Pamela C. Powell

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang II-forming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient KitW/KitW-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

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.

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 Wv (dominant negative) mutations of c-kit. In vivo, adult W/Wv cardiomyocytes are phenotypically indistinguishable from their wild-type counterparts. After acute pressure overload adult W/Wv cardiomyocytes reenter the cell cycle and proliferate, leading to left ventricular growth; furthermore in transgenic mice with cardiomyocyte-restricted overexpression of the dominant negative Wv 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/Wv mice with pressure overload–induced cardiomyocyte hyperplasia had improved left ventricular function and survival. In W/Wv mice, c-kit dysfunction also resulted in an ≈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.

Microarray identifies extensive downregulation of noncollagen extracellular matrix and profibrotic growth factor genes in chronic isolated mitral regurgitation in the dog.

Background— The volume overload of isolated mitral regurgitation (MR) in the dog results in left ventricular (LV) dilatation and interstitial collagen loss. To better understand the mechanism of collagen loss, we performed a gene array and overlaid regulated genes into ingenuity pathway analysis. Methods and Results— Gene arrays from LV tissue were compared in 4 dogs before and 4 months after MR. Cine-magnetic resonance–derived LV end-diastolic volume increased 2-fold (P=0.005), and LV ejection fraction increased from 41% to 53% (P<0.007). LV interstitial collagen decreased 40% (P<0.05) compared with controls, and replacement collagen was in short strands and in disarray. Ingenuity pathway analysis identified Marfan syndrome, aneurysm formation, LV dilatation, and myocardial infarction, all of which have extracellular matrix protein defects and/or degradation. Matrix metalloproteinase-1 and -9 mRNA increased 5- (P=0.01) and 10-fold (P=0.003), whereas collagen I did not change and collagen III mRNA increased 1.5-fold (P=0.02). However, noncollagen genes important in extracellular matrix structure were significantly downregulated, including decorin, fibulin 1, and fibrillin 1. In addition, connective tissue growth factor and plasminogen activator inhibitor were downregulated, along with multiple genes in the transforming growth factor-β signaling pathway, resulting in decreased LV transforming growth factor-β1 activity (P=0.03). Conclusions— LV collagen loss in isolated, compensated MR is chiefly due to posttranslational processing and degradation. The downregulation of multiple noncollagen genes important in global extracellular matrix structure, coupled with decreased expression of multiple profibrotic factors, explains the failure to replace interstitial collagen in the MR heart.

Cardiac O-GlcNAcylation blunts autophagic signaling in the diabetic heart

AIMS Increased O-linked attachment of β-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.

Dynamic molecular and histopathological changes in the extracellular matrix and inflammation in the transition to heart failure in isolated volume overload

Left ventricular (LV) volume overload (VO) causes eccentric remodeling with inflammatory cell infiltration and extracellular matrix (ECM) degradation, for which there is currently no proven therapy. To uncover new pathways that connect inflammation and ECM homeostasis with cellular dysfunction, we determined the cardiac transciptome in subacute, compensated, and decompensated stages based on in vivo hemodynamics and echocardiography in the rat with aortocaval fistula (ACF). LV dilatation at 5 wk was associated with a normal LV end-diastolic dimension-to-posterior wall thickness ratio (LVEDD/PWT; compensated), whereas the early 2-wk (subacute) and late 15-wk (decompensated) ACF groups had significant increases in LVEDD/PWT. Subacute and decompensated stages had a significant upregulation of genes related to inflammation, the ECM, the cell cycle, and apoptosis. These changes were accompanied by neutrophil and macrophage infiltration, nonmyocyte apoptosis, and interstitial collagen loss. At 15 wk, there was a 40-fold increase in the matricellular protein periostin, which inhibits connections between collagen and cells, thereby potentially mediating a side-to-side slippage of cardiomyocytes and LV dilatation. The majority of downregulated genes was composed of mitochondrial enzymes whose suppression progressed from 5 to 15 wk concomitant with LV dilatation and systolic heart failure. The profound decrease in gene expression related to fatty acid, amino acid, and glucose metabolism was associated with the downregulation of peroxisome proliferator associated receptor (PPAR)-α-related and bioenergetic-related genes at 15 wk. In VO, an early phase of inflammation subsides at 5 wk but reappears at 15 wk with marked periostin production along with the suppression of genes related to PPAR-α and energy metabolism.

Chymase Inhibition Prevents Fibronectin and Myofibrillar Loss and Improves Cardiomyocyte Function and LV Torsion Angle in Dogs With Isolated Mitral Regurgitation

Background— The left ventricular (LV) dilatation of isolated mitral regurgitation (MR) is associated with an increase in chymase and a decrease in interstitial collagen and extracellular matrix. In addition to profibrotic effects, chymase has significant antifibrotic actions because it activates matrix metalloproteinases and kallikrein and degrades fibronectin. Thus, we hypothesize that chymase inhibitor (CI) will attenuate extracellular matrix loss and LV remodeling in MR. Methods and Results— We studied dogs with 4 months of untreated MR (MR; n=9) or MR treated with CI (MR+CI; n=8). Cine MRI demonstrated a >40% increase in LV end-diastolic volume in both groups, consistent with a failure of CI to improve a 25% decrease in interstitial collagen in MR. However, LV cardiomyocyte fractional shortening was decreased in MR versus normal dogs (3.71±0.24% versus 4.81±0.31%; P<0.05) and normalized in MR+CI dogs (4.85±0.44%). MRI with tissue tagging demonstrated an increase in LV torsion angle in MR+CI versus MR dogs. CI normalized the significant decrease in fibronectin and FAK phosphorylation and prevented cardiomyocyte myofibrillar degeneration in MR dogs. In addition, total titin and its stiffer isoform were increased in the LV epicardium and paralleled the changes in fibronectin and FAK phosphorylation in MR+CI dogs. Conclusions— These results suggest that chymase disrupts cell surface–fibronectin connections and FAK phosphorylation that can adversely affect cardiomyocyte myofibrillar structure and function. The greater effect of CI on epicardial versus endocardial titin and noncollagen cell surface proteins may be responsible for the increase in torsion angle in chronic MR.

Cardiac Kallikrein-Kinin System Is Upregulated in Chronic Volume Overload and Mediates an Inflammatory Induced Collagen Loss

Background The clinical problem of a “pure volume overload” as in isolated mitral or aortic regurgitation currently has no documented medical therapy that attenuates collagen loss and the resultant left ventricular (LV) dilatation and failure. Here, we identify a potential mechanism related to upregulation of the kallikrein-kinin system in the volume overload of aortocaval fistula (ACF) in the rat. Methodology/Principal Findings LV interstitial fluid (ISF) collection, hemodynamics, and echocardiography were performed in age-matched shams and 4 and 15 wk ACF rats. ACF rats had LV dilatation and a 2-fold increase in LV end-diastolic pressure, along with increases in LV ISF bradykinin, myocardial kallikrein and bradykinin type-2 receptor (BK2R) mRNA expression. Mast cell numbers were increased and interstitial collagen was decreased at 4 and 15 wk ACF, despite increases in LV ACE and chymase activities. Treatment with the kallikrein inhibitor aprotinin preserved interstitial collagen, prevented the increase in mast cells, and improved LV systolic function at 4 wk ACF. To establish a cause and effect between ISF bradykinin and mast cell-mediated collagen loss, direct LV interstitial bradykinin infusion in vivo for 24 hrs produced a 2-fold increase in mast cell numbers and a 30% decrease in interstitial collagen, which were prevented by BK2R antagonist. To further connect myocardial stretch with cellular kallikrein-kinin system upregulation, 24 hrs cyclic stretch of adult cardiomyocytes and fibroblasts produced increased kallikrein, BK2R mRNA expressions, bradykinin protein and gelatinase activity, which were all decreased by the kallikrein inhibitor-aprotinin. Conclusions/Significance A pure volume overload is associated with upregulation of the kallikrein-kinin system and ISF bradykinin, which mediates mast cell infiltration, extracellular matrix loss, and LV dysfunction–all of which are improved by kallikrein inhibition. The current investigation provides important new insights into future potential medical therapies for the volume overload of aortic and mitral regurgitation.

Tumor Necrosis Factor-α 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.

Dissociation between cardiomyocyte function and remodeling with β-adrenergic receptor blockade in isolated canine mitral regurgitation

The low-pressure volume overload of isolated mitral regurgitation (MR) is associated with increased adrenergic drive, left ventricular (LV) dilatation, and loss of interstitial collagen. We tested the hypothesis that beta1-adrenergic receptor blockade (beta1-RB) would attenuate LV remodeling after 4 mo of MR in the dog. beta1-RB did not attenuate collagen loss or the increase in LV mass in MR dogs. Using MRI and three-dimensional (3-D) analysis, there was a 70% increase in the LV end-diastolic (LVED) volume-to-LV mass ratio, a 23% decrease in LVED midwall circumferential curvature, and a >50% increase in LVED 3-D radius/wall thickness in MR dogs that was not attenuated by beta1-RB. However, beta1-RB caused a significant increase in LVED length from the base to apex compared with untreated MR dogs. This was associated with an increase in isolated cardiomyocyte length (171+/-5 microm, P<0.05) compared with normal (156+/-3 microm) and MR (165+/-4 microm) dogs. Isolated cardiomyocyte fractional shortening was significantly depressed in MR dogs compared with normal dogs (3.73+/-0.31 vs. 5.02+/-0.26%, P<0.05) and normalized with beta1-RB (4.73+/-0.48%). In addition, stimulation with the beta-adrenergic receptor agonist isoproterenol (25 nM) increased cardiomyocyte fractional shortening by 215% (P<0.05) in beta1-RB dogs compared with normal (56%) and MR (50%) dogs. In summary, beta1-RB improved LV cardiomyocyte function and beta-adrenergic receptor responsiveness despite further cell elongation. The failure to attenuate LV remodeling associated with MR could be due to a failure to improve ultrastructural changes in extracellular matrix organization.