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

Department of Surgery, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 15 April 2004 ; accepted in final form 20 September 2004

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-, IL-1, 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 µg/ml) for 1) 45 min at room temperature to measure total cellular Ca²⁺, 2) 45 min at 30°C followed by incubation at 37°C for 2 h to eliminate cytosolic fluorescence, and 3) 20 min at 37°C in MnCl₂ (200 µM)-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 µg/5 x 10⁴ cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca²⁺ and promoted myocyte secretion of TNF-, IL-1, 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.

intracellular calcium; cardiomyocytes; fura 2-acetyoxymethyl ester; mitochondrial calcium; adult rats; tumor necrosis factor-; interleukin-1; interleukin-6; inflammatory cytokines

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