INTRODUCTION: This study was designed to examine the role of mitochondrial calcium homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial calcium is a primary modulator of cardiomyocyte TNF-, IL-1, and IL-6 responses to injury and infection. METHODS: Ventricular myocytes were prepared by perfusing hearts (Langendorff) from adult rats given either sham burn or burn injury over 40% TBSA to produce enzymatic digestion (collagenase). Isolated cardiomyocytes were suspended in minimum essential medium (MEM), cell number determined, and aliquots of myocytes from each experimental group were loaded with Fura-2AM (2 µg/ml) under three experimental conditions: 45 min at room temperature to measure total cellular calcium, 45 min at 30°C followed by incubation at 37°C for two hrs to eliminate cytosolic fluorescence, 20 min at 37°C in MnCl₂ (200 µM) -containing buffer to quench cytosolic Fura-2AM signal. In vitro studies included preparation of myocytes from control hearts and challenge of myocytes with either LPS or burn serum (BS), interventions shown previously to increase cytosolic calcium. Additional aliquots of myocytes were challenged with LPS or BS in the presence or absence of the selective inhibitor of mitochondrial calcium (ruthenium red, RR). All cells were examined on a stage inverted microscope which was interfaced with InCyt Im2^TM Fluorescence Imaging System. RESULTS: Either heat treatment or MnCl₂ challenge eliminated myocyte cytosolic fluorescence while cells maintained at room temperature retained 95% of their initial fluorescence. Compared to calcium levels measured in shams myocytes, burn trauma increased cytosolic calcium ([Ca²⁺]_C, 90±3 to 293±6 nM, p<0.05) as well as mitochondrial calcium levels ([Ca²⁺]_MT, 24±1 to 75±2 nM, p<0.05). Either LPS (25 µg/5x10⁴ cells) or BS challenge (10% by volume) for 18 hrs increased cardiomyocyte [Ca²⁺]_C and [Ca²⁺]_MT and promoted myocyte secretion of TNF-, IL-1 and IL-6. RR pretreatment decreased LPS and BS related rise in [Ca²⁺]_MT and cytokine secretion but had no effect on [Ca²⁺]_C. 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 [Ca²⁺]_MT is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis or burn trauma.