The landscape of human cardiac biology was transformed by the discovery that adult somatic cells can be reprogrammed into induced pluripotent stem cells, enabling patient-specific disease modeling, drug testing, and regenerative strategies without the prior ethical or biological constraints. Subsequent advances in directed differentiation made the generation of human iPSC-derived cardiac myocytes reliable and scalable. Despite this progress, a central limitation has remained: these cells are developmentally immature, resembling fetal cardiac myocytes in structure, metabolism, and function. This immaturity restricts their utility for modeling adult-onset disease, predicting drug responses, and achieving clinical translation. Maturation is now understood as a multifactorial symphony, requiring coordinated molecular, structural, and environmental inputs rather than single interventions. As a result, the field is shifting toward integrative approaches that incorporate 3D architecture, multicellular systems, and biomimetic environments to better replicate native cardiac tissue. While fully adult-like myocardium remains an ongoing goal, advances in bioengineering and system-level design are narrowing the gap, with success increasingly defined by the generation of functional cardiac tissue rather than isolated cell maturity.

OBJECTIVE To evaluate the ability of a proprietary artificial intelligence (AI) model to predict the number of days until delivery using ultrasound images alone and to assess the continuous improvement of prediction accuracy, particularly for preterm births, through model retraining.METHODS An AI software was developed and trained using de-identified ultrasound images from a cohort of women who delivered at the University of Kentucky from 2017 to 2021. Initially, 5,714 pregnant women, with 19,940 unique ultrasound exams and 877,141 total ultrasound images were utilized from this timeframe. Images from 79% of this cohort (4,505 patients) trained the AI to estimate the number of days until delivery and secondarily optimize predictions related to preterm birth (<37 weeks gestational age). The output consisted of days until delivery which was subsequently categorized as either preterm or term birth.The remaining 21% of the cohort (1,209 patients) was reserved for derivation and validation of test characteristics. Delivery outcomes for this subgroup were blinded from the AI by an independent third-party data monitor. Unique predictions were made for each patient after each ultrasound exam, and the AI’s performance was evaluated against the actual delivery date using metrics such as R2 values and mean absolute error (MAE) compared to actual days until delivery. After initial testing, the AI was retrained x3 more using the same data (Version 2, V2) and later with an additional 1,165,618 images obtained by extension of the study to include data from our center until 2023 (Version 3 (V3), Version 4 (V4)- consisted of retraining on V3)RESULTS Preterm birth rates were similar between the training (18.4%) and validation (18.6%) sets in the initial study set. The initial AI model exhibited a sensitivity of 39% and specificity of 93% for preterm birth prediction, with an AUC of 0.757. The AI’s predictions of days to delivery versus actual in the validation set yielded R2 of 0.90 for term births, 0.88 for spontaneous preterm birth plus term births, and 0.48 for spontaneous preterm birth alone. The MAE in predicting the number of days until delivery showed similar accuracy across all trimesters that were assessed by image analysis. Finally, retraining with improvements in AI architecture and training methodology using additional images provided improved preterm birth prediction, with R2 values for all births increasing from 0.85 (V1) to 0.88 (V3) to 0.92 (V4). For spontaneous PTB, MAE was 19.99 days in V4.CONCLUSIONS AI can predict timing until delivery from ultrasound data alone. This technology can also predict preterm delivery with limited sensitivity. Retraining the AI with supervised and unsupervised learning has the potential to further improve performance.

Introduction: Early prediction of preeclampsia remains a critical unmet need in obstetric care. The renin-angiotensin aldosterone system (RAAS) plays a central role in blood pressure regulation and placental development, yet its utility for the prediction of pre-eclampsia is understudied. ALDO+ (previously RAAS-Triple A) is a mass spectrometry-based platform for quantifying equilibrium concentrations of angiotensin I (Ang I), angiotensin II (Ang II), and aldosterone, enabling derivation of physiologically meaningful RAAS biomarkers. Purpose: The purpose of this study was to evaluate whether RAAS biomarkers measured using the ALDO+ platform could distinguish patients with preeclampsia from non-pre-eclamptic pregnancies and identify those at risk for developing preeclampsia later in gestation. Methods: We conducted a gestational age–matched case-control study of pregnant patients with and without preeclampsia (mean 28 weeks gestation; 51 cases, 49 controls). ALDO+ was used to measure Ang I, Ang II, and aldosterone. Receiver operating characteristics analyses were performed to evaluate predictive performance of individual biomarkers. A cut-off for the Ang II to Ang I ratio (ACE-R) was identified in the case-control study. This cut-off was then applied to an independent longitudinal cohort of 130 pregnant patients sampled at 28 weeks, 14 of whom subsequently developed preeclampsia. Results: In the case-control cohort, ACE-R was significantly elevated in preeclampsia cases. A cut-off of ACE-R >2.05 predicted preeclampsia with 84% sensitivity and 61.2% specificity (AUC=0.85). When applied to the longitudinal cohort, this cut-off identified 7 of 14 individuals who later developed preeclampsia (50% sensitivity) and correctly excluded 81 of 116 who did not (70% specificity). Other RAAS markers, including individual concentrations of Ang I, Ang II, and aldosterone, showed good but lower predictive performance. Conclusions: RAAS activity biomarkers derived using ALDO+ demonstrated potential for predicting preeclampsia in non-optimized cohorts. The ACE-R in particular showed promising sensitivity and specificity for disease identification and prediction. These results support further investigation of RAAS-based biomarkers in prospective cohorts for risk stratification in pregnancy (alone or in combination with current tools) and provide a mechanistically grounded framework for preeclampsia prediction.

Introduction:Neonatal hypoxic respiratory failure is commonly assessed with the oxygenation index (OI) to determine severity and guide ECMO initiation. Calculation of the OI requires arterial blood sampling which can be difficult to obtain. A non-invasive alternative, the oxygen saturation index (OSI), has shown promise, but its utility in ECMO determination is not well-described. We aimed to evaluate the correlation between the OI and OSI in neonates requiring ECMO. Methods:We pursued a retrospective chart review of 64 neonatal ECMO patients at Kentucky Children's Hospital (2012-2022) and analyzed OI and OSI values in the 12 h preceding ECMO initiation. Results:A moderate correlation was observed between the OI and OSI. An OSI >17.41 predicted ECMO initiation, and OI can be estimated with the equation: OI = 1.978(OSI)-6.743. Conclusion:These findings suggest OSI may be a useful adjunct to OI for assessing neonatal respiratory failure and could be beneficial when arterial sampling is impractical.

Duchenne muscular dystrophy (DMD) is a fatal genetic disease of progressive muscle deterioration with no cure. DMD treatment requires a body-wide approach to target all diseased striated muscles: limb, respiratory, and heart. To address this, we focus studies on blocking the onset of muscle membrane instability, the primary defect in DMD, as a promising yet unmet druggable target. Here, data show the remarkable potency of a synthetic poly(ethylene oxide)/poly(propylene oxide) side chain-based bottlebrush block copolymer, ~150,000 times more potent than linear polymers, to rapidly restore contractile function to DMD skeletal muscle fibers in vitro. Strikingly, upon bottlebrush polymer delivery to DMD animals, results show highly efficacious prevention of the onset of skeletal and diaphragm muscle damage and the blocking of stress-induced cardiac injury and death in vivo. These data suggest bottlebrush polymers as a potent stand-alone muscle membrane-stabilizing therapeutic for DMD. Given DMD's early childhood onset, together with newborn screening for DMD, bottlebrush macromolecules could be envisioned as an early therapy to preserve and protect viable muscle and potentially for other acquired or inherited diseases involving membrane damage.

Cancer cachexia is a highly debilitating clinical syndrome of involuntary body mass loss featuring profound muscle wasting leading to high mortality. Notably, cardiac wasting is prominent in cancer patients and cancer survivors. Cachexia studies present significant challenges due to the absence of human models and mainly short-term animal studies. To address this translational gap, we have developed a robust human-based cachexia experimental approach characterized by marked cardiac muscle wasting and contractile dysfunction, with increased expression of protein degradation markers. Using human iPSC-derived cardiac muscle, we investigated morphological, functional, and metabolic alterations in the key stages of cachexia and in the post-cachexia phase. C26 and HCT116 tumor cell lines were used to induce cachexia by two methods, pulse addition of cancer cell conditioned media or in transwell-adapted co-culture. Cachectic cardiac myocytes exhibited reduced contraction amplitude, prolonged relaxation time, and increased oxygen consumption rate (OCR), as assessed by video-based and Seahorse analyses. Mechanistic investigations centered on the Atrogin-1/Calcineurin A/NFAT axis revealed this signaling pathway as a central driver of cachexia-induced cardiac atrophy. Cachectic cardiac myocytes exhibited significant upregulation of Atrogin-1, leading to a marked decrease in Calcineurin A protein levels. This, in turn, impaired nuclear translocation of NFAT, thereby suppressing its transcriptional activity and downstream cell growth signaling. These molecular changes were accompanied by increased autophagic flux, as indicated by elevated LC3BII/LC3BI ratios. Furthermore, withdrawal of cachexia-inducing stimuli followed by regular media changes for one week led to normalization of Atrogin-1 and autophagy markers; however, functional impairments and metabolic dysregulation persisted, highlighting delayed recovery. Our new findings establish the Atrogin-1/Calcineurin A/NFAT axis as a key regulatory mechanism in cardiac muscle wasting and suggest this aberrant signaling axis may serve as a targetable mechanism for treatment of cachexia-induced cardiac dysfunction.

Background: Left ventricular hypertrophy (LVH) is a risk factor for cardiovascular disease in adults. In youth, elevated blood pressure (BP) is associated with LVH, but independent effects of adiposity and hypertension on LVH are not well defined. The purpose of our study was to investigate relationships among LVH, LV mass, BMI, and other clinical parameters in pediatric patients referred for elevated BP. We hypothesized both BP and BMI to be associated with LV mass. Methods: We conducted an IRB-approved retrospective chart review of all patients ages 6-18 years who underwent 24-hour ambulatory BP monitoring via the pediatric nephrology clinic at Kentucky Children’s Hospital from August 2012-December 2023 (excluding those with secondary HTN). Obesity was defined by BMI and weight-for-length percentiles according to CDC guidelines. LV mass was assessed by standard echocardiography. We compared LVMI and BP measures among groups stratified by obesity stage. Associations among BP, obesity, and LVMI were determined using descriptive statistics, correlational analyses, and multivariable logistic modeling using a backwards elimination variables selection criteria with α=0.05. Results: Of the 520 patients, 66% (n=342) were male, 76% (n=395) were obese, and 46% (n=238) had LVH. LVMI was strongly correlated with 24-hr systolic BP, however there was no difference in mean LVMI across BP severity category. In contrast, there was a near stepwise increase in LVMI across obesity stage with the strongest effect in girls – Normal: 29.7 ± 5.3 (girls) v. 34.8 ± 11.4 (boys) g/m 2.7 ; OB-1: 36.5 ± 6.6 (girls) v. 43.2 ± 10.3 (boys) g/m 2.7 ; OB-2: 41 ± 11.6 (girls) v. 44.2 ± 9.2 (boys) g/m 2.7 ; OB 3: 48 ± 11.6 (girls) v. 43.5 ± 9.4 (boys) g/m 2.7 . LVMI was positively correlated with TSH, creatinine, uric acid, cystatin, and inversely correlated with HDL. Multivariable logistic modeling revealed obesity stage and HDL cholesterol, but not BP or laboratory variables, as significant independent predictors of LVH. Conclusions: We report a high incidence of LVH in youth with obesity and demonstrate a strong association between obesity and LVMI. Our data, in agreement with previous studies, indicate that adiposity is a significant determinant of left ventricular mass, and suggest the need for cardiac screening of youth with obesity even in the absence of elevated blood pressure.

Background: Mechanisms underlying the development of pre-eclampsia in humans are not well-understood. Activation of the RAAS with markedly increased secretion of aldosterone is a key feature of normotensive pregnancy, and thought to be impaired in pre-eclampsia. The purpose of our study was to perform RAAS profiling in pre-eclampsia, where we hypothesized reduced concentrations of aldosterone in serum of patients with pre-eclampsia compared to pregnant patients without pre-eclampsia. Methods: This is a case-control study of 50 patients with pre-eclampsia and 50 gestational age-matched controls. Clinical characteristics (blood pressure, biochemical laboratory values) and blood samples were collected upon enrollment, and maternal and fetal outcomes were collected at delivery. Blood samples were analyzed for angiotensin I, angiotensin II, and aldosterone by LC-MS/MS using RAAS-Triple A. All patients have been recruited, and preliminary data are available for the cases. Results: The mean gestational age of pre-eclampsia cases was 27.9 + 6.2 weeks. RAAS profiling revealed two distinct subpopulations, where 35 patients had concentrations of aldosterone in serum that were characteristically low for pre-eclampsia: below 204 pmol/L, with median and interquartile range (IQR) of 120.0 and 35.0 – 132.2 pmol/L, and an aldosterone to angiotensin II ratio (AA2-R) of 1.05 (compared to previously published results where the median and IQR of aldosterone in pregnant women at 28 weeks was 724.8 and 376.6 – 1197 pmol/L, and median AA2-R of 4.15). In contrast, 15 patients had aldosterone concentrations more characteristic of normal pregnancy, with a median and IQR of 589.8 and 402.2– 769.6 pmol/L, and median AA2-R of 4.8). For comparison, a recent population study of non-pregnant adults determined median and IQR of aldosterone concentrations of 93 and 57.9 – 153.9 pmol/L using RAAS Triple-A. Conclusions: These data suggest two distinct phenotypes of pre-eclampsia based on RAAS profiling. Consistent with previous literature, our data indicate that pre-eclampsia is predominately characterized by reduced secretion of aldosterone, where concentrations are comparable to non-pregnant individuals. In contrast, we report that approximately one-third of patients with pre-eclampsia have elevated concentrations of aldosterone expected for pregnancy, suggesting multiple mechanisms for pre-eclampsia in humans defined by the presence or absence of elevated aldosterone secretion.

Objective: The obesity rate in children is rising in the United States, and this is particularly true in children located in rural regions. Rural communities are at greater risk for health disparities, such as metabolic disease, due to socioeconomic factors and access to food and healthcare. Previous studies in rural youth have focused on lifestyle intervention and behavior modification to improve obesity rates. However, there are limited studies comparing clinical parameters of metabolic disease in rural versus non-rural regions in youth. The objective of this study was to examine blood markers of glucose and lipid status among rural vs non-rural adolescents to identify risk factors for metabolic disease. Design and method: This is a single timepoint pilot study of n = 82 children and adolescents ages 6–17 recruited from a Pediatric High BMI Clinic. Subjects were grouped according to region: rural (n = 12), micropolitan (n = 20), and metropolitan (n = 50). Weight, adiposity, clinical data, and blood samples were collected at the time of initial clinic visit. Data were analyzed via one-way ANOVA. Results: BMI and waist circumference (WC) was highest in rural subjects (BMI - rural: 38.5 ± 5.4 versus micropolitan: 37.7 ± 9.1 versus metropolitan: 33.5 ± 5.5 kg/m2; P < 0.05. WC - rural: 116 ± 12.5 versus micropolitan: 115.1 ± 19.8 versus metropolitan: 105.2 ± 15.6 cm; P < 0.05). In contrast, there was no difference in markers of glucose homeostasis or lipids between regions. Serum concentrations of 25(OH)D 3 were different among groups, with the lowest in subjects from the metropolitan area, and highest in the rural area (rural: 34.9 ± 9.6; micropolitan: 30.2 ± 9.7; metropolitan: 23.0 ± 9.3 ng/mL; P < 0.001). Conclusions: In agreement with literature, adiposity was greatest in subjects living in non-metropolitan areas. However, this was not associated with elevated markers of metabolic risk, and rural subjects had the greatest serum vitamin D 3 concentrations. Further understanding of differences in lifestyle in rural versus non-rural areas may improve opportunities for targeted health interventions.

Hypertensive disorders of pregnancy (HDP) are a leading cause of maternal and infant morbidities and mortalities. There is a lack of clinical tools identifying subtypes of this heterogenous group of disorders, which limits optimization of individual patient care. The RAAS, a hormonal system activated during pregnancy, is suppressed in pregnancies with hypertension, but key RAAS biomarkers in subgroups of HDP have not been defined. We quantified serum biomarkers of the RAAS using LC-MS/MS in the first and third trimesters of pregnancy in a cohort of n=92 women at high-risk for preeclampsia. Data are expressed as median [IQR] of the delta of the third - first trimester. There were n=15 women that developed gestational hypertension (GH) and n=12 women that developed pre-eclampsia (Pre-e). Serum aldosterone levels increased with pregnancy in high-risk women with no adverse outcomes, but not in women that developed GH (GH, n=15: 19.4 [-321.6 - 661.6] versus Control, n=22: 502.5 [161-1019]; Data are pmol/L; P<0.01). PRA-S, a marker for activity of the RAAS that normally increases in healthy pregnancy, did not increase with pregnancy in patients that developed GH, and was further reduced in patients that developed Pre-e (GH: -17.4 [-57.1 to 2.2] versus Pre-E: -56.3 [-160.9 to -3.5] pmol/L; P<0.05). In high-risk patients that developed pre-eclampsia, aldosterone levels varied widely, depending pre-existing risk factors, such as chronic hypertension. Serum levels of RAAS biomarkers differed by pregnancy outcome and were influenced by pre-existing risk. Declining aldosterone and PRA-S over gestation may be indicative of development of Pre-e in women with clinical risk factors for Pre-e.

Numerous inherited and acquired cardiac diseases are directly linked to a deficiency in the protein dystrophin, such as the development of severe cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients. Dilated cardiomyopathy, arrhythmias, and congestive heart failure represent the most important life-limiting condition in DMD. Currently, there is great excitement in the muscular dystrophy community as multiple ongoing gene-based therapies, are advancing through clinical trials. These therapies utilize miniaturized dystrophin constructs, and these modified dystrophins are the focus of this study. We implemented an innovative and valuable system to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin constructs. This system provides a novel means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac muscle in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic micro-dystrophin gene excision. We examined the time course of full-length and micro-dystrophin mRNA as a biologically relevant surrogate for intact gene excision efficiency. Our data showed evidence of significant full-length dystrophin cDNA and micro-dystrophin gene excision, with a complete loss of micro-dystrophin mRNA and a gene excision efficiency of 80% for full-length dystrophin. Heart tissues were then extracted for protein quantitative analysis of micro-dystrophin (5, 10 and 30 days) and of full-length dystrophin (1, 3 and 6 months) post tamoxifen administration. Results reported a fast turnover rate for micro-dystrophin in the heart, with calculated half-life of between 5-7 days. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Studies of in vivo full-length and micro-dystrophin protein half-life in skeletal muscle, are ongoing. This work will provide key information required for long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.

McNamara, maria; Beech, Lauren; Nelson, David; Krishna, Ashwin; Schadler, Aric; Bacon, Matthew; Bauer, John; Shenoi, Asha Author Information

Background: Hypertension is a recognized concern in obese children and can include short-term and long-term consequences. Early life hypertension is linked to increased risks for other cardiovascular disease states during adulthood, with large social and medical costs. 24-hr ambulatory blood pressure monitoring (ABPM) has become one of the most important tools in diagnosing hypertension in children and circadian patterns of blood pressure may be important disease-risks predictors. Design/Methods: We conducted a retrospective chart review with patients aged 7-21 …