Cell-based therapies reverse the heart failure-altered right ventricular proteome.

Background: Congenital heart defects can lead to right ventricular (RV) pressure-overload and heart failure. Cell-based therapies, including mesenchymal stromal cells (MSCs) and c-kit positive cells (CPCs) have been studied clinically as options to restore heart function in disease states. Many studies have indicated these cells act through paracrine mechanisms to prevent apoptosis, promote cellular function, and regulate gene/protein expression. We aimed to determine the proteomic response of diseased hearts to cell therapy. Methods: We utilized an animal model of RV pressure overload created by banding the pulmonary artery (PAB). Two weeks post-banding, bone marrow-derived mesenchymal stromal cells (MSCs) and 3 populations of CPCs (nCPCs, cCPCs, ES-CPCs) were delivered to the RV free wall. RV function and cellular retention were measured for four weeks post-injection, at which point hearts were extracted and the RV was excised for liquid chromatography and tandem mass spectrometry. Resulting RV proteomes were compared and analyzed using systems biology and bioinformatics. Results: Proteomic profiling identified 1156 total proteins from the RV, of which 5.97% were significantly changed after PAB. This disease-altered proteome was responsive to cellular therapy, with 72% of the PAB-altered proteome being fully or partially reversed by MSC therapy. This was followed by nCPCs (54%), ES-CPCs (52%), and cCPCs (39%). Systems biology and bioinformatics analysis showed MSC, nCPC, or ES-CPC cell therapy is associated with a decrease in predicted adverse cardiac effects. We also observed an effect of cell therapy on the non-altered RV proteome, however, this was associated with minor predicted pathological endpoints. Conclusions: Our data indicate MSCs, ES-CPCs, and nCPCs significantly reverse the PAB-altered proteome towards a pre-disease state. These results indicate cell-based therapies show promise in improving RV function after pressure overload through partial restoration of the disease-altered cardiac proteome.

Loss of cardiac myosin light chain kinase contributes to contractile dysfunction in right ventricular pressure overload.

Nearly 1 in every 100 children born have a congenital heart defect. Many of these defects primarily affect the right heart causing pressure overload of the right ventricle (RV). The RV maintains function by adapting to the increased pressure; however, many of these adaptations eventually lead to RV hypertrophy and failure. In this study, we aim to identify the cellular and molecular mechanisms of these adaptions. We utilized a surgical animal model of pulmonary artery banding (PAB) in juvenile rats that has been shown to accurately recapitulate the physiology of right ventricular pressure overload in young hearts. Using this model, we examined changes in cardiac myocyte protein expression as a result of pressure overload with mass spectrometry 4 weeks post-banding. We found pressure overload of the RV induced significant downregulation of cardiac myosin light chain kinase (cMLCK). Single myocyte calcium and contractility recordings showed impaired contraction and relaxation in PAB RV myocytes, consistent with the loss of cMLCK. In the PAB myocytes, calcium transients were of smaller amplitude and decayed at a slower rate compared to controls. We also identified miR-200c, which has been shown to regulate cMLCK expression, as upregulated in the RV in response to pressure overload. These results indicate the loss of cMLCK is a critical maladaptation of the RV to pressure overload and represents a novel target for therapeutic approaches to treat RV hypertrophy and failure associated with congenital heart defects.

Anesthesia-Induced Neuronal Apoptosis in the Developing Retina: A Window of Opportunity.

BACKGROUND: Anesthetics cause widespread apoptosis in the developing brain, resulting in neurocognitive abnormalities. However, it is unknown whether anesthesia-induced neurotoxicity occurs in humans because there is currently no modality to assess for neuronal apoptosis in vivo. The retina is unique in that it is the only portion of the central nervous system that can be directly visualized noninvasively. Thus, we aimed to determine whether isoflurane induces apoptosis in the developing retina. METHODS: CD-1 male mouse pups underwent 1-hour exposure to isoflurane (2%) or air. After exposure, activated caspase-3, caspase-9, and caspase-8 were quantified in the retina, cytochrome c release from retinal mitochondria was assessed, and the number and types of cells undergoing apoptosis were identified. Retinal uptake and the ability of fluorescent-labeled annexin V to bind to cells undergoing natural cell death and anesthesia-induced apoptosis in the retina were determined after systemic injection. RESULTS: Isoflurane activated the intrinsic apoptosis pathway in the inner nuclear layer (INL) and activated both the intrinsic and extrinsic pathways in the ganglion cell layer. Immunofluorescence demonstrated that bipolar and amacrine neurons within the INL underwent physiologic cell death in both cohorts and that amacrine cells were the likely targets of isoflurane-induced apoptosis. After injection, fluorescent-labeled annexin V was readily detected in the INL of both air-exposed and isoflurane-exposed mice and colocalized with activated caspase-3-positive cells. CONCLUSIONS: These findings indicate that isoflurane-induced neuronal apoptosis occurs in the developing retina and lays the groundwork for development of a noninvasive imaging technique to detect anesthesia-induced neurotoxicity in infants and children.

Superresolution imaging of human cytomegalovirus vMIA localization in sub-mitochondrial compartments.

The human cytomegalovirus (HCMV) viral mitochondria-localized inhibitor of apoptosis (vMIA) protein, traffics to mitochondria-associated membranes (MAM), where the endoplasmic reticulum (ER) contacts the outer mitochondrial membrane (OMM). vMIA association with the MAM has not been visualized by imaging. Here, we have visualized this by using a combination of confocal and superresolution imaging. Deconvolution of confocal microscopy images shows vMIA localizes away from mitochondrial matrix at the Mitochondria-ER interface. By gated stimulated emission depletion (GSTED) imaging, we show that along this interface vMIA is distributed in clusters. Through multicolor, multifocal structured illumination microscopy (MSIM), we find vMIA clusters localize away from MitoTracker Red, indicating its OMM localization. GSTED and MSIM imaging show vMIA exists in clusters of ~100-150 nm, which is consistent with the cluster size determined by Photoactivated Localization Microscopy (PALM). With these diverse superresolution approaches, we have imaged the clustered distribution of vMIA at the OMM adjacent to the ER. Our findings directly compare the relative advantages of each of these superresolution imaging modalities for imaging components of the MAM and sub-mitochondrial compartments. These studies establish the ability of superresolution imaging to provide valuable insight into viral protein location, particularly in the sub-mitochondrial compartments, and into their clustered organization.