E. Christopher Orton

A canine model of heart failure by intracoronary adriamycin injection: Hemodynamic and energetic results☆

BACKGROUND To eliminate the noncardiac toxicities associated with Adriamycin (Pharmacia Upjohn, Kalamazoo, MI) and to avoid the need for surgical implantation of a coronary artery catheter, we proposed serial intracoronary infusions of Adriamycin (Pharmacia Upjohn) with a Judkins catheter to develop a nonsurgical but selective model of dilated cardiomyopathy in dogs. METHODS AND RESULTS Twenty-two dogs were entered onto the study. Each dog received four weekly injections of Adriamycin (Pharmacia Upjohn; 15 mg/wk) through a Judkins catheter and verapamil orally (5 mg/kg twice daily) for 4 weeks. Dogs were evaluated with echocardiography, right-sided cardiac catheterization, and endomyocardial biopsy at times 0 and 10 weeks. The left ventricular ejection fraction decreased from 0.65 +/- 0.04 to 0.48 +/- 0.11 (P = .0006). The left ventricular end-diastolic and end-systolic diameter indices increased from 42.00 +/- 2.17 to 46.92 +/- 5.81 mm (P = .0029) and from 27.65 +/- 2.59 to 35.00 +/- 6.84 mm, respectively (P = .0210). The mechanical cardiac efficiency decreased from 28.55% +/- 13.30% at baseline and 15.64% +/- 9.09% at 10 weeks (P = .001). Cardiac myocyte vacuolation was present on histological examination at 10 weeks. The mortality rate was 41%. Minimal systemic Adriamycin (Pharmacia Upjohn) toxicity was noticed. CONCLUSION Intracoronary injection of Adriamycin (Pharmacia Upjohn) in dogs induced a chronic dilated cardiomyopathy.

Signaling pathways in mitral valve degeneration.

Heart valves exhibit a highly-conserved stratified structure exquisitely designed to counter biomechanical forces delivered over a lifetime. Heart valve structure and competence is maintained by heart valve cells through a process of continuous turnover extracellular matrix (ECM). Degenerative (myxomatous) mitral valve disease (DMVD) is an important disease associated with aging in both dogs and humans. DMVD is increasingly regarded as a disease with identifiable signaling mechanisms that control key genes associated with regulation and dysregulation of ECM homeostasis. Initiating stimuli for these signaling pathways have not been fully elucidated but likely include both mechanical and chemical stimuli. Signaling pathways implicated in DMVD include serotonin, transforming growth factor β (TGFβ), and heart valve developmental pathways. High circulating serotonin (carcinoid syndrome) and serotoninergic drugs are known to cause valvulopathy that shares pathologic features with DMVD. Recent evidence supports a local serotonin signaling mechanism, possibly triggered by high tensile loading on heart valves. Serotonin initiates TGFβ signaling, which in turn has been strongly implicated in canine DMVD. Recent evidence suggests that degenerative aortic and mitral valve disease may involve pathologic processes that mimic osteogenesis and chondrogenesis, respectively. These processes may be mediated by developmental pathways shared by heart valves, bone, and cartilage. These pathways include bone morphogenic protein (BMP) and Wnt signaling. Other signaling pathways implicated in heart valve disease include Notch, nitric oxide, and angiotensin II. Ultimately, increased understanding of signaling mechanisms could point to therapeutic strategies aimed at slowing or halting disease progression.

Static and cyclic tensile strain induce myxomatous effector proteins and serotonin in canine mitral valves

OBJECTIVES Degenerative (myxomatous) mitral valve disease is an important cardiac disease in dogs and humans. The mechanisms that initiate and propagate myxomatous pathology in mitral valves are poorly understood. We investigated the hypothesis that tensile strain initiates expression of proteins that mediate myxomatous pathology. We also explored whether tensile strain could induce the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1), serotonin synthesis, and markers of chondrogenesis. ANIMALS Mitral valves were obtained postmortem from dogs without apparent cardiovascular disease. METHODS Mitral valves were placed in culture and subjected to 30% static or cyclic tensile strain and compared to cultured mitral valves subjected to 0% strain for 72 h. Abundance of target effector proteins, TPH1, and chondrogenic marker proteins was determined by immunoblotting. Serotonin was measured in the conditioned media by ELISA. RESULTS Both static and cyclic strain increased (p < 0.05) expression of myxomatous effector proteins including markers of an activated myofibroblast phenotype, matrix catabolic and synthetic enzymes in canine mitral valves compared to unstrained control. Expression of TPH1 was increased in statically and cyclically strained mitral valves. Expression of chondrogenic markers was increased in statically strained mitral valves. Serotonin levels were higher (p < 0.05) in media of cyclically strained valves compared to unstrained valves after 72 h of culture. CONCLUSION Static or cyclic tensile strain induces acute increases in the abundance of myxomatous effector proteins, TPH1, and markers of chondrogenesis in canine mitral valves. Canine mitral valves are capable of local serotonin synthesis, which may be influenced by strain.

Canine cardiomyopathy after whole heart and partial lung irradiation

The late radiation response of the heart is of concern because of many reports of heart disease following radiation therapy of thoracic tumors. This study was done because of the clinical relevance of the pathophysiology of cardiopulmonary irradiation and because the heart is a good model for late effects of vasculoconnective tissue due to its lack of acutely responding parenchymal cells. Thoracic irradiation of adult beagle dogs including the heart and one third of the lung volume produced an early response in the heart at 1 and 3 months which consisted of an increase in left ventricle and septal wall thickness, decreased left ventricle ejection fraction, increased heart rates, intraventricular conduction disturbances and a high probability for pericardial effusion at 3 months. Radiation doses were 36, 44, or 52 Gy given in 4 Gy fractions in 4 weeks. Premature atrial contractions, paroxysmal atrial tachycardia, sustained atrial tachycardia and atrial fibrillation occurred at all dose levels. Evidence suggests that both early and late responses were due, at least in part, to direct injury to the cardiac microvasculature. The later effects appeared to be enhanced by injury to the lung. The early response appeared to resolve in 6 to 9 months, after which there was thinning of the myocardium at higher doses and resolution of pericardial effusions. At 12 months, elevations in right atrial pressure, but not pulmonary wedge pressure, were suggestive of right-sided congestive heart failure. Pulmonary hypertension was also present at 12 months presumably due to partial lung irradiations, and may have exacerbated right-sided congestive heart failure. The radiation injury may continue to increase with time leading to serious deficits in cardiopulmonary function. The functional studies may aid in predicting late effects and evaluating residual injury.

Local serotonin mediates cyclic strain-induced phenotype transformation, matrix degradation, and glycosaminoglycan synthesis in cultured sheep mitral valves

This study addressed the following questions: 1) Does cyclic tensile strain induce protein expression patterns consistent with myxomatous degeneration in mitral valves? 2) Does cyclic strain induce local serotonin synthesis in mitral valves? 3) Are cyclic strain-induced myxomatous protein expression patterns in mitral valves dependent on local serotonin? Cultured sheep mitral valve leaflets were subjected to 0, 10, 20, and 30% cyclic strain for 24 and 72 h. Protein levels of activated myofibroblast phenotype markers, α-smooth muscle actin (α-SMA) and nonmuscle embryonic myosin (SMemb); matrix catabolic enzymes, matrix metalloprotease (MMP) 1 and 13, and cathepsin K; and sulfated glycosaminoglycan (GAG) content in mitral valves increased with increased cyclic strain. Serotonin was present in the serum-free media of cultured mitral valves and concentrations increased with cyclic strain. Expression of the serotonin synthetic enzyme tryptophan hydroxylase 1 (TPH1) increased in strained mitral valves. Pharmacologic inhibition of the serotonin 2B/2C receptor or TPH1 diminished expression of phenotype markers (α-SMA and SMemb) and matrix catabolic enzyme (MMP1, MMP13, and cathepsin K) expression in 10- and 30%-strained mitral valves. These results provide first evidence that mitral valves synthesize serotonin locally. The results further demonstrate that tensile loading modulates local serotonin synthesis, expression of effector proteins associated with mitral valve degeneration, and GAG synthesis. Inhibition of serotonin diminishes strain-mediated protein expression patterns. These findings implicate serotonin and tensile loading in mitral degeneration, functionally link the pathogeneses of serotoninergic (carcinoid, drug-induced) and degenerative mitral valve disease, and have therapeutic implications.

Aortic thrombosis in dogs: presentation, therapy, and outcome in 26 cases.

OBJECTIVES The pathogenesis and presentation of aortic thrombosis (AT) in dogs is not well characterized and an effective antithrombotic therapy for AT in dogs has not been identified. Our goal is to report the clinical presentation and results of therapies in dogs with AT. ANIMALS Twenty-six client-owned dogs. METHODS Retrospective review of medical records of dogs diagnosed with AT between 2003 and 2010. RESULTS Twenty-six dogs had an apparent primary mural aortic thrombus. None had structural heart disease at diagnosis. Twenty dogs were ambulatory with varying degrees of pelvic limb dysfunction. Duration of ambulatory dysfunction was 7.8 weeks (range 1 day-52 weeks). A majority of dogs (58%) had no concurrent conditions at diagnosis. Fourteen dogs were treated with a standard warfarin protocol for a median period of 22.9 months (range 0.5-53 months). Ambulatory function improved in all dogs treated with warfarin. Time until clinical improvement was 13.9 days (range 2-49 days). Dogs treated with warfarin did not become non-ambulatory, die or undergo euthanasia related to AT, or have a known serious hemorrhagic event. CONCLUSIONS The pathogenesis of AT in dogs is distinct from that of aortic thromboembolism (ATE) in cats. Aortic thrombosis in dogs is more likely to involve local thrombosis in the distal aorta with embolization to the arteries of the pelvic limb resulting in chronic progressive ambulatory dysfunction. Chronic warfarin administration is well-tolerated and appears to be an effective short-term and long-term therapy for dogs with AT.

Immunoproteomic identification of bovine pericardium xenoantigens.

Bovine pericardium is an important biomaterial with current application in glutaraldehyde-fixed bioprosthetic heart valves and possible future application as an unfixed biological scaffold for tissue engineering. The importance of both humoral and cell-mediated rejection responses toward fixed and unfixed xenogeneic tissues has become increasingly apparent. However, the full scope and specific identities of bovine pericardium proteins that can elicit an immune response remain largely unknown. In this study, an immunoproteomic approach was used to survey bovine pericardium proteins for their ability to elicit a humoral immune response in rabbits. A two-stage protein extraction protocol was used to separate bovine pericardium proteins into water- and lipid-soluble fractions. Two-dimensional (2-D) gel electrophoresis was performed to separate the proteins from each fraction. Western blots were generated from 2-D gels of both bovine pericardium protein fractions. These blots were probed with serum from rabbits immunized with bovine pericardium and a secondary antibody was used to assess for IgG positivity. Western blots were compared to duplicate 2-D gels and proteins in matched spots were identified by tandem mass spectrometry. Thirty-one putative protein antigens were identified, eight of which are known to be antigenic from previous studies. All of the putative antigens demonstrated progressive staining intensity with increasing days of post-exposure serum. Identified antigenic proteins represented a variety of functional and structural protein types, and included both cellular and matrix proteins. The results of this study have implications for the use of bovine pericardium as a biomaterial in bioprostheses and tissue engineering applications, as well as xenotransplantation in general.

Small animal thoracic surgery

A comprehensive reference for the small animal surgeon, covering all aspects of thoracic surgical conditions. Taking a full perioperative approach, it covers: anaesthesia and complications; critical care; cardiology; cancer; swallowed objects and colour doppler echocardiography.

Serotonin transmembrane transporter is down-regulated in late-stage canine degenerative mitral valve disease

OBJECTIVE To compare expression of the serotonin transmembrane transporter (SERT) in normal, early-stage degenerative, and late-stage degenerative canine mitral valve disease. ANIMALS 24 post-mortem canine mitral valves. METHODS SERT expression was determined in canine normal (n = 8), early-stage degenerative (n = 8), and late-stage degenerative (n = 8) mitral valves by immunohistochemistry (IHC) and immunoblot (IB) analyses. RESULTS SERT was expressed in valve interstitial cells of all layers of normal and early-stage degenerative mitral valves based on IHC. SERT was markedly down-regulated in valve interstitial cells, but not valve endothelial cells, of late-stage degenerative mitral valves. SERT expression was significantly decreased in late-stage compared to normal and early-stage degenerative mitral valves based on IB analysis (P < 0.05). CONCLUSIONS Down-regulation of SERT expression occurs in valve interstitial cells of late-stage, but not early-stage, canine degenerative mitral valves. Down-regulation of SERT could enhance the recently speculated role of serotonin in canine DMVD by decreasing serotonin metabolism and increasing interaction with its receptor. Down-regulation of SERT likely does not play an initiating role in canine DMVD since it does not occur in early-stage disease.

Differential protein expression between normal, early-stage, and late-stage myxomatous mitral valves from dogs.

Valvular heart disease accounts for over 20 000 deaths and 90 000 hospitalizations yearly in the United States. Myxomatous valve disease (MVD) is the most common disease of the mitral valve in humans and dogs. MVD is pathologically identical in these species and its pathogenesis is poorly understood. The objectives of this study were to (i) develop proteomic methodology suitable for analysis of extracellular matrix‐rich heart valve tissues and (ii) survey over‐ and under‐expressed proteins that could provide mechanistic clues into the pathogenesis of MVD. Normal, early‐stage, and late‐stage myxomatous mitral valves from dogs were studied. A shotgun proteomic analysis was used to quantify differential protein expression. Proteins were classified by function and clustered according to differential expression patterns. More than 300 proteins, with 117 of those being differentially expressed, were identified. Hierarchical sample clustering of differential protein profiles showed that early‐ and late‐stage valves were closely related. This finding suggests that proteome changes occur in early degeneration stages and these persist in late stages, characterizing a diseased proteome that is distinct from normal. Shotgun proteome analysis of matrix‐rich canine heart valves is feasible, and should be applicable to human heart valves. This study provides a basis for future investigations into the pathogenesis of MVD.