It has been found that maximum specific force (F(max); force per cross-sectional area) of rat diaphragm muscle doubles from birth to 84 days (adult). We hypothesize that this developmental change in F(max) reflects an increase in myosin heavy chain (MHC) content per half-sarcomere (an estimate of the number of cross bridges in parallel) and/or a greater force per cross bridge in fibers expressing fast MHC isoforms compared with slow and neonatal MHC isoforms (MHC(slow) and MHC(neo), respectively). Single Triton 100-X-permeabilized fibers were activated at a pCa of 4.0. MHC isoform expression was determined by SDS-PAGE. MHC content per half-sarcomere was determined by densitometric analysis and comparison to a standard curve of known MHC concentrations. MHC content per half-sarcomere progressively increased during early postnatal development. When normalized for MHC content per half-sarcomere, fibers expressing MHC(slow) and coexpressing MHC(neo) produced less force than fibers expressing fast MHC isoforms. We conclude that lower force per cross bridge in fibers expressing MHC(slow) and MHC(neo) contributes to the lower F(max) seen in early postnatal development.

Background and Purpose Although intracellular zinc accumulation has been shown to contribute to neuronal death following cerebral ischemia, the mechanism by which zinc keeps on accumulating to cause severe brain damage remains unclear. Herein the dynamic cause-effect relationships between zinc accumulation and ROS production during cerebral ischemia/reperfusion are investigated. Methods Rats were treated with zinc chelator, ROS scavenger, mitochondria-targeted ROS inhibitor, or NADPH oxidase inhibitor during a 90-minute middle cerebral artery occlusion. Cytosolic labile zinc, ROS level, cerebral infarct volume, and neurological functions were assessed after ischemia/reperfusion. Results Zinc and ROS were colocalized in neurons, leading to neuronal apoptotic death. Chelating zinc reduced ROS production at 6 and 24h after reperfusion, whereas eliminating ROS reduced zinc accumulation only at 24h. Furthermore suppression of mitochondrial ROS production reduced the total ROS level and brain damage at 6h after reperfusion, but did not change zinc accumulation, indicating that ROS is produced mainly from mitochondria during early reperfusion and the initial zinc release is upstream of ROS generation following ischemia. Suppression of NADPH oxidase decreased ROS generation, zinc accumulation and brain damage only at 24h after reperfusion, indicating that the majority of ROS is produced by NADPH oxidase at later reperfusion time. Conclusions This study provides the direct evidence that there exists a positive feedback loop between zinc accumulation and NADPH oxidase-induced ROS production, which greatly amplifies the damaging effects of both. These findings reveal that different ROS-generating source contributes to ischemia-generated ROS at different time, underscoring the critical importance of spatial and temporal factors in the interaction between ROS and zinc accumulation, and the consequent brain injury, following cerebral ischemia/reperfusion.

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