Comparative study of ECG and echocardiographic parameters indicative of cardiac hypertrophy in athletes

The identification of left ventricular hypertrophy (LVH) through electrocardiographic voltage criteria has been widely studied in patients. However, their validity in “athlete’s heart” remains quite unknown. The aim of this study was to evaluate the most common electrocardiographic indices indicative of LVH compared to the known echocardiographic ones in athletes. The study group comprised 150 male adult competitive athletes (group A) and 50 sedentary participants (group B). Thirteen accepted electrocardiographic voltage criteria indicative of LVH were calculated and correlated with the common echocardiographic indices of left ventricular mass (LVM). Nine of the 13 ECG voltage criteria were significantly increased in athletes compared to controls. Statistically, the Sokolow–Lyon index, which is the most commonly used voltage index was found to be affected by the body mass index (10.7 %, p < 0.05), the group (7.3 %, p < 0.05) and systolic blood pressure (4.5 %, p < 0.05) in total variance of 16.6 % (p < 0.05). No electrocardiographic voltage criterion was significantly correlated with any echocardiographic index, except for the Cornell index that was correlated with end-diastolic volume index (r = 0.29, p < 0.05) and the Sokolow index (V6) with LVMI (r = 0.26, p < 0.05) in group A. We suggested that ECG voltage indices should not be considered valid when assessing LVH in athletes. Thus, the echocardiographic study is preferable in studying training-induced structural cardiac changes.     

Non-invasive cardiac electrophysiological indices in soccer players with mitral valve prolapse.

BACKGROUND: Cardiac disorders constitute the most common causes of sudden cardiac death (SCD) and mitral valve prolapse (MVP) is one of the cardiac structural causes in young populations. The aim of this study was to evaluate some reliable non-invasive electrophysiological variables of SCD in young athletes with mild or moderate MVP and to compare them with their cardiorespiratory adaptations. DESIGN: The study population comprised of 40 amateur male soccer players, who were equally subdivided into those with (group A, aged 20.4+/-4.5 years) and without (group B, 18.4+/-2.4 years) MVP and 20 healthy age-matched sedentary individuals (group C). METHODS: All subjects underwent echo study for left ventricular mass index (LVMI) estimation, treadmill spiro-ergometric test for maximal oxygen uptake (VO2max) measurement and continuous ambulatory 24-h ECG recordings for arrhythmias detection and heart rate variability (HRV) analysis. Furthermore, groups A and B were also submitted to signal-averaged electrocardiogram for late potentials (LP) assessment and to submaximal exercise test for T-wave alternans (TWA) detection. RESULTS: Maximal oxygen uptake, LVMI and HRV index were enhanced in all athletes compared to group C (P<0.05). Resting QTc interval was significantly prolonged only in group B (by 3.7%) compared to C (P<0.05), whereas no significant difference was found between A and B. No positive LP or TWA were observed in all subjects. Significant correlation was found only between HRV index and VO2max in all groups. CONCLUSIONS: Athletes with mild or moderate MVP do not present any significant difference in non-invasive cardiac electrophysiological indices as well as in cardiorespiratory performance, compared to healthy ones. It seems that long-term exercise induced adaptations overlap any cardiac autonomic disorders in cases of mild-to-moderate MVP severity.     

Increased Ca 2+-sensitivity of myofibrillar tension in heart failure and its functional implication

In human failing myocardium, an increased Ca ²⁺-sensitivity of myofilament tension development has been described in Triton X skinned cardiac myocytes compared to cardiomyocytes obtained from non-failing human donor hearts. The present study aimed to investigate whether there are functional implications of the increased Ca ²⁺-sensitivity in heart failure and whether alterations of myofilament function are already obvious at earlier stages of heart failure, such as in cardiac hypertrophy or whether alterations of the intracellular Ca ²⁺-homeostasis are able to induce alterations in myofilament function. Ca ²⁺-activated tension development was measured in Triton X-skinned fibers from human failing and non-failing myocardium. Ca ²⁺-sensitivity of myofilament tension development was significantly shifted to the left in human failing myocardium. Plots of diastolic free Ca ²⁺ versus diastolic tension development showed that in a range of similar diastolic Ca ²⁺-concentrations, diastolic tension was significantly enhanced in the failing hearts. The Ca ²⁺/tension relationship was shifted to the right in Triton X-skinned fiber preparations from transgenic renin overexpressing rats (TG(mREN2)27), shown to have concentric hypertrophy. In addition, the Ca ²⁺/tension relationship was unchanged in phospholamban knock-out mice with an increased systolic Ca ²⁺ (and enhanced diastolic Ca ²⁺-load). It is concluded that the increased Ca ²⁺-sensitivity of myofilament tension observed in single cardiomyocytes from failing human myocardium may be a phenomenon also present in multicellular preparations and may contribute to the diastolic dysfunction observed in human heart failure. Alterations of myofilament function occur at very early stages of heart failure and may be species dependent, or dependent on intracellular free Ca ²⁺-levels.     

Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during beta-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine(43/45) and threonine(144) mutated to alanine) and for PKA (serine(23/24) mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by beta-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the beta-agonist isoproterenol decreased twitch duration and relaxation time constant (tau) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after beta-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased tau by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that beta-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.     

Stress dose of hydrocortisone is not beneficial in patients with classic congenital adrenal hyperplasia undergoing short-term, high-intensity exercise

Classic congenital adrenal hyperplasia (CAH) is associated with impaired function of the adrenal cortex and medulla leading to decreased production of cortisol and epinephrine. As a result, the normal exercise-induced rise in blood glucose is markedly blunted in such individuals. We examined whether an extra dose of hydrocortisone, similar to that given during other forms of physical stress such as intercurrent illness, would normalize blood glucose levels during exercise in patients with CAH. We studied hormonal, metabolic, and cardiorespiratory parameters in response to a standardized high-intensity exercise protocol in nine adolescent patients with classic CAH. Patients were assigned to receive either an additional morning dose of hydrocortisone or placebo, in addition to their usual glucocorticoid and mineralocorticoid replacement in a randomized, double-blind, crossover design 1 h before exercising. Although plasma cortisol levels approximately doubled after administration of the additional hydrocortisone dose compared with the usual single dose, fasting and exercise-induced blood glucose levels did not differ. In addition, no differences were observed in the serum concentrations of the glucose-modulating hormones epinephrine, insulin, glucagon, and GH and of the metabolic parameters lactate and free fatty acids. Although maximal heart rate was slightly higher after stress dosing (193 +/- 3 vs. 191 +/- 3 beats/min, mean +/- sem, P < 0.05), this did not affect exercise performance or perceived exertion. We conclude that patients with classic CAH do not benefit from additional hydrocortisone during short-term, high-intensity exercise. Although this has not been tested with long-term exercise, a high degree of caution should be used when considering the frequent use of additional hydrocortisone administration with exercise, given the adverse side effects of glucocorticoid excess.     

Role of cavities and hydration in the pressure unfolding of T4 lysozyme

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T₄ lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T₄ lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.The destabilization of proteins by pressure is a fundamental and highly informative probe of their structural free energy landscape but remains inadequately understood (1). The underlying determinants of pressure-induced unfolding have recently been a subject of several detailed investigations (2–10). Fundamentally, pressure-induced unfolding of proteins results from the population of nonnative conformations having a lower total system volume than the native structure seen at ambient pressure. Various mechanisms for pressure-induced unfolding have been proposed including changes in water structure that weaken the hydrophobic effect at high pressure (11, 12), increases in solvent density at the protein surface that contribute to a reduction in the total volume of the protein–water system (13, 14), and the elimination of cavities in the protein interior through exposure to solvent (3). With the development of high-pressure sample cells compatible with modern solution NMR probes (15), detailed measurements of proteins unfolding under pressure with atomic resolution have now become possible (5, 16–18). Recent studies of staphylococcal nuclease (SNase) compellingly argue that the filling of void volumes present in the native state is the primary determinant of pressure-induced unfolding (4–6). A critical aspect of a “destruction of voids” mechanism for pressure-induced unfolding of proteins is whether the voids or cavities are occupied with water in the folded state. Early investigations of buried hydrophobic pockets indicated that even large cavities are typically not hydrated, whereas hydrophilic cavities generally are occupied by water (19, 20). Many of the key studies impacting this question used the L99A single-point mutant of the model enzyme T₄ lysozyme (20).The L99A mutation creates an internal cavity with an estimated volume of ∼150–160 ų, large enough to accommodate three or four water molecules (21) (Fig. 1). Crystallographic investigation found no electron density within this pocket at ambient pressure (22, 23). In contrast, solution NMR and molecular-dynamics simulations suggest that the region of the protein around the hydrophobic pocket is highly dynamic, possibly to the extent that the pocket may be transiently accessible to solvent (22, 24–27). Crystallographic studies conducted at high pressure conversely suggested that the region around the pocket is rigid and exhibits increasing rigidity with increased pressure (23). Electron density also increased within the cavity as the hydrostatic pressure was increased (22), consistent with a pressure-induced filling of the hydrophobic cavity with water molecules. In contrast, fluorescence and small-angle X-ray scattering studies in bulk solution demonstrated that the protein is unfolded at these elevated pressures (2), suggesting that the crystal packing effects stabilize the protein. The hydrophobic cavity also provides a general, moderate-affinity binding site for small, relatively nonpolar ligands (28).Open in a separate windowFig. 1.Hydration of T₄ lysozyme L99A at ambient pressure (∼1 bar). A backbone ribbon representation of L99A [Protein Data Bank (PDB) ID code 1L90 (63)] is shown with the N-terminal domain (residues 13–65) illustrated in blue, and the C-terminal domain (residues 1–12 and 66–164) is colored green. The hydrophobic pocket created by the L99A mutation is shown as orange mesh, and the three tryptophan side chains are shown as stick representations. The helices are numbered as a reference for discussion in the text. Cyan spheres are shown at the positions of amide hydrogens where an NOE to the water resonance was detected. Yellow spheres indicate the positions of amide hydrogens within NOE distance (5 Å) of the interior of the hydrophobic pocket, but outside NOE distance to the protein surface. These are the sites where detection of NOEs to the water resonance would indicate hydration of the pocket. No NOE cross-peaks from these sites to the water resonance were observed, suggesting that the pocket is not hydrated at ambient pressure.T₄ lysozyme is one of the smallest known proteins to contain more than one cooperative folding unit. The folding of WT T₄ lysozyme has been examined in detail by using hydrogen–deuterium exchange approaches and has been shown to contain two domains that fold cooperatively and with distinct free energy profiles (29–32). The N-terminal domain is ∼6 kcal/mol less stable than the C-terminal domain. The cavity created by the L99A mutation is in the center of the C-terminal domain. The thermal stability of the L99A mutant is reduced compared with the WT protein by 16 °C (5 kcal/mol) (33), an effect that is partially abrogated by binding hydrophobic ligands to the cavity (28, 34).The L99A mutant of T₄ lysozyme provides a unique system to examine the hydration of internal pockets and the details of pressure-induced unfolding. In principle, protein–water interactions can be characterized by solution NMR methods (35), but severe artifacts often render the approach quite limited (36). Recently, it has been shown that various advantageous properties of proteins and water encapsulated within reverse micelles largely overcome these artifacts (37, 38). Here, we use this approach to directly measure the hydration of the internal cavity. High-pressure NMR is used to examine the pressure-induced response of the protein in bulk solution and under confinement by the reverse micelle. We demonstrate that the hydrophobic pocket appears to be essentially dehydrated at ambient pressure (∼1 bar) and that the pressure response of the protein is an unfolding of the C-terminal domain only, representing an inversion of the relative stability of the domains as a result of the cavity-creating mutation. This result is in contrast to the unfolding of the cysteine-free WT (WT*) protein, which shows only the earliest stages of pressure-induced subglobal unfolding. Furthermore, the L99A mutant with benzene occupying the cavity shows no evidence of pressure unfolding. Nanoscale confinement of the protein also suppresses the L99A pressure-induced unfolding transition (P_u) as a result of the restriction of conformational space imposed by the reverse micelle. In lieu of the pressure unfolding transition, the volume reduction imposed by increasing pressure is compensated for in the reverse micelle by progressively increasing incorporation of water into the cavity interior, essentially recapitulating the observations from high-pressure crystallography in a solution measurement. These findings have important implications with respect to the nature of pressure-induced unfolding, the roles of cavities in protein structural stability, and the effects of confinement, a critical parameter when considering the intracellular milieu, in which proteins must fold and carry out their functions.