In the early minutes of myocardial reperfusion a sudden influx of calcium activates numerous pathways resulting in myocyte dysfunction and injury, and even myocyte death secondary to contracture (16
). Calcium overload associated with ischemia and reperfusion activates calpains and reduces calpastatin levels (17
). Until recently, no in vivo
studies have examined calpain regulation of the NF-κB pathway during reperfusion of immature myocardium. In prior studies we demonstrated an association between calpain inhibition, maintenance of IκBα protein in the cytosol, and decreased NF-κB activity (3
). These changes were associated with reduced markers of inflammation (2
) and apoptotic cell injury (19
). Having demonstrated an effect of calpain inhibition on NF-κB activation, we attempted to compare the functional recovery seen with calpain inhibition with changes in NF-κB activity. In the current study, the cardiopulmonary functional recovery with NF-κB inhibition was similar to that observed in calpain inhibition studies.
Glucocorticoid treatment, which prevented the breakdown of calpastatin in myocardium after CPB/DHCA (1
) and direct administration of a peptide calpain inhibitor (2
), maintained IκBα protein levels in the cytosol and reduced NF-κB binding to DNA in the nucleus. With various modes of activation, IκBα underwent serine phosphorylation, ubiquitination, and degradation through a well-characterized proteosomal pathway (20
). However, recent studies indicated that two parallel pathways of IκB degradation exist: the proteosomal pathway and a calpain-dependent pathway that degraded IκBα after phosphorylation (21
), probably regulated by tyrosine instead of serine phosphorylation (9
). In specific cell types, such as IgM+
B cells, IκBα degradation through calpain-regulated mechanisms was more important than the proteosome pathway (8
). Tyrosine kinase regulation of NF-κB activation was especially evident during ischemic preconditioning in the heart (23
). As neonatal hearts have higher levels of calpain than adults, a possible reliance on calpain-mediated IκB regulation could exist. However the administration of high doses of corticosteroids to patients during CPB remains controversial (25
). A number of studies indicate that high dose steroids can result in detrimental postoperative alterations. Therefore the goal would be to target specific therapies to the beneficial steroid pathways that do not activate the broad spectrum of effects associated with steroid administration.
A basal level of NF-κB might be necessary to maintain the pro-survival signal in some cell types, for instance, NF-κBp65−/−
mice die at mid-gestation due to severe apoptosis in the liver (26
). Stimuli inducing NF-κB above this basal level, however, may overpower the pro-survival signal and result in cell injury or death. Although targeting a specific activity level for NF-κB would be clinically challenging the potential to affect one pathway activating NF-κB, such as the calpain-mediated pathway, while leaving other activators intact, might provide a mechanism to regulate NF-κB downstream genes without ablating basal levels.
Effect of NF-κB inhibition on pulmonary function
These data indicated a significant attenuation of the typical increase in CPB/DHCA-associated PVR by suppressing the elevation in NF-κB activity with SN50. The reduction in PVR was associated with a marked reduction in the elevated levels of endothelin-1 detected in untreated animals undergoing CPB/DHCA. Interestingly, these changes were similar to those seen with calpain inhibition (3
). Little is known about the role of calpain in regulation of PVR or pulmonary function. However, endothelin-1 is a potent and important regulator of PVR in the perioperative period. Administration of bosentan, an endothelin-1 receptor antagonist, reduces pulmonary hypertension in a very similar piglet model of hypoxia and reoxygenation on CPB (14
). While endothelin-1 production can be regulated through several pathways, NF-κB has a direct role in stimulating endothelin-1 transcription. It was not surprising therefore that NF-κB reduction with SN50 could decrease plasma endothelin-1, resulting in lower PVR after CPB/DHCA. The effect of calpain inhibition on PVR and endothelin-1 might be through regulation of NF-κB, although a direct effect of calpain on endothelin-1 levels cannot be ruled out.
Effect of NF-κB inhibition on cardiac function
The current study demonstrated a significant benefit of SN50 on myocardial function, as well as, clinically relevant signs of cardiac output. Blocking the rise in NF-κB associated with CPB/DHCA resulted in both improved systolic and diastolic function measured by maximum and minimum dP/dt, Tau, and preload recruitable stroke work. Furthermore, the reduction in cardiac dysfunction was associated with improved oxygen delivery and would be considered relevant in the clinical setting. The data are in agreement with prior studies that showed inhibition of NF-κB attenuates inflammation and apoptosis associated with myocardial ischemia and reperfusion (27
Effect of NF-κB inhibition on markers of myocardial injury
Troponin is a major contractile protein in the mammalian heart that is a target for calpain proteolytic activity. Inhibition of calpain activity prevents TnI degradation in rodent hearts (28
) and in this model (1
). The current study demonstrated a reduction in both total troponin I degradation products and the major 26 kD degradation product in SN50-treated animals. However, calpain cleavage of TnI was thought to be a direct proteolytic effect and not necessarily related to NF-κB. Nonetheless in this study, NF-κB inhibition resulted in decreased TnI degradation and was associated with preservation of calpastatin levels, indicating a possible regulation of calcium-dependent calpain activity by NF-κB. Reducing calcium influx could modulate cardiomyocyte calpain activity and the calpain-mediated degradation of calpastatin. The higher calpastatin levels with NF-κB inhibition might then be protective of troponin proteins susceptible to calpain degradation. Modalities that preserve intact myofilament proteins may reduce myocardial dysfunction after reperfusion.
NF-κB mediation of calpain activation
There are very limited indications that NF-κB can affect calpain activation, possibly through regulation of calcium influx. Nuclear factor-κB can suppress expression of Cav1.2 calcium channels in smooth muscle cells with kappaB binding sites on the L-type calcium channel gene that prevent the contractile response to acetylcholine (30
). In addition, NF-κB binds the promoter of the transient receptor potential channel 1 (TRPC1) gene in endothelial cells, which induces calcium influx after depletion of intracellular calcium stores (31
). These mechanisms of NF-κB calcium regulation might provide a link for NF-κB mediation of calpain activity, higher calpastatin levels in the heart, and NF-κB’s reduction in TnI degradation.