Christina A. Pacak

Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury

Mitochondrial damage and dysfunction occur during ischemia and modulate cardiac function and cell survival significantly during reperfusion. We hypothesized that transplantation of autologously derived mitochondria immediately prior to reperfusion would ameliorate these effects. New Zealand White rabbits were used for regional ischemia (RI), which was achieved by temporarily snaring the left anterior descending artery for 30 min. Following 29 min of RI, autologously derived mitochondria (RI-mitochondria; 9.7 ± 1.7 × 10(6)/ml) or vehicle alone (RI-vehicle) were injected directly into the RI zone, and the hearts were allowed to recover for 4 wk. Mitochondrial transplantation decreased (P < 0.05) creatine kinase MB, cardiac troponin-I, and apoptosis significantly in the RI zone. Infarct size following 4 wk of recovery was decreased significantly in RI-mitochondria (7.9 ± 2.9%) compared with RI-vehicle (34.2 ± 3.3%, P < 0.05). Serial echocardiograms showed that RI-mitochondria hearts returned to normal contraction within 10 min after reperfusion was started; however, RI-vehicle hearts showed persistent hypokinesia in the RI zone at 4 wk of recovery. Electrocardiogram and optical mapping studies showed that no arrhythmia was associated with autologously derived mitochondrial transplantation. In vivo and in vitro studies show that the transplanted mitochondria are evident in the interstitial spaces and are internalized by cardiomyocytes 2-8 h after transplantation. The transplanted mitochondria enhanced oxygen consumption, high-energy phosphate synthesis, and the induction of cytokine mediators and proteomic pathways that are important in preserving myocardial energetics, cell viability, and enhanced post-infarct cardiac function. Transplantation of autologously derived mitochondria provides a novel technique to protect the heart from ischemia-reperfusion injury.

Pompe disease gene therapy

Pompe disease is an autosomal recessive metabolic myopathy caused by the deficiency of the lysosomal enzyme acid alpha-glucosidase and results in cellular lysosomal and cytoplasmic glycogen accumulation. A wide spectrum of disease exists from hypotonia and severe cardiac hypertrophy in the first few months of life due to severe mutations to a milder form with the onset of symptoms in adulthood. In either condition, the involvement of several systems leads to progressive weakness and disability. In early-onset severe cases, the natural history is characteristically cardiorespiratory failure and death in the first year of life. Since the advent of enzyme replacement therapy (ERT), the clinical outcomes have improved. However, it has become apparent that a new natural history is being defined in which some patients have substantial improvement following ERT, while others develop chronic disability reminiscent of the late-onset disease. In order to improve on the current clinical outcomes in Pompe patients with diminished clinical response to ERT, we sought to address the cause and potential for the treatment of disease manifestations which are not amenable to ERT. In this review, we will focus on the preclinical studies that are relevant to the development of a gene therapy strategy for Pompe disease, and have led to the first clinical trial of recombinant adeno-associated virus-mediated gene-based therapy for Pompe disease. We will cover the preliminary laboratory studies and rationale for a clinical trial, which is based on the treatment of the high rate of respiratory failure in the early-onset patients receiving ERT.

Tissue specific promoters improve specificity of AAV9 mediated transgene expression following intra-vascular gene delivery in neonatal mice.

The AAV9 capsid displays a high natural affinity for the heart following a single intravenous (IV) administration in both newborn and adult mice. It also results in substantial albeit relatively lower expression levels in many other tissues. To increase the overall safety of this gene delivery method we sought to identify which one of a group of promoters is able to confer the highest level of cardiac specific expression and concurrently, which is able to provide a broad biodistribution of expression across both cardiac and skeletal muscle. The in vivo behavior of five different promoters was compared: CMV, desmin (Des), alpha-myosin heavy chain (α-MHC), myosin light chain 2 (MLC-2) and cardiac troponin C (cTnC). Following IV administration to newborn mice, LacZ expression was measured by enzyme activity assays. Results showed that rAAV2/9-mediated gene delivery using the α-MHC promoter is effective for focal transgene expression in the heart and the Des promoter is highly suitable for achieving gene expression in cardiac and skeletal muscle following systemic vector administration. Importantly, these promoters provide an added layer of control over transgene activity following systemic gene delivery.

Injection of isolated mitochondria during early reperfusion for cardioprotection

Previously, we demonstrated that ischemia induces mitochondrial damage and dysfunction that persist throughout reperfusion and impact negatively on postischemic functional recovery and cellular viability. We hypothesized that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, would enhance postischemic functional recovery and limit infarct size. New Zealand White rabbits (n = 52) were subjected to 30 min of equilibrium and 30 min of regional ischemia (RI) induced by snaring the left anterior descending coronary artery. At 29 min of RI, the RI zone was injected with vehicle (sham control and RI vehicle) or vehicle containing mitochondria (7.7 x 10(6) +/- 1.5 x 10(6)/ml) isolated from donor rabbit left ventricular tissue (RI-Mito). The snare was released at 30 min of RI, and the hearts were reperfused for 120 min. Our results show that left ventricular peak developed pressure and systolic shortening in RI-Mito hearts were significantly enhanced (P < 0.05 vs. RI-vehicle) to 75% and 83% of equilibrium value, respectively, at 120 min of reperfusion compared with 57% and 62%, respectively, in RI-vehicle hearts. Creatine kinase-MB, cardiac troponin I, and infarct size relative to area at risk were significantly decreased in RI-Mito compared with RI-vehicle hearts (P < 0.05). Confocal microscopy showed that injected mitochondria were present and viable after 120 min of reperfusion and were distributed from the epicardium to the subendocardium. These results demonstrate that viable respiration-competent mitochondria, isolated from tissue unaffected by ischemia and then injected into the ischemic zone just before reperfusion, significantly enhance postischemic functional recovery and cellular viability.

Actin-dependent mitochondrial internalization in cardiomyocytes: evidence for rescue of mitochondrial function

Previously, we have demonstrated that the transplantation of viable, structurally intact, respiration competent mitochondria into the ischemic myocardium during early reperfusion significantly enhanced cardioprotection by decreasing myocellular damage and enhancing functional recovery. Our in vitro and in vivo studies established that autologous mitochondria are internalized into cardiomyocytes following transplantation; however, the mechanism(s) modulating internalization of these organelles were unknown. Here, we show that internalization of mitochondria occurs through actin-dependent endocytosis and rescues cell function by increasing ATP content and oxygen consumption rates. We also show that internalized mitochondria replace depleted mitochondrial (mt)DNA. These results describe the mechanism for internalization of mitochondria within host cells and provide a basis for novel therapeutic interventions allowing for the rescue and replacement of damaged or impaired mitochondria.

Relative persistence of AAV serotype 1 vector genomes in dystrophic muscle

The purpose of this study was to assess the behavior of pseudotyped recombinant adeno-associated virus type 1 (rAAV2/1) vector genomes in dystrophic skeletal muscle. A comparison was made between a therapeutic vector and a reporter vector by injecting the hindlimb in a mouse model of Limb Girdle Muscular Dystrophy Type 2D (LGMD-2D) prior to disease onset. We hypothesized that the therapeutic vector would establish long-term persistence through prevention of myofiber turnover. In contrast, the reporter vector genome copy number would diminish over time due to disease-associated muscle degradation.One day old alpha sarcoglycan knockout mice (sgca-/-) were injected with 1 × 1011 vector genomes of rAAV2/1-tMCK-sgca in one hindlimb and the same dose of rAAV2/1-tMCK-LacZ in the contra lateral hindlimb. Newborn mice are tolerant of the foreign transgene allowing for long-term expression of both the marker and the therapeutic gene in the null background. At 2 time-points following vector administration, hindlimb muscles were harvested and analyzed for LacZ or sarcoglycan expression. Our data demonstrate prolonged vector genome persistence in skeletal muscle from the hindlimbs injected with the therapeutic transgene as compared to hindlimbs injected with the reporter gene. We observed loss of vector genomes in skeletal muscles that were there were not protected by the benefits of therapeutic gene transfer. In comparison, the therapeutic vector expressing sarcoglycan led to reduction or elimination of myofiber loss. Mitigating the membrane instability inherent in dystrophic muscle was able to prolong the life of individual myofibers.

Superparamagnetic Iron Oxide Nanoparticles Function as a Long-Term, Multi-Modal Imaging Label for Non- Invasive Tracking of Implanted Progenitor Cells

The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (μCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by μCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged in vivo and ex vivo for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while μCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and μCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and μCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.

Straightening of curved pattern of collagen fibers under load controls aortic valve shape.

The network of collagen fibers in the aortic valve leaflet is believed to play an important role in the strength and durability of the valve. However, in addition to its stress-bearing role, such a fiber network has the potential to produce functionally important shape changes in the closed valve under pressure load. We measured the average pattern of the collagen network in porcine aortic valve leaflets after staining for collagen. We then used finite element simulation to explore how this collagen pattern influences the shape of the closed valve. We observed a curved or bent pattern, with collagen fibers angled downward from the commissures toward the center of the leaflet to form a pattern that is concave toward the leaflet free edge. Simulations showed that these curved fiber trajectories straighten under pressure load, leading to functionally important changes in closed valve shape. Relative to a pattern of straight collagen fibers running parallel to the leaflet free edge, the concave pattern of curved fibers produces a closed valve with a 40% increase in central leaflet coaptation height and with decreased leaflet billow, resulting in a more physiological closed valve shape. Furthermore, simulations show that these changes in loaded leaflet shape reflect changes in leaflet curvature due to modulation of in-plane membrane stress resulting from straightening of the curved fibers. This effect appears to play an important role in normal valve function and may have important implications for the design of prosthetic and tissue engineered replacement valves.

Biological characterization of F-18-labeled rhodamine B, a potential positron emission tomography perfusion tracer

INTRODUCTION Myocardial infarction is the leading cause of death in western countries, and positron emission tomography (PET) plays an increasing role in the diagnosis and treatment planning for this disease. However, the absence of an (18)F-labeled PET myocardial perfusion tracer hampers the widespread use of PET in myocardial perfusion imaging (MPI). We recently reported a potential MPI agent based on (18)F-labeled rhodamine B. The goal of this study was to more completely define the biological properties of (18)F-labeled rhodamine B with respect to uptake and localization in an animal model of myocardial infarction and to evaluate the uptake (18)F-labeled rhodamine B by cardiomyocytes. METHODS A total of 12 female Sprague Dawley rats with a permanent ligation of the left anterior descending artery (LAD) were studied with small-animal PET. The animals were injected with 100-150 μCi of (18)F-labeled rhodamine B diethylene glycol ester ([(18)F]RhoBDEGF) and imaged two days before ligation. The animals were imaged again two to ten days post-ligation. After the post-surgery scans, the animals were euthanized and the hearts were sectioned into 1mm slices and myocardial infarct size was determined by phosphorimaging and 2,3,5-triphenyltetrazolium chloride staining (TTC). In addition, the uptake of [(18)F]RhoBDEGF in isolated rat neonatal cardiomyocytes was determined by fluorescence microscopy. RESULTS Small-animal PET showed intense and uniform uptake of [(18)F]RhoBDEGF throughout the myocardium in healthy rats. After LAD ligation, well defined perfusion defects were observed in the PET images. The defect size was highly correlated with the infarct size as determined ex vivo by phosphorimaging and TTC staining. In vitro, [(18)F]RhoBDEGF was rapidly internalized into rat cardiomyocytes with ~40 % of the initial activity internalized within the 60 min incubation time. Fluorescence microscopy clearly demonstrated localization of [(18)F]RhoBDEGF in the mitochondria of rat cardiomyocytes. CONCLUSION Fluorine-18-labeled rhodamine B diethylene glycol ester ([(18)F]RhoBDEGF) provides excellent image quality and clear delineation of myocardial infarcts in a rat infarct model. In vitro studies demonstrate localization of the tracer in the mitochondria of cardiac myocytes. In combination, these results support the continued evaluation of this tracer for the PET assessment of myocardial perfusion.

(18)F-labeled rhodamines as potential myocardial perfusion agents: comparison of pharmacokinetic properties of several rhodamines.

INTRODUCTION We recently reported the development of the [(18)F]fluorodiethylene glycol ester of rhodamine B as a potential positron emission tomography (PET) tracer for myocardial perfusion imaging (MPI). This compound was developed by optimizing the ester moiety on the rhodamine B core, and its pharmacokinetic properties were found to be superior to those of the prototype ethyl ester. The goal of the present study was to optimize the rhodamine core while retaining the fluorodiethyleneglycol ester prosthetic group. METHODS A series of different rhodamine cores (rhodamine 6G, rhodamine 101, and tetramethylrhodamine) were labeled with (18)F using the corresponding rhodamine lactones as the precursors and [(18)F]fluorodiethylene glycol ester as the prosthetic group. The compounds were purified by semipreparative HPLC, and their biodistribution was measured in rats. Additionally, the uptake of the compounds was evaluated in isolated rat cardiomyocytes. RESULTS As was the case with the different prosthetic groups, we found that the rhodamine core has a significant effect on the in vitro and in vivo properties of this series of compounds. Of the rhodamines evaluated to date, the pharmacologic properties of the (18)F-labeled diethylene glycol ester of rhodamine 6G are superior to those of the (18)F-labeled diethylene glycol esters of rhodamine B, rhodamine 101, and tetramethylrhodamine. As with (18)F-labeled rhodamine B, [(18)F]rhodamine 6G was observed to localize in the mitochondria of isolated rat cardiomyocytes. CONCLUSIONS Based on these results, the (18)F-labeled diethylene glycol ester of rhodamine 6G is the most promising potential PET MPI radiopharmaceutical of those that have evaluated to date, and we are now preparing to carry out first-in-human clinical studies with this compound.