Adeno-associated virus serotype 9 antibody seroprevalence for patients in the United States with spinal muscular atrophy.

Onasemnogene abeparvovec is a recombinant adeno-associated virus serotype 9 (AAV9) vector-based gene therapy for spinal muscular atrophy (SMA). Patients with elevated titers of anti-AAV9 antibodies (AAV9-Ab) should not receive onasemnogene abeparvovec because of potential safety and efficacy implications. We conducted a retrospective study to describe the seroprevalence of anti-AAV9 binding antibodies for pediatric patients with SMA in the United States. At initial testing, 13.0% (115 of 882) of patients (mean [SD] age, 26.29 [33.66] weeks) had elevated AAV9-Ab titers. The prevalence of elevated titers decreased as age increased, with 18.2% (92 of 507) of patients ≤3 months old but only 1.1% (1 of 92) of patients ≥21 months old having elevated titers. This suggests transplacental maternal transfer of antibodies. No patterns of geographic variations in AAV9-Ab prevalence were confirmed. Elevated AAV9-Ab titers in children <6 weeks old decreased in all circumstances. Lower magnitudes of elevated titers declined more rapidly than greater magnitudes. Retesting was completed at the discretion of the treating clinician, so age at testing and time between tests varied. AAV9-Ab retesting should be considered when patients have elevated titers, and elevations at a young age are not a deterrent to eventual onasemnogene abeparvovec administration. Early disease-modifying treatment for SMA leads to optimal outcomes.

NFATc3-dependent loss of I(to) gradient across the left ventricular wall during chronic beta adrenergic stimulation.

In heart, pore-forming Kv4 alpha channel subunits underlie the K(+) transient outward current (I(to)). Expression of Kv4 is greater in left ventricular epicardial (EPI) than in endocardial (ENDO) cells, resulting in larger I(to) in EPI than in ENDO cells. In adult ventricular myocytes, the transcription factor NFATc3 suppresses Kv4 expression. NFATc3 activity is higher in ENDO than in EPI cells and this has been proposed to contribute to heterogeneous Kv4 expression across the left ventricular free wall. Here, we tested the hypothesis that regional activation of NFATc3 signaling dissipates the gradient of I(to) density across the mouse left ventricle during chronic activation of beta adrenergic signaling. [Ca(2+)](i), calcineurin, and NFAT activity were larger in ENDO than in EPI myocytes. Infusion of the beta adrenergic receptor agonist isoproterenol increased [Ca(2+)](i), calcineurin, and NFAT activity in EPI, but not in ENDO myocytes, leading to equalization of these parameters in EPI and ENDO cells. This was accompanied by dissipation of the transmural gradient in Kv4.2 expression and I(to) density. Unlike wild type, ENDO or EPI myocytes from beta1 adrenergic receptor-null and NFATc3-null mice did not undergo changes in I(to) density during isoproterenol infusion. Collectively, these data suggest that calcineurin and NFATc3 signaling contributes to the loss of heterogeneous Kv4 expression, and hence I(to) density, in the mouse left ventricle during chronic beta adrenergic stimulation.

Differential calcineurin/NFATc3 activity contributes to the Ito transmural gradient in the mouse heart.

Kv4 channels are differentially expressed across the mouse left ventricular free wall. Accordingly, the transient outward K+ current (Ito), which is produced by Kv4 channels, is greater in left ventricular epicardial (EPI) than in endocardial (ENDO) cells. However, the mechanisms underlying heterogeneous Kv4 expression in the heart are unclear. Here, we tested the hypothesis that differential [Ca2+]i and calcineurin/NFATc3 signaling in EPI and ENDO cells contributes to the gradient of Ito function in the mouse left ventricle. In support of this hypothesis, we found that [Ca2+]i, calcineurin, and NFAT activity were greater in ENDO than in EPI myocytes. However, the amplitude of Ito was the same in ENDO and EPI cells when [Ca2+]i, calcineurin, and NFAT activity were equalized. Consistent with this, we observed complete loss of Ito and Kv4 heterogeneity in NFATc3-null mice. Interestingly, Kv4.3, Kv4.2, and KChIP2 genes had different apparent thresholds for NFATc3-dependent suppression and were ordered as Kv4.3 approximately KChIP2>Kv4.2. Based on these data, we conclude that calcineurin and NFATc3 constitute a Ca(2+)-driven signaling module that contributes to the nonuniform distribution of Kv4 expression, and hence Ito function, in the mouse left ventricle.

Mechanisms underlying variations in excitation-contraction coupling across the mouse left ventricular free wall.

Ca(2+) release during excitation-contraction (EC) coupling varies across the left ventricular free wall. Here, we investigated the mechanisms underlying EC coupling differences between mouse left ventricular epicardial (Epi) and endocardial (Endo) myocytes. We found that diastolic and systolic [Ca(2+)](i) was higher in paced Endo than in Epi myocytes. Our data indicated that differences in action potential (AP) waveform between Epi and Endo cells only partially accounted for differences in [Ca(2+)](i). Rather, we found that the amplitude of the [Ca(2+)](i) transient, but not its trigger - the Ca(2+) current - was larger in Endo than in Epi cells. We also found that spontaneous Ca(2+) spark activity was about 2.8-fold higher in Endo than in Epi cells. Interestingly, ryanodine receptor type 2 (RyR2) protein expression was nearly 2-fold higher in Endo than in Epi myocytes. Finally, we observed less Na(+)-Ca(2+) exchanger function in Endo than in Epi cells, which was associated with decreased Ca(2+) efflux during the AP; this contributed to higher diastolic [Ca(2+)](i) and SR Ca(2+) in Endo than in Epi cells during pacing. We propose that transmural differences in AP waveform, SR Ca(2+) release, and Na(+)-Ca(2+) exchanger function underlie differences in [Ca(2+)](i) and EC coupling across the left ventricular free wall.

Overexpression of beta2-adrenergic receptors cAMP-dependent protein kinase phosphorylates and modulates slow delayed rectifier potassium channels expressed in murine heart: evidence for receptor/channel co-localization.

The cardiac slow delayed rectifier potassium channel (IKs), comprised of (KCNQ1) and beta (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of beta-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of 2-adrenergic receptors (beta2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hbeta2-ARs. KCNQ1 and beta2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, beta2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and beta2-ARs and that beta2-AR signaling can modulate the function of IKs channels under conditions of increased beta2-AR expression, even in the absence of exogenous beta-AR agonist.

Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death.

Mutations in ion channels involved in the generation and termination of action potentials constitute a family of molecular defects that underlie fatal cardiac arrhythmias in inherited long-QT syndrome. We report here that a loss-of-function (E1425G) mutation in ankyrin-B (also known as ankyrin 2), a member of a family of versatile membrane adapters, causes dominantly inherited type 4 long-QT cardiac arrhythmia in humans. Mice heterozygous for a null mutation in ankyrin-B are haploinsufficient and display arrhythmia similar to humans. Mutation of ankyrin-B results in disruption in the cellular organization of the sodium pump, the sodium/calcium exchanger, and inositol-1,4,5-trisphosphate receptors (all ankyrin-B-binding proteins), which reduces the targeting of these proteins to the transverse tubules as well as reducing overall protein level. Ankyrin-B mutation also leads to altered Ca2+ signalling in adult cardiomyocytes that results in extrasystoles, and provides a rationale for the arrhythmia. Thus, we identify a new mechanism for cardiac arrhythmia due to abnormal coordination of multiple functionally related ion channels and transporters.

Defective intracellular Ca(2+) signaling contributes to cardiomyopathy in Type 1 diabetic rats.

The goal of the study was to determine whether defects in intracellular Ca(2+) signaling contribute to cardiomyopathy in streptozotocin (STZ)-induced diabetic rats. Depression in cardiac systolic and diastolic function was traced from live diabetic rats to isolated individual myocytes. The depression in contraction and relaxation in myocytes was found in parallel with depression in the rise and decline of intracellular free Ca(2+) concentration ([Ca(2+)](i)). The sarcoplasmic reticulum (SR) Ca(2+) store and rates of Ca(2+) release and resequestration into SR were depressed in diabetic rat myocytes. The rate of Ca(2+) efflux via sarcolemmal Na(+)/Ca(2+) exchanger was also depressed. However, there was no change in the voltage-dependent L-type Ca(2+) channel current that triggers Ca(2+) release from the SR. The depression in SR function was associated with decreased SR Ca(2+)-ATPase and ryanodine receptor proteins and increased total and nonphosphorylated phospholamban proteins. The depression of Na(+)/Ca(2+) exchanger activity was associated with a decrease in its protein level. Thus it is concluded that defects in intracellular Ca(2+) signaling caused by alteration of expression and function of the proteins that regulate [Ca(2+)](i) contribute to cardiomyopathy in STZ-induced diabetic rats. The increase in phospholamban, decrease in Na(+)/Ca(2+) exchanger, and unchanged L-type Ca(2+) channel activity in this model of diabetic cardiomyopathy are distinct from other types of cardiomyopathy.

Termination of cardiac Ca(2+) sparks: an investigative mathematical model of calcium-induced calcium release.

A Ca(2+) spark arises when a cluster of sarcoplasmic reticulum (SR) channels (ryanodine receptors or RyRs) opens to release calcium in a locally regenerative manner. Normally triggered by Ca(2+) influx across the sarcolemmal or transverse tubule membrane neighboring the cluster, the Ca(2+) spark has been shown to be the elementary Ca(2+) signaling event of excitation-contraction coupling in heart muscle. However, the question of how the Ca(2+) spark terminates remains a central, unresolved issue. Here we present a new model, "sticky cluster," of SR Ca(2+) release that simulates Ca(2+) spark behavior and enables robust Ca(2+) spark termination. Two newly documented features of RyR behavior have been incorporated in this otherwise simple model: "coupled gating" and an opening rate that depends on SR lumenal [Ca(2+)]. Using a Monte Carlo method, local Ca(2+)-induced Ca(2+) release from clusters containing between 10 and 100 RyRs is modeled. After release is triggered, Ca(2+) flux from RyRs diffuses into the cytosol and binds to intracellular buffers and the fluorescent Ca(2+) indicator fluo-3 to produce the model Ca(2+) spark. Ca(2+) sparks generated by the sticky cluster model resemble those observed experimentally, and Ca(2+) spark duration and amplitude are largely insensitive to the number of RyRs in a cluster. As expected from heart cell investigation, the spontaneous Ca(2+) spark rate in the model increases with elevated cytosolic or SR lumenal [Ca(2+)]. Furthermore, reduction of RyR coupling leads to prolonged model Ca(2+) sparks just as treatment with FK506 lengthens Ca(2+) sparks in heart cells. This new model of Ca(2+) spark behavior provides a "proof of principle" test of a new hypothesis for Ca(2+) spark termination and reproduces critical features of Ca(2+) sparks observed experimentally.