New Stretch Dependent Signaling Pathway Discovered in Cardiac Ventricular Myocytes termed “X-ROS” MUSCLE PHYSIOLOGY STRETCH DEPENDENT XROS SIGNALING: RAPID MECHANO-TRANSDUCTION IN HEART, Benjamin L. Prosser1, Christopher W. Ward2, Brian Hagen1, Ramzi J. Khairallah1, W. Jonathan Lederer1. University of Maryland, Baltimore, MD, USA. The manipulators tilt up and fold back to facilitate system setup. There are two programmable memory positions (home and target) for easy exchange of the 35mm dishes, providing access to the bath so you can add the live cells. The combination of these features enables high experimental throughput. The SI-CTS200 system utilizes a unique rotating bath to dramatically improve experimental throughput. It is designed to orient cells in the XY plane so that no physical manipulation of the position of the cell itself is required prior to capture by the grabbing devices attached to the force sensor and linear actuator. This bath has two interchangeable inserts. The first holds any 35mm glass bottom dish (WPI #FD35-100). The second is a native cuvette insert containing the live cells. A confocal microscope in linescan mode is used to record calcium spikes. The healthy cell in the top image shows a marked increase in number and intensity of calcium sparks over the duration of the stretch. The diseased muscle in the bottom image shows fewer sparks when at rest and a profound increase in sparks of short duration during the stretch. Introduction ●● Control of cardiac Ca2+ release is critical for the regulation of contraction and maintenance of electrical activity. ●● A new signaling pathway was discovered: X-ROS signaling. ●● It regulates normal Ca2+ release in healthy heart cells and may drive pathologic Ca2+ release in diseased cells. Results ●● Stretch triggers the generation of ROS by NOX2 ●● Stretch-dependent process activates NOX2 production of ROS which reversibly oxidizes nearby RyR2 ryanodine receptors. ●● X-ROS oxidation tunes the sensitivity of the RyR2s, increasing the Ca2+ spark rate and enhancement of Ca2+ signaling. ●● During a sustained stretch of a cardiomyocyte in a Cell Tester, a rapid elevation of ROS production subsides over the duration of stretch. ●● During a repetitive cyclical stretch, a new level of steady-state ROS production is maintained. Conclusion The level of steady state ROS generation in the cell may be graded by diastolic length or pre-load. REFERENCE: X-ROS signaling: rapid mechano-chemo transduction in heart. Prosser BL, Ward CW, Lederer WJ. Science. 2011 Sep 9;333(6048):1440-5. PMID: Signal Conditioning Amplifier System with CTS200 Electronics Sarc. Len: 1.95µm SI-BAM21-LCB Optical Transducer Amplifier SI-CISB Position Control Module SI-AOSUB Anti Oscillation Unit SI-TCM2B Temperature Control Module Expansion Slots Power Switch SI-CTS200 Complete Cell Tester System Sarc. Len: 2.38µm System Includes: Rotating Cuvette System; Micromanipulator System; Signal Conditioning Amplifier with four modules: Optical Transducer Amplifier; Temperature Controller; AntiOscillation Unit; Position Controller; data acquisition system with MDAC Software for recording, controlling stimulation and nanomotor position; Force Transducer of choice; Nanomotor; Glass Fiber Cell Mounts (1 set); MyoTak Bio-adhesive kit (5-week supply) SI-CTS200B SI-CTS200A Cell Tester, Non-Rotating, No Micromanipulators Cell Tester, Manual Platform, No Micromanipulators UK: Tel: +44 (0)1462 424700 • wpiuk@wpi-europe.com A single skeletal muscle cell is held with microtweezers. The top image shows the unstretched cell, and the bottom shows the stretched cell. 3 World Precision Instruments www.wpiinc.com Germany: Tel: +49 (0)30-6188845 • wpide@wpi-europe.com US: Tel: 941-371-1003 • sales@wpiinc.com