Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Dissecting the understanding of students about our teaching methods: A single institute, analytical cross-sectional study.

OBJECTIVE: To measure the perception of medical students regarding different methods of active learning, and its association with the year of study. METHODS: The analytical cross-sectional study was conducted at Shalamar Medical and Dental College, Lahore, Pakistan, from May to September 2020, and comprised medical students of either gender from first to final year of studies. Data was collected using an online questionnaire regarding different methods of active learning and e-learning. Perceptions and their association with the year of study were worked out. Data was analysed using SPSS 16. RESULTS: Of the 270 subjects, 155(57.4%) were females and 115(42.5%) were males. Overall, 39(14.4%) students were from the first year of studies, 32(11.9%) second, 47(17.4%) third, 120(44.4%) fourth and 32(11.9%) were from the final year of medical studies. Most students preferred class lectures as the teaching method of choice 240(89%), followed by small group discussions 156(58%). Students showed positive perception of different learning methods except e-learning 78(28.89%). The association between perceptions and the year of study was statistically significant (p< 0.05). CONCLUSIONS: Students apparently enjoyed using different interactive methods, but were apprehensive about online learning.

Identification of Cardiac Fibrosis in Young Adults With a Homozygous Frameshift Variant in SERPINE1.

Importance: Cardiac fibrosis is exceedingly rare in young adults. Identification of genetic variants that cause early-onset cardiomyopathy may inform novel biological pathways. Experimental models and a single case report have linked genetic deficiency of plasminogen activator inhibitor-1 (PAI-1), a downstream target of cardiac transforming growth factor ß, with cardiac fibrosis. Objective: To perform detailed cardiovascular phenotyping and genotyping in young adults from an Amish family with a frameshift variant (c.699_700dupTA) in SERPINE1, the gene that codes for PAI-1. Design, Setting, and Participants: This observational study included participants from 3 related nuclear families from an Amish community in the primary analysis and participants from the extended family in the secondary analysis. Participants were recruited from May 2015 to December 2016, and analysis took place from June 2015 to June 2020. Main Outcomes and Measures: (1) Multimodality cardiovascular imaging (transthoracic echocardiography and cardiac magnetic resonance imaging), (2) whole-exome sequencing, and (3) induced pluripotent stem cell-derived cardiomyocytes. Results: Among 17 participants included in the primary analysis, the mean (interquartile range) age was 23.7 (20.9-29.9) years and 9 individuals (52.9%) were confirmed to be homozygous for the SERPINE1 c.699_700dupTA variant. Late gadolinium enhancement was present in 6 of 9 homozygous participants (67%) with absolute PAI-1 deficiency vs 0 of 8 in the control group (P = .001). Late gadolinium enhancement patterns tended to be dense and linear, usually subepicardial but also midmyocardial and transmural with noncoronary distributions. Targeted whole-exome sequencing analysis identified that homozygosity for c.699_700dupTA SERPINE1 was the only shared pathogenic variant or variant of uncertain significance after examination of cardiomyopathy genes among those with late gadolinium enhancement. Induced pluripotent stem cell-derived cardiomyocytes from participants homozygous for the SERPINE1 c.699_700dupTA variant exhibited susceptibility to cardiomyocyte injury in response to angiotensin II (increased transforming growth factor ß1 secretion and release of lactate dehydrogenase) compared with control induced pluripotent stem cell-derived cardiomyocytes. In a secondary analysis based on echocardiography in 155 individuals across 3 generations in the extended family, no difference in global longitudinal strain was observed in carriers for the SERPINE1 c.699_700dupTA variant compared with wild-type participants, supporting an autosomal recessive inheritance pattern. Conclusions and Relevance: In this study, a highly penetrant, autosomal recessive, cardiac fibrosis phenotype among young adults with homozygous frameshift variant for SERPINE1 was identified, suggesting an optimal range of PAI-1 levels are needed for cardiac homeostasis.

Generation of human iPSCs from urine derived cells of a non-affected control subject.

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from non-affected control subjects to use as a comparison group for the iPSC lines containing a Plasminogen Activator Inhibitor-1 (PAI-1 homozygous/heterozygous) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the UCs.

Generation of human iPSCs from urine derived cells of patient with a novel heterozygous PAI-1 mutation.

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from subjects heterozygous for a novel Plasminogen Activator Inhibitor-1 (PAI-1) mutation. The Sendai Virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the PAI-1 UCs.

Generation of human iPSCs from urine derived cells of a patient with a novel homozygous PAI-1 mutation.

We have generated a human induced pluripotent stem cell (iPSC) line under feeder-free culture conditions using the urine derived cells (UCs) collected from subject with a novel homozygous Plasminogen Activator Inhibitor-1 (PAI-1 null) mutation. The Sendai virus (SeV) vector encoding pluripotent Yamanaka transcription factors was used at a low multiplicity of infection to reprogram the PAI-1 UCs.

Pre-conditioned mesenchymal stem cells ameliorate renal ischemic injury in rats by augmented survival and engraftment.

BACKGROUND: Ischemia is the major cause of acute kidney injury (AKI), associated with high mortality and morbidity. Mesenchymal stem cells (MSCs) have multilineage differentiation potential and can be a potent therapeutic option for the cure of AKI. METHODS: MSCs were cultured in four groups SNAP (S-nitroso N-acetyl penicillamine), SNAP + Methylene Blue (MB), MB and a control for in vitro analysis. Cultured MSCs were pre-conditioned with either SNAP (100 µM) or MB (1 µM) or both for 6 hours. Renal ischemia was induced in four groups (as in in vitro study) of rats by clamping the left renal padicle for 45 minutes and then different pre-conditioned stem cells were transplanted. RESULTS: We report that pre-conditioning of MSCs with SNAP enhances their proliferation, survival and engraftment in ischemic kidney. Rat MSCs pre-conditioned with SNAP decreased cell apoptosis and increased proliferation and cytoprotective genes' expression in vitro. Our in vivo data showed enhanced survival and engraftment, proliferation, reduction in fibrosis, significant improvement in renal function and higher expression of pro-survival and pro-angiogenic factors in ischemic renal tissue in SNAP pre-conditioned group of animals. Cytoprotective effects of SNAP pre-conditioning were abrogated by MB, an inhibitor of nitric oxide synthase (NOS) and guanylate cyclase. CONCLUSION: The results of these studies demonstrate that SNAP pre-conditioning might be useful to enhance therapeutic potential of MSCs in attenuating renal ischemia reperfusion injury.