These data provide a mechanism by which glucocorticoids might preserve myocardial function in neonatal myocytes subjected to CPB and DHCA. Although the benefits of high dose steroids in conjuction with DHCA remains controversial, especially in regards to cerebral protection [27
], there is evidence that glucocorticoids are cardioprotective after CPB and DHCA in animal models [20
]. The pre- and intra-operative doses used in this study have also been shown to decrease post-operative inflammatory markers in pediatric cardiac surgery patients [5
]. The load-independent measures used to assess ventricular function using pressure-volume relationships reaffirm the ventricular dysfunction associated with CPB and DHCA and the benefit of glucocorticoid therapy detected in our previous studies [20
]. The ability to associate these measures of myocardial contractility in an intact heart with calcium cycling in myocytes harvested from the same hearts is a unique feature of this study. For example, PRSW, a measure of the systolic function of the heart, and Tau, a measure of the rate of relaxation and diastolic function, were unchanged from controls in glucocorticoid-treated animals, as was calcium transient amplitude and time of transient decline. In contrast, untreated animals had decreased PRSW and longer Tau in the intact heart that was associated with a drop in calcium transient amplitude and extended calcium transient time in myocytes isolated from untreated hearts after undergoing CPB. The rate of relaxation in the intact heart is reflective of the intracellular calcium transient decline [30
]. Global ischemia reduces the rate of relaxation in the myocyte with subsequent diastolic dysfunction and is closely related to intracellular calcium homeostasis [30
]. In addition, diastolic stiffness, evident in the increased EDPVR after CPB and DHCA in this study, can be ascribed to incomplete diastolic clearance of calcium resulting in persistent tension in the ventricles [30
Regulation of cytosolic calcium occurs through extracellular calcium transport by membrane proteins, such as L-type calcium channels, and intracellular cycling through the SR, mediated by SERCA uptake and ryanodine SR-release channels [32
]. Specifically, calcium reuptake from the cytosol into the SR depends upon the expression level of the SERCA calcium pump and the pump’s affinity for calcium, which is mediated by the phosphorylation state of PLB [17
]. Phospholamban in the phosphorylated state increases the activity of SERCA and uptake of calcium into the sarcoplasmic reticulum [17
]. Phospholamban serine-16 and threonine-17 are phosphorylated by protein kinase A and calcium/calmodulin-dependent protein kinase, respectively. Phosphorylation at either amino acid, separately or in tandem, is sufficient to increase cardiac relaxation rates in vivo
Levels of SERCA2a protein in the myocardium are linked to ventricular function with reduced SERCA2a expression correlating with heart failure. The decreased SERCA2a levels and dephosphorylation of PLB after CPB and DHCA in this study agrees with other models of ischemia and reperfusion including Langendorff-perfused hearts [30
] and isolated cardiac myocytes [31
]. The increase in dephosphorylated PLB at both serine-16 and threonine-17 after CPB and DHCA might be responsible, at least in part, for the decline in calcium transient amplitude and the increase in transient time. In addition, the maintenance of PLB in the phosphorylated state with glucocorticoid therapy is reflected in the prevention of the decline in vitro
calcium transients and the in vivo
ventricular function associated with CPB and DHCA.
Our prior findings of calpastatin preservation and a reduction in calpain activity with glucocorticoid therapy prior to and during CPB and DHCA may help to elucidate the mechanisms underlying the beneficial effects of glucocorticoids [20
]. Calpain, which in addition to directly degrading cytoskeletal and contractile proteins, can degrade calcium transport proteins, as SERCA2a [14
], and calcium channels [34
]. French and colleagues demonstrated that ischemia and reperfusion induced calpain activation that resulted in SERCA2a degradation in rats. In addition, administration of a calpain inhibitor protected against myocardial SERCA2a degradation and maintained cardiac function after ischemia and reperfusion [14
]. The maintenance of SERCA2a content with glucocorticoid therapy in this study might be, in part, a result of the prevention of calpain activation after CPB and DHCA.
In addition to affects on SR proteins, calpastatin, the endogenous inhibitor of calpains, might also have a direct effect on maintaining L-type calcium channel function. The L-domain of calpastatin can prevent L-type calcium channel run-down [35
] and reprime channels [36
] by interacting with the calmodulin binding site of the L-type Cav1.2 channel [38
]. Furthermore, the effects of calpastatin on calcium channel activity are independent of any calpain inhibitory actions [35
]. Hence, glucocorticoid-mediated preservation of calpastatin after ischemia and reperfusion might also directly improve calcium dynamics in cardiac myocytes.
One limitation of this study is that we have examined only the SR calcium re-uptake regulatory proteins. Further studies will investigate calcium release from the SR and the contribution of extracellular calcium through ion channel activation.
In summary, these data demonstrated that glucocorticoids preserve intracellular calcium cycling proteins in isolated cardiac myocytes. Although the mechanisms by which glucocorticoids maintain calcium handling are not entirely clear, glucocorticoids can reduce ischemia and reperfusion-induced calpain activation and subsequent inactivation of calcium transport proteins. This study provides further evidence of the multiple and diverse mechanisms by which glucocorticoids might protect the myocardium and continues to support the clinical use of glucocorticoids to minimize ischemia and reperfusion injury.