Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.
Chondrocytes are the resident cells of articular cartilage and are responsible for synthesizing a range of collagenous and non-collagenous extracellular matrix macromolecules. Whilst chondrocytes exist at low densities in the tissue (1–10% of the total tissue volume in mature cartilage) they are extremely active cells and are capable of responding to a range of mechanical and biochemical stimuli. These responses are necessary for the maintenance of viable cartilage and may be compromised in inflammatory diseases such as arthritis. Although chondrocytes are non-excitable cells their plasma membrane contains a rich complement of ion channels. This diverse channelome appears to be as complex as one might expect to find in excitable cells although, in the case of chondrocytes, their functions are far less well understood. The ion channels so far identified in chondrocytes include potassium channels (KATP, BK, Kv, and SK), sodium channels (epithelial sodium channels, voltage activated sodium channels), transient receptor potential calcium or non-selective cation channels and chloride channels. In this review we describe this emerging channelome and discuss the possible functions of a range of chondrocyte ion channels.
Many cell types have significant negative resting membrane potentials (RMPs) resulting from the activity of potassium-selective and chloride-selective ion channels. In excitable cells, such as neurones, rapid changes in membrane permeability underlie the generation of action potentials. Chondrocytes have less negative RMPs and the role of the RMP is not clear. Here we examine the basis of the chondrocyte RMP and possible physiological benefits. We demonstrate that maintenance of the chondrocyte RMP involves gadolinium-sensitive cation channels. Pharmacological inhibition of these channels causes the RMP to become more negative (100 µM gadolinium: ΔVm = −30 ± 4 mV). Analysis of the gadolinium-sensitive conductance reveals a high permeability to calcium ions (PCa/PNa ≈80) with little selectivity between monovalent ions; similar to that reported elsewhere for TRPV5. Detection of TRPV5 by PCR and immunohistochemistry and the sensitivity of the RMP to the TRPV5 inhibitor econazole (ΔVm = −18 ± 3 mV) suggests that the RMP may be, in part, controlled by TRPV5. We investigated the physiological advantage of the relatively positive RMP using a mathematical model in which membrane stretch activates potassium channels allowing potassium efflux to oppose osmotic water uptake. At very negative RMP potassium efflux is negligible, but at more positive RMP it is sufficient to limit volume increase. In support of our model, cells clamped at −80 mV and challenged with a reduced osmotic potential swelled approximately twice as much as cells at +10 mV. The positive RMP may be a protective adaptation that allows chondrocytes to respond to the dramatic osmotic changes, with minimal changes in cell volume. J. Cell. Physiol. 226: 2979–2986, 2011. © 2011 Wiley-Liss, Inc.
Whilst the phenomenon of an electrical resting membrane potential (RMP) is a central tenet of biology, it is nearly always discussed as a phenomenon that facilitates the propagation of action potentials in excitable tissue, muscle, and nerve. However, as ion channel research shifts beyond these tissues, it became clear that the RMP is a feature of virtually all cells studied. The RMP is maintained by the cell’s compliment of ion channels. Transcriptome sequencing is increasingly revealing that equally rich compliments of ion channels exist in both excitable and non-excitable tissue. In this review, we discuss a range of critical roles that the RMP has in a variety of cell types beyond the action potential. Whereas most biologists would perceive that the RMP is primarily about excitability, the data show that in fact excitability is only a small part of it. Emerging evidence show that a dynamic membrane potential is critical for many other processes including cell cycle, cell-volume control, proliferation, muscle contraction (even in the absence of an action potential), and wound healing. Modulation of the RMP is therefore a potential target for many new drugs targeting a range of diseases and biological functions from cancer through to wound healing and is likely to be key to the development of successful stem cell therapies.
Background and PurposeTransient receptor potential vanilloid type 4 (TRPV4) and calcium-activated potassium channels (KCa) mediate osmosensing in many tissues. Both TRPV4 and KCa channels are found in the paraventricular nucleus (PVN) of the hypothalamus, an area critical for sympathetic control of cardiovascular and renal function. Here, we have investigated whether TRPV4 channels functionally couple to KCa channels to mediate osmosensing in PVN parvocellular neurones and have characterized, pharmacologically, the subtype of KCa channel involved.Experimental ApproachWe investigated osmosensing roles for TRPV4 and KCa channels in parvocellular PVN neurones using cell-attached and whole-cell electrophysiology in mouse brain slices and rat isolated PVN neurons. Intracellular Ca2+ was recorded using Fura-2AM. The system was modelled in the NEURON simulation environment.Key ResultsHypotonic saline reduced action current frequency in hypothalamic slices; a response mimicked by TRPV4 channel agonists 4αPDD (1 μM) and GSK1016790A (100 nM), and blocked by inhibitors of either TRPV4 channels (RN1734 (5 μM) and HC067047 (300 nM) or the low-conductance calcium-activated potassium (SK) channel (UCL-1684 30 nM); iberiotoxin and TRAM-34 had no effect. Our model was compatible with coupling between TRPV4 and KCa channels, predicting the presence of positive and negative feedback loops. These predictions were verified using isolated PVN neurons. Both hypotonic challenge and 4αPDD increased intracellular Ca2+ and UCL-1684 reduced the action of hypotonic challenge.Conclusions and ImplicationsThere was functional coupling between TRPV4 and SK channels in parvocellular neurones. This mechanism contributes to osmosensing in the PVN and may provide a novel pharmacological target for the cardiovascular or renal systems.
Chondrocytes are the cells within cartilage which produce and maintain the extracellular matrix. Volume regulation in these cells is vital to their function and occurs in several different physiological and pathological contexts. Firstly, chondrocytes exist within an environment of changing osmolarity and compressive loads. Secondly, in osteoarthritic joint failure, cartilage water content changes and there is a notable increase in chondrocyte apoptosis. Thirdly, endochondral ossification requires chondrocyte swelling in association with hypertrophy. Regulatory volume decrease (RVD) and regulatory volume increase (RVI) have both been observed in articular chondrocytes and this review focuses on the mechanisms identified to account for these. There has been evidence so far to suggest TRPV4 is central to RVD; however other elements of the pathway have not yet been identified. Unlike RVD, RVI appears less robust in articular chondrocytes and there have been fewer mechanistic studies; the primary focus being on the Na+-K+-2Cl- co-transporter. The clinical significance of chondrocyte volume regulation remains unproven. Importantly however, transcript abundances of several ion channels implicated in volume control are changed in chondrocytes from osteoarthritic cartilage. A critical question is whether disturbances of volume regulation mechanisms lead to, result from or are simply coincidental to cartilage damage.
Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm2). The mean slope conductance of the principal single channels resolved within the total stretch-activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, EK+, suggesting it was a high-conductance potassium channel. Activation of these high-conductance potassium channels was inhibited by extracellular TEA (Kd approx. 900 µM) and iberiotoxin (Kd approx. 40 nM). This suggests that the current was largely carried by BK-like potassium (MaxiK) channels. To further characterize these BK-like channels, we used inside-out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1- and β1-subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction.
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