Dysregulation of catalase activity in newborn myocytes during hypoxia is mediated by c-Abl tyrosine kinase.

In the adult heart, catalase (CAT) activity increases appropriately with increasing levels of hydrogen peroxide, conferring cardioprotection. This mechanism is absent in the newborn for unknown reasons. In the present study, we examined how the posttranslational modification of CAT contributes to its activation during hypoxia/ischemia and the role of c-Abl tyrosine kinase in this process. Hypoxia studies were carried out using primary cardiomyocytes from adult (>8 weeks) and newborn rats. Following hypoxia, the ratio of phosphorylated to total CAT and c-Abl in isolated newborn rat myocytes did not increase and were significantly lower (1.3- and 4.2-fold, respectively; P < .05) than their adult counterparts. Similarly, there was a significant association (P < .0005) between c-Abl and CAT in adult cells following hypoxia (30.9 ± 8.2 to 70.7 ± 13.1 au) that was absent in newborn myocytes. Although ubiquitination of CAT was higher in newborns compared to adults following hypoxia, inhibition of this did not improve CAT activity. When a c-Abl activator (5-(1,3-diaryl-1H-pyrazol-4-yl)hydantoin [DPH], 200 µmol/L) was administered prior to hypoxia, not only CAT activity was significantly increased (P < .05) but also phosphorylation levels were also significantly improved (P < .01) in these newborn myocytes. Additionally, ischemia-reperfusion (IR) studies were performed using newborn (4-5 days) rabbit hearts perfused in a Langendorff method. The DPH given as an intracardiac injection into the right ventricle of newborn rabbit resulted in a significant improvement (P < .002) in the recovery of developed pressure after IR, a key indicator of cardiac function (from 74.6% ± 6.6% to 118.7% ± 10.9%). In addition, CAT activity was increased 3.92-fold (P < .02) in the same DPH-treated hearts. Addition of DPH to adult rabbits in contrast had no significant effect (from 71.3% ± 10.7% to 59.4% ± 12.1%). Therefore, in the newborn, decreased phosphorylation of CAT by c-Abl potentially mediates IR-induced dysfunction, and activation of c-Abl may be a strategy to prevent ischemic injury associated with surgical procedures.

The modulation of cardiac progenitor cell function by hydrogel-dependent Notch1 activation.

Myocardial infarction is the leading cause of death worldwide and phase I clinical trials utilizing cardiac progenitor cells (CPCs) have shown promising outcomes. Notch1 signaling plays a critical role in cardiac development and in the survival, cardiogenic lineage commitment, and differentiation of cardiac stem/progenitor cells. In this study, we functionalized self-assembling peptide (SAP) hydrogels with a peptide mimic of the Notch1 ligand Jagged1 (RJ) to evaluate the therapeutic benefit of CPC delivery in the hydrogels in a rat model of myocardial infarction. The behavior of CPCs cultured in the 3D hydrogels in vitro including gene expression, proliferation, and growth factor production was evaluated. Interestingly, we observed Notch1 activation to be dependent on hydrogel polymer density/stiffness with synergistic increase in presence of RJ. Our results show that RJ mediated Notch1 activation depending on hydrogel concentration differentially regulated cardiogenic gene expression, proliferation, and growth factor production in CPCs in vitro. In rats subjected to experimental myocardial infarction, improvement in acute retention and cardiac function was observed following cell therapy in RJ hydrogels compared to unmodified or scrambled peptide containing hydrogels. This study demonstrates the potential therapeutic benefit of functionalizing SAP hydrogels with RJ for CPC based cardiac repair.

Over-expression of catalase in myeloid cells confers acute protection following myocardial infarction.

Cardiovascular disease is the leading cause of death in the United States and new treatment options are greatly needed. Oxidative stress is increased following myocardial infarction and levels of antioxidants decrease, causing imbalance that leads to dysfunction. Therapy involving catalase, the endogenous scavenger of hydrogen peroxide (H2O2), has been met with mixed results. When over-expressed in cardiomyocytes from birth, catalase improves function following injury. When expressed in the same cells in an inducible manner, catalase showed a time-dependent response with no acute benefit, but a chronic benefit due to altered remodeling. In myeloid cells, catalase over-expression reduced angiogenesis during hindlimb ischemia and prevented monocyte migration. In the present study, due to the large inflammatory response following infarction, we examined myeloid-specific catalase over-expression on post-infarct healing. We found a significant increase in catalase levels following infarction that led to a decrease in H2O2 levels, leading to improved acute function. This increase in function could be attributed to reduced infarct size and improved angiogenesis. Despite these initial improvements, there was no improvement in chronic function, likely due to increased fibrosis. These data combined with what has been previously shown underscore the need for temporal, cell-specific catalase delivery as a potential therapeutic option.

Targeting extracellular DNA to deliver IGF-1 to the injured heart.

There is a great need for the development of therapeutic strategies that can target biomolecules to damaged myocardium. Necrosis of myocardium during a myocardial infarction (MI) is characterized by extracellular release of DNA, which can serve as a potential target for ischemic tissue. Hoechst, a histological stain that binds to double-stranded DNA can be conjugated to a variety of molecules. Insulin-like growth factor-1 (IGF-1), a small protein/polypeptide with a short circulating-half life is cardioprotective following MI but its clinical use is limited by poor delivery, as intra-myocardial injections have poor retention and chronic systemic presence has adverse side effects. Here, we present a novel delivery vehicle for IGF-1, via its conjugation to Hoechst for targeting infarcted tissue. Using a mouse model of ischemia-reperfusion, we demonstrate that intravenous delivery of Hoechst-IGF-1 results in activation of Akt, a downstream target of IGF-1 and protects from cardiac fibrosis and dysfunction following MI.

Functionalized dendrimer-based delivery of angiotensin type 1 receptor siRNA for preserving cardiac function following infarction.

Cardiovascular disease (CVD) is the leading cause of death throughout the world and much pathology is associated with upregulation of inflammatory genes. Gene silencing using RNA interference is a powerful tool in regulating gene expression, but its application in CVDs has been prevented by the lack of efficient delivery systems. We report here the development of tadpole dendrimeric materials for siRNA delivery in a rat ischemia-reperfusion (IR) model. Angiotensin II (Ang II) type 1 receptor (AT1R), the major receptor that mediates most adverse effects of Ang II, was chosen to be the silencing targeting. Among the three tadpole dendrimers synthesized, the oligo-arginine conjugated dendrimer loaded with siRNA demonstrated effective down-regulation in AT1R expression in cardiomyocytes in vitro. When the dendrimeric material was applied in vivo, the siRNA delivery prevented the increase in AT1R levels and significantly improved cardiac function recovery compared to saline injection or empty dendrimer treated groups after IR injury. These experiments demonstrate a potential treatment for dysfunction caused by IR injury and may represent an alternative to AT1R blockade.

Age- and chamber-specific differences in oxidative stress after ischemic injury.

Each year, tens of thousands of children undergo cardiopulmonary bypass (CPB) to correct congenital heart defects. Although necessary for surgery, CPB involves stopping the heart and exposing it to ischemic conditions. On reoxygenation, the heart can experience effects similar to that of acute myocardial infarction. Although much is known about adult injury, little is known about the effects of global ischemia on newborn ventricles. We studied newborn (2 to 4 days old) and adult (>8 weeks old) rabbit hearts subjected to ischemia-reperfusion (30 min of ischemia and 60 min of reperfusion). Our data demonstrated chamber- and age-specific changes in oxidative stress. During ischemia, hydrogen peroxide (H(2)O(2)) increased in both right-ventricular (RV) and left-ventricular (LV) myocytes of the newborn, although only the RV change was significant. In contrast, there was no significant increase in H(2)O(2) in either RV or LV myocytes of adults. There was a fivefold increase in H(2)O(2) formation in newborn RV myocytes compared with adults (P = 0.006). In whole-heart tissue, superoxide dismutase activity increased from sham versus ischemia in the left ventricle of both adult and newborn hearts, but it was increased only in the right ventricle of the newborn heart. Catalase activity was significantly increased after ischemia in both adult ventricles, whereas no increase was seen in newborn compared with sham hearts. In addition, catalase levels in newborns were significantly lower, indicating less scavenging potential. Nanoparticle-encapsulated ebselen, given as an intracardiac injection into the right or left ventricle of newborn hearts, significantly increased functional recovery of developed pressure only in the right ventricle, indicating the potential for localized antioxidant therapy during and after pediatric surgical procedures.