Differentiation of COVID-19-Associated Multisystem Inflammatory Syndrome From Kawasaki Disease With the Use of Cardiac Biomarkers.

BACKGROUND: Multisystem inflammatory syndrome in children (MIS-C) after COVID-19 shares clinical similarities to Kawasaki disease (KD). We sought to determine whether cardiac biomarker levels differentiate MIS-C from KD and their association with cardiac involvement. METHODS: Subjects included 38 MIS-C patients with confirmed prior COVID-19 and 32 prepandemic and 38 contemporaneous KD patients with no evidence of COVID-19. Patient, clinical, echocardiographic, electrocardiographic, and laboratory data timed within 72 hours of cardiac biomarker assessment were abstracted. Groups were compared, and regression analyses were used to determine associations between biomarker levels, diagnosis and cardiac involvement, adjusting for clinical factors. RESULTS: MIS-C patients had fewer KD clinical features, with more frequent shock, intensive care unit admission, inotrope requirement, and ventricular dysfunction, with no difference regarding coronary artery involvement. Multivariable regression analysis showed that both higher N-terminal pro-B-type natriuretic peptide (NT-proBNP) and cardiac troponin I (TnI) were associated with MIS-C vs KD, after adjusting for significant covariates. Receiver operating characteristic curves for diagnosis showed that any detectable TnI greater than 10 ng/L was predictive of MIS-C vs KD with 91% sensitivity and 76% specificity. NT-proBNP > 2000 ng/L predicted MIS-C vs KD with 82% sensitivity and 82% specificity. Higher TnI but not NT-proBNP was associated with lower LV ejection fraction. Neither biomarker was associated with coronary artery involvement. CONCLUSIONS: Positive TnI and higher NT-proBNP may differentiate MIS-C from KD, which may become more relevant as evidence of prior COVID-19 becomes more challenging to determine. Cardiac biomarkers may have limited associations with cardiac involvement in this setting.

Off-Pump Double Coronary Artery Bypass in a 14-Year-Old With Kawasaki Disease.

A 14-year-old male patient with a history of atypical Kawasaki disease at age 2 presents with triple vessel giant coronary aneurysms. Over the last several years, he began experiencing angina and dyspnea on exertion, which was a result of fully occluded right coronary and left circumflex arteries and 90% stenosis in the left anterior descending artery. He underwent off-pump coronary artery bypass using the left and right internal mammary arteries. At 18-month follow-up, there is no evidence of ischemia. Off-pump bypass is a feasible option for surgical management of the stenotic and occlusive complications of Kawasaki disease.

Perspective on precision medicine in paediatric heart failure.

In 2015, President Obama launched the Precision Medicine Initiative (PMI), which introduced new funding to a method of research with the potential to study rare and complex diseases. Paediatric heart failure, a heterogeneous syndrome affecting approximately 1 in 100000 children, is one such condition in which precision medicine techniques may be applied with great benefit. Current heart failure therapies target downstream effects of heart failure rather than the underlying cause of heart failure. As such, they are often ineffective in paediatric heart failure, which is typically of primary (e.g. genetic) rather than secondary (e.g. acquired) aetiology. It is, therefore, important to develop therapies that can target the causes of heart failure in children with greater specificity thereby decreasing morbidity, mortality and burden of illness on both patients and their families. The benefits of co-ordinated research in genomics, proteomics, metabolomics, transcriptomics and phenomics along with dietary, lifestyle and social factors have led to novel therapeutic and prognostic applications in other fields such as oncology. Applying such co-ordinated research efforts to heart failure constitutes an important step in advancing care and improving the lives of those affected.

Microinjection Technique for Assessment of Gap Junction Function.

Gap junctions are essential for the proper function of many native mammalian tissues including neurons, cardiomyocytes, embryonic tissues, and muscle. Assessing these channels is therefore fundamental to understanding disease pathophysiology, developing therapies for a multitude of acquired and genetic conditions, and providing novel approaches to drug delivery and cellular communication. Microinjection is a robust, albeit difficult, technique, which provides considerable information that is superior to many of the simpler techniques due to its ability to isolate cells, quantify kinetics, and allow cross-comparison of multiple cell lines. Despite its user-dependent nature, the strengths of the technique are considerable and with the advent of new, automation technologies may improve further. This text describes the basic technique of microinjection and briefly discusses modern automation advances that can improve the success rates of this technique.

Robotic adherent cell injection for characterizing cell-cell communication.

Compared to robotic injection of suspended cells (e.g., embryos and oocytes), fewer attempts were made to automate the injection of adherent cells (e.g., cancer cells and cardiomyocytes) due to their smaller size, highly irregular morphology, small thickness (a few micrometers thick), and large variations in thickness across cells. This paper presents a robotic system for automated microinjection of adherent cells. The system is embedded with several new capabilities: automatically locating micropipette tips; robustly detecting the contact of micropipette tip with cell culturing surface and directly with cell membrane; and precisely compensating for accumulative positioning errors. These new capabilities make it practical to perform adherent cell microinjection truly via computer mouse clicking in front of a computer monitor, on hundreds and thousands of cells per experiment (versus a few to tens of cells as state of the art). System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4000 cells. This paper also reports the use of the new robotic system to perform cell-cell communication studies using large sample sizes. The gap junction function in a cardiac muscle cell line (HL-1 cells), for the first time, was quantified with the system.

TMEM43 mutation p.S358L alters intercalated disc protein expression and reduces conduction velocity in arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a myocardial disease characterized by fibro-fatty replacement of myocardium in the right ventricular free wall and frequently results in life-threatening ventricular arrhythmias and sudden cardiac death. A heterozygous missense mutation in the transmembrane protein 43 (TMEM43) gene, p.S358L, has been genetically identified to cause autosomal dominant ARVC type 5 in a founder population from the island of Newfoundland, Canada. Little is known about the function of the TMEM43 protein or how it leads to the pathogenesis of ARVC. We sought to determine the distribution of TMEM43 and the effect of the p.S358L mutation on the expression and distribution of various intercalated (IC) disc proteins as well as functional effects on IC disc gap junction dye transfer and conduction velocity in cell culture. Through Western blot analysis, transmission electron microscopy (TEM), immunofluorescence (IF), and electrophysiological analysis, our results showed that the stable expression of p.S358L mutation in the HL-1 cardiac cell line resulted in decreased Zonula Occludens (ZO-1) expression and the loss of ZO-1 localization to cell-cell junctions. Junctional Plakoglobin (JUP) and α-catenin proteins were redistributed to the cytoplasm with decreased localization to cell-cell junctions. Connexin-43 (Cx43) phosphorylation was altered, and there was reduced gap junction dye transfer and conduction velocity in mutant TMEM43-transfected cells. These observations suggest that expression of the p.S358L mutant of TMEM43 found in ARVC type 5 may affect localization of proteins involved in conduction, alter gap junction function and reduce conduction velocity in cardiac tissue.

High-throughput measurement of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

Cell surface expression of human ether-a-go-go-related gene (hERG) channels is regulated by caveolin-3 protein via the ubiquitin ligase Nedd4-2.

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (I(Kr)) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native I(Kr). Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology.

Interaction between the cardiac rapidly (IKr) and slowly (IKs) activating delayed rectifier potassium channels revealed by low K+-induced hERG endocytic degradation.

Cardiac repolarization is controlled by the rapidly (I(Kr)) and slowly (I(Ks)) activating delayed rectifier potassium channels. The human ether-a-go-go-related gene (hERG) encodes I(Kr), whereas KCNQ1 and KCNE1 together encode I(Ks). Decreases in I(Kr) or I(Ks) cause long QT syndrome (LQTS), a cardiac disorder with a high risk of sudden death. A reduction in extracellular K(+) concentration ([K(+)](o)) induces LQTS and selectively causes endocytic degradation of mature hERG channels from the plasma membrane. In the present study, we investigated whether I(Ks) compensates for the reduced I(Kr) under low K(+) conditions. Our data show that when hERG and KCNQ1 were expressed separately in human embryonic kidney (HEK) cells, exposure to 0 mM K(+) for 6 h completely eliminated the mature hERG channel expression but had no effect on KCNQ1. When hERG and KCNQ1 were co-expressed, KCNQ1 significantly delayed 0 mM K(+)-induced hERG reduction. Also, hERG degradation led to a significant reduction in KCNQ1 in 0 mM K(+) conditions. An interaction between hERG and KCNQ1 was identified in hERG+KCNQ1-expressing HEK cells. Furthermore, KCNQ1 preferentially co-immunoprecipitated with mature hERG channels that are localized in the plasma membrane. Biophysical and pharmacological analyses indicate that although hERG and KCNQ1 closely interact with each other, they form distinct hERG and KCNQ1 channels. These data extend our understanding of delayed rectifier potassium channel trafficking and regulation, as well as the pathology of LQTS.