Energetics of Ca2+ homeostasis during ischemia–reperfusion on neonatal rat hearts under high-[K+] cardioplegia

Abstract

The mechanocalorimetric consequences and mechanisms involved in Ca2+ homeostasis during ischemia–reperfusion (I/R) as well as the protective role of cardioplegic pretreatment with high [K+] (25 mmol/L) and low or near-normal [Ca2+] (0.5 or 2 mmol/L) were evaluated in a model of neonatal rat heart. Beating hearts from 10–12-day-old rats were perfused with Krebs solution (2 mmol/L Ca2+) under both isotonic and isometric conditions. During pretreatment, hearts were exposed for 20 min to either Krebs (control) or cardioplegia (CPG) before 15 min ischemia and 45 min reperfusion while being continuously measured for either contractility or total heat rate (Ht) in a flow calorimeter. Contractile recovery after reperfusion in hearts exposed to ischemia only (control) was higher in the isometric hearts under optimal length (87.9% ± 8.1%) than in the isotonic hearts (57.3% ± 10.6%). This same behavior was found in hearts pretreated with CPG-0.5 mmol/L Ca2+. Ht in controls was reduced from 11.5 ± 0.8 mW/g in the initial beating condition to 1.11 ± 0.33 mW/g during ischemia and was increased to 13.02 ± 0.93 mW/g (113.8% ± 5.0% of preischemic) after reperfusion. Hearts pretreated with CPG-0.5 mmol/L Ca2+ showed the same behavior. However, when extracellular calcium ([Ca]o) was increased to 2 mmol/L under CPG, isotonic hearts, but not isometric hearts, significantly increased the contractile recovery to a maximum of 88.7% ± 10.8% of preischemic levels. Ht was recovered to 92.1% ± 4.3% of preischemic, suggesting that contractile recovery was less energetically expensive after CPG-2 mmol/L Ca2+ than it was in postischemic hearts exposed to control or CPG-0.5 mmol/L Ca2+. The role of the sarcoplasmic reticulum store was evaluated by pretreating hearts with 10 mmol/L caffeine, which reduced contractile recovery only under isometric conditions or after increasing [Ca]o in CPG under isotonic conditions, suggesting that the contribution of the sarcoplasmic reticulum was dependent on the fibre length or the [Ca]o. The inhibition of the reverse mode of the sarcolemmal Na/Ca exchanger (NCX) and the mitochondrial Ca uniporter (CaU) by KB-R7943 (KBR) at 5 µmol/L in CPG-0.5 mmol/L Ca2+ improved contractile recovery of isotonic hearts, whereas it decreased Ht at the start of reperfusion, suggesting that mitochondria could uptake Ca2+ vía the mitochondrial CaU. Neither the positive inotropism nor Ht were changed by inhibiting the mitochondrial NCX with 10 µmol/L clonazepam in CPG-0.5 mmol/L Ca2+ + 5 µmol/L KBR, which suggests that the mitochondrial NCX does not have a role. Finally, the role of the forward mode of the sarcolemmal NCX was evidenced by the fall in contractile recovery with increased Ht when KBR was increased to 20 µmol/L and added to CPG-2 mmol/L Ca2+ + 10 mmol/L caffeine before I/R. Thus the sarcolemmal NCX was essential for removing the diastolic Ca2+ during the periods of CPG and I/R. In summary, Ca2+ homeostasis during I/R of neonatal rat hearts is different from that of adult rats. High-[K+] CPG protected neonatal hearts only under isotonic conditions, at a near-normal [Ca]o, or by exposure to KBR. Mitochondria were able to uptake Ca2+ via the mitochondrial CaU and reduce the Ca2+ available for contractile recovery. Nevertheless, after increasing [Ca]o in CPG, the sarcoplasmic reticulum had a main role in restoring contractility during reperfusion, as it does in adults. Thus, the degree of maturation of the heart must be taken into account to evaluate the effects of CPG and drugs on I/R.