Impact of early pericardial fluid chymase activation after cardiac surgery.

Introduction: Chymase is a highly destructive serine protease rapidly neutralized in the circulation by protease inhibitors. Here we test whether pericardial fluid (PCF) chymase activation and other inflammatory biomarkers determine intensive care unit length of stay, and explore mechanisms of chymase delivery by extracellular vesicles to the heart. Methods: PCF was collected from adult patients (17 on-pump; 13 off-pump) 4 h after cardiac surgery. Extracellular vesicles (EVs) containing chymase were injected into Sprague-Dawley rats to test for their ability to deliver chymase to the heart. Results: The mean intensive care unit (ICU) stay and mean total length of stay was 2.17 ± 3.8 days and 6.41 ± 1.3 days respectively. Chymase activity and 32 inflammatory markers did not differ in on-pump vs. off-pump cardiac surgery. Society of Thoracic Surgeons Predicted Risk of Morbidity and Mortality Score (STS-PROM), 4-hour post-surgery PCF chymase activity and C-X-C motif chemokine ligand 6 (CXCL6) were all independent predictors of ICU and total hospital length of stay by univariate analysis. Mass spectrometry of baseline PCF shows the presence of serine protease inhibitors that neutralize chymase activity. The compartmentalization of chymase within and on the surface of PCF EVs was visualized by immunogold labeling and transmission electron microscopy. A chymase inhibitor prevented EV chymase activity (0.28 fmol/mg/min vs. 14.14 fmol/mg/min). Intravenous injection of PCF EVs obtained 24 h after surgery into Sprague Dawley rats shows diffuse human chymase uptake in the heart with extensive cardiomyocyte damage 4 h after injection. Discussion: Early postoperative PCF chymase activation underscores its potential role in cardiac damage soon after on- or off-pump cardiac surgery. In addition, chymase in extracellular vesicles provides a protected delivery mechanism from neutralization by circulating serine protease inhibitors.

Mitochondrial haplotype modulates genome expression and mitochondrial structure/function in cardiomyocytes following volume overload.

Mitochondrial DNA (mtDNA) haplotype regulates mitochondrial structure/function and reactive oxygen species in aortocaval fistula (ACF) in mice. Here, we unravel the mitochondrial haplotype effects on cardiomyocyte mitochondrial ultrastructure and transcriptome response to ACF in vivo. Phenotypic responses and quantitative transmission electron microscopy (TEM) and RNA sequence at 3 days were determined after sham surgery or ACF in vivo in cardiomyocytes from wild-type (WT) C57BL/6J (C57n:C57mt) and C3H/HeN (C3Hn:C3Hmt) and mitochondrial nuclear exchange mice (C57n:C3Hmt or C3Hn:C57mt). Quantitative TEM of cardiomyocyte mitochondria C3HWT hearts have more electron-dense compact mitochondrial cristae compared with C57WT. In response to ACF, mitochondrial area and cristae integrity are normal in C3HWT; however, there is mitochondrial swelling, cristae lysis, and disorganization in both C57WT and MNX hearts. Tissue analysis shows that C3HWT hearts have increased autophagy, antioxidant, and glucose fatty acid oxidation-related genes compared with C57WT. Comparative transcriptomic analysis of cardiomyocytes from ACF was dependent upon mtDNA haplotype. C57mtDNA haplotype was associated with increased inflammatory/protein synthesis pathways and downregulation of bioenergetic pathways, whereas C3HmtDNA showed upregulation of autophagy genes. In conclusion, ACF in vivo shows a protective response of C3Hmt haplotype that is in large part driven by mitochondrial nuclear genome interaction.NEW & NOTEWORTHY The results of this study support the effects of mtDNA haplotype on nuclear gene expression in cardiomyocytes. Currently, there is no acceptable therapy for volume overload due to mitral regurgitation. The findings of this study could suggest that mtDNA haplotype activates different pathways after ACF warrants further investigations on human population of heart disease from different ancestry backgrounds.

Plasma Exosome Hemoglobin Released During Surgery Is Associated With Cardiac Injury in Animal Model.

BACKGROUND: Patients with valvular heart disease require cardiopulmonary bypass and cardiac arrest. Here, we test the hypothesis that exosomal hemoglobin formed during cardiopulmonary bypass mediates acute cardiac injury in humans and in an animal model system. METHODS: Plasma exosomes were collected from arterial blood at baseline and 30 minutes after aortic cross-clamp release in 20 patients with primary mitral regurgitation and 7 with aortic stenosis. These exosomes were injected into Sprague-Dawley rats and studied at multiple times up to 30 days. Tissue was examined by hematoxylin and eosin stain, immunohistochemistry, transmission electron microscopy, and brain natriuretic peptide. RESULTS: Troponin I levels increased from 36 ± 88 ng/L to 3622 ± 3054 ng/L and correlated with exosome hemoglobin content (Spearman r = 0.7136, < .0001, n = 24). Injection of exosomes isolated 30 minutes after cross-clamp release into Sprague-Dawley rats resulted in cardiomyocyte myofibrillar loss at 3 days. Transmission electron microscopy demonstrated accumulation of electron dense particles of ferritin within cardiomyocytes, in the interstitial space, and within exosomes. At 21 days after injection, there was myofibrillar and myosin breakdown, interstitial fibrosis, elevated brain natriuretic peptide, and left ventricle diastolic dysfunction measured by echocardiography/Doppler. Pericardial fluid exosomal hemoglobin content is fourfold higher than simultaneous plasma exosome hemoglobin, suggesting a cardiac source of exosomal hemoglobin. CONCLUSIONS: Red blood cell and cardiac-derived exosomal hemoglobin may be involved in myocardial injury during cardiopulmonary bypass in patients with valvular heart disease.

Red blood cell exosome hemoglobin content increases after cardiopulmonary bypass and mediates acute kidney injury in an animal model.

OBJECTIVE: Hemolysis, characterized by formation of free hemoglobin (Hb), occurs in patients undergoing cardiopulmonary bypass (CPB). However, there is no study of the dynamic changes in red blood cell (RBC)-derived exosomes (Exos) released during CPB, nor whether these particles mediate acute kidney injury (AKI). METHODS: This study is a comprehensive time-course analysis, at baseline, 30 minutes, to 24 hours post-crossclamp release (XCR) to determine (1) Exos Hb content; (2) free Hb/heme, haptoglobin, hemopexin; and (3) urinary markers of AKI over the same time period. In addition, we developed a model system in Sprague-Dawley rats to test for AKI after intravenous injection of Exos Hb released during CPB. RESULTS: In 30 patients undergoing CPB, there is a significant increase in plasma Hb-positive Exos but not microvesicles 30 minutes post-XCR versus other time points, with a simultaneous decrease in the haptoglobin/Hb ratio. These changes presage a significant increase in urine neutrophil gelatinase-associated lipocalin and kidney injury molecule-1 at 24 hours. Intravenous injection of plasma Exos (109-10 particles obtained 30 minutes post-XCR) into rats causes AKI at 72 hours, manifested by multifocal degeneration of proximal tubular epithelium. At 21 days, there is persistent tubular injury and interstitial fibrosis. Intravenous injection of Exos from 35-day-old stored RBCs into rats results in glomerular-tubular injury, increased kidney ferritin and hemoxygenase-1 expression, and significant elevation of kidney injury molecule-1 and proteinuria at 72 hours. CONCLUSIONS: These combined studies raise the potential for RBC-derived Exos, released during CPB, to target the kidney and mediate AKI.

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats.

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 min) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase 2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure.

Heart failure due to chronic volume overload (VO) in rats and humans is characterized by disorganization of the cardiomyocyte desmin/mitochondrial network. Here, we tested the hypothesis that desmin breakdown is an early and continuous process throughout VO. Male Sprague-Dawley rats had aortocaval fistula (ACF) or sham surgery and were examined 24 h and 4 and 12 wk later. Desmin/mitochondrial ultrastructure was examined by transmission electron microscopy (TEM) and immunohistochemistry (IHC). Protein and kinome analysis were performed in isolated cardiomyocytes, and desmin cleavage was assessed by mass spectrometry in left ventricular (LV) tissue. Echocardiography demonstrated a 40% decrease in the LV mass-to-volume ratio with spherical remodeling at 4 wk with ACF and LV systolic dysfunction at 12 wk. Starting at 24 h and continuing to 4 and 12 wk, with ACF there is TEM evidence of extensive mitochondrial clustering, IHC evidence of disorganization associated with desmin breakdown, and desmin protein cleavage verified by Western blot analysis and mass spectrometry. IHC results revealed that ACF cardiomyocytes at 4 and 12 wk had perinuclear translocation of αB-crystallin from the Z disk with increased α, ß-unsaturated aldehyde 4-hydroxynonelal. Use of protein markers with verification by TUNEL staining and kinome analysis revealed an absence of cardiomyocyte apoptosis at 4 and 12 wk of ACF. Significant increases in protein indicators of mitophagy were countered by a sixfold increase in p62/sequestosome-1, which is indicative of an inability to complete autophagy. An early and continuous disruption of the desmin/mitochondrial architecture, accompanied by oxidative stress and inhibition of apoptosis and mitophagy, suggests its causal role in LV dilatation and systolic dysfunction in VO.NEW & NOTEWORTHY This study provides new evidence of early onset (24 h) and continuous (4-12 wk) desmin misarrangement and disruption of the normal sarcomeric and mitochondrial architecture throughout the progression of volume overload heart failure, suggesting a causal link between desmin cleavage and mitochondrial disorganization and damage.

Disruption of desmin-mitochondrial architecture in patients with regurgitant mitral valves and preserved ventricular function.

OBJECTIVE: Recent studies have demonstrated improved outcomes in patients receiving early surgery for degenerative mitral regurgitation (MR) rather than adhering to conventional guidelines for surgical intervention. However, studies providing a mechanistic basis for these findings are limited. METHODS: Left ventricular (LV) myocardium from 22 patients undergoing mitral valve repair for American Heart Association class I indications was evaluated for desmin, the voltage-dependent anion channel, α-B-crystallin, and α, ß-unsaturated aldehyde 4-hydroxynonenal by fluorescence microscopy. The same was evaluated in 6 normal control LV autopsy specimens. Cardiomyocyte ultrastructure was examined by transmission electron microscopy. Magnetic resonance imaging with tissue tagging was performed in 55 normal subjects and 22 MR patients before and 6 months after mitral valve repair. RESULTS: LV end-diastolic volume was 1.5-fold (P < .0001) higher and LV mass-to-volume ratio was lower in MR (P = .004) hearts versus normal hearts and showed improvement 6 months after mitral valve surgery. However, LV ejection fraction decreased from 65% ± 7% to 52% ± 9% (P < .0001) and LV circumferential (P < .0001) and longitudinal strain decreased significantly below normal values (P = .002) after surgery. Hearts with MR had a 53% decrease in desmin (P < .0001) and a 2.6-fold increase in desmin aggregates (P < .0001) versus normal, along with substantial, intense perinuclear staining of α, ß-unsaturated aldehyde 4-hydroxynonenal in areas of mitochondrial breakdown and clustering. Transmission electron microscopy demonstrated numerous electron-dense deposits, myofibrillar loss, Z-disc abnormalities, and extensive granulofilamentous debris identified as desmin-positive by immunogold transmission electron microscopy. CONCLUSIONS: Despite well-preserved preoperative LV ejection fraction, severe oxidative stress and disruption of cardiomyocyte desmin-mitochondrial sarcomeric architecture may explain postoperative LV functional decline and further supports the move toward earlier surgical intervention.

Increased sarcolipin expression and adrenergic drive in humans with preserved left ventricular ejection fraction and chronic isolated mitral regurgitation.

BACKGROUND: There is currently no therapy proven to attenuate left ventricular (LV) dilatation and dysfunction in volume overload induced by isolated mitral regurgitation (MR). To better understand molecular signatures underlying isolated MR, we performed LV gene expression analyses and overlaid regulated genes into ingenuity pathway analysis in patients with isolated MR. METHODS AND RESULTS: Gene arrays from LV tissue of 35 patients, taken at the time of surgical repair for isolated MR, were compared with 13 normal controls. Cine-MRI was performed in 31 patients before surgery to measure LV function and volume from serial short-axis summation. LV end-diastolic volume was 2-fold (P=0.005) higher in MR patients than in normal controls, and LV ejection fraction was 64±7% (50%-79%) in MR patients. Ingenuity pathway analysis identified significant activation of pathways involved in ß-adrenergic, cAMP, and G-protein-coupled signaling, whereas there was downregulation of pathways associated with complement activation and acute phase response. SERCA2a and phospholamban protein were unchanged in MR versus control left ventricles. However, mRNA and protein levels of the sarcoplasmic reticulum Ca2+ ATPase (SERCA) regulatory protein sarcolipin, which is predominantly expressed in normal atria, were increased 12- and 6-fold, respectively. Immunofluorescence analysis confirmed the absence of sarcolipin in normal left ventricles and its marked upregulation in MR left ventricles. CONCLUSIONS: These results demonstrate alterations in multiple pathways associated with ß-adrenergic signaling and sarcolipin in the left ventricles of patients with isolated MR and LV ejection fraction>50%, suggesting a beneficial role for ß-adrenergic blockade in isolated MR.

Cardiac O-GlcNAcylation blunts autophagic signaling in the diabetic heart.

AIMS: Increased O-linked attachment of ß-N-acetylglucosamine (O-GlcNAc) to proteins has been implicated in the adverse effects of diabetes on the heart, although this has typically been based on models of severe hyperglycemia. Diabetes has also been associated with dysregulation of autophagy, a critical cell survival process; however, little is known regarding autophagy in the diabetic heart or whether this is influenced by O-GlcNAcylation or hemodynamic stress. MAIN METHODS: Young male rats were assigned to control (12% kcal fat/19% protein/69% carbohydrate), high fat diet (60/19/21%) and type 2 diabetic (high fat diet+low dose streptozotocin) groups for 8 weeks, followed by sham or pressure overload surgeries; animals were sacrificed 8 weeks after surgery. KEY FINDINGS: A modest increase in arterial pressure resulted in no significant effects on cardiac function in control or high fat groups, while diabetic hearts exhibited contractile dysfunction and increased apoptosis and scar formation. Immunoprecipitation studies revealed, for the first time, that Beclin-1, which plays a critical early role in autophagy, and the anti-apoptotic Bcl-2, are targets for O-GlcNAcylation. Interestingly, we also found that cardiomyocytes isolated from type 2 diabetic db/db mice exhibited a blunted autophagic response and this was at least partially reversed by inhibiting glucose entry into the hexosamine biosynthesis pathway, which regulates O-GlcNAc synthesis. We also found that acutely augmenting O-GlcNAc levels in non-diabetic cardiomyocytes mimicked the effects of diabetes by blunting autophagic signaling. SIGNIFICANCE: These data suggest that O-GlcNAc-mediated inhibition of autophagy may contribute to the abnormal response of diabetic hearts to hemodynamic stress.

Increased oxidative stress and cardiomyocyte myofibrillar degeneration in patients with chronic isolated mitral regurgitation and ejection fraction >60%.

OBJECTIVES: This study assessed myocardial damage in patients with chronic isolated mitral regurgitation (MR) and left ventricular ejection fraction (LVEF) >60%. BACKGROUND: Typically, MR patients have decreased LVEF after mitral valve (MV) repair despite normal pre-operative LVEF. METHODS: Twenty-seven patients with isolated MR had left ventricular (LV) biopsies taken at time of MV repair. Magnetic resonance imaging with tissue tagging was performed in 40 normal subjects and in MR patients before and 6 months after MV repair. RESULTS: LVEF (66 +/- 5% to 54 +/- 9%, p < 0.0001) and LV end-diastolic volume index (108 +/- 28 ml/m(2) to 78 +/- 24 ml/m(2), p < 0.0001) decreased, whereas left ventricular end-systolic (LVES) volume index was 60% above normal pre- and post-MV repair (p < 0.05). The LV circumferential and longitudinal strain rates decreased below normal following MV repair (6.38 +/- 1.38 vs. 5.11 +/- 1.28, p = 0.0009, and 7.51 +/- 2.58 vs. 5.31 +/- 1.61, percentage of R to R interval, p < 0.0001), as LVES stress/LVES volume index ratio was depressed at baseline and following MV repair versus normal subjects (0.25 +/- 0.10 and 0.28 +/- 0.05 vs. 0.33 +/- 0.12, p < 0.01). LV biopsies demonstrated cardiomyocyte myofibrillar degeneration versus normal subjects (p = 0.035). Immunostaining and immunoblotting demonstrated increased xanthine oxidase in MR versus normal subjects (p < 0.05). Lipofuscin deposition was increased in cardiomyocytes of MR versus normal subjects (0.62 +/- 0.20 vs. 0.33 +/- 0.11, percentage of area: p < 0.01). CONCLUSIONS: Decreased LV strain rates and LVES wall stress/LVES volume index following MV repair indicate contractile dysfunction, despite pre-surgical LVEF >60%. Increased oxidative stress could cause myofibrillar degeneration and lipofuscin accumulation resulting in LV contractile dysfunction in MR.

Tumor necrosis factor-alpha produced in cardiomyocytes mediates a predominant myocardial inflammatory response to stretch in early volume overload.

Acute stretch caused by volume overload (VO) of aorto-caval fistula (ACF) induces a variety of myocardial responses including mast cell accumulation, matrix metalloproteinase (MMP) activation, and collagen degradation, all of which are critical in dictating long-term left ventricle (LV) outcome to VO. Meanwhile, these responses can be part of myocardial inflammation dictated by tumor necrosis factor-alpha (TNF-alpha), which is elevated after acute ACF. However, it is unknown whether TNF-alpha mediates a major myocardial inflammatory response to stretch in early VO. In 24-h ACF and sham rats, microarray gene expression profiling and subsequent Ingenuity Pathway Analysis identified a predominant inflammatory response and a gene network of biologically interactive genes strongly linked to TNF-alpha. Western blot demonstrated increased local production of TNF-alpha in the LV (1.71- and 1.66-fold in pro- and active-TNF-alpha over control, respectively, P<0.05) and cardiomyocytes (2- and 4-fold in pro- and active-TNF-alpha over control, respectively, P<0.05). TNF-alpha neutralization with infliximab (5.5 mg/kg) attenuated the myocardial inflammatory response to acute VO, as indicated by inhibition of inflammatory gene upregulation, myocardial infiltration (total CD45+ cells, mast cells, and neutrophils), MMP-2 activation, collagen degradation, and cardiac cell apoptosis, without improving LV remodeling and function. These results indicate that TNF-alpha produced by cardiomyocytes mediates a predominant inflammatory response to stretch in the early VO in the ACF rat, suggesting an important role of TNF-alpha in initiating pathophysiological response of myocardium to VO.

c-kit is required for cardiomyocyte terminal differentiation.

c-kit, the transmembrane tyrosine kinase receptor for stem cell factor, is required for melanocyte and mast cell development, hematopoiesis, and differentiation of spermatogonial stem cells. We show here that in the heart, c-kit is expressed not only by cardiac stem cells but also by cardiomyocytes, commencing immediately after birth and terminating a few days later, coincident with the onset of cardiomyocyte terminal differentiation. To examine the function of c-kit in cardiomyocyte terminal differentiation, we used compound heterozygous mice carrying the W (null) and W(v) (dominant negative) mutations of c-kit. In vivo, adult W/W(v) cardiomyocytes are phenotypically indistinguishable from their wild-type counterparts. After acute pressure overload adult W/W(v) cardiomyocytes reenter the cell cycle and proliferate, leading to left ventricular growth; furthermore in transgenic mice with cardiomyocyte-restricted overexpression of the dominant negative W(v) mutant, pressure overload causes cardiomyocytes to reenter the cell cycle. In contrast, in wild-type mice left ventricular growth after pressure overload results mainly from cardiomyocyte hypertrophy. Importantly, W/W(v) mice with pressure overload-induced cardiomyocyte hyperplasia had improved left ventricular function and survival. In W/W(v) mice, c-kit dysfunction also resulted in an approximately 14-fold decrease (P<0.01) in the number of c-kit(+)/GATA4(+) cardiac progenitors. These findings identify novel functions for c-kit: promotion of cardiac stem cell differentiation and regulation of cardiomyocyte terminal differentiation.

Moderate hypoxia induces xanthine oxidoreductase activity in arterial endothelial cells.

Xanthine oxidoreductase (XOR) activity has been previously noted to be responsive to changes in O2 tension. While prior studies have focused on the extremes (0-3% and 95-100%) of O2 tensions, we report the influence of 10% O2 on endothelial cell XOR, a concentration resembling modest arterial hypoxia commonly found in patients with chronic cardiopulmonary diseases. Exposure of bovine aortic endothelial cells to 10% O2 increased XOR mRNA and protein abundance by 50%. Concomitantly, there was a 3-fold increase in XOR activity, XOR-dependent reactive oxygen species production, and cellular export of active enzyme. Although increases in mRNA and immunoreactive protein levels were observed, inhibition of transcription, translation, or protein degradation did not significantly alter cellular XOR specific activity, suggesting only modest contributions to 10% O2-induced effects. Exposure to 10% O2 did not increase cellular HIF-1alpha protein levels and hypoxia mimics did not alter XOR activity. Treatment of control cells with adenosine resulted in increased XOR activity similar to hypoxia. Exposure to the adenosine receptor agonist NECA increased enzymatic activity 4-fold while 8SPT, an adenosine receptor antagonist, reduced hypoxic induction of XOR activity approximately 50%. Combined, these data reveal that moderate hypoxia significantly enhances endothelial XOR specific activity, release, and XOR-derived reactive oxygen species generation. These effects appear to be mediated in part via adenosine-dependent processes.

Cardiac interstitial bradykinin and mast cells modulate pattern of LV remodeling in volume overload in rats.

In the current study, interstitial fluid (ISF), bradykinin (BK), and angiotensin II (ANG II) levels were measured using cardiac microdialysis in conscious, nonsedated rats at baseline and at 48 h and 5 days after each of the following: sham surgery (sham, n = 6), sham + administration of ANG-converting enzyme inhibitor ramipril (R, n = 6), creation of aortocaval fistula (ACF, n = 6), ACF + R (n = 6), and ACF + R + BK2 receptor antagonist (HOE-140) administration (n = 6). At 5 days, both ISF ANG II and BK increased in ACF rats (P < 0.05); however, in ACF + R rats, ISF ANG II did not differ from basal levels and ISF BK increased greater than threefold above baseline at 2 and 5 days (P < 0.05). Five days after ACF, the left ventricular (LV) weight-to-body weight ratio increased 30% (P < 0.05) in ACF but did not differ from sham in ACF + R and ACF + R + HOE-140 rats despite similar systemic arterial pressures across all ACF groups. However, ACF + R + HOE-140 rats had greater postmortem wall thickness-to-diameter ratio and smaller cross-sectional diameter compared with ACF + R rats. There was a significant increase in mast cell density in ACF and ACF + R rats that decreased below sham in ACF + R + HOE-140 rats. These results suggest a potentially important interaction of mast cells and BK in the cardiac interstitium that modulates the pattern of LV remodeling in the acute phase of volume overload.