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Science 333 (6048): 1440-1445

Copyright © 2011 by the American Association for the Advancement of Science

X-ROS Signaling: Rapid Mechano-Chemo Transduction in Heart

Benjamin L. Prosser1, Christopher W. Ward2,*, and W. J. Lederer1,*

1 Center for Biomedical Engineering and Technology (BioMET), University of Maryland School of Medicine, Baltimore, MD 21209, USA.
2 School of Nursing, University of Maryland, Baltimore, MD 21209, USA.


Figure 1
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Fig. 1. Stretch-activated Ca2+ release in healthy and dystrophic cardiac myocytes. (A) Single rat ventricular myocyte attached to stiff glass micro-rods coated with MyoTak (8). (B) Fluorescent surface plot of stretch-activated Ca2+ sparks in myocyte subject to 8% axial stretch. (C) (Top) Average positional output from piezo-electric length controller upon 8% diastolic stretch, which imposes a sarcomere length change of 1.84 ± 0.02 to 1.99 ± 0.03 μm. (Bottom) Ca2+ spark histogram (500ms bins) before (black), during (red), and after (blue) stretch (n = 52 rat cells). (D) Frequency of Ca2+ waves before and during stretch, under control conditions (1.8 mM [Ca2+]o, n = 52 rat cells) and under conditions of Ca2+ overload (5 mM [Ca2+]o, n = 10 rat cells). (E) Fluorescent surface plot demonstrating stretch-induced Ca2+ wave in dystrophic (mdx) myocyte. (F) Frequency of Ca2+ waves in mdx myocytes (n = 38 cells) compared with WT myocytes (n = 25 cells) before and during stretch. *P < 0.05 compared with rest value. #P < 0.05 compared with control conditions value (D) or WT value (F); paired t test.

 

Figure 2
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Fig. 2. ROS generation and microtubule integrity underlie stretch-activated Ca2+ sparks. (A) Stretch-activated Ca2+ sparks in 14 rat cardiomyocytes in normal Tyrode’s (NT), after 5 min local superfusion with 10 mM NAC-NT, and upon washout in NT. (B) (Top) Ca2+ spark histogram in untreated control rat myocytes (n = 11 cells, filled boxes) and in myocytes treated with 3 μM DPI (n = 11 cells, hashed boxes) before (black), during (red), and after (blue) stretch. (Bottom) Quantitation of spark rate in these cells. (C) Average baseline-corrected (fig. S5) DCF fluorescence time course in 36 rat myocytes subjected to 8% stretch (black). The derivative (red trace) of a polynomial fit (green trace, r2 = 0.96) to the DCF data reveals the time course of ROS production. (D) Average DCF fluorescence time course in control (black, n = 19 cells), colchicine-treated (10 μM, blue, n = 9 cells), and DPI-treated (3 μM, red, n = 14 cells) rat myocytes. (E) Quantification of DCF slope before (black), during (red), and after (blue) stretch. Data normalized to pre-stretch slope. *P < 0.05 compared with rest value; paired t test.

 

Figure 3
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Fig. 3. NOX2 in the t-system produces stretch-dependent ROS that sensitizes nearby RyR2s. (A) Quantification of DCF slope before (black), during (red), and after (blue) stretch in rat myocytes treated with 1 μM gp91ds-tat–scramble peptide (n = 6 cells), 1 μM gp91ds-tat (n = 10 cells), 3 μM gp91ds-tat (n = 9 cells), 20 μM rac1 inhibitor (n = 7 cells), or in the nonstretched region of cells treated with scramble peptide (n = 6 cells). Data was normalized to prestretch slope. (B) DCF fluorescence time course with stretch in WT and NOX2–/– myocytes (n = 6 cells). (C) Quantification of DCF slope before (black), during (red), and after (blue) stretch. (D) (Top) WT myocyte pre-incubated with 1 μM FAM-labeled gp91ds-tat shows sarcolemma, intercalated disc, and Z-line staining. (Bottom) High-magnification image of WT myocyte co-stained with Di-D (t-tubules) and FAM-labeled gp91ds-tat. Fluorescence plot-profile from region enclosed by dotted lines shows overlay of t-tubules and NOX2 inhibitory peptide. (E) (Top) NOX2–/– myocyte pre-incubated with 1 μM FAM-labeled gp91ds-tat and imaged with identical settings as (D) shows only diffuse staining. (Bottom) High-magnification image of NOX2–/– myocyte co-stained with Di-D and FAM-labeled gp91ds-tat shows loss of t-tubule staining pattern for gp91ds. (F and G) Line-scan image of Fluo-4 signal from electrically stimulated rat ventricular myocyte in the presence of 2.5 μM nifedipine at resting length (F) and upon 8% diastolic stretch (G). Red mark denotes electrical stimulation (fig. S9, protocol details). White bars mark active calcium release units (CRUs) rapidly triggered by depolarization. (H) Quantification of the percentage of active CRUs triggered immediately (within 10 ms) upon depolarization in cells held at resting length (black) or upon stretch (red) in control myocytes (n = 11 cells) or those treated with 1 μM gp91ds-tat (n = 6 cells). *P < 0.05 compared with rest values; paired t test.

 

Figure 4
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Fig. 4. Stretch-induced ROS generation and aberrant Ca2+ release in dystrophic myocytes. (A) Average DCF fluorescence time course in WT myocytes treated with 1 μM gp91ds-tat–scramble (black, n = 10 cells) or 1 μM gp91ds-tat (red, n = 9 cells) and mdx myocytes treated with 1 μM gp91ds-tat–scramble (blue, n = 9 cells) or 1 μM gp91ds-tat (green, n = 9 cells). (B) Quantification of DCF slope before (black), during (red), and after (blue) stretch. Data normalized to prestretch slope. (C) Ca2+ wave frequency before (black), during (red), and after (blue) stretch. (D) Percentage of cells demonstrating Ca2+ oscillations during stretch protocols. (E) Fluorescence surface plot of mdx myocyte treated with 1 μM gp91ds-tat–scramble demonstrating stretch-induced Ca2+ oscillations. *P < 0.05 compared with rest value; paired t test. #P < 0.05 compared with WT value; paired t test.

 


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