Chondrogenesis is known to be regulated by calcium-dependent signalling pathways in which temporal aspects of calcium homeostasis are of key importance. We aimed to better characterise calcium influx and release functions with respect to rapid calcium oscillations in cells of chondrifying chicken high density cultures. We found that differentiating chondrocytes express the ␣ 1 subunit of voltage-operated calcium channels (VOCCs) at both mRNA and protein levels, and that these ion channels play important roles in generating Ca 2+ influx for oscillations as nifedipine interfered with repetitive calcium transients. Furthermore, VOCC blockade abrogated chondrogenesis and almost completely blocked cell proliferation. The contribution of internal Ca 2+ stores via store-operated Ca 2+ entry (SOCE) seems to be indispensable to both Ca 2+ oscillations and chondrogenesis. Moreover, this is the first study to show the functional expression of STIM1/STIM2 and Orai1, molecules that orchestrate SOCE, in chondrogenic cells. Inhibition of SOCE combined with ER calcium store depletion abolished differentiation and severely diminished proliferation, suggesting the important role of internal pools in calcium homeostasis of differentiating chondrocytes. Finally, we present an integrated model for the regulation of calcium oscillations of differentiating chondrocytes that may have important implications for studies of chondrogenesis induced in various stem cell populations.

Articular cartilage damage occurring during early osteoarthritis (OA) is a key event marking the development of the disease. Here, we modeled early human OA by gathering detailed spatiotemporal data from surgically induced knee OA development in sheep. We identified a specific topographical pattern of osteochondral changes instructed by a defined meniscal injury, showing that both cartilage and subchondral bone degeneration are initiated from the region adjacent to the damage. Alterations of the subarticular spongiosa arising locally and progressing globally disturbed the correlations of cartilage with subchondral bone seen at homeostasis and were indicative of disease progression. We validated our quantitative findings against human OA, showing a similar pattern of early OA correlating with regions of meniscal loss and an analogous late critical disturbance within the entire osteochondral unit. This translational model system can be used to elucidate mechanisms of OA development and provides a roadmap for investigating regenerative therapies.

Store-operated Ca entry (SOCE) is a Ca-entry process activated by the depletion of intracellular stores and has an important role in many cell types. In skeletal muscle, however, its role during physiological muscle activation has been controversial. To address this question, sarcoplasmic reticulum (SR) calcium release in a mouse strain with a naturally occurring mutation in the myostatin gene (Compact (Cmpt)) leading to a hypermuscular yet reduced muscle-force phenotype was compared to that in wild-type mice. To elicit Ca release from the SR of flexor digitorum brevis (FDB) fibers, either a ryanodine receptor agonist (4-chloro-meta-cresol) or depolarizing pulses were used. In muscles from Cmpt mice, endogenous protein levels of STIM1 and Orai1 were reduced, and consequently, SOCE after 4-chloro-meta-cresol-induced store depletion was suppressed. Although the voltage dependence of SR calcium release was not statistically different between wild-type and Cmpt fibers, the amount of releasable calcium was significantly reduced in the latter, indicating a smaller SR content. To assess the immediate role of SOCE in replenishing the SR calcium store, the evolution of intracellular calcium concentration during a train of long-lasting depolarizations to a maximally activating voltage was monitored. Cmpt mice exhibited a faster decline in calcium release, suggesting a compromised ability to refill the SR. A simple model that incorporates a reduced SOCE as an important partner in regulating immediate calcium influx through the surface membrane readily accounts for the steady-state reduction in SR calcium content and its more pronounced decline after calcium release.

BackgroundUnderstanding the key elements of signaling of chondroprogenitor cells at the earliest steps of differentiation may substantially improve our opportunities for the application of mesenchymal stem cells in cartilage tissue engineering, which is a promising approach of regenerative therapy of joint diseases. Ion channels, membrane potential and Ca2+-signaling are important regulators of cell proliferation and differentiation. Our aim was to identify such plasma membrane ion channels involved in signaling during chondrogenesis, which may serve as specific molecular targets for influencing chondrogenic differentiation and ultimately cartilage formation.Methodology/Principal FindingsUsing patch-clamp, RT-PCR and Western-blot experiments, we found that chondrogenic cells in primary micromass cell cultures obtained from embryonic chicken limb buds expressed voltage-gated NaV1.4, KV1.1, KV1.3 and KV4.1 channels, although KV1.3 was not detectable in the plasma membrane. Tetrodotoxin (TTX), the inhibitor of NaV1.4 channels, had no effect on cartilage formation. In contrast, presence of 20 mM of the K+ channel blocker tetraethyl-ammonium (TEA) during the time-window of the final commitment of chondrogenic cells reduced KV currents (to 27±3% of control), cell proliferation (thymidine incorporation: to 39±4.4% of control), expression of cartilage-specific genes and consequently, cartilage formation (metachromasia: to 18.0±6.4% of control) and also depolarized the membrane potential (by 9.3±2.1 mV). High-frequency Ca2+-oscillations were also suppressed by 10 mM TEA (confocal microscopy: frequency to 8.5±2.6% of the control). Peak expression of TEA-sensitive KV1.1 in the plasma membrane overlapped with this period. Application of TEA to differentiated chondrocytes, mainly expressing the TEA-insensitive KV4.1 did not affect cartilage formation.Conclusions/SignificanceThese data demonstrate that the differentiation and proliferation of chondrogenic cells depend on rapid Ca2+-oscillations, which are modulated by KV-driven membrane potential changes. KV1.1 function seems especially critical during the final commitment period. We show the critical role of voltage-gated cation channels in the differentiation of non-excitable cells with potential therapeutic use.

Osteoarthritis (OA) is considerably affected by joint alignment. Here, we investigate the patterns of spatial osteochondral heterogeneity in patients with advanced varus knee OA together with clinical data. We report strong correlations of osteochondral parameters within individual topographical patterns, highlighting their fundamental and location-dependent interactions in OA. We further identify site-specific effects of varus malalignment on the lesser loaded compartment and, conversely, an unresponsive overloaded compartment. Last, we trace compensatory mechanisms to the overloaded subarticular spongiosa in patients with additional high body weight. We therefore propose to consider and to determine axial alignment in clinical trials when selecting the location to assess structural changes in OA. Together, these findings broaden the scientific basis of therapeutic load redistribution and weight loss in varus knee OA.

Accumulating evidence supports the role of astrocytes in endocannabinoid mediated modulation of neural activity. It has been reported that some astrocytes express the cannabinoid type 1 receptor (CB1-R), the activation of which is leading to Ca2+ mobilization from internal stores and a consecutive release of glutamate. It has also been documented that astrocytes have the potential to produce the endocannabinoid 2-arachidonoylglycerol, one of the best known CB1-R agonist. However, no relationship between CB1-R activation and 2-arachidonoylglycerol production has ever been demonstrated. Here we show that rat spinal astrocytes co-express CB1-Rs and the 2-arachidonoylglycerol synthesizing enzyme, diacylglycerol lipase-alpha in close vicinity to each other. We also demonstrate that activation of CB1-Rs induces a substantial elevation of intracellular Ca2+ concentration in astrocytes. Finally, we provide evidence that the evoked Ca2+ transients lead to the production of 2-arachidonoylglycerol in cultured astrocytes. The results provide evidence for a novel cannabinoid induced endocannabinoid release mechanism in astrocytes which broadens the bidirectional signaling repertoire between astrocytes and neurons.

Key pointsr Hypermuscularity associated with naturally occurring mutations in the myostatin gene as found in Compact mice results in increased muscle mass but reduced specific force.r The calcium sensitivity of the contractile apparatus as assessed on chemically skinned skeletal muscle fibres under isometric conditions is not altered in these animals.r While the resting calcium concentration remains unaffected, depolarization-evoked increases in intracellular calcium concentration are suppressed.r Spontaneous calcium release events from sarcoplasmic reticulum are also decreased in frequency, amplitude and spatial spread.r Our results suggest that mutations in the myostatin gene are accompanied by alterations in excitation contraction coupling, which manifest as a reduction in sarcoplasmic calcium release.Abstract Myostatin, a member of the transforming growth factor β family, is a potent negative regulator of skeletal muscle growth, as myostatin-deficient mice show a great increase in muscle mass. Yet the physical performance of these animals is reduced. As an explanation for this, alterations in the steps in excitation-contraction coupling were hypothesized and tested for in mice with the 12 bp deletion in the propeptide region of the myostatin precursor (Mstn or Cmpt). In voluntary wheel running, control C57BL/6 mice performed better than the mutant animals in both maximal speed and total distance covered. Despite the previously described lower specific force of Cmpt animals, the p Ca -force relationship, determined on chemically permeabilized fibre segments, did not show any significant difference between the two mouse strains. While resting intracellular Ca 2+ concentration ([Ca 2+ ] i ) measured on single intact flexor digitorum brevis (FDB) muscle fibres using Fura-2 AM was similar to control (72.0 ± 1.7 vs. 78.1 ± 2.9 nM, n = 38 and 45), the amplitude of KCl-evoked calcium transients was smaller (360 ± 49 vs. 222 ± 45 nM, n = 22) in the mutant strain. Similar results were obtained using tetanic stimulation and Rhod-2 AM, which gave calcium transients that were smaller (2.42 ± 0.11 vs. 2.06 ± 0.10 F/F 0 , n = 14 and 13, respectively) on Cmpt mice. Sarcoplasmic reticulum (SR) calcium release flux calculated from these transients showed a reduced peak (23.7 ± 3.0 vs. 15.8 ± 2.1 mMs −1 ) and steady level (5.7 ± 0.7 vs. 3.7 ± 0.5 mM s −1 ) with no change in the peak-to-steady ratio. The amplitude and spatial spread of calcium release events detected on permeabilized FDB fibres were also significantly smaller in mutant mice. These results suggest that reduced SR calcium release underlies the reduced muscle force in Cmpt animals.

a b s t r a c tWe have previously demonstrated that elevation of free cytosolic Ca 2+ concentration at the time of differentiation of chondroblasts was mainly due to a Ca 2+ influx and it was indispensable to cartilage formation in chicken high density mesenchymal cell cultures (HDC) [C. Matta, J. Fodor, Z. Szijgyarto, T. Juhasz, P. Q1 Gergely, L. Csernoch, R. Zakany, Cytosolic free Ca 2+ concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium 44 (2008) 310-323]. Here, we report that chondrogenic cells secreted ATP and administration of ATP to the culture medium evoked Ca 2+ transients exclusively in the presence of extracellular Ca 2+ and only on day 3 of culturing, when the final commitment of chondroblasts occurs. Moreover, ATP caused elevated protein expression of the chondrogenic transcription factor Sox9 and stimulated cartilage matrix production. Expression pattern of different types of both ionotropic and metabotropic purinergic receptors was detected. Agonists of metabotropic receptors, ADP and UDP did not evoke any Ca 2+ transients and had no influence on cartilage formation, while UTP caused transient elevation of cytosolic Ca 2+ concentration in 3-day-old HDC without stimulating matrix production. Suramin, which blocks all P2X receptors but not P2X 4 did not impede the effects of ATP, furthermore, P2X 4 appeared in the plasma membrane fraction and gave signals with immunocytochemistry only from day 3. In summary, we suggest a role of ionotropic purinergic signalling of P2X 4 in the generation of ATP-dependent Ca 2+ transients of differentiating chondroblasts.