Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.

Transcription factors are known to play multiple roles in cellular function. Investigators report that factors such as early growth response (Egr) protein and nuclear factor kappa B (NF-κB) are activated in the brain during cancer, brain injury, inflammation, and/or memory. To explore NF-κB activity further, we investigated the transcriptomes of hippocampal slices following electrical stimulation of NF-κB p50 subunit knockout mice (p50-/-) versus their controls (p50+/+). We found that the early growth response gene Egr-2 was upregulated by NF-κB activation, but only in p50+/+ hippocampal slices. We then stimulated HeLa cells and primary cortical neurons with tumor necrosis factor alpha (TNFα) to activate NF-κB and increase the expression of Egr-2. The Egr-2 promoter sequence was analyzed for NF-κB binding sites and chromatin immunoprecipitation (ChIP) assays were performed to confirm promoter occupancy in vivo. We discovered that NF-κB specifically binds to an NF-κB consensus binding site within the proximal promoter region of Egr-2. Luciferase assay demonstrated that p50 was able to transactivate the Egr-2 promoter in vitro. Small interfering RNA (siRNA)-mediated p50 knockdown corroborated other Egr-2 expression studies. We show for the first time a novel link between NF-κB activation and Egr-2 expression with Egr-2 expression directly controlled by the transcriptional activity of NF-κB.

Peripheral nerve regeneration following injury is often incomplete, resulting in significant personal and socioeconomic costs. Although a conditioning crush lesion prior to surgical nerve transection and repair greatly promotes nerve regeneration and functional recovery, feasibility and ethical considerations have hindered its clinical applicability. In a recent proof of principle study, we demonstrated that conditioning electrical stimulation (CES) had effects on early nerve regeneration, similar to that seen in conditioning crush lesions (CCL). To convincingly determine its clinical utility, establishing the effects of CES on target reinnervation and functional outcomes is of utmost importance. In this study, we found that CES improved nerve regeneration and reinnervation well beyond that of CCL. Specifically, compared to CCL, CES resulted in greater intraepidermal skin and NMJ reinnervation, and greater physiological and functional recovery including mechanosensation, compound muscle action potential on nerve conduction studies, normalization of gait pattern, and motor performance on the horizontal ladder test. These findings have direct clinical relevance as CES could be delivered at the bedside before scheduled nerve surgery.

Tetrahydrobiopterin (BH4) is an essential cofactor for the production of nitric oxide (NO) and supplementation with BH4 improves NO-dependent vasodilation. NO also reduces sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle. Thus, we hypothesized that supplementation with BH4 would blunt sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle. Sprague-Dawley rats (n = 15, 399 ± 57 g) were anesthetized and instrumented with an indwelling brachial artery catheter, femoral artery flow probe, and a stimulating electrode on the lumbar sympathetic chain. Triceps surae muscles were stimulated to contract rhythmically at 30% and 60% of maximal contractile force (MCF). The percentage change of femoral vascular conductance (%FVC) in response to sympathetic stimulations delivered at 2 and 5 Hz was determined at rest and during muscle contraction in control and acute BH4 supplementation (20 mg·kg(-1) + 10 mg·kg(-1)·h(-1), IA) conditions. BH4 reduced (P < 0.05) the vasoconstrictor response to sympathetic stimulation (i.e., decrease in FVC) at rest (Control: 2 Hz: -28 ± 5%FVC; 5 Hz: -45 ± 5%; BH4: 2 Hz: -17 ± 4%FVC; 5 Hz: -34 ± 7%FVC) and during muscular contraction at 30% MCF (Control: 2 Hz: -14 ± 6%FVC; 5 Hz: -28 ± 11%; BH4: 2 Hz: -6 ± 6%FVC; 5 Hz: -16 ± 10%) and 60% MCF (Control: 2 Hz: -7 ± 3%FVC; 5 Hz: -16 ± 6%FVC; BH4: 2 Hz: -2 ± 3%FVC; 5 Hz: -11 ± 6%FVC). These data are consistent with our hypothesis that acute BH4 supplementation decreases sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle.

Adenosine, through activation of its A(1) receptors, has neuroprotective effects during hypoxia and ischemia. Recently, using transgenic mice with neuronal expression of human equilibrative nucleoside transporter 1 (hENT1), we reported that nucleoside transporter-mediated release of adenosine from neurons was not a key mechanism facilitating the actions of adenosine at A(1) receptors during hypoxia/ischemia. The present study was performed to test the importance of CD73 (ecto-5'-nucleotidase) for basal and hypoxic/ischemic adenosine production. Hippocampal slice electrophysiology was performed with CD73(+/+) and CD73(-/-) mice. Adenosine and ATP had similar inhibitory effects in both genotypes, with IC(50) values of approximately 25 µM. In contrast, ATP was a less potent inhibitor (IC(50) = 100 µM) in slices from mice expressing hENT1 in neurons. The inhibitory effects of ATP in CD73(+/+) and CD73(-/-) slices were blocked by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and were enhanced by the nucleoside transport inhibitor S-(4-nitrobenzyl)-6-thioinosine (NBTI), consistent with effects that are mediated by adenosine after metabolism of ATP. AMP showed a similar inhibitory effect to ATP and adenosine, indicating that the response to ATP was not mediated by P2 receptors. In comparing CD73(-/-) and CD73(+/+) slices, hypoxia and oxygen-glucose deprivation produced similar depression of synaptic transmission in both genotypes. An inhibitor of tissue non-specific alkaline phosphatase (TNAP) was found to attenuate the inhibitory effects of AMP and ATP, increase basal synaptic activity and reduce responses to oxygen-glucose deprivation selectively in slices from CD73(-/-) mice. These results do not support an important role for CD73 in the formation of adenosine in the CA1 area of the hippocampus during basal, hypoxic or ischemic conditions, but instead point to TNAP as a potential source of extracellular adenosine when CD73 is absent.

Exercise training (ET) improves endurance capacity by increasing both skeletal muscle mitochondrial number and function, as well as contributing to favourable cardiac remodelling.Interestingly, some of the benefits of regular exercise can also be mimicked by the naturally occurring polyphenol, resveratrol (RESV). However, it is not known whether RESV enhances physiological adaptations to ET. To investigate this, male Wistar rats were randomly assigned to a control chow diet or a chow diet that contained RESV (4 g kg⁻¹ of diet) and subsequently subjected to a programme of progressive treadmill running for 12 weeks. ET-induced improvements in exercise performance were enhanced by 21% (P <0.001) by the addition of RESV to the diet. In soleus muscle, ET+RESV increased both the twitch (1.8-fold; P <0.05) and tetanic(1.2-fold; P <0.05) forces generated during isometric contraction, compared to ET alone. In vivo echocardiography demonstrated that ET+RESV also increased the resting left ventricular ejection fraction by 10% (P <0.05), and reduced left ventricular wall stress compared to ET alone.These functional changes were accompanied by increased cardiac fatty acid oxidation (1.2-fold;P <0.05) and favourable changes in cardiac gene expression and signal transduction pathways that optimized the utilization of fatty acids in ET+RESV compared to ET alone. Overall, our findings provide evidence that the capacity for fatty acid oxidation is augmented by the addition of RESV to the diet during ET, and that this may contribute to the improved physical performance of rats following ET.

Hypoxia exerts broad effects on cardiomyocyte function and viability, ranging from altered metabolism and mitochondrial physiology to apoptotic or necrotic cell death. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of cardiomyocyte metabolism and mitochondrial function and is down-regulated in hypoxia; however, the underlying mechanism is incompletely resolved. Using primary rat cardiomyocytes coupled with electrophoretic mobility shift and luciferase assays, we report that hypoxia impaired mitochondrial energetics and resulted in an increase in nuclear localization of the Nuclear Factor-κB (NF-κB) p65 subunit, and the association of p65 with the PGC-1α proximal promoter. Tumor necrosis factor α (TNFα), an activator of NF-κB signaling, similarly reduced PGC-1α expression and p65 binding to the PGC-1α promoter in a dose-dependent manner, and TNFα-mediated down-regulation of PGC-1α expression could be reversed by the NF-κB inhibitor parthenolide. RNA-seq analysis revealed that cardiomyocytes isolated from p65 knockout mice exhibited alterations in genes associated with chromatin remodeling. Decreased PGC-1α promoter transactivation by p65 could be partially reversed by the histone deacetylase inhibitor trichostatin A. These results implicate NF-κB signaling, and specifically p65, as a potent inhibitor of PGC-1α expression in cardiac myocyte hypoxia.

Experimental and clinical therapies in the field of Alzheimer's disease (AD) have focused on elimination of extracellular amyloid beta aggregates or prevention of cytoplasmic neuronal fibrillary tangles formation, yet these approaches have been generally ineffective. Interruption of nuclear lamina integrity, or laminopathy, is a newly identified concept in AD pathophysiology. Unraveling the molecular players in the induction of nuclear lamina damage may lead to identification of new therapies. Here, using 3xTg and APP/PS1 mouse models of AD, and in vitro model of amyloid beta42 (Aβ42) toxicity in primary neuronal cultures and SH-SY5Y neuroblastoma cells, we have uncovered a key role for cathepsin L in the induction of nuclear lamina damage. The applicability of our findings to AD pathophysiology was validated in brain autopsy samples from patients. We report that upregulation of cathepsin L is an important process in the induction of nuclear lamina damage, shown by lamin B1 cleavage, and is associated with epigenetic modifications in AD pathophysiology. More importantly, pharmacological targeting and genetic knock out of cathepsin L mitigated Aβ42 induced lamin B1 degradation and downstream structural and molecular changes. Affirming these findings, overexpression of cathepsin L alone was sufficient to induce lamin B1 cleavage. The proteolytic activity of cathepsin L on lamin B1 was confirmed using mass spectrometry. Our research identifies cathepsin L as a newly identified lamin B1 protease and mediator of laminopathy observed in AD. These results uncover a new aspect in the pathophysiology of AD that can be pharmacologically prevented, raising hope for potential therapeutic interventions.

Alzheimer's disease (AD) is the most common late onset neurodegenerative disorder with indications that women are disproportionately affected. Mitochondrial dysfunction has been one of the most discussed hypotheses associated with the early onset and progression of AD, and it has been attributed to intraneuronal accumulation of amyloid β (Aβ). It was suggested that one of the possible mediators for Aβ-impaired mitochondrial function is the nuclear factor kappa B (NF-κB) signaling pathway. NF-κB plays important roles in brain inflammation and antioxidant defense, as well as in the regulation of mitochondrial function, and studies have confirmed altered NF-κB signaling in AD brain. In this study, we looked for sex-based differences in impaired bioenergetic processes and NF-κB signaling in the AD-like brain using transgenic (Tg) CRND8 mice that express excessive brain Aβ, but without tau pathology. Our results show that mitochondrial dysfunction is not uniform in affected brain regions. We observed increased basal and coupled respiration in the hippocampus of TgCRND8 females only, along with a decreased Complex II-dependent respiratory activity. Cortical mitochondria from TgCRND8 mice have reduced uncoupled respiration capacity, regardless of sex. The pattern of changes in NF-κB signaling was the same in both brain structures, but was sex specific. Whereas in females there was an increase in all three subunits of NF-κB, in males we observed increase in p65 and p105, but no changes in p50 levels. These results demonstrate that mitochondrial function and inflammatory signaling in the AD-like brain is region- and sex-specific, which is an important consideration for therapeutic strategies.

Remodeling of the cardiac extracellular matrix is responsible for a number of the detrimental effects on heart function that arise secondary to hypertension, diabetes and myocardial infarction. This remodeling consists both of an increase in new matrix protein synthesis, and an increase in the expression of matrix metalloproteinases (MMPs) that degrade existing matrix structures. Previous studies utilizing knockout mice have demonstrated clearly that MMP2 plays a pathogenic role during matrix remodeling, thus it is important to understand the mechanisms that regulate MMP2 gene expression. We have shown that the transcription factor scleraxis is an important inducer of extracellular matrix gene expression in the heart that may also control MMP2 expression. In the present study, we demonstrate that scleraxis directly transactivates the proximal MMP2 gene promoter, resulting in increased histone acetylation, and identify a specific E-box sequence in the promoter to which scleraxis binds. Cardiac myo-fibroblasts isolated from scleraxis knockout mice exhibited dramatically decreased MMP2 expression; however, scleraxis over-expression in knockout cells could rescue this loss. We further show that regulation of MMP2 gene expression by the pro-fibrotic cytokine TGFβ occurs via a scleraxis-dependent mechanism: TGFβ induces recruitment of scleraxis to the MMP2 promoter, and TGFβ was unable to up-regulate MMP2 expression in cells lacking scleraxis due to either gene knockdown or knockout. These results reveal that scleraxis can exert control over both extracellular matrix synthesis and breakdown, and thus may contribute to matrix remodeling in wound healing and disease.

It is widely recognized that propagation of electrophysiological signals between the soma and dendrites of neurons differs depending on direction, i.e. it is asymmetric. How this asymmetry influences the activation of voltage-gated dendritic channels, and consequent neuronal behavior, remains unclear. Based on the analysis of asymmetry in several types of motoneurons, we extended our previous methodology for reducing a fully reconstructed motoneuron model to a two-compartment representation that preserved asymmetric signal propagation. The reduced models accurately replicated the dendritic excitability and the dynamics of the anatomical model involving a persistent inward current (PIC) dispersed over the dendrites. The relationship between asymmetric signal propagation and dendritic excitability was investigated using the reduced models while varying the asymmetry in signal propagation between the soma and the dendrite with PIC density constant. We found that increases in signal attenuation from soma to dendrites increased the activation threshold of a PIC (hypo-excitability), whereas increases in signal attenuation from dendrites to soma decreased the activation threshold of a PIC (hyper-excitability). These effects were so strong that reversing the asymmetry in the soma-to-dendrite vs. dendrite-to-soma attenuation, reversed the correlation between PIC threshold and distance of this current source from the soma. We propose the tight relation of the asymmetric signal propagation to the input resistance in the dendrites as a mechanism underlying the influence of the asymmetric signal propagation on the dendritic excitability. All these results emphasize the importance of maintaining the physiological asymmetry in dendritic signaling not only for normal function of the cells but also for biophysically realistic simulations of dendritic excitability.

Much is known about the electrophysiological variation in motoneuron somata across different motor units. However, comparatively less is known about electrophysiological variation in motor axons and how this could impact function or electrodiagnosis in healthy or diseased states. We performed nerve excitability testing on two groups of motor axons in Sprague-Dawley rats that are known to differ significantly in their chronic daily activity patterns and in the relative proportion of motor unit types: one group innervating the soleus ("slow motor axons") and the other group innervating the tibialis anterior ("fast motor axons") muscles. We found that slow motor axons have significantly larger accommodation compared to fast motor axons upon application of a 100 ms hyperpolarizing conditioning stimulus that is 40% of axon threshold (Z = 3.24, p = 0.001) or 20% of axon threshold (Z = 2.67, p = 0.008). Slow motor axons had larger accommodation to hyperpolarizing currents in the current-threshold measurement (-80% Z = 3.07, p = 0.002; -90% Z = 2.98, p = 0.003). In addition, we found that slow motor axons have a significantly smaller rheobase than fast motor axons (Z = -1.99, p = 0.047) accompanied by a lower threshold in stimulus-response curves. The results provide evidence that slow motor axons have greater activity of the hyperpolarization-activated inwardly rectifying cation conductance (IH) than fast motor axons. It is possible that this difference between fast and slow axons is caused by an adaptation to their chronic differences in daily activity patterns, and that this adaptation might have a functional effect on the motor unit. Moreover, these findings indicate that slow and fast motor axons may react differently to pathological conditions.

We previously introduced a machine learning-based Alzheimer's Disease Designation (MAD) framework for identifying AD-related metabolic patterns among neurodegenerative subjects.

The brain has a high demand for energy, of which creatine (Cr) is an important regulator. Studies document neurocognitive benefits of oral Cr in mammals, yet little is known regarding their physiological basis. This study investigated the effects of Cr supplementation (3%, w/w) on hippocampal function in male C57BL/6 mice, including spatial learning and memory in the Morris water maze and oxygen consumption rates from isolated mitochondria in real time. Levels of transcription factors and related proteins (CREB, Egr1, and IκB to indicate NF-κB activity), proteins implicated in cognition (CaMKII, PSD-95, and Egr2), and mitochondrial proteins (electron transport chain Complex I, mitochondrial fission protein Drp1) were probed with Western blotting. Dietary Cr decreased escape latency/time to locate the platform (P < 0.05) and increased the time spent in the target quadrant (P < 0.01) in the Morris water maze. This was accompanied by increased coupled respiration (P < 0.05) in isolated hippocampal mitochondria. Protein levels of CaMKII, PSD-95, and Complex 1 were increased in Cr-fed mice, whereas IκB was decreased. These data demonstrate that dietary supplementation with Cr can improve learning, memory, and mitochondrial function and have important implications for the treatment of diseases affecting memory and energy homeostasis.

The creatine (Cr) energy system has been implicated in Alzheimer's disease (AD), including reductions in brain phosphoCr and Cr kinase, yet no studies have examined the neurobehavioral effects of Cr supplementation in AD, including the 3xTg mouse model. This studied investigated the effects of Cr supplementation on spatial cognition, plasticity- and disease-related protein levels, and mitochondrial function in the 3xTg hippocampus. Here, 3xTg mice were fed a control or Cr-supplemented (3% Cr (w/w)) diet for 8-9 weeks and tested in the Morris water maze. Mitochondrial oxygen consumption (Seahorse) and protein levels (Western blots) were measured in the hippocampus in subsets of mice. Overall, 3xTg females exhibited impaired memory as compared to males. In females, Cr supplementation decreased escape latency and was associated with increased spatial search strategy use. In males, Cr supplementation decreased the use of spatial search strategies. Pilot data indicated mitochondrial enhancements with Cr supplementation in both sexes. In females, Cr supplementation increased CREB phosphorylation and levels of IκB (NF-κB suppressor), CaMKII, PSD-95, and high-molecular-weight amyloid β (Aβ) species, whereas Aβ trimers were reduced. These data suggest a beneficial preventative effect of Cr supplementation in females and warrant caution against Cr supplementation in males in the AD-like brain.

Alzheimer's disease (AD) is a major public health concern worldwide. Advanced age and female sex are two of the most prominent risk factors for AD. AD is characterized by progressive neuronal loss, especially in the cortex and hippocampus, and mitochondrial dysfunction has been proposed to be an early event in the onset and progression of the disease. Our results showed early perturbations in mitochondrial function in 3xTg mouse brain, with the cortex being more susceptible to mitochondrial changes than the hippocampus. In the cortex of 3xTg females, decreased coupled and uncoupled respiration were evident early (at 2 months of age), while in males it appeared later at 6 months of age. We observed increased coupled respiration in the hippocampus of 2-month-old 3xTg females, but no changes were detected later in life. Changes in mitochondrial dynamics were indicated by decreased mitofusin (Mfn2) and increased dynamin related protein 1 (Drp1) (only in females) in the hippocampus and cortex of 3xTg mice. Our findings highlight the importance of controlling and accounting for sex, brain region, and age in studies examining brain bioenergetics using this common AD model in order to more accurately evaluate potential therapies and improve the sex-specific translatability of preclinical findings.

Research has identified several transcription factors that regulate activity-dependent plasticity and memory, with cAMP-response element binding protein (CREB) being the most well-studied. In neurons, CREB activation is influenced by the transcription factor nuclear factor kappa B (NF-κB), considered central to immunity but more recently implicated in memory. The transcription factor early growth response-2 (Egr-2), an NF-κB gene target, is also associated with learning and memory. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an antioxidant transcription factor linked to NF-κB in pathological conditions, has not been studied in normal memory. Given that numerous transcription factors implicated in activity-dependent plasticity demonstrate connections to NF-κB, this study simultaneously evaluated protein levels of NF-κB, CREB, Egr-2, Nrf2, and actin in hippocampi from young (1 month-old) weanling CD1 mice after training in the Morris water maze, a hippocampal-dependent spatial memory task. After a 6-day acquisition period, time to locate the hidden platform decreased in the Morris water maze. Mice spent more time in the target vs. non-target quadrants of the maze, suggestive of recall of the platform location. Western blot data revealed a decrease in NF-κB p50 protein after training relative to controls, whereas NF-κB p65, Nrf2 and actin increased. Nrf2 levels were correlated with platform crosses in nearly all tested animals. These data demonstrate that training in a spatial memory task results in alterations in and associations with particular transcription factors in the hippocampus, including upregulation of NF-κB p65 and Nrf2. Training-induced increases in actin protein levels caution against its use as a loading control in immunoblot studies examining activity-dependent plasticity, learning, and memory.

Resident fibroblasts synthesize the cardiac extracellular matrix, and can undergo phenotype conversion to myofibroblasts to augment matrix production, impairing function and contributing to organ failure. A significant gap in our understanding of the transcriptional regulation of these processes exists. Given the key role of this phenotype conversion in fibrotic disease, the identification of such novel transcriptional regulators may yield new targets for therapies for fibrosis.

During fictive locomotion the excitability of adult cat lumbar motoneurones is increased by a reduction (a mean hyperpolarization of approximately 6.0 mV) of voltage threshold (Vth) for action potential (AP) initiation that is accompanied by only small changes in AP height and width. Further examination of the experimental data in the present study confirms that Vth lowering is present to a similar degree in both the hyperpolarized and depolarized portions of the locomotor step cycle. This indicates that Vth reduction is a modulation of motoneurone membrane currents throughout the locomotor state rather than being related to the phasic synaptic input within the locomotor cycle. Potential ionic mechanisms of this locomotor-state-dependent increase in excitability were examined using three five-compartment models of the motoneurone innervating slow, fast fatigue resistant and fast fatigable muscle fibres. Passive and active membrane conductances were set to produce input resistance, rheobase, afterhyperpolarization (AHP) and membrane time constant values similar to those measured in adult cat motoneurones in non-locomoting conditions. The parameters of 10 membrane conductances were then individually altered in an attempt to replicate the hyperpolarization of Vth that occurs in decerebrate cats during fictive locomotion. The goal was to find conductance changes that could produce a greater than 3 mV hyperpolarization of Vth with only small changes in AP height (< 3 mV) and width (< 1.2 ms). Vth reduction without large changes in AP shape could be produced either by increasing fast sodium current or by reducing delayed rectifier potassium current. The most effective Vth reductions were achieved by either increasing the conductance of fast sodium channels or by hyperpolarizing the voltage dependency of their activation. These changes were particularly effective when localized to the initial segment. Reducing the conductance of delayed rectifier channels or depolarizing their activation produced similar but smaller changes in Vth. Changes in current underlying the AHP, the persistent Na(+) current, three Ca(2+) currents, the "h" mixed cation current, the "A" potassium current and the leak current were either ineffective in reducing Vth or also produced gross changes in the AP. It is suggested that the increased excitability of motoneurones during locomotion could be readily accomplished by hyperpolarizing the voltage dependency of fast sodium channels in the axon hillock by a hitherto unknown neuromodulatory action.

Many etiologies of heart disease are characterized by expansion and remodeling of the cardiac extracellular matrix (ECM or matrix) which results in cardiac fibrosis. Cardiac fibrosis is mediated in cardiac fibroblasts by TGF-β1 /R-Smad2/3 signaling. Matrix component proteins are synthesized by activated resident cardiac fibroblasts known as myofibroblasts (MFB). These events are causal to heart failure with diastolic dysfunction and reduced cardiac filling. We have shown that exogenous Ski, a TGF-β1 /Smad repressor, localizes in the cellular nucleus and deactivates cardiac myofibroblasts. This deactivation is associated with reduction of myofibroblast marker protein expression in vitro, including alpha smooth muscle actin (α-SMA) and extracellular domain-A (ED-A) fibronectin. We hypothesize that Ski also acutely regulates MMP expression in cardiac MFB. While acute Ski overexpression in cardiac MFB in vitro was not associated with any change in intracellular MMP-9 protein expression versus LacZ-treated controls,exogenous Ski caused elevated MMP-9 mRNA expression and increased MMP-9 protein secretion versus controls. Zymographic analysis revealed increased MMP-9-specific gelatinase activity in myofibroblasts overexpressing Ski versus controls. Moreover, Ski expression was attended by reduced paxillin and focal adhesion kinase phosphorylation (FAK - Tyr 397) versus controls. As myofibroblasts are hypersecretory and less motile relative to fibroblasts, Ski's reduction of paxillin and FAK expression may reflect the relative deactivation of myofibroblasts. Thus, in addition to its known antifibrotic effects, Ski overexpression elevates expression and extracellular secretion/release of MMP-9 and thus may facilitate internal cytoskeletal remodeling as well as extracellular ECM components. Further, as acute TGF-β1 treatment of primary cardiac MFB is known to cause rapid translocation of Ski to the nucleus, our data support an autoregulatory role for Ski in mediating cardiac ECM accumulation.