Efficacy of the stand-alone Cox-Maze IV procedure in patients with longstanding persistent atrial fibrillation.

INTRODUCTION: Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, and results in significant morbidity and mortality. The Cox-Maze IV procedure (CMP-IV) has been shown to have excellent efficacy in returning patients to sinus rhythm, but there have been few reports of late follow-up in sizable cohorts of patients with longstanding persistent AF, the most difficult type of AF to treat. METHODS AND RESULTS: Between May 2003 and March 2020, 174 consecutive patients underwent a stand-alone CMP-IV for longstanding persistent AF. Rhythm outcome was assessed postoperatively for up to 10 years, primarily via prolonged monitoring (Holter monitor, pacemaker interrogation, or implantable loop recorder). Fine-Gray regression was used to investigate factors associated with atrial tachyarrhythmia (ATA) recurrence, with death as a competing risk. Median duration of preoperative AF was 7.8 years (interquartile range: 4.0-12.0 years), with 71% (124/174) having failed at least one prior catheter-based ablation. There were no 30-day mortalities. Freedom from ATAs was 94% (120/128), 83% (53/64), and 88% (35/40) at 1, 5, and 7 years, respectively. On regression analysis, preoperative AF duration and early postoperative ATAs were associated with late ATAs recurrence. CONCLUSION: Despite the majority of patients having a long-duration of preoperative AF and having failed at least one catheter-based ablation, the stand-alone CMP-IV had excellent late efficacy in patients with longstanding persistent AF, with low morbidity and no mortality. We recommend consideration of stand-alone CMP-IV for patients with longstanding persistent AF who have failed or are poor candidates for catheter ablation.

Update on stem cell technologies in congenital heart disease.

BACKGROUND: Congenital heart disease (CHD) continues to be among the most common birth defects, affecting an estimated 40 000 births annually in the United States. The most common complication of CHD is heart failure. With improved medical management and surgical outcomes, survival for complex congenital heart defects has dramatically improved, but consequentially there are more adults with CHD than children with CHD. Due to longer-term sequelae of CHD, surgical and medical treatment previously thought to be curative is now realized at best to be palliative, and there is a considerable burden of CHD-related heart failure. Stem cell therapy as an adjunct to current surgical and medical strategies is being explored in an effort to ameliorate CHD-related heart failure. This review aims to explore the current literature with regard to stem cell therapy for CHD as well as ongoing trials. METHODS: A MEDLINE (Ovid), MEDLINE (Pubmed), and clinicaltrials.gov search were performed using the medical subject headings congenital heart defects combined with hematopoietic stem cells, stem cell transplantation, mesenchymal stem cells (MSC), cell- or tissue-based therapy, or MSC transplantation. Articles must have been published after 2010. RESULTS: Twenty three articles and 9 ongoing trials met all inclusion criteria. CONCLUSIONS: Areas of interest include myocardiocyte regeneration, tissue graft development to minimize reoperations, and methods of stem cell delivery. While several small trials are showing promise, it is too soon to make definitive statements about the future of stem cell therapies in this field.

Extraction of high-quality RNA from human articular cartilage.

Extracting high-quality RNA from articular cartilage is challenging due to low cellularity and high proteoglycan content. This problem hinders efficient application of RNA sequencing (RNA-seq) analysis in studying cartilage homeostasis. Here we developed a method that purifies high-quality RNA directly from cartilage. Our method optimized the collection and homogenization steps so as to minimize RNA degradation, and modified the conventional TRIzol protocol to enhance RNA purity. Cartilage RNA purified using our method has appropriate quality for RNA-seq experiments including an RNA integrity number of ∼8. Our method also proved efficient in extracting high-quality RNA from subchondral bone.

Pain management in home hospice patients: A retrospective descriptive study.

The development and evaluation of evidence-based, safe, and effective home-based pain management models for caregivers implementation is receiving greater attention in the literature because of international initiatives intended to increase the number of people who receive end-of-life care in home-based settings. The purpose of this "retrospective descriptive design" study was to describe pharmacological pain management and outcomes for 40 cancer and non-cancer patients receiving hospice care at home. While the median pain score was higher at admission in the cancer group than in the hospice care at home group, the difference was not significant at or within 48 hour of admission. Overall, there was a significant decrease in pain from the first measurement to the second. Within the last seven days of life, the majority of participants were not able to provide a pain severity score when asked to evaluate the effectiveness of pain management, thus their caregiver provided a proxy evaluation. Pain management was effective in the home setting. More research is needed on the best methods to teach lay caregivers to assess pain and evaluate the effectiveness of pharmacological modalities to manage pain.

Biological Augmentation of Flexor Tendon Repair: A Challenging Cellular Landscape.

Advances in surgical technique and rehabilitation have transformed zone II flexor tendon injuries from an inoperable no-man's land to a standard surgical procedure. Despite these advances, many patients develop substantial range of motion-limiting adhesions after primary flexor tendon repair. These suboptimal outcomes may benefit from biologic augmentation or intervention during the flexor tendon healing process. However, there is no consensus biological approach to promote satisfactory flexor tendon healing; we propose that insufficient understanding of the complex cellular milieu in the healing tendon has hindered the development of successful therapies. This article reviews recent advances in our understanding of the cellular components of flexor tendon healing and adhesion formation, including resident tendon cells, synovial sheath, macrophages, and bone marrow-derived cells. In addition, it examines molecular approaches that have been used in translational animal models to improve flexor tendon healing and gliding function, with a specific focus on progress made using murine models of healing. This information highlights the importance of understanding and potentially exploiting the heterogeneity of the cellular environment during flexor tendon healing, to define rational therapeutic approaches to improve healing outcomes.

Transcriptomes of post-mitotic neurons identify the usage of alternative pathways during adult and embryonic neuronal differentiation.

BACKGROUND: Understanding the mechanisms by which neurons are generated and specified, and how they integrate into functional circuits is key to being able to treat disorders of the nervous system and acute brain trauma. Much of what we know about neuronal differentiation has been studied in developing embryos, but differentiation steps may be very different during adult neurogenesis. For this reason, we compared the transcriptomes of newly differentiated neurons in zebrafish embryos and adults. RESULTS: Using a 4tU RNA labeling method, we isolated and sequenced mRNA specifically from cells of one day old embryos and adults expressing the transgene HA-uprt-mcherry under control of the neuronal marker elavl3. By categorizing transcript products into different protein classes, we identified similarities and differences of gene usage between adult and embryonic neuronal differentiation. We found that neurons in the adult brain and in the nervous system of one day old embryos commonly use transcription factors - some of them identical - during the differentiation process. When we directly compared adult differentiating neurons to embryonic differentiating neurons, however, we found that during adult neuronal differentiation, the expression of neuropeptides and neurotransmitter pathway genes is more common, whereas classical developmental signaling through secreted molecules like Hedgehog or Wnt are less enriched, as compared to embryonic stages. CONCLUSIONS: We conclude that both adult and embryonic differentiating neurons show enriched use of transcription factors compared to surrounding cells. However, adult and embryonic developing neurons use alternative pathways to differentiate. Our study provides evidence that adult neuronal differentiation is distinct from the better characterized embryonic neuronal differentiation process. This important insight and the lists of enriched genes we have identified will now help pave the way to a better understanding of the mechanisms of embryonic and adult neuronal differentiation and how to manipulate these processes.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex- or integrin alpha7-deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin alpha6 to reduce muscle degeneration. Taken together, these results define a novel cell adhesion pathway that may have future therapeutic relevance for a broad spectrum of muscular dystrophies.

Visual communication design: a neglected factor in nutrition promotion via packaged food labels.

Packaging design is a communication device and a critical component in branding strategy, and has relevance for food policy. Presently, packaging-related nutrition policy initiatives focus on the role of regulated claims, nutrition information panels and front-of-pack nutrition labels to help guide consumer food choices and address high prevalences of discretionary and ultra-processed food consumption in many countries. However, these nutrition labelling systems are not optimized as public health policy tools as many consumers do not use them to inform their food choices. Visual communication design theory posits that a designer orders the elements and principles of design into hierarchies that prioritize certain elements over others, and that some of these elements are more dominant and given more emphasis than others. The overall design of the package thereby directs consumer attention to some aspects of pack design (e.g., characters, contents of the package) and away from others (e.g., nutrition details). Dual processing frameworks propose that food decisions are made with the interplay between automatic and rational thinking processes. Packaging designs affect whether consumers rely predominantly on automatic or rational thinking to select a food. This narrative review outlines the role of food packaging design and how it impacts the clear communication of nutrition aspects of food products and how the use of nutrition information by consumers to make decisions may depend upon design structures in packaging. This article attests that nutrition scientists and policy makers should incorporate visual communication design into research on the food packaging as a public health promotion tool. A stronger focus on the communication of regulated front-of-pack nutrition information can be made with a re-evaluation of the hierarchy of elements in the front-of-pack design enabling consumers to make healthier decisions.

Clinical Outcomes Following Percutaneous Ankle Fusion With Bone Graft Substitute.

Background: Percutaneous ankle fusion is an emerging technique with minimal published outcome data. The goal of the present study is to retrospectively review clinical and radiographic outcomes following percutaneous ankle fusion and provide technique tips to perform percutaneous ankle fusion. Methods: Patients >18 years of age, treated by a single surgeon, from February 2018 to June 2021, who underwent primary isolated percutaneous ankle fusion supplemented with platelet-derived growth factor B (rhPDGF-BB) and beta-tricalcium phosphate, with at least 1-year follow-up were included. Surgical technique consisted of percutaneous ankle preparation followed by fixation with 3 headless compression screws. Pre- and postoperative visual analog scale (VAS) and Foot Function Index (FFI) were compared using paired t tests. Fusion was assessed radiographically by the surgeon on postoperative radiographs and computed tomography (CT) at 3 months postoperatively. Results: Twenty-seven consecutive adult patients were included in the study. Mean follow-up was 21 months. Mean age was 59.8 years. Mean preoperative and postoperative VAS scores were 7.4 and 0.2, respectively (P < .01). Mean preoperative FFI pain domain, disability domain, activity restriction domain, and total score were 20.9, 16.7, 18.5, and 56.4, respectively. Mean postoperative FFI pain domain, disability domain, activity restriction domain, and total score were 4.3, 4.7, 6.7, and 15.8, respectively (P < .01). Fusion was achieved in 26 of 27 patients (96.3%) at 3 months. Four patients (14.8%) had complications. Conclusion: We found in this cohort with surgery performed by a surgeon highly experienced in minimally invasive surgery that percutaneous ankle fusion augmented with a bone graft supplement achieved a high rate of fusion (96.3%) and a significant improvement in pain and function postoperatively while associated with minimal complications. Level of Evidence: Level IV, case series.

Extubation of Patients Receiving Vasopressor Infusions: Results of a Survey on Statewide Practices.

Current guidelines recommend extubation only if a patient is not receiving vasopressor therapy or is receiving minimal doses of vasopressors. However, recent data indicate that extubation of patients receiving higher vasopressor doses may be safe. This study was undertaken to examine practices regarding extubation of patients receiving vasopressor therapy reported by clinician respondents to a survey by the Michigan Health and Hospital Association Keystone Center. One-third of respondents indicated that they would extubate a patient receiving vasopressors, and one-quarter indicated that it depended on the agent used, but more than half reported that their unit did not have a vasopressor use protocol or they did not know whether it did. Practices regarding extubation of patients receiving vasopressor therapy differed significantly by unit type and by role as a direct care provider. These data indicate that patient and clinician factors may drive practice patterns. Additional research to inform guidelines and local protocols is warranted.

An experimental model of chronic severe mitral regurgitation.

Objective: To develop a minimally invasive, reproducible model of chronic severe mitral regurgitation (MR) that replicates the clinical phenotype of left atrial (LA) and left ventricular dilation and susceptibility to atrial fibrillation. Methods: Under transesophageal echocardiographic guidance, chordae tendinae were avulsed using endovascular forceps until the ratio of regurgitant jet area to LA area was ≥70%. Animals survived for an average of 8.6 ± 1.6 months (standard deviation) and imaged with monthly transthoracic echocardiography (TTE). Animals underwent baseline and preterminal magnetic resonance imaging. Terminal studies included TTE, transesophageal echocardiography, and rapid atrial pacing to test inducibility of atrial tachyarrhythmias. Results: Eight dogs underwent creation of severe MR and interval monitoring. Two were excluded-one died from acute heart failure, and the other had resolution of MR. Six dogs underwent the full experimental protocol; only one required medical management of clinical heart failure. MR remained severe over time, with a mean terminal regurgitant jet area to LA area of 71 ± 14% (standard deviation) and regurgitant fraction of 52 ± 11%. Mean LA volume increased over 130% (TTE: 163 ± 147%, P = .039; magnetic resonance imaging: 132 ± 54%, P = .011). Mean left ventricular end-diastolic volume increased by 38 ± 21% (P = .008). Inducible atrial tachyarrhythmias were seen in 4 of 6 animals at terminal surgery, and none at baseline. Conclusions: Within the 6 dogs that successfully completed the full experimental protocol, this model replicated the clinical phenotype of severe MR, which led to marked structural and electrophysiologic cardiac remodeling. This model allowed for precise measurements at repeated time points and will facilitate future studies to elucidate the mechanisms of atrial and ventricular remodeling secondary to MR and the pathophysiology of valvular atrial fibrillation.

Pregnant women's knowledge, practices, and needs related to food safety and listeriosis: a study in British Columbia.

OBJECTIVE: To understand the knowledge, attitudes, practices, and needs of pregnant women regarding food safety, including the risk of listeriosis, in order to develop targeted messages and educational resources in British Columbia (BC). DESIGN: Qualitative study using focus groups and quantitative study using a standardized questionnaire. SETTING: Seven family practice clinics in BC. Focus groups were conducted in 3 program groups for new mothers. PARTICIPANTS: Pregnant women and women who had recently delivered babies. METHODS: Three focus groups were conducted with women who had recently delivered. Qualitative analysis to identify common themes was conducted. A questionnaire was completed by pregnant women at their health care providers' (HCPs') offices. Statistical analysis was done to assess associations between demographic features, knowledge, and practices. Results from both study methods were compared and common findings were presented. MAIN FINDINGS: Participants reported that food safety and the risk of listeriosis were important to them during pregnancy; however, their knowledge of high-risk foods and safe food practices was limited. Although they identified their HCPs as a valuable source of information, they explained there were barriers to getting information from them. Participants reported doing their own research using books, websites, and social networks. They made recommendations to improve food safety messages, as well as the availability and format of resources. CONCLUSION: Women in BC identified a gap between the information on food safety and listeriosis that they needed during pregnancy and the resources that were available. Using the information collected from this study, resources that are targeted at women of childbearing years, as well as their HCPs, are under development in BC.

Ligand-Directed GPCR Antibody Discovery.

Developing affinity reagents recognizing and modulating G-protein coupled receptors (GPCR) function by traditional animal immunization or in vitro screening methods is challenging. Some anti-GPCR antibodies exist on the market, but the success rate of development is still poor compared with antibodies targeting soluble or peripherally anchored proteins. More importantly, most of these antibodies do not modulate GPCR function. The current pipeline for antibody development primarily screens for overall affinity rather than functional epitope recognition. We developed a new strategy utilizing natural ligand affinity to generate a library of antibody variants with an inherent bias toward the active site of the GPCR. Instead of using phage libraries displaying antibodies with random CDR sequences at polymorphism sites observed in natural immune repertoire sequences, we generated focused antibody libraries with a natural ligand encoded within or conjugated to one of the CDRs or the N-terminus. To tailor antibody binding to the active site, we limited the sequence randomization of the antibody in regions holstering the ligand while leaving the ligand-carrying part unaltered in the first round of randomization. With hits from the successful first round, the second round of randomization of the ligand-carrying part was then performed to eliminate the bias of the ligand. Based on our results on three different GPCR targets, the proposed pipeline will enable the rapid generation of functional antibodies (both agonists and antagonists) against high-value targets with poor function epitope exposures including GPCR, channels, transporters as well as cell surface targets whose binding site is heavily masked by glycosylation.

Impact of aortic valve effective height following valve-sparing root replacement on postoperative insufficiency and reoperation.

BACKGROUND: This study evaluated the impact of anatomic aortic root parameters during valve-sparing root replacement on the probability of postoperative aortic insufficiency and freedom from aortic valve reoperation. METHODS: From 1995 to 2020, 177 patients underwent valve-sparing root replacement (163 reimplantations, 14 remodeling). Preoperative and postoperative echocardiograms were analyzed to measure annulus and sinus diameters, effective height of leaflet coaptation, and degree of aortic insufficiency. Logistic regression was used to evaluate predictors of 2+ or greater late postoperative aortic insufficiency. Fine-Gray regression determined predictors for aortic valve reintervention. RESULTS: The study population included 122 (69%) men with a mean age of 43 ± 15 years. A total of 119 patients (67%) had an identified connective tissue disorder. The cumulative incidence of aortic valve reoperation was estimated as 7% at 5 years and 12% at 10 years. The probability of 2+ or greater late postoperative aortic insufficiency was inversely related to effective height during valve-sparing root replacement (P = .018). As postoperative effective height fell below 11 mm, the probability of 2+ or greater aortic insufficiency exceeded 10%. On multivariable logistic regression, effective height (odds ratio, 0.53; 0.33-0.86; P = .010), preoperative annulus diameter (odds ratio, 1.44; 1.13-1.82; P = .003), and degree of preoperative aortic insufficiency (odds ratio, 2.57; 1.45-4.52; P = .001) were associated with increased incidence of 2+ or greater late postoperative aortic insufficiency. On multivariable Fine-Gray regression, risk factors for aortic valve reintervention included preoperative annulus diameter (subdistribution hazard ratio, 1.28 [1.03-1.59], P = .027), history of 3+ or greater aortic insufficiency (subdistribution hazard ratio, 4.28; 1.60-11.44; P = .004), and 2+ or greater early postoperative aortic insufficiency (subdistribution hazard ratio, 5.22; 2.29-11.90; P < .001). CONCLUSIONS: Measures to increase effective height during valve-sparing root replacement may decrease the risk of more than mild postoperative aortic insufficiency after repair and the need for aortic valve reoperation.

Concomitant surgical ablation for atrial fibrillation is associated with increased risk of acute kidney injury but improved late survival.

BACKGROUND: Acute kidney injury (AKI) after cardiac surgery remains a common complication that has been associated with increased morbidity and mortality. This study implemented Kidney Disease Improving Global Outcomes criteria to evaluate renal outcomes after concomitant surgical ablation for atrial fibrillation. METHODS: Patients with a history of atrial fibrillation who underwent elective cardiac surgery at our institution from 2008 to 2018 were retrospectively reviewed. Those with preoperative renal dysfunction were excluded. Patients were classified as those who underwent concomitant Cox-Maze IV (CMP-IV) (n = 376) or no surgical ablation (n = 498). Nearest neighbor 1:1 propensity matching was conducted on fourteen covariates. AKI was evaluated by mixed effects logistic regression analysis. Long-term survival was evaluated by proportional hazards regression. RESULTS: Propensity matching yielded 308 patients in each group (n = 616). All preoperative variables were similar between groups. The concomitant CMP-IV group had a greater incidence of AKI: 32% (n = 99) versus 16% (n = 49), P < .001. After accounting for bypass time and nonablation operations on mixed effects analysis, concomitant CMP-IV was associated with increased risk of AKI (odds ratio, 1.89; confidence interval, 1.12-3.18; P = .017). While AKI was associated with decreased late survival (P < .001), patients who received a concomitant CMP-IV maintained superior 7-year survival to patients who received no ablation (P < .001). No patients required permanent dialysis. CONCLUSIONS: Concomitant CMP-IV was independently associated with increased risk of AKI in the acute postoperative period. However, the long-term risks of AKI were offset by the significant survival benefit of CMP-IV. Concerns regarding new-onset renal dysfunction should not prohibit recommendation of this procedure in appropriate patients.

MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.

Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed 'MILKSHAKE,' is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue's modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates.

Surgical ablation for atrial fibrillation is efficacious in patients with giant left atria.

OBJECTIVE: The Cox-Maze IV procedure (CMP-IV) is the most effective treatment for atrial fibrillation. Increased left atrial (LA) size has been identified as a risk factor for failure to restore sinus rhythm. This has biased many surgeons against ablation in patients with giant left atrium (GLA), defined as LA diameter >6.5 cm. In this study we aimed to define the efficacy of the CMP-IV in patients with GLA. METHODS: From April 2004 through March 2020, 786 patients with a documented LA diameter underwent elective CMP-IV, 72 of whom had GLA. Median follow-up duration was 4 years (interquartile range, 1-7 years). Recurrence was defined as any documented atrial tachyarrhythmia (ATA) lasting 30 seconds. ATA recurrence and survival were analyzed across GLA versus non-GLA groups. RESULTS: Median age at surgery was 65 (interquartile range, 56-73) years. Median LA diameter within the GLA group was 7.0 (range, 6.6-10.0) cm. There were no differences in rates of postoperative complications for the 2 groups, including rate of postoperative stroke and pacemaker placement (GLA 14%; non-GLA 12%; P = .682). A trend toward increased 30-day mortality in the GLA group did not reach statistical significance (GLA 6%; non-GLA 2%; P = .051). Freedom from ATAs at 5 years postoperatively was comparable for the 2 groups (GLA 82%; non-GLA 84%). CONCLUSIONS: The CMP-IV had good efficacy in patients with GLA. Our results suggest that LA diameter >6.5 cm should not preclude a patient from undergoing surgical ablation for atrial fibrillation.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not beta-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis.

Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.

Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.