The Popeye domain-containing 1 (POPDC1) gene encodes a plasma membrane-localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases

Using digital motion analysis, the ontogeny of the cholinergic, tachykinin and pituitary adenylate cyclaseactivating polypeptide (PACAP) control systems was studied in zebrafish Danio rerio larvae, in vivo.

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe hyperventilation and cardiophysiologic response. The vhl mutants develop polycythemia with concomitantly increased epo/epor mRNA levels and erythropoietin signaling. In situ hybridizations reveal global up-regulation of both red and white hematopoietic lin-

Cardiac activity and anaerobic metabolism were analyzed in zebrafish larvae raised under normoxia (PO(2) = 20 kPa) and under chronic hypoxia (PO(2) = 10 kPa) at three different temperatures (25, 28, and 31 degrees C). Heart rate increased with development and with temperature. Under normoxia, cardiac output increased significantly at high temperature (31 degrees C), but not at 28 or at 25 degrees C. Under chronic hypoxia, however, heart rate as well as cardiac output increased at all temperatures in larvae at about hatching time or shortly thereafter. Cardiac activity of larvae raised for 2 wk after fertilization with a reduced hemoglobin oxygen-carrying capacity in their blood (hypoxemia; due to the presence of CO or of phenylhydrazine in the incubation water) was not different from control animals. Whole body lactate content of these animals did not increase. Thus there was no indication of a stimulated anaerobic energy metabolism. The increase in cardiac activity observed during hypoxia suggests that at about hatching time receptors are present that sense hypoxic conditions, and this information can be used to induce a stimulation of convective oxygen transport to compensate for a reduction in bulk oxygen diffusion in the face of a reduced oxygen gradient between environmental water and tissues. Under normoxia, however, the PO(2) gradient between environmental water and tissues and diffusional oxygen transport assure sufficient oxygen supply even if hemoglobin oxygen transport in the blood is severely impaired. Thus, under normoxic conditions and with a normal metabolic rate of the tissues, convective oxygen transport is not required until approximately 2 wk after fertilization.

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) ␤ 1a subunit. Lack of ␤ 1a results in (i) reduced membrane expression of the pore forming DHPR ␣ 1S subunit, (ii) elimination of ␣ 1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscletype excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of ␤ 1a from rather general functions of ␤ isoforms. Zebrafish and mammalian ␤ 1a subunits quantitatively restored ␣ 1S triad targeting and charge movement as well as intracellular Ca 2؉ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal ␤ 2a as the phylogenetically closest, and the ancestral housefly ␤ M as the most distant isoform to ␤ 1a also completely recovered ␣ 1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca 2؉ transients and very limited tetrad formation compared with ␤ 1a . Consequently, larval motility was either only partially restored (␤ 2a -injected larvae) or not restored at all (␤ M ). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested ␤ subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the ␤ 1a isoform. Consequently, we postulate a model that presents ␤ 1a as an allosteric modifier of ␣ 1S conformation enabling skeletal muscle-type EC coupling. Excitation-contraction (EC)3 coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca 2ϩ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR ␣ 1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca 2ϩ influx through the DHPR (5). The release of Ca 2ϩ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca 2ϩ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming ␣ 1S subunit and auxiliary subunits ␤ 1a , ␣ 2 ␦-1, and ␥ 1 (7). While gene knock-out of t...