SR Ca2+ ATPase 2a (SERCA2a) is a critical ATPase responsible for Ca2+ re-uptake during excitation-contraction coupling. Impaired SR Ca2+ uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure (HF)1. Accordingly, restoration of SERCA2a expression by gene transfer has proven to be effective in improving cardiac function in HF patients2 as well as in animal models3. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins4, which is involved in most cellular process5. Here, we show that SERCA2a is SUMOylated at lysine 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability. The levels of SUMO1 and SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated virus-mediated gene delivery maintained protein abundance of SERCA2a and significantly improved cardiac function in HF mice. This effect was comparable to SERCA2a gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca2+ decay. Transgene-mediated SUMO1 overexpression rescued pressure overload-induced cardiac dysfunction concomitantly with increased SERCA2a function. By contrast, down-regulation of SUMO1 using shRNA accelerated pressure overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function and provides a platform for the design of novel therapeutic strategies for HF.

Background-Stem cell labeling with iron oxide (ferumoxide) particles allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment. However, the validity of MRI for distinguishing surviving ferumoxide-labeled cells from other sources of MRI signal, for example, macrophages containing ferumoxides released from nonsurviving cells, has not been thoroughly investigated. We sought to determine the relationship between the persistence of iron-dependent MRI signals and cell survival 3 weeks after injection of syngeneic or xenogeneic ferumoxides-labeled stem cells (cardiac-derived stem cells) in rats. Methods and Results-We studied nonimmunoprivileged human and rat cardiac-derived stem cells and human mesenchymal stem cells doubly labeled with ferumoxides and ␤-galactosidase and injected intramyocardially into immunocompetent Wistar-Kyoto rats. Animals were imaged at 2 days and 3 weeks after stem cell injection in a clinical 3-T MRI scanner. At 2 days, injection sites of xenogeneic and syngeneic cells (cardiac-derived stem cells and mesenchymal stem cells) were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks (50% to 90% of initial signal). Histology (at 3 weeks) revealed the presence of iron-containing macrophages at the injection site, identified by CD68 staining, but very few or no ␤-galactosidase-positive stem cells in the animals transplanted with syngeneic or xenogeneic cells, respectively. Conclusions-The persistence of significant iron-dependent MRI signal derived from ferumoxide-containing macrophages despite few or no viable stem cells 3 weeks after transplantation indicates that MRI of ferumoxide-labeled cells does not reliably report long-term stem cell engraftment in the heart. (Circulation. 2008;117:1555-1562.)

Objectives We examined the sodium-iodide symporter (NIS) which promotes in vivo cellular uptake of 99mTc or 124I, as a reporter gene for cell tracking by SPECT or PET imaging. Background Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo. Methods The human NIS cDNA was transduced into rat cardiac-derived stem cells (rCDCs) using lentiviral vectors. Rats were injected intra-myocardially with up to 4 million NIS+-rCDCs immediately following LAD ligation. Dual isotope SPECT (or PET) imaging was performed, using 99mTc (or 124I) for cell detection and 201Tl (or 13NH3) for myocardial delineation. In a subset of animals, high resolution ex vivo SPECT scans of explanted hearts were obtained to confirm that in vivo signals were derived from the cell injection site. Results NIS expression in rCDCs did not affect cell viability and proliferation. NIS activity was verified in isolated transduced cells by measuring 99mTc uptake. NIS+ rCDCs were visualized in vivo as regions of 99mTc or 124I uptake within a perfusion deficit in the SPECT and PET images, respectively. Cells could be visualized by SPECT up to day 6 post-injection. Ex vivo SPECT confirmed that in vivo 99mTc signals were localized to the cell injection sites. Conclusion Ectopic NIS expression allows non invasive in vivo stem cell tracking in the myocardium, using either SPECT or PET. The general approach shows significant promise in tracking the fate of transplanted cells participating in cardiac regeneration, given its ability to observe living cells using clinically-applicable imaging modalities.