One explanation for irreversible inhibition is the destruction of the heme catalytic center leading to decreased binding in a manner that cannot be reversed. Therefore, heme a,a3 content in cytochrome c oxidase subunit I was examined. Heme a,a3 content decreased significantly 48 h after 2CLP (Fig. 2), whereas values after sham operation and CLP were unchanged.
Previous studies in liver have demonstrated a persistent decrease in the expression of key proteins in liver and lung after 2CLP (12–14,16,17). Persistently decreased expression of subunit I could also explain the alteration in Vmax observed at 48 h after 2CLP. Therefore, Northern blot analysis was used to examine steady-state levels of mRNA encoding subunit I (Fig. 3). After sham operation, there was a transient decrease in subunit I mRNA levels 3 h after intervention. From 6 h onward, levels after sham operation were equivalent or higher than values at T0. After CLP, mRNA levels decreased but returned to baseline by 16 h after operation. This is similar to observations in the liver (12–14,16,17). In contrast, steady-state levels of subunit I mRNA decreased and remained depressed at all time points after 2CLP. Again, this is consistent with observations in lung and liver (12–14,16,17).
For altered steady-state mRNA levels to be meaningful, protein levels must be altered also. Therefore, immunoblotting was used to examine cytochrome c oxidase subunit I abundance (Fig. 4). After sham operation, levels decreased early and approached baseline by 24 h. The decrease in levels after CLP and 2CLP was persistent and more pronounced in double-puncture animals during the late phase (Fig. 4).
In previous studies, we and others have used the CLP model in rodents to investigate a number of sepsis-associated abnormalities in different organ systems (15,17,24). After CLP, mice and rats develop inflammation, but recovery is the rule (14,17,21). In contrast, 2CLP results in a predictable mortality of 50% at 24 h, 75% at 48 h, and 85% to 90% at 72 h postprocedure (17,21). Chaudry and coworkers (25) have characterized cardiodynamic dysfunction in this model, showing that, up to about 20 h after 2CLP, cardiac output is increased. At subsequent time points, however, contractility and cardiac output decline. These sepsis-associated alterations in cardiac performance are well documented, but the etiology of the hypodynamic state remains obscure. Our findings offer a potential explanation: an initial reversible and later irreversible inability to generate the energy required for contraction and cellular homeostasis. In addition, although the cause of death in this model of sepsis is unknown, several investigators have speculated that it is secondary to cardiovascular collapse (26). The irreversible defect in myocardial ATP generation late in the course of severe sepsis correlates temporally with the development of late myocardial depression, supporting this proposal. Indeed, the irreversible impairment in cytochrome c oxidase function and, as a result, in ATP generation, also correlates with the high mortality seen after 2CLP and contrasts with the recovery characteristic of CLP.
One limitation of our work is the lack of a functional correlate to the kinetic abnormalities and alterations in expression of cytochrome oxidase we have reported. Chaudry's work (25,27) compared 2CLP with SO in rats and mice at 5 and 20 h postprocedure. This work has been generally accepted and universally cited. Because our methodology duplicates that of Chaudry et al. almost exactly, we believe that the function should be the same. Nevertheless, it will be important to demonstrate a correlation between altered activity and expression of cytochrome oxidase and cardiac performance at each time point after 2CLP, CLP, and SO.
Similar dysfunction in tissues other than the heart could easily reflect a loss of integrity of mitochondrial function. For example, we have recently demonstrated regenerative failure in the liver of septic mice (35). Regeneration would clearly be compromised by a defect in oxidative phosphorylation. Further studies will examine specific etiologies of myocardial cytochrome c oxidase impairment, examine other enzymes in the pathway, and seek to demonstrate a defect in other organ systems. A unified mechanism to explain the diverse abnormalities observed in sepsis, which has thus far eluded identification, may emerge.
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