogenesis of rheumatoid arthritis (RA). Our goal was to determine the effects of selectively scavenging superoxide (O2•-) and hydroxyl radicals with antioxidant nanoparticles, called poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), on the pathogenic functions of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and on the progression of an animal model of RA. We used human FLS from patients with RA to determine PEG-HCC internalization and effects on FLS cytotoxicity, invasiveness, proliferation, and production of proteases. We used the pristane-induced arthritis (PIA) rat model of RA to assess the benefits of PEG-HCCs on reducing disease severity. PEG-HCCs were internalized by RA-FLS, reduced their intracellular O2•-, and reduced multiple measures of their pathogenicity in vitro, including proliferation and invasion. In PIA, PEG-HCCs caused a 65% reduction in disease severity, as measured by a standardized scoring system of paw inflammation and caused a significant reduction in bone and tissue damage, and circulating rheumatoid factor. PEG-HCCs did not induce lymphopenia during PIA. Our study demonstrated a role for O2•- and hydroxyl radicals in the pathogenesis of a rat model of RA and showed efficacy of PEG-HCCs in treating a rat model of RA.

Modulation of Lymphocyte Potassium Channel KV1.3 by Membrane-Penetrating, Joint-Targeting Immunomodulatory Plant Defensin.

We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.

AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9.

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo. However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8+ T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.

Inhibition of Upf2-Dependent Nonsense-Mediated Decay Leads to Behavioral and Neurophysiological Abnormalities by Activating the Immune Response.

In humans, disruption of nonsense-mediated decay (NMD) has been associated with neurodevelopmental disorders (NDDs) such as autism spectrum disorder and intellectual disability. However, the mechanism by which deficient NMD leads to neurodevelopmental dysfunction remains unknown, preventing development of targeted therapies. Here we identified novel protein-coding UPF2 (UP-Frameshift 2) variants in humans with NDD, including speech and language deficits. In parallel, we found that mice lacking Upf2 in the forebrain (Upf2 fb-KO mice) show impaired NMD, memory deficits, abnormal long-term potentiation (LTP), and social and communication deficits. Surprisingly, Upf2 fb-KO mice exhibit elevated expression of immune genes and brain inflammation. More importantly, treatment with two FDA-approved anti-inflammatory drugs reduced brain inflammation, restored LTP and long-term memory, and reversed social and communication deficits. Collectively, our findings indicate that impaired UPF2-dependent NMD leads to neurodevelopmental dysfunction and suggest that anti-inflammatory agents may prove effective for treatment of disorders with impaired NMD.

KCa1.1 and Kv1.3 channels regulate the interactions between fibroblast-like synoviocytes and T lymphocytes during rheumatoid arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLS) and CCR7- effector memory T (TEM) cells are two of the major cell types implicated in the progression of rheumatoid arthritis (RA). In particular, FLS become highly invasive, whereas TEM cells proliferate and secrete proinflammatory cytokines, during RA. FLS and T cells may also interact and influence each other's phenotypes. Inhibition of the pathogenic phenotypes of both FLS and TEM cells can be accomplished by selectively blocking the predominant potassium channels that they upregulate during RA: KCa1.1 (BK, Slo1, MaxiK, KCNMA1) upregulated by FLS and Kv1.3 (KCNA3) upregulated by activated TEM cells. In this study, we investigated the roles of KCa1.1 and Kv1.3 in regulating the interactions between FLS and TEM cells and determined if combination therapies of KCa1.1- and Kv1.3-selective blockers are more efficacious than monotherapies in ameliorating disease in rat models of RA. METHODS: We used in vitro functional assays to assess the effects of selective KCa1.1 and Kv1.3 channel inhibitors on the interactions of FLS isolated from rats with collagen-induced arthritis (CIA) with syngeneic TEM cells. We also used flow cytometric analyses to determine the effects of KCa1.1 blockers on the expression of proteins used for antigen presentation on CIA-FLS. Finally, we used the CIA and pristane-induced arthritis models to determine the efficacy of combinatorial therapies of KCa1.1 and Kv1.3 blockers in reducing disease severity compared with monotherapies. RESULTS: We show that the interactions of FLS from rats with CIA and of rat TEM cells are regulated by KCa1.1 and Kv1.3. Inhibiting KCa1.1 on FLS reduces the ability of FLS to stimulate TEM cell proliferation and migration, and inhibiting Kv1.3 on TEM cells reduces TEM cells' ability to enhance FLS expression of KCa1.1 and major histocompatibility complex class II protein, as well as stimulates their invasion. Furthermore, we show that combination therapies of selective KCa1.1 and Kv1.3 blockers are more efficacious than monotherapies at reducing signs of disease in two rat models of RA. CONCLUSIONS: Our results demonstrate the importance of KCa1.1 and Kv1.3 in regulating FLS and TEM cells during RA, as well as the value of combined therapies targeting both of these cell types to treat RA.

Enhanced Cardiomyocyte NLRP3 Inflammasome Signaling Promotes Atrial Fibrillation.

BACKGROUND: Atrial fibrillation (AF) is frequently associated with enhanced inflammatory response. The NLRP3 (NACHT, LRR, and PYD domain containing protein 3) inflammasome mediates caspase-1 activation and interleukin-1ß release in immune cells but is not known to play a role in cardiomyocytes (CMs). Here, we assessed the role of CM NLRP3 inflammasome in AF. METHODS: NLRP3 inflammasome activation was assessed by immunoblot in atrial whole-tissue lysates and CMs from patients with paroxysmal AF or long-standing persistent (chronic) AF. To determine whether CM-specific activation of NLPR3 is sufficient to promote AF, a CM-specific knockin mouse model expressing constitutively active NLRP3 (CM-KI) was established. In vivo electrophysiology was used to assess atrial arrhythmia vulnerability. To evaluate the mechanism of AF, electric activation pattern, Ca2+ spark frequency, atrial effective refractory period, and morphology of atria were evaluated in CM-KI mice and wild-type littermates. RESULTS: NLRP3 inflammasome activity was increased in the atrial CMs of patients with paroxysmal AF and chronic AF. CM-KI mice developed spontaneous premature atrial contractions and inducible AF, which was attenuated by a specific NLRP3 inflammasome inhibitor, MCC950. CM-KI mice exhibited ectopic activity, abnormal sarcoplasmic reticulum Ca2+ release, atrial effective refractory period shortening, and atrial hypertrophy. Adeno-associated virus subtype-9-mediated CM-specific knockdown of Nlrp3 suppressed AF development in CM-KI mice. Finally, genetic inhibition of Nlrp3 prevented AF development in CREM transgenic mice, a well-characterized mouse model of spontaneous AF. CONCLUSIONS: Our study establishes a novel pathophysiological role for CM NLRP3 inflammasome signaling, with a mechanistic link to the pathogenesis of AF, and establishes the inhibition of NLRP3 as a potential novel AF therapy approach.

Targeting KCa1.1 Channels with a Scorpion Venom Peptide for the Therapy of Rat Models of Rheumatoid Arthritis.

Fibroblast-like synoviocytes (FLSs) are a key cell type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLSs and found that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects. In this study, we aimed to determine whether the KCa1.1 ß1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch-clamp and functional assays were used to determine whether IbTX can regulate FLSs through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined whether IbTX causes side effects including incontinence or tremors in rats, compared with those treated with the small-molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 were expressed by FLSs in joints of rats with experimental arthritis. IbTX inhibited KCa1.1 channels expressed by FLSs from patients with RA and by FLSs from rat models of RA and reduced FLS invasiveness. IbTX significantly reduced disease severity in two rat models of RA. Unlike paxilline, IbTX did not induce tremors or incontinence in rats. Overall, IbTX inhibited KCa1.1 channels on FLSs and treated rat models of RA without inducing side effects associated with nonspecific KCa1.1 blockade and could become the basis for the development of a new treatment of RA.

Differences in ion channel phenotype and function between humans and animal models.

Ion channels play crucial roles in regulating a broad range of physiological processes. They form a very large family of transmembrane proteins. Their diversity results from not only a large number of different genes encoding for ion channel subunits but also the ability of subunits to assemble into homo- or heteromultimers, the existence of splice variants, and the expression of different regulatory subunits. These characteristics and the existence of very selective modulators make ion channels very attractive targets for therapy in a wide variety of pathologies. Some ion channels are already being targeted in the clinic while many more are being evaluated as novel drug targets in both clinical and preclinical studies. Advancing ion channel modulators from the bench to the clinic requires their assessment for safety and efficacy in animal models. While extrapolating results from one species to another is tempting, doing such without careful evaluation of the ion channels in different species presents a risk as the translation is not always straightforward. Here, we discuss differences between species in terms of ion channels expressed in selected tissues, differing roles of ion channels in some cell types, variable response to pharmacological agents, and human channelopathies that cannot fully be replicated in animal models.

Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

KCa1.1 channels regulate ß1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.

Large-conductance calcium-activated potassium channel (KCa1.1; BK, Slo1, MaxiK, KCNMA1) is the predominant potassium channel expressed at the plasma membrane of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) isolated from the synovium of patients with RA. It is a critical regulator of RA-FLS migration and invasion and therefore represents an attractive target for the therapy of RA. However, the molecular mechanisms by which KCa1.1 regulates RA-FLS invasiveness have remained largely unknown. Here, we demonstrate that KCa1.1 regulates RA-FLS adhesion through controlling the plasma membrane expression and activation of ß1 integrins, but not α4, α5, or α6 integrins. Blocking KCa1.1 disturbs calcium homeostasis, leading to the sustained phosphorylation of Akt and the recruitment of talin to ß1 integrins. Interestingly, the pore-forming α subunit of KCa1.1 coimmunoprecipitates with ß1 integrins, suggesting that this physical association underlies the functional interaction between these molecules. Together, these data outline a new signaling mechanism by which KCa1.1 regulates ß1-integrin function and therefore invasiveness of RA-FLSs.-Tanner, M. R., Pennington, M. W., Laragione, T., Gulko, P. S., Beeton, C. KCa1.1 channels regulate ß1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog.

Effector memory T lymphocytes (TEM cells) that lack expression of CCR7 are major drivers of inflammation in a number of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. The Kv1.3 potassium channel is a key regulator of CCR7- TEM cell activation. Blocking Kv1.3 inhibits TEM cell activation and attenuates inflammation in autoimmunity, and as such, Kv1.3 has emerged as a promising target for the treatment of TEM cell-mediated autoimmune diseases. The scorpion venom-derived peptide HsTX1 and its analog HsTX1[R14A] are potent Kv1.3 blockers and HsTX1[R14A] is selective for Kv1.3 over closely-related Kv1 channels. PEGylation of HsTX1[R14A] to create a Kv1.3 blocker with a long circulating half-life reduced its affinity but not its selectivity for Kv1.3, dramatically reduced its adsorption to inert surfaces, and enhanced its circulating half-life in rats. PEG-HsTX1[R14A] is equipotent to HsTX1[R14A] in preferential inhibition of human and rat CCR7- TEM cell proliferation, leaving CCR7+ naïve and central memory T cells able to proliferate. It reduced inflammation in an active delayed-type hypersensitivity model and in the pristane-induced arthritis (PIA) model of rheumatoid arthritis (RA). Importantly, a single subcutaneous dose of PEG-HsTX1[R14A] reduced inflammation in PIA for a longer period of time than the non-PEGylated HsTX1[R14A]. Together, these data indicate that HsTX1[R14A] and PEG-HsTX1[R14A] are effective in a model of RA and are therefore potential therapeutics for TEM cell-mediated autoimmune diseases. PEG-HsTX1[R14A] has the additional advantages of reduced non-specific adsorption to inert surfaces and enhanced circulating half-life.

Detection of Matrix Metalloproteinases by Zymography.

Matrix metalloproteinases (MMPs) represent more than 20 zinc-containing endopeptidases that cleave internal peptide bonds, leading to protein degradation. They play a critical role in many physiological cell functions, including tissue remodeling, embryogenesis, and angiogenesis. They are also involved in the pathogenesis of a vast array of diseases, including but not limited to systemic inflammation, various cancers, and cardiovascular, neurological, and autoimmune diseases. Here, we describe gel zymography to detect MMPs in cell and tissue samples and in cell culture supernatants.

Changes in Gene Expression and Metabolism in the Testes of the Rat following Spinal Cord Injury.

Spinal cord injury (SCI) results in devastating changes to almost all aspects of a patient's life. In addition to a permanent loss of sensory and motor function, males also will frequently exhibit a profound loss of fertility through poorly understood mechanisms. We demonstrate that SCI causes measureable pathology in the testis both acutely (24 h) and chronically up to 1.5 years post-injury, leading to loss in sperm motility and viability. SCI has been shown in humans and rats to induce leukocytospermia, with the presence of inflammatory cytokines, anti-sperm antibodies, and reactive oxygen species found within the ejaculate. Using messenger RNA and metabolomic assessments, we describe molecular and cellular changes that occur within the testis of adult rats over an acute to chronic time period. From 24 h, 72 h, 28 days, and 90 days post-SCI, the testis reveal a distinct time course of pathological events. The testis show an acute drop in normal sexual organ processes, including testosterone production, and establishment of a pro-inflammatory environment. This is followed by a subacute initiation of an innate immune response and loss of cell cycle regulation, possibly due to apoptosis within the seminiferous tubules. At 1.5 years post-SCI, there is a chronic low level immune response as evidenced by an elevation in T cells. These data suggest that SCI elicits a wide range of pathological processes within the testes, the actions of which are not restricted to the acute phase of injury but rather extend chronically, potentially through the lifetime of the subject. The multiplicity of these pathological events suggest a single therapeutic intervention is unlikely to be successful.

Functional KCa1.1 channels are crucial for regulating the proliferation, migration and differentiation of human primary skeletal myoblasts.

Myoblasts are mononucleated precursors of myofibers; they persist in mature skeletal muscles for growth and regeneration post injury. During myotonic dystrophy type 1 (DM1), a complex autosomal-dominant neuromuscular disease, the differentiation of skeletal myoblasts into functional myotubes is impaired, resulting in muscle wasting and weakness. The mechanisms leading to this altered differentiation are not fully understood. Here, we demonstrate that the calcium- and voltage-dependent potassium channel, KCa1.1 (BK, Slo1, KCNMA1), regulates myoblast proliferation, migration, and fusion. We also show a loss of plasma membrane expression of the pore-forming α subunit of KCa1.1 in DM1 myoblasts. Inhibiting the function of KCa1.1 in healthy myoblasts induced an increase in cytosolic calcium levels and altered nuclear factor kappa B (NFκB) levels without affecting cell survival. In these normal cells, KCa1.1 block resulted in enhanced proliferation and decreased matrix metalloproteinase secretion, migration, and myotube fusion, phenotypes all observed in DM1 myoblasts and associated with disease pathogenesis. In contrast, introducing functional KCa1.1 α-subunits into DM1 myoblasts normalized their proliferation and rescued expression of the late myogenic marker Mef2. Our results identify KCa1.1 channels as crucial regulators of skeletal myogenesis and suggest these channels as novel therapeutic targets in DM1.