Wilson W. Cui

Stac3 is a component of the excitation–contraction coupling machinery and mutated in Native American myopathy

Excitation-contraction coupling, the process that regulates contractions by skeletal muscles, transduces changes in membrane voltage by activating release of Ca2+ from internal stores to initiate muscle contraction. Defects in EC coupling are associated with muscle diseases. Here we identify Stac3 as a novel component of the EC coupling machinery. Using a zebrafish genetic screen, we generate a locomotor mutation that is mapped to stac3. We provide electrophysiological, Ca2+ imaging, immunocytochemical and biochemical evidence that Stac3 participates in excitation-contraction coupling in muscles. Furthermore, we reveal that a mutation in human STAC3 as the genetic basis of the debilitating Native American myopathy (NAM). Analysis of NAM stac3 in zebrafish shows that the NAM mutation decreases excitation-contraction coupling. These findings enhance our understanding of both excitation-contraction coupling and the pathology of myopathies.

Zebrafish relatively relaxed mutants have a ryanodine receptor defect, show slow swimming and provide a model of multi-minicore disease

Wild-type zebrafish embryos swim away in response to tactile stimulation. By contrast, relatively relaxed mutants swim slowly due to weak contractions of trunk muscles. Electrophysiological recordings from muscle showed that output from the CNS was normal in mutants, suggesting a defect in the muscle. Calcium imaging revealed that Ca2+ transients were reduced in mutant fast muscle. Immunostaining demonstrated that ryanodine and dihydropyridine receptors, which are responsible for Ca2+ release following membrane depolarization, were severely reduced at transverse-tubule/sarcoplasmic reticulum junctions in mutant fast muscle. Thus, slow swimming is caused by weak muscle contractions due to impaired excitation-contraction coupling. Indeed, most of the ryanodine receptor 1b (ryr1b) mRNA in mutants carried a nonsense mutation that was generated by aberrant splicing due to a DNA insertion in an intron of the ryr1b gene, leading to a hypomorphic condition in relatively relaxed mutants. RYR1 mutations in humans lead to a congenital myopathy, multi-minicore disease (MmD), which is defined by amorphous cores in muscle. Electron micrographs showed minicore structures in mutant fast muscles. Furthermore, following the introduction of antisense morpholino oligonucleotides that restored the normal splicing of ryr1b, swimming was recovered in mutants. These findings suggest that zebrafish relatively relaxed mutants may be useful for understanding the development and physiology of MmD.

accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1.

When wild-type zebrafish embryos are touched at 24 hours post-fertilization (hpf), they typically perform two rapid alternating coils of the tail. By contrast, accordion (acc) mutants fail to coil their tails normally but contract the bilateral trunk muscles simultaneously to shorten the trunk, resulting in a pronounced dorsal bend. Electrophysiological recordings from muscles showed that the output from the central nervous system is normal in mutants, suggesting a defect in muscles is responsible. In fact, relaxation in acc muscle is significantly slower than normal. In vivo imaging of muscle Ca2+ transients revealed that cytosolic Ca2+ decay was significantly slower in acc muscle. Thus, it appears that the mutant behavior is caused by a muscle relaxation defect due to the impairment of Ca2+ re-uptake. Indeed, acc mutants carry a mutation in atp2a1 gene that encodes the sarco(endo)plasmic reticulum Ca2+-ATPase 1 (SERCA1), a Ca2+ pump found in the muscle sarcoplasmic reticulum (SR) that is responsible for pumping Ca2+ from the cytosol back to the SR. As SERCA1 mutations in humans lead to Brody disease, an exercise-induced muscle relaxation disorder, zebrafish accordion mutants could be a useful animal model for this condition.

TRPM7 Is Required within Zebrafish Sensory Neurons for the Activation of Touch-Evoked Escape Behaviors

Mutations in the gene encoding TRPM7 (trpm7), a member of the Transient Receptor Potential (TRP) superfamily of cation channels that possesses an enzymatically active kinase at its C terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations that abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons, we found that TRPM7s kinase activity and selectivity for divalent cations over monovalent cations were dispensable for touch-evoked activation of escape behaviors in zebrafish. Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively, these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses.

touché Is Required for Touch-Evoked Generator Potentials within Vertebrate Sensory Neurons

The process by which light touch in vertebrates is transformed into an electrical response in cutaneous mechanosensitive neurons is a largely unresolved question. To address this question we undertook a forward genetic screen in zebrafish (Danio rerio) to identify mutants exhibiting abnormal touch-evoked behaviors, despite the presence of sensory neurons and peripheral neurites. One family, subsequently named touché, was found to harbor a recessive mutation which produced offspring that were unresponsive to light touch, but responded to a variety of other sensory stimuli. The optogenetic activation of motor behaviors by touché mutant sensory neurons expressing channelrhodopsin-2 suggested that the synaptic output of sensory neurons was intact, consistent with a defect in sensory neuron activation. To explore sensory neuron activation we developed an in vivo preparation permitting the precise placement of a combined electrical and tactile stimulating probe upon eGFP-positive peripheral neurites. In wild-type larva electrical and tactile stimulation of peripheral neurites produced action potentials detectable within the cell body. In a subset of these sensory neurons an underlying generator potential could be observed in response to subthreshold tactile stimuli. A closer examination revealed that the amplitude of the generator potential was proportional to the stimulus amplitude. When assayed touché mutant sensory neurons also responded to electrical stimulation of peripheral neurites similar to wild-type larvae, however tactile stimulation of these neurites failed to uncover a subset of sensory neurons possessing generator potentials. These findings suggest that touché is required for generator potentials, and that cutaneous mechanoreceptors with generator potentials are necessary for responsiveness to light touch in zebrafish.

NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non‐active (navmi89 and navmi130). nav embryos displayed diminished spontaneous and touch‐evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding NaV1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in NaV1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild‐type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In‐vivo electrophysiological analysis of the touch‐evoked escape circuit indicated that voltage‐dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that NaV1.6a was required for touch‐induced activation of the swim locomotor network.

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish

Background: The existence of gap junctions in differentiated skeletal muscles has been recently appreciated in vertebrates. Results: Connexin 39.9-mediated gap junctions in slow-twitch muscles are necessary for robust activation of muscle in zebrafish. Conclusion: Gap junction-mediated electrical coupling in skeletal muscle plays an essential role in coordinated behavior. Significance: Gap junctions ensure robust muscle activation despite unreliable neural outputs during early motor development. In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca2+ transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers.

Pharmacologic approaches to weaning from cardiopulmonary bypass and extracorporeal membrane oxygenation

Cardiopulmonary bypass (CBP) and extracorporeal membrane oxygenation (ECMO) are two modalities of mechanical circulatory support. They provide hemodynamic stability for patients undergoing invasive cardiothoracic interventions, and they can be lifesaving in emergencies resulting from cardiogenic shock or respiratory failure. Unlike implantable ventricular assist devices, CPB and ECMO are short-term solutions meant to last from hours to days, and the patient will need to be weaned from the mechanical support once the intervention has completed or when the underlying condition has improved. Weaning imposes major physiological strain upon the recovering cardiovascular and pulmonary systems, and it usually requires pharmacological support. This article focuses on the proper diagnosis of the underlying pathophysiology, an understanding of the pharmacology of available agents, and a rational approach to the management of patients weaning from CPB and ECMO.

Aorta-right atrial tunnel in an elderly patient.

December 2013 • Volume 117 • Number 6 A 71-year-old man with decompensated heart failure due to mitral regurgitation, tricuspid regurgitation, and aortic insufficiency was admitted before his valvular surgery. Preoperative coronary angiography revealed a vessel that was near the right coronary artery (RCA) ostium (Fig. 1). Magnetic resonance imaging showed a tortuous vessel that abutted the RCA anteriorly, coursed posteriorly, and terminated near the pulmonary artery. It was thought to be an arteriovenous fistula originating from the sinus of Valsalva. Intraoperative transesophageal echocardiography (TEE) confirmed the diagnosis of incompetent mitral, tricuspid, and aortic valves. In addition, in the midesophageal shortand long-axis views of the aortic valve, there was a large vessel exiting from the RCA ostium (Fig. 2A; Video 1, see Supplemental Digital Content 1, http://links.lww.com/ AA/A594). The modified bicaval view showed a large continuous jet entering the right atrium (RA) near the superior vena cava (SVC) (Fig. 2B; Video 2, see Supplemental Digital Content 2, http://links.lww.com/AA/A595). The continuous flow was due to the constant pressure gradient between the aortic root and the RA, which differed from the tricuspid regurgitation that only occurred during systole. The proximity of the aortic root and RA suggested a fistula, possibly due to a ruptured sinus of Valsalva aneurysm. However, withdrawing the TEE probe slightly while keeping a shortaxis view of the ascending aorta detected color flow Doppler signal in the extra-cardiac space between the aorta and RA (Fig. 3A). Withdrawing further, a tubular structure became apparent (Fig. 3B). After surgical exposure, a vessel was found originating from a dilated ostium that was shared by the RCA. It coursed rightward, before turning posteriorly under the RA appendage and entering the RA near the SVC, consistent with a diagnosis of aorta-right atrial tunnel. The resection of the tunnel, patch closure of its RA opening, mitral and aortic replacement, tricuspid annuloplasty, and reimplantation of RCA were successfully performed on cardiopulmonary bypass. Aorta-right atrial tunnel is a rare congenital condition. Its incidence is unknown, because most patients are asymptomatic with only a continuous murmur on examination. Symptoms, when present, are usually due to significant left-to-right shunt, pulmonary overcirculation, arrhythmia, and heart failure due to volume overload.1 A review of published reports showed that patients may present at any age, from neonates2 to young adults.1 Our patient was unusual in his advanced age at presentation because there is only 1 previous report of aorta-right atrial tunnel in a septuagenarian.3 Furthermore, in this case, the tunnel was undiagnosed despite a previous transthoracic echocardiogram. A number of conditions can lead to an abnormal blood return to the RA (Table 1). Coronary-cameral fistula occurs when an aberrant branch of coronary artery drains directly into one of the cardiac chambers causing ischemia and heart failure, usually in utero. Adults with aneurysmal coronary arteries have also been described, either causing external compression of the heart4 or forming a fistula that drains into the RA.5 In our patient, the RCA was of normal caliber and followed a normal course. However, we cannot dismiss the possibility that this was a very proximal fistula originated at the RCA ostium. Also in older patients, a sinus of Valsalva aneurysm and subsequent rupture into the RA is more common than aorta-right atrial tunnel. One institution reported 9 cases of aorta-right atrial tunnel compared with 66 cases of ruptured aneurysm during a 10-year period.1 The key distinction between the 2 is the extra-cardiac location of aorta-right atrial tunnel and its tortuous course. Other causes include trauma, endocarditis, and complications from surgeries involving the aortic valve, ascending aorta, and ventricular and atrial septal defects. A fistula between a saphenous vein graft and RA

23. Are persistent changes in SEPs and MEPs sensitive for predicting postoperative limb motor deficit during cerebral aneurysm surgery

Introduction Motor evoked potentials (MEPs) can be used as an adjunct to somatosensory evoked potentials (SEPs) to identify brain ischemia during surgical treatment of cerebral aneurysms. Persistent changes (failure to return to baseline by end of surgery) can be used to predict postoperative deficit. The sensitivity, specificity, positive and negative predictive value of persistent changes in SEPs/MEPs for predicting neurologic deficit is still being defined. In a separate study, the sensitivity of persistent changes in SEPs for predicting new limb paresis was 13%, and MEPs was 32%. In this study we evaluated those patients with new postoperative motor deficits to determine the reason for the low sensitivity. Material and methods This was a retrospective study based on 446 cerebral aneurysm patients who underwent clipping at our institution from 2009–2011. Twenty-eight patients with new postoperative motor deficits were identified. The neuromonitoring reports and medical records were reviewed in more detail in these patients. Results In the 28 patients, ten patients (36%, sensitivity) were predicted by persistent SEPs/MEPs changes. In the 18 patients whose deficits were not predicted, five patients (18%) had delayed deficits; eight (29%) had mild temporary motor deficits; in two patients (7%), monitoring was stopped less than ten minutes after aneurysms were clipped; in one patient (3.6%), no reliable SEPs were recorded on the surgery side at baseline. If the last three cases are eliminated, the sensitivity of predicting for all deficit at time when patient wake up is 50% (10/20 patients); to severe and persistent motor deficit is 83% (10/12 patients). Conclusion This study demonstrates that the reason for the low sensitivity of SEPs/MEPs is that these methods are not sensitive to delayed and/or mild temporary deficits. However, the study also shows that SEPs/MEPs have better predictability in cases of severe and persistent postoperative limb paresis.