Regulation of cardiomyocyte maturation during critical perinatal window

Abstract

A primary limitation in the use of pluripotent stem cell‐derived cardiomyocytes (PSC‐CMs) for both patient health and scientific investigation is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive structural, functional, and metabolic changes during maturation By contrast, PSC‐CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. There is thus a significant need to understand the biological processes underlying proper CM maturation in vivo. Here, we discuss what is known regarding the initiation and coordination of CM maturation. We postulate that there is a critical perinatal window, ranging from embryonic day 18.5 to postnatal day 14 in mice, in which the maturation process is exquisitely sensitive to perturbation. While the initiation mechanisms of this process are unknown, it is increasingly clear that maturation proceeds through interconnected regulatory circuits that feed into one another to coordinate concomitant structural, functional, and metabolic CM maturation. We highlight PGC1α, SRF, and the MEF2 family as transcription factors that may potentially mediate this cross‐talk. We lastly discuss several emerging technologies that will facilitate future studies into the mechanisms of CM maturation. Further study will not only produce a better understanding of its key processes, but provide practical insights into developing a robust strategy to produce mature PSC‐CMs.

Here, we postulate that there is a critical window, ranging from embryonic day 18.5 to postnatal day 14 in mice, in which interconnected regulatory circuits enable coordinated, concomitant structural, functional, and metabolic cardiomyocyte maturation.2

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What grows together, goes together: assessing variability in cardiomyocyte function

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Ionotropic and metabotropic kainate receptor signaling regulates Cl− homeostasis and GABAergic inhibition

Key Points

KCC2 plays a critical role in regulating chloride homeostasis, which is essential for hyperpolarizing inhibition in the mature nervous system. KCC2 interacts with many proteins involved in excitatory neurotransmission, including the GluK2 subunit of the kainate receptor (KAR). We show that activation of KARs hyperpolarizes the reversal potential for GABA (E_GABA) through both ionotropic and metabotropic signalling mechanisms. KCC2 is required for the metabotropic KAR‐mediated regulation of E_GABA, but ionotropic KAR signalling can hyperpolarize E_GABA independent of KCC2 transporter function. The KAR‐mediated hyperpolarization of E_GABA is absent in the GluK1/2^−/− mouse and is independent of zinc release from mossy fibre terminals. The ability of KARs to regulate KCC2 function may have implications in diseases with disrupted excitation: inhibition balance, such as epilepsy, neuropathic pain, autism spectrum disorders, and down syndrome.

Abstract

Potassium‐chloride co‐transporter 2 (KCC2) plays a critical role in the regulation of chloride (Cl⁻) homeostasis within mature neurons. KCC2 is a secondarily active transporter that extrudes Cl⁻ from the neuron, which maintains a low intracellular Cl⁻ concentration [Cl⁻]. This results in a hyperpolarized reversal potential of GABA (E_GABA), which is required for fast synaptic inhibition in the mature central nervous system. KCC2 also plays a structural role in dendritic spines and at excitatory synapses, and interacts with 'excitatory' proteins, including the GluK2 subunit of kainate receptors (KARs). KARs are glutamate receptors that display both ionotropic and metabotropic signalling. We show that activating KARs in the hippocampus hyperpolarizes E_GABA, thus strengthening inhibition. This hyperpolarization occurs through both ionotropic and metabotropic KAR signalling in the CA3 region, is absent in the GluK1/2^−/− mouse, and is independent of zinc release from mossy fibre terminals. The metabotropic signalling mechanism is dependent on KCC2, but the ionotropic signalling mechanism produces a hyperpolarization of E_GABA even in the absence of KCC2 transporter function. These results demonstrate a novel functional interaction between a glutamate receptor and KCC2, a transporter critical for maintaining inhibition, suggesting that the KAR: KCC2 complex may play an important role in excitatory: inhibitory (E:I) balance in the hippocampus. Additionally, the ability of KARs to regulate chloride homeostasis independently of KCC2 suggests that KAR signalling can regulate inhibition through multiple mechanisms. Activation of kainate‐type glutamate receptors could serve as an important mechanism for increasing the strength of inhibition during periods of strong glutamatergic activity.

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Inspiratory pressure‐generating capacity is preserved during ventilatory and non‐ventilatory behaviours in young dystrophic mdx mice despite profound diaphragm muscle weakness

Key points

Respiratory muscle weakness is a major feature of Duchenne muscular dystrophy (DMD), yet little is known about the neural control of the respiratory muscles in DMD and animal models of dystrophic disease. Substantial diaphragm muscle weakness is apparent in young (8‐week‐old) mdx mice, but ventilatory capacity in response to maximum chemostimulation in conscious mice is preserved. Peak volume‐ and flow‐related measures during chemoactivation are equivalent in anaesthetized, vagotomized wild‐type and mdx mice. Diaphragm and T3 external intercostal EMG activities are lower during protracted sustained airway occlusion in mdx compared with wild‐type mice. Yet, peak inspiratory pressure generation is remarkably well preserved. Despite profound diaphragm weakness and lower muscle activation during maximum non‐ventilatory efforts, inspiratory pressure‐generating capacity is preserved in young adult mdx mice, revealing compensation in support of respiratory system performance that is adequate, at least early in dystrophic disease.

Abstract

Diaphragm dysfunction is recognized in the mdx mouse model of muscular dystrophy, however there is a paucity of information concerning the neural control of dystrophic respiratory muscles. In young adult (8 weeks of age) male wild‐type and mdx mice, we assessed ventilatory capacity, neural activation of the diaphragm and external intercostal (EIC) muscles and inspiratory pressure‐generating capacity during ventilatory and non‐ventilatory behaviours. We hypothesized that respiratory muscle weakness is associated with impaired peak inspiratory pressure‐generating capacity in mdx mice. Ventilatory responsiveness to hypercapnic hypoxia was determined in conscious mice by whole‐body plethysmography. Diaphragm isometric and isotonic contractile properties were determined ex vivo. In anaesthetized mice, thoracic oesophageal pressure, and diaphragm and EIC electromyogram (EMG) activities were recorded during baseline conditions and sustained tracheal occlusion for 30–40s. Despite substantial diaphragm weakness, mdx mice retain the capacity to enhance ventilation during hypercapnic hypoxia. Peak volume‐ and flow‐related measures were also maintained in anaesthetized, vagotomized mdx mice. Peak inspiratory pressure was remarkably well preserved during chemoactivated breathing, augmented breaths, and maximal sustained efforts during airway obstruction in mdx mice. Diaphragm and EIC EMG activities were lower during airway obstruction in mdx compared with wild‐type mice. We conclude that ventilatory capacity is preserved in young mdx mice. Despite profound respiratory muscle weakness and lower diaphragm and EIC EMG activities during high demand in mdx mice, peak inspiratory pressure is preserved, revealing adequate compensation in support of respiratory system performance, at least early in dystrophic disease. We suggest that a progressive loss of compensation during advancing disease, combined with diaphragm dysfunction, underpins the development of respiratory system morbidity in dystrophic diseases.

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Genome editing for disease locus dissection

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Validity of Field and Laboratory Three-Compartment Models in Healthy Adults

Modified laboratory- and field-based multi-compartment models have been found valid for estimating body composition. However, the comparability between these models is unknown. Purpose This study determined the validity of field and laboratory three-compartment (3C) models in healthy adults. Methods One-hundred twenty participants (63 men and 57 women; age = 22 ± 5 years; BMI = 24.9 ± 3.9 kg/m2) participated in this study. A criterion four-compartment model (4C-Criterion) was determined with underwater weighing for body volume (BV), bioimpedance spectroscopy (BIS) for total body water (TBW), and dual energy X-ray absroptiometry (DXA) for bone mineral content (BMC). Modified laboratory-based 3C models were determined using BIS for TBW and two separate DXA BV equations (3C-DXASR and 3C-DXAW) whereas a field-based 3C model (3C-Field) was obtained using single-frequency bioimpedance analysis for TBW and skinfold-derived BV. In addition, a stand-alone DXA assessment was evaluated. Results The effect size of the mean differences when compared to the 4C-Criterion were trivial to small for all modified 3C models and DXA when estimating fat mass, fat-free mass, and body fat percentage. The standard error of estimate and 95% limtis of agreement for all modified 3C models and DXA were similar and considered acceptable. However, 3C-Field produced the lowest total error (TE) values and 3C-DXASR produced slightly lower TE values than 3C-DXAW and DXA. Conclusion The present study found that all modified 3C models and DXA exhibited acceptable errors. When performed by expert personnel, a field-based 3C model appears to be a viable alternative to laboratory-derived models in the young healthy adults. Corresponding Author: Brett S. Nickerson, Ph.D., CSCS*D, EP-C, College of Nursing and Health Sciences, Texas A&M International University, 5201 University Boulevard, Laredo, TX 78041. 956-326-2696. brett.nickerson@tamiu.edu Results of the present study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. The authors did not receive any funding for this project and have no conflict of interest to declare in relation to any products used in the current study. Results of the present study do not constitute endorsement by the American College of Sports Medicine. Accepted for publication: 13 December 2018. © 2018 American College of Sports Medicine

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pQCT- and Ultrasound-based Muscle and Fat Estimate Errors after Resistance Exercise

Purpose Resistance exercise increases blood flow, induces osmotic and hydrostatic fluid shifts during and immediately after exercise, and may trigger inflammatory responses for several days in the working muscle. The resultant muscle swelling can subsequently affect muscle size and quality assessments. However, the effects of muscle swelling on x-ray attenuation of adipose estimate errors are unknown. Methods Peripheral quantitative computed tomography (pQCT) and ultrasonography were used to assess muscle and adipose tissue properties of both upper arms before, 24, 48 and 72 h after unilateral resistance exercise. Recreationally active participants (n=17) completed the exercise while their contralateral limb served as a control. Results Resistance exercise resulted in a significant increase in pQCT-derived muscle CSA (includes intermuscular adipose tissue [IMAT] area), muscle area (excludes IMAT area) and IMAT area, and ultrasound-derived muscle thickness at 24, 48, and 72 h. A significant decrease in pQCT-derived muscle density was also detected as well as an increase in ultrasound-derived echo intensity at 48 and 72 h. The changes in muscle area, IMAT area and muscle density were significantly correlated with changes in echo intensity, while the changes in muscle CSA and IMAT area were significantly correlated with changes in muscle thickness. Conclusion Unaccustomed resistance exercise can cause errors in pQCT- and ultrasound-based muscle and adipose estimates for at least 72 h. These errors are the result of muscle swelling likely caused by muscle blood flow and inflammation-dependent fluid shifts in muscle. These findings may have implications for measurements in other inflammatory conditions. Corresponding author: Grant S. Rowe, Centre for Exercise and Sports Science Research (CESSR), School of Medical and Health Sciences, Edith Cowan University, 270 Joondalup Drive, Joondalup, Perth, Australia 6027. Email: g.rowe@ecu.edu.au Funding Sources: No funds were received to complete this work. Compliance with the ethical standards Conflict of Interest: The authors declare that they have no conflict of interest. Ethical approval: All procedures performed herein were in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Declaration: The present study does not constitute endorsement by ACSM. The results of this study are presented clearly, honestly, and without fabrication, falsification, or inappropriate data manipulation. Accepted for publication: 7 December 2018. © 2018 American College of Sports Medicine

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