Alejandro Garbino

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 Ca2+-induced Ca2+ 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.

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.

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 (Ca2+) signaling in cardiac myocytes. Downregulation of JPH2 expression in a model of cardiac hypertrophy was recently associated with defective coupling between plasmalemmal L-type Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+-handling proteins were unchanged, shJPH2 cells demonstrated depressed maximal Ca2+ transient amplitudes that were insensitive to L-type Ca2+ channel activation with JPH2 knockdown. Further, reduced caffeine-triggered sarcoplasmic reticulum store Ca2+ levels were observed with potentially increased total Ca2+ stores. Spontaneous Ca2+ oscillations were elicited at a higher extracellular [Ca2+] 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 Ca2+ homeostasis, which may be associated with prohypertrophic remodeling.

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.

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 Ca2+-induced Ca2+ 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.

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.

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.

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.

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.

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.