Pulmonary fluid shifts occur as a result of scuba diving at NASA's Neutral Buoyancy Lab.

Introduction: Pulmonary fluid shifts can occur while scuba diving. Such shifts, generally thought to be rare, may result in a life-threatening phenomenon known as immersion pulmonary edema (IPE). This study aims to better classify the normal physiology of diving using ultrasound (US) to determine if these fluid shifts occur routinely during commercial diving work at the NASA Neutral Buoyancy Laboratory (NBL). Methods: Chest US was performed on commercial divers prospectively pre- and post-dive to evaluate the presence of B-lines in a total of 12 intercostal points on the anterior, posterior, and lateral chest wall. The number of B-lines at each anatomic site was recorded and scored by two independent reviewers. An increase in the number of B-lines post-dive was considered a positive result. Results: There were 67 exposures; 39 (58%) had an increase of one or more B-lines post dive; 64% of the female exposures and 57% of the male exposures were positive for B-lines post-dive, suggesting no difference across gender (Fisher's exact; p = 0.763). After the dive, all divers remained asymptomatic. Conclusion: From our results, fluid shifts can be viewed as a normal, transient, and physiologic process in commercial divers. This correlation can be compared to the formation of low-grade venous gas emboli (VGE) from decompression that does not result in decompression sickness. Further study of US B-lines in symptomatic divers may define the utility of field US in the diagnosis and management of IPE, and help identify associated risk factors.

The Partial Pressure of Inspired Carbon Dioxide Exposure Levels in the Extravehicular Mobility Unit.

BACKGROUND: NASA has been making efforts to assess the carbon dioxide (CO2) washout capability of spacesuits using a standard CO2 sampling protocol. This study established the methodology for determining the partial pressure of inspired CO2 (PIco2) in a pressurized spacesuit. We applied the methodology to characterize PIco2 for the extravehicular mobility unit (EMU).METHODS: We suggested an automated and mathematical algorithm to find the end-tidal CO2 and the end of inspiration. We provided objective and standardized guidelines to identify acceptable breath traces, which are essential to accurate and reproducible calculation of the in-suit inhaled and exhaled partial pressure of CO2 (Pco2). The mouth guard-based method for measurement of inhaled and exhaled dry-gas Pco2 was described. We calculated all individual concentrations of PIco2 inhaled by 19 healthy subjects classified into 3 fitness groups. The transcutaneous Pco2 was monitored as a secondary measure to validate washout performance.RESULTS: Mean and standard deviation values for the data collection performance and the CO2 metrics were presented (e.g., minimum time weighted average Pco2 at suited workloads of resting, 1000, 2000, and 3000 (BTU h1) were 4.75 1.03, 8.09 1.39, 11.39 1.26, and 14.36 1.29 (mmHg s1). All CO2 metrics had a statistically significant association and all positive slopes with increasing metabolic rate. No significant differences in CO2 metrics were found between the three fitness groups.DISCUSSION: A standardized and automated methodology to calculate PIco2 exposure level is presented and applied to characterize CO2 washout in the EMU. The EMU has been operated successfully in over 400 extravehicular activities (EVAs) and is considered to provide acceptable CO2 washout performance. Results provide a basis for establishing verifiable Pco2 requirements for current and future EVA spacesuits.Kim KJ, Bekdash OS, Norcross JR, Conkin J, Garbino A, Fricker J, Young M, Abercromby AFJ. The partial pressure of inspired carbon dioxide exposure levels in the extravehicular mobility unit. Aerosp Med Hum Perform. 2020; 91(12):923931.

Oxygen exposures at NASA's Neutral Buoyancy Lab: a 20-year experience.

Astronauts training for extravehicular activity (EVA) operations can spend many hours submerged underwater in a pressurized suit, called an extravehicular mobility unit (EMU), exposed to pressures exceeding 2 atmospheres absolute (ATA). To minimize the risk of decompression sickness (DCS) a 46% nitrox mixture is used. This limits the nitrogen partial pressure, decreasing the risk of DCS. The trade-off with using a 46% nitrox mixture is the increased potential for oxygen toxicity, which can lead to severe neurologic symptoms including seizures. Suited runs, which typically expose astronauts of 0.9-1.1 ATA for longer than six hours, routinely exceed the recommendation for central nervous system oxygen toxicity limits (CNSOTL) published by the National Oceanic and Atmospheric Administration (NOAA). Fortunately, in over 50,000 hours of suited training dives spanning 20 years of EVA training operations at NASA's Neutral Buoyancy Laboratory (NBL) there has never been an occurrence of oxygen toxicity. This lends support to anecdotal sentiment among certain members of the hyperbaric community that the NOAA CNSOTL recommendations might be overly conservative, at least for the oxygen pressure and time regime in which NBL operates. The NOAA CNSOTL recommendations are the result of expert consensus with a focus on safety and do not necessarily reflect rigorous experimental evidence. The data from the NBL suited dive operations provide a foundation of evidence that can help inform the expert discussion on dive-related neurologic oxygen toxicity performance and overnight recovery in young, healthy males.

Hypertension screening of commercial divers from an occupational medicine perspective.

Hypertension is one of the most common chronic diseases in the world: in the United States, it affects one-third of the adult population. Most cases are primary hypertension - with no identified cause - and significantly increase risk for cardiovascular disease and stroke. At NASA's Neutral Buoyancy Laboratory (NBL), there is a cadre of professional divers who provide underwater support during extravehicular activity training (EVAs, commonly referred to as "spacewalks") for astronauts. The NBL occupational health program requires regular "work-site" physical exams, including blood pressure checks, on at least a weekly basis. Although some blood pressures measured during such regular checks were elevated, the diagnosis and management of blood pressure was outside the scope of that occupational medicine program. As a result, there was no protocol to analyze and manage asymptomatic elevated blood pressure if and when it warranted physician follow-up. We therefore analyzed the blood pressure data from all divers in 2015, vetted the worksite physical and developed a referral pathway to ensure divers have access to effective management of their blood pressure while minimizing impact of such a program on daily operations.

Emergency Medical Considerations in a Space-Suited Patient.

INTRODUCTION: The Stratex Project is a high altitude balloon flight that culminated in a freefall from 41,422 m (135,890 ft), breaking the record for the highest freefall to date. Crew recovery operations required an innovative approach due to the unique nature of the event as well as the equipment involved. The parachutist donned a custom space suit similar to a NASA Extravehicular Mobility Unit (EMU), with life support system mounted to the front and a parachute on the back. This space suit had a metal structure around the torso, which, in conjunction with the parachute and life support assembly, created a significant barrier to extraction from the suit in the event of a medical emergency. For this reason the Medical Support Team coordinated with the pressure suit assembly engineer team for integration, training in suit removal, definition of a priori contingency leadership on site, creation of color-coded extraction scenarios, and extraction drills with a suit mock-up that provided insight into limitations to immediate access. This paper discusses novel extraction processes and contrasts the required medical preparation for this type of equipment with the needs of the prior record-holding jump that used a different space suit with easier immediate access. Garbino A, Nusbaum DM, Buckland DM, Menon AS, Clark JB, Antonsen EL. Emergency medical considerations in a space-suited patient. Aerosp Med Hum Perform. 2016; 87(11):958-962.

Crew Recovery and Contingency Planning for a Manned Stratospheric Balloon Flight - the StratEx Program.

The StratEx program used a self-contained space suit and balloon system to loft pilot Alan Eustace to a record-breaking altitude and skydive from 135,897 feet (41,422 m). After releasing from the balloon and a stabilized freefall, the pilot safely landed using a parachute system based on a modified tandem parachute rig. A custom spacesuit provided life support using a similar system to NASA's (National Aeronautics and Space Administration; Washington, DC USA) Extravehicular Mobility Unit. It also provided tracking, communications, and connection to the parachute system. A recovery support team, including at least two medical personnel and two spacesuit technicians, was charged with reaching the pilot within five minutes of touchdown to extract him from the suit and provide treatment for any injuries. The team had to track the flight at all times, be prepared to respond in case of premature release, and to operate in any terrain. Crew recovery operations were planned and tailored to anticipate outcomes during this novel event in a systematic fashion, through scenario and risk analysis, in order to minimize the probability and impact of injury. This analysis, detailed here, helped the team configure recovery assets, refine navigation and tracking systems, develop procedures, and conduct training. An extensive period of testing and practice culminated in three manned flights leading to a successful mission and setting the record for exit altitude, distance of fall with stabilizing device, and vertical speed with a stabilizing device. During this mission, recovery teams reached the landing spot within one minute, extracted the pilot, and confirmed that he was not injured. This strategy is presented as an approach to prehospital planning and care for improved safety during crew recovery in novel, extreme events. Menon AS , Jourdan D , Nusbaum DM , Garbino A , Buckland DM , Norton S , Clark JB , Antonsen EL . Crew recovery and contingency planning for a manned stratospheric balloon flight - the StratEx program. Prehosp Disaster Med. 2016;31(5):524-531.

Emergency medical support for a manned stratospheric balloon test program.

INTRODUCTION: Red Bull Stratos was a commercial program that brought a test parachutist, protected by a full-pressure suit, in a stratospheric balloon with pressurized capsule to over 127,582 ft (38,969 m), from which he free fell and subsequently parachuted to the ground. Given that the major risks to the parachutist included ebullism, negative Gz (toe-to-head) acceleration exposure from an uncontrolled flat spin, and trauma, a comprehensive plan was developed to recover the parachutist under nominal conditions and to respond to any medical contingencies that might have arisen. In this report, the project medical team describes the experience of providing emergency medical support and crew recovery for the manned balloon flights of the program. METHODS: The phases of flight, associated risks, and available resources were systematically evaluated. RESULTS: Six distinct phases of flight from an Emergency Medical Services (EMS) standpoint were identified. A Medical Support Plan was developed to address the risks associated with each phase, encompassing personnel, equipment, procedures, and communications. DISCUSSION: Despite geographical, communications, and resource limitations, the medical team was able to implement the Medical Support Plan, enabling multiple successful manned balloon flights to 71,615 ft (21,828 m), 97,221 ft (29,610 m), and 127,582 ft (38,969 m). The experience allowed refinement of the EMS and crew recovery procedures for each successive flight and could be applied to other high altitude or commercial space ventures.

Tolerance of centrifuge-simulated suborbital spaceflight by medical condition.

INTRODUCTION: We examined responses of volunteers with known medical disease to G forces in a centrifuge to evaluate how potential commercial spaceflight participants (SFPs) might tolerate the forces of spaceflight despite significant medical history. METHODS: Volunteers were recruited based upon suitability for each of five disease categories (hypertension, cardiovascular disease, diabetes, lung disease, back or neck problems) or a control group. Subjects underwent seven centrifuge runs over 2 d. Day 1 consisted of two +G(z) runs (peak = +3.5 G(z), Run 2) and two +G(x), runs (peak = +6.0 G(x), Run 4). Day 2 consisted of three runs approximating suborbital spaceflight profiles (combined +G(x) and +G(z), peak = +6.0 G(x)/+4.0 G(z)). Data collected included blood pressure, electrocardiogram, pulse oximetry, neurovestibular exams, and post-run questionnaires regarding motion sickness, disorientation, grayout, and other symptoms. RESULTS: A total of 335 subjects registered for participation, of which 86 (63 men, 23 women, age 20-78 yr) participated in centrifuge trials. The most common causes for disqualification were weight and severe and uncontrolled medical or psychiatric disease. Five subjects voluntarily withdrew from the second day of testing: three for anxiety reasons, one for back strain, and one for time constraints. Maximum hemodynamic values recorded included HR of 192 bpm, systolic BP of 217 mmHg, and diastolic BP of 144 mmHg. Common subjective complaints included grayout (69%), nausea (20%), and chest discomfort (6%). Despite their medical history, no subject experienced significant adverse physiological responses to centrifuge profiles. DISCUSSION: These results suggest that most individuals with well-controlled medical conditions can withstand acceleration forces of launch and re-entry profiles of current commercial spaceflight vehicles.

Physiological monitoring and analysis of a manned stratospheric balloon test program.

INTRODUCTION: The Red Bull Stratos Project consisted of incremental high altitude parachute jumps [maximum altitude 127,852 ft (38,969 m)] from a pressurized capsule suspended from a stratospheric helium-filled balloon. A physiological monitoring system was worn by the parachutist to provide operational medical and acceleration data and to record a unique set of data in a supersonic environment. METHODS: Various physiological parameters, including heart rate (HR), respiratory rate (RR), skin temperature, and triaxial acceleration, were collected during the ascent, high altitude float, free fall, and parachute opening and descent stages of multiple low- and high altitude jumps. Physiologic data were synchronized with global positioning system (GPS) and audiovisual data for a comprehensive understanding of the environmental stressors experienced. RESULTS: HR reached maximum during capsule egress and remained elevated throughout free fall and landing. RR reached its maximum during free fall. Temperature data were unreliable and did not provide useful results. The highest accelerations parameters were recorded during parachute opening and during landing. During each high altitude jump, immediately after capsule egress, the parachutist experienced a few seconds of microgravity during which some instability occurred. Control was regained as the parachutist entered denser atmosphere. DISCUSSION: The high altitude environment resulted in extremely high vertical speeds due to little air resistance in comparison to lower altitude jumps with similar equipment. The risk for tumbling was highest at initial step-off. Physiological responses included elevated HR and RR throughout critical phases of free fall. The monitoring unit performed well despite the austere environment and extreme human performance activities.

Flat spin and negative Gz in high-altitude free fall: pathophysiology, prevention, and treatment.

INTRODUCTION: Red Bull Stratos was a commercial program that brought a test parachutist protected by a full pressure suit to 127,852 ft (38,964 m), via a stratospheric balloon with a pressurized capsule, from which he free fell and subsequently parachuted to the ground. In light of the uniqueness of the operation and the medical threats faced, medical protocols specific to distinctive injury patterns were developed. One unique threat was that of a flat spin during free fall with resultant exposure to -Gz (toe-to-head) acceleration. In preparation for stratospheric free fall, the medical team conducted a review of the literature on the spectrum of human and animal injury patterns attributable to -Gz exposures. Based on the findings, an emergency medical field response protocol was developed for the rapid assessment, diagnosis, and treatment of individuals suspected of -Gz injury. METHODS: A systematic review was conducted on available literature on human and animal studies involving significant -Gz exposure, with subsequent development of an applicable field treatment protocol. RESULTS: The literature review identified pathophysiologic processes and mitigation strategies that were used to develop a prevention and treatment protocol, outlining appropriate interventions using current best medical practices. A medical field treatment protocol was successfully established for the high-altitude balloon program. DISCUSSION: Available literature provided insight into best medical practices for the prevention and treatment of significant -Gz exposures during high-altitude parachute activity. Using the protocol developed for the field medical response, injuries from sustained -Gz exposure can be effectively managed in similar high-altitude and space operations.

Mutation E169K in junctophilin-2 causes atrial fibrillation due to impaired RyR2 stabilization.

OBJECTIVES: This study sought to study the role of junctophilin-2 (JPH2) in atrial fibrillation (AF). BACKGROUND: JPH2 is believed to have an important role in sarcoplasmic reticulum (SR) Ca(2+) handling and modulation of ryanodine receptor Ca(2+) channels (RyR2). Whereas defective RyR2-mediated Ca(2+) release contributes to the pathogenesis of AF, nothing is known about the potential role of JPH2 in atrial arrhythmias. METHODS: Screening 203 unrelated hypertrophic cardiomyopathy patients uncovered a novel JPH2 missense mutation (E169K) in 2 patients with juvenile-onset paroxysmal AF (pAF). Pseudoknock-in (PKI) mouse models were generated to determine the molecular defects underlying the development of AF caused by this JPH2 mutation. RESULTS: PKI mice expressing E169K mutant JPH2 exhibited a higher incidence of inducible AF than wild type (WT)-PKI mice, whereas A399S-PKI mice expressing a hypertrophic cardiomyopathy-linked JPH2 mutation not associated with atrial arrhythmias were not significantly different from WT-PKI. E169K-PKI but not A399A-PKI atrial cardiomyocytes showed an increased incidence of abnormal SR Ca(2+) release events. These changes were attributed to reduced binding of E169K-JPH2 to RyR2. Atrial JPH2 levels in WT-JPH2 transgenic, nontransgenic, and JPH2 knockdown mice correlated negatively with the incidence of pacing-induced AF. Ca(2+) spark frequency in atrial myocytes and the open probability of single RyR2 channels from JPH2 knockdown mice was significantly reduced by a small JPH2-mimicking oligopeptide. Moreover, patients with pAF had reduced atrial JPH2 levels per RyR2 channel compared to sinus rhythm patients and an increased frequency of spontaneous Ca(2+) release events. CONCLUSIONS: Our data suggest a novel mechanism by which reduced JPH2-mediated stabilization of RyR2 due to loss-of-function mutation or reduced JPH2/RyR2 ratios can promote SR Ca(2+) leak and atrial arrhythmias, representing a potential novel therapeutic target for AF.

Overview of medical operations for a manned stratospheric balloon flight.

INTRODUCTION: Red Bull Stratos was a commercial program designed to bring a test parachutist protected by a full-pressure suit via a stratospheric balloon with a pressurized capsule to 120,000 ft (36,576 m), from which he would freefall and subsequently parachute to the ground. On March 15, 2012, the Red Bull Stratos program successfully conducted a preliminary manned balloon test flight and parachute jump, reaching a final altitude of 71,581 ft (21,818 m). In light of the uniqueness of the operation and medical threats faced, a comprehensive medical plan was needed to ensure prompt and efficient response to any medical contingencies. This report will serve to discuss the medical plans put into place before the first manned balloon flight and the actions of the medical team during that flight. METHODS: The medical operations developed for this program will be systematically evaluated, particularly, specific recommendations for improvement in future high-altitude and commercial space activities. RESULTS: A multipronged approach to medical support was developed, consisting of event planning, medical personnel, equipment, contingency-specific considerations, and communications. DISCUSSION: Medical operations were found to be highly successful when field-tested during this stratospheric flight, and the experience allowed for refinement of medical operations for future flights. The lessons learned and practices established for this program can easily be used to tailor a plan specific to other aviation or spaceflight events.

Routine inpatient provider-initiated HIV testing in Malawi, compared with client-initiated community-based testing, identifies younger children at higher risk of early mortality.

OBJECTIVE: To determine how routine inpatient provider-initiated HIV testing differs from traditional community-based client-initiated testing with respect to clinical characteristics of children identified and outcomes of outpatient HIV care. DESIGN: Prospective observational cohort. METHODS: Routine clinical data were collected from children identified as HIV-infected by either testing modality in Lilongwe, Malawi, in 2008. After 1 year of outpatient HIV care at the Baylor College of Medicine Clinical Center of Excellence, outcomes were assessed. RESULTS: Of 742 newly identified HIV-infected children enrolling into outpatient HIV care, 20.9% were identified by routine inpatient HIV testing. Compared with community-identified children, hospital-identified patients were younger (median 25.0 vs 53.5 months), with more severe disease (22.2% vs 7.8% WHO stage IV). Of 466 children with known outcomes, 15.0% died within the first year of HIV care; median time to death was 15.0 weeks for community-identified children vs 6.0 weeks for hospital-identified children. The strongest predictors of early mortality were severe malnutrition (hazard ratio, 4.3; 95% confidence interval, 2.2-8.3), moderate malnutrition (hazard ratio, 3.2; confidence interval, 1.6-6.6), age < 12 months (hazard ratio, 3.2; 95% confidence interval, 1.4-7.2), age 12 to 24 months (hazard ratio, 2.5; 95% confidence interval, 1.1-5.7), and WHO stage IV (hazard ratio, 2.2; 95% confidence interval, 1.1-4.6). After controlling for other variables, hospital identification did not independently predict mortality. CONCLUSIONS: Routine inpatient HIV testing identifies a subset of younger HIV-infected children with more severe, rapidly progressing disease that traditional community-based testing modalities are currently missing.

History of suborbital spaceflight: medical and performance issues.

The development of manned sub-orbital commercial space vehicles is rapidly occurring and flight testing followed by operational flights will soon begin. The experience of manned suborbital spaceflight at the designated altitude (100 km/62.14 mi) is very limited--two Mercury-Redstone flights, two X-15 flights, one inadvertent Soyuz launch abort, and three recent SpaceShipOne flights, with only 15 min of critical flight time each. All indications were that the sequence of acceleration-weightlessness-deceleration was well tolerated with minimal neurovestibular dysfunction. However, there are some indications that distraction and spatial disorientation did occur. Vertigo on transition from the boost phase to weightlessness was reported on most high-altitude X-15 flights. +Gz tolerance to re-entry deceleration forces (as high as 6 + Gz) after 4 min of weightlessness is still unknown. Only further suborbital spaceflight experience will clarify if pilot performance will be affected.

Disrupted junctional membrane complexes and hyperactive ryanodine receptors after acute junctophilin knockdown in mice.

BACKGROUND: Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum within junctional membrane complexes. Although previous studies revealed a loss of junctional membrane complexes and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in junctional membrane complex formation and the Ca(2+)-induced Ca(2+) release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. METHODS AND RESULTS: To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of junctional membrane complexes and increased variability in the plasmalemma-sarcoplasmic reticulum distance. CONCLUSIONS: Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific short hairpin RNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart.

Junctophilin-2 expression silencing causes cardiocyte hypertrophy and abnormal intracellular calcium-handling.

BACKGROUND: Junctophilin-2 (JPH2), a protein expressed in the junctional membrane complex, is necessary for proper intracellular calcium (Ca(2+)) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca(2+) channels and sarcoplasmic reticular ryanodine receptors. However, it remains unclear whether JPH2 expression is altered in patients with hypertrophic cardiomyopathy (HCM). In addition, the effects of downregulation of JPH2 expression on intracellular Ca(2+) handling are presently poorly understood. We sought to determine whether loss of JPH2 expression is noted among patients with HCM and whether expression silencing might perturb Ca(2+) handling in a prohypertrophic manner. METHODS AND RESULTS: JPH2 expression was reduced in flash-frozen human cardiac tissue procured from patients with HCM compared with ostensibly healthy traumatic death victims. Partial silencing of JPH2 expression in HL-1 cells by a small interfering RNA probe targeted to murine JPH2 mRNA (shJPH2) resulted in myocyte hypertrophy and increased expression of known markers of cardiac hypertrophy. Whereas expression levels of major Ca(2+)-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca(2+) transient amplitudes that were insensitive to L-type Ca(2+) channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca(2+) levels were observed with potentially increased total Ca(2+) stores. Spontaneous Ca(2+) oscillations were elicited at a higher extracellular [Ca(2+)] and with decreased frequency in JPH2 knockdown cells. CONCLUSIONS: Our results show that JPH2 levels are reduced in patients with HCM. Reduced JPH2 expression results in reduced excitation-contraction coupling gain as well as altered Ca(2+) homeostasis, which may be associated with prohypertrophic remodeling.

Emerging role of junctophilin-2 as a regulator of calcium handling in the heart.

Junctophilin-2 (JPH2) is a membrane-binding protein that plays a key role in the organization of the junctional membrane complex (JMC) in cardiac myocytes. JPH2 is believed to keep the plasma membrane and sarcoplasmic reticulum at a fixed distance within the JMC, which is essential for proper Ca(2+)-induced Ca(2+) release during the excitation-contraction process. Recent studies have revealed that mutations in the JPH2 gene are associated with hypertrophic cardiomyopathy, highlighting the importance of this protein for normal cardiac physiology. In this paper, we review current knowledge about the structure and function of junctophilin-2 in the heart.

Molecular evolution of the junctophilin gene family.

Junctophilins (JPHs) are members of a junctional membrane complex protein family important for the physical approximation of plasmalemmal and sarcoplasmic/endoplasmic reticulum membranes. As such, JPHs facilitate signal transduction in excitable cells between plasmalemmal voltage-gated calcium channels and intracellular calcium release channels. To determine the molecular evolution of the JPH gene family, we performed a phylogenetic analysis of over 60 JPH genes from over 40 species and compared conservation across species and different isoforms. We found that JPHs are evolutionary highly conserved, in particular the membrane occupation and recognition nexus motifs found in all species. Our data suggest that an ancestral form of JPH arose at the latest in a common metazoan ancestor and that in vertebrates four isoforms arose, probably following two rounds of whole genome duplications. By combining multiple prediction techniques with sequence alignments, we also postulate the presence of new important functional regions and candidate sites for posttranslational modifications. The increasing number of available sequences yields significant insight into the molecular evolution of JPHs. Our analysis is consistent with the emerging concept that JPHs serve dual important functions in excitable cells: structural assembly of junctional membrane complexes and regulation of intracellular calcium signaling pathways.