In the experimental setting, 5 μg of PP2Cα siRNA and scrambled siRNA were electroporated into contralateral Gas muscles (alternating right and left between animals). Three days later, mice were implanted subcutaneously with osmotic minipumps (Alzet 1007D) continuously infusing either sterile saline (control) or 1,000 ng/kg/min AngII (Phoenix Pharmaceuticals). This dose of AngII yields a 2.8-fold increase in plasma AngII that is within the pathophysiological ranges observed in patients with congestive heart failure (CHF) and chronic kidney disease [12 (link),14 (link),27 (link)-30 (link)]. Mice were sacrificed after another 4 days, muscles were collected, weighed, and either used fresh or embedded in Allprotect Tissue Reagent (Qiagen) and stored at -80°C until processing. Cryosections were prepared by incubating Gas muscles in 50% optimal cutting temperature (OCT) compound (Tissue-Tek, USA) for 15 min on ice followed by freezing in 100% OCT. Eight micron serial cross sections were prepared from the middle of each muscle and kept at -80°C until processing. The animal protocols were approved by Tulane University Institutional Animal Care and Use Committee.
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