a) Main Findings
This computational work, based on our published model of the healthy adult chondrocyte membrane potential 38, confirms that, the electrogenic current generated by the Na+/K+ pump is strongly temperature-dependent. This net outward current is relatively small. However, it can have substantial hyperpolarizing influences on the resting membrane potential of the chondrocyte due mainly to the very high input resistance, approximately 10 Giga-ohms 14,55,56 of this cell. In both physiological and pathophysiological settings it is likely that the Na+/K+ pump in the chondrocyte is strongly activated due to the relatively high intracellular Na+ levels (~20 mM or more) in this cell type 10,43,70.
b) Previous Findings
Hall and colleagues first reported evidence for energy-requiring active transport of K+ across the surface membrane of healthy adult mammalian chondrocytes 71. This initial observation was supported and put into a conventional cell physiology context by Mobasheri and colleagues 39 who adapted 3H-labeled ouabain binding methods to demonstrate substantial expression of the Na+/K+ pump alpha subunit in mammalian chondrocytes. Mobasheri et al. then confirmed and extended these findings based on experimental 3H-labeled ouabain binding and confocal immunofluorescence microscopy work 39,40. They demonstrated regulation of surface expression by changes in levels of intra- and extracellular Na+ in isolated cells 44,72 and in the extracellular matrix of articular cartilage from healthy bovine joints 43 and also reported a scheme for overall Na+ regulation based on data that identified functional roles in the chondrocyte for Na+/Ca2+ exchange, Na+/H+ exchange, and antiporter exchange due to Na+/K+/Cl- expression 10. These papers and subsequent work have specified the essential role of the Na+/K+ pump and overall regulation of intracellular Na+ levels in volume regulation of the chondrocyte. It is also known that regulation of volume in the chondrocyte depends, in part, on the membrane potential of these cells 20,73.
c) Physiological Effects of the Electrogenic Na+/K+ Pump in Chondrocytes
Insights gained from our mathematical modeling support the working hypothesis that the Na+/K+ pump can strongly regulate the resting membrane potential in chondrocytes from healthy and diseased adult articular cartilage. Specifically, it is plausible that due to its expression levels, turnover rates 74 and intrinsic voltage dependence 75,76 this pump mechanism produces a hyperpolarizing influence that can be as large as 30 mV. This hyperpolarization would be expected to modulate volume regulation; in addition, however, it is also likely to markedly alter the overall electrophysiological function or electrophysiological operating point of the chondrocyte 77. This is because a number of the other ion channels that are expressed in the chondrocyte, e.g., L-type Ca2+ channels 15,27,78–80, delayed rectifier K+ channels 55,57, and 2-pore K+ channels 56 exhibit strong intrinsic voltage dependence in the range -40 to -80 mV. Accordingly, the hyperpolarizing influence of the Na+/K+ pump significantly regulates the activation and/or deactivation of these (and perhaps other) ion channels in the chondrocyte.
We note, however, that the singular focus on the physiological and pathophysiological effects of the electrogenic current produced by the Na+/K+ pump in this study could be somewhat misleading. When the chondrocyte is stimulated by stretch, or activated by ligands such as histamine or ATP, agonist-induced ion fluxes through either piezo 81, Cl- 77, or TRP channels 14,77,82,83 will reduce the input resistance of the cell. The resulting parallel conductance will partially ‘shunt’ the influence of the electrogenic current generated by the Na+/K+ pump. In addition, in both health and disease, the chondrocyte exists and functions in a relatively hypoxic environment 84–86. In this setting, the supply of ATP as the principal energy source of energy for chondrocytes 87 may limit pump activity to a range that is less than the maximal currents shown in the Figures 88,89.
d) Concluding Remarks
Although we acknowledge the possibility that the Na+/K+ pump function in mammalian chondrocytes may be modulated by altered expression levels of selected isoforms of the a, b, and g subunits, our analysis is not sufficiently complete to fully examine the consequences as has been done in other tissues e.g., skeletal muscle 74. It is known that the Na+/K+ pump can be strongly modulated by altered redox conditions 90 such as those that occur in sterile inflammation, or “low grade inflammation” in the context of chondrocyte biology and OA 91,92. Our work provides a basis for this type of analysis but important pathophysiological effects such as this have not been studied. Finally, both classical findings and more recent detailed analyses have drawn attention to conditions under which changes in intracellular Ca2+ can markedly alter the function of the Na+/K+ pump 93. Our model, at its present state of development, cannot be used to simulate these Ca2+-dependent effects due to the simplistic formulations now used for intracellular Ca2+ buffering and Na+/Ca2+ exchange; in the absence of mathematical descriptors for Ca2+ pumps and Ca2+-sensitive channels localized to the endoplasmic reticulum 94. These additions and other improvements will be needed before the mathematical modeling approach used in this study can be extended to analysis of ion homeostasis and the chondrocyte channelome 16,22 in a more physiological context, specifically in chondron units, which represent the chondrocyte and its immediate pericellular environment 95. Further model development is also needed before our simulations can provide insights into the altered articular joint electrolyte homeostasis resulting from disease-producing point mutations in one or more of the Na+/K+ pump subunits or accessory proteins 69.
From a tissue engineering perspective, there is ongoing interest in the Na+/K+ pump, ion transport and the modulation of intracellular Na+ by pharmacological agents such as ouabain and bumetanide as in vitro treatments for altering intracellular ion concentrations as a viable method for manipulating ECM synthesis by chondrocytes and enhancing the mechanical properties of engineered articular cartilage 96.
Finally, in the context of drug development and screening for diseases such as OA, which is known to be characterized by cellular senescence, or “chondrosenescence” 29,97, the Na+/K+ pump has already been identified as a candidate target for modulation by cardiac glycosides, re-entering the limelight as classical cardiotonic drugs re-invented as senolytic compounds 98. Thus, the modeling approaches described in this paper can support drug development for OA and related osteoarticular disorders as well as understanding of the basic cellular physiology and pathophysiology of joint tissues.