SARS-CoV-2, the virus underlying COVID-19, has now been recognized to cause multiorgan disease with a systemic effect on the host. To effectively combat SARS-CoV-2 and the subsequent development of COVID-19, it is critical to detect, monitor, and model viral pathogenesis. In this review, we discuss recent advancements in microfluidics, organ-on-a-chip, and human stem cell-derived models to study SARS-CoV-2 infection in the physiological organ microenvironment, together with their limitations. Microfluidic-based detection methods have greatly enhanced the rapidity, accessibility, and sensitivity of viral detection from patient samples. Engineered organ-on-a-chip models that recapitulate in vivo physiology have been developed for many organ systems to study viral pathology. Human stem cell-derived models have been utilized not only to model viral tropism and pathogenesis in a physiologically relevant context but also to screen for effective therapeutic compounds. The combination of all these platforms, along with future advancements, may aid to identify potential targets and develop novel strategies to counteract COVID-19 pathogenesis.

BACKGROUND: Postnatal corticosteroids are used in the critical care of preterm infants for the prevention and treatment of bronchopulmonary dysplasia. We aimed to investigate the effects of early postnatal dexamethasone therapy and dose on cardiac maturation and morphology in preterm lambs.
METHODS: Lambs were delivered prematurely at ~128 days of gestational age and managed postnatally according to best clinical practice. Preterm lambs were administered dexamethasone daily at either a low-dose (n = 9) or a high-dose (n = 7), or were naïve to steroid treatment and administered saline (n = 9), over a 7-day time-course. Hearts were studied at postnatal Day 7 for gene expression and assessment of myocardial structure.
RESULTS: High-dose dexamethasone treatment in the early postnatal period led to marked differences in cardiac gene expression, altered cardiomyocyte maturation and reduced cardiomyocyte endowment in the right ventricle, as well as increased inflammatory infiltrates into the left ventricle. Low-dose exposure had minimal effects on the preterm heart.
CONCLUSIONS: Neonatal dexamethasone treatment led to adverse effects in the preterm heart in a dose-dependent manner within the first week of life. The observed cardiac changes associated with high-dose postnatal dexamethasone treatment may influence postnatal growth and remodeling of the preterm heart and subsequent long-term cardiac function.

Pulsed electrical field (PEF) energy is a promising technique for catheter ablation of cardiac arrhythmias. In this article, the key aspects that need to be considered for safe and effective PEF delivery are reviewed, and their impact on clinical feasibility is discussed. The most important benefit of PEF appears to be the ability to kill cells through mechanisms that do not alter stromal proteins, sparing sensitive structures to improve safety, without sacrificing cardiomyocyte ablation efficacy. Many parameters affect PEF treatment outcomes, including pulse intensity, waveform shape, and number of pulses, as well as electrode configuration and geometry. These physical and electrical characteristics must be titrated carefully to balance target tissue effects with collateral implications (muscle contraction, temperature rise, risk of electrical arcing events). It is important to note that any combination of parameters affecting PEF needs to be tested for clinical efficacy and safety. Applying PEF clinically requires knowledge of the fundamentals of this technology to exploit its opportunities and generate viable, durable health improvements for patients.

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Activation of AT1 (type 1 Ang) receptors stimulates cardiomyocyte hypertrophy in vitro. Accordingly, it has been suggested that regression of cardiac hypertrophy associated with renin-Ang system blockade is due to inhibition of cellular actions of Ang II in the heart, above and beyond their effects to reduce pressure overload. We generated 2 distinct mouse lines with cell-specific deletion of AT1A receptors, from cardiomyocytes. In the first line (C-SMKO), elimination of AT1A receptors was achieved using a heterologous Cre recombinase transgene under control of the Sm22 promoter, which expresses in cells of smooth muscle lineage including cardiomyocytes and vascular smooth muscle cells of conduit but not resistance vessels. The second line (R-SMKO) utilized a Cre transgene knocked-in to the Sm22 locus, which drives expression in cardiac myocytes and vascular smooth muscle cells in both conduit and resistance arteries. Thus, although both groups lack AT1 receptors in the cardiomyocytes, they are distinguished by presence (C-SMKO) or absence (R-SMKO) of peripheral vascular responses to Ang II. Similar to wild-types, chronic Ang II infusion caused hypertension and cardiac hypertrophy in C-SMKO mice, whereas both hypertension and cardiac hypertrophy were reduced in R-SMKOs. Thus, despite the absence of AT1A receptors in cardiomyocytes, C-SMKOs develop robust cardiac hypertrophy. By contrast, R-SMKOs developed identical levels of hypertrophy in response to pressure overload-induced by transverse aortic banding. Our findings suggest that direct activation of AT1 receptors in cardiac myocytes has minimal influence on cardiac hypertrophy induced by renin-Ang system activation or pressure overload.

The sarcolemma of cardiomyocytes contains many proteins that are essential for electromechanical function in general, and excitation-contraction coupling in particular. The distribution of these proteins is nonuniform between the bulk sarcolemmal surface and membrane invaginations known as transverse tubules (TT). TT form an intricate network of fluid-filled conduits that support electromechanical synchronicity within cardiomyocytes. Although continuous with the extracellular space, the narrow lumen and the tortuous structure of TT can form domains of restricted diffusion. As a result of unequal ion fluxes across cell surface and TT membranes, limited diffusion may generate ion gradients within TT, especially deep within the TT network and at high pacing rates.
We postulate that there may be an advective component to TT content exchange, wherein cyclic deformation of TT during diastolic stretch and systolic shortening serves to mix TT luminal content and assists equilibration with bulk extracellular fluid.
Using electron tomography, we explore the 3-dimensional nanostructure of TT in rabbit ventricular myocytes, preserved at different stages of the dynamic cycle of cell contraction and relaxation. We show that cellular deformation affects TT shape in a sarcomere length-dependent manner and on a beat-by-beat time-scale. Using fluorescence recovery after photobleaching microscopy, we show that apparent speed of diffusion is affected by the mechanical state of cardiomyocytes, and that cyclic contractile activity of cardiomyocytes accelerates TT diffusion dynamics.
Our data confirm the existence of an advective component to TT content exchange. This points toward a novel mechanism of cardiac autoregulation, whereby the previously implied increased propensity for TT luminal concentration imbalances at high electrical stimulation rates would be countered by elevated advection-assisted diffusion at high mechanical beating rates. The relevance of this mechanism in health and during pathological remodeling (eg, cardiac hypertrophy or failure) forms an exciting target for further research.

The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined.
Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation.
We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos.
We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

After birth, cycling mammalian CMs (cardiomyocytes) progressively lose the ability to undergo cytokinesis and hence they become binucleated, which leads to cell cycle exit and loss of regenerative capacity. During late embryonic and early postnatal heart growth, CM development is accompanied by an expansion of the cardiac fibroblast (cFb) population and compositional changes in the ECM (extracellular matrix). Whether and how these changes influence cardiomyocyte cytokinesis is currently unknown.
To elucidate the role of postnatal cFbs and the ECM in cardiomyocyte cytokinesis and identify ECM proteins that promote cardiomyocyte cytokinesis.
Using primary rat cardiomyocyte cultures, we found that a proportion of postnatal, but not embryonic, cycling cardiomyocytes fail to progress through cytokinesis and subsequently binucleate, consistent with published reports of in vitro and in vivo observations. Direct coculture with postnatal cFbs increased cardiomyocyte binucleation, which could be inhibited by RGD peptide treatment. In contrast, cFb-conditioned medium or transwell coculture did not significantly increase cardiomyocyte binucleation, suggesting that cFbs inhibit cardiomyocyte cytokinesis through ECM modulation rather than by secreting diffusible factors. Furthermore, we found that both embryonic and postnatal CMs binucleate at a significantly higher rate when cultured on postnatal cFb-derived ECM compared with embryonic cFb-derived ECM. These cytokinetic defects correlate with cardiomyocyte inefficiency in mitotic rounding, a process which is key to successful cytokinesis. To identify ECM proteins that modulate cardiomyocyte cytokinesis, we compared the composition of embryonic and postnatal cFb-derived ECM by mass spectrometry followed by functional assessment. We found that 2 embryonically enriched ECM proteins, SLIT2 and NPNT (nephronectin), promote cytokinesis of postnatal CMs in vitro and in vivo.
We identified the postnatal cardiac ECM as a nonpermissive environment for cardiomyocyte cytokinesis and uncovered novel functions for the embryonic ECM proteins SLIT2 and NPNT (nephronectin) in promoting postnatal cardiomyocyte cytokinesis. Graphic Abstract: A graphic abstract is available for this article.

Cardiotoxic β1 adrenergic receptor (β1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β1AR and organizes a receptor signalosome.
We aim to elucidate the dynamics of β1AR-SAP97 signalosome and its potential role in chronic cardiotoxic β1AR-CaMKII signaling that contributes to development of heart failure.
The integrity of cardiac β1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β1AR-SAP97 complex and increases in CaMKII activity in hearts.
These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.