Carbon dioxide elimination by cardiomyocytes: a tale of high carbonic anhydrase activity and membrane permeability.

Abstract

Life as a multi-cellular organism requires a heart with a coordinated contractile activity that must never cease, and in this regard, it is very much like the brain with a high mass-specific ‘resting’ metabolic rate. To maintain this constant activity requires a high rate of oxygen consumption and resultant carbon dioxide generation and, pari passu, the rapid exchange of large amounts of these gases with capillary blood. While oxygen has been far more the focus of the two gases in cardiac physiology, the work of Arias-Hidalgo et al.1 in this issue of the journal offers new insights and better understanding of how CO2 uniquely traverses its path from production in the mitochondria to the capillary blood and does so with much smaller driving gradients than necessary for oxygen to diffuse from the blood to mitochondria (20vs. <5mmHg). This work builds on many important and ground-breaking studies of CO2 and the heart that Gerolf Gros and his colleagues over the decades have performed. The cardiac output necessary to sustain whole body resting metabolism requires a myocardial O2 consumption and resultant CO2 production on the order of 0.1 mL g−1 min−1, roughly 20-fold greater than that of the body when compared on a g min−1 basis or 20 times that of unstimulated cultured cells.1 With maximum exercise, cardiac output must increase 5–10 times depending upon the athletic capacity of the specific creature and accordingly myocardial O2 consumption and CO2 production rise proportionately. In terms of both O2 delivery and CO2 clearance, cardiac muscle is richly vascularized and perfused to diminish the diffusing distance between cells and blood to bring oxygen to the cells and carry away carbon dioxide and other metabolic end products. To sustain a functioning intracellular acid–base milieu to permit such high rates of activity, cardiomyocytes must have mechanisms that permit CO2 to easily exit the cell into blood. A variety of proteins including several isozymes of carbonic anhydrase (CA), membrane acid–base ion transporters and membrane channel proteins all function in this capacity. Arias-Hidalgo and colleagues have notably found that isolated cardiomyocytes have a much greater CA activity than generally appreciated. This is largely due to intracellular membrane bound CA isozymes with their activity available to the cytoplasm. Furthermore, their sarcolemmal plasma membranes (second only to red cell plasma membrane) have the highest CO2 permeability of any known tissue owing to a very low cholesterol content, such that other channel proteins (aquaporins and Rh proteins) through which carbon dioxide can leave the cell have not been detected and would appear to be unnecessary. The authors employed a highly accurate and sensitive mass spectroscopy method to determine intracellular myocardial CA activity by measuring the rate of exchange of labelled 18-oxygen between water, bicarbonate and carbon dioxide. This method measures CA activity in intact cells under near-equilibrium conditions with the enzyme in its native state and environment. Their finding of intracellular CA activity in isolated cardiomyocytes is sufficient to catalyse the rate of CO2 hydration and HCO3 dehydroxylation reactions by more than 5000-fold matches that calculated by Villafuerte et al.2 by a different methodology. These catalytic values put the cardiomyocyte in the same league as other cells known to have high CA content including erythrocytes (~20 000-fold), and parietal and proximal tubular cells of the stomach and kidney respectively. Unlike the latter organs, in which CA was detected within the first decade of discovery of red cell CA in 1932, CA activity was not reported in the heart until the 1960s, first by rather indirect methods and ultimately and more definitively by Gros and colleagues in a series of papers in the 1990s (reviewed in ref. 3). Given the high flux rates of CO2 and acid production in these cells, it is still somewhat a mystery why it took so long for the heart with its very high metabolic rate to have its high CA activity established. The functions of CA in the heart are numerous with different isozymes located critically in various membranes, organelles and regions of the cell. These include enhancing the rapid egress of carbon dioxide produced by mitochondrial oxidative phosphorylation through the cytoplasm and across membranes by a process termed ‘facilitated diffusion’, which Gros and colleagues established many decades ago.4 By rapidly interconverting CO2 and HCO3−, CA permits considerable amounts of bicarbonate to co-diffuse with CO2 if adequate buffering substances such as phosphate and small proteins and peptides are present to handle the necessary H+ production and consumption. Under in vitro conditions approaching that of the cardiomyocyte, CA can enhance CO2 flux by as much as fivefold. In this sense, intracellular CA acts at a microscopic level like that of red cells with their CA, that of enhancing CO2 carriage by utilizing bicarbonate as a transport form. Other roles for CA include rapid dissipation of proton gradients and heterogeneities in regional intracellular pH, rapid release and re-uptake of calcium across the sarcolemmal and sarcoplasmic membranes for optimal cardiac contraction and relaxation,5 and supporting rapid transmembrane H+ and HCO3− exchanges for Na+ and Cl− in intracellular pH defence.2 Surprisingly, administration of acetazolamide, the prototypical CA inhibitor, does not impair cardiac function in vivo even during maximal exercise at clinical doses generally capable of inhibiting up to 99% of all CA activity in most tissues. In these studies of maximal exercise, doses of acetazolamide generally achieve free plasma drug concentrations of 10–20 μm, which in most tissues causes >99.9% inhibition.6 This either means that even a small fractional amount of remaining CA activity is sufficient or that the penetrance of acetazolamide into cardiomyocytes at these doses is less than most tissues. Although Villafuerte et al.2 did not test these lower concentrations, they did show that 100 μm was sufficient to inhibit intracellular CA activity involved in membrane Na+-HCO3− cotransport after only brief exposure times. The difficulty of studying the effect of CA inhibitors during exercise is that if higher concentrations of drug are required to fully inhibit myocardial CA, it will be difficult to determine whether any reduction in cardiac output or exercise capacity is related to a cardiac limitation or due to generalized tissue CO2 retention in the muscle occurring with red cell CA inhibition.6 Relevant to the ability of the cardiomyocyte to effectively dissipate metabolically produced CO2 by facilitated diffusion through the cell is the requirement that CO2 also easily pass out of the cell at the sarcolemmal membrane, the last barrier before entering the blood to be returned to lungs for elimination. Whereas until recently, it was thought that CO2 readily moves readily across lipid-rich bilayer cell membranes by passive diffusion alone, now it has been clearly established that at least two classes of membrane proteins, the aquaporins and Rh proteins, act as gas channels for CO2 and even possibly for other gases such as nitric oxide, oxygen and ammonia.7 Without these ‘capnoporins’, the average cholesterol-rich cell membrane is not very permeable to CO2 as discussed by Endeward et al.7 Thus, red cells that carry CO2 and must facilitate its rapid conversion to HCO3 for effective transport to the lungs have both aquaporin-1 and Rh proteins, which Gros and colleagues have demonstrated provide the major transmembrane route (>90%) for CO2 exchange.7 Interestingly, in cardiomyocytes that lack aquaporin-1 in their sarcolemmal membranes, Arias-Hidalgo et al.1 estimate from their measurements that the CO2 permeability (PCO2) of the cardiomyocyte is on the order of 0.10 cm s−1, a figure nearly equal to the cholesterol-rich red cell membrane, which however is reliant on both aquaporin 1 and the Rh proteins. The high PCO2 in the absence of gas channels is due to the very low cholesterol content (0.2 mol mol−1 total membrane lipid) of cardiomyocytes and is very like that of mitochondrial membranes (0.33 cm s−1) which also have very low cholesterol content and lack gas channel proteins.8 Figure 1 shows in simplified terms the essential elements of high rates of CO2 movement from mitochondria to capillary red cells. These involve nearly matched degrees of transmembrane passage, and passive as well as cytosolic CA-mediated facilitated CO2 diffusion. This work by Arias-Hidalgo et al.1 along with the work from the laboratory of Gerolf Gros referenced here and much earlier work that space does not permit citation clearly establishes the unique physiology and efficiency of the heart that permits high O2 consumption and power output. A combination of high intracellular CA activity in red cells and cardiomyocytes with extremely high CO2 permeability of the mitochondrial, sarcolemmal and red cell membranes allows for the large consequent production of carbon dioxide without rate-limiting build-up. To further understand this physiology, studies, if feasible, in which cardiac CA activity can be inhibited selectively leaving red cell CA activity intact are needed to test whether maximal cardiac output with normal exercise is dependent on CA activity. It is also interesting to speculate if changes with ageing, inactivity, poor diet and atherosclerosis alter membrane CO2 permeability and might underlie the reduced exercise capacity and cardiovascular health in modern society. I have no conflict of interest to declare.