Mechanotransduction, the process of converting mechanical stimuli into cellular responses, enables cells to produce signals that regulate a wide range of physiological responses. In the beating heart, for example, the stretching of muscle cells causes the release of chemical signals that regulate heart function, and studies in mice and humans have suggested a connection between faulty stretch-sensing mechanisms and heart disease ( 1). The mechanisms underlying such processes, however, have been unclear. On page 1440 of this issue, Prosser et al. ( 2) use a novel method that involves precisely stretching single heart muscle cells (cardiomyocytes) that have been glued to microscopic glass rods to provide some clarity. They demonstrate that a moderate stretch during the cell’s relaxed state (diastole) can trigger a burst of calcium “sparks.” They also show that this process is defective in a lifethreatening muscle disease. Mechanotransduction has an important role in the myocardium, the heart’s muscular tissue. Each contraction phase (systole) of the cardiac cycle causes sarcomeres (the basic unit of muscle) to shorten; the sarcomeres then lengthen again during diastole. In the early 1900s, European researchers Otto Frank and Ernest Starling showed that an increased length change during diastole produces a stronger contraction in the following systole. To better understand the mechanisms underlying cardiac mechanotransduction, Prosser et al. developed new tools to apply a controlled and moderate stretch (8%) to isolated rat or mice cardiomyocytes during diastole, and then measured intracellular levels of calcium ions (Ca2+) and reactive oxygen species (ROS) before, during, and after stretch. They observed that stretch initiated, within milliseconds, a burst of Ca2+ sparks—highly localized and temporary increases in intracellular Ca2+ concentration—and a nearly instantaneous increase in the rate of ROS production. Both Ca2+ spark generation and ROS production immediately returned to baseline levels after the cell was restored to its initial length. They demonstrated that the stretch-induced burst of Ca2+ sparks requires ROS; introducing an antioxidant molecule prevented the sparks, whereas a mild oxidant enhanced them. Prosser et al. also studied the behavior of single cardiomyocytes from mice that have a genetic mutation that causes a muscle disease similar to human Duchenne muscular dystrophy. Myocytes from these mdx mice display greater nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity— and higher cellular ROS levels—than myocytes from wild-type mice ( 3). Prosser et al. report that moderately stretching mdx cells produced Ca2+ waves instead of sparks; waves are typical responses of abnormal cardiac Ca2+ signaling. Prosser et al. propose that the process they call X-ROS signaling produces these results. Past studies had described increased Ca2+ spark generation in response to stretching of single cardiomyocytes ( 4– 6), and implicated ROS in the enhanced Ca2+ sensitivity of mdx cardiomyocytes ( 3, 7), but the molecular mechanisms were unresolved. Now, using pharmacological and molecular techniques, Prosser et al. show that moderate diastolic stretch activates the enzyme complex NADPH oxidase 2 (NOX2), which they found colocalized with markers for the transverse tubule (T-T) system formed by invaginations of the muscle fi ber’s plasma membrane. NOX2, in turn, directly mediates ROS-dependent Ca2+ spark generation (see the fi gure). The fi nding that dystrophic heart muscle has an excessive X-ROS signaling response advances our knowledge of the mechanisms underlying abnormal Ca2+ signaling in this disease. Prosser et al. also propose that stretchinduced ROS production increases Ca2+ spark generation by sensitizing ryanodine receptor type 2 (RyR2) channels located in the nearby sarcoplasmic reticulum (SR), the intra cellular membrane network that surrounds myofi brils. The SR releases and recaptures Ca2+ in each contraction-relaxation cycle that underlies the heartbeat. The cycle starts with Ca2+ entry into cardiomyocytes through voltage-activated channels located in the T-T system. Next, Ca2+ entry stimulates the opening of RyR2 Ca2+ release channels. This cellular response, known as Ca2+-induced Ca2+ release (CICR), causes muscle contraction. The cycle ends with relaxation, which occurs when intracellular Ca2+ returns to resting levels ( 8). The Ca2+ sensitivity of RyR2 channels is a key feature in CICR regulation. Alterations in RyR2 Ca2+ sensitivity, which is infl uenced by cellular factors and RyR2 redox state ( 9, 10), may underlie subcellular changes in Ca2+ signaling that contribute to disease ( 11). Although researchers reported more than a decade ago that the Ca2+ sensitivity of single RyR2 channels is redox-dependent ( 12), Prosser et al. demonstrate that a Ca2+ spark burst results from the very fast and reversible X-ROS signaling, which requires an intact microtubule network. Previous studies indicated that activation of cardiac NOX2 increases RyR2 S-glutathionylation, a reversible redox modifi cation that enhances RyR2 activity and hence promotes SR Ca2+ release ( 13). Tachycardia (accelerated heart rate) and exercise augment these effects ( 14), suggesting a direct correlation between increased heart activity, NOX2 activation, increased RyR2 S-glutathionylation, and enhanced Ca2+ release. It remains unclear, however, if the Ca2+ spark burst induced by controlled stretch entails RyR2 S-glutathionylation. In addition, the cellular mechanisms that so effi ciently return ROS production and RyR2 activity to baseline levels after stretch remain unclear, as do the molecular mechanisms that enable extremely fast microtubule-dependent NOX2 activation. For example, do angiotensin receptors mediate this response, as they mediate the slow stretch response of the myocardium ( 15, 16)?
From : SCIENCE 9 SEPTEMBER 2011
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