Glucocorticoids Preserve Calpastatin and Troponin I during Cardiopulmonary Bypass in Immature Pigs

Abstract

Degradation of troponin I (TnI) by calpain occurs with myocardial stunning in ischemia-reperfusion injury. Glucocorticoids attenuate myocardial ischemia-reperfusion injury, but their effect on TnI degradation is unknown. A piglet model was used to test the hypotheses that cardiopulmonary bypass (CPB) and deep hypothermic circulatory arrest (DHCA) are associated with TnI degradation and that TnI alterations could be prevented by glucocorticoid treatment. Piglets were cooled to 18°C, subjected to 2 h of circulatory arrest, rewarmed to 37°C, and allowed to recover for 2 h. Methylprednisolone was administered 6 h before surgery (3 0 mg/kg) and at initiation of CPB (30 mg/kg). The untreated group received saline. Left ventricular tissue was collected after recovery and analyzed by Western blot for TnI, calpain, and calpastatin (the natural inhibitor of calpain). CPB/ DHCA animals had 27.4 Ϯ 0.2% of total detected TnI present in degraded form. Glucocorticoid treatment significantly decreased the percentage of degraded TnI (12.0 Ϯ 0.1%, p Ͻ 0.05). Calpain I and calpain II increased after CPB/DHCA compared with non-CPB/DHCA controls (p Ͻ 0.05), with or without glucocorticoid treatment. Calpastatin significantly decreased in untreated CPB/DHCA animals compared with non-CPB/DHCA controls (p Ͻ 0.05), but levels were preserved by glucocorticoids. Glucocorticoids were associated with preservation of maximum rate of increase of left ventricular pressure at 95 Ϯ 10% of baseline, whereas maximum rate of increase of left ventricular pressure decreased to 62 Ϯ 12% of baseline without steroids. TnI degradation occurs after CPB/DHCA in neonatal pigs. Reduction in reperfusion injury by glucocorticoids may depend partly on preservation of calpastatin activity and intact TnI. Surgery for congenital heart disease often requires CPB and periods of myocardial ischemia. A period of total circulatory arrest may also be required, particularly for complex neonatal repairs. In the first 8 to 12 h after CPB there is a transient decrease in myocardial function consistent with reperfusion injury (1). Although the exact mechanisms of CPB-related myocardial injury remain unknown, there is substantial evidence that CPB can produce a systemic inflammatory response or free radical injury (2-4). The cysteine protease calpain exists in the myocardium in two isoforms, calpain I and calpain II. Recently, it has been suggested that activation of calpain during reperfusion of ischemic myocardium may be a key event in the process of myocardial stunning (5, 6). Calcium influx that occurs when ischemic myocardium is reperfused promotes dissociation of calpain from its inhibitor, calpastatin. Activated calpain then degrades the contractile regulatory protein TnI by cleavage at the C-terminus to form the fragment . Additional systematic degradation of the amino terminus can lead to subsequent smaller degradation products. Degradation products and covalent complexes of TnI have been observed in humans with ischemic heart disease (7,8).The use of glucocorticoids ...

Corticosteroid effects on ventilator-induced diaphragm dysfunction in anesthetized rats depend on the dose administered

“…Interestingly, our results reveal that administration of a relatively low dose of methylprednisolone (5 mg/kg, corresponding ~0.8 mg/kg in humans [19]) exacerbates the CMV-induced diaphragm dysfunction, whereas a higher dose (30 mg/kg, corresponding ~5 mg/kg in humans [19]) completely protected the diaphragm against VIDD. The dose-depending effect of corticosteroids are in agreement with previous studies [7,8,31]. Our finding of a negative correlation between calpain activity and either diaphragmatic force production or diaphragm fiber CSA further supports the notion that calpain activation plays an important role in CMV-induced diaphragmatic atrophy and contractile dysfunction.…”

“…The positive correlation found in our study between calpastatin and diaphragm force or fiber dimensions further stress the potentially important role of calpastatin in this model. Inhibition of calpain by MP through a preservation of calpastatin levels has been previously reported in a model of cardiopulmonary bypass [31]. These findings coupled with our data suggest that high doses of corticosteroids may prevent loss of calpastatin and therefore prevent the activation of calpain in skeletal muscle.…”

“…In patients with Duchenne muscular dystrophy, treatment with prednisolone significantly improved muscle strength and this beneficial effect appeared to be associated with an increase in muscle mass probably mediated by inhibition of muscle proteolysis rather than by stimulation of muscle protein synthesis [27]. Inhibition of muscle proteolysis, in particular the calpain system, by corticosteroids has been suggested in several in vitro [7,19,28,29] and in vivo [14,30,31] studies. In addition, treatment with methylprednisolone has been shown to reduce caspase-3 mRNA and protein expression in several animal models [11-13].…”

“…In a rat model of ischemia-induced liver injury pretreatment of animals with 10 mg/kg of prednisolone (corresponding ~1.6 mg/kg in humans [19]) significantly inhibited calpain activation in the liver while lower doses (1 mg/kg, corresponding ~0.2 mg/kg in humans [19]and 3 mg/kg, corresponding ~0.5 mg/kg in humans [19]) did not [8]. Also a dose of 30 mg/kg of corticosteroids administered to piglets (corresponding ~6.5 mg/kg in humans [19]) before and during cardiopulmonary bypass was able to reduce the percentage of degraded troponin I while preserving calpastatin activity levels [31]. This is interesting knowing that the dose of 30 mg/kg is currently used in patients undergoing cardio-pulmonary bypass [32-34].…”

“…In mdx muscle fibers, a condition in which cytosolic Ca 2+ is increased, treatment with methylprednisolone attenuated the rise in cytosolic free calcium following hypo-osmotic stress[35]. On the other hand, preservation of calpastatin levels was associated with calpain inhibition after MP treatment in a piglet model of cardiopulmonary bypass[31]. Therefore, in the current experiments, it is possible that the MP treatment inhibited CMV-induced calpain activation in the diaphragm by preventing an increase in cytosolic Ca 2+ levels, preservation of calpastatin levels, or some combination of both.…”