Effect of racing on cardiac troponin I concentration and associations with cardiac rhythm disturbances in Standardbred racehorses.

INTRODUCTION/OBJECTIVES: Accumulating evidence indicates intense exercise can be associated with myocardial damage. Investigating the impact of maximal effort on myocardium and exploring possible association of injury with rhythm disturbance requires a high-sensitivity cardiac troponin assay. The objectives of this study were: (1) to determine the effect of racing on serum cardiac troponin I (cTnI) in Standardbred horses using a high-sensitivity assay; (2) to determine the 99th percentile of cTnI in healthy horses and investigate the effect of demographic variables on cTnI prevailing pre-race in Standardbred horses using a validated high-sensitivity assay and a contemporary assay, and; (3) to explore associations between exercise-associated arrhythmia and cTnI concentration. ANIMALS: Racehorses (n = 145). MATERIALS AND METHODS: ≤ 2 h pre-race, cTnI concentrations were measured in 158 race starts. Electrocardiogram (ECG) monitoring was applied during racing and race recovery and screened for complex ventricular arrhythmia. Associations between cTnI prevailing before racing concentration, age, sex, and gait were investigated. Demographic and performance variables were evaluated for associations with cTnI concentration post-race and rhythm disturbance. RESULTS: Incidence of arrhythmia was 11.6% (16 horses). A significant increase in median (interquartile range) cTnI concentration of 1.36 (0.49-2.81) ng/L was found post-race (p < 0.0001). Serum cardiac troponin I (cTnI) concentration prevailing pre-race was positively associated with increasing age, and gait. Serum cardiac troponin I prevailing post-race was positively associated with concentration prevailing pre-race. Interaction between arrhythmia and finishing distanced revealed horses finishing distanced and experiencing arrhythmia displayed higher cTnI release than with the presence of either alone. CONCLUSIONS: Racing increased cTnI concentration. Horses finishing distanced and also exhibiting arrhythmia may be experiencing myocardial compromise.

Contractile function of the excised hagfish heart during anoxia exposure.

Pacific hagfish, Eptatretus stoutii, can recover from 36 h of anoxia and their systemic hearts continue to work throughout the exposure. Recent work demonstrates that glycogen stores are utilized in the E. stoutii heart during anoxia but that these are not sufficient to support the measured rate of ATP production. One metabolic fuel that could supplement glycogen during anoxia is glycerol. This substrate can be derived from lipid stores, stored in the heart, or delivered via the blood. The purpose of this study was to determine the effect of glycerol on the contractile function of the excised E. stoutii heart during anoxia exposure. When excised hearts, perfused with metabolite free saline (mf-saline), were exposed to anoxia for 12 h, there was no difference in heart rate, pressure generation (max-dP), rate of contraction (max-dP/dtsys), or rate of relaxation (max-dP/dtdia) compared to hearts perfused with mf-saline in normoxia. However, hearts perfused with saline containing glycerol (gly-saline) in anoxia had higher max-dP, max-dP/dtsys, and max-dP/dtdia than hearts perfused with mf-saline in anoxia. Tissue levels of glycerol increased when hearts were perfused with gly-saline in normoxia, but not when perfused with gly-saline in anoxia. Anoxia exposure did not affect the activities of triglyceride lipase, glycerol kinase, or glycerol-3-phosphate dehydrogenase. This study suggests that glycerol stimulates cardiac function in the hagfish but that it is not derived from stored lipids. How glycerol may stimulate contraction is not known. This could be as an energy substrate, as an allosteric factor, or a combination of the two.

Post-exercise cardiac troponin I release and clearance in normal Standardbred racehorses.

BACKGROUND: There are currently no studies detailing cardiac troponin I (cTnI) release in normal horses post-exercise using an analytically validated assay. These data are essential for selecting appropriate sampling times in equine athletes with suspected myocardial injury. OBJECTIVE: To plot the magnitude and time course of cTnI release after maximal effort, using validated cTnI assays. STUDY DESIGN: Descriptive longitudinal study. METHODS: Five clinically normal Standardbred racehorses in race training were included in the study. Horses were exercised in harness at near-race intensity. Blood samples were taken immediately pre- and post-exercise and then hourly for 24 h. Samples were analysed using the validated high-sensitivity cTnI assay and a contemporary sensitivity cTnI assay. RESULTS: Mean resting cTnI was 1.33 ± 0.6 s.d. ng/L (range, 0.82-2.33 ng/L) using assay A. All horses were below the detection limit at rest using assay B. Peak elevation occurred 2-6 h post-exercise with both assays (mean, 4.6 ± 1.7 and 4.0 ± 2 h, respectively). Mean peak increase in cTnI was 11.96 ± 9.41 ng/L (range, 1.72-23.76 ng/L) using assay A. Peak concentrations were detectable in three of the horses using assay B and were between 0.039 and 0.051 µg/L (mean: 0.043 ± 0.006 µg/L). All horses returned to baseline within 24 h. MAIN LIMITATIONS: A small (n = 5) convenience sample was used as random sampling was not logistically possible. CONCLUSIONS: All horses experienced an increase in cTnI post-exercise, with peak occurring 2-6 h post-exercise. Cardiac troponin I elevation was detected earlier using the high-sensitivity assay, which may convey a diagnostic advantage. Targeted studies are needed to determine the significance of these increases.

Troponin assays in the assessment of the equine myocardium.

In 2000, troponin assays were adopted as the test of choice for detection of myocardial injury in man. This decision was made after extensive testing and followed a 60 year search for a biomarker of myocardial damage with sufficient analytical sensitivity and specificity. This has led to proliferation of assays for use in human medicine, each requiring extensive testing and validation before it could be made available on the open market for human use. The search for ever-more analytically sensitive assays and for a standard reference material continues. The adoption of troponin testing in veterinary medicine followed shortly after its development for use in man, providing a much-needed means of detecting and monitoring myocardial damage in horses. However, application of these tests in veterinary medicine has exclusively involved use of assays designed for and clinically validated in human patients. There is no mandated requirement for test validation in veterinary medicine and, while many of these assays have been shown to be capable of detecting equine troponin, the wide diversity of available tests, lack of validation, absence of protocols for their use and lack of standardisation make their application problematic. The objective of this review article is to address this issue, offering guidance where data are available and encouraging caution where there are none. Ultimately, the overall goal of this review is to examine critically the use of troponin assays in the horse and to promote the accurate and appropriate interpretation of valid results.

Identifying a role of the actin capping protein CapZ in ß-adrenergic receptor signalling.

AIM: ß-Adrenergic receptor activation increases myocardial contractility, in part through protein kinase A (PKA)-dependent modification of cardiac myofilaments. PKA regulation of cardiac myofilaments is contingent influenced by protein kinase C (PKC) phosphorylation of troponin I (TnI). Reductions in the cardiac Z-disc protein CapZ attenuate PKC regulation of myofilament activation. We hypothesized that CapZ-deficient transgenic mouse hearts respond poorly to ß-adrenergic receptor activation, as a result of impaired PKC activation. METHODS: Wild-type and CapZ-deficient transgenic mice were treated with the ß-adrenergic receptor agonist isoproterenol (ISO) and whole heart function assessed by echocardiography. Cardiac myofilaments were isolated post-ISO treatment and subjected to an actomyosin MgATPase assay and protein phosphorylation gels. RESULTS: CapZ-deficient transgenic mouse hearts exhibited increased contractility and myofilament calcium sensitivity at baseline, as compared to wild-type mice. In wild-type mice, ISO increased myocardial contractility and decreased myofilament calcium sensitivity, along with an increase in TnI phosphorylation. CapZ-deficient transgenic mice responded to ISO treatment, and myocardial functional differences between transgenic and wild-type mice were abolished. ISO-dependent changes in myofilament activation in transgenic mice were similar to those observed in wild-type. TnI phosphorylation was similarly increased in wild-type and transgenic mice following ISO treatment, while CapZ-deficient transgenic mouse myofilaments also exhibited increased myosin-binding protein C phosphorylation. Differences in myofilament protein phosphorylation patterns suggest the intracellular mechanisms utilized by ß-adrenergic receptor activation are different than that seen in wild-type hearts. CONCLUSIONS: These data further support the concept that the cardiac Z-disc protein is a regulator of myofilament function and intracellular signalling transduction.

Effects of kappa-opioid receptor activation on myocardium.

Kappa-opioid receptor stimulation of the heart transiently increases twitch amplitude and decreases Ca2+-dependent actomyosin Mg2+-ATPase activity through an undetermined mechanism. One purpose of the present study was to determine if the increase in twitch amplitude is due to changes in myofilament Ca2+ sensitivity. We also wanted to determine if kappa-opioid receptor activation alters maximum actin-myosin ATPase activity and Ca2+ sensitivity of tension in a way consistent with protein kinase A or protein kinase C (PKC) action. Rat hearts were treated with U50,488H (a kappa-opioid receptor agonist), phenylephrine plus propranolol (alpha-adrenergic receptor stimulation), isoproterenol (a beta-adrenergic receptor agonist), or phorbol 12-myristate 13-acetate (PMA, receptor independent activator of PKC) or were untreated (control), and myofibrils were isolated. U50,488H, phenylephrine plus propranolol, and PMA all decreased maximum Ca2+-dependent actomyosin Mg2+-ATPase activity, whereas isoproterenol treatment increased maximum Ca2+-dependent actomyosin Mg2+- ATPase activity. Untreated myofibrils exposed to exogenous PKC-epsilon, but not PKC-delta, decreased maximum actomyosin Mg2+-ATPase activity. Langendorff-perfused hearts treated with U50,488H, phenylephrine plus propranolol, or isoproterenol had significantly higher ventricular ATP levels compared with control hearts. PKC inhibitors abolished the effects of U50,488H on Ca2+-dependent actomyosin Mg2+-ATPase activity and myocardial ATP levels. U50,488H and PMA treatment of isolated ventricular myocytes increased Ca2+ sensitivity of isometric tension compared with control myocytes at pH 7.0. The U50,488H-dependent increase in Ca2+ sensitivity of tension was retained at pH 6.6. Together, these findings are consistent with the hypotheses that 1) the positive inotropy associated with kappa-opioid receptor activation may be due in part to a PKC-mediated increase in myofilament Ca2+-sensitivity of tension and 2) the kappa-opioid receptor-PKC pathway is a modulator of myocardial energy status through reduction of actomyosin ATP consumption.

Cardioprotection with kappa-opioid receptor stimulation is associated with a slowing of cross-bridge cycling.

Opioid and alpha-adrenergic receptor activation protect the heart from ischemic damage. One possible intracellular mechanism to explain this is that an improvement in ATP availability contributes to cardioprotection. We tested this hypothesis by correlating postischemic left ventricular developed pressure (LVDP) and myofibrillar Ca(2+)-dependent actomyosin Mg(2+)-ATPase from isolated rat hearts treated with the kappa-opioid receptor agonist U-50488H (1 microM) or the alpha-adrenergic receptor agonist phenylephrine (10 microM) + propranolol (3 microM). Preischemic treatment with U-50488H or phenylephrine + propranolol improved postischemic LVDP recovery by 25-30% over control hearts. Ca(2+)-dependent actomyosin Mg(2+)-ATPase was found to be 20% lower in both U-50488H- and phenylephrine + propranolol-treated hearts compared with control hearts. The kappa-opioid receptor antagonist nor-binaltorphimine (1 microM) abolished the effects of U-50488H on postischemic LVDP and actomyosin Mg(2+)-ATPase activity. Reduced actomyosin ATP utilization was also suggested in single ventricular myocytes treated with either U-50488H or the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), because U-50488H and PMA lowered maximum velocity of unloaded shortening by 15-25% in myocytes. U-50488H and phenylephrine + propranolol treatment both resulted in increased phosphorylation of troponin I and C protein. These findings are consistent with the hypothesis that kappa-opioid and alpha-adrenergic receptors decrease actin-myosin cycling rate, leading to a conservation of ATP and cardioprotection during ischemia.