Stretch-activated cation channels in skeletal muscle myotubes from sarcoglycan-deficient hamsters.

Deficiency of delta-sarcoglycan (delta-SG), a component of the dystrophin-glycoprotein complex, causes cardiomyopathy and skeletal muscle dystrophy in Bio14.6 hamsters. Using cultured myotubes prepared from skeletal muscle of normal and Bio14.6 hamsters (J2N-k strain), we investigated the possibility that the delta-SG deficiency may lead to alterations in ionic conductances, which may ultimately lead to myocyte damage. In cell-attached patches (with Ba(2+) as the charge carrier), an approximately 20-pS channel was observed in both control and Bio14.6 myotubes. This channel is also permeable to K(+) and Na(+) but not to Cl(-). Channel activity was increased by pressure-induced stretch and was reduced by GdCl(3) (>5 microM). The basal open probability of this channel was fourfold higher in Bio14.6 myotubes, with longer open and shorter closed times. This was mimicked by depolymerization of the actin cytoskeleton. In intact Bio14.6 myotubes, the unidirectional basal Ca(2+) influx was enhanced compared with control. This Ca(2+) influx was sensitive to GdCl(3), signifying that stretch-activated cation channels may have been responsible for Ca(2+) influx in Bio14.6 hamster myotubes. These results suggest a possible mechanism by which cell damage might occur in this animal model of muscular dystrophy.

Sodium-calcium exchanger (NCX-1) and calcium modulation: NCX protein expression patterns and regulation of early heart development.

Ouabain-induced inhibition of early heart development indicated that Na/K-ATPase plays an important role in maintaining normal ionic balances during differentiation of cardiomyocytes (Linask and Gui [1995] Dev Dyn 203:93-105). Inhibition of the sodium pump is generally accepted to affect the activity of the Na(+)-Ca(++) exchanger (NCX) to increase intracellular [Ca(++)]. These previous findings suggested that Ca(++) signaling may be an important modulator during differentiation of cardiomyocytes. In order to identify a connection between heart development and NCX-mediated Ca(++) regulation, we determined the embryonic spatiotemporal protein expression pattern of NCX-1 during early developmental stages. In both chick and mouse embryos, NCX-1 (the cardiac NCX isoform) is asymmetrically expressed during gastrulation; in the right side of the Hensen's node in the chick, in the right lateral mesoderm in the mouse. At slightly later stages, NCX-1 is expressed in the heart fields at comparable stages of heart development, in the chick at stage 7 and in the mouse at embryonic day (ED) 7.5. By ED 8 in the mouse, the exchanger protein displays a rostrocaudal difference in cardiac expression and an outer curvature-inner curvature ventricular difference. By ED 9.5, cardiac expression has increased from that seen at ED8 and NCX-1 is distributed throughout the myocardium consistent with the possibility that it is important in regulating initial cardiac contractile function. Only a low level of expression is detected in inflow and outflow regions. To substantiate a role for the involvement of calcium-mediated signaling, using pharmacologic approaches, ionomycin (a Ca(++) ionophore) was shown to perturb cardiac cell differentiation in a manner similar to ouabain as assayed by cNkx2.5 and sarcomeric myosin heavy chain expression. In addition, we show that an inhibitor of NCX, KB-R7943, can similarly and adversely affect early cardiac development at stage 4/5 and arrests cardiac cell contractility in 12-somite embryos. Thus, based upon NCX-1 protein expression patterns in the embryo, experimental Ca(++) modulation, and inhibition of NCX activity by KB-R7943, these results suggest an early and central role for calcium-mediated signaling in cardiac cell differentiation and NCX's regulation of the initial heartbeats in the embryo.

Different effects of the Ca(2+)-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2.

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels.

The New York University Pediatric Heart Failure Index: a new method of quantifying chronic heart failure severity in children.

OBJECTIVE: The assessment of the severity of heart failure in pediatric patients is handicapped by the subjectivity of diagnostic parameters. This study evaluated the feasibility of a new standardized heart failure index, the New York University Pediatric Heart Failure Index (NYU PHFI), to quantify the degree of heart failure in a selected pediatric population. METHODS AND RESULTS: The index is a weighted, linear combination of scores based on symptoms, physical signs, and medical regimen. Overall, healthy children (n = 12) scored very low (0 to 2) on this index. Mean scores of children (<2 years; mean age, 4.8 months; n = 12) with a left-to-right shunt lesion declined from 11.4 (SD +/- 4.1, P <.001, 2-tailed test) before surgery to 1.8 (SD +/- 1.3) after surgical correction of their cardiac defects. The average inter-observer correlation coefficient was 0.95 (P <.001), despite a wide range of scores. CONCLUSIONS: The NYU PHFI appears to be a reliable and convenient instrument for measuring heart failure severity in children. These initial results support further testing in broader diagnostic and age groups and over longer periods.

Thyroid hormone increases pacemaker activity in rat neonatal atrial myocytes.

The effects of thyroid hormone (3,3',5-triiodo- L -thyronine, T3) on pacemaker activity were studied with electrophysiological and pharmacological approaches using spontaneously beating neonatal atrial myocytes cultured from 2-day-old rats. Treatment with T3 (10(-8)m) for 24-48 h led to a positive chronotropic effect. The beating rate of T3-treated cells was 244+/-19 beats/min and for control cells it was 122+/-10 beats/min (P<0.05). Action potentials were recorded and showed that the predominant effect of T3 was to increase the diastolic depolarization rate (99.5+/-9.8 in T3-treated group v 44.0+/-7.8 mV/s in untreated group). Some cells that exhibited pacemaker activity lacked a pacemaker current (I(f)) under voltage clamp conditions I(f)was recorded in 5 of 12 spontaneously active control cells and in 6 of 10 T3-treated cells. In those cells exhibiting the pacemaker current, the I(f)density was significantly larger in T3-treated cells (-7.9+/-2.6 pA/pF v-1.8+/-0.5 pA/pF in control). The L-type Ca2+ current density was similar in the two groups (at -7 mV, -7.5+/-1.5 in treated group v-8.6+/-1.0 pA/pF in control). In the presence of T3, the Na+-Ca2+ exchanger current (I(Na/Ca)) density was larger (e.g. at +60 mV, it was 4.8+/-0.5 v 3.5+/-0.2 pA/pF in control cells, P<0.05). As intracellular Ca2+ is extruded from the cell, the electrogenic Na+-Ca2+ exchanger causes a declining inward current, which may contribute to the pacemaker potential-this declining inward current was demonstrated using the action potential voltage clamp technique and was shown to be larger in T3-treated myocytes. Our data demonstrate that thyroid hormone enhances pacemaker activity and that this may be due in part to an increased Na+-Ca2+ exchanger activity.

Abnormal right coronary artery flow and multiple right ventricular myocardial infarctions associated with severe right ventricular systolic hypertension.

The impact of left ventricular hypertension on coronary flow patterns in adult patients has been well described. In contrast, few reports exist regarding the association of right coronary flow abnormalities with right ventricular hypertension. The observation of myocardial infarcts, right ventricular hypertension, and abnormal coronary flow pattern-as well as its normalization with relief of right ventricular hypertension-lends support to a causal relation between ventricular hypertension and coronary flow abnormalities.

Toxic exposures.

Adolescence is a time of immense physical and social change. Risk-taking behavior is commonplace and often includes experimentation with various illicit drugs. Toxic ingestions remain a significant medical problem in this population. The major objective of this review is to provide an overview of the more common toxic exposures in adolescents. A framework is presented for understanding some of the factors that result in substance abuse in adolescents. Signs, symptoms and clinical manifestations of commonly abused drugs are presented along with general approaches to management of acute overdose. As the population of adolescents with underlying heart disease continues to grow, it is important for primary care providers to individualize treatment of adolescents with congenital or acquired heart disease. The impact of pre-existing cardiac disease is briefly reviewed.

Amiodarone inhibits cardiac ATP-sensitive potassium channels.

INTRODUCTION: ATP-sensitive K+ channels (K(ATP)) are expressed abundantly in cardiovascular tissues. Blocking this channel in experimental models of ischemia can reduce arrhythmias. We investigated the acute effects of amiodarone on the activity of cardiac sarcolemmal K(ATP) channels and their sensitivity to ATP. METHODS AND RESULTS: Single K(ATP) channel activity was recorded using inside-out patches from rat ventricular myocytes (symmetric 140 mM K+ solutions and a pipette potential of +40 mV). Amiodarone inhibited K(ATP) channel activity in a concentration-dependent manner. After 60 seconds of exposure to amiodarone, the fraction of mean patch current relative to baseline current was 1.0 +/- 0.05 (n = 4), 0.8 +/- 0.07 (n = 4), 0.6 +/- 0.07 (n = 5), and 0.2 +/- 0.05 (n = 7) with 0, 0.1, 1.0, or 10 microM amiodarone, respectively (IC50 = 2.3 microM). ATP sensitivity was greater in the presence of amiodarone (EC50 = 13 +/- 0.2 microM in the presence of 10 microM amiodarone vs 43 +/- 0.1 microM in controls, n = 5; P < 0.05). Kinetic analysis showed that open and short closed intervals (bursting activity) were unchanged by 1 microM amiodarone, whereas interburst closed intervals were prolonged. Amiodarone also inhibited whole cell K(ATP) channel current (activated by 100 microM bimakalim). After a 10-minute application of amiodarone (10 microM), relative current was 0.71 +/- 0.03 vs 0.92 +/- 0.09 in control (P < 0.03). CONCLUSION: Amiodarone rapidly inhibited K(ATP) channel activity by both promoting channel closure and increasing ATP sensitivity. These actions may contribute to the antiarrhythmic properties of amiodarone.

Cellular basis for age-related differences in cardiac excitation-contraction coupling.

Clinical experience indicates that infants and young children respond to a variety of cardiovascular pharmacological and physiological interventions differently than adults. What is less clear, however, are the cellular and molecular mechanisms that contribute to these age-related differences. Based largely upon results from animal models, it is apparent that developmental changes occur in numerous pathways and proteins involved in the regulation of contractile function and in the determinants of inotropic responsiveness. The purposes of this review are to provide a brief overview of cardiac excitation-contraction and to illustrate some of the important age-related differences in the mechanisms involved in calcium regulation in the heart. This scientific foundation may help to explain certain clinical observations in the very young. Furthermore, it is hoped that a better understanding of the fundamental processes involved in controlling cardiac contractile function will stimulate additional research in the search for more specific, rational and age-appropriate cardiovascular therapeutics.

Overview of the management of pediatric heart failure.

For the most part of this the century heart failure syndrome was understood as a pump failure disorder with hemodynamic consequences stemming from the same myocardial dysfunction. In addition supply and demand theories were used to explain the nature of symptoms. As a result, therapeutic strategies were directed at correcting the abnormal hemodynamic conditions and normalizing the delivery of the much needed nutrients. Improvement of cardiac pump function with inotropic drugs and abnormal circulatory conditions with afterload and preload modifications became therapeutic goals and standards of care. However, while vasodilators and inotropic drugs immediately improved symptoms, hemodynamics and functional status, in the long term they either did not affect or worsen the natural history of heart failure. In pediatrics, this is further complicated by the lack of large scale trials addressing issues pertinent to the particularities that affect heart failure in children. In the late 1980s and 1990s heart failure has evolved into a more complex, multiple and interactive pathophysiologic disorder. Today not only the abnormal hemodynamics but also the biological disorders are pharmacologic targets. The reversal or slowing of myocardial maladaptation has become one of the most important therapeutic goals. With this end in mind therapeutic strategies may seem counterintuitive and paradoxical, such as the use of beta-blockers. This review will address the current thinking and therapeutic modalities used today in the treatment of heart failure syndrome in the adult population. We also discuss some of the issues why we think that these principles can be extrapolated to the pediatric population.

Impact of heparin bonding on pediatric cardiopulmonary bypass: a prospective randomized study.

BACKGROUND: Heparin-coated circuits reduce the inflammatory response to cardiopulmonary bypass in adult patients; however, little is known about its effects in the pediatric population. Two studies were performed to assess this technology's impact on inflammation and clinical outcomes. METHODS: In a pilot study, complement and interleukins were measured in 19 patients who had either uncoated cardiopulmonary bypass circuits or heparin-bonded circuits. Subsequently, 23 additional patients were studied in a randomized fashion. Respiratory function and blood product utilization were recorded. RESULTS: In the pilot study, heparin-bonded circuit patients had less complement 3a (p < 0.001) and interleukin-8 (p < 0.05) compared with uncoated cardiopulmonary bypass circuit patients. The randomized study revealed that the heparin-bonded circuit was associated with reduced complement 3a (p = 0.02). Multiple variable analysis revealed that the following postoperative variables were increased with bypass time (p = 0.01) and diminished with heparin-bonded circuits: interleukins (p = 0.01), peak airway pressures (p = 0.05), and prothrombin time (p = 0.03). CONCLUSIONS: Heparin-bonded circuits significantly reduce cytokines and complement during cardiopulmonary bypass and lower interleukin levels postbypass; they were also associated with improved pulmonary and coagulation function. Heparin-bonded circuits ameliorate the systemic inflammatory response in pediatric patients from cardiopulmonary bypass.

Intraoperative myocardial ischemia recognized by transesophageal echocardiography monitoring in the pediatric population: a report of 3 cases.

We used continuous intraoperative transesophageal echocardiography (TEE) monitoring to detect intraoperative myocardial ischemia in children after they had been weaned from cardiopulmonary bypass for cardiac surgery. Three pediatric patients are described here to illustrate the usefulness of such TEE monitoring in surgical procedures involving coronary arteries. The indications for intraoperative TEE monitoring and a simplified scheme for immediate qualitative interpretation are discussed.

Effects of thyroid hormone on action potential and repolarizing currents in rat ventricular myocytes.

Thyroid hormones play an important role in cardiac electrophysiology through both genomic and nongenomic mechanisms of action. The effects of triiodothyronine (T(3)) on the electrophysiological properties of ventricular myocytes isolated from euthyroid and hypothyroid rats were studied using whole cell patch clamp techniques. Hypothyroid ventricular myocytes showed significantly prolonged action potential duration (APD(90)) compared with euthyroid myocytes, APD(90) of 151 +/- 5 vs. 51 +/- 8 ms, respectively. Treatment of hypothyroid ventricular myocytes with T(3) (0.1 microM) for 5 min significantly shortened APD by 24% to 115 +/- 10 ms. T(3) similarly shortened APD in euthyroid ventricular myocytes, but only in the presence of 4-aminopyridine (4-AP), an inhibitor of the transient outward current (I(to)), which prolonged the APD by threefold. Transient outward current (I(to)) was not affected by the acute application of T(3) to either euthyroid or hypothyroid myocytes; however, I(to) density was significantly reduced in hypothyroid compared with euthyroid ventricular myocytes.

Subcellular [Ca2+]i gradients during excitation-contraction coupling in newborn rabbit ventricular myocytes.

The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes.

The role of Kir2.1 in the genesis of native cardiac inward-rectifier K+ currents during pre- and postnatal development.

Our results demonstrate that (a) the Kir2.1 gene encodes a native K+ channel protein with a 21-pS conductance; (b) this channel has an important role in the genesis of adult ventricular 1K1; and (c) the contribution of Kir2.1 channel proteins to 1K1 changes during development. The lack of contribution of Kir2.1 to fetal 1K1 channels is interesting from the point of view of possible future generation of knockout mice lacking Kir2.1, since cardiac abnormalities would not be expected to result in fetal lethality. These observations provide further support for a generalized hypothesis that different genes may code for 1K1 channel proteins at various developmental stages. However, the effects of these AS-oligos must first be examined on native 1K1 channels in cardiac myocytes before definite conclusions can be reached.

Identification and cloning of TWIK-originated similarity sequence (TOSS): a novel human 2-pore K+ channel principal subunit.

We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK-originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK-originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK-originated similarity sequence shares a low (< 30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK-1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue-specific expression patterns. Expression of TWIK-originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK-originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK-originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK-originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit.

Noninvasive, in utero imaging of mouse embryonic heart development with 40-MHz echocardiography.

BACKGROUND: The increasing number of transgenic and targeted mutant mice with embryonic cardiac defects has resulted in the need for noninvasive techniques to examine cardiac structure and function in early mouse embryos. We report the first use of a novel 40-MHz ultrasound imaging system in the study of mouse cardiac development in utero. METHODS AND RESULTS: Transabdominal scans of mouse embryos staged between 8.5 and 13.5 days of gestation (E8.5 to E13.5) were obtained in anesthetized mice. Atrial and ventricular contractions could be discerned from E9.5, and changes in cardiac morphology were observed from E9.5 to E13.5. Hyperechoic streaming patterns delineated flow through the umbilical, vitelline, and other major blood vessels. Diastolic and systolic ventricular areas were determined by planimetry of the epicardial borders, and fractional area change was measured as an index of contractile function. Significant increases in ventricular size were documented at each stage between E10.5 and E13.5, and the ability to perform serial imaging studies over 3 days of embryonic development is described. Finally, the detection of vascular cell adhesion molecule 1 (VCAM-1) homozygous null mutant embryos demonstrates the first example of noninvasive, in utero analysis of cardiac structure and function in a targeted mouse mutant. CONCLUSIONS: We used 40-MHz echocardiography to identify key elements of the early mouse embryonic cardiovascular system and for noninvasive dimensional analysis of developing cardiac ventricles. The ability to perform serial measurements and to detect mutant embryos with cardiac defects highlights the usefulness of the technique for investigating normal and abnormal cardiovascular development.

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes.

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth.