Hugo Aguilar,* Eduardo Fricovsky,* Sang Ihm, Magdalena Schimke, Lisandro Maya-Ramos, Nakon Aroonsakool, Guillermo Ceballos, Wolfgang Dillmann, Francisco Villarreal, and Israel Ramirez-Sanchez Seccion de Posgrado, Escuela Superior de Medicina, Instituto Politecnico Nacional, Ciudad de Mexico, Mexico, Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California; Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California; University of Innsbruck, Innsbruck, Austria; and The Catholic University of Korea, Seoul, South Korea

Eduardo S. Fricovsky, Jorge Suarez, Sang-Hyun Ihm, Brian T. Scott, Jorge A. Suarez-Ramirez, Indroneal Banerjee, Moises Torres-Gonzalez, Hong Wang, Irina Ellrott, Lisandro Maya-Ramos, Francisco Villarreal, and Wolfgang H. Dillmann Department of Medicine, University of California, San Diego, La Jolla, California; and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, California

Cardiac relaxation partially depends on the expression of the sarcoplasmic, reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on cardiac SR Ca2+ handling under normal and pathological conditions we generated a new transgenic rats model expressing a human cytomegalovirus enhancer/chicken beta-actin promotor-controlled rat SERCA2a transgene. Characterization of a heterozygous transgenic rat line (L1167) showed that the steady-state SERCA2 mRNA and protein levels increased by +69% and +25%, respectively, relative to wild-type rats. The levels of mRNA encoded by some of the other genes involved in cardiac Ca2+ control, such as phospholamban and Na+/Ca2+ exchanger, remained unchanged. Functional analysis of SR Ca2+ handling in isolated membranes in the presence of the synthetic protein kinase A inhibitor peptide [PKI(6-22)amide] indicated that the rate of oxalate-supported Ca2+ uptake was increased in average by 49% at free Ca2+ concentrations ranging from 0.5 to 3.7 muM if compared to wildtype controls. The sensitivity of uptake to the specific SR Ca2+-ATPase inhibitor thapsigargin was similar in transgenic and wild-type animals (IC50:3.4 +/- 0.7 vs. 3.8 +/- 0.4 nM, respectively). Cardiac expression of the SERCA2a transgene also occurred in streptozotocin-induced diabetes mellitus and propylthiouracil-induced hypothyroidism and this rescued, at least partially, the compromised cardiac SR Catransport in these diseased conditions. At 3.7 muM free Ca2+, homc genate SR Ca2+ uptake of hypothyroid and diabetic transgenic animals was 42% and 33% higher than in respective diseased hearts of wild-type rats (p < 0.05, respetively). Our results suggest that transgenic rats overexpressing SERCA2a can serve as a valid model for further evaluation concerning the possible therapeutic impact of specifically targeting gene expression of the SR Ca2+-ATPase under pathological conditions with compromised cardiac SR Ca2+ transport.

The protein phosphatase 2A (PP2A) holoenzyme consists of a catalytic subunit, C, and two regulatory subunits, A and B. The PP2A core enzyme is composed of subunits A and C. Both the holoenzyme and the core enzyme are similarly abundant in heart tissue. Transgenic mice were generated expressing high levels of a dominant negative mutant of the A subunit (A delta 5) in the heart, skeletal muscle, and smooth muscle that competes with the endogenous A subunit for binding the C subunit but does not bind B subunits. We found that the ratio of core enzyme to holoenzyme was increased in A delta 5-expressing hearts. Importantly, already at day 1 after birth, A delta 5-transgenic mice had an increased heart weight-to-body weight ratio that persisted throughout life. Echocardiographic analysis of A delta 5-transgenic hearts revealed increased end-diastolic and end-systolic dimensions and decreased fractional shortening. In addition, the thickness of the septum and of the left ventricular posterior wall was significantly reduced. On the basis of these findings, we consider the heart phenotype of A delta 5-transgenic mice to be a form of dilated cardiomyopathy that frequently leads to premature death.

Increased expression of heat shock protein 70 (HSP70) in the brain has been extensively documented in association with a variety of insults, including ischemia, and is suggested to play a role in cell survival and recovery after ischemic injury. To more directly assess the protective role of HSP70 during ischemic brain damage, we used transgenic mice overexpressing the rat HSP70 (HSP70tg mice). In contrast to wild-type (wt) littermates, high levels of HSP70 messenger RNA and protein were detected in brains of HSP70tg mice under normal conditions, and immunohistochemical analysis revealed primarily neuronal expression of HSP70. Heterozygous HSP70tg mice and their wt littermates were subjected to permanent focal cerebral ischemia by intraluminal blockade of the middle cerebral artery. Cerebral infarction after 6 hours of ischemia, as evaluated by Nissl staining, was significantly less in HSP70tg mice compared with wt mice. This reduction in infarction volume in HSP70tg mice was not attributable to an altered cardiovascular anatomy or to initial differences in body temperature or hemodynamic parameters. The HSP70tg mice were still protected against cerebral infarction 24 hours after permanent focal ischemia. The data suggest that HSP70 can markedly protect the brain against ischemic damage and that approaches aimed at inducing HSP70 may lead to new therapeutic interventions in cerebrovascular injuries.

Previous studies have demonstrated that overexpression of myocardial heat shock protein (HSP72) in transgenic rats or mice correlates with reduced infarct size and enhanced myocardial function, following cardiac ischemia. In the present study, using immunohistochemical technique and monoclonal HSP72 antibody we provide anatomical mapping of HSP72 in CNS in rats genetically modified to overexpress HSP72. In this strain of animals the highest expression of HSP72 was found in the following structures i) brain: nucleus caudatus, hippocampal CA1 region, cerebral cortex (layers V-VI), occasionaly also in Purkinje cells of cerebellum and in ii) spinal cord: superficial dorsal horn (laminae II-IV), alpha-motoneurons, dorsal column; ascending pathways - fasciculus gracilis. fasciculus cuneatus and descending pathways - corticospinal tract. These results demonstrate the ability of adenoviral - HSPi construct to transfer and achieve permanent overexpression of inducible HSP72 in specific CNS regions. This regional specificity of HSP72 expression also suggests that this strain of animals represents a valuable experimental tool to define a potential protective role of HSP72 in a variety of pathological conditions such as ischemia, trauma or neuronal excitotoxicity.

SE describes a method for transport regulation LIVE IN CALCIUM IN CARDIAC MUSCLE animals suffering from Congestive Heart Failure. UNDER method, the ATPase activity CALCIUM (DECREASES TO DEVELOP AS Congestive Heart Failure) AND CARDIAC MUSCLE contractility increased by administration of a gene operably encoding enzyme at heart. ARE PROVIDED management systems (INCLUDING RECOMBINANT EXPRESSION VECTORS) AND METHODS FOR CONTROLLING THE EXPRESSION AND EFFECT ON GENE PRODUCT cardiac output.