Slowed relaxation in diabetic cardiomyopathy (CM) is partially related to diminished expression of the sarcoplasmic reticulum (SR) Ca2+-ATPase SERCA2a. To evaluate the impact of SERCA2a overexpression on SR Ca2+ handling in diabetic CM, we 1) generated transgenic rats harboring a human cytomegalovirus enhancer/chicken beta-actin promotor-controlled rat SERCA2 transgene (SERCA2-TGR), 2) characterized their SR phenotype, and 3) examined whether transgene expression may rescue SR Ca2+ transport in streptozotocin-induced diabetes. The transgene was expressed in all heart chambers. Compared to wild-type (WT) rats, a heterozygous line exhibited increased SERCA2 mRNA (1.5-fold), SERCA2 protein (+26%) and SR Ca2+ uptake (+37%). Phospholamban expression was not altered. In SERCA2-TGR, contraction amplitude (+48%) and rates of contraction (+34%) and relaxation (+35%) of isolated papillary muscles (PM) were increased (P2+ uptake and SERCA2 protein of SERCA2-TGR were 1.3-fold higher (P2+ uptake, accelerates relaxation and compensates, in part, for depressed Ca2+ uptake in diabetic CM. Therefore, SERCA2 expression might constitute an important therapeutic target to rescue cardiac SR Ca2+ handling in diabetes.

The bradykinin-forming enzyme kallikrein-1 is expressed in the heart. To examine whether contractile performance and sarcoplasmic reticulum Ca2+ transport of the diabetic heart can be rescued by targeting the kallikrein-kinin system, we studied left ventricular function and sarcoplasmic reticular Ca2+ uptake after induction of streptozotocin-induced diabetes mellitus in transgenic rats expressing the human tissue kallikrein-1 gene. Six weeks after a single injection of either streptozotocin (70 mg/kg ip) or vehicle, left ventricular performance was determined using a Millar-Tip catheter system. The Ca2+-transporting activity of reticulum-derived membrane vesicles was determined in left ventricular homogenates as oxalate-supported 45Ca2+ uptake. Western blot analysis was used to quantify the reticular Ca2+-ATPase SERCA2a, phospholamban, and the phosphorylation status of the latter. Contractile performance and Ca2+ uptake activity were similar in nondiabetic wild-type and transgenic rats. Severely diabetic wild-type animals exhibited impaired left ventricular performance and decreased reticular Ca2+ uptake (-39% vs. wild-type rats, P<0.05, respectively). These changes were attenuated in diabetic transgenic rats that, in addition, exhibited a markedly increased phospholamban phosphorylation at the Ca2+/calmodulin kinase-specific site threonine17 (2.2-fold vs. diabetic wild-type rats, P<0.05). These transgene-related effects were abolished after treatment with the bradykinin B2 receptor antagonist icatibant (Hoe 140). The SERCA2-to-phospholamban ratio, phosphoserine16-phospholamban levels, and the apparent affinity for Ca2+ of the uptake reaction did not differ between the groups. Increasing the activity of the kallikrein-kinin system by expressing a human kallikrein-1 transgene protects rat heart against diabetes-induced contractile and reticular Ca2+ transport dysfunctions. An increased phosphorylation of the SERCA2 regulatory protein phospholamban at threonine17 via a B2 receptor-mediated mechanism is thereby involved.

The sarco/endoplasmic reticulum (SR) Ca 2ϩ -ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca 2ϩ reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca 2ϩ handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dtmax, and dP/dtmin in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P Ͻ 0.05). In parallel, a 1.4-fold higher Vmax value of homogenate SR Ca 2ϩ uptake was observed in hypothyroid TG (P Ͻ 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by Ϫ24% was markedly less than the decrease of Ϫ49% in WT (P Ͻ 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the Vmax values of SR Ca 2ϩ uptake when the respective data of all experimental groups were plotted together (r ϭ 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca 2ϩ uptake and in vivo heart function were only partially rescued. sarcoplasmic reticulum; Ca 2ϩ -ATPase; female; thyroid hormone; atrophy THE SARCO-/ENDOPLASMIC RETICULUM Ca 2ϩ pump SERCA2a has a key role in Ca 2ϩ regulation during the contraction-relaxation cycle of the myocardium. The SERCA2a-mediated transport of Ca 2ϩ from the cytosol of the cardiomyocyte to the lumen of the sarcoplasmic reticulum (SR) is the major mechanism for lowering the cytosolic Ca 2ϩ concentration during muscle relaxation. Decreases in SERCA2a protein levels and/or its Ca 2ϩ

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