optogenetic-activation-intrinsic-cardiac-autonomic-neurons-excised

Optogenetic Activation of Intrinsic Cardiac Autonomic Neurons in Excised Perfused Mouse Hearts

A balance of cholinergic and catecholaminergic activation is necessary to maintain heart health. Interrogating the interaction between these pathways can ...

The George Washington University

Obstructive sleep apnea is a prevalent and poorly treated cardiovascular disease that leads to hypertension and autonomic imbalance. Recent studies that restore cardiac parasympathetic tone using selective activation of hypothalamic oxytocin neurons have shown beneficial cardiovascular outcomes in animal models of cardiovascular disease. This study aimed to determine if chemogenetic activation of hypothalamic oxytocin neurons in animals with existing obstructive sleep apnea-induced hypertension would reverse or blunt the progression of autonomic and cardiovascular dysfunction.

Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653nm vs. 510nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, Ksv, were observed during the initial 40 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4 to 8, with a small ratio reduction for pH>10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6 to 0.8s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels < 5% within 3 -10 hours for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics in vitro for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.

High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) represent a scalable experimental model relevant to human physiology. Oxygen consumption of hiPSC-CMs has not been studied in high-throughput (HT) format plates used in pre-clinical studies. Here, we provide comprehensive characterization and validation of a system for HT long-term optical measurements of peri-cellular oxygen in cardiac syncytia (human iPSC-CM and human cardiac fibroblasts), grown in glass-bottom 96-well plates. Laser-cut oxygen sensors having a ruthenium dye and an oxygen-insensitive reference dye were used. Ratiometric measurements (409 nm excitation) reflected dynamic changes in oxygen, as validated with simultaneous Clark electrode measurements. Emission ratios (653 nm vs. 510 nm) were calibrated for percent oxygen using two-point calibration. Time-dependent changes in the Stern-Volmer parameter, , were observed during the initial 40-90 min of incubation, likely temperature-related. Effects of pH on oxygen measurements were negligible in the pH range of 4-8, with a small ratio reduction for pH > 10. Time-dependent calibration was implemented, and light exposure time was optimized (0.6-0.8 s) for oxygen measurements inside an incubator. Peri-cellular oxygen dropped to levels <5% within 3-10 h for densely-plated hiPSC-CMs in glass-bottom 96-well plates. After the initial oxygen decrease, samples either settled to low steady-state or exhibited intermittent peri-cellular oxygen dynamics. Cardiac fibroblasts showed slower oxygen depletion and higher steady-state levels without oscillations, compared to hiPSC-CMs. Overall, the system has great utility for long-term HT monitoring of peri-cellular oxygen dynamics  for tracking cellular oxygen consumption, metabolic perturbations, and characterization of the maturation of hiPSC-CMs.

Pediatric obstructive sleep apnea poses a significant health risk, with potential long-term consequences on cardiovascular health. This study explores the dichotomous nature of neonatal cardiac response to chronic intermittent hypoxia (CIH) between males and females, aiming to fill a critical knowledge gap in the understanding of sex-specific cardiovascular consequences of sleep apnea in early life. Neonates were exposed to CIH until p28 and underwent comprehensive in vivo physiological assessments, including whole-body plethysmography, treadmill stress-tests, and echocardiography. Results indicated that male CIH rats weighed 13.7% less than age-matched control males (p = 0.0365), while females exhibited a mild yet significant increased respiratory drive during sleep (93.94 ± 0.84 vs. 95.31 ± 0.81;p = 0.02). Transcriptomic analysis of left ventricular tissue revealed a substantial sex-based difference in the cardiac response to CIH, with males demonstrating a more pronounced alteration in gene expression compared to females (5986 vs. 3174 genes). The dysregulated miRNAs in males target metabolic genes, potentially predisposing the heart to altered metabolism and substrate utilization. Furthermore, CIH in males was associated with thinner left ventricular walls and dysregulation of genes involved in the cardiac action potential, possibly predisposing males to CIH-related arrhythmia. These findings emphasize the importance of considering sex-specific responses in understanding the cardiovascular implications of pediatric sleep apnea.

Article	Year
Hypothalamic Oxytocin Neuron Activation Attenuates Intermittent Hypoxia-Induced Hypertension and Cardiac Dysfunction in an Animal Model of Sleep Apnea. Hypertension (Dallas, Tex. : 1979)\| PubMed ID: 36794581	2023
High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing. bioRxiv : the preprint server for biology\| PubMed ID: 37163022	2023
High-throughput optical sensing of peri-cellular oxygen in cardiac cells: system characterization, calibration, and testing. Frontiers in bioengineering and biotechnology\| PubMed ID: 37397961	2023
Sex differences in cardiac transcriptomic response to neonatal sleep apnea. Physiological reports\| PubMed ID: 38981849	2024

Rebekah Russo

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