Marc S. Kraus

Inactivation of focal adhesion kinase in cardiomyocytes promotes eccentric cardiac hypertrophy and fibrosis in mice

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that plays a major role in integrin signaling pathways. Although cardiovascular defects were observed in FAK total KO mice, the embryonic lethality prevented investigation of FAK function in the hearts of adult animals. To circumvent these problems, we created mice in which FAK is selectively inactivated in cardiomyocytes (CFKO mice). We found that CFKO mice develop eccentric cardiac hypertrophy (normal LV wall thickness and increased left chamber dimension) upon stimulation with angiotensin II or pressure overload by transverse aortic constriction as measured by echocardiography. We also found increased heart/body weight ratios, elevated markers of cardiac hypertrophy, multifocal interstitial fibrosis, and increased collagen I and VI expression in CFKO mice compared with control littermates. Spontaneous cardiac chamber dilation and increased expression of hypertrophy markers were found in the older CFKO mice. Analysis of cardiomyocytes isolated from CFKO mice showed increased length but not width. The myocardium of CFKO mice exhibited disorganized myofibrils with increased nonmyofibrillar space filled with swelled mitochondria. Last, decreased tyrosine phosphorylation of FAK substrates p130Cas and paxillin were observed in CFKO mice compared with the control littermates. Together, these results provide strong evidence for a role of FAK in the regulation of heart hypertrophy in vivo.

Cardiac developmental defects and eccentric right ventricular hypertrophy in cardiomyocyte focal adhesion kinase (FAK) conditional knockout mice.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that plays an important role in integrin-mediated signal transduction. To explore the role and mechanisms of FAK in cardiac development, we inactivated FAK in embryonic cardiomyocytes by crossing the floxed FAK mice with myosin light chain-2a (MLC2a) Cre mice, which expressed Cre as early as embryonic day 9.5 in the heart. The majority of conditional FAK knockout mice generated from MLC2a-Cre (CFKO-2a) died in the embryonic stage with thin ventricular wall and ventricular septal defects. A small fraction of CFKO-2a mice survived to adulthood with spontaneous eccentric right ventricle hypertrophy. Transmission electron microscopy analysis displayed swelling in the rough endoplasmic reticulum in CFKO-2a embryonic cardiomyocytes. We found that decreased cell proliferation, but not increased cell apoptosis or differentiation, is the reason for the thin ventricular wall in CFKO-2a mice. Microarray analysis suggests that myocyte enhancer factor 2a (MEF2a) can be regulated by FAK and that inactivation of FAK in the embryonic heart compromised MEF2a expression. Last, we found that Src, but not PI3K, is important in mediating signal transduction for the regulation of MEF2a by FAK. Together, these results identified the role and mechanisms of FAK in embryonic cardiac development.

Measurement of plasma cardiac troponin I concentration by use of a point-of-care analyzer in clinically normal horses and horses with experimentally induced cardiac disease

OBJECTIVE To compare cardiac troponin I (cTnI) concentrations determined by use of a point-of-care analyzer with values determined by use of a bench-top immunoassay in plasma samples obtained from clinically normal horses with and without experimentally induced cardiac disease, and to establish a reference range for plasma equine cTnI concentration determined by use of the point-of-care analyzer. ANIMALS 83 clinically normal horses, 6 of which were administered monensin to induce cardiac disease. PROCEDURES A blood sample was collected from each of the 83 clinically normal horses to provide plasma for analysis by use of the point-of-care analyzer; some of the same samples were also analyzed by use of the immunoassay. All 83 samples were used to establish an analyzer-specific reference range for plasma cTnI concentration in clinically normal horses. In 6 horses, blood samples were also collected at various time points after administration of a single dose of monensin (1.0 to 1.5 mg/kg) via nasogastric intubation; plasma cTnI concentration in those samples was assessed by use of both methods. RESULTS The analyzer-specific reference range for plasma cTnI concentration in clinically normal horses was 0.0 to 0.06 ng/mL. Following monensin treatment in 5 horses, increases in plasma cTnI concentration determined by use of the 2 methods were highly correlated (Pearson correlation, 0.83). Peak analyzer-determined plasma cTnI concentrations in monensin-treated horses ranged from 0.08 to 3.68 ng/mL. CONCLUSIONS AND CLINICAL RELEVANCE In horses with and without experimentally induced cardiac disease, the point-of-care analyzer and bench-top immunoassay provided similar values of plasma cTnI concentration.

Hypercoagulability in Cats with Cardiomyopathy

BACKGROUND Arterial thromboembolism (ATE) is a common complication of feline cardiomyopathy; however, the pathogenesis of ATE is unknown. HYPOTHESIS Systemic activation of the coagulation cascade (hypercoagulability) and endothelial injury promote ATE in cardiomyopathic cats. ANIMALS Healthy cats (n = 30) and 3 groups of cardiomyopathic cats: Group (1) left atrial enlargement only (LAE [n = 11]), ie, left atrial to aortic ratio >1.4; Group (2) LAE with spontaneous echocardiographic contrast, atrial thrombi or both (SEC-T [n = 16]); and Group (3) acute ATE with LAE (n = 16). METHODS Hypercoagulability was defined by 2 or more laboratory abnormalities reflecting coagulation factor excess (high fibrinogen concentration or Factor VIII coagulant activity), inhibitor deficiency (low antithrombin activity), or thrombin generation (high thrombin-antithrombin complex [TAT] and d-dimer concentrations). High von Willebrand factor antigen concentration (vWF : Ag) was considered a marker of endothelial injury. Data were analyzed using nonparametric statistics. RESULTS The 3 groups of cats with cardiac disease had higher median fibrinogen concentrations than did the healthy cats. Criteria of hypercoagulability were found exclusively in cats with SEC-T (50%) and ATE (56%). Hypercoagulability was not associated with left atrial size or congestive heart failure (CHF). ATE cats had significantly higher median vWF : Ag concentration than did the other groups. CONCLUSION AND CLINICAL IMPORTANCE Systemic hypercoagulability is evident in many cardiomyopathic cats, often without concurrent CHF or overt ATE. Hypercoagulabilty may represent a risk factor for ATE. High vWF : Ag in ATE cats was attributed to downstream endothelial injury from the occlusive thrombus.

Assessment of serum N-terminal pro-B-type natriuretic peptide concentration for differentiation of congestive heart failure from primary respiratory tract disease as the cause of respiratory signs in dogs

OBJECTIVE To determine whether serum N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentration is useful in discriminating between cardiac and noncardiac (ie, primary respiratory tract disease) causes of respiratory signs (ie, coughing, stertor, stridor, excessive panting, increased respiratory effort, tachypnea, or overt respiratory distress) in dogs. DESIGN Multicenter cross-sectional study. ANIMALS P 115 dogs with respiratory signs. PROCEDURES Dogs with respiratory signs were solicited for study. Physical examination, thoracic radiography, and echocardiography were used to determine whether respiratory signs were the result of cardiac (ie, congestive heart failure) or noncardiac (ie, primary respiratory tract disease) causes. Serum samples for NT-proBNP assay were obtained at time of admission for each dog. Receiver-operating characteristic curves were constructed to determine the ability of serum NT-proBNP concentration to discriminate between cardiac and noncardiac causes of respiratory signs. RESULTS Serum NT-proBNP concentration was significantly higher in dogs with cardiac versus noncardiac causes of respiratory signs. In dogs with primary respiratory tract disease, serum NT-proBNP concentration was significantly higher in those with concurrent pulmonary hypertension than in those without. A serum NT-proBNP cutoff concentration > 1,158 pmol/L discriminated between dogs with congestive heart failure and dogs with primary respiratory tract disease with a sensitivity of 85.5% and a specificity of 81.3%. CONCLUSIONS AND CLINICAL RELEVANCE Measuring serum NT-proBNP concentration in dogs with respiratory signs helps to differentiate between congestive heart failure and primary respiratory tract disease as an underlying cause.

Morphology of Ventricular Arrhythmias in the Boxer as Measured by 12-Lead Electrocardiography with Pace-Mapping Comparison

The QRS amplitude and polarity were determined in 12-lead electrocardiograms recorded from 22 Boxers with ventricular arrhythmias. Eighty-one percent (18/22) of dogs displayed a positive QRS morphology in the caudoventral leads (II, III, and aVF) and 77% (17/22) of dogs displayed a positive QRS morphology in the left precordial leads (V2-V6). In leads I and V1, the polarity of the QRS complex was variable (positive or negative). To determine if these morphologic features were suggestive of ventricular complexes arising from the right or left ventricle, a comparison was made to the QRS complexes in a pace-mapping study performed in 7 healthy mixed-breed dogs. A total of 3 right and 4 left ventricular sites were paced. None of the left ventricular paced sites resulted in a QRS morphology similar to the most common spontaneous ventricular arrhythmia in the Boxers. In contrast, QRS morphology in each of the 3 right ventricular sites was similar to that observed in the Boxers (P < .033). Each of these produced positive deflections in the caudoventral and left precordial leads, but both positive and negative QRS complexes were observed in leads I and V1 only when the right ventricular septum was paced. This finding suggested that the right ventricular septum might be a site of origin for the ventricular rhythm observed in the Boxers because in the Boxers the polarity of leads I and V1 also varied. Pacing the right ventricular outflow tract always resulted in a negative QRS complex in lead 1, whereas pacing the right ventricular apex always resulted in a positive QRS complex in lead I and a negative QRS complex in V1. However, these locations cannot be excluded as possible sites of origin for the spontaneous ventricular arrhythmias in the Boxers because the arrhythmias could be originating from both of these locations. The spontaneous ventricular arrhythmia of the Boxer is most similar to that of paced ventricular rhythms arising from the right ventricle. More precise localization to a region of the right ventricle such as outflow tract, septal, or apical could not be made.

Macrocyclic lactone resistance in Dirofilaria immitis: Failure of heartworm preventives and investigation of genetic markers for resistance

Macrocyclic lactone (ML) endectocides are used as chemoprophylaxis for heartworm infection (Dirofilaria immitis) in dogs and cats. Claims of loss of efficacy (LOE) of ML heartworm preventives have become common in some locations in the USA. We directly tested whether resistance to MLs exists in LOE isolates of D. immitis and identified genetic markers that are correlated with, and therefore can predict ML resistance. ML controlled studies showed that LOE strains of D. immitis established infections in dogs despite chemoprophylaxis with oral ivermectin or injectable moxidectin. A whole genome approach was used to search for loci associated with the resistance phenotype. Many loci showed highly significant differences between pools of susceptible and LOE D. immitis. Based on 186 potential marker loci, Sequenom(®) SNP frequency analyses were conducted on 663 individual parasites (adult worms and microfilariae) which were phenotypically characterized as susceptible (SUS), confirmed ML treatment survivors/resistant (RES), or suspected resistant/loss of efficacy (LOE) parasites. There was a subset of SNP loci which appears to be promising markers for predicting ML resistance, including SNPs in some genes that have been associated with ML resistance in other parasites. These data provide unequivocal proof of ML resistance in D. immitis and identify genetic markers that could be used to monitor for ML resistance in heartworms.

Clinical Findings and Serum Cardiac Troponin I Concentrations in Horses after Intragastric Administration of Sodium Monensin

Six adult horses were administered sodium monensin, 1.0–1.5 mg/kg, via gastric gavage. Anorexia and/or diarrhea occurred within 24 hr after monensin administration in all 6 horses. Cardiac disease and dysfunction were evaluated by both elevations in heart rate, echocardiography, and an increase in serum concentrations of cardiac troponin I (cTnI), occurred in 4 horses. The development and severity of cardiac disease was likely affected by the monensin dose, vehicle (water or corn oil) mixed with monensin, and/or whether the monensin was administered to fed or fasted horses. Initial increases in cTnI concentrations occurred between 24 and 72 hr after monensin administration. The 2 horses with the highest cTnI concentrations died or were euthanized within 5 days after monensin administration and had severe cardiac disease. One horse had increased cTnI concentrations from day 2 to day 16, but no apparent change in ventricular contractile function was evident on echocardiography. The fourth diseased horse did not return to cTnI reference intervals until day 27 after monensin administration, and the ventricular function was still abnormal just before euthanasia 9 months later. Cardiac troponin I measurements could be useful in managing farm outbreaks of accidental monensin feeding by the early identification of horses with cardiac disease.

Evaluation of L-lactate and cardiac troponin I in horses undergoing emergency abdominal surgery.

OBJECTIVE To evaluate changes in plasma cardiac troponin I (cTnI) and L-lactate (LLt) as prognostic indicators in horses undergoing emergency abdominal surgery. DESIGN Prospective observational study. SETTING Veterinary teaching hospital. ANIMALS Thirty-four horses undergoing emergency abdominal surgery. INTERVENTIONS Serial blood sampling during various times during hospitalization (hospital admission, and 12, 24, 48, and 72 h postoperatively) evaluating cTnI and LLt concentrations. MEASUREMENTS AND MAIN RESULTS All horses required surgery for correction of a strangulating (n = 29) or nonstrangulating obstruction (n = 5) of the small or large intestine. Twenty-seven horses survived to discharge; 7 were euthanized either during (n = 1) or after (n = 6) surgery due to disease severity or systemic complications associated with the primary gastrointestinal lesion. Preoperative cTnI concentrations were increased above the normal reference interval in 24% of horses (8/34, median = 0.01 ng/mL, range = 0-12.23 ng/mL), whereas LLt concentrations were increased above the normal reference interval in 88% of horses (30/34, median = 3.37 mmol/L, range = 0.77-13.26 mmol/L). The LLt concentration was significantly higher (P < 0.05) in nonsurviving compared with surviving horses at admission, and at 24 and 72 hours postoperatively. No significant difference in the cTnI concentration was detected between groups at admission. However, the cTnI concentration was significantly higher (P<0.05) in nonsurviving compared with surviving horses at all time points postoperatively. CONCLUSIONS Measurement of both LLt and cTnI concentrations may provide information for prognostication in surgical colic horses. Marked increases in admission concentrations of LLt (median 7.56 mmol/L) and even moderate postoperative increases in cTnI concentration (median 0.97 ng/mL) may both indicate a poor prognosis in critically ill horses following abdominal surgery.Objective To evaluate changes in plasma cardiac troponin I (cTnI) and L-lactate (LLt) as prognostic indicators in horses undergoing emergency abdominal surgery. Design Prospective observational study. Setting Veterinary teaching hospital. Animals Thirty-four horses undergoing emergency abdominal surgery. Interventions Serial blood sampling during various times during hospitalization (hospital admission, and 12, 24, 48, and 72 h postoperatively) evaluating cTnI and LLt concentrations. Measurements and Main Results All horses required surgery for correction of a strangulating (n = 29) or nonstrangulating obstruction (n = 5) of the small or large intestine. Twenty-seven horses survived to discharge; 7 were euthanized either during (n = 1) or after (n = 6) surgery due to disease severity or systemic complications associated with the primary gastrointestinal lesion. Preoperative cTnI concentrations were increased above the normal reference interval in 24% of horses (8/34, median = 0.01 ng/mL, range = 0–12.23 ng/mL), whereas LLt concentrations were increased above the normal reference interval in 88% of horses (30/34, median = 3.37 mmol/L, range = 0.77–13.26 mmol/L). The LLt concentration was significantly higher (P < 0.05) in nonsurviving compared with surviving horses at admission, and at 24 and 72 hours postoperatively. No significant difference in the cTnI concentration was detected between groups at admission. However, the cTnI concentration was significantly higher (P<0.05) in nonsurviving compared with surviving horses at all time points postoperatively. Conclusions Measurement of both LLt and cTnI concentrations may provide information for prognostication in surgical colic horses. Marked increases in admission concentrations of LLt (median 7.56 mmol/L) and even moderate postoperative increases in cTnI concentration (median 0.97 ng/mL) may both indicate a poor prognosis in critically ill horses following abdominal surgery.

Effect of a tart cherry juice blend on exercise-induced muscle damage in horses.

OBJECTIVE To evaluate whether administering a tart cherry juice blend (TCJB) prior to exercise would reduce skeletal and cardiac muscle damage by decreasing the inflammatory and oxidative stress response to exercise in horses. ANIMALS 6 horses. PROCEDURES Horses were randomly allocated into 2 groups in a crossover study with a 2-week washout period and orally administered either TCJB or a placebo solution (1.42 L, twice daily) in a double-masked protocol for 2 weeks prior to a stepwise incremental exercise protocol. Horses were tested for serum activities of creatine kinase and aspartate aminotransferase (AST) and concentrations of cardiac troponin I (cTnI), thiobarbituric acid reactive substances (TBARS; an indicator of oxidative stress), and serum amyloid A (SAA; an indicator of inflammation). To ensure that treatment would not result in positive results of an equine drug-screening protocol, serum samples obtained from each horse prior to and after 2 weeks of administration of TCJB or the placebo solution were tested. RESULTS All horses had negative results of drug screening at both sample times. The exercise protocol resulted in a significant increase in TBARS concentration, SAA concentration, and serum AST activity in all horses. Administration of TCJB or placebo solution was not associated with an effect on malondialdehyde or SAA concentrations. However, administration of TCJB was associated with less serum activity of AST, compared with administration of placebo solution. CONCLUSIONS AND CLINICAL RELEVANCE Administration of TCJB may diminish muscle damage induced by exercise.