This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.
Specific Aim #1. Which components of the beta-1AR/PI3 kinase or the fetal gene program are expressed by newly proliferating cardiomyocytes in the regenerating myocardium betaCardiac myocytes display two developmentally regulated modes of growth. During fetal and early postnatal life cardiomyocytes proliferate activity. Later in development, mitosis is irreversibly blocked and growth occurs solely via hypertrophy, limiting potential for regeneration following injury. During the period of proliferative growth, there is a fetal gene program characterized by increased expression of c-fos and c-myc, atrial natriuretic factor and specific isoforms of myosin heavy chain contractile proteins. We have demonstrated an important role of beta-1AR signaling in regulation of cardiac myocytes proliferation during the fetal and early postnatal period. We have demonstrated a constitutively elevated level of signaling of all the components of PI3 kinase/P70 ribosomal protein S6 kinase activity (p70 S6K) pathway during the proliferative period of cardiac growth (Tseng et al).
Specific Aim #2. Is the beta-1AR/PI3 kinase signaling pathway required for myocardial regeneration?Experimental protocol and functional examination: Using a well-established chronic myocardial infarction model by ligation of left descending artery (LAD), we sought to determine if human CD 34 (+) hematopoietic stem cells (HSC) targeted with bivalent antibodies directed against myosin light chain antigen will exhibit the protective effect against myocardial infarction. Animals were divided into: 1) MI heart group (n = 4) received RPMI medium through tail vein injection; 2) MI+CD34(+)HSC group (n = 7) received 0.5x10 6 CD 34 (+)HSC through tail vein injection; 3) Sham group (n = 4): animals underwent thoractomy without creation of infarction. Our results showed that MI heart exhibited deteriorated cardiac remodeling, which was prevented by administration of armed stem cell. Cardiac functional recovery was restored by infusion of the armed stem cells. We are now examining if the infused CD34(+) HSC differentiate into myocyte-like structures and/or exhibit enhanced angiogenesis. Using double labeling these sections with human specific antigen HLA and cardiac troponin as well as factor VIII, we will obtain the evidence whether or not the protection results from HSC-derived regeneration. These experiments also formed the basis for us to investigate the role of PI3K/Akt-1 in myocardial remodeling and repair in the following experiments. We sought to examine the role of the PI3K signaling component Akt1 in cardiac regeneration by examining the ability of HSCs from Akt1 knock out animals to support recovery from experimental MI. We first established the isolation of mouse Lin-/c-kit+ HSCs. Briefly, bone marrow was collected from tibias, femurs and iliac crests in mice, and depleted by pre-incubation with lineage-specific biotin-conjugated monoclonal antibodies followed by magnetic cell sorting in the magnetic field of a MACS separator. Anti-c-kit x anti-ICAM armed stem cells were delivered intravenously to recipients. SCID mice were used as recipients in the myocardial infarction model. After 48 hrs, animals were divided into 4 groups: 1) Sham group: animals underwent thoractomy without MI. 2) Ischemia: animals underwent LAD but received cell medium alone; 3) LAD + Wild type Lin-/c-kit+: animals received 5x105 wild type Lin-/c-kit+ cells after LAD; 4) LAD + (Akt1-/-) Lin-/c-kit+: animals received 5x105 Akt-1-/- Lin-/c-kit+ cells after LAD. The Ischemia hearts showed a significant reduction in left ventricular systolic pressure as compared with the Sham controls. Administration of Wt stem cells restored the recovery of left ventricular systolic pressure as well as ventricular developed pressure compare to the ischemic control group. Functional restoration was eliminated by administration of stem cells derived from Akt1-/- animals. The functional data show that restoration of cardiac performance in the infarcted heart is dependent on Akt1.Akt1 and angiogenesis following MI: It is well known that revascularization plays an important role in myocardial functional recovery following MI. To see whether or not Akt1 was essential for angiogenesis, we used immunofluorescent staining to detect the density of alpha-smooth muscle actin in the cardiac sections. As shown in Figure 1, compared to MI group, infusion of wild type stem cell significantly increased the density of alpha-SMA staining, but the increase in angiogenic response was diminished by infusion of Akt1-/- stem cells. The data suggest that Akt1 is essential to improve the angiogenic response in the injured heart post MI.The anti-apoptotic effect of Lin-c-kit+ stem cells following MI: As shown above, we demonstrated that Akt-1 was indispensable for stem cells to achieve cardiac protection and prevent remodeling. We sought to see if infusion of stem cells could reduce the number of apoptotic cells in the damaged hearts. As shown in Figure 2, Terminal dUTP nick-end labeling (TUNEL) analysis showed abundant apoptotic positive cells following myocardial infarction. The increase in apoptosis was also reduced by infusion of wild type stem cells. Such anti-apoptotic effect of stem cells infusion was significantly abrogated by deletion of Akt1, suggesting that Akt1 is essential to prevent apoptosis in the injured heart post MI. Cell tracking and cellular mechanisms: Because we have demonstrated the importance of Akt-1 in Lin-c-kit+ stem cells in restoration of cardiac function, increase in angiogenic response as well as anti-apoptosis, we next need to examine the cellular mechanism(s) by tracking the stem cells. By taking advantage of Fluorescence in situ hybridization (FISH) analysis, we will use gender mismatch strategy to detect the Y-chromosome in MI heart. In this regard, we have infused Lin-c-kit stem cell from the male mice into female recipients SCID mice which have been subjected to ischemia. Detection of Y-chromosome will support our expectation that Lin-c-kit stem cells migrate into the damaged area and co-localization of Y-chromosome with the specific markers such as alpha-sarcomeric actinin or factor VIII will reveals the potentiality of myocardial repair. In addition, we are using real time polymerase chain reaction analysis to detect the Y-chromosome genome in cardiac section. The data will further confirm the results of FISH.Summary: We have demonstrated that infusion of Lin-c-kit+ stem cells restored myocardial function flowing myocardial infarction, reduced apoptosis as well as increased angiogenic response. These effects were abolished using Lin-c-kit stem cells from Akt1-/- deficient animals, suggesting the essential role of Akt-1 in this effect. We are endeavoring to determine the cell fate by FISH analysis in combination with the double labeling in gender mismatched animals. Additionally, we have already obtained preliminary data to successfully obtain detectable genomic DNA in experimental samples, which provides the basis to decipher the cellular mechanism(s) involved. This study reveals the cellular mechanism(s) of Akt1 in regulation of myocardial repair and supports the importance of the beta-1AR/ PI3K pathway in myocardial regeneration.
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