Role of cytosolic vs. mitochondrial Ca2+ accumulation in burn injury-related myocardial inflammation and function

This study was designed to examine the role of mitochondrial Ca2+ homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial Ca2+ is a primary modulator of cardiomyocyte TNF-alpha, IL-1beta, and IL-6 responses to injury and infection. Ventricular myocytes were prepared by Langendorff perfusion of hearts from adult rats subjected to sham burn or burn injury over 40% of total body surface area to produce enzymatic ( collagenase) digestion. Isolated cardiomyocytes were suspended in MEM, cell number was determined, and aliquots of myocytes from each experimental group were loaded with fura 2-AM ( 2 mug/ml) for 1) 45 min at room temperature to measure total cellular Ca2+, 2) 45 min at 30 degreesC followed by incubation at 37 degreesC for 2 h to eliminate cytosolic fluorescence, and 3) 20 min at 37 degreesC in MnCl2 ( 200 muM)-containing buffer to quench cytosolic fura 2-AM signal. In vitro studies included preparation of myocytes from control hearts and challenge of myocytes with LPS or burn serum (BS), which have been shown to increase cytosolic Ca2+. Additional aliquots of myocytes were challenged with LPS or BS with or without a selective inhibitor of mitochondrial Ca2+, ruthenium red (RR). All cells were examined on a stage-inverted microscope that was interfaced with the InCyt Im2 fluorescence imaging system. Heat treatment or MnCl2 challenge eliminated myocyte cytosolic fluorescence, whereas cells maintained at room temperature retained 95% of their initial fluorescence. Compared with Ca2+ levels measured in sham myocytes, burn trauma increased cytosolic Ca2+ from 90 +/- 3 to 293 +/- 6 nM ( P < 0.05) and mitochondrial Ca2+ from 24 +/- 1 to 75 +/- 2 nM ( P < 0.05). LPS ( 25 mug/5 x 10(4) cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca2+ and promoted myocyte secretion of TNF-alpha, IL-1beta, and IL-6. RR pretreatment decreased LPS- and BS-related rise in mitochondrial Ca2+ and cytokine secretion but had no effect on cytosolic Ca2+. BS challenge in perfused control hearts impaired myocardial contraction/relaxation, and RR pretreatment of hearts prevented BS-related myocardial contractile dysfunction. Our data suggest that a rise in mitochondrial Ca2+ is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis and burn trauma.