(* em P /em 0.05 vs Tyr, + em P /em 0.05 vs MI+DZX). Discussion Isolated animal myocytes demonstrate significant swelling and reduced contractility during exposure to hypothermic hyperkalemic cardioplegia, MI, or hyposmotic stress.1C4,15,18 DZX prevents these detrimental consequences secondary to all three stresses in two animal species.1C4 These detrimental consequences may be potentiated when the stresses are combined in situations such as cardiac surgery, and these changes may underlie one mechanism of postoperative myocardial stunning. 20 minutes). Test solutions (at 4C for 5 minutes. The supernatant was discarded and the pellet resuspended in 15 mL of 37C calcium-free Tyr with 1000 mg/L bovine serum albumin (Sigma Chemical Corporation) and 925 mg/L collagenase type II (Worthington Biomedical) and agitated in a 37C water bath at 100 rpms for 20 Triisopropylsilane minutes. This solution was then centrifuged at 100at 4C for 5 minutes. The previous two steps were then repeated. The supernatant was discarded and the pellet resuspended in a 37C cell isolation solution containing (in mmol/L): potassium glutamate 120, KCl 10, KH2PO4 10, MgSO4 1.8, K2EGTA 0.5, taurine 10, HEPES 10, and glucose 20, and triturated to separate the cells. This solution was then filtered through 300 micron nylon mesh to remove large debris and centrifuged at 100at 4C for 10 minutes. The supernatant was discarded and the pellet resuspended in cell isolation solution and centrifuged again at 100at 4C for 10 minutes, three times. The supernatant was then discarded and the pellet resuspended in cell isolation solution and allowed to settle for 30 minutes.15C17 Myocyte Imaging Myocytes were used immediately on the day of isolation and were not cultured. Myocytes were visualized on a slide on a glass-bottom chamber on an inverted microscope stage (Leitz, Wetzlar, Germany) as previously described.4 An aliquot of the isolated cells was placed into the chamber and allowed to stabilize for 5 minutes, after which the chamber was perfused at a rate of 3 mL/min with Tyrode’s physiological control solution (in mmol/L): NaCl Triisopropylsilane 130, KCl 5, CaCl2 2.5, MgSO4 1.2, NaHCO3 24, Na2HPO4 1.75, and glucose 10 (buffered to a pH of 7.4 using 95% O2 to 5% CO2). Cells were evaluated for viability based on the following criteria: normal rod shape, smooth edges, sharp borders, clear striations, absence of blebbing, and lack of spontaneous contractions.18 Only viable cells were used. Cell length, width, and area were manually traced using Scion Image software (Scion Corporation, Frederick, MD) and estimated as previously described.4,18 Experimental Protocol Cells were perfused for 20 minutes with 37C control Tyr to obtain baseline volume. Any changes in cell volume secondary to the isolation or imaging protocol would be evident during this period. Myocytes were then perfused for 20 minutes with test solution followed by a 20 minutes reexposure period with 37C control Tyr. Test solutions included control Tyr (Tyr 37C, axis) vs time (axis). (*axis) vs time (axis). (*axis) vs time (axis). (*axis) vs time (axis). (* em P /em Rabbit polyclonal to OSBPL6 0.05 vs Tyr, + em P /em 0.05 vs MI+DZX). Discussion Isolated animal myocytes demonstrate significant swelling and reduced contractility during exposure to hypothermic hyperkalemic cardioplegia, MI, or hyposmotic stress.1C4,15,18 DZX prevents these detrimental consequences secondary to all three stresses in two animal species.1C4 These detrimental consequences may be potentiated when Triisopropylsilane the stresses are combined in situations such as cardiac surgery, and these changes may underlie one mechanism of postoperative myocardial stunning. This study was conducted to investigate if the same phenomena are observed in human myocytes. This study confirmed that significant myocyte swelling occurs in isolated human myocytes secondary to exposure to hyperkalemic cardioplegia, hyposmotic stress, and MI. This significant swelling was eliminated or lessened by the addition of DZX (a known KATP channel opener) with or without pharmacological inhibition of the KATP channel. This confirmation of responses in human myocytes is vital to any future translation to clinical use. Hypothermic hyperkalemic cardioplegia or exposure to hyposmotic stress results in myocyte swelling because of exposure to a hyposmolar extracellular environment. In contrast, MI results in myocyte swelling because of the development of a hyperosmolar intracellular environment. Interestingly, DZX (by an unknown mechanism) provides cellular volume homeostasis by lessening or eliminating myocyte swelling during exposure to all three stresses. It is not known if the beneficial effect of DZX observed in isolated myocytes is related to cardioprotective effects that have been documented at the whole organ or the organism level. We propose that myocyte swelling (which we have shown to be associated with decreased contractility) may be Triisopropylsilane one mechanism of myocardial stunning. DZX may therefore provide protection by maintaining.