Widespread intracoronary allogeneic cardiosphere-derived cell therapy with and without cyclosporine in reperfused myocardial infarction.

Allogeneic cardiosphere-derived cell (CDC) therapy has been demonstrated to improve myocardial function when administered to reperfused myocardial infarcts. We previously pretreated animals with low-dose cyclosporine immunosuppression to limit allogeneic CDC rejection, but whether it is necessary and, if so, can be initiated at the time of reperfusion remains uncertain. Closed-chest swine (n = 29 animals) were subjected to a 90-min left anterior descending (LAD) coronary artery occlusion. Using a three-way blinded design, we randomized two groups to receive global intracoronary infusions of 20 × 106 CDCs 30 min after reperfusion. A third control group was treated with saline. One CDC group received cyclosporine 10 min before reperfusion (2.5 mg/kg iv and 100 mg/day po), whereas the other groups received placebos. After 1 mo, neither chronic infarct size relative to area at risk (saline control, 46.2 ± 4.0%; CDCs, 46.4 ± 2.1%; and CDCs + cyclosporine, 49.2 ± 3.1%; P = 0.79) nor ejection fraction (saline control, 51 ± 2%; CDCs, 51 ± 2%; and CDC + cyclosporine, 48 ± 2%; P = 0.42) were different among treatment groups. Multiple histological measures of cellular remodeling, myocyte proliferation, and apoptosis were also not different among treatment groups. In contrast to previous studies, we were unable to reproduce the cardioprotective effects demonstrated by allogeneic CDCs without cyclosporine. Furthermore, initiation of intravenous cyclosporine at the time of reperfusion followed by oral therapy was not sufficient to elicit the functional improvement observed in studies where cyclosporine was started 72 h before CDC therapy. This suggests that oral cyclosporine pretreatment may be necessary to effect cardiac repair with allogeneic CDCs.NEW & NOTEWORTHY In a three-way blinded, randomized design, we determined whether allogeneic CDCs administered at reperfusion improved myocardial function and whether intravenous cyclosporine enhanced their efficacy. In contrast to prior studies using oral cyclosporine, CDCs with or without intravenous cyclosporine had no effect on function or infarct size. This indicates that CDCs may be most efficacious for treating chronic LV dysfunction where cyclosporine can be initiated at least 72 h before cell therapy.

Quantitative proteomic and phosphoproteomic profiling of ischemic myocardial stunning in swine.

Despite decades of research on the pathophysiology of myocardial stunning, protein changes and/or phosphorylation status underlying alterations in cardiac function/structure remain inadequately understood. Here, we utilized comprehensive and quantitative proteomic and phosphoproteomic approaches to explore molecular mechanisms of myocardial stunning in swine. The closed-chest swine (n = 5 pigs) were subjected to a 10-min left anterior descending coronary artery (LAD) occlusion producing regional myocardial stunning. Tissues from the ischemic LAD region and a remote nonischemic area of the left ventricle were collected 1 h after reperfusion. Ion current-based proteomics (IonStar) and quantitative phosphoproteomics were employed in parallel to identify alterations in protein level and site-specific phosphorylation changes. A novel swine heart protein database exhibiting high accuracy and low redundancy was developed here to facilitate comprehensive study. Further informatic investigations identified potential protein-protein interactions in stunned myocardium. In total, we quantified 2,099 protein groups and 4,699 phosphorylation sites with only 0.4% missing values. Proteomic analyses revealed downregulation of contractile function and extracellular matrix remodeling. Meanwhile, alterations in phosphorylation linked with contractile dysfunction and apoptotic cell death were uncovered. NetworKIN/STRING analysis predicted regulatory kinases responsible for altered phosphosites, such as protein kinase C-mediated phosphorylation of cardiac troponin I-S199 and CaMKII-mediated phosphorylation of phospholamban-T17. In summary, the ion current-based proteomics and phosphoproteomics reliably identified novel alterations in protein content and phosphorylation contributing to contractile dysfunction, extracellular matrix (ECM) damage, and programmed cell death in stunned myocardium, which corroborate well with our physiological observations. Moreover, this work developed a comprehensive database of the swine heart proteome, a highly valuable resource for future translational research in porcine models with cardiovascular diseases.NEW & NOTEWORTHY We first used ion current-based proteomics and phosphoproteomics to reliably identify novel alterations in protein expression and phosphorylation contributing to contractile dysfunction, extracellular matrix (ECM) damage, and programmed cell death in stunned myocardium and developed a comprehensive swine heart-specific proteome database, which provides a valuable resource for future research in porcine models of cardiovascular diseases.

Nonocclusive multivessel intracoronary infusion of allogeneic cardiosphere-derived cells early after reperfusion prevents remote zone myocyte loss and improves global left ventricular function in swine with myocardial infarction.

Intracoronary cardiosphere-derived cells (icCDCs) infused into the infarct-related artery reduce scar volume but do not improve left ventricular (LV) ejection fraction (LVEF). We tested the hypothesis that this reflects the inability of regional delivery to prevent myocyte death or promote myocyte proliferation in viable myocardium remote from the infarct. Swine (n = 23) pretreated with oral cyclosporine (200 mg/day) underwent a 1-h left anterior descending coronary artery (LAD) occlusion, which reduced LVEF from 61.6 ± 1.0 to 45.3 ± 1.5% 30 min after reperfusion. At that time, animals received global infusion of allogeneic icCDCs (n = 8), regional infusion of icCDCs restricted to the LAD using the stop-flow technique (n = 8), or vehicle (n = 7). After 1 mo, global icCDCs increased LVEF from 44.8 ± 1.9 to 60.8 ± 3.8% (P < 0.05) with no significant change after LAD stop-flow icCDCs (44.8 ± 3.6 to 50.9 ± 3.1%) or vehicle (46.5 ± 2.5 to 47.7 ± 2.6%). In contrast, global icCDCs did not alter infarct volume (%LV mass) assessed at 2 days (11.2 ± 2.3 vs. 12.6 ± 2.3%), whereas it was reduced after LAD stop-flow icCDCs (7.1 ± 1.1%, P < 0.05). Histopathological analysis of remote myocardium after global icCDCs demonstrated a significant increase in myocyte proliferation (147 ± 32 vs. 14 ± 10 nuclei/106 myocytes, P < 0.05) and a reduction in myocyte apoptosis (15 ± 9 vs. 46 ± 10 nuclei/106 myocytes, P < 0.05) that increased myocyte nuclear density (1,264 ± 39 vs. 1,157 ± 33 nuclei/mm2, P < 0.05) and decreased myocyte diameter (13.2 ± 0.2 vs. 14.5 ± 0.3 µm, P < 0.05) compared with vehicle-treated controls. In contrast, remote zone changes after regional LAD icCDCs were no different from vehicle. These data indicate that changes in global LVEF after icCDCs are dependent upon preventing myocyte loss and hypertrophy in myocardium remote from the infarct. These arise from stimulating myocyte proliferation and reducing myocyte apoptosis indicating the importance of directing cell therapy to viable remote regions.NEW & NOTEWORTHY Administration of allogeneic cardiosphere-derived cells to the entire heart via global intracoronary infusion shortly after myocardial infarction favorably influenced left ventricular ejection fraction by preventing myocyte death and promoting myocyte proliferation in remote, noninfarcted myocardium in swine. In contrast, regional intracoronary cell infusion did not significantly affect remote zone myocyte remodeling. Global cell administration targeting viable myocardium remote from the infarct may be an effective approach to prevent adverse ventricular remodeling after myocardial infarction.

Troponin Release and Reversible Left Ventricular Dysfunction After Transient Pressure Overload.

BACKGROUND: The authors previously demonstrated that brief ischemia elicits cardiac troponin I (cTnI) release and myocyte apoptosis in the absence of necrosis. It remains uncertain whether other pathophysiological stresses can produce apoptosis and transient cTnI release without ischemia. OBJECTIVES: This study sought to determine whether a transient increase in left ventricular (LV) preload elicits cTnI release in the absence of ischemia. METHODS: Propofol-anesthetized swine (N = 13) received intravenous phenylephrine (PE) (300 µg/min) for 1 h to increase left ventricular end-diastolic pressure (LVEDP) to ∼30 mm Hg. Serial cTnI and echocardiographic function were assessed for 24 h, and myocardial tissue was analyzed for apoptosis and necrosis. RESULTS: PE infusion increased systolic blood pressure from 137 ± 14 mm Hg to 192 ± 11 mm Hg (mean ± SD; p < 0.001) and increased LVEDP from 17 ± 2 mm Hg to 30 ± 5 mm Hg (p < 0.001). Myocardial flow measurements demonstrated no evidence of ischemia. Hemodynamics normalized rapidly after PE, but LV ejection fraction remained depressed (32 ± 21% vs. 58 ± 7%; p < 0.01) with normalization after 24 h (51 ± 16%; p = 0.31). Baseline transcoronary cTnI release was low (16 ± 20 ng/l) but increased to 856 ± 956 ng/l (p = 0.01) 1 h after LVEDP elevation. Circulating cTnI rose above the 99th percentile within 30 min and remained elevated at 24 h (1,462 ± 1,691 ng/l). Pathological analysis demonstrated myocyte apoptosis at 3 h (31.3 ± 11.9 myocytes/cm2 vs. 4.6 ± 3.7 myocytes/cm2; p < 0.01), that normalized after 24 h (6.2 ± 5.6 myocytes/cm2; p = 0.46) without histological necrosis. CONCLUSIONS: Transient elevations of LVEDP lead to cTnI release, apoptosis, and reversible stretch-induced stunning in the absence of ischemia. Thus, preload-induced myocyte injury may explain many cTnI elevations seen in the absence of clinical signs or symptoms of myocardial ischemia.

Sensitive, High-Throughput, and Robust Trapping-Micro-LC-MS Strategy for the Quantification of Biomarkers and Antibody Biotherapeutics.

For LC-MS-based targeted quantification of biotherapeutics and biomarkers in clinical and pharmaceutical environments, high sensitivity, high throughput, and excellent robustness are all essential but remain challenging. For example, though nano-LC-MS has been employed to enhance analytical sensitivity, it falls short because of its low loading capacity, poor throughput, and low operational robustness. Furthermore, high chemical noise in protein bioanalysis typically limits the sensitivity. Here we describe a novel trapping-micro-LC-MS (T-µLC-MS) strategy for targeted protein bioanalysis, which achieves high sensitivity with exceptional robustness and high throughput. A rapid, high-capacity trapping of biological samples is followed by µLC-MS analysis; dynamic sample trapping and cleanup are performed using pH, column chemistry, and fluid mechanics separate from the µLC-MS analysis, enabling orthogonality, which contributes to the reduction of chemical noise and thus results in improved sensitivity. Typically, the selective-trapping and -delivery approach strategically removes >85% of the matrix peptides and detrimental components, markedly enhancing sensitivity, throughput, and operational robustness, and narrow-window-isolation selected-reaction monitoring further improves the signal-to-noise ratio. In addition, unique LC-hardware setups and flow approaches eliminate gradient shock and achieve effective peak compression, enabling highly sensitive analyses of plasma or tissue samples without band broadening. In this study, the quantification of 10 biotherapeutics and biomarkers in plasma and tissues was employed for method development. As observed, a significant sensitivity gain (up to 25-fold) compared with that of conventional LC-MS was achieved, although the average run time was only 8 min/sample. No appreciable peak deterioration or loss of sensitivity was observed after >1500 injections of tissue and plasma samples. The developed method enabled, for the first time, ultrasensitive LC-MS quantification of low levels of a monoclonal antibody and antigen in a tumor and cardiac troponin I in plasma after brief cardiac ischemia. This strategy is valuable when highly sensitive protein quantification in large sample sets is required, as is often the case in typical biomarker validation and pharmaceutical investigations of antibody therapeutics.

Brief Myocardial Ischemia Produces Cardiac Troponin I Release and Focal Myocyte Apoptosis in the Absence of Pathological Infarction in Swine.

In a porcine model of brief ischemia leading to reversible stunning in the absence of tissue necrosis, we demonstrated delayed release of cTnI that exceeded the 99th percentile for normals 60-minutes after reperfusion and rose to readily detectable levels 24-hours later. While tissue analysis at 60-minutes showed no evidence of infarction, TUNEL staining demonstrated isolated myocytes undergoing apoptosis, which was absent after 24-hours. These results demonstrate that cTnI elevations occur after ischemia of a duration that is insufficient to produce myocyte necrosis and reflect myocyte injury associated with delayed apoptosis in the absence of pathological evidence of infarction.

Inhibiting Extracellular Vesicle Release from Human Cardiosphere Derived Cells with Lentiviral Knockdown of nSMase2 Differentially Effects Proliferation and Apoptosis in Cardiomyocytes, Fibroblasts and Endothelial Cells In Vitro.

Numerous studies have shown a beneficial effect of cardiosphere-derived cell (CDC) therapy on regeneration of injured myocardium. Paracrine signaling by CDC secreted exosomes may contribute to improved cardiac function. However, it has not yet been demonstrated by a genetic approach that exosome release contributes to the therapeutic effect of transplanted CDCs. By employing a lentiviral knockdown (KD) strategy against neutral spingomyelinase 2 (nSMase2), a crucial gene in exosome secretion, we have defined the role of physiologically secreted human CDC-derived exosomes on cardiac fibroblast, endothelial cell and primary cardiomyocyte proliferation, cell death, migration and angiogenesis using a series of in vitro coculture assays. We found that secretion of hCDC-derived exosomes was effectively inhibited by nSMase2 lentiviral KD and shRNAi expression was stable and constitutive. hCDC exosome release contributed to the angiogenic and pro-migratory effects of hCDCs on HUVECs, decreased proliferation of fibroblasts, and decreased apoptosis of cardiomyocytes. These in vitro reactions support a role for exosome secretion as a paracrine mechanism of stem cell-mediated cardiac repair in vivo. Importantly, we have established a novel tool to test constitutive inhibition of exosome secretion in stem cell populations in animal models of cardiac disease.

Heart-Derived Stem Cells in Miniature Swine with Coronary Microembolization: Novel Ischemic Cardiomyopathy Model to Assess the Efficacy of Cell-Based Therapy.

A major problem in translating stem cell therapeutics is the difficulty of producing stable, long-term severe left ventricular (LV) dysfunction in a large animal model. For that purpose, extensive infarction was created in sinclair miniswine by injecting microspheres (1.5 × 106 microspheres, 45 µm diameter) in LAD. At 2 months after embolization, animals (n = 11) were randomized to receive allogeneic cardiosphere-derived cells derived from atrium (CDCs: 20 × 106, n = 5) or saline (untreated, n = 6). Four weeks after therapy myocardial function, myocyte proliferation (Ki67), mitosis (phosphor-Histone H3; pHH3), apoptosis, infarct size (TTC), myocyte nuclear density, and cell size were evaluated. CDCs injected into infarcted and remodeled remote myocardium (global infusion) increased regional function and global function contrasting no change in untreated animals. CDCs reduced infarct volume and stimulated Ki67 and pHH3 positive myocytes in infarct and remote regions. As a result, myocyte number (nuclear density) increased and myocyte cell diameter decreased in both infarct and remote regions. Coronary microembolization produces stable long-term ischemic cardiomyopathy. Global infusion of CDCs stimulates myocyte regeneration and improves left ventricular ejection fraction. Thus, global infusion of CDCs could become a new therapy to reverse LV dysfunction in patients with asymptomatic heart failure.

LKB1 knockout mouse develops spontaneous atrial fibrillation and provides mechanistic insights into human disease process.

BACKGROUND: Atrial fibrillation (AF) is a complex disease process, and the molecular mechanisms underlying initiation and progression of the disease are unclear. Consequently, AF has been difficult to model. In this study, we have presented a novel transgenic mouse model of AF that mimics human disease and characterized the mechanisms of atrial electroanatomical remodeling in the genesis of AF. METHODS AND RESULTS: Cardiac-specific liver kinase B1 (LKB1) knockout (KO) mice were generated, and 47% aged 4 weeks and 95% aged 12 weeks developed spontaneous AF from sinus rhythm by demonstrating paroxysmal and persistent stages of the disease. Electrocardiographic characteristics of sinus rhythm were similar in KO and wild-type mice. Atrioventricular block and atrial flutter were common in KO mice. Heart rate was slower with persistent AF. In parallel with AF, KO mice developed progressive biatrial enlargement with inflammation, heterogeneous fibrosis, and loss of cardiomyocyte population with apoptosis and necrosis. Atrial tissue was infiltrated with inflammatory cells. C-reactive protein, interleukin 6, and tumor necrosis factor α were significantly elevated in serum. KO atria demonstrated elevated reactive oxygen species and decreased AMP-activated protein kinase activity. Cardiomyocyte and myofibrillar ultrastructure were disrupted. Intercellular matrix and gap junction were interrupted. Connexins 40 and 43 were reduced. Persistent AF caused left ventricular dysfunction and heart failure. Survival and exercise capacity were worse in KO mice. CONCLUSIONS: LKB1 KO mice develop spontaneous AF from sinus rhythm and progress into persistent AF by replicating the human AF disease process. Progressive inflammatory atrial cardiomyopathy is the genesis of AF, through mechanistic electrical and structural remodeling.

Revascularization of chronic hibernating myocardium stimulates myocyte proliferation and partially reverses chronic adaptations to ischemia.

BACKGROUND: The time course and extent of recovery after revascularization of viable dysfunctional myocardium are variable. Although fibrosis is a major determinant, myocyte structural and molecular remodeling may also play important roles. OBJECTIVES: This study sought to determine whether persistent myocyte loss and/or irreversibility of protein changes that develop in hibernating myocardium have an impact on functional recovery in the absence of infarction. METHODS: Swine implanted with a chronic left anterior descending artery (LAD) stenosis to produce hibernating myocardium underwent percutaneous revascularization, with serial functional recovery evaluated for 1 month (n = 12). Myocardial tissue was evaluated to assess myocyte size, nuclear density, and proliferation indexes in comparison with those of normal animals and nonrevascularized controls. Proteomic analysis by 2-dimensional differential in-gel electrophoresis was used to determine the reversibility of molecular adaptations of hibernating myocytes. RESULTS: At 3 months, physiological features of hibernating myocardium were confirmed, with depressed LAD wall thickening and no significant infarction. Revascularization normalized LAD flow reserve, with no immediate change in LAD wall thickening. Regional LAD wall thickening slowly improved but remained depressed 1 month post-percutaneous coronary intervention. Surprisingly, revascularization was associated with histological evidence of myocytes re-entering the growth phase of the cell cycle and increases in the number of c-Kit(+) cells. Myocyte nuclear density returned to normal, whereas regional myocyte hypertrophy regressed. Proteomic analysis demonstrated heterogeneous effects of revascularization. Up-regulated stress and cytoskeletal proteins normalized, whereas reduced contractile and metabolic proteins persisted. CONCLUSIONS: Delayed recovery of hibernating myocardium in the absence of scar may reflect persistent reductions in the amounts of contractile and metabolic proteins. Although revascularization appeared to stimulate myocyte proliferation, the persistence of small immature myocytes may have contributed to delayed functional recovery.

Global intracoronary infusion of allogeneic cardiosphere-derived cells improves ventricular function and stimulates endogenous myocyte regeneration throughout the heart in swine with hibernating myocardium.

BACKGROUND: Cardiosphere-derived cells (CDCs) improve ventricular function and reduce fibrotic volume when administered via an infarct-related artery using the "stop-flow" technique. Unfortunately, myocyte loss and dysfunction occur globally in many patients with ischemic and non-ischemic cardiomyopathy, necessitating an approach to distribute CDCs throughout the entire heart. We therefore determined whether global intracoronary infusion of CDCs under continuous flow improves contractile function and stimulates new myocyte formation. METHODS AND RESULTS: Swine with hibernating myocardium from a chronic LAD occlusion were studied 3-months after instrumentation (n = 25). CDCs isolated from myocardial biopsies were infused into each major coronary artery (∼ 33 × 10(6) icCDCs). Global icCDC infusion was safe and while ∼ 3% of injected CDCs were retained, they did not affect ventricular function or myocyte proliferation in normal animals. In contrast, four-weeks after icCDCs were administered to animals with hibernating myocardium, %LADWT increased from 23 ± 6 to 51 ± 5% (p<0.01). In diseased hearts, myocyte proliferation (phospho-histone-H3) increased in hibernating and remote regions with a concomitant increase in myocyte nuclear density. These effects were accompanied by reductions in myocyte diameter consistent with new myocyte formation. Only rare myocytes arose from sex-mismatched donor CDCs. CONCLUSIONS: Global icCDC infusion under continuous flow is feasible and improves contractile function, regresses myocyte cellular hypertrophy and increases myocyte proliferation in diseased but not normal hearts. New myocytes arising via differentiation of injected cells are rare, implicating stimulation of endogenous myocyte regeneration as the primary mechanism of repair.

Quantitative proteomics in cardiovascular research: global and targeted strategies.

Extensive technical advances in the past decade have substantially expanded quantitative proteomics in cardiovascular research. This has great promise for elucidating the mechanisms of cardiovascular diseases and the discovery of cardiac biomarkers used for diagnosis and treatment evaluation. Global and targeted proteomics are the two major avenues of quantitative proteomics. While global approaches enable unbiased discovery of altered proteins via relative quantification at the proteome level, targeted techniques provide higher sensitivity and accuracy, and are capable of multiplexed absolute quantification in numerous clinical/biological samples. While promising, technical challenges need to be overcome to enable full utilization of these techniques in cardiovascular medicine. Here, we discuss recent advances in quantitative proteomics and summarize applications in cardiovascular research with an emphasis on biomarker discovery and elucidating molecular mechanisms of disease. We propose the integration of global and targeted strategies as a high-throughput pipeline for cardiovascular proteomics. Targeted approaches enable rapid, extensive validation of biomarker candidates discovered by global proteomics. These approaches provide a promising alternative to immunoassays and other low-throughput means currently used for limited validation.

Reproducible ion-current-based approach for 24-plex comparison of the tissue proteomes of hibernating versus normal myocardium in swine models.

Hibernating myocardium is an adaptive response to repetitive myocardial ischemia that is clinically common, but the mechanism of adaptation is poorly understood. Here we compared the proteomes of hibernating versus normal myocardium in a porcine model with 24 biological replicates. Using the ion-current-based proteomic strategy optimized in this study to expand upon previous proteomic work, we identified differentially expressed proteins in new molecular pathways of cardiovascular interest. The methodological strategy includes efficient extraction with detergent cocktail; precipitation/digestion procedure with high, quantitative peptide recovery; reproducible nano-LC/MS analysis on a long, heated column packed with small particles; and quantification based on ion-current peak areas. Under the optimized conditions, high efficiency and reproducibility were achieved for each step, which enabled a reliable comparison of 24 the myocardial samples. To achieve confident discovery of differentially regulated proteins in hibernating myocardium, we used highly stringent criteria to define "quantifiable proteins". These included the filtering criteria of low peptide FDR and S/N > 10 for peptide ion currents, and each protein was quantified independently from ≥2 distinct peptides. For a broad methodological validation, the quantitative results were compared with a parallel, well-validated 2D-DIGE analysis of the same model. Excellent agreement between the two orthogonal methods was observed (R = 0.74), and the ion-current-based method quantified almost one order of magnitude more proteins. In hibernating myocardium, 225 significantly altered proteins were discovered with a low false-discovery rate (∼3%). These proteins are involved in biological processes including metabolism, apoptosis, stress response, contraction, cytoskeleton, transcription, and translation. This provides compelling evidence that hibernating myocardium adapts to chronic ischemia. The major metabolic mechanisms include a down-regulation of mitochondrial respiration and an increase in glycolysis. Meanwhile, cardioprotective and cytoskeletal proteins are increased, while cardiomyocyte contractile proteins are reduced. These intrinsic adaptations to regional ischemia maintain long-term cardiomyocyte viability at the expense of contractile function.

LDL cholesterol modulates human CD34+ HSPCs through effects on proliferation and the IL-17 G-CSF axis.

BACKGROUND: Hypercholesterolemia plays a critical role in atherosclerosis. CD34+ CD45dim Lineage- hematopoietic stem/progenitor cells (HSPCs) give rise to the inflammatory cells linked to atherosclerosis. In mice, high cholesterol levels mobilize HSPCs into the bloodstream, and promote their differentiation to granulocytes and monocytes. The objective of our study was to determine how cholesterol levels affect HSPC quantity in humans. METHODS: We performed a blinded, randomized hypothesis generating study in human subjects (n=12) treated sequentially with statins of differing potencies to vary lipid levels. CD34+ HSPC levels in blood were measured by flow cytometry. Hematopoietic colony forming assays confirmed the CD34+ population studied as HSPCs with multlineage differentiation potential. Mobilizing cytokine levels were measured by ELISA. RESULTS: The quantity of HSPCs was 0.15 ± 0.1% of buffy coat leukocytes. We found a weak, positive correlation between CD34+ HSPCs and both total and LDL cholesterol levels (r(2)=0.096, p < 0.025). Additionally, we tested whether cholesterol modulates CD34+ HSPCs through direct effects or on the levels of mobilizing cytokines. LDL cholesterol increased cell surface expression of CXCR4, G-CSFR affecting HSPC migration, and CD47 mediating protection from phagocytosis by immune cells. LDL cholesterol also increased proliferation of CD34+ HSPCs (28 ± 5.7%, n=6, p < 0.03). Finally, the HSPC mobilizing cytokine G-CSF (r(2)=0.0683, p < 0.05), and its upstream regulator IL-17 (r(2)=0.0891, p < 0.05) both correlated positively with LDL cholesterol, while SDF-1 levels were not significantly affected. CONCLUSIONS: Our findings support a model where LDL cholesterol levels positively correlate with CD34+ HSPC levels in humans through effects on the levels of G-CSF via IL-17 promoting mobilization of HSPCs, and by direct effects of LDL cholesterol on HSPC proliferation. The findings are provocative of further study to determine if HSPCs, like cholesterol levels, are linked to CVD events.

The physiological significance of a coronary stenosis differentially affects contractility and mitochondrial function in viable chronically dysfunctional myocardium.

The reversibility of viable dysfunctional myocardium after revascularization is variable and the reasons for this are unknown. Using 2D-DIGE, we tested the hypothesis that this could reflect the extent of molecular remodeling of myocardial tissue in the absence of infarction. Swine with a progressive left anterior descending (LAD) stenosis were studied 2 months (n = 18) or 3 months (n = 22) post-instrumentation. Coronary flow reserve (vasodilated/rest) was severely reduced at 2 months (LAD 2.6 ± 0.4 versus 5.1 ± 0.4 in normal, p < 0.05) and became critically impaired after 3 months (LAD 1.1 ± 0.2, p < 0.05 vs. 2 months). Despite progression in stenosis severity, reductions in wall thickening at 2 months (LAD 37 ± 4% vs. remote 86 ± 9%, p < 0.05) were unchanged at 3 months (LAD 32 ± 3%, p = ns). Contractile dysfunction was primarily related to reductions (LAD/normal) in contractile proteins which were not affected by stenosis severity (e.g., troponin T, 2 months 0.82 ± 0.03 vs. 0.74 ± 0.03 at 3 months, p-ns). In contrast, mitochondrial function and proteins were normal at 2 months but declined with progression to a critical stenosis (state 3 respiration at 3 months 145 ± 13 vs. 216 ± 5 ng-atoms O2 mg(-1) min(-1) at 2 months, p < 0.05). In a similar fashion, increases in stress (e.g., αB-crystalline 2.13 ± 0.2 vs. 1.17 ± 0.13 at 2 months, p < 0.05) and cytoskeletal proteins (e.g., desmin 1.63 ± 0.12 vs. 1.24 ± 0.10 at 2 months, p < 0.05) only developed with more advanced remodeling from a critical stenosis. We conclude that similar degrees of chronic contractile dysfunction can have diverse intrinsic molecular adaptations to ischemia. This spectrum of adaptations may underlie variability in the time course and extent of reversibility in viable chronically dysfunctional myocardium after revascularization.

Combinatorial peptide ligand library treatment followed by a dual-enzyme, dual-activation approach on a nanoflow liquid chromatography/orbitrap/electron transfer dissociation system for comprehensive analysis of swine plasma proteome.

The plasma proteome holds enormous clinical potential, yet an in-depth analysis of the plasma proteome remains a daunting challenge due to its high complexity and the extremely wide dynamic range in protein concentrations. Furthermore, existing antibody-based approaches for depleting high-abundance proteins are not adaptable to the analysis of the animal plasma proteome, which is often essential for experimental pathology/pharmacology. Here we describe a highly comprehensive method for the investigation of the animal plasma proteome which employs an optimized combinatorial peptide ligand library (CPLL) treatment to reduce the protein concentration dynamic range and a dual-enzyme, dual-activation strategy to achieve high proteomic coverage. The CPLL treatment enriched the lower abundance proteins by >100-fold when the samples were loaded in moderately denaturing conditions with multiple loading-washing cycles. The native and the CPLL-treated plasma were digested in parallel by two enzymes (trypsin and GluC) carrying orthogonal specificities. By performing this differential proteolysis, the proteome coverage is improved where peptides produced by only one enzyme are poorly detectable. Digests were fractionated with high-resolution strong cation exchange chromatography and then resolved on a long, heated nano liquid chromatography column. MS analysis was performed on a linear triple quadrupole/orbitrap with two complementary activation methods (collisionally induced dissociation (CID) and electron transfer dissociation). We applied this optimized strategy to investigate the plasma proteome from swine, a prominent animal model for cardiovascular diseases (CVDs). This large-scale analysis results in identification of a total of 3421 unique proteins, spanning a concentration range of 9-10 orders of magnitude. The proteins were identified under a set of commonly accepted criteria, including a precursor mass error of <15 ppm, Xcorr cutoffs, and ≥2 unique peptides at a peptide probability of ≥95% and a protein probability of ≥99%, and the peptide false-positive rate of the data set was 1.8% as estimated by searching the reversed database. CPLL treatment resulted in 55% more identified proteins over those from native plasma; moreover, compared with using only trypsin and CID, the dual-enzyme/activation approach enabled the identification of 2.6-fold more proteins and substantially higher sequence coverage for most individual proteins. Further analysis revealed 657 proteins as significantly associated with CVDs (p < 0.05), which constitute five CVD-related pathways. This study represents the first in-depth investigation of a nonhuman plasma proteome, and the strategy developed here is adaptable to the comprehensive analysis of other highly complex proteomes.

Reductions in mitochondrial O(2) consumption and preservation of high-energy phosphate levels after simulated ischemia in chronic hibernating myocardium.

We performed the present study to determine whether hibernating myocardium is chronically protected from ischemia. Myocardial tissue was rapidly excised from hibernating left anterior descending coronary regions (systolic wall thickening = 2.8 +/- 0.2 vs. 5.4 +/- 0.3 mm in remote myocardium), and high-energy phosphates were quantified by HPLC during simulated ischemia in vitro (37 degrees C). At baseline, ATP (20.1 +/- 1.0 vs. 26.7 +/- 2.1 micromol/g dry wt, P < 0.05), ADP (8.1 +/- 0.4 vs. 10.3 +/- 0.8 micromol/g, P < 0.05), and total adenine nucleotides (31.2 +/- 1.3 vs. 40.1 +/- 2.9 micromol/g, P < 0.05) were depressed compared with normal myocardium, whereas total creatine, creatine phosphate, and ATP-to-ADP ratios were unchanged. During simulated ischemia, there was a marked attenuation of ATP depletion (5.6 +/- 0.9 vs. 13.7 +/- 1.7 micromol/g at 20 min in control, P < 0.05) and mitochondrial respiration [145 +/- 13 vs. 187 +/- 11 ng atoms O(2).mg protein(-1).min(-1) in control (state 3), P < 0.05], whereas lactate accumulation was unaffected. These in vitro changes were accompanied by protection of the hibernating heart from acute stunning during demand-induced ischemia. Thus, despite contractile dysfunction at rest, hibernating myocardium is ischemia tolerant, with reduced mitochondrial respiration and slowing of ATP depletion during simulated ischemia, which may maintain myocyte viability.

A straightforward and highly efficient precipitation/on-pellet digestion procedure coupled with a long gradient nano-LC separation and Orbitrap mass spectrometry for label-free expression profiling of the swine heart mitochondrial proteome.

For label-free expression profiling of tissue proteomes, efficient protein extraction, thorough and quantitative sample cleanup and digestion procedures, as well as sufficient and reproducible chromatographic separation, are highly desirable but remain challenging. However, optimal methodology has remained elusive, especially for proteomes that are rich in membrane proteins, such as the mitochondria. Here, we describe a straightforward and reproducible sample preparation procedure, coupled with a highly selective and sensitive nano-LC/Orbitrap analysis, which enables reliable and comprehensive expression profiling of tissue mitochondria. The mitochondrial proteome of swine heart was selected as a test system. Efficient protein extraction was accomplished using a strong buffer containing both ionic and nonionic detergents. Overnight precipitation was used for cleanup of the extract, and the sample was subjected to an optimized 2-step, on-pellet digestion approach. In the first step, the protein pellet was dissolved via a 4 h tryptic digestion under vigorous agitation, which nano-LC/LTQ/ETD showed to produce large and incompletely cleaved tryptic peptides. The mixture was then reduced, alkylated, and digested into its full complement of tryptic peptides with additional trypsin. This solvent precipitation/on-pellet digestion procedure achieved significantly higher and more reproducible peptide recovery of the mitochondrial preparation than observed using a prevalent alternative procedure for label-free expression profiling, SDS-PAGE/in-gel digestion (87% vs 54%). Furthermore, uneven peptide losses were lower than observed with SDS-PAGE/in-gel digestion. The resulting peptides were sufficiently resolved by a 5 h gradient using a nano-LC configuration that features a low-void-volume, high chromatographic reproducibility, and an LTQ/Orbitrap analyzer for protein identification and quantification. The developed method was employed for label-free comparison of the mitochondrial proteomes of myocardium from healthy animals versus those with hibernating myocardium. Each experimental group consisted of a relatively large number of animals (n = 10), and samples were analyzed in random order to minimize quantitative false-positives. With this approach, 904 proteins were identified and quantified with high confidence, and those mitochondrial proteins that were altered significantly between groups were compared with the results of a parallel 2D-DIGE analysis. The sample preparation and analytical strategy developed here represents an advancement that can be adapted to analyze other tissue proteomes.

Persistent regional downregulation in mitochondrial enzymes and upregulation of stress proteins in swine with chronic hibernating myocardium.

Hibernating myocardium is accompanied by a downregulation in energy utilization that prevents the immediate development of ischemia during stress at the expense of an attenuated level of regional contractile function. We used a discovery based proteomic approach to identify novel regional molecular adaptations responsible for this phenomenon in subendocardial samples from swine instrumented with a chronic LAD stenosis. After 3 months (n=8), hibernating myocardium was present as reflected by reduced resting LAD flow (0.75+/-0.14 versus 1.19+/-0.14 mL x min(-1) x g(-1) in remote) and wall thickening (1.93+/-0.46 mm versus 5.46+/-0.41 mm in remote, P<0.05). Regionally altered proteins were quantified with 2D Differential-in-Gel Electrophoresis (2D-DIGE) using normal myocardium as a reference with identification of candidates using MALDI-TOF mass spectrometry. Hibernating myocardium developed a significant downregulation of many mitochondrial proteins and an upregulation of stress proteins. Of particular note, the major entry points to oxidative metabolism (eg, pyruvate dehydrogenase complex and Acyl-CoA dehydrogenase) and enzymes involved in electron transport (eg, complexes I, III, and V) were reduced (P<0.05). Multiple subunits within an enzyme complex frequently showed a concordant downregulation in abundance leading to an amplification of their cumulative effects on activity (eg, "total" LAD PDC activity was 21.9+/-3.1 versus 42.8+/-1.9 mU, P<0.05). After 5-months (n=10), changes in mitochondrial and stress proteins persisted whereas cytoskeletal proteins (eg, desmin and vimentin) normalized. These data indicate that the proteomic phenotype of hibernating myocardium is dynamic and has similarities to global changes in energy substrate metabolism and function in the advanced failing heart. These proteomic changes may limit oxidative injury and apoptosis and impact functional recovery after revascularization.