Investigating the cardiac pathology of SCO2-mediated hypertrophic cardiomyopathy using patients induced pluripotent stem cell-derived cardiomyocytes.

Mutations in SCO2 are among the most common causes of COX deficiency, resulting in reduced mitochondrial oxidative ATP production capacity, often leading to hypertrophic cardiomyopathy (HCM). To date, none of the recent pertaining reports provide deep understanding of the SCO2 disease pathophysiology. To investigate the cardiac pathology of the disease, we were the first to generate induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) from SCO2-mutated patients. For iPSC generation, we reprogrammed skin fibroblasts from two SCO2 patients and healthy controls. The first patient was a compound heterozygote to the common E140K mutation, and the second was homozygote for the less common G193S mutation. iPSC were differentiated into cardiomyocytes through embryoid body (EB) formation. To test the hypothesis that the SCO2 mutation is associated with mitochondrial abnormalities, and intracellular Ca2+ -overload resulting in functional derangements and arrhythmias, we investigated in SCO2-mutated iPSC-CMs (compared to control cardiomyocytes): (i) the ultrastructural changes; (ii) the inotropic responsiveness to ß-adrenergic stimulation, increased [Ca2+ ]o and angiotensin-II (AT-II); and (iii) the Beat Rate Variability (BRV) characteristics. In support of the hypothesis, we found in the mutated iPSC-CMs major ultrastructural abnormalities and markedly attenuated response to the inotropic interventions and caffeine, as well as delayed afterdepolarizations (DADs) and increased BRV, suggesting impaired SR Ca2+ handling due to attenuated SERCA activity caused by ATP shortage. Our novel results show that iPSC-CMs are useful for investigating the pathophysiological mechanisms underlying the SCO2 mutation syndrome.

Genotype-Specific Cortisol Reserve in a Cohort of Subjects With Nonclassic Congenital Adrenal Hyperplasia (NCCAH).

CONTEXT: Recent guidelines suggest that patients with nonclassic congenital adrenal hyperplasia (NCCAH) stop glucocorticoid therapy after achieving adult height. However, these guidelines do not differentiate between NCCAH genotype groups. OBJECTIVE: Compare ACTH-stimulated cortisol and 17-hydroxyprogesterone (17OHP) levels, and the rate of partial cortisol insufficiency in subjects with NCCAH carrying one mild and one severe (mild/severe) mutation vs subjects with biallelic mild (mild/mild) mutations. METHODS: Retrospective evaluation of the medical records of 122 patients who presented with postnatal virilization and were diagnosed with NCCAH. Patients underwent standard intravenous 0.25 mg/m2 ACTH stimulation testing. Those with stimulated 17OHP level ≥40 nmol/L were screened for the 9 most frequent CYP21A2 gene mutations followed by multiplex ligation-dependent probe amplification. A stimulated cortisol level below 500 nmol/L was defined as partial cortisol deficiency. RESULTS: Patients were subdivided into 3 genotype groups: 77 carried the mild/mild genotype, mainly homozygous for p.V281L mutation; 29 were compound heterozygous for mild/severe mutation, mainly p.V281L/p.I2Splice, and 16 were heterozygous for p.V281L, and were excluded from statistical evaluation. Stimulated cortisol levels were significantly lower in the mild/severe than in the mild/mild group (mean ± SD, 480 ± 90 vs 570 ± 125 nmol/L, P < .001). The mild/severe group exhibited a significantly higher rate of partial cortisol insufficiency (21/28, 75% vs 28/71, 39%, P = .004). Peak 17OHP was significantly higher in the mild/severe group (198 ± 92 vs 118 ± 50 nmol/L, P < .001). CONCLUSION: The high rate of partial adrenal insufficiency in the mild/severe group underscores the need to carefully consider the value of glucocorticoid therapy cessation and the importance of stress coverage in this group.

B-cell depletion reactivates B lymphopoiesis in the BM and rejuvenates the B lineage in aging.

Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. These changes decrease the body's ability to mount protective antibody responses. We show here that age-related changes in the B lineage are mediated by the accumulating long-lived B cells. Thus, depletion of B cells in old mice was followed by expansion of multipotent primitive progenitors and common lymphoid progenitors, a revival of B-lymphopoiesis in the bone marrow, and generation of a rejuvenated peripheral compartment that enhanced the animal's immune responsiveness to antigenic stimulation. Collectively, our results suggest that immunosenescence in the B-lineage is not irreversible and that depletion of the long-lived B cells in old mice rejuvenates the B-lineage and enhances immune competence.

Depletion of B cells rejuvenates the peripheral B-cell compartment but is insufficient to restore immune competence in aging.

Aging is associated with increasing prevalence and severity of infections caused by a decline in bone marrow (BM) lymphopoiesis and reduced B-cell repertoire diversity. The current study proposes a strategy to enhance immune responsiveness in aged mice and humans, through rejuvenation of the B lineage upon B-cell depletion. We used hCD20Tg mice to deplete peripheral B cells in old and young mice, analyzing B-cell subsets, repertoire and cellular functions in vitro, and immune responsiveness in vivo. Additionally, elderly patients, previously treated with rituximab healthy elderly and young individuals, were vaccinated against hepatitis B (HBV) after undergoing a detailed analysis for B-cell compartments. B-cell depletion in old mice resulted in rejuvenated B-cell population that was derived from de novo synthesis in the bone marrow. The rejuvenated B cells exhibited a "young"-like repertoire and cellular responsiveness to immune stimuli in vitro. Yet, mice treated with B-cell depletion did not mount enhanced antibody responses to immunization in vivo, nor did they survive longer than control mice in "dirty" environment. Consistent with these results, peripheral B cells from elderly depleted patients showed a "young"-like repertoire, population dynamics, and cellular responsiveness to stimulus. Nevertheless, the response rate to HBV vaccination was similar between elderly depleted and nondepleted subjects, although antibody titers were higher in depleted patients. This study proposes a proof of principle to rejuvenate the peripheral B-cell compartment in aging, through B-cell depletion. Further studies are warranted in order to apply this approach for enhancing humoral immune responsiveness among the elderly population.

Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

BACKGROUND: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. METHODS AND RESULTS: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 µg kg-1 min-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. CONCLUSIONS: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Developmental changes in electrophysiological characteristics of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Previous studies proposed that throughout differentiation of human induced Pluripotent Stem Cell-derived cardiomyocytes (iPSC-CMs), only 3 types of action potentials (APs) exist: nodal-, atrial-, and ventricular-like. OBJECTIVES: To investigate whether there are precisely 3 phenotypes or a continuum exists among them, we tested 2 hypotheses: (1) During culture development a cardiac precursor cell is present that-depending on age-can evolve into the 3 phenotypes. (2) The predominant pattern is early prevalence of a nodal phenotype, transient appearance of an atrial phenotype, evolution to a ventricular phenotype, and persistence of transitional phenotypes. METHODS: To test these hypotheses, we (1) performed fluorescence-activated cell sorting analysis of nodal, atrial, and ventricular markers; (2) recorded APs from 280 7- to 95-day-old iPSC-CMs; and (3) analyzed AP characteristics. RESULTS: The major findings were as follows: (1) fluorescence-activated cell sorting analysis of 30- and 60-day-old cultures showed that an iPSC-CMs population shifts from the nodal to the atrial/ventricular phenotype while including significant transitional populations; (2) the AP population did not consist of 3 phenotypes; (3) culture aging was associated with a shift from nodal to ventricular dominance, with a transient (57-70 days) appearance of the atrial phenotype; and (4) beat rate variability was more prominent in nodal than in ventricular cardiomyocytes, while pacemaker current density increased in older cultures. CONCLUSION: From the onset of development in culture, the iPSC-CMs population includes nodal, atrial, and ventricular APs and a broad spectrum of transitional phenotypes. The most readily distinguishable phenotype is atrial, which appears only transiently yet dominates at 57-70 days of evolution.

Development of ALS-like disease in SOD-1 mice deficient of B lymphocytes.

Several recent studies proposed a role for innate immunity and inflammation in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, possible links, if any, between disease and adaptive immunity are poorly understood. The present study probed for the role of B cells in ALS disease using the G93A-SOD-1 transgenic mouse model. In agreement with other studies, we show here that autoantibodies are detectable in SOD-1 mice. However, SOD-1 B cells did not express any altered phenotype and exhibited indistinguishable responsiveness to immunogenic stimuli relative to wild-type B cells. This was obtained for B cells isolated before, during and after the onset of ALS-like disease. Finally, to obtain an in vivo conclusion, we generated SOD-1 mice that are deficient of B cells, by crossing SOD-1 mice with Igmu-deficient mice (muMT), where B cell development is blocked at the proB stage. The meteoric assays performed on a rota-rod clearly showed the development of ALS-like disease in SOD-1 mice that are deficient of B cells not differently than in control SOD-1 mice. Our results propose that B lymphocytes do not have a major role in the pathogenesis of ALS-like disease in SOD-1 mice.