Albuterol increases lean body mass in ambulatory boys with Duchenne or Becker muscular dystrophy.

BACKGROUND: Albuterol is a beta-2 agonist that has been demonstrated to increase muscle strength in studies in animals and humans. Based on a pilot study of extended-release albuterol Repetabs in children with dystrophinopathies, the authors conducted a randomized, double-blind, placebo-controlled study with a crossover design. METHODS: Fourteen boys with Duchenne or Becker muscular dystrophy, 6 to 11 years old, completed two treatment periods (albuterol and placebo), 12 weeks each, separated by a 12-week washout period. As the albuterol Repetab formulation was no longer available, an alternate extended release albuterol was used (Volmax, 12 mg per day). Outcome measurements included 1) lean body mass, 2) fat mass, 3) isometric knee extensor and flexor moments, 4) manual muscle testing, and 5) timed functional tests. RESULTS: Lean body mass was significantly higher for subjects following albuterol treatment compared to placebo treatment, while fat mass was significantly lower. No differences were found in isometric knee moments or manual muscle tests. Time to run/walk 30 feet was improved following albuterol. CONCLUSIONS: Short-term treatment with extended release albuterol may increase lean body mass, decrease fat mass, and improve functional measures in patients with dystrophinopathies. However, the significant change in strength of specific muscle groups found in the pilot study was not observed in the present study. These findings may be attributed to differences in the drug release and kinetics between Repetab and Volmax formulations as they affect the concentration of available beta-2 receptors on the muscle cell surface differently.

Predictors of graft longevity in pediatric heart transplantation.

Given the volume of pediatric orthotopic heart transplants (OHTs) at several centers, it is now possible to generate pediatric-specific, single-center OHT survival data. The transplant experience for 152 pediatric OHT patients at our institution was reviewed. The following were noted for each patient: graft survival; immunosuppressant therapy; initial diagnosis; cause of graft failure; clinical status at time of transplant; donor and recipient blood type, sex, weight, and age; ischemic time; previous cardiac surgery; race; and immune status. A series of Kaplan-Meier survival curves were constructed. Univariate comparisons of survival curves were performed with the Breslow test to determine equality of each pair of curves. Only immunosuppression with tacrolimus and an initial diagnosis of noncongenital heart disease positively influenced survival in pediatric OHT patients (p < or = 0.021 and p < or = 0.03, respectively). The more recently transplanted patients, managed with tacrolimus, had less mortality early after OHT (acute rejection) and less mortality during the period 2 or 3 years after OHT. No other factors, including prior cardiothoracic surgery, sex matching, and race matching, significantly influenced survival. Recently transplanted patients managed with tacrolimus-based immunosuppression and patients with noncongenital cardiomyopathy have significantly superior graft survival.

Pilot trial of albuterol in Duchenne and Becker muscular dystrophy.

The authors conducted a randomized, crossover, double-blind, placebo-controlled pilot study of albuterol in nine boys with dystrophinopathies. Primary outcomes were 1) isometric knee extensor and flexor strength; and 2) manual muscle testing (MMT). Isometric knee extensor strength and MMT scores were significantly higher in patients taking albuterol vs placebo. Therefore, 12-week treatment with extended-release albuterol may increase strength in patients with dystrophinopathies. A larger, double-blind, randomized study is necessary to confirm these results.

Inhibition of L-type Ca2+ channel current in Xenopus oocytes by amiodarone.

BACKGROUND: Although amiodarone has been referred to as a class III antiarrhythmic agent, it also possesses electrophysiologic characteristics of the three other classes (classes I and IV and minor class II effects). Previous studies have demonstrated that amiodarone inhibits Ca2+ channel current in intact cardiac myocytes. However, it is not clear whether this response reflects a pure class IV effect (direct Ca2+ channel inhibition) or a class II effect (beta-adrenergic receptor blockade) of amiodarone. METHODS: In the current study, the effects of amiodarone on Ca2+ current were studied in the absence of sympathetic regulation using a Xenopus oocyte expression system. The L-type Ca2+ channel alpha1C subunit was coexpressed with the alpha2delta and beta2a subunits in enzymatically digested Xenopus oocytes. Ca2+ currents were recorded using the cut-open oocyte preparation. RESULTS: We found that perfusion of 10 microM isoproterenol produced no significant change in peak Ca2+ current (from 223+/-33 to 210+/-29 nA, mean+/-SEM, n=5, P=not significant), indicating the absence of a functional stimulatory sympathetic signal pathway in these oocytes. After 10 minutes of exposure to 10 microM amiodarone, Ca2+ current amplitude was significantly decreased from 174+/-33 to 100+/-26 nA (n=8, P<0.01; control group: 220+/-33 to 212+/-29 nA, n=5, P=not significant). These effects were similar to those of 10 microM nifedipine (201+/-48 to 108+/-48 nA, n=6, P<0.05), a typical Ca2+ channel blocker. On the other hand, neither amiodarone nor nifedipine significantly altered the Ca2+ current activation or inactivation kinetics. CONCLUSIONS: These results demonstrate that amiodarone inhibits Ca2+ current in the absence of a functional intrinsic beta-adrenergic stimulatory system and, therefore, represents a true class IV effect.

Lack of muscarinic regulation of Ca(2+) channels in G(i2)alpha gene knockout mouse hearts.

The purpose of the present study was to examine the role of G(i2)alpha in Ca(2+) channel regulation using G(i2)alpha gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the beta-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca(2+) currents in both 129Sv wild-type (WT) and G(i2)alpha gene knockout (G(i2)alpha-/-) mice. Perfusion with CCh significantly inhibited the Ca(2+) current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca(2+) currents in G(i2)alpha-/- ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca(2+) current in WT cardiomyocytes but not in G(i2)alpha-/- cells. These findings demonstrate that, whereas the Iso-G(s)alpha signal pathway is intact in G(i2)alpha gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca(2+) channels by CCh. Therefore, G(i2)alpha is necessary for the muscarinic regulation of Ca(2+) channels in the mouse heart. Further studies are needed to delineate the possible interaction of G(i) and other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca(2+) current in the heart.

Molecular cloning of junctin from human and developing rabbit heart.

Canine junctin is a 26-kDa transmembrane protein found in the sarcoplasmic reticulum (SR) membrane in cardiac and skeletal muscle. Junctin has recently been shown to bind directly to calsequestrin, the ryanodine receptor, and triadin. Junctin is thought to play a role in facilitating (and perhaps regulating) Ca(2+) release from the SR. Immature heart exhibits decreased utilization of SR Ca(2+) stores for cell contraction. We have cloned human and rabbit cardiac junctin and investigated the expression of junctin in developing rabbit heart. Human junctin was cloned from an adult cardiac cDNA library. Rabbit junctin was cloned by RT-PCR. Northern blot analysis demonstrates a single primary mRNA transcript of approximately 2.8 kb in hearts from both species. Sequence analysis demonstrates greater than 97% homology between the predicted amino acid sequences of human, rabbit, and canine junctin in the putative transmembrane domain and subsequent initial 61 amino acid portion of the putative luminal domain. These domains also exhibit sequence homology with triadin. The C-terminal region shows much lower (72 to 75%) sequence homology among the three species. In addition, Northern blot analysis demonstrates that the expression of junctin increases markedly in postnatal rabbit myocardium. These findings suggest that the putative transmembrane domain and subsequent initial portion of the putative luminal domain of junctin play an important role in the binding of junctin to calsequestrin, the ryanodine receptor, and triadin in the postnatal heart. Furthermore, the previously described increase in SR Ca(2+) release with development is associated with the increased expression of junctin.

Sarcoplasmic reticulum Ca(2+)ATPase and cell contraction in developing rabbit heart.

The purpose of the present study was to determine whether age-related changes in the expression and function of the cardiac isoform of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA2a) play a role in SR Ca(2+)release and cell contraction. SERCA2a protein levels and subcellular localization were compared between fetal, neonatal, juvenile and adult New Zealand White rabbits. Studies of SERCA function in isolated myocytes were performed in situ by examining the rate of reloading of the SR Ca(2+)stores following caffeine-induced depletion. We found that significant quantities of SERCA2a were present early in immature heart and that SERCA2a expression reached adult levels within 15-30 days after birth. Furthermore, SERCA2a protein is present as a series of transverse striations within the cell as early as 1 day of age. In contrast to previous studies of SERCA in vitro, the SERCA protein function in situ was found to be comparable between neonatal and adult myocytes in maintaining SR Ca(2+)stores. These results indicate that the paucity of SR Ca(2+)release in immature ventricular cardiac myocytes is not the result of immaturity in SERCA2a expression.

The relationship between temperature and calcium in acute cell damage after exposure to radiofrequency or thermal energy in isolated neonatal and adult rabbit cardiac myocytes.

Radiofrequency (RF) ablation is a nonsurgical technique using catheter-directed RF energy for treating cardiac arrhythmias in children and adults. Previous reports have suggested that sequestration of calcium (Ca2+) by the sarcoplasmic reticulum may partially protect mature cardiac myocytes from the effects of RF energy. The purposes of this study were to determine whether differences exist between neonatal and adult myocyte responses to RF energy and if myocyte damage is a Ca2+-dependent process. Because immature myocardium is functionally deficient in sarcoplasmic reticulum, we hypothesized that immature myocytes would be more susceptible to damage induced by RF energy. Isolated ventricular myocytes were obtained from neonatal and adult New Zealand White rabbits by enzymatic dissociation, then placed in a perfusion chamber designed to deliver RF energy or a heated perfusate solution. Measurements of bath temperature, cell morphology, and contractile response to electrical stimuli were recorded. RF energy application associated with increased perfusate temperature resulted in cell death, but not when the temperature rise was inhibited. Thus, the acute damage to cells exposed to RF energy appears to be mediated by thermal energy. After exposure to thermal energy, neonatal cells underwent contracture at lower temperatures than did adult cells. Perfusion with solutions containing low Ca2+ concentrations, comparable to intracellular diastolic Ca2+ levels, had a protective effect for both neonatal and adult myocytes. These findings indicate that acute cell damage after exposure to RF energy is mediated by a Ca2+-dependent process. Furthermore, immature myocardium is particularly susceptible to RF-mediated cell damage, possibly secondary to reduced Ca2+ sequestration by the sarcoplasmic reticulum.

Caffeine-induced contractions in developing rabbit heart.

Mature myocardium utilizes calcium released by the sarcoplasmic reticulum (SR) for cell contraction. Transient exposure of mature myocytes to caffeine is known to directly trigger Ca2+ release from the SR. In contrast, neonatal rabbit heart cells rely on transsarcolemmal Ca2+ influx for tension generation. SR function is decreased in immature heart and appears to play a minimal role as a calcium source. Accordingly, we hypothesized that neonatal rabbit myocytes would not respond to a caffeine pulse. Isolated neonatal and adult myocytes were paced to load the SR with calcium and then exposed to a 1-s pulse of 10 mM caffeine. As previously described, adult myocytes exhibited a brisk contraction in response to caffeine. Unexpectedly, neonatal myocytes also exhibited a similar, brisk response. These caffeine-induced contractions were not dependent on extracellular Ca2+ but were dependent upon the loading of SR Ca2+ stores. When SR Ca2+ stores were depleted by exposure to caffeine, mature myocytes exhibited only small, slow contractions in response to electrical field stimulation. Replenishing the SR Ca2+ stores resulted in normal, brisk contractions. In contrast, electrically stimulated contractions in immature myocytes were largely unaffected by caffeine-induced SR depletion. Thus, although neonatal myocytes are capable of loading and releasing calcium from the SR, such SR calcium release is not normally required for contraction in the developing heart. The minor role of SR Ca2+ release in immature rabbit heart may not result from immaturity of the SR, but rather from an inadequate mechanism to trigger SR calcium release.

Mechanisms of amiodarone-induced inhibition of Ca2+ current in isolated neonatal rabbit ventricular myocytes.

BACKGROUND: Amiodarone is an effective antiarrhythmic drug used to treat a wide variety of ventricular and supraventricular tachyarrhythmias. Recent voltage clamp studies indicate that amiodarone may possess a variety of antiarrhythmic effects. METHODS: The tight-seal, whole-cell voltage clamp technique was used to investigate the acute effects of amiodarone on L-type Ca2+ channel kinetics in isolated neonatal ventricular myocytes. RESULTS: We found that acute perfusion with 1 mumol/L amiodarone inhibited peak inward Ca2+ current by 39.1% (4.85 +/- 0.42 to 2.95 +/- 0.6 pA/pF, n = 10, p < 0.001) without changing the shape of the current-voltage relation. In addition, amiodarone shifted Ca2+ channel steady-state inactivation to more negative membrane potentials. In the absence of amiodarone, half inactivation of the Ca2+ current occurred at a membrane potential of -23.8 +/- 0.2 mV compared to -34.2 +/- 0.6 mV after addition of amiodarone (n = 11, p < 0.01). Furthermore, amiodarone significantly delayed Ca2+ current recovery from previous inactivation. CONCLUSIONS: These results provide evidence that amiodarone blocks voltage-dependent Ca2+ current in isolated neonatal rabbit ventricular myocytes by a variety of different mechanisms. The inhibitory effect of amiodarone on L-type Ca2+ current may represent an important facet of amiodarone's acute antiarrhythmic activity in the immature heart.

Effects of isoflurane on electrophysiological measurements in children with the Wolff-Parkinson-White syndrome.

This study was designed to assess the effects of isoflurane (ISO) on the electrophysiological properties of the accessory pathway, atrium, ventricle, and AV node in children with the Wolff-Parkinson-White (WPW) syndrome. The results of programmed electrical stimulation were analyzed in 51 patients (4 months to 17 years of age) with WPW. The study population was divided into two groups. Twenty-seven patients received local anesthesia and intramuscular injection of meperidine, promethazine, and chlorpromazine (MPC group). Twenty-four patients received general anesthesia with ISO inhalation (ISO group). We compared the antegrade effective refractory period of the accessory pathway (antegrade APERP), ventricular effective refractory period (VERP), atrial effective refractory period (AERP), AH interval, and cycle length of circus movement tachycardia (CMT-CL) in 12 pairs of age and sex matched patients selected from the MPC and ISO groups. Of the 12 pairs of age and sex matched patients, antegrade APERP in patients who received ISO (299 +/- 17 ms, mean +/- SEM) was significantly longer as compared with matched patients in the MPC group (262 +/- 5 ms, P < 0.025). The VERP and AERP in patients from the ISO group were significantly prolonged compared with the MPC patients (239 +/- 7 vs 210 +/- 8 ms, P < 0.025, and 228 +/- 11 vs 180 +/- 6 ms, P < 0.01, respectively). There was no significant difference in the AH interval or CMT-CL between the two subgroups. Thus, ISO prolongs the antegrade APERPs as well as the effective refractory periods of atrial and ventricular muscle in children with WPW, while the AH interval and CMT-CL appear to be unaffected. Care must be taken in interpreting measurements of the antegrade APERP made in patients under general anesthesia for RF ablation of accessory pathways.

Developmental changes in the effects of pH on contraction and Ca2+ current in rabbit heart.

Protons inhibit Ca2+ current and contraction in heart muscle. The present study compares the effects of lowering the pH of the bath solution on single-cell contraction, action potential configuration and Ca2+ currents between neonatal and adult rabbit hearts. We found that a reduction of extracellular pH from 7.3 to 6.3 decreased cell contraction amplitude to 84.3% of control in isolated neonatal myocytes. A comparable change in extracellular pH resulted in a decrease in cell contraction to 56.2% of control in adult cells. Similarly, tension generation in intact neonatal papillary muscles was less sensitive to a decrease in external pH as compared to papillary muscles from adult animals. In addition, acidosis caused a less pronounced inhibition of Ca2+ current in neonatal cells than in adult cells (85 +/- 4% nu 62 +/- 4% of control, pH = 6.3, P < 0.001; 63 +/- 5% nu 32 +/- 5% of control, pH = 5.8, P < 0.001). Thus, the effect of external acidosis on myocardial contractility is commensurate with the effect on trans-sarcolemmal Ca2+ current. The membrane potential at which peak Ca2+ current occurred was not altered by low pH in neonatal cells but was shifted toward positive potentials by 17.7 mV in adult myocytes. Further, low external pH solution reduced Ca2+ current conductance more in adult cells than in neonatal cells. Moreover, action potential configuration in neonatal cells was altered less by acidosis as compared with adult cells. These findings may help explain the greater resistance of neonatal hearts to extracellular acidosis.

Developmental cardiac electrophysiology recent advances in cellular physiology.

This overview of cardiac ion channel development does not suggest any particular theme underlying the expression or regulation of all channel subtypes. Calcium and potassium channels generally exhibit increased expression in more mature hearts. However, this increase in channel number or activity as determined under voltage clamp conditions may not be translated into increased activity in vivo. Concomitant changes in other physiological factors such as local intracellular Ca2+ accumulation, increased resting membrane potential and decreased heart rate in mature heart may inhibit or augment channel activity. Na(+)-Ca2+ exchange activity appears to decrease with development, possibly reflecting its decreasing role in both systolic and diastolic Ca2+ regulation. Na+ channel activity follows a middle course, exhibiting little change in channel conductance. The reported shift in the voltage dependence of channel inactivation toward more negative membrane potentials may reflect a concomitant shift in the resting membrane potential in mature heart. However, this change is in the direction opposite to that reported for L-type Ca2+ channel inactivation, suggesting that the regulation of these channels is not modulated by a common factor such as membrane surface charge. A detailed characterization of multiple channel subtypes in mature myocardium has resulted in significant advances in models of the cardiac action potential and excitation-contraction coupling. Recently, developmental changes in ion channel physiology have been described, setting the stage for a comparable elucidation of the ontogeny of the cardiac action potential. Ca2+ and K+ channel currents generally become more prominent with development. In contrast, developmental changes in Na+ currents are less dramatic and Na(+)-Ca2+ exchange currents appear to decrease with age. These changes may, in part, be reflected by the increasingly important role of transsarcolemmal Ca2+ influx in triggering Ca2+ release from the SR in mature heart as compared to its direct role of providing Ca2+ for cell contraction in immature heart. These developmental changes in ion channel expression and function are likely to have a significant effect on the generation of the action potential in individual myocytes. Developmental changes in the characteristics of the action potential may then have a major influence on the initiation, propagation and termination of autonomic, triggered, and re-entrant arrhythmias. Progress in this area provides an essential foundation for the evolution of a systematic approach to pediatric arrhythmias comparable to that under development for mature heart [3].

Steady-state mRNA levels of G protein subunits in developing rabbit myocardium.

Cardiac responsiveness to beta-adrenergic stimulation changes with age. Developmental changes in expression of guanine nucleotide-binding coupling protein (G protein) subunits may account for these physiologic changes. We measured steady-state levels of mRNA encoding the alpha-subunit of the specific G protein that stimulates adenylyl cyclase (Gs alpha) and three isoforms of beta-subunit of G proteins (G beta) in developing myocardium. Total RNA prepared from the right and left ventricles of fetal, neonatal, juvenile, and adult rabbits was size-fractionated, blotted, and probed with 32P-labeled cDNAs encoding rat Gs alpha, bovine G beta-1, human G beta-2, and human G beta-3. For standardization, these blots were subsequently hybridized with a 32P-labeled cDNA encoding glyceraldehyde 3-phosphate dehydrogenase (GAPD). Two-dimensional densitometric analysis of autoradiographs was used to quantify relative hybridization intensities. An age-dependent decrease in mRNAs encoding Gs alpha, G beta-1, and G beta-2 relative to mRNA encoding GAPD was observed in both ventricles, while G beta-3 mRNA was not detected. At all ages studied, levels of Gs alpha and G beta-1 mRNA were similar in the two ventricles. However, G beta-2 mRNA declined more in the left ventricle than in the right ventricle during maturation. Our results demonstrate developmental control in heart for mRNAs encoding several G protein subunits. In addition, differential declines in G beta-1 and G beta-2 mRNA in the right ventricle suggest that these G beta isoforms are regulated uniquely and may reflect functional roles for these G beta isoforms in different signaling cascades.

Adenylyl cyclase integrates multiple G protein signals to modulate calcium currents in neonatal rabbit heart.

We investigated the effects of added beta gamma subunits of G proteins (G beta gamma) on beta-adrenergic responsiveness of transmembrane Ca2+ currents (ICa) in ventricular myocytes from neonatal rabbits. G beta 1 gamma 1 purified from retinal rods was dialyzed into cells via the voltage clamp micro-electrode. Stimulation of ICa by isoproterenol was not affected by added intracellular G beta 1 gamma 1 or by carbachol alone but was completely blocked by combined G beta 1 gamma 1 and carbachol. Pretreatment of cells with pertussis toxin or temporal separation of carbachol and isoproterenol allowed stimulation of ICa by isoproterenol in cells dialyzed with G beta 1 gamma 1. Carbachol and G beta 1 gamma 1 together also did not prevent stimulation of ICa by dibutyryl-cyclic AMP. Thus, rather than simply inactivating Gs alpha by mass action, G beta 1 gamma 1 acts in concert with carbachol to inhibit isoproterenol stimulation of ICa.

Age- and anesthesia-related changes in accessory pathway conduction in children with Wolff-Parkinson-White syndrome.

The anterograde effective refractory period of the accessory pathway is age-dependent in pediatric patients with the WPW syndrome. Thus, age should be considered when developing electrophysiologic criteria for the risk of hypotensive arrhythmias in these patients. In addition, general anesthesia must also be considered in interpreting age-related changes in the anterograde APERP, especially in children.

Na+/Ca2+ exchange and cell contraction in isolated neonatal and adult rabbit cardiac myocytes.

Recent studies have demonstrated a relative deficiency in voltage-gated Ca2+ currents (ICa) in immature myocardium. We hypothesized that contraction in developing heart results in part from Ca2+ influx via "reverse" Na+/Ca2+ exchange current (INa/Ca). Accordingly, INa/Ca and cell contraction amplitude were measured in single neonatal and adult rabbit ventricular myocytes. INa/Ca was dependent on Ca2+ concentration, Na+ concentration, and membrane potential and was blocked by 5 mM Ni2+ but not by the Ca(2+)-channel blocker nifedipine. In neonatal cells, contraction amplitude reached a plateau for depolarizations positive to 0 mV. In adult myocytes, contraction amplitude was maximal at 0 mV and decreased at positive membrane potentials. Inhibition of ICa with nifedipine did not affect maximal contraction amplitude in neonatal myocytes but almost completely suppressed contraction of adult cells. These data suggest that Ca2+ influx via ICa is not required for contraction of neonatal rabbit cardiac myocytes. Moreover, Ca2+ influx via reversal of the Na+/Ca2+ exchange mechanism may provide a significant portion of the Ca2+ regulating cell contraction, especially during depolarization to positive membrane potentials.

Sodium-calcium exchange in neonatal myocardium: reversible inhibition by halothane.

Neonatal myocardium is distinctly more sensitive to extracellular calcium levels than is mature myocardium. This has been ascribed to the poorly developed sarcoplasmic reticulum of neonatal myocardium. Recent evidence has suggested that there is an increased dependence of neonatal myocardium on the sodium-calcium exchange current, and that sodium-calcium exchange may be a major source of calcium influx in neonatal myocardial cells. We determined the effect of halothane on the sodium-calcium exchange current on single neonatal (2- to 5-day-old) rabbit ventricular myocytes by means of the whole cell voltage clamp. Lower (1.5%) halothane decreased sodium-calcium exchange current by 49%, from 29 +/- 3 to 15 +/- 6 pA. Higher (3%) halothane decreased this current by 66%, from 50 +/- 9 to 17 +/- 9 pA. Thus halothane has a reversible inhibition of sodium-calcium exchange current in neonatal myocardium. Inhibition of sodium-calcium exchange current would be expected to have a magnified effect on contractility in neonatal as opposed to adult myocardium, and could theoretically ameliorate reperfusion injury due to influx of calcium via the sodium-calcium exchanger.