Rebeccah F. Young

Regional alterations in SR Ca2+-ATPase, phospholamban, and HSP-70 expression in chronic hibernating myocardium

We sought to identify mechanisms for chronic dysfunction in hibernating myocardium. Pigs were instrumented with a left anterior descending artery stenosis for 3 mo. Angiography demonstrated high-grade stenoses and hibernating myocardium with 1) severe anterior hypokinesis ( P < 0.001 vs. shams), 2) reduced subendocardial perfusion [0.73 ± 0.05 (SE) vs. 1.01 ± 0.06 ml ⋅ min-1 ⋅ g-1in normal, P < 0.001], and 3) critically reduced adenosine flow (1.0 ± 0.17 vs. 3.84 ± 0.26 ml ⋅ min-1 ⋅ g-1in normal, P < 0.001). Histology did not reveal necrosis. Northern blot analysis of hibernating myocardium demonstrated regional downregulation in mRNAs for sarcoplasmic reticulum (SR) proteins phospholamban (0.76 ± 0.08 vs. 1.07 ± 0.06, P < 0.02) and SR Ca2+-ATPase (0.83 ± 0.06 vs. 1.02 ± 0.06, P < 0.05) with no change in calsequestrin (1.08 ± 0.06 vs. 0.96 ± 0.05, P = not significant). Heat shock protein (HSP)-70 mRNA was regionally induced in hibernating myocardium (2.4 ± 0.3 vs. 1.0 ± 0.11, P < 0.01). Directionally similar changes were confirmed by Western blot analysis of respective proteins. Our results indicate that hibernating myocardium exhibits a molecular phenotype that on a regional basis is similar to end-stage ischemic cardiomyopathy. This supports the hypothesis that SR dysfunction from reversible ischemia may be an early defect in the progression of left ventricular dysfunction.

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 · min−1 · 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.

Proteomic analysis of cisplatin-induced cochlear damage: Methods and early changes in protein expression

To identify early changes in protein expression associated with cisplatin ototoxicity, we used two dimensional-difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption-time-of-flight (MALDI-TOF) mass spectrometry to analyze proteins from P3 rat cochleae that were cultured for 3h with or without 1mM cisplatin. Replicate analysis of fluorescent images from six gels revealed significant (p<0.01) cisplatin-induced changes (greater than 1.5-fold) in expression of 22 cochlear proteins. These include increases in the expression of five proteins, four of which were identified as nucleobindin 1, a nuclear calcium signaling and homeostasis protein (2.1-fold), heterogeneous nuclear ribonucleoprotein C, an RNA processing protein (1.8-fold), a 55 kDa protein that is either endothelial differentiation-related factor 1 or alpha-6 tubulin (1.7-fold), and calreticulin, a calcium binding chaperone of the endoplasmic reticulum (ER, 1.6-fold). The expression of 17 proteins was significantly (p<0.01) decreased by greater than 1.5-fold. These include ribonuclease/angiogenin inhibitor 1 (1.6-fold), RAS-like, family 12 (predicted), ras association (RalGDS/AF-6) domain family 5 (4.5-fold), homologous the RAS family of GTPase signaling proteins (2.4-fold), and Protein tyrosine phosphatase domain containing 1 (predicted, 6.1-fold). We identified seven cochlear proteins with either smaller (1.2-1.5-fold) or less significant (p<0.05) cisplatin-induced changes in expression. Notably, heat shock 70 kDa protein 5 (Hspa5, Grp78, and BiP), an ER chaperone protein involved in stress response, decreased 1.7-fold. We observed changes consistent with phosphorylation in the level of isoforms of another ER stress-induced protein, glucose-regulated protein Grp58. Changes in cisplatin-induced protein expression are discussed with respect to known or hypothesized functions of the identified proteins.

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.

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 (∼33×106 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.

Reductions in mitochondrial O2 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.

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

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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.

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.

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.