Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts

Transcription

1 Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts Abeel A Mangi, Nicolas Noiseux, Deling Kong, Huamei He, Mojgan Rezvani, Joanne S Ingwall & Victor J Dzau Transplantation of adult bone marrow derived mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of cells overexpressing Akt into the ischemic rat myocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition and cardiac myocyte hypertrophy, regenerated 80 90% of lost myocardial volume, and completely normalized systolic and diastolic cardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1 restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacz. Thus, mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance. Prolonged interruption of myocardial blood flow initiates events that culminate in cardiac myocyte death 1.Proposed endogenous reparative mechanisms include cardiac myocyte hypertrophy 2 and hyperplasia 3 and the trafficking of bone marrow derived stem cells (BMCs) to the myocardium for repair and angiogenesis 4,5.None of these proposed mechanisms are adequate in restoring lost myocardium or sustaining cardiac function. Harvesting BMCs followed by direct injection into ischemic myocardium results in angiogenesis and myogenesis 6 8,but functional improvement is incomplete. It is unclear whether transplantation of BMCs affects repair of the ischemic myocardium primarily by angioblast-mediated vasculogenesis 9,10, which prevents apoptosis of native cardiac myocytes, or by direct regeneration of the lost myocytes. Mesenchymal stem cells (MSCs) are self-renewing, clonal precursors of non-hematopoietic tissues. They are expandable in culture and multipotent, and can differentiate into osteoblasts 11,chondrocytes 11, astrocytes 12,neurons 13 and skeletal muscle 14.Several groups have reported that putative MSCs derived from bone marrow can differentiate into cardiac muscle in vitro 15 and in vivo 16,17.However,it has been observed that transplantation of as many as of these putative MSCs into infarcted porcine hearts yielded only marginal improvement in cardiac function 17.This is explained at least in part by poor viability of the transplanted cells. It has been estimated that >99% of MSCs die 4 d after transplantation into uninjured nudemouse hearts 16. Cell transplantation strategies to replace lost myocardium are limited by the inability to deliver large numbers of cells that resist peritransplantation graft cell death Accordingly, we set out to isolate and expand a highly purified population of adult rat bone marrow derived MSCs, and to engineer these cells to overexpress Akt, a serine threonine kinase and powerful survival signal in many systems 21,22,to test the hypothesis that Aktengineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic rat heart. Our results documented significant retention of Akt-MSCs in the ischemic heart that was associated with inhibition of cardiac remodeling, a greater volume of regenerated myocardium, and near-complete normalization of systolic and diastolic cardiac function. RESULTS MSCs express markers distinct from hematopoietic stem cells MSCs proliferated in mixed culture with hematopoietic cells, yielding cells by day 15 of culture. We separated MSCs from hematopoietic cells based on their preferential attachment to polystyrene surfaces 23.By immunocytochemistry, over 99% of MSCs expressed CD29, CD71, CD90, CD106 and CD117 (Fig. 1); 60% expressed Ki67 and 15% expressed the transcription factors Nkx2.5 (Fig. 1) and Gata-4 (data not shown). MSCs did not express the hematopoietic markers CD34 and CD45 (Fig. 1) or the cardiac-specific markers myosin heavy chain (MHC), myosin light chain (MLC), cardiac troponin I (CTnI), α-sarcomeric-actin (αsa) or MEF-2 (data not shown). In addition, MSCs also expressed the gap junction protein connexin-43 as verified by RT- PCR (data not shown). We further purified MSCs using negative paramagnetic bead sorting targeting CD34, resulting in a population that was >99.9% pure. We were unable to induce these MSCs to differentiate into megakaryocytes and erythroid cells using described methods 24. Increased Akt activity protects against MSC apoptosis We used retroviruses to transduce MSCs with genes expressing green Department of Medicine, Brigham and Women s Hospital and Harvard Medical School, 75 Francis Street, Boston, Massachusetts, USA. Correspondence should be addressed to V.J.D. (vdzau@partners.org). Published online 10 August 2003; doi: /nm912 NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

2 Figure 1 Immunocytochemical characterization of MSCs. Immunostaining was performed with antibodies to c-kit, CD29, CD106, Ki-67, ctni, MHC, MLC, Nkx2.5, GATA-4, MEF-2C, N-cadherin, CD90, CD34, CD45, connexin-43, GFP, α-sa and CD71. Appropriate fluorochrome-linked secondary monoclonal antibodies were used. Negative controls for cell type included NIH-3T3 fibroblasts and human umbilical vein endothelial cells. Negative controls for antibody type were performed on MSCs using appropriate blocking peptide when available, or by omitting the primary antibody. fluorescent protein (GFP), LacZ or murine Akt, with over 90% efficiency. Using PCR for genomic DNA amplification with probes designed to detect both endogenous rat Akt1 and exogenous murine Akt1, we observed a threefold increase in the total Akt signal in Akt- a c MSCs, suggesting the successful incorporation of copies of the exogenous Akt1 gene (data not shown). The amount of murine Akt1 mrna was, on average, 7.8-fold higher in Akt-MSCs than the amounts of endogenous rat Akt1 mrna in MSCs, GFP-MSCs or LacZ-MSCs b Figure 2 Effect of Akt1 transduction in MSC. (a) Real-time RT-PCR showing that 24 h of hypoxia and serum deprivation (+) had no effect on the level of the endogenous Akt1 mrna expression as compared with normoxia ( ). Note transduction of exogenous murine Akt1 (filled bar) resulted in significant suppression of the endogenous rat Akt mrna (open bar) in the Akt-MSC group (*, P < 0.05). Hspa1b mrna abundance was increased after hypoxia in all cells, except the Akt-MSCs (top). (b) Representative western blots showing that at normoxia ( ), the abundances of total and phospho- Akt protein were very low in all groups, but high in Akt-MSCs. Phospho-Akt increased in all groups after exposure to hypoxia (+). The antibody is known to detect phospho-akt isoforms 44,45. Akt activity was also higher in the Akt-MSCs at normoxia, and increased significantly in all groups in response to hypoxia. (c) Akt overexpression reduces MSC apoptosis in vitro by 79% as judged by TUNEL assay for apoptosis. (d) Quantification of intramyocardial c-kit + cells and percentage of TUNEL-positive c-kit + cells in heart of animals 24 h after Akt-MSC injection as compared to GFP-MSC injection. The fraction of c-kit + apoptotic cells was significantly smaller in Akt-MSCs than in GFP-MSCs (19% of 82 ± versus 68% of 33 ± , *, P < 0.001, after 24 h). After 72 h, the fraction of c-kit + apoptotic cells was even smaller in Akt-MSCs than in GFP-MSCs (17% of 66 ± versus 37% of the 13 ± P < 0.001, after 72 h; data not shown). d 1196 VOLUME 9 NUMBER 9 SEPTEMBER 2003 NATURE MEDICINE

3 a c b Figure 3 Analysis of myocardial repair 3 weeks after MSC injection into ischemic rat hearts. (a) H&E staining demonstrates the difference in scar size and infiltration of viable, mature cardiac myocytes from the border zone into the scarred area (arrow), comparing Akt-MSC groups with the saline (control) group. (b) There was no blue staining of X-gal seen in remote areas of the infarcted heart or in saline-injected heart (negative control), but blue staining is seen in what would have been the infarct area after injection of LacZ-MSCs into border zone of ischemic myocardium. Examination of the border zone ( 10) of LacZ-MSC injected sections showed cells with phenotypic characteristics of cardiac myocytes (large, elongated, centrally multinucleated cells) with blue nuclei (arrows) in the border zone. (c) Merged images of double staining of sections for GFP (green) and for cardiac-specific proteins: MHC, CTn1, α-sa and MLC (red) demonstrated the colocalization of the reporter with these cardiacspecific proteins (yellow). (d) Merged images of double staining of sections for GFP (green) and for connexin-43 (Cx-43) (red) or N-cadherin (red) demonstrated that MSC-derived cardiac myocytes express connexin-43 and that the expression abuts native cardiac myocytes (yellow). This suggests that MSC-derived cardiac myocytes are capable of electromechanical coupling with native cardiac myocytes. (e) FISH staining for the Y chromosome (green), which is colocalized with α-sa (red), confirming the male origin of donor cells. d e (P < 0.05; Fig. 2a). Overexpression of murine Akt was associated with suppression of endogenous rat Akt1 mrna. Twenty-four hours of exposure to hypoxia (1% ambient O 2 ) and serum deprivation had no effect on the amount of endogenous Akt1 mrna. On the other hand, the amount of Hspa1b (formerly Hsp70) mrna increased significantly, as expected, in response to hypoxia (Fig. 2a). Total Akt protein was on average 19.7-fold more abundant in Akt-MSCs than in the other groups at 21% O 2, and these relative abundances were unchanged after exposure to hypoxia (Fig. 2b). At normoxia, phosphorylated Akt protein was elevated in Akt-MSCs, but remained very low in the other groups. With hypoxia, phospho-akt increased further in all groups, suggesting posttranslational regulation. Similarly, Akt activity in Akt-MSCs was higher at normoxia and increased further after 24 h of hypoxia than in the other groups. In response to hypoxia, endogenous Akt activity also increased in MSCs, GFP-MSCs and LacZ-MSCs (Fig. 2b). This increase in Akt activity in MSCs translated into a reduction of DNA laddering, a 27% reduction in the pro-apoptotic gene Bax and a 50% upregulation in the apoptotic gene Bcl2 (data not shown). As a result, we observed an 80% reduction in apoptosis of Akt-MSCs as compared with GFP-MSCs in vitro after 24 h of NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

4 Figure 4 Injection of MSCs into ischemic rat heart reduced infarct volume. Area at risk (arbitrary units) for infarction in hearts of all groups of animals was equivalent (top). Infarct volume was greatest after saline injection and was reduced in dose-dependent fashion after injection of LacZ-MSCs. Injection of equivalent numbers of Akt-MSCs resulted in a smaller infarct volume by a factor of 2 5 (*, P < 0.01). Saline, LacZ and Akt represent animals injected with saline, LacZ-MSCs and Akt-MSCs, respectively ( or cells). hypoxia (from 53 ± 9% apoptotic cells to 9 ± 0.5%, P < 0.01; Fig. 2c). We assessed the cytoprotective effects of Akt overexpression in vivo by injecting Akt or GFP-MSCs into the left ventricle (LV) of adult female rats 60 min after myocardial infarction. At 24 h after injection, we double-stained LV sections for c-kit immunoreactivity and terminal deoxynucleotidyl transferase mediated dutp nick end-labeling (TUNEL). In the Akt-MSC group, we observed that a greater number of c-kit + cells were retained in the myocardium and a smaller percentage of these cells were TUNEL+ (Fig. 2d). We were unable to detect c-kit + cells in the myocardium after 2 weeks, suggesting that MSCs did not persist in the undifferentiated state in the ischemic myocardium. MSCs develop into cardiac myocyte-like cells after transplantation H&E staining of post myocardial infarction hearts injected with MSCs showed infiltration of finger-like extensions of organized cardiac myocytes (Fig. 3a) into the myocardial scar. β-galactosidase staining of thick sections of whole hearts injected with LacZ-MSCs showed intense blue coloration in the peri-infarct zone resulting from the blue nuclear staining of cells that bore the phenotype of cardiac myocytes (Fig. 3b). In ischemic hearts injected with GFP-MSCs, we observed large, multinucleated syncytiae oriented in the same direction as the native cardiac myocytes in the border zone (data not shown). Double staining of the sections for cardiac-specific proteins showed that GFP colocalized with MHC, CtnI, α-sa and MLC (Fig. 3c). Cardiac myocytes expressing GFP also expressed connexin-43 and N-cadherin (Fig. 3d) at contact points with native cardiac myocytes. We verified the male donor origin of these cardiac myocyte like cells in female recipient hearts by fluorescent in situ hybridization for the Y chromosome, which colocalized with the above-mentioned cardiac-specific proteins (Fig. 3e). Three weeks after transplantation, cardiac myocytes expressing GFP and/or staining positively for the presence of the Y chromosome no longer expressed c-kit or CD90. We examined the contribution of ischemic myocardium to the localization of MSC-derived cardiac myocytes and were unable to detect GFP-containing cardiac myocytes after injection of GFP- MSCs into uninjured myocardium. We also did not detect GFP in endothelium, smooth muscle, hematopoietic elements or remote areas of the heart after injection of GFP-MSCs into ischemic myocardium. As a control for cell type, we injected equivalent numbers of GFP-transduced c-kit CD34 + cells into ischemic myocardium and could not detect GFP in cardiac myocytes (data not shown). Intramyocardial MSC injection reduces infarct volume The area at risk of infarction after coronary artery ligation was equivalent in all groups. Three weeks after coronary ligation, the volume of left ventricular infarct (V infarct ) varied based on the type and number of cells injected (Fig. 4). V infarct was maximal after saline injection. Injection of LacZ-MSCs yielded a 9.8% smaller V infarct and injection of LacZ-MSCs yielded a 12.9% smaller V infarct. Genetic modification with Akt exerted a powerful inhibitory effect on V infarct :V infarct was 44.8% smaller after injection of Akt- MSCs, whereas Akt-MSCs resulted in almost complete abolition of V infarct. MSC transplantation normalizes cardiac function To avoid the confounding effects of preload, afterload, in vivo sympathetic activity and anesthesia, we measured ventricular performance using isolated, perfused, isovolumetrically contracting hearts 2 weeks after MSC transplantation. LV systolic performance in post-infarct, saline-injected control hearts decreased to 58% of sham-operated hearts. Transplantation of or LacZ-MSCs did not improve LV systolic performance, but transplantation of Akt- MSCs resulted in a 37% increase in baseline LV systolic pressure as compared with controls. Transplantation of Akt-MSCs resulted in a further increase in systolic performance to a level that was indistinguishable from sham-operated animals (Fig. 5a). These differences persisted during inotropic challenge with dobutamine (Fig. 5b). Diastolic function, as assessed by dp/dt, also deteriorated in the post myocardial infarction saline control group as compared with sham, but improved in the Akt-MSC group at baseline and with dobutamine (Fig. 5c). a b c Figure 5 MSC injection improved cardiac function. (a) Left ventricular systolic pressure (LVSP) per gram of viable tissue was lowest in control infarct animals, and did not change after injection of LacZ-MSCs or LacZ-MSCs. Injection of or Akt-MSCs increased LVSP in a dose-dependent fashion (*, P < as compared with corresponding LacZ-MSCs). LVSP was normalized after injection of Akt-MSCs. (b) Further improvements of LVSP were seen consistently after infusion of the inotrope dobutamine (*, P < as compared with corresponding LacZ-MSCs). (c) Left ventricular dp/dt (in the presence of dobutamine) deteriorated in the saline group as compared with sham (*, P < 0.05), suggesting that diastolic relaxation in saline-injected hearts is impaired. This improved with transplantation of Akt-MSCs (, P < 0.05 as compared with saline group), suggesting that cell implantation rescued diastolic relaxation VOLUME 9 NUMBER 9 SEPTEMBER 2003 NATURE MEDICINE

5 defining the exact mechanism of therapeutic cardiac repair by MSCs are clearly necessary. Generating functional cardiac myocytes from autologous MSCs seems to be a superior strategy to the intramyocardial implantation of skeletal myoblasts 27 31,which lack the capacity for electromechanical coupling 31 and have the potential to proliferate in an uncontrolled fashion 32 ; and to the transplantation of adult, fetal 33 or neonatal cardiac myocytes 34 or of embryonic stem cell derived cardiac myocytes 35,all of which are difficult to obtain in clinically meaningful numbers and are susceptible to immunologic rejection. Our data also suggest that MSC transplantation (especially transplantation of Akt- MSCs) exerts a marked inhibitory effect on pathological myocardial remodeling that may be important in mediating the therapeutic benefit. Whether the effect on remodeling is due to mechanical improvement resulting from cell transplantation or to paracrine mediators released by MSCs requires further investigation. Our approach to myocardial repair is enhanced by the ability to genetically engineer stem cells addressing, in this case, the problem of cell death. The causes of cell death in this setting are multifactorial and are influenced by the ischemic environment, which is devoid of nutrients and oxygen 19,coupled with the loss of survival signals from matrix attachments and cell cell interactions. Akt is activated by hypoxia and a variety of other stimuli, including cytokines 22.It is a general mediator of survival signals and is both necessary and sufficient for cell survival 22.Akt achieves this by targeting apoptotic family members Ced- 9/Bcl-2 and Ced-3/caspases, forkhead transcription factors, IKK-α and IKK-β. In addition, Akt also has a role in modulating intracellular glucose metabolism 22, thereby enhancing energy production during hypoxia. Thus, Akt is an excellent therapeutic gene for preserving MSC viability in the early post-transplant period. As shown in our study, use of wild-type Akt whose gene product was not constitutively active 36, but was activated in response to hypoxia, protected cells from apoptosis while avoiding the potential detrimental effects of constitutive activated Akt expression 37.As a result, intracardiac retention and engraftment of MSCs genetically enhanced to overexpress Akt are superior to that of control MSCs expressing reporter genes alone. We have described genetic modification of mesenchymal stem cells with a therapeutic gene before transplantation as a strategy for regenerative medicine. We speculate that future therapy for acute myocara b c MSC transplantation prevents remodeling At 5 d after induction of myocardial infarction and injection of Akt-MSCs, we observed that CD45 + infiltration of the myocardium was significantly lower than that of the post-myocardial infarction saline control (36 ± 1.1 versus ± 3 cell per high-power field; P < 0.01) and was at the level seen in sham-operated animals. Two weeks after transplantation of Akt-MSCs, the increases in whole heart collagen-area fraction (Fig. 6a,b) and cardiac myocyte diameter in histologic cross-sections (Fig. 6c) seen in saline-injected myocardial infarction controls were completely inhibited, to levels observed in sham-operated animals. Transplantation of LacZ- MSCs did not result in the same degree of reduction. Three weeks after myocardial infarction, we observed that saline-injected controls had larger LV than did sham-operated animals (LV mass, excluding infarcted tissue, increased from 537 ± 39 to 672 ± 4.2 mg; P < 0.05). Transplantation of Akt-MSCs prevented ventricular enlargement, whereas LacZ-MSCs had a more moderate effect (533 ± 77 and 597 ± 50 mg, respectively). DISCUSSION We have shown here that CD117 + CD90 + CD34 MSCs can be isolated based on adhesion to polystyrene surfaces and highly purified by immunoselection, and are amenable to genetic engineering with retroviruses. MSCs injected into the border zone migrate specifically toward the ischemic myocardium and develop into cardiac myocyte like cells that form connections with native myocytes. These cellular responses seem to be unique to the microenvironment of the ischemic myocardium because they are not activated after transplantation of MSCs into normal uninjured myocardium, nor are MSCs discovered in remote, uninjured parts of coronary ligated hearts. Finally, in this setting, MSCs do not seem to have the potential to differentiate into new blood vessels or to form new capillaries from existing arterioles. The mechanism by which MSCs develop into cardiac myocyte like cells remains controversial. Although several investigators have claimed that these cells differentiate into cardiac myocytes, previous in vitro studies have shown that stem cells may fuse with existing native cells 25,26, theoretically improving function by contributing their own genetic and cellular materials. Our experiments were not designed to address fusion versus differentiation. Future experiments Figure 6 Injection of MSCs inhibits ventricular remodeling. (a,b) Masson s trichrome staining showing that collagen deposition within the whole heart was reduced 2 weeks after injection of Akt-MSCs to a level that was not significantly different from that of sham-operated heart. Collagen deposition was higher in LacZ-MSCs than in Akt-MSC treated hearts (*, P < 0.001). (c) Cardiac myocyte diameter as measured in histologic cross-section was significantly lower in hearts injected with Akt-MSCs (, P < 0.01) and was not significantly different from that in sham-operated hearts. Cardiac myocyte diameter was higher in hearts treated with LacZ-MSCs as opposed to Akt-MSCs (*, P < 0.01). NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER

6 dial infarction may involve transplantation of MSCs overexpressing Akt to facilitate the repair of the damaged heart. This novel, cell-based, gene-therapy approach has the potential to address cell availability and clinical scalability, and to make cell-based therapy an effective treatment for human cardiac disease. METHODS Animals and surgical procedures. We used 250-g Sprague-Dawley rats with approval of the Harvard Medical Area Standing Committee on Animals. We used 17 animals for MSC isolation and characterization. Eight animals per group (vehicle, c-kit CD34 + cells, LacZ-MSCs, Akt-MSCs, sham ligation) were used for morphologic analysis at 2 weeks. Additional animals were prepared (n = 8 per group) for studies requiring different time points. For functional studies, we used four animals per group for echocardiography at 2 weeks, followed by isolated Langendorff preparations to assess ventricular performance. Each experiment was performed independently, in triplicate. Sixty minutes after suture ligation of the left anterior descending artery 35 of female rat hearts, MSCs suspended in saline were injected into five sites in the border zone of the ischemic LV. Controls underwent coronary ligation and only saline injection. Sham animals underwent placement of the suture without ligation. Retroviral design and transduction. IRES-GFP (Clontech) was cloned into the Murine Stem Cell Virus vector (pmscv; Clontech) after digestion with XhoI and BamHI. We PCR-amplified cdna for murine Akt 38 (forward, 5 -GCAA- GATCTGATACCATGAACGACGTAGCC-3 ; reverse, 5 -CGGTCACCGT- GTCGGACTCCTAGGATC-3 ) and cloned it into pmscv using BglII and BamH1. Nuclear-localized LacZ expression plasmid was obtained from the Harvard Gene Therapy Initiative. We exposed MSCs three times to particles of high-titer VSV-G pseudotyped retrovirus with 6 µg/ml polybrene (Sigma-Aldrich) for 6 h. Purification of mesenchymal stem cells. Whole bone marrow was flushed from the tibia and femur of adult male rats. MSCs preferentially attached to the polystyrene surface 23 and were further purified by incubating the mixed culture with CD34 antibody (Santa Cruz Biotechnology) linked to biotin (Sigma- Aldrich) and collecting the negative fraction after exposure to avidin-coated magnetic beads (Beckman Coulter) in the presence of a magnet. This CD34- negative fraction was further propagated in alpha minimum essential medium (αmem), with Glutamax (Invitrogen) plus 10% fetal bovine serum (Invitrogen) and antibiotics (Invitrogen). Assays for Akt mrna, protein, activity and cell death. MSCs were cultured in serum-free medium in 1% O 2 at 37 C for 24 h. Ten minutes after normoxia in complete medium, protein extracts were prepared for western blot analysis. Membranes were incubated according to manufacturer s protocols with specific antibodies to Akt and phospho-akt (Ser-473; Cell Signaling) and to actin (Santa Cruz Biotechnology). The Akt kinase activity was assayed, using GSK-3 α/β fusion protein as substrate, by the Akt Kinase Assay Kit (Cell Signaling) on immunoprecipitated Akt. RNA was extracted with TRIzol reagent (Invitrogen). Real-time RT-PCR was carried out using SuperScript One-Step RT-PCR with Platinum Taq kits (Invitrogen). The primers were designed to match both rat and mouse Akt (total Akt) (forward, 5 -AACGGACTTCGGGCTGTG-3 ; reverse, 5 -TTGTCCTCCAGCACCTCAGG-3 ), but the probes were designed to be specific for rat (5 -CGTTCTGCGGGACACCCGAGTACC-3 ) and mouse (5 -AAGACATTCTGCGGAACGCCGGAGTA-3 ). The fluorogenic probes contained a 5 -FAM report dye and a 3 -BHQ1 quencher dye. TaqMan 18S Ribosomal RNA (Applied Biosystems) was used as housekeeping gene. RT-PCR reactions were carried out in icycler IQ Real-Time Detection Systems (Bio-Rad). Conventional RT-PCR was carried out using the Superscript first-strand synthesis system (Invitrogen) to transcribe cdna that was ultimately used for PCR amplification of Hspa1b and Gapd using the following primer sets: Hspa1b, forward, 5 -TGCTGACCAAGATGAAG- 3, reverse, 5 -AGAGTCGATCTCCAGGC-3 ; Gapd, forward, 5?-GGTGAT- GCTGGTGCTGAGTATGTC-3, reverse, 5 -CACCAGTGGATGCAGGG- ATGA-3. TUNEL for detection of apoptotic nuclei was performed according to the manufacturer s protocol (Roche). DNA was isolated by glass-fiber column elution (Roche) and electrophoresed. Genomic DNA PCR amplification. Genomic DNA was isolated using Easy-DNA Kit (Invitrogen) and PCR amplified using primer sets for Akt1 (forward, 5 -GTGCTGGAGGACAACGACT-3 ; reverse, 5 -GTGTAGGGTC- CTTCTTGAGCA-3 ), α-actin (forward, 5 -GTTTGCCGGAATCAATTTTC-3 ; reverse, 5 -AGCCAGAGCTGTGATCTCCTT-3 ) and murine Akt from plasmid pmscv-akt (forward, 5 -CTCGATCCTCCCTTTATCCAG-3 ; reverse, 5 -TGT GCCACTGAGAAGTTGTTG-3 ). Morphometry. Area at risk was estimated by Evans blue retrograde perfusion 39. LV volume was calculated by dividing wet weight by density (1.06 g/ml) 40.On Masson s Trichrome staining, all blue staining was quantified morphometrically and divided by all nonwhite area. This yielded the area of infarct per 5-µm section. This area of infarct was then added up for all 15 sections. To convert this to the volume of infarct for the thick slice from which the sections were obtained, we multiplied it by the appropriate multiplier based on the thickness of the slice. We then added up the volume of infarct (mm 3 ) of the whole heart. Collagen area fraction was measured as described 41.Cardiac myocyte diameter in histologic cross-section was measured using morphometric techniques as described 42. Analysis of cardiac function. Isolated rat hearts were perfused in the Langendorff model 43.Indices of isovolumic contractile and relaxation performance were measured by placing a polyvinylchloride balloon in the LV connected to a data acquisition system at baseline and in the presence of 300 nm dobutamine. Statistics. Student s t-test was used for two-group comparison and ANOVA followed by an unpaired Student s t-test with Bonferroni s correction was used for multiple group comparisons. ACKNOWLEDGMENTS We thank D.G. Phinney, D.J. Prockop and R. Pratt for helpful discussions and assistance in establishing culture conditions for propagation of bone marrow derived MSCs; M.A. Perrella for sharing Akt1 constructs and for helpful discussions on Akt biology; and S. Colgan for the use of a hypoxia chamber. This work was supported by grants HL35610 (V.J.D.), HL (V.J.D.), HL (V.J.D.), HL (V.J.D.) and HL52320 (J.S.I.) from the National Heart, Lung and Blood Institute, US National Institutes of Health. A.A.M. is the recipient of a National Research Service Award (1 F32 NHL ) from the National Heart, Lung and Blood Institute, National Institutes of Health; and the Robert R. Linton Research Fellowship from the Department of Surgery, Massachusetts General Hospital. N.N. is a recipient of a scholarship from the Canadian Institutes of Health Research. M.R. is the recipient of an American Heart Association Research Award ( T). COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Received 29 May; accepted 17 July 2003 Published online at 1. Williams, R.S. & Benjamin, I.J. Protective responses in the ischemic myocardium. J. Clin. Invest. 106, (2000). 2. Pfeffer, J.M., Pfeffer, M.A., Fletcher, P.J. & Braunwald, E. Progressive ventricular remodeling in rat with myocardial infarction. Am. J. Physiol. 260, H (1991). 3. Beltrami, A.P. et al. Evidence that human cardiac myocytes divide after myocardial infarction. N. Engl. J. Med. 344, (2001). 4. Jackson, K.A. et al. Regeneration of ischemic cardiac muscle and vascular endothelium by adult stem cells. J. Clin. Invest. 107, (2001). 5. Quaini, F. et al. Chimerism of the transplanted heart. N. Engl. J. Med. 346, 5 15 (2002). 6. Tomita, S. et al. Autologous transplantation of bone marrow cells improves damaged heart function. Circulation 100, II (1999). 7. Wang, J.S. et al. Marrow stromal cells for cellular cardiomyoplasty: feasibility and potential clinical advantages. J. Thorac. Cardiovasc. Surg. 120, (2000). 8. Orlic, D. et al. Bone marrow cells regenerate infarcted myocardium. Nature 410, (2001) VOLUME 9 NUMBER 9 SEPTEMBER 2003 NATURE MEDICINE

7 9. Kocher, A.A. et al. Neovascularization of ischemic myocardium by human bonemarrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat. Med. 7, (2001). 10. Kawamoto, A. et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation 103, (2001). 11. Pereira, R.F. et al. Cultured adherent cells from marrow can serve as long-lasting precursor cells for bone, cartilage, and lung in irradiated mice. Proc. Natl. Acad. Sci. USA 92, (1995). 12. Azizi, S.A. et al. Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats similarities to astrocyte grafts. Proc. Natl. Acad. Sci. USA 95, (1998). 13. Chen, J. et al. Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats. J. Neurol. Sci. 189, (2001). 14. Ferrari, G. et al. Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279, (1998). 15. Makino, S. et al. Cardiomyocytes can be generated from marrow stromal cells in vitro. J. Clin. Invest. 103, (1999). 16. Toma, C. et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 105, (2002). 17. Shake, J.G. et al. Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann. Thorac. Surg. 73, , 1926 (2002). 18. Reinecke, H., Zhang, M., Bartosek, T. & Murry, C.E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, (1999). 19. Zhang, M. et al. Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies. J. Mol. Cell. Cardiol. 33, (2001). 20. Muller-Ehmsen, J. et al. Survival and development of neonatal rat cardiomyocytes transplanted into adult myocardium. J. Mol. Cell. Cardiol. 34, (2002). 21. Franke, T.F., Kaplan, D.R. & Cantley, L.C. PI3K: downstream AKTion blocks apoptosis. Cell 88, (1997). 22. Datta, S.R., Brunet, A. & Greenberg, M.E. Cellular survival: a play in three Akts. Genes Dev. 13, (1999). 23. Colter, D.C., Class, R., DiGirolamo, C.M. & Prockop, D.J. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc. Natl. Acad. Sci. USA 97, (2000). 24. Hunnestad, J.A., Steen, R., Tjonnfjord, G.E. & Egeland, T. Thrombopoietin combined with early-acting growth factors effectively expands human hematopoietic progenitor cells in vitro. Stem Cells 17, (1999). 25. Terada, N. et al. Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature 416, (2002). 26. Ying, Q.L., Nichols, J., Evans, E.P. & Smith, A.G. Changing potency by spontaneous fusion. Nature 416, (2002). 27. Taylor, D.A. et al. Regenerating functional myocardium: improved performance after skeletal myoblast transplantation. Nat. Med. 4, (1998). 28. Murry, C.E., Wiseman, R.W., Schwartz, S.M. & Hauschka, S.D. Skeletal myoblast transplantation for repair of myocardial necrosis. J. Clin. Invest. 98, (1996). 29. Atkins, B.Z. et al. Cellular cardiomyoplasty improves diastolic properties of injured heart. J. Surg. Res. 85, (1999). 30. Menasche, P. et al. Myoblast transplantation for heart failure. Lancet 357, (2001). 31. Reinecke, H., MacDonald, G.H., Hauschka, S.D. & Murry, C.E. Electromechanical coupling between skeletal and cardiac muscle. Implications for infarct repair. J. Cell. Biol. 149, (2000). 32. Reinecke, H. & Murry, C.E. Transmural replacement of myocardium after skeletal myoblast grafting into the heart. Too much of a good thing? Cardiovasc. Pathol. 9, 337 (2000). 33. Li, R.K. et al. Natural history of fetal rat cardiomyocytes transplanted into adult rat myocardial scar tissue. Circulation 96, II , (1997). 34. Watanabe, E. et al. Cardiomyocyte transplantation in a porcine myocardial infarction model. Cell Transplant. 7, (1998). 35. Min, J.Y. et al. Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats. J. Appl. Physiol. 92, (2002). 36. Fujio, Y. et al. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation 101, (2000). 37. Matsui, T. et al. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J. Biol. Chem. 277, (2002). 38. Fukumoto, S. et al. Akt participation in the Wnt signaling pathway through Dishevelled. J. Biol. Chem. 276, (2001). 39. Melo, L.G. et al. Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene. Circulation 105, (2002). 40. Orlic, D. et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc. Natl. Acad. Sci. USA 98, (2001). 41. Nwogu, J. et al. Inhibition of collagen synthesis with prolyl 4-hydroxylase inhibitor improves left ventricular function and alters the pattern of left ventricular dilatation after myocardial infarction. Circulation 104, (2001). 42. Choukroun, G. et al. Regulation of cardiac hypertrophy in vivo by the stress-activated protein kinases/c-jun NH 2 -terminal kinases. J. Clin. Invest. 104, (1999). 43. Spindler, M. et al. Diastolic dysfunction and altered energetics in the αmhc403/+ mouse model of familial hypertrophic cardiomyopathy. J. Clin. Invest. 101, (1998). 44. Okano, J. et al. Akt/protein kinase B isoforms are differentially regulated by epidermal growth factor stimulation. J. Biol. Chem. 275, (2000). 45. Teruel, T., Hernandez, R. & Lorenzo, M. Ceramide mediates insulin resistance by tumor necrosis factor-α in brown adipocytes by maintaining Akt in an inactive dephosphorylated state. Diabetes 50, (2001). NATURE MEDICINE VOLUME 9 NUMBER 9 SEPTEMBER