This study examined whether HSP70 could bind to and protect against thermal inactivation of SERCA1a, the SERCA isoform expressed in adult fast-twitch skeletal muscle. Sarcoplasmic reticulum vesicles prepared from rat gastrocnemius muscle were incubated with purified HSP70 at both 37 and 41°C for either 30, 60, or 120 min. Maximal SERCA1a activity (mol/g protein/min) in the absence of HSP70 was reduced progressively with time, with greater reductions occurring at 41°C compared with 37°C. HSP70 protected against thermal inactivation of SERCA1a activity at 37°C but not at 41°C and only at 30 and 60 min but not at 120 min. HSP70 also protected against reductions in binding capacity for fluorescein isothiocyanate, a fluorescent probe that binds to Lys 515 in the nucleotide binding domain of SERCA, at 30 and 60 min but not at 120 min, an effect that was independent of temperature. HEK-293 cells were cotransfected with cDNAs encoding rabbit SERCA1a and human HSP-EYFP and subjected to 40°C for 1 h. Immunohistochemistry revealed nearly complete co-localization of SERCA1a with HSP70 under these conditions. Co-immunoprecipitation showed physical interaction between HSP70 and SERCA1a under all thermal conditions both in vitro and in HEK-293 cells. Modeling showed that the fluorescein isothiocyanate-binding site of intact SERCA1a in the E2 form lies in its close proximity to a potential interaction site between SERCA1a and HSP70. These results indicate that HSP70 can bind to SERCA1a and, depending on the severity of heat stress, protect SERCA1a function by stabilizing the nucleotide binding domain. Sarco(endo)plasmic reticulum Ca2ϩ -ATPases (SERCAs) 1 are 110-kDa integral membrane proteins that catalyze the ATPdependent transport of Ca 2ϩ from the cytosol to the lumen of the sarco(endo)plasmic reticulum (SR) (1). Two SERCA isoforms predominate in adult striated muscle, namely SERCA1a and SERCA2a. SERCA1a accounts for more than 99% of the SERCA isoforms expressed in adult fast-twitch skeletal muscle, whereas SERCA2a is the major isoform expressed in heart and slow-twitch skeletal muscle (2). In muscle, SERCAs perform at least two crucial functions obligatory for precise control of intracellular Ca 2ϩ . In conjunction with plasma membrane Ca 2ϩ -ATPases, SERCAs are responsible for setting resting cytoplasmic Ca 2ϩ concentrations, and during repetitive muscle contractions, they induce muscle relaxation through the rapid sequestration of large Ca 2ϩ loads from the cytoplasm into the lumen of the SR, thereby determining the size of the SR Ca 2ϩ

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Uniformly-sized preparations with average microbubble (MB) diameters from 1 µm to 7 µm were produced reliably by sonicating decafluorobutane-saturated solutions of serum albumin and dextrose. Detailed protocols for producing and size-separating the MBs are presented, along with the effects that changing each production parameter (serum albumin concentration, sonication power, sonication time, etc.) had on MB size distribution and acoustic stability. These protocols can be used to produce MBs for experimental applications or serve as templates for developing new protocols that yield MBs with physical and acoustic properties better suited to specific applications. Size stability and ultrasonic performance quality control tests were developed to assure that successive MB preparations perform identically and to distinguish the physical and acoustic properties of identically sized MBs produced with different serum albumin-dextrose formulations and sonication parameters. MBs can be stored at 5°C for protracted periods (2 weeks to one year depending on formulation).

Background Microbubbles (MB) combined with ultrasound (US) have been shown to lyse clots without tissue plasminogen activator (tPA) both in vitro and in vivo. We evaluated sonothrombolysis with three types of MB using a rabbit embolic stroke model. Methods New Zealand White rabbits (n=74) received internal carotid angiographic embolization of single 3 day-old cylindrical clots (0.6×4.0-mm). Groups included: 1) control (n=11) embolized without treatment, 2) tPA (n=20), 3) tPA+US (n=10), 4) Perflutren Lipid MB+US (n=16), 5) albumin 3µm MB+US (n=8), and 6) tagged albumin 3µm MB+US (n=9). Treatment began 1 hour post-embolization. Ultrasound was pulsed-wave (1 MHz; 0.8 W/cm2) for 1 hour; rabbits with tPA received intravenous tPA (0.9 mg/kg) over 1 hour. Lipid MB dose was intravenous (0.16 mg/kg) over 30 minutes. Dosage of 3µm MB was 5×109 MB intravenously alone or tagged with eptifibatide and fibrin antibody over 30 minutes. Rabbits were euthanized at 24 hours. Infarct volume was determined using vital stains on brain sections. Hemorrhage was evaluated on H&E sections. Results Infarct volume percent was lower for rabbits treated with Lipid MB+US (1.0%±0.6%; P=0.013), 3µm MB+US (0.7%±0.9%; P=0.018), and tagged 3µm MB+US (0.8%±0.8%; P=0.019) compared with controls (3.5%±0.8%). The three MB types collectively had lower infarct volumes (P=0.0043) than controls. Infarct volume averaged 2.2%±0.6% and 1.7%±0.8% for rabbits treated with tPA alone and tPA+US, respectively (P=NS). Conclusions Sonothrombolysis without tPA using these MB is effective in decreasing infarct volumes. Study of human application and further MB technique development are justified.