key: cord-0076108-it469lv5
authors: nan
title: Full Issue PDF
date: 2022-03-28
journal: JACC Basic Transl Sci
DOI: 10.1016/s2452-302x(22)00064-x
sha: f15acf4a21b039413f85b88a0f0aaa9520e390f7
doc_id: 76108
cord_uid: it469lv5

nan

Current knowledge regarding mechanisms underlying cardiovascular complications in patients with COVID-19 is limited and urgently needed. We shed light on a previously unrecognized mechanism and unravel a key role of red blood cells, driving vascular dysfunction in patients with COVID-19 infection. We establish the presence of profound and persistent endothelial dysfunction in vivo in patients with COVID-19. Mechanistically, we show that targeting reactive oxygen species or arginase 1 improves vascular dysfunction mediated by red blood cells.

These translational observations hold promise that restoring the redox balance in red blood cells might alleviate the clinical complications of COVID-19-associated vascular dysfunction. Early in the pandemic, one hypothesis explaining endothelial injury was proposed to be via a direct viral entry of the virus through its decoy receptor angiotensin-converting enzyme 2 (ACE2). 1 This has recently been challenged by the observation that ACE2 is not expressed in endothelial cells. 6 Thus, it is more likely that endothelial injury arises because of systemic inflammation, which is supported by the observation that plasma isolated from patients with COVID-19 induces a toxic effect on cultured endothelial cells. 7 Another cell type suggested to be affected in COVID-19 is the red blood cell (RBC). RBCs are equipped with a fascinating battery of antioxidants, which are able to resist oxidative insult for maintenance of functional integrity and cell structure. 8 In certain circumstances with excess in oxidative stress, the RBCs may switch to a redox balance promoting oxidative stress and become harmful for both resident and circulatory adjacent cell types. 8 Epidemiological data support the notion that elevated red cell distribution width represents an independent predictor for mortality from COVID-19. 9 RBCs from patients with COVID-19 also display alterations in the lipid architecture, increased oxidative metabolites, and reduction in antioxidants. 10 The functional implications of these changes in COVID-19 are unknown, however.

We recently demonstrated that oxidative stress is substantially increased in RBCs from patients with type 2 diabetes and that this alteration induces endothelial dysfunction through upregulation of arginase 1 and reactive oxygen species (ROS). 11, 12 It is therefore reasonable to hypothesize that altered function of RBCs in COVID-19 is involved in the development of endothelial dysfunction via increased oxidative stress and arginase 1. Indeed arginase 1 has been implicated in the pathogenesis of COVID-19. 13 Based on the preceding, this study was designed to test the hypothesis that patients hospitalized for COVID-19 have impairment in endothelial function and that RBCs from those patients contribute to vascular injury through alterations in redox signaling.

We observed a pronounced impairment in endothelial function in patients with COVID-19 in vivo irrespective of underlying comorbidities. Furthermore,

RBCs, but not plasma, from these patients induced impairment in both endothelium-dependent and Healthy controls were examined once. RBCs were isolated from whole blood after a washing procedure and incubated to a final hematocrit of w45% with isolated aortic rings from wild-type rats for 18 hours. 11 Endothelium-dependent (EDR) and -independent relaxations (EIR) were subsequently evaluated by application of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, in isolated organ chambers. Separate aortic segments that underwent similar incubation protocol were fixed and immunohistochemically stained for arginase 1 and Oxygen requirements anytime N/A 17 (100) Max oxygen requirement, L/min N/A 2.5 (2.0-4.0) HFNC anytime N/A 4 (24) Oxygen demand in total, d N/A 8 (3) (4) (5) (6) (7) (8) (9) Oxygen requiring at inclusion N/A 13 (76) Liters/min among those N/A 1. 11 and nitrate export with high-performance liquid chromatography. 15 , 16 The reader is referred to the Supplemental Appendix for a detailed methods section. All experimental conditions, which involved the addition of pharmacological drugs or compounds, are presented as paired observations. Two-tailed P < 0.05 was considered statistically different for all analyses.

All analyses were carried out with GraphPad Prism version 7.02 (GraphPad Software, Inc).

STUDY SUBJECTS. Study subject characteristics at inclusion are shown in Table 1 . Table 1 ).

ACUTE PHASE OF COVID-19. Previous reports from in situ postmortem studies have reported disruption of the endothelial cell lining in microvascular beds in patients with COVID-19. 4, 5 To confirm that this is reflected by endothelial dysfunction in vivo, we assessed microvascular digital endothelial function.

RHI was w33% lower in patients with COVID-19 in the acute phase compared with healthy control subjects ( Figure 1A) . Furthermore, RHI in patients with COVID-19 remained significantly impaired and did not significantly improve at follow-up 4 months after the infection ( Figures 1A and 1B) . There was no difference in RHI between patients with COVID-19 with (n ¼ 6) and without comorbidities (n ¼ 9) (Supplemental Figure 1A) . RHI was significantly lower among the patients with COVID-19 without comorbidities than in the healthy subjects (P < 0.001). Procalcitonin, mg/L -0.14 (0.08-0. 19) LD, mkat/L It is further unlikely that the observations are caused by hemolysis, as free hemoglobin in plasma did not differ between healthy subjects and patients with COVID-19 (Supplemental Figure 3 ).

THROUGH VASCULAR ARGINASE 1 AND ROS. Arginase, which is known to be a regulator of endothelial function and nitric oxide (NO) bioactivity, has been implicated in COVID-19. 13 Based on this, we hypoth- Figure 5B ). Because ROS production reciprocally regulates NO bioactivity, we hypothesized that export of NO bioactivity from C19-RBCs is compromised compared with H-RBCs. Because export of NO bioactivity from RBCs is tightly regulated by arginase, ABH was used to increase NO bioactivity with extracellular levels of the NO metabolite nitrate as the readout according to previous results. 16 There was no difference in baseline nitrate production between the groups (Supplemental Figure 4 ). Extracellular nitrate levels were significantly increased following incubation of H-RBCs with ABH, whereas they were completely unchanged following incubation of C19-RBCs ( Figure 5C ). These observations suggest that export of NO bioactivity is markedly compromised in C19-RBCs.

INTERFERONg TRIGGERS ENDOTHELIAL DYSFUNCTION INDUCED BY RBCS. As the cytokine storm is clearly a major contributor to illness in COVID-19, we investigated the impact of cytokines on the impairment in endothelial function induced by RBCs. RBCs have been proposed to represent a dynamic reservoir of cytokines. 17 We investigated whether this represents a putative mechanism behind the observed interaction between RBCs and endothelial function in COVID- 19 . We performed a Luminex technology- Given the wide range of symptoms arising from COVID- 19 , it has been speculated that many of the cardiovascular complications are attributed to endothelial injury. 1, 3 Whether vascular dysfunction and more specifically endothelial dysfunction actually is present in patients with ongoing COVID- 19 infection has not been unequivocally demonstrated, however.

The mechanisms by which such injury occurs are also poorly understood. Evidence provided so far is limited to autopsy studies, 4,5 evaluation of endothelial function several weeks after a positive PCR test, 18 and assessment of microvascular function 19, 20 tion. 22 , 23 The impairment in microvascular digital endothelial function in this cohort of patients with COVID-19 in the acute phase is even more pronounced than that previously detected using the same methodology in patients with type 2 diabetes having microand macrovascular complications. 14 This illustrates the severity of microvascular endothelial dysfunction among these patients with COVID-19.

The implications of structural 9 and oxidative 10 A feature of RBCs is their central involvement in redox regulation with prominent pro-oxidative as well as antioxidant capacities. 8 Thus, an increase in oxidative stress by the RBCs may have important biological effects. It is therefore of interest that we found a clear increase in ROS production in C19-RBCs compared with H-RBCs. This switch in redox balance toward a pro-oxidative phenotype in COVID-19 contributes to the development of endothelial dysfunction, which was evident from the rescue of endothelial function by application of a superoxide dismutase mimetic. This is corroborated by recent observations that oxidative metabolites are markedly elevated in C19-RBCs. 10 However, the levels of antioxidants in C19-RBCs are largely intact. Interestingly, one of the few antioxidants that are reduced in C19-RBCs, is superoxide dismutase. This, together with our observations, suggests that the oxidative phenotype is driven by not only increased superoxide anion radical but also a decrease in its anti-oxidative capacity. The increased formation of ROS is also reflected by the attenuated export of nitrate, the stable metabolite of NO, and a marker of NO bioactivity, from C19-RBCs. In these experiments, arginase, the reciprocal inhibitor of NO production in RBCs, 16 was inhibited to enhance the formation and export of NO bioactivity. In accordance with previous results, 16 nitrate was markedly increased in the supernatant following incubation of H-RBCs with the arginase inhibitor. By contrast, there was no increase in nitrate levels in the supernatant from C19-RBCs in the presence of arginase inhibitor. It is still under debate how and in which form RBCs are able to export NO. 12 Together with the increased ROS production, one possibility is that uncoupling of eNOS with attenuated NO formation results in less production of nitrate. Another possible explanation is that NO is exported in the form of nitrate, which is reduced in C19-RBCs. given the endothelial involvement and specific changes in COVID-19 compared with other viral infections, such as influenza, 4 11 indicating that the rat aorta is representative for this type of investigation.

The present study advances our knowledge regarding vascular complications in COVID- 19 is that all patients were on various levels of oxygen therapy, which makes it impossible to discern COVID-19Àspecific alteration versus oxygen-induced alteration of the red cell membrane and function. It is known that oxygen therapy promotes oxidation of membrane lipids, as shown by increased hydroperoxide values in the erythrocyte membrane of preterm infants exposed to oxygen therapy, 4 and that erythrocyte viscosity and aggregation are increased in patients with COVID-19 who require oxygen supplementation. 5 The changes in erythrocyte viscosity and aggregation are largely mediated by increased fibrinogen, suggesting that they may be secondary to hypercoagulability and vascular changes associated with pulmonary lesions. 5 Studies in patients with type 1 diabetes mellitus and who were exposed to hyperbaric oxygen showed decreased iNOS activity and/or expression in lymphocytes and increased plasma arginase activity, 6 which suggested that a NO-restricted state could be a consequence of oxygen therapy.

In conclusion, this is a well done and provocative study, which suggests the need for additional indepth mechanistic studies to determine the role of erythrocyte abnormalities in the vasculopathy associated with COVID-19.

The author has reported that he has no relationships relevant to the contents of this paper to disclose. CELLULARIZATION. WJ-MSCs were isolated from umbilical cord of newborn female piglets by mechanic dissociation, as previously described. 19 The isolated cells were fed with fresh medium every 3 days and expanded until passages 3 to 5. Fluorescenceactivated cell sorting analysis was used to determine cell surface marker expression. 12, 19 Expanded WJ-MSCs (passage 2 to passage 5) were seeded onto decellularized porcine SIS at a density of 2.5 Â 10 5 /cm 2 and cultured until graft maturation according to the protocol previously optimized by our group. [11] [12] 19 IN VIVO EXPERIMENTS. A total of 11 4-week-old healthy Landrace female piglets (mean weight: 20.5 AE 4.1 kg) were used in this study (9 for comparison between seeded and unseeded grafts, and 2 to define as shown in Figure 1A . Four animals were randomized to receive a graft made from commercially available SIS, whereas 5 animals received a WJ-MSC-seeded graft; the surgeon was blinded to the graft seeding. Figure 2D ).

CMR showed that laterolateral and superoinferior MPA diameters increased significantly at follow-up in the seeded group, whereas no significant growth was surements on cardiovascular magnetic resonance at baseline and at 6-month follow-up, in unseeded and seeded animals. MPA area growth was significant in seeded graft, but not in unseeded grafts (n ¼ 4, AE SD). Abbreviations as in Figure 2 . Figure 3D ).

TISSUES. At macroscopic inspection ( Figure 4A ) and on Von Kossa staining ( Figure 4B) , explanted grafts presented a smooth luminal surface with no signs of obstruction or tissue calcification and degradation in both groups.

Although hematoxylin and eosin images showed extensive nucleation throughout both seeded and unseeded grafts, a remarkably more organized cellular structure was detected in the seeded group ( Figure 4C) . Histological analysis showed a signifi- Figure 6 ). Therefore, it is most likely that This study is part of a stepwise process whose ultimate goal will be to develop a growing valved graft leaflets; therefore, we believe that in a preclinical setting the 2 components must be addressed and analyzed separately as a first step.

In a proof-of-concept study, we have previously shown the feasibility of using a mesenchymal stem cell (MSC)-seeded SIS tissue-engineered graft for MPA and RVOT reconstruction in healthy piglets. 12, 19 Additionally, we upgraded our in vitro manufacturing process of this graft to clinical grade, Good Manufacturing Practice-compliant standards. 19 Here, we assessed its function with a randomized controlled preclinical trial. Our study has 4 main findings.

First, when seeded with WJ-MSC and using our This hypothesis is also supported by works previously published by ours and other groups. 11, 12, 15 Sugiura et al 15 

This study was supported by grants from the Sir Jules Thorn Chari- However, the lack of availability and the high costs of 

The authors have reported that they have no relationships relevant to the contents of this paper to disclose. 

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined.

We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, Damaged mitochondria also leak mitochondrial DNA, which is a potent trigger for immune activation due to its physical features that mimic bacterial DNA. 1 Indeed, mitochondrial damage resulting in sterile myocardial inflammation and cardiac myocyte death is implicated in causing cardiomyopathy under pressure overload stress 2,3 and with ischemia-reperfusion injury, 4,5 whereby targeting cell death as well as deleterious myocardial inflammation are regarded as key therapeutic targets to prevent and treat heart failure. 1, 6 Mitophagy is a lysosomal process to sequester damaged mitochondria (or parts thereof) to facilitate their removal via lysosomal degradation, and serves as a defense mechanism against mitochondrial DNA sensing to drive inflammation and cell death. 2, 7 Mitophagy plays a critical role in the myocardium during perinatal cardiac growth and development, 8 with aging, 9, 10 with exhaustive exercise, 7 and in pathologic states, such as after injury. 2, [9] [10] [11] [12] [13] [14] [15] Despite these discoveries, its role in physiological function of the adult heart remains unknown. Moreover, although studies with contemporary reporters demonstrate robust rates of cardiac myocyte mitophagy in the unstressed myocardium, 16, 17 ablation of canonical mitophagy mediators such as PTENinduced kinase 1 (PINK1) and PARKIN 18 does not alter mitophagy in young adult mouse hearts. 12, 15, [19] [20] [21] Prior studies indicate that lysosome function is critical for preventing myocardial inflammation under stress, as ablation of lysosomal DNase II in cardiac myocytes, which degrades mitochondrial DNA, exacerbates myocardial inflammation to accelerate development of cardiomyopathy with pressure overload. 2 Whether physiological mitophagy suppresses myocardial inflammatory signaling in the unstressed heart remains unknown. It is also noteworthy that all currently defined mitophagy pathways are intrinsic to damaged mitochondria and recognize mitochondrial damage or dysfunction to facilitate lysosomal removal of damaged mitochondria via mitophagy or other mechanisms, 22 whereby participation of innate immune mechanisms in facilitating mitophagy remains a missing piece of this conceptual framework.

In this paper, we reveal evidence that tumor necrosis factor receptor-associated factor-2 (TRAF2), an innate immunity effector, acts as a first line of defense Representative immunoblot is shown depicting TRAF2 expression in hearts from male C57BL/6J mice (8 weeks old) subjected to ex vivo ischemia-reperfusion injury (IR) or sham (S) treatment as control. Expression of VDAC, FACL4, calreticulin, VAPB, and IRE1a is shown to evaluate cosegregation with mitochondria (mito, with VDAC), mitochondria-associated membranes (MAM, with FACL4), endoplasmic reticulum (ER, with calreticulin, VAPB, and IRE1a), and cytosol (cyto, with GAPDH). *P < 0.05 and ***P < 0.001, by t-test. No statistically significant differences were observed between the groups for TRAF2 abundance in the cyto fraction (by t-test) or ER (by Mann-Whitney test). Table 1 ). Hearts were categorized as either nonfailing (no history of heart failure, obtained from nonfailing brain-dead donors) or ischemic cardiomyopathy (obtained at time of orthotopic heart transplantation). After in situ cold cardioplegia, all hearts were placed on wet ice at 4 C in Krebs-Henseleit buffer. Transmural left ventricular (LV) samples were obtained from the LV free wall with epicardial fat excluded. The mitoKeima construct was generously provided by Dr. Atsushi Miyawaki, RIKEN brain science institute in Saitama, Japan.

ECHOCARDIOGRAPHY AND MYOCARDIAL CHARAC-TERIZATION. Two-dimensional-directed M-mode echocardiography was performed as we have previously described. 25 Histologic assessment for myocyte registration was performed with hematoxylin and eosin staining, and myocardial fibrosis was assessed with Masson's trichrome staining, as we have previously described. 25 Quantitative assessment of fibrosis was performed with Picrosirius Red staining, and images were assessed for area of the red signal as a fraction of total tissue area by Visiomorph software (Visiopharm). Transmission electron microscopy was performed in cardiac tissue as previously described. 25 

Adeno-associated virus serotype 9 [AAV9] particles coding for TRAF2 or its Rm mutant constructs (which we have described previously 26 Hearts were isolated and perfused as previously described. 28 After a 20-minute stabilization period, mouse hearts were subjected to no-flow ischemia (time [t] ¼ 0 minutes) for 30 minutes followed by reperfusion for 60 minutes (total t ¼ 90 minutes).

ADULT CARDIAC MYOCYTE ISOLATION. Adult mouse hearts were subjected to enzymatic digestion to isolate adult cardiac myocytes, as described. 29 The remaining cellular fraction was isolated as the nonmyocyte fraction. The cells were subsequently homogenized in RIPA buffer and subjected to SDS-PAGE gel electrophoresis followed by immunoblotting as we have previously described. 26 PREPARATION OF MURINE EMBRYONIC FIBRO-BLASTS. Traf2 floxed or Pink1 floxed mice were subjected to timed mating and murine embryonic fibroblasts were prepared from embryonic day 14 pups following standard protocols.

Mitochondria-enriched and cytosolic fractions were prepared from hearts and cells following the protocols we have previously described. 26 IMMUNOFLUORESCENCE ANALYSIS. We followed the protocol we have previously described. 25 Paraffinembedded heart sections (10-mm thick) were subjected to heat-induced epitope retrieval, followed by blocking, and then incubated overnight with primary antibody. After serial washes, samples were stained with Alexa Fluor 594 (Invitrogen) and mounted with fluorescent DAPI mounting medium (Vector Labs, with sequential excitation at wavelengths at 458 and 561 nm, followed by emission at 580 nm using a Nikon A1R confocal system at the Washington University

Center for Cellular Imaging. Adenoviruses expressing Cre and rat PARKIN were generated as previously described. 26 The images were quantitated using ImageJ software (NIH). Mitophagy was also assessed after injection of AAV9-cTnT-TRAF2 or AAV9-cTnTnull viral particles (with 3.5 Â 10 11 viral particles per mouse) in young adult mitoQC mice. Mitophagy index was calculated as a ratio of the average intensity of the red/green signal using ImageJ software.

ANALYSES. Assessment of transcript abundance was performed as previously described 25 using SYBR Green with primers reported in Supplemental Table 2 .

Assessment of Pink1 transcript expression was performed using TaqMan gene expression assays (Applied Biosystems) for mouse Pink1 (Mm00550827_m1).

IMMUNOBLOTTING. Immunoblotting was performed as previously described. 25 Enzyme-linked immunosorbent assays were performed on sera collected from mice at terminal bleed with the following kits per the manufacturers' transcripts in mice modeled as in A. *P < 0.05, **P < 0.01, and ***P < 0.001 by post hoc test after 1-way ANOVA for all panels except by Kruskal-Wallis test for H and J.

Abbreviations as in Figure 2 . Figure S1 ). These observations suggest that the stress-induced increase in TRAF2 localization to mitochondria may play a functional role in its cytoprotective effects, as suggested by prior studies

showing that TRAF2 is both necessary 33 and sufficient 28, 34, 35 to enhance cardiomyocyte survival after ischemia-reperfusion injury.

To examine the mechanisms for recruitment of TRAF2 to the mitochondria, we evaluated whether its Abbreviations as in Figure 2 .

mitochondrial localization depends upon PINK1. Figure 7H ). These data demonstrate that the E3 ligase domain of TRAF2 is required for myocardial homeostasis and mitochondrial quality control, mirroring prior observations in isolated cultured primary cardiomyocyte. 26 We next examined whether TRAF2 overexpression was sufficient to induce mitophagy in cardiac myocytes in the mouse heart. To test this, we employed AAV9-cTnT promoter-driven TRAF2 expression ( Figures 7C and 7D ) and compared that with expression of empty AAV9-cTnT null viral particles (as control) in hearts from mice expressing the mitoQC mitophagy reporter. 17 26 We examined PARKIN levels in TRAF2-icKO mice and did not note them to be significantly altered ( Figure 3A) . Figure S10 ). These data are consistent with a lack of role for Parkin in basal mitophagy in cardiomyocytes in young adult mouse hearts. 8, 21 To determine whether the defective mitophagy induced by TRAF2 deficiency was responsible for increased cell death, we studied Traf2 floxed MEFs Figure 8D) . Importantly, ablation of TRAF2 did not alter expression of LC3 or p62 two markers of macroautophagy (Supplemental Figure S11B) administrative assistance with conducting the study.

Graphical abstract was created with BioRender.com. 

Among the 26 patients (Table 1) those collected outside of these events ( Figure 1G ).

Together, these data suggest that EVs in plasma from patients at the baseline activated ECs to increase permeability and that this activity increased in patients with bleeding and was associated with pEVs, which were significantly increased in post-LVAD samples.

EVs PROMOTED VWF-DEPENDENT ANGIOGENESIS.

The results reported in Figure 1 led us to hypothesize that VWF þ pEVs carry angiogenic activity because increased permeability is considered to be an early stage of angiogenesis. When plasma was fractionated, we found that EVFP ( To specifically examine the effects of pEVs, we Figure 3C ), which were reduced by the GP Iba antibody AK2. This antibody blocks VWF binding to GP Iba on platelets. 29 We also detected sheardependent hemolysis (Supplemental Figure 3 ). The pEVs from platelets exposed to HSS promoted angiogenesis in a VWF-dependent manner, but those from unsheared platelet-rich plasma did not ( Figures 3D to 3F ). The vascular sprouts from AVSs cultured in pEV-supplemented GPM were shorter, denser, and hairlike ( Figure 3E ), similar to those induced by EVs from LVAD patients ( Figure 2) . These pEVs contained 537.7 AE 36.6 pg/mL of VEGF (n ¼ 6), significantly higher than that in the heterogeneous EVs from LVAD patients (n ¼ 16; t test, P ¼ 0.011).

The CD31 þ microvessels from AVSs cultured in pEVsupplemented GPM were small and often lacked intact vascular lumens ( Figure 3I ), whereas those in GRM were fully developed ( Figure 3J ). The number of CD31 þ vessels was higher in AVSs cultured with pEV and was reduced by the VWF antibody ( Figure 3K) . Figure 4G ). We also measured VWF cleaved by ADAMTS-13 and uncleaved VWF in EV and EV-free fractions of plasma samples collected at discharge of LVAD patients using mass spectrometry. 30 The ratio of cleaved VWF in EVFP to that in EV fraction was 2.27 after adjustment for total VWF, suggesting that platelet-and pEV-bound VWF was significantly less cleaved. Together, these results suggest that: 1) the ECs underwent persistent exocytosis of VWF after LVAD implants and were subjected to additional stress at the time of severe bleeding or thrombosis; 2) plasma VWF in LVAD patients was techniques. First, the co-immunoprecipitation assay showed that a recombinant A2 protein bound to the exposed A1 domain 28 to form a complex with VWF in the plasma samples of LVAD patients ( Figure 5C ). In contrast, A2 bound VWF from healthy subjects only in the presence of ristocetin ( Figure 5C ), which activates VWF to bind its platelet receptor. 29 Second, A2 blocked SIPA ( Figure 5D ), which is induced by the binding of shear stress-activated VWF to platelets. 25 Third, the thiol-containing VWF accounted for 86.4% AE 7.1% in healthy subjects but was reduced to 61.0% AE 8.1% in patients at baseline and decreased further to 42.3% AE 13.0% after LVAD implantation ( Figure 5E) , with a parallel increase of VWF in the supernatant (Supplemental Figure 6) . These results suggest that VWF multimers in LVAD patients were oxidized and underwent conformational changes to expose the A1 domain.

Exposing normal platelet-rich plasma to HSS for 5 minutes failed to induce VWF cleavage ( Figure 5F ), but it did induce significant platelet activation and aggregation by VWF (Figures 3A to 3C) . As a control, VWF was cleaved in static conditions after incubation for 16 hours in the presence of 1.5 M urea and 1 mM of BaCl 2 . An isolated A2 required 1 hour to be cleaved without added chemicals ( Figure 5G ). In contrast, VWF was partially cleaved after exposure to HSS for 60 minutes ( Figure 5H ). The cleavage was not affected by platelets (2 Â 10 5 /mL) or erythrocytes (2 Â 10 6 /mL), but it was prevented by collagen (10 mg/mL). The cleavage was similarly induced under a turbulent flow generated in a vortex Figure 6A) ; and 2) we have shown that VWF maintains its shear-induced active conformation for more than 5 hours after shear exposure has stopped, 32 allowing sufficient time for experiments to be conducted. Hemostasis was restored partially in VWF -/mice infused with VWF and completely with the VWF that was exposed to HSS for 5 minutes at 37 C ( Figure 6B, Supplemental Figure 8 ). Platelets from VWF -/mice infused with sheared VWF expressed CD62p ( Figure 6C) , developed moderate thrombocytopenia ( Figure 6D ) and generated more VWF þ pEVs ( Figure 6E ). These mice also had elevated levels of endothelial EVs ( Figure 6F) . These results suggest that VWF exposed to HSS fully restored the hemostasis of VWF -/mice. VWF before and after LVAD implantation. We made several novel observations.

First, the EVs from LVAD patients induced significant vascular permeability and aberrant angiogenesis in a VWF-dependent fashion (Figures 1 and 2) .

This finding is consistent with previous reports that EVs can transmigrate through the endothelial barrier 38, 39 and that VWF plays a role in the process. 28 is reduced in LVAD patients. We found that VWF:CB was moderately reduced post-LVAD, but it remained higher than that of healthy subjects.

The finding that VWF lost large multimers but remained hyperadhesive, albeit at reduced levels ( Figure 4) , defines a GOF phenotype with large VWF multimers lost to enhanced binding to platelets, consistent with increased VWF on platelets and pEVs found in the post-LVAD samples (Figure 1) . Consistent with the notion, VWF found on pEVs was significantly less cleaved than VWF found in plasma, suggesting that platelets were activated to generate pEVs by less cleaved and thus more adhesive VWF, which also mediated EV-EC interaction to promote angiogenesis.

This GOF phenotype is supported by the finding that the VWF exposed to HSS was more effective in restoring hemostasis in VWF-null mice (Figure 6) 65 We show that the A1 and A2

complexes are disassociated to expose the A1 domain of VWF in LVAD patients ( Figure 5C ), as schematically illustrated in Figure 7B , but did not induce cleavage likely because the VWF was oxidized ( Figure 5E A2 complex so that the A1 exposed (activated) VWF can form a complex with isolated A2. (C) Cysteine thiols of VWF can be oxidized to form intermultimeric disulfide bonds under HSS and thus cannot be precipitated by the active thiol beads, which form mix disulfide bonds with surface exposed free thiols on VWF. 68 Abbreviations as in Figures 1 and 3 . compare VWF profiles so that we will be able to more precisely define the impact of shear stress on VWF.

Third, because of a limited plasma volume, we were unable to map the specific amino acids involved in forming intermultimer disulfide bonds or being oxidized. We are developing a new mass spectrometric protocol to overcome this technical difficulty.

Finally, because of the lack of suitable research on LVADs in mouse models, we were unable to test our hypothesis of LVAD-induced GOF VWF and its synergistic actions with pEVs to promote angiogenesis in vivo. We can overcome this obstacle in experiments on large animals in the future.

In summary, we demonstrate that VWF was not 

The authors have reported that they have no relationships relevant to the contents of this paper to disclose. Normal cardiopulmonary reflexes are disrupted in disease, leading to increased sympathetic and decreased parasympathetic transmission.

Injury activates afferent nerves that mediate sympathoexcitatory-positive feedback reflexes that contribute to myocardial and/or lung injury.

We do not adequately understand (clockwise from top) the electrophysiological and biophysical properties of autonomic ganglia; the impact of sex as a biological variable; how to distinguish the roles of ganglionic versus systemic inflammation in neural remodeling; the mechanisms that drive afferent and efferent remodeling during disease; how to integrate clinical data from a variety of sources, scales, and modalities to guide therapy for specific patients; and the nature of interactions between cardiac and pulmonary nerves. and removal becomes disrupted, with elevated release and suppressed reuptake leading to high extracellular NE that is detrimental to the heart (29).

As the disease progresses, acetylcholine (Ach) replaces NE in some sympathetic neurons, and NPY synthesis and release increase, potentially contributing to pathology (30, 31) . It is now recognized that neural remodeling occurs throughout the nervous system in HF. In addition to neurochemical plasticity, morphologic changes include remodeling of axon arbors in the heart-both degeneration and regrowthas well as increased size of cell bodies and dendritic arbors within stellate and intracardiac ganglia (27, 28) .

Electrical remodeling includes enhanced excitability in stellate ganglia (32) , altered firing frequencies of sensory afferents (33) and parasympathetic efferents, as well as remodeling of connections within intracardiac ganglia (34) . These neural changes influence the heart, causing down-regulation of b1, upregulation of b2 adrenergic receptors, and modulating cardiac electrophysiology (35) . A recent clinical trial showed that propranolol, which blocks both b1 and b2 receptors, is much more effective than metoprolol in managing patients with electrical storm (36) . (201) shows a concept diagram relating stress to chronic disorders such as heart failure that involve autonomic effectors. Stress is a condition in which a homeostatic comparator senses a discrepancy between afferent information to the brain about a monitored variable and a set point or other instructions for responding. The error signal drives effectors including components of the autonomic nervous system in a manner that reduces the discrepancy. Cumulative wear and tear (allostatic load) decreases effector efficiency, eventually precipitating dyshomeostatic vicious cycles. Feed-forward anticipatory processes shift input-output curves determined by the "Regulator." The right panel shows major types of sensory afferent nerves and the corresponding abnormalities in autonomic reflexes observed in heart failure are illustrated. Sympathoexcitatory afferents are shown in green; sympathoinhibitory afferents in blue. Examples of underlying mechanisms acting at sensory, central, efferent, and effector organ sites that contribute to the reflex cardiovascular/respiratory dysregulation are noted. Aff ¼ afferent;

DRG ¼ dorsal root ganglia; HR ¼ heart rate; ParaSNA ¼ parasympathetic nerve activity; SNA ¼ sympathetic nerve activity. The link between activity of the ANS and atrial arrhythmias has been known for over a century.

Recent clinical trials have tested autonomic (122) and later reproduced in large prospective community studies (123, 124) . These studies show that SCD has a nighttime nadir followed by a morning and smaller evening peak. Subsequent studies show that the 24-hour pattern in SCD or life-threatening events is different in people living with certain types of CVD.

People with HF tend to have an early morning dip followed by an increase in the incidence of SCD and tachyarrhythmias during the daytime hours (125) .

People with hypertrophic cardiomyopathy have a bimodal distribution in the incidence of SCD that peaks in the morning and again in the evening hours (126) . for daytime hypertension and CVD (172, 173) . Insomnia and other disorders characterized by sleep fragmentation also lead to SNSA and are associated with CVD (174) . However, the roles of treating these disorders as a strategy for CVD risk reduction or for treating associated AD have not been systematically assessed.

A summary of the ANS alterations association with sleep and circadian processes and the related knowledge gaps and research priorities are depicted in Figure 5 . Figure 6 ). Consequences include perturbations of resting cardiac physiology (increased heart rate, loss of HRV, increased nocturnal blood pressure) as well as exercise physiology (diminished maximal heart rate, slower heart rate increase and recovery, decreased contractility, decreased functional capacity) (188) . Over the course of years after transplantation, there can be a process of reinnervation (which may improve functional capacity and outcomes) (189, 190) , but this is highly variable and not well-understood.

Pulmonary parasympathetic activity is enhanced in COPD and may play a critical role in the airway obstruction, airway hyper-responsiveness, and increased mucus production characteristic of this condition (184, 191) . Inhaled anticholinergic therapies have thus been the mainstay of COPD treatment (192, 193) . Similarly, disruption of pulmonary parasympathetic nerves in patients with COPD has the potential to provide long-lasting anticholinergic effects with reduction of symptoms and exacerbations.

Targeted lung denervation (TLD) is a novel bronchoscopic therapy that disrupts the afferent and efferent pulmonary branches of the vagus nerve along the outside of the main stem bronchi using radiofrequency ablation ( Figure 6 ). Initial studies have demonstrated the safety and feasibility of TLD and have shown trends toward improvements in symptoms and exacerbations (194, 195) ; its efficacy is currently being assessed in the pivotal AIRFLOW-3

(Evaluation of the Safety and Efficacy of TLD in Patients With COPD) trial (196) . Determining the appropriate radiofrequency ablation parameters in TLD is challenging because there are few direct assessments of afferent/efferent function. Because TLD also targets lung afferents, evaluation of cardiopulmonary reflexes such as respiratory sinus arrhythmia pre-and post-treatment may have the potential to assess the extent of pulmonary vagal denervation achieved with this approach (197, 198) .

1. There are critical gaps in our understanding of the contribution of specific afferent and efferent nerve subsets (199, 200) (Figure 1) . 22 These suggestions have been refined according to the evolving evidence gradually being made available from autopsy series reported from centers around the globe. One early suggestion was direct cell injury by means of SARSmechanisms may be relevant and may coexist in some patients, such as, venous thromboembolism, with pulmonary embolism being an important cause of adverse outcomes associated with COVID-19. 32 An autopsy report from Germany reported deep venous thrombosis in 58% of patients (7 of 12) in whom venous thromboembolism was not suspected before death, 13 and the incidence of alveolar capillary microthrombi was significantly higher than following H1N1 influenza. 12 Microthrombi have also been identified in the cardiac vasculature of patients with COVID-19; nevertheless, the study lacks cardiac imaging and clinical correlation to corroborate the postmortem findings, which may well be incidental rather than causal of the myocardial injury. 33 The patients. 36 Elevated interleukin-6, which was found to be one of the predictors of more rapid COVID-19 deterioration, may partly explain the acute and long-term consequences of COVID-19, including myocardial inflammation, as it is known to be a mediator of the prothrombotic state, platelet function, and antibody production. 37 Other interleukins may also be involved in acute heart failure and myocarditis. For instance, intense NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome In the acute setting, both direct (viral) and indirect (immune-mediated) damage to the myocardial and other heart tissues can give rise to perimyocarditis or myocarditis. Moreover, endothelial tissue injury, by means of endotheliitis and microthrombus formation, leads to type 2 myocardial infarction. TF ¼ tissue factor; TNF¼ tumor necrosis factor; other abbreviations as in Figure 1 .

Siripanthong et al formation in the heart is observed in myocarditis, producing the proinflammatory cytokine interleukin-1 which exacerbates the myocardial injury. 38 Figure 2 shows the proposed molecular mechanism for the indirect and indirect myocardial injury due to SARS-CoV-2. One patient had a stroke several days after being discharged in stable condition following a myocarditis episode. 65 Other milder complications include reduced exercise tolerance and persistent reduction in systolic ejection fraction. 66, 67 A full summary of the 77 case reports can be found in Supplemental Table 1 .

POST-COVID-19 SYNDROME. There is a distinct lack of data on the long-term sequelae and prognosis healthy control subjects and risk factor-matched control subjects. 17 Abnormal CMR findings were present in 78% of patients and ongoing myocardial inflammation in 60%. Native T1 and T2 mapping provided the best discriminatory ability to detect COVID-19-associated myocardial disease. These findings correlated with higher levels of high-sensitivity troponin T and active lymphocytic inflammation on endomyocardial biopsy specimens. 17 In summary, this study also highlights that myocardial inflammation can be observed radiologically (on CMR) in patients recovering from COVID-19, in both asymptomatic and symptomatic patients. 75 The long-term cardiac consequences of COVID-19 may also be dependent on the severity of initial COVID-19 itself. A recent study of health workers seropositive for SARS-CoV-2 and either mild or no symptoms detected no differences in the CMR find- following hospital discharge. 11 The investigators re- imaging, determining antecedence and prior myocardial scar or structural abnormalities is difficult, despite the use of matched non-COVID-19 control subjects and healthy control subjects. 77 In a US study of 26 competitive athletes recovering from COVID-19, 4 male athletes (15%) had CMR findings suggestive of myocarditis on the basis of the Lake Louise criteria. 75 Long-Term Consequences of COVID-19 Cardiac Injury

Autonomic modulation for cardiovascular disease

Effects of transcutaneous auricular vagus nerve stimulation on cardiovascular autonomic control in health and disease

Preclinical and clinical evaluation of autonomic function in humans

The autonomic nervous system and cardiac arrhythmias: current concepts and emerging therapies

Clinical neurocardiology defining the value of neuroscience-based cardiovascular therapeutics

Molecular and cellular neurocardiology: development, and cellular and molecular adaptations to heart disease

An atlas of vagal sensory neurons and their molecular specialization

Mapping of sensory nerve subsets within the vagal ganglia and the brainstem using reporter mice for Pirt, TRPV1, 5-HT3, and Tac1 expression

Modeling beta-adrenergic control of cardiac myocyte contractility in silico

Mechanisms of cardiac pain

Intrapericardial capsaicin and bradykinin induce different cardiacsomatic and cardiovascular reflexes in rats

Cardiac TRPV1 afferent signaling promotes arrhythmogenic ventricular remodeling after myocardial infarction

Cardiac sympathetic afferent denervation attenuates cardiac remodeling and improves cardiovascular dysfunction in rats with heart failure

Innervation and neuronal control of the mammalian sinoatrial node a comprehensive atlas

Characterization of cardiovascular reflexes evoked by airway stimulation with allylisothiocyanate, capsaicin, and ATP in Sprague-Dawley rats

Nociceptive pulmonary-cardiac reflexes are altered in the spontaneously hypertensive rat

Cardiac ganglionitis associated with sudden unexpected death

Inflammation, oxidative stress, and glial cell activation characterize stellate ganglia from humans with electrical storm

The effects of interleukin 17A on left stellate ganglion remodeling are mediated by neuroimmune communication in normal structural hearts

Neuro-immune crosstalk and allergic inflammation

Cardiac exosomes in ischemic heart disease-a narrative review. Diagnostics (Basel)

Insights into the proteomic profiling of extracellular vesicles for the identification of early biomarkers of neurodegeneration

Cardiac noradrenergic nerve terminal abnormalities in dogs with experimental congestive heart failure

Relationship between regional cardiac hyperinnervation and ventricular arrhythmia

Nerve sprouting and sudden cardiac death

Cardiac sympathetic nerve function in congestive heart failure

Coronary sinus neuropeptide Y levels and adverse outcomes in patients with stable chronic heart failure

Electroanatomic remodeling of the left stellate ganglion after myocardial infarction

Cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states

Cardiac sympathetic denervation for refractory ventricular arrhythmias

Beta 1-and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure

Propranolol versus metoprolol for treatment of electrical storm in patients with implantable cardioverter-defibrillator

Neurotransmitter switching coupled to betaadrenergic signaling in sympathetic neurons in prehypertensive states

Cardiopulmonary baroreflex control of renal sympathetic nerve activity is impaired in dogs with left ventricular dysfunction

Sympathetic activation in congestive heart failure: an updated overview

Influence of brain-derived neurotrophic factortyrosine receptor kinase B signalling in the nucleus tractus solitarius on baroreflex sensitivity in rats with chronic heart failure

Altered ENaC is associated with aortic baroreceptor dysfunction in chronic heart failure

Cellular and molecular mechanisms underlying arterial baroreceptor remodeling in cardiovascular diseases and diabetes

Inhibition of chemoreceptor inputs to nucleus of tractus solitarius neurons during baroreceptor stimulation

0 2 2 sympathetic afferents inhibits baroreflex in rats with heart failure

Human carotid bodies as a therapeutic target: new insights from a clinician's perspective

Altered arterial baroreflexmuscle metaboreflex interaction in heart failure

Sympathetic and baroreflex alterations in congestive heart failure with preserved, midrange and reduced ejection fraction

Nitrosative stress drives heart failure with preserved ejection fraction

Female sex is protective in a preclinical model of heart failure with preserved ejection fraction

Nicotinamide for the treatment of heart failure with preserved ejection fraction

Striking volume intolerance is induced by mimicking arterial baroreflex failure in normal left ventricular function

Baroreflex sensitivity impairment is associated with cardiac diastolic dysfunction in rats

Splanchnic nerve block for chronic heart failure

The ion channel ASIC2 is required for baroreceptor and autonomic control of the circulation

PIEZOs mediate neuronal sensing of blood pressure and the baroreceptor reflex

Arterial baroreceptors sense blood pressure through decorated aortic claws

Tentonin 3/ TMEM150C senses blood pressure changes in the aortic arch

Astrocytes monitor cerebral perfusion and control systemic circulation to maintain brain blood flow

Astrocytes modulate baroreflex sensitivity at the level of the nucleus of the solitary tract

Heterogeneous neurochemical responses to different stressors: a test of Selye's doctrine of nonspecificity

Adrenaline and the Inner World: An Introduction to Scientific Integrative Medicine

How does homeostasis happen? Integrative physiological, systems biological, and evolutionary perspectives

Neurohumoral features of myocardial stunning due to sudden emotional stress

Vagal nerve stimulation markedly improves long-term survival after chronic heart failure in rats

Chronic vagus nerve stimulation: a new and promising therapeutic approach for chronic heart failure

Spinal cord stimulation improves ventricular function and reduces ventricular arrhythmias in a canine postinfarction heart failure model

Spinal cord stimulation is safe and feasible in patients with advanced heart failure: early clinical experience

Thoracic Spinal Cord Stimulation for HEArt Failure as a Restorative Treatment (SCS HEART study): firstin-man experience

Chronic baroreceptor activation enhances survival in dogs with pacing-induced heart failure. Hypertension

Baroreflex activation therapy for the treatment of heart failure with a reduced ejection fraction

Baroreflex activation therapy in patients with heart failure with reduced ejection fraction

Determining the Feasibility of Spinal Cord Neuromodulation for the Treatment of Chronic Systolic Heart Failure: the DEFEAT-HF study

Vagus nerve stimulation for the treatment of heart failure: the INOVATE-HF trial

Betablocker therapy for long QT syndrome and catecholaminergic polymorphic ventricular tachycardia: are all beta-blockers equivalent? Heart Rhythm

Prevalence of diagnosed atrial fibrillation in adults: national implications for rhythm management and stroke prevention: the AnTicoagulation and Risk Factors in Atrial Fibrillation (ATRIA) study

AHA/ACC/HRS guideline for the management of patients with atrial fibrillation: executive summary: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines and the Heart Rhythm Society

Role of the autonomic nervous system in atrial fibrillation: pathophysiology and therapy

The role of the autonomic ganglia in atrial fibrillation

Low-level transcutaneous electrical vagus nerve stimulation suppresses atrial fibrillation

Transcutaneous Electrical Vagus Nerve Stimulation to Suppress Atrial Fibrillation): a randomized clinical trial

Neuromodulation for the treatment of heart rhythm disorders

The role of the autonomic nervous system in cardiac arrhythmias: the neuro-cardiac axis, more foe than friend?

Ganglion plexus ablation in advanced atrial fibrillation: the AFACT study

Calcium-induced autonomic denervation in patients with postoperative atrial fibrillation

Vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis

Obstructive sleep apnea, obesity, and the risk of incident atrial fibrillation

Association of atrial fibrillation and obstructive sleep apnea

Obstructive sleep apnea and the recurrence of atrial fibrillation

Central sleep apnea in atrial fibrillation: risk factor or marker of untreated underlying disease?

Association of nocturnal arrhythmias with sleepdisordered breathing: the Sleep Heart Health Study

Nocturnal arrhythmias across a spectrum of obstructive and central sleep-disordered breathing in older men: outcomes of sleep disorders in older men (MrOS sleep) study

Triggering of nocturnal arrhythmias by sleepdisordered breathing events

The role of ganglionated plexi in apnea-related atrial fibrillation

Effect of renal denervation on neurohumoral activation triggering atrial fibrillation in obstructive sleep apnea. Hypertension

Low-level Cardiovasc Electrophysiol

Atrial fibrillation in acute obstructive sleep apnea: autonomic nervous mechanism and modulation

Effect of obstructive sleep apnea and its treatment of atrial fibrillation recurrence after radiofrequency catheter ablation: a meta-analysis

Shift in the pattern of autonomic atrial innervation in subjects with persistent atrial fibrillation

Targeted nonviral gene-based inhibition of Galpha(i/o)-mediated vagal signaling in the posterior left atrium decreases vagal-induced atrial fibrillation

Targeted G-protein inhibition as a novel approach to decrease vagal atrial fibrillation by selective parasympathetic attenuation

Cardiac glial cells release neurotrophic S100B upon catheter-based treatment of atrial fibrillation

Alteration of parasympathetic/sympathetic heart: a novel antiarrhythmic therapy. Circulation

Augmentation of left ventricular contractility by cardiac sympathetic neural stimulation

Efferent sympathetic and vagal innervation of the canine right ventricle

Disruption of cardiac cholinergic neurons enhances susceptibility to ventricular arrhythmias

The cardiac sympathetic co-transmitter neuropeptide Y is proarrhythmic following ST-elevation myocardial infarction despite beta-blockade

Sympathetic neural recording-it is all in the details

Simultaneous noninvasive recording of skin sympathetic nerve activity and electrocardiogram

Simultaneous noninvasive recording of electrocardiogram and skin sympathetic nerve activity (neuECG)

Atrial fibrillation and ventricular arrhythmias: sex differences in electrophysiology, epidemiology, clinical presentation, and clinical outcomes

Incidence of atrial fibrillation in whites and African-Americans: the Atherosclerosis Risk in Communities (ARIC) study

Age, race, and sex differences in autonomic cardiac function measured by spectral analysis of heart rate variability-the ARIC study. Atherosclerosis Risk in Communities

Wave syndromes expert consensus conference report: Emerging concepts and gaps in knowledge

Circadian variation in the incidence of sudden cardiac death in the Framingham Heart Study population

Prospective evidence of a circadian rhythm for out-of-hospital cardiac arrests

Temporal differences in out-of-hospital cardiac arrest incidence and survival

Reduction in inappropriate ICD therapy in MADIT-RIT patients without history of atrial tachyarrhythmia

Circadian variability in the occurrence of sudden cardiac death in patients with hypertrophic cardiomyopathy

The circadian pattern of the development of ventricular fibrillation in patients with Brugada syndrome

Circadian variation of ventricular arrhythmias in catecholaminergic polymorphic ventricular tachycardia

An international, multicentered, evidence-based reappraisal of genes reported to cause congenital long QT syndrome

Seasonal and circadian distributions of cardiac events in genotyped patients with congenital long QT syndrome

Genotype-phenotype correlation in the long-QT syndrome: gene-specific triggers for lifethreatening arrhythmias

Role of the circadian system in cardiovascular disease

Circadian variation and triggers of onset of acute cardiovascular disease

Role of the autonomic nervous system in modulating cardiac arrhythmias

Ventricular tachycardia in ischemic heart disease: the sympathetic heart and its scars

Cardiac sympathetic denervation in patients with refractory ventricular arrhythmias or electrical storm: intermediate and long-term follow-up

Wireless and battery-free technologies for neuroengineering

Circadian rhythm of cardiac electrophysiology, arrhythmogenesis, and the underlying mechanisms

Circadian variation in human ventricular refractoriness

Circadian variation of heart rate variability

Circadian rhythm: potential therapeutic target for atherosclerosis and thrombosis

Circadian rhythms and cardiovascular health

Diurnal heart rate patterns in inappropriate sinus tachycardia

Blood pressure non-dipping and obstructive sleep apnea syndrome: a metaanalysis

Circadian mechanisms: cardiac ion channel remodeling and arrhythmias

Circadian rhythms of cardiovascular autonomic function: Physiology and clinical implications in neurodegenerative diseases

Sleep, circadian rhythms and health

Heart-rate variability and cardiac autonomic function in diabetes

Circadian and gender effects on repolarization in healthy adults: a study using harmonic regression analysis

Association of sleep characteristics with cardiovascular and metabolic risk factors in a population sample: the Chicago Area Sleep study

Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression

A circadian clock in the sinus node mediates daynight rhythms in Hcn4 and heart rate

The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization

Clock genes alterations and endocrine disorders

Deficient of a clock gene, brain and muscle Arnt-like protein-1 (BMAL1), induces dyslipidemia and ectopic fat formation

Role of mutation of the circadian clock gene Per2 in cardiovascular circadian rhythms

Cardiovascular physiology and coupling with respiration: central and autonomic regulation

Impact of sleep on arrhythmogenesis

Sympathetic-nerve activity during sleep in normal subjects

Sleep, dreams, and sudden death: the case for sleep as an autonomic stress test for the heart

Sleep apnea: types, mechanisms, and clinical cardiovascular consequences

Regulation of breathing and autonomic outflows by chemoreceptors

Effect of autonomic blocking agents on the respiratory-related oscillations of ventricular action potential duration in humans

O2 sensing, mitochondria and ROS signaling: the fog is lifting

Hypoxia and aging

Obstructive sleep apnoea and hypertension: the role of the central nervous system

Obstructive sleep apnea and systemic hypertension: gut dysbiosis as the mediator?

Hypoglossal nerve stimulation and heart rate variability: analysis of STAR trial responders

Sex differences in obstructive sleep apnea phenotypes, the Multi-Ethnic Study of Atherosclerosis

More than the sum of the respiratory events: personalized medicine approaches for obstructive sleep apnea

The sleep apnea-specific pulse rate response predicts cardiovascular morbidity and mortality

Osteoporotic Fractures in Men (MrOS) Study Group. Individual periodic limb movements with arousal are temporally associated with nonsustained ventricular tachycardia: a case-crossover analysis

Associations of incident cardiovascular events with restless legs syndrome and periodic leg movements of sleep in older men, for the outcomes of sleep disorders in older men study (mros sleep study)

Insomnia and risk of cardiovascular disease

Vonk-Noordegraaf A. Neurohormonal axis in patients with pulmonary arterial hypertension: friend or foe?

Right ventricular diastolic impairment in patients with pulmonary arterial hypertension

Contribution of impaired parasympathetic activity to right ventricular dysfunction and pulmonary vascular remodeling in pulmonary arterial hypertension

Effect of inhaled corticosteroids on bronchial responsiveness in patients with "corticosteroid naive" mild asthma: a meta-analysis

Persistent airway inflammation and bronchial hyperwith uncontrolled asthma

Lung eosinophils increase vagus nerve-mediated airway reflex bronchoconstriction in mice

Reflex regulation of airway smooth muscle tone

Autonomic neural control of intrathoracic airways

Vagotomy reverses established allergen-induced airway hyperreactivity to methacholine in the mouse

Population of sensory neurons essential for asthmatic hyperreactivity of inflamed airways

Early denervation and later reinnervation of the heart following cardiac transplantation: a review

Normalization of the heart rate response to exercise 6 months after cardiac transplantation

Reinnervation post-heart transplantation

Cholinergic and neurogenic mechanisms in obstructive airways disease

Prevention of exacerbations of chronic obstructive pulmonary disease with tiotropium, a oncedaily inhaled anticholinergic bronchodilator: a randomized trial

Tiotropium versus salmeterol for the prevention of exacerbations of COPD

Safety and Adverse Events after Targeted Lung Denervation for Symptomatic Moderate to Severe Chronic Obstructive Pulmonary Disease (AIRFLOW). A multicenter randomized controlled clinical trial

Two-year outcomes for the double-blind, randomized, sham-controlled study of targeted lung denervation in patients with moderate to severe COPD: AIRFLOW-2

Design for a multicenter, randomized, sham-controlled study to evaluate

Physiologic and histopathologic effects of targeted lung denervation in an animal model

Respiratory sinus arrhythmia attenuation via targeted lung denervation in sheep and humans

Vagal afferent innervation of the airways in health and disease

Molecular identity, anatomy, gene expression and function of neural crest vs. placode-derived nociceptors in the lower airways

Concepts of scientific integrative medicine applied to the physiology and pathophysiology of catecholamine systems

Autonomic nervous system dysfunction: JACC focus seminar

Pathogenesis and Long-Term Consequences of COVID-19 Cardiac Injury Bhurint Siripanthong

p,q HIGHLIGHTS COVID-19 myocardial injury results from immune and hypercoagulability responses

Long-term cardiac consequences of COVID-19 include structural and functional changes

Myocarditis after COVID-19 vaccination is uncommon (highest risk in teenage males)

These include persistent myocardial fibrosis, edema, and intraventricular thrombi with embolic events, while functionally, the left ventricle is enlarged, with a reduced ejection fraction and new-onset arrhythmias reported in a number of patients. Myocarditis post-COVID-19 vaccination is rare but more common among young male patients. Larger studies, including prospective data from biobanks, will be useful in expanding these early findings and determining their validity

St Bartholomew's Hospital

United Kingdom; g NIHR Barts Biomedical Research Centre

United Kingdom; h Department of Neuroscience, Imaging and Clinical Sciences

Santa Maria Della Misericordia

Division of Cardiovascular Medicine, Perelman School of Medicine at the University of Pennsylvania

COVID-19 pandemic live update

Follow-up of adults with noncritical COVID-19 two months after symptom onset

for the Gemelli Against COVID-19 Post-Acute Care Study Group. Persistent symptoms in patients after acute COVID-19

Patient outcomes after hospitalisation with COVID-19 and implications for follow-up: results from a prospective UK cohort

Sixty-day outcomes among patients hospitalized with COVID-19

Cardiac troponin for assessment of myocardial injury in COVID-19: JACC review topic of the week

Cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (COVID-19)

Association of cardiac injury with mortality in hospitalized patients with COVID-19 in Wuhan, China

High sensitivity troponin and COVID-19 outcomes

Patterns of myocardial injury in recovered troponin-positive COVID-19 patients assessed by cardiovascular magnetic resonance

Coronavirus disease 2019 is delaying the diagnosis and management of chest pain, acute coronary syndromes, myocarditis and heart failure

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

Thrombosis in COVID-19

COVID-19 and the cardiovascular system: implications for risk assessment, diagnosis, and treatment options

Cardiac involvement in COVID-19 patients: mid-term follow up by cardiovascular magnetic resonance

Outcomes of cardiovascular magnetic resonance imaging in patients recently recovered from coronavirus disease 2019 (COVID-19)

Pathological evidence for SARS-CoV-2 as a cause of myocarditis: JACC review topic of the week

New heart failure diagnoses among patients hospitalized for COVID-19

Association between COVID-19 and myocarditis using hospital-based administrative data-United States

SARS-CoV-2 cardiac involvement in young competitive athletes

Recognizing COVID-19-related myocarditis: the possible pathophysiology and proposed guideline for diagnosis and management

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

Human iPSC-derived cardiomyocytes are susceptible to SARS-CoV-2 infection

Humaninduced pluripotent stem cell-derived cardiomyocytes platform to study SARS-CoV-2 related myocardial injury

Forty years with coronaviruses

Coronavirus disease (COVID-19) associated delayed-onset fulminant myocarditis in patient with a history of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection

Association of cardiac infection with SARS-CoV-2 in confirmed COVID-19 autopsy cases

Endothelial cell infection and endotheliitis in COVID-19

Presence of active myocarditis at the 6 month follow-up appointment for a severe form of COVID-19: a case report

Focal myocarditis after mild COVID-19 infection in athletes

Pulmonary embolism in patients with coronavirus disease-2019 (COVID-19) pneumonia: a narrative review

COVID-19-associated nonocclusive fibrin microthrombi in the heart

High thrombus burden in patients with COVID-19 presenting with ST-segment elevation myocardial infarction

Profiling serum cytokines in COVID-19 patients reveals IL-6 and IL-10 are disease severity predictors

Cytokine storm in COVID-19: the current evidence and treatment strategies

Interleukin 6 and haemostasis

Interleukin-1 and the inflammasome as therapeutic targets in cardiovascular disease

Myocarditis and coronary aneurysms in a child with acute respiratory syndrome coronavirus 2

COVID-19-associated suspected myocarditis as the etiology for recurrent and protracted fever in an otherwise healthy adult

Atypical manifestation of COVID-19-induced myocarditis

Patients recovered from COVID-19 show ongoing subclinical myocarditis as revealed by cardiac magnetic resonance imaging

Fatal eosinophilic myocarditis in a healthy 17-year-old male with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2c)

COVID-19-related fatal myocarditis in a 42-year-old female patient

Fatal SARS-CoV-2 inflammatory syndrome and myocarditis in an adolescent: a case report

Acute myocarditis presenting as a reverse tako-tsubo syndrome in a patient with SARS-CoV-2 respiratory infection

Acute myocarditis with ventricular noncompaction in a COVID-19 patient

A recovered case of COVID-19 myocarditis and ARDS treated with corticosteroids, tocilizumab

Fulminant COVID-19-related myocarditis in an infant

Acute fulminant myocarditis in a pediatric patient with COVID-19 infection

Fulminant myocarditis due to COVID-19

COVID-19 fulminant myocarditis: a case report

SARS-CoV-2 fulminant myocarditis

Approach to acute cardiovascular complications in COVID-19 infection

Recognition and initial management of fulminant myocarditis: a scientific statement from the

Tocilizumab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, openlabel, platform trial

Institute for Health and Care Excellence. COVID-19 rapid guideline: managing COVID-19. NG191. Accessed

Treating lifethreatening myocarditis by blocking interleukin-1

Fulminant myocarditis and cardiogenic shock during SARS-CoV-2 infection

Case report: high-grade atrioventricular block in suspected COVID-19 myocarditis

First case of COVID-19 complicated with fulminant myocarditis: a case report and insights

Cardioembolic stroke in a patient with coronavirus disease of 2019 (COVID-19) myocarditis: a case report

COVID-19) fulminant myopericarditis and acute respiratory distress syndrome (ARDS) in a middle-aged male patient

National Institute for Health Research. Living with COVID19

The lingering consequences of sepsis: a hidden public health disaster?

Sir Isaac Newton, sepsis, SIRS, and CARS

Associations between immune-suppressive and stimulating drugs and novel COVID-19-a systematic review of current evidence

Cardiovascular considerations for patients, health care workers, and health systems during the COVID-19 pandemic

T1 mapping in discrimination of hypertrophic phenotypes: hypertensive heart disease and hypertrophic cardiomyopathy: findings from the international T1

Tissue characterization by mapping and strain cardiac MRI to evaluate myocardial inflammation in fulminant myocarditis

Cardiovascular magnetic resonance findings in competitive athletes recovering from COVID-19 infection

Prospective case-control study of cardiovascular abnormalities 6 months following mild COVID-19 in healthcare workers

Adverse cardiovascular magnetic resonance phenotypes are associated with greater likelihood of incident coronavirus disease 2019: findings from the UK Biobank

Prevalence of inflammatory heart disease among professional athletes with prior COVID-19 infection who received systematic return-to-play cardiac screening

Twelve-month systemic consequences of COVID-19 in patients discharged from hospital: a prospective cohort study in Wuhan, China

COVID-19-associated myocarditis presenting as new-onset heart failure and atrial fibrillation

Fulminant myocarditis and atrial fibrillation in child with acute COVID-19

Long-term health sequelae and quality of life at least 6 months after infection with SARS-CoV-2: design and rationale of the COVIDOM-study as part of the NAPKON population-based cohort platform (POP)

Myocarditis following immunization with mRNA COVID-19 vaccines in members of the US military

Myocarditis and pericarditis after vaccination for COVID-19

Myocarditis after BNT162b2 mRNA vaccine against COVID-19 in Israel

Ventricular arrhythmias in myocarditis: characterization and relationships with myocardial inflammation

Long-term arrhythmic risk assessment in biopsy-proven myocarditis

Postacute COVID-19 syndrome

COVID-19 and the UK Biobank-opportunities and challenges for research and collaboration with other large population studies

Medium-term effects of SARS-CoV-2 infection on multiple vital organs, exercise capacity, cognition, quality of life and mental health, posthospital discharge

Malignant ventricular arrhythmias in patients with severe acute respiratory distress syndrome due to COVID-19 without significant structural heart disease

Heart block in patients with coronavirus disease 2019: a case series of 3 patients infected with SARS-CoV-2

Atrial fibrillation is an independent predictor for in-hospital mortality in patients admitted with SARS-CoV-2 infection

The possible association between COVID-19 and postural tachycardia syndrome

COVID-19, inflammation, myocardial injury, myocarditis, sudden cardiac death, troponin APPENDIX For supplemental tables and references, please see the online version of this paper