Reduced Expression of Septin7 Hinders Skeletal Muscle Regeneration.

Septins are considered the fourth component of the cytoskeleton with the septin7 isoform playing a critical role in the formation of diffusion barriers in phospholipid bilayers and intra- and extracellular scaffolds. While its importance has already been confirmed in different intracellular processes, very little is known about its role in skeletal muscle. Muscle regeneration was studied in a Sept7 conditional knock-down mouse model to prove the possible role of septin7 in this process. Sterile inflammation in skeletal muscle was induced which was followed by regeneration resulting in the upregulation of septin7 expression. Partial knock-down of Sept7 resulted in an increased number of inflammatory cells and myofibers containing central nuclei. Taken together, our data suggest that partial knock-down of Sept7 hinders the kinetics of muscle regeneration, indicating its crucial role in skeletal muscle functions.

Genetic Manipulation of CB1 Cannabinoid Receptors Reveals a Role in Maintaining Proper Skeletal Muscle Morphology and Function in Mice.

The endocannabinoid system (ECS) refers to a widespread signaling system and its alteration is implicated in a growing number of human diseases. Cannabinoid receptors (CBRs) are highly expressed in the central nervous system and many peripheral tissues. Evidence suggests that CB1Rs are expressed in human and murine skeletal muscle mainly in the cell membrane, but a subpopulation is present also in the mitochondria. However, very little is known about the latter population. To date, the connection between the function of CB1Rs and the regulation of intracellular Ca2+ signaling has not been investigated yet. Tamoxifen-inducible skeletal muscle-specific conditional CB1 knock-down (skmCB1-KD, hereafter referred to as Cre+/-) mice were used in this study for functional and morphological analysis. After confirming CB1R down-regulation on the mRNA and protein level, we performed in vitro muscle force measurements and found that peak twitch, tetanus, and fatigue were decreased significantly in Cre+/- mice. Resting intracellular calcium concentration, voltage dependence of the calcium transients as well as the activity dependent mitochondrial calcium uptake were essentially unaltered by Cnr1 gene manipulation. Nevertheless, we found striking differences in the ultrastructural architecture of the mitochondrial network of muscle tissue from the Cre+/- mice. Our results suggest a role of CB1Rs in maintaining physiological muscle function and morphology. Targeting ECS could be a potential tool in certain diseases, including muscular dystrophies where increased endocannabinoid levels have already been described.

Astaxanthin Exerts Anabolic Effects via Pleiotropic Modulation of the Excitable Tissue.

Astaxanthin is a lipid-soluble carotenoid influencing lipid metabolism, body weight, and insulin sensitivity. We provide a systematic analysis of acute and chronic effects of astaxanthin on different organs. Changes by chronic astaxanthin feeding were analyzed on general metabolism, expression of regulatory proteins in the skeletal muscle, as well as changes of excitation and synaptic activity in the hypothalamic arcuate nucleus of mice. Acute responses were also tested on canine cardiac muscle and different neuronal populations of the hypothalamic arcuate nucleus in mice. Dietary astaxanthin significantly increased food intake. It also increased protein levels affecting glucose metabolism and fatty acid biosynthesis in skeletal muscle. Inhibitory inputs innervating neurons of the arcuate nucleus regulating metabolism and food intake were strengthened by both acute and chronic astaxanthin treatment. Astaxanthin moderately shortened cardiac action potentials, depressed their plateau potential, and reduced the maximal rate of depolarization. Based on its complex actions on metabolism and food intake, our data support the previous findings that astaxanthin is suitable for supplementing the diet of patients with disturbances in energy homeostasis.

Septins, a cytoskeletal protein family, with emerging role in striated muscle.

Appropriate organization of cytoskeletal components are required for normal distribution and intracellular localization of different ion channels and proteins involved in calcium homeostasis, signal transduction, and contractile function of striated muscle. Proteins of the contractile system are in direct or indirect connection with the extrasarcomeric cytoskeleton. A number of other molecules which have essential role in regulating stretch-, voltage-, and chemical signal transduction from the surface into the cytoplasm or other intracellular compartments are already well characterized. Sarcomere, the basic contractile unit, is comprised of a precisely organized system of thin (actin), and thick (myosin) filaments. Intermediate filaments connect the sarcomeres and other organelles (mitochondria and nucleus), and are responsible for the cellular integrity. Interacting proteins have a very diverse function in coupling of the intracellular assembly components and regulating the normal physiological function. Despite the more and more intense investigations of a new cytoskeletal protein family, the septins, only limited information is available regarding their expression and role in striated, especially in skeletal muscles. In this review we collected basic and specified knowledge regarding this protein group and emphasize the importance of this emerging field in skeletal muscle biology.

Synthesis and cellular effects of a mitochondria-targeted inhibitor of the two-pore potassium channel TASK-3.

The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.

Electrophysiological Effects of the Transient Receptor Potential Melastatin 4 Channel Inhibitor (4-Chloro-2-(2-chlorophenoxy)acetamido) Benzoic Acid (CBA) in Canine Left Ventricular Cardiomyocytes.

Transient receptor potential melastatin 4 (TRPM4) plays an important role in many tissues, including pacemaker and conductive tissues of the heart, but much less is known about its electrophysiological role in ventricular myocytes. Our earlier results showed the lack of selectivity of 9-phenanthrol, so CBA ((4-chloro-2-(2-chlorophenoxy)acetamido) benzoic acid) was chosen as a new, potentially selective inhibitor. Goal: Our aim was to elucidate the effect and selectivity of CBA in canine left ventricular cardiomyocytes and to study the expression of TRPM4 in the canine heart. Experiments were carried out in enzymatically isolated canine left ventricular cardiomyocytes. Ionic currents were recorded with an action potential (AP) voltage-clamp technique in whole-cell configuration at 37 °C. An amount of 10 mM BAPTA was used in the pipette solution to exclude the potential activation of TRPM4 channels. AP was recorded with conventional sharp microelectrodes. CBA was used in 10 µM concentrations. Expression of TRPM4 protein in the heart was studied by Western blot. TRPM4 protein was expressed in the wall of all four chambers of the canine heart as well as in samples prepared from isolated left ventricular cells. CBA induced an approximately 9% reduction in AP duration measured at 75% and 90% of repolarization and decreased the short-term variability of APD90. Moreover, AP amplitude was increased and the maximal rates of phase 0 and 1 were reduced by the drug. In AP clamp measurements, CBA-sensitive current contained a short, early outward and mainly a long, inward current. Transient outward potassium current (Ito) and late sodium current (INa,L) were reduced by approximately 20% and 47%, respectively, in the presence of CBA, while L-type calcium and inward rectifier potassium currents were not affected. These effects of CBA were largely reversible upon washout. Based on our results, the CBA induced reduction of phase-1 slope and the slight increase of AP amplitude could have been due to the inhibition of Ito. The tendency for AP shortening can be explained by the inhibition of inward currents seen in AP-clamp recordings during the plateau phase. This inward current reduced by CBA is possibly INa,L, therefore, CBA is not entirely selective for TRPM4 channels. As a consequence, similarly to 9-phenanthrol, it cannot be used to test the contribution of TRPM4 channels to cardiac electrophysiology in ventricular cells, or at least caution must be applied.

Ca2+-activated Cl- current is antiarrhythmic by reducing both spatial and temporal heterogeneity of cardiac repolarization.

The role of Ca2+-activated Cl- current (ICl(Ca)) in cardiac arrhythmias is still controversial. It can generate delayed afterdepolarizations in Ca2+-overloaded cells while in other studies incidence of early afterdepolarization (EAD) was reduced by ICl(Ca). Therefore our goal was to examine the role of ICl(Ca) in spatial and temporal heterogeneity of cardiac repolarization and EAD formation. Experiments were performed on isolated canine cardiomyocytes originating from various regions of the left ventricle; subepicardial, midmyocardial and subendocardial cells, as well as apical and basal cells of the midmyocardium. ICl(Ca) was blocked by 0.5mmol/L 9-anthracene carboxylic acid (9-AC). Action potential (AP) changes were tested with sharp microelectrode recording. Whole-cell 9-AC-sensitive current was measured with either square pulse voltage-clamp or AP voltage-clamp (APVC). Protein expression of TMEM16A and Bestrophin-3, ion channel proteins mediating ICl(Ca), was detected by Western blot. 9-AC reduced phase-1 repolarization in every tested cell. 9-AC also increased AP duration in a reverse rate-dependent manner in all cell types except for subepicardial cells. Neither ICl(Ca) density recorded with square pulses nor the normalized expressions of TMEM16A and Bestrophin-3 proteins differed significantly among the examined groups of cells. The early outward component of ICl(Ca) was significantly larger in subepicardial than in subendocardial cells in APVC setting. Applying a typical subepicardial AP as a command pulse resulted in a significantly larger early outward component in both subepicardial and subendocardial cells, compared to experiments when a typical subendocardial AP was applied. Inhibiting ICl(Ca) by 9-AC generated EADs at low stimulation rates and their incidence increased upon beta-adrenergic stimulation. 9-AC increased the short-term variability of repolarization also. We suggest a protective role for ICl(Ca) against risk of arrhythmias by reducing spatial and temporal heterogeneity of cardiac repolarization and EAD formation.

Sarcolemmal Ca(2+)-entry through L-type Ca(2+) channels controls the profile of Ca(2+)-activated Cl(-) current in canine ventricular myocytes.

Ca(2+)-activated Cl(-) current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca(2+) cycling and AP voltage-clamp) as well as in conditions designed to change [Ca(2+)]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied. The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca(2+)]i was monitored using Fura-2-AM. Setting [Ca(2+)]i to the systolic level measured in the bulk cytoplasm (1.1µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca(2+)-entry through L-type Ca(2+) channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium. Activation of ICl(Ca) in canine ventricular cells requires Ca(2+)-entry through neighboring L-type Ca(2+) channels and is only augmented by SR Ca(2+)-release. Substantial activation of ICl(Ca) requires high Ca(2+) concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Septin 7 interacts with Numb to preserve sarcomere structural organization and muscle contractile function.

Here, we investigated the mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development, including asymmetric cell division, cell-type specification, and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (Numbl) in mouse myofibers caused weakness, disorganization of sarcomeres, and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, Numbl knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb that Septin 7 is a potential Numb-binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets, and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb-binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Migration of Myogenic Cells Is Highly Influenced by Cytoskeletal Septin7.

Septin7 as a unique member of the GTP binding protein family, is widely expressed in the eukaryotic cells and considered to be essential in the formation of hetero-oligomeric septin complexes. As a cytoskeletal component, Septin7 is involved in many important cellular processes. However, its contribution in striated muscle physiology is poorly described. In skeletal muscle, a highly orchestrated process of migration is crucial in the development of functional fibers and in regeneration. Here, we describe the pronounced appearance of Septin7 filaments and a continuous change of Septin7 protein architecture during the migration of myogenic cells. In Septin7 knockdown C2C12 cultures, the basic parameters of migration are significantly different, and the intracellular calcium concentration change in migrating cells are lower compared to that of scrambled cultures. Using a plant cytokinin, forchlorfenuron, to dampen septin dynamics, the altered behavior of the migrating cells is described, where Septin7-depleted cells are more resistant to the treatment. These results indicate the functional relevance of Septin7 in the migration of myoblasts, implying its contribution to muscle myogenesis and regeneration.

SEPTIN 7 INTERACTS WITH NUMB TO PRESERVE SARCOMERE STRUCTURAL ORGANIZATION AND MUSCLE CONTRACTILE FUNCTION.

Here, we investigated mechanisms by which aging-related reductions of the levels of Numb in skeletal muscle fibers contribute to loss of muscle strength and power, two critical features of sarcopenia. Numb is an adaptor protein best known for its critical roles in development including asymmetric cell division, cell-type specification and termination of intracellular signaling. Numb expression is reduced in old humans and mice. We previously showed that, in mouse skeletal muscle fibers, Numb is localized to sarcomeres where it is concentrated near triads; conditional inactivation of Numb and a closely related protein Numb-like (NumbL) in mouse myofibers caused weakness, disorganization of sarcomeres and smaller mitochondria with impaired function. Here, we found that a single knockout of Numb in myofibers causes reduction in tetanic force comparable to a double Numb, NumbL knockout. We found by proteomics analysis of protein complexes isolated from C2C12 myotubes by immunoprecipitation using antibodies against Numb, that Septin 7 is a potential Numb binding partner. Septin 7 is a member of the family of GTP-binding proteins that organize into filaments, sheets and rings, and is considered part of the cytoskeleton. Immunofluorescence evaluation revealed a partial overlap of staining for Numb and Septin 7 in myofibers. Conditional, inducible knockouts of Numb led to disorganization of Septin 7 staining in myofibers. These findings indicate that Septin 7 is a Numb binding partner and suggest that interactions between Numb and Septin 7 are critical for structural organization of the sarcomere and muscle contractile function.

Alternatively spliced exon regulates context-dependent MEF2D higher-order assembly during myogenesis.

During muscle cell differentiation, the alternatively spliced, acidic ß-domain potentiates transcription of Myocyte-specific Enhancer Factor 2 (Mef2D). Sequence analysis by the FuzDrop method indicates that the ß-domain can serve as an interaction element for Mef2D higher-order assembly. In accord, we observed Mef2D mobile nuclear condensates in C2C12 cells, similar to those formed through liquid-liquid phase separation. In addition, we found Mef2D solid-like aggregates in the cytosol, the presence of which correlated with higher transcriptional activity. In parallel, we observed a progress in the early phase of myotube development, and higher MyoD and desmin expression. In accord with our predictions, the formation of aggregates was promoted by rigid ß-domain variants, as well as by a disordered ß-domain variant, capable of switching between liquid-like and solid-like higher-order states. Along these lines, NMR and molecular dynamics simulations corroborated that the ß-domain can sample both ordered and disordered interactions leading to compact and extended conformations. These results suggest that ß-domain fine-tunes Mef2D higher-order assembly to the cellular context, which provides a platform for myogenic regulatory factors and the transcriptional apparatus during the developmental process.

Age-dependent changes in ion channel mRNA expression in canine cardiac tissues.

The expression pattern of cardiac ion channels displays marked changes during ontogeny. This study was designed to follow the developmental changes in the expression of major ventricular and atrial ion channel proteins (including both pore forming and regulatory subunits) in canine cardiac tissues at the mRNA level using competitive reverse transcription polymerase chain reaction. Therefore, the corresponding mRNA levels were compared in myocardial tissues excised from embryonic (25-60 days of gestation) and adult (2-3 years old) canine hearts. Expression level of Kv4.3, Kv1.4, KChIP2, KvLQT1, and Cav3.2 mRNAs were higher in the adult than in the embryonic hearts, while expression of Nav1.5 and minK mRNAs were higher in the embryonic than in the adult myocardium. No change in Kir2.1, HERG, Kv1.5, and Cav1.2 mRNA was observed during ontogeny. Direction of the developmental change in the mRNA level, determined for any specific channel protein, was identical in the atrial and ventricular samples. The age-dependent increase observed in the expression of Kv4.3, Kv1.4, KChIP2, and KvLQT1 is congruent with the greater repolarization reserve of the adult myocardium, associated with higher densities of Ito and IKs. The results indicate that age-dependent changes in the expression pattern of many ion channels are similar in canine and healthy human myocardium, therefore, canine cardiac muscle can be considered as a good model of studying developmental changes in the human heart.

The mechanosensitive Piezo1 channels contribute to the arterial medial calcification.

Vascular calcification (VC) is associated with a number of cardiovascular diseases, as well as chronic kidney disease. The role of smooth muscle cells (SMC) has already been widely explored in VC, as has the role of intracellular Ca2+ in regulating SMC function. Increased intracellular calcium concentration ([Ca2+]i) in vascular SMC has been proposed to stimulate VC. However, the contribution of the non-selective Piezo1 mechanosensitive cation channels to the elevation of [Ca2+]i, and consequently to the process of VC has never been examined. In this work the essential contribution of Piezo1 channels to arterial medial calcification is demonstrated. The presence of Piezo1 was proved on human aortic smooth muscle samples using immunohistochemistry. Quantitative PCR and Western blot analysis confirmed the expression of the channel on the human aortic smooth muscle cell line (HAoSMC). Functional measurements were done on HAoSMC under control and calcifying condition. Calcification was induced by supplementing the growth medium with inorganic phosphate (1.5 mmol/L, pH 7.4) and calcium (CaCl2, 0.6 mmol/L) for 7 days. Measurement of [Ca2+]i using fluorescent Fura-2 dye upon stimulation of Piezo1 channels (either by hypoosmolarity, or Yoda1) demonstrated significantly higher calcium transients in calcified as compared to control HAoSMCs. The expression of mechanosensitive Piezo1 channel is augmented in calcified arterial SMCs leading to a higher calcium influx upon stimulation. Activation of the channel by Yoda1 (10 µmol/L) enhanced calcification of HAoSMCs, while Dooku1, which antagonizes the effect of Yoda1, reduced this amplification. Application of Dooku1 alone inhibited the calcification. Knockdown of Piezo1 by siRNA suppressed the calcification evoked by Yoda1 under calcifying conditions. Our results demonstrate the pivotal role of Piezo1 channels in arterial medial calcification.

Septin7 is indispensable for proper skeletal muscle architecture and function.

Today septins are considered as the fourth component of the cytoskeleton, with the Septin7 isoform playing a critical role in the formation of higher-order structures. While its importance has already been confirmed in several intracellular processes of different organs, very little is known about its role in skeletal muscle. Here, using Septin7 conditional knockdown (KD) mouse model, the C2C12 cell line, and enzymatically isolated adult muscle fibers, the organization and localization of septin filaments are revealed, and an ontogenesis-dependent expression of Septin7 is demonstrated. KD mice displayed a characteristic hunchback phenotype with skeletal deformities, reduction in in vivo and in vitro force generation, and disorganized mitochondrial networks. Furthermore, knockout of Septin7 in C2C12 cells resulted in complete loss of cell division while KD cells provided evidence that Septin7 is essential for proper myotube differentiation. These and the transient increase in Septin7 expression following muscle injury suggest that it may be involved in muscle regeneration and development.

Astaxanthin: A Potential Mitochondrial-Targeted Antioxidant Treatment in Diseases and with Aging.

Oxidative stress is characterized by an imbalance between prooxidant and antioxidant species, leading to macromolecular damage and disruption of redox signaling and cellular control. It is a hallmark of various diseases including metabolic syndrome, chronic fatigue syndrome, neurodegenerative, cardiovascular, inflammatory, and age-related diseases. Several mitochondrial defects have been considered to contribute to the development of oxidative stress and known as the major mediators of the aging process and subsequent age-associated diseases. Thus, mitochondrial-targeted antioxidants should prevent or slow down these processes and prolong longevity. This is the reason why antioxidant treatments are extensively studied and newer and newer compounds with such an effect appear. Astaxanthin, a xanthophyll carotenoid, is the most abundant carotenoid in marine organisms and is one of the most powerful natural compounds with remarkable antioxidant activity. Here, we summarize its antioxidant targets, effects, and benefits in diseases and with aging.

Altered expression of triadin 95 causes parallel changes in localized Ca2+ release events and global Ca2+ signals in skeletal muscle cells in culture.

The 95 kDa triadin (Trisk 95), an integral protein of the sarcoplasmic reticular membrane in skeletal muscle, interacts with both the ryanodine receptor (RyR) and calsequestrin. While its role in the regulation of calcium homeostasis has been extensively studied, data are not available on whether the overexpression or the interference with the expression of Trisk 95 would affect calcium sparks the localized events of calcium release (LCRE). In the present study LCRE and calcium transients were studied using laser scanning confocal microscopy on C2C12 cells and on primary cultures of skeletal muscle. Liposome- or adenovirus-mediated Trisk 95 overexpression and shRNA interference with triadin translation were used to modify the level of the protein. Stable overexpression in C2C12 cells significantly decreased the amplitude and frequency of calcium sparks, and the frequency of embers. In line with these observations, depolarization-evoked calcium transients were also suppressed. Similarly, adenoviral transfection of Trisk 95 into cultured mouse skeletal muscle cells significantly decreased both the frequency and amplitude of spontaneous global calcium transients. Inhibition of endogenous triadin expression by RNA interference caused opposite effects. Primary cultures of rat skeletal muscle cells expressing endogenous Trisk 95 readily generated spontaneous calcium transients but rarely produced calcium sparks. Their transfection with specific shRNA sequence significantly reduced the triadin-specific immunoreactivity. Functional experiments on these cells revealed that while caffeine-evoked calcium transients were reduced, LCRE appeared with higher frequency. These results suggest that Trisk 95 negatively regulates RyR function by suppressing localized calcium release events and global calcium signals in cultured muscle cells.

Altered calcium handling following the recombinant overexpression of protein kinase C isoforms in HaCaT cells.

Both changes in intracellular calcium concentration ([Ca(2+)](i)) and activation of certain protein kinase C (PKC) isoforms play a crucial role in keratinocyte functions. To better understand the interaction between these two signalling pathways we investigated the resting [Ca(2+)](i) and the extracellular ATP-induced changes in [Ca(2+)](i) on HaCaT cell clones overexpressing either the classical alpha or the beta PKC isoform. These PKC isoenzymes were previously shown to decrease (alpha) or increase (beta) cell proliferation and augment (alpha) or suppress (beta) cell differentiation. Keratinocyte clones with decreased proliferation rate were found to have unaltered resting [Ca(2+)](i), but responded with greater calcium transients to the application of 180 mum of ATP. In contrast, clones with increased proliferation rate had elevated resting [Ca(2+)](i) and suppressed calcium responses to ATP. Calcium transients on PKCbeta clones displayed a faster falling phase. Each clone had a distinct purinergic receptor expression pattern, some of which paralleled the altered proliferation rate and calcium handling. Keratinocytes overexpressing PKCbeta revealed decreased P2X1 and increased P2Y1 receptor expression as compared with the control or PKCalpha clones. The expression level of P2X7 was significantly increased in keratinocytes overexpressing PKCalpha. On the other hand neither the P2X2 nor the P2Y2 expression was altered significantly in the cell types investigated. These data indicate that a modified proliferation and differentiation pattern is associated with altered calcium handling in keratinocytes. The observations also suggest that different PKC isoenzymes have different effects on the phosphatidyl-inositol signalling pathway.

Mitochondrial expression of the two-pore domain TASK-3 channels in malignantly transformed and non-malignant human cells.

The presence of TASK-3 channels has been described in a number of healthy and malignantly transformed cells, showing mainly intracellular distribution with relatively insignificant labelling of the cell surface membrane. In this work, immunochemical and molecular biology methods were utilised to establish the intracellular organelle whose TASK-3 expression accounts for this strong intracellular labelling using cultured melanoma and HaCaT cells. Before the immunocytochemical experiments, the presence of TASK-3 mRNA was also confirmed in melanoma cells. Comparison of the results of the TASK-3- and mitochondrion-specific labelling indicated that the TASK-3 channel subunits were strongly expressed by mitochondria in both investigated cell types. Moreover, prominent TASK-3 expression of keratinocytes could also be demonstrated in histological sections excised from the human skin. These results indicate that TASK-3 channels are present in the mitochondria in both malignantly transformed and healthy cells, suggesting that they might have roles in ensuring mitochondrial functions.

Glycogen phosphorylase inhibition improves beta cell function.

BACKGROUND AND PURPOSE: Glycogen phosphorylase (GP) is the key enzyme for glycogen degradation. GP inhibitors (GPi-s) are glucose lowering agents that cause the accumulation of glucose in the liver as glycogen. Glycogen metabolism has implications in beta cell function. Glycogen degradation can maintain cellular glucose levels, which feeds into catabolism to maintain insulin secretion, and elevated glycogen degradation levels contribute to glucotoxicity. The purpose of this study was to assess whether influencing glycogen metabolism in beta cells by GPi-s affects the function of these cells. EXPERIMENTAL APPROACH: The effects of structurally different GPi-s were investigated on MIN6 insulinoma cells and in a mouse model of diabetes. KEY RESULTS: GPi treatment increased glycogen content and, consequently, the surface area of glycogen in MIN6 cells. Furthermore, GPi treatment induced insulin receptor ß (InsRß), Akt and p70S6K phosphorylation, as well as pancreatic and duodenal homeobox 1(PDX1) and insulin expression. In line with these findings, GPi-s enhanced non-stimulated and glucose-stimulated insulin secretion in MIN6 cells. The InsRß was shown to co-localize with glycogen particles as confirmed by in silico screening, where components of InsR signalling were identified as glycogen-bound proteins. GPi-s also activated the pathway of insulin secretion, indicated by enhanced glycolysis, mitochondrial oxidation and calcium signalling. Finally, GPi-s increased the size of islets of Langerhans and improved glucose-induced insulin release in mice. CONCLUSION AND IMPLICATIONS: These data suggest that GPi-s also target beta cells and can be repurposed as agents to preserve beta cell function or even ameliorate beta cell dysfunction in different forms of diabetes. LINKED ARTICLES: This article is part of a themed section on Inventing New Therapies Without Reinventing the Wheel: The Power of Drug Repurposing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.2/issuetoc.