Role of cytosolic vs. mitochondrial Ca 2+ accumulation in burn injury-related myocardial inflammation and function

• Published in: American journal of physiology. Heart and circulatory physiology 2005-02, Vol.288 (2), p.H744-H751
• Main Authors: Maass, David L., White, Jean, Sanders, Billy, Horton, Jureta W.
• Format: Article
• Language: English
• ISSN: 0363-6135; 1522-1539
• DOI: 10.1152/ajpheart.00367.2004

This study was designed to examine the role of mitochondrial Ca 2+ homeostasis in burn-related myocardial inflammation. We hypothesized that mitochondrial Ca 2+ is a primary modulator of cardiomyocyte TNF-α, IL-1β, 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 Ca 2+ , 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 MnCl 2 (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 Ca 2+ . Additional aliquots of myocytes were challenged with LPS or BS with or without a selective inhibitor of mitochondrial Ca 2+ , 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 MnCl 2 challenge eliminated myocyte cytosolic fluorescence, whereas cells maintained at room temperature retained 95% of their initial fluorescence. Compared with Ca 2+ levels measured in sham myocytes, burn trauma increased cytosolic Ca 2+ from 90 ± 3 to 293 ± 6 nM ( P < 0.05) and mitochondrial Ca 2+ from 24 ± 1 to 75 ± 2 nM ( P < 0.05). LPS (25 μg/5 × 10 4 cells) or BS (10% by volume) challenge for 18 h increased cardiomyocyte cytosolic and mitochondrial Ca 2+ and promoted myocyte secretion of TNF-α, IL-1β, and IL-6. RR pretreatment decreased LPS- and BS-related rise in mitochondrial Ca 2+ and cytokine secretion but had no effect on cytosolic Ca 2+ . 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 Ca 2+ is one modulator of myocardial inflammation and dysfunction in injury states such as sepsis and burn trauma.