Michael Tillson

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.

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.

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.

Left ventricular remodeling in preclinical experimental mitral regurgitation of dogs

Dogs with experimental mitral regurgitation (MR) provide insights into the left ventricular remodeling in preclinical MR. The early preclinical left ventricular (LV) changes after mitral regurgitation represent progressive dysfunctional remodeling, in that no compensatory response returns the functional stroke volume (SV) to normal even as total SV increases. The gradual disease progression leads to mitral annulus stretch and enlargement of the regurgitant orifice, further increasing the regurgitant volume. Remodeling with loss of collagen weave and extracellular matrix (ECM) is accompanied by stretching and hypertrophy of the cross-sectional area and length of the cardiomyocyte. Isolated ventricular cardiomyocytes demonstrate dysfunction based on decreased cell shortening and reduced intracellular calcium transients before chamber enlargement or decreases in contractility in the whole heart can be clinically appreciated. The genetic response to increased end-diastolic pressure is down-regulation of genes associated with support of the collagen and ECM and up-regulation of genes associated with matrix remodeling. Experiments have not demonstrated any beneficial effects on remodeling from treatments that decrease afterload via blocking the renin-angiotensin system (RAS). Beta-1 receptor blockade and chymase inhibition have altered the progression of the LV remodeling and have supported cardiomyocyte function. The geometry of the LV during the remodeling provides insight into the importance of regional differences in responses to wall stress.

Volume overload induces differential spatiotemporal regulation of myocardial soluble guanylyl cyclase in eccentric hypertrophy and heart failure

Nitric oxide activation of soluble guanylyl cyclase (sGC) blunts the cardiac stress response, including cardiomyocyte hypertrophy. In the concentric hypertrophied heart, oxidation and re-localization of myocardial sGC diminish cyclase activity, thus aggravating depressed nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signaling in the pressure-overloaded failing heart. Here, we hypothesized that volume-overload differentially disrupts myocardial sGC activity during early compensated and late decompensated stages of eccentric hypertrophy. To this end, we studied the expression, redox state, subcellular localization, and activity of sGC in the left ventricle of dogs subjected to chordal rupture-induced mitral regurgitation (MR). Unoperated dogs were used as Controls. Animals were studied at 4weeks and 12months post chordal rupture, corresponding with early (4wkMR) and late stages (12moMR) of eccentric hypertrophy. We found that the sGC heterodimer subunits relocalized away from caveolae-enriched lipid raft microdomains at different stages; sGCβ1 at 4wkMR, followed by sGCα1 at 12moMR. Moreover, expression of both sGC subunits fell at 12moMR. Using the heme-dependent NO donor DEA/NO and NO-/heme-independent sGC activator BAY 60-2770, we determined the redox state and inducible activity of sGC in the myocardium, within caveolae and non-lipid raft microdomains. sGC was oxidized in non-lipid raft microdomains at 4wkMR and 12moMR. While overall DEA/NO-responsiveness remained intact in MR hearts, DEA/NO responsiveness of sGC in non-lipid raft microdomains was depressed at 12moMR. Caveolae-localization protected sGC against oxidation. Further studies revealed that these modifications of sGC were also reflected in caveolae-localized cGMP-dependent protein kinase (PKG) and MAPK signaling. In MR hearts, PKG-mediated phosphorylation of vasodilator-stimulated phosphoprotein (VASP) disappeared from caveolae whereas caveolae-localization of phosphorylated ERK5 increased. These findings show that differential oxidation, re-localization, and expression of sGC subunits distinguish eccentric from concentric hypertrophy as well as compensated from decompensated heart failure.

High-resolution computed tomography bronchial lumen to pulmonary artery diameter ratio in anesthetized ventilated cats with normal lungs.

High-resolution computed tomography (CT) is the preferred noninvasive tool for diagnosing bronchiectasis in people. A criterion for evaluating dilation of the bronchus is the bronchial lumen to pulmonary artery diameter (bronchoarterial ratio [BA ratio]). A ratio of > 1.0 in humans or > 2.0 in dogs has been suggested as a threshold for identifying bronchiectasis. The purpose of this study was to establish the BA ratio in normal cats. Fourteen specific pathogen-free cats were selected for analysis of thoracic CT images. The BA ratios of the lobar bronchi of the left cranial (cranial and caudal parts), right cranial, right middle, left caudal, and right caudal lung lobes were measured. The mean of the mean BA ratio of all lung lobes was 0.71 +/- 0.05. Individual BA ratios ranged from 0.5 to 1.11. Comparing individual lobes for each cat, there was no significant difference (P = 0.145) in mean BA ratio between lung lobes. A mean BA ratio for these normal cats was 0.71 +/- 0.1, which suggests an upper cut-off normal value > 0.91 (mean +/- 2 standard deviations) between normal and abnormal cats.

Mast cell stabilization decreases cardiomyocyte and LV function in dogs with isolated mitral regurgitation.

BACKGROUND Mast cells are increased in isolated mitral regurgitation (MR) in the dog and may mediate extracellular matrix loss and left ventricular (LV) dilatation. We tested the hypothesis that mast cell stabilization would attenuate LV remodeling and improve function in the MR dog. METHODS AND RESULTS MR was induced in adult dogs randomized to no treatment (MR, n = 5) or to the mast cell stabilizer, ketotifen (MR + MCS, n = 4) for 4 months. LV hemodynamics were obtained at baseline and after 4 months of MR and magnetic resonance imaging (MRI) was performed at sacrifice. MRI-derived, serial, short-axis LV end-diastolic (ED) and end-systolic (ES) volumes, LVED volume/mass ratio, and LV 3-dimensional radius/wall thickness were increased in MR and MR + MCS dogs compared with normal dogs (n = 6) (P < .05). Interstitial collagen was decreased by 30% in both MR and MR + MCS versus normal dogs (P < .05). LV contractility by LV maximum time-varying elastance was significantly depressed in MR and MR + MCS dogs. Furthermore, cardiomyocyte fractional shortening was decreased in MR versus normal dogs and further depressed in MR + MCS dogs (P < .05). In vitro administration of ketotifen to normal cardiomyocytes also significantly decreased fractional shortening and calcium transients. CONCLUSIONS Chronic mast cell stabilization did not attenuate eccentric LV remodeling or collagen loss in MR. However, MCS therapy had a detrimental effect on LV function because of a direct negative inotropic effect on cardiomyocyte function.

Temporal Pattern of Circulating Antigens and Antibody Responses in Cats Experimentally Infected with Dirofilaria immitis

Heartworm disease in cats has been attributed to immature adult heartworms reaching the pulmonary arteries approximately 2.5 – 4 months post infection and the presence of mature adult worms in the cardiopulmonary system approximately 6 months after infection. The arrival and death of immature heartworms causes significant lung pathology, a condition referred to as heartwormassociated respiratory disease (HARD). To evaluate the humoral immune response to Dirofilaria immitis, 12 cats were each infected by subcutaneous injection of 100 third-stage larvae. Postinfection serum samples were collected weekly for 4 weeks, every other week for the following 4 weeks, then monthly to day 270 for evaluation of antibodies to D. immitis recombinant antigens HWAg-1 and HWAg-2 and circulating heartworm antigen (HWAg) using the PetChek® HTWM PF Test Kit (IDEXX Laboratories). Necropsies were performed 278 – 299 days post infection for collection of adult worms. Eleven cats were HWAg1 antibody-positive 68 days post-infection, and the remaining cat became positive by day 140. Eleven cats were positive for HWAg-2 antibody 42 – 84 days post infection; all 11 remained HWAg-2 antibodypositive through day 270. Circulating HWAg was detected in 10 cats, 2 by day 140 and 8 others by day 168. The 2 antigen-negative cats had no adult worms at necropsy. This study demonstrates that decomposition of immature adult heartworms can result in detectible levels of circulating antigen prior to sexual maturation. In this experimental Original Article WAAVP_2017.indb 91 29.06.17 15:34 S92 EndoparasitEs EndoparasitEs model, D. immitis antigen was detectable in cats at time points (days 140 and 168) associated with HARD from dying immature adult heartworms.