key: cord-0007722-33jxxp1b authors: nan title: Full Issue PDF date: 2018-06-25 journal: JACC Basic Transl Sci DOI: 10.1016/s2452-302x(18)30124-4 sha: fd663ef4239b5e3f4a14768eda5c0f6f5111ac74 doc_id: 7722 cord_uid: 33jxxp1b nan Titin-isoform expression, titin phosphorylation, and myocardial fibrosis were studied in 30 patients with severe symptomatic aortic stenosis (AS). Patients were grouped into "classical" high-gradient, normal-flow AS with preserved ejection fraction (EF); "paradoxical" low-flow, low-gradient AS with preserved EF; and AS with reduced EF. Nonfailing donor hearts served as controls. AS was associated with increased fibrosis, titin-isoform switch toward compliant N2BA, and both total and site-specific titin hypophosphorylation compared with control hearts. All AS subtypes revealed titin and matrix alterations. The extent of myocardial remodeling in "paradoxical" AS was no less severe than in other AS subtypes, thus explaining the unfavorable prognosis. S evere symptomatic aortic stenosis (AS) is a common cardiac disease associated with poor prognosis in the absence of heart valve replacement (1) . In AS, chronically elevated afterload induces left ventricular (LV) hypertrophy, increased myocardial stiffness, and impaired relaxation (2, 3) . This myocardial remodeling is associated with progressive dyspnea, the most common symptom of AS, and poor prognosis (1) . Patients with severe, high-gradient, normal-flow AS with preserved ejection fraction ("classical" AS) benefit with a high probability from aortic valve replacement (1) . However, there are other hemodynamic types of AS, such as low-flow, low-gradient severe AS with preserved ejection fraction (EF) ("paradoxical" AS) and aortic stenosis with reduced ejection fraction (ASrEF), which occur in more than 30% of all cases (4, 5) . Probably due to advanced myocardial remodeling, the positive effect of aortic valve replacement is more uncertain in these patients (1, 5) . In particular, the pathophysiology of "paradoxical" AS is not fully understood. There is controversy about whether it is merely a moderate form of AS with favorable prognosis or an advanced form of AS. It could be a pseudo-severe AS due to inaccuracies in the measurements or inconsistencies in the guidelines (6) (7) (8) , or it could be a true AS associated with pronounced myocardial stiffening, severe intrinsic myocardial damage, impaired relaxation, and therefore, worse prognosis than "classical" AS (5, 9, 10) . Pathological myocardial relaxation and stiffness in AS are determined, in part, by fibrosis (11) (12) (13) . More recently, the giant sarcomere protein titin has emerged as a main contributor to myocardial relaxation and stiffness (14) . Titin properties can be severely altered in heart failure, including AS (14) . Titin is expressed in 2 major isoforms in adult hearts: stiff N2B and compliant N2BA isoforms (15) . In recent years, there has been growing evidence for a titin isoform switch toward the more compliant N2BA isoform in various cardiac diseases, for example, heart failure with reduced or preserved EF (14) . This isoform transition may be compensatory, to counteract the increased stiffening from myocardial fibrosis. In contrast, for AS patients, it is not clear whether titin isoforms switch toward N2BA, N2B, or not at all (16) (17) (18) . In addition, there are no studies of titin isoform expression in different hemodynamic subtypes of AS. Phosphorylation of titin is another parameter that determines myocardial passive stiffness (14) , but likewise, has not been studied in hemodynamic subtypes of AS. To our knowledge, site-specific titin phosphorylation has never been investigated in human AS, but would be important to measure in particular at 2 mechanically active regions within the titin springs present in both the N2B and N2BA isoforms, the N2B-unique sequence (N2Bus) and the proline/glutamate/valine/lysine-rich (PEVK) region, because the stiffness of these segments is altered when they become phosphorylated (14) or dephosphorylated (19) . In the present study, we investigated titin-isoform expression, total-titin and site-specific N2Bus/PEVK phosphorylation, and the degree of fibrosis in endomyocardial biopsy samples from AS patients compared with nonfailing donor heart samples. We 1) more than moderate valve regurgitation or stenosis; 2) previous cardiac surgery; and 3) a previous myocardial infarction. A previous myocardial infarction was defined according to current recommendations (20) . Coronary angiography was performed pre-operatively. Coronary heart disease was diagnosed when coronary artery stenosis was $50%. The presence of coronary artery occlusions, including those that were subclinical, was considered an exclusion criterion for the study. All patients gave written consent to participate in this study. The study protocol was approved by the local ethics committee (approval were used (24) : anti-P-S4010, anti-P-S4099, anti-P-S4185 (all N2Bus), and P-S11878 (PEVK). Except for anti-P-S4185 (generated by Immunoglobe, Himmelstadt, Germany), these antibodies were made by Eurogentec (Brussels, Belgium). To measure the titin to myosin heavy chain (MHC) ratio, Coomassie-stained 2.5% polyacrylamide/1% agarose gels were prepared. We note that a substantial number of AS biopsies revealed no or barely measurable signals in the titin isoform/phosphorylation analysis, which was probably due to the fact that the biopsy material sometimes contained a low number of cardiomyocytes, whereas fibrotic regions were abundant. Bands were visualized using the LAS-4000 Image CLINICAL FOLLOW-UP. Clinical and echocardiographic follow-up evaluations were performed after 3 months. During this time, 6 patients died due to acute cardiogenic shock (n ¼ 1), sepsis (n ¼ 2), ventricular fibrillation (n ¼ 1), and heart pump failure (n ¼ 2). The 30-day mortality rate was 3%. There was no significant difference in outcome between patients with or without coronary artery bypass grafting (p ¼ 0.750). In the 24 surviving patients, NYHA functional class improved; aortic mean gradient, valvuloarterial impedance Z, and LV mass index decreased; and AVA increased significantly ( Table 2) . Among the different AS subgroups, significant differences were found only for LVEF and aortic mean gradient ( Table 3 ). The 1-year survival rates were 93% in patients with classical AS, 75% in patients with ASrEF, and 63% in patients with paradoxical AS. Although the differences in survival were not significant, there was a trend toward worse survival in patients with paradoxical AS. In endomyocardial biopsies, significantly more cases of severe fibrosis were found in patients with severe AS than in the control group (severe AS: no/mild fibrosis, n ¼ 1; moderate fibrosis, n ¼ 13; severe fibrosis, n ¼ 14; vs. control group: no/mild fibrosis, n ¼ 8; p ¼ 0.001). After adjusting for age and sex, aortic stenosis was the only predictor for the degree of fibrosis. The degree of myocardial fibrosis appeared to be similar among patients with classical AS, paradoxical AS, and ASrEF ( Figure 1 ). In patients with or without coronary heart disease, there was no significant difference in the degree of fibrosis (coronary artery disease: no/ mild fibrosis, n ¼ 3; moderate fibrosis, n ¼ 5; severe fibrosis, n ¼ 8; vs. no coronary artery disease: no/mild fibrosis, n ¼ 4; moderate fibrosis, n ¼ 7; severe fibrosis, n ¼ 6; p ¼ 0.231). Comparing the AS subgroups to the donor group, the median (first quartile, third quartile) N2BA/N2B Values are % or mean AE SD. *cAS vs. ASrEF (p < 0.05). †cAS vs. pAS (p < 0.05). D indicates comparison of baseline parameters to parameters at 3-month follow-up. Abbreviations as in Table 1 . Table 4 , Figure 4 ). Phosphorylation of these titin spring elements affects cardiomyocyte and myocardial passive stiffness (14) . The phosphoserines within N2Bus measured here are known to be phosphorylated by protein kinase (PK)A or extracellular signal-regulated protein kinase 2 (P-S4010), cGMPactivated PKG (P-S4099), and PKG or PKA (P-S4185), and they can be dephosphorylated by protein phosphatase 5 (19) , whereas phosphoserine P-S11878 within the PEVK domain can be phosphorylated by PKCa (14) . In AS versus donor samples, no difference in median phosphorylation was observed for P-S4010, P-S4099, and P-11878 ( Figure 4B ). Using multivariable analysis, both aortic stenosis and age were found to predict phosphorylation of the titin phosphosite P-S4185 (p < 0.01 for both). The subgroup pAS showed a trend for hypophosphorylation at S4185, compared with donor hearts and cAS, whereas the ASrEF subgroup was significantly hypophosphorylated at S4185 versus donors (Table 4, Figure 4C ). In summary, the PKA/PKG-dependent phosphoserine P-S4185 within human N2Bus was hypophosphorylated in AS, explaining part of the total titin phosphorylation deficit in AS versus nonfailing control hearts. Mann-Whitney U test revealed statistically significant differences between control hearts and all AS patients (p ¼ 0.005). (C) Comparison was made between study patients' subgroups using the Kruskal-Wallis test (p ¼ 0.042). Post hoc multiple pairwise comparisons revealed significant differences between the control group and paradoxical AS (*p < 0.05). AS ¼ aortic In this study, we examined patients with severe symptomatic AS who underwent surgical aortic valve replacement. There were no significant differences between the subgroups of AS in terms of clinical symptoms and AVA. However, the hemodynamic analysis revealed fundamental differences between classical high-gradient, normal-flow AS; paradoxical low-flow, low-gradient AS with preserved LV ejection fraction; and AS with reduced EF. In particular, there were significant differences in aortic mean gradient, stroke volume, valvuloarterial impedance Z, LVEF, and BNP ( Table 1) . A trend toward worse outcome was observed in patients with paradoxical AS after aortic valve replacement (1-year survival was 63%, compared with 93% in classical AS). This trend is consistent with earlier findings (9) . MYOCARDIAL FIBROSIS. The myocardium in AS is known to be characterized by increased cell mass and increased fibrosis (1) (2) (3) (11) (12) (13) . In our study, we confirmed that patients with severe symptomatic AS have significant myocardial fibrosis. Patients with paradoxical AS showed a degree of fibrosis similar to that of patients with classical AS or ASrEF ( Figure 1 ). Our findings are consistent with those of Herrmann et al. (12) , who examined different forms of AS (12) , and underscore the view that paradoxical AS is a disease with severe intrinsic myocardial damage (9, 10) . Diverging published data exist regarding the ratio of the more compliant titin isoform N2BA to the stiffer isoform N2B in patients with AS. One study demonstrated a reduced N2BA/N2B expression ratio in 19 patients with AS, compared to donor hearts (18) . Another study reported no significant alterations in titin-isoform composition in 14 AS patients versus healthy hearts (17) . Yet, another study with 9 AS patients, from the same group, detected a significant shift of the titin-isoform expression ratio toward more compliant N2BA (16) . The reason for these diverging results is unknown. We investigated the largest number of AS patients yet for titin-isoform expression. In our patient group, we found a significant shift toward the more compliant N2BA titin isoform versus nonfailing donor heart samples ( Figure 2) . The magnitude and direction of this isoform transition were similar to those seen in end-stage failing human hearts with ischemic or dilated cardiomyopathy, compared with nonfailing donor hearts (26, 27) . hypophosphorylation of titin at the cardiac-specific N2B element in human heart failure (29) (30) (31) and in animal models of heart failure with preserved EF (32, 33) . Some studies also showed an increased ratio of phosphorylated N2BA over phosphorylated N2B in human heart failure with reduced or preserved ejection fraction (16, 17 (8, 9) . Titin's contribution can be modulated by isoform variation and phosphorylation (10, 11) . The splice factor RBM20 is responsible for generation of 2 isoforms: the larger, more compliant N2BA, and the smaller N2B isoform (12) . Titin phosphorylation occurs at multiple sites in its N2B and PEVK segments (10, 11 Figure 1F , Table 1 ). survival post-I/R in STZ-induced diabetic mice compared with noninsulin treated mice (survival 77% vs. 31%; p ¼ 0.0223) ( Figure 1F , Table 1 , Supplemental Figure 1D ). In contrast, there was a 45% decrease in survival post-I/R between insulin-and noninsulintreated db/db mice (survival 69% vs. 25%; p ¼ 0.0241) ( Figure 1F , Table 1 , Supplemental Figure 1B ). These suggest both T1DM and T2DM contributed to worse outcomes in myocardial ischemia/reperfusion (I/R), and the difference in response to insulin therapy between them was possibly due to their baseline insulin level and/or insulin sensitivity. Figures 2A and 2B) . Level of miR-24 decreased despite progressing from hyperglycemia to euglycemia (from 0 to 50 min), supporting that the insulin infusion rather than glucose levels is the direct cause for the reduction in miR-24 level ( Figure 2C ). As further supporting evidence, in patients with T1DM (where patients are on various forms of exogenous insulin therapy for low insulin levels) we observed an inverse correlation between circulating miR-24 and plasma levels of insulin ( Figure 2D) . In a small pilot on myocardial function, we first compared the myocardial infarction size in response to I/R between WT and db/db mice ( Figure 3A ). As mentioned in the previous text, we observed a significantly increased infarct size in diabetic hearts ( Figures 1C to 1E ). into db/db mice (2 weeks prior to I/R), we observed significant restoration of cardiac miR-24 levels ( Figure 3B ) (miR-126 served as a control) and a reduction in infarct size ( Figures 3C to 3E ). The increase in infarct size in DM mice (low miR-24) and its rescue by exogenous miR-24 (restoring levels of miR-24) further support a myocardial protective role for miR-24. Notably, we also observed a significant reduction in plasma insulin level and fasting glucose level with intravenously delivery of miR-24 to db/db mice ( Figures 3F and 3G ), which could contribute to the decrease in infarct size in DM mice. To demonstrate a cardiac-specific effect, we used a cardiac-specific miR-24 overexpression mouse model. Figure 2B ). c-Myc is known to be downstream of Akt (31) . We have previously demonstrated that c-Myc negatively regulates miR-24 (20) . Wang et al. from WT mice or mice with cardiomyocyte-specific overexpression of miR-24, and heart lysates from in db/db mice with control or miR-24 mimic (Life Technologies, 5 mg/kg) delivery. Abbreviations as in Figure 1 . In the heart, miR-24 has been reported to target BIM and junctophilin-2 in cardiomyocytes, and targeting transcription factor GATA2, p21-activated kinase PAK4, and eNOS in endothelial cells (36) . In the vascular system, miR-24 was found to target chitinase 3-like 1 to limit vascular inflammation and matrix metalloproteinase-14 in macrophage to retard atherosclerotic plaque progression (35 We have previously demonstrated that high glucose levels can also reduce miR-24 from the endothelium predisposing to thrombosis (20) . Together, high glucose and high insulin, as observed most dramatically in T2DM, lead to a severe reduction in miR-24 level and an enlarged infarct through promoting apoptosis and excessive O-GlcNAcylation ( Figure 6 ). Our data suggest that overload insulin is detrimental to myocardial function through reducing the protective miR-24, particularly because the heart is nor- Wang et al. MiR-24 is also part of a pri-miR cluster, together with miR-23 and miR-27 (7) . Increased expression of miR-24 but also of miR-27b has been associated with cardiac dysfunction (8, 9) . Previously, the au- Tetrahydrobiopterin is an essential cofactor for NO production. Limitation of endogenous tetrahydrobiopterin reduces NO bioavailability, enhances oxidative stress, and impairs vascular function. Orally supplemented tetrahydrobiopterin has therapeutic challenges because it is rapidly oxidized in vivo. Here, the authors demonstrate that Reduced nitric oxide (NO) bioavailability correlates with impaired cardiovascular function. NO is extremely labile and has been challenging to develop as a therapeutic agent. However, NO bioavailability could be enhanced by pharmacologically targeting endogenous NO regulatory pathways. Tetrahydrobiopterin, an essential cofactor for NO production, is synthesized by GTP cyclohydrolase-1 (GCH1), which complexes with GCH1 feedback regulatory protein (GFRP). The dietary amino acid L-phenylalanine activates this complex, elevating vascular Thus, raising endothelial BH 4 levels has been suggested as a strategy to maintain healthy NO production and bioavailability at the level of the endothelium (9,10). To support this, intra-arterial administration of BH 4 improves endothelial dysfunction in patients with hypertension (11), coronary artery disease (12) , and hypercholesterolemia (13). However, due to its unstable nature, orally administered BH 4 (or its analogue sapropterin) has limited efficacy in improving vascular hemodynamics (14) (15) (16) . Therefore, other strategies to raise endogenous BH 4 bioavailability at the level of the endothelium are desirable. BH 4 is synthesized from GTP in a reaction where the committing step is mediated by GTP cyclohydrolase-1 (GCH1) (17) . Modulation of GCH1 expression has been shown to regulate BH 4 , NO, and cardiovascular function (18) (19) (20) . GCH1 is subject to feed-forward regulation by L-phenylalanine (L-phe), via an allosteric protein interaction with GCH1 feedback regulatory protein (GFRP) (21, 22) . This GCH1-GFRP complex is operative in humans because oral challenge with L-phe leads to a 3-fold rise in plasma biopterin levels (a correlate of BH 4 )-an effect that is attenuated in patients with a loss-of-function GCH1 mutation (23 To support this hypothesis, it is known the GCH1-GFRP axis regulates BH 4 and NO in endothelial cells (25) . Overexpression of GFRP reduces basal BH 4 levels (26) and attenuates the rise in BH 4 and NO that occurs in response to a proinflammatory stimulus (27) . Additionally, the primary source of BH 4 appears to be derived from GCH1 localized within the vascular endothelium, and GFRP is coexpressed within these cells (28, 29) . Finally, oral L-phe elicits a rise in vascular BH 4 -an effect that is absent in mice lacking endothelial GCH1 (24) . legend, or in the Supplemental Table 2 . As previously described (24, 30) , the combination of purified recombinant GFRP with GCH1 protein had a higher basal activity than GCH1 alone in vitro. The addition of L-phe (2 mmol$l À1 ) had no effect on purified GCH1 activity alone but caused a significant rise in GCH1 activity when coincubated with GFRP, confirming that L-phe is an allosteric regulator of the GCH1-GFRP complex only ( Figure 1A ). In vivo, oral L-phe (100 mg$kg À1 ) bolus to WKY and SHR significantly increased plasma BH 4 levels within 30 min, and levels returned back to baseline within 4 h ( Figure 1B) . Correspondingly, a significant rise in nitrite levels was also detected within 30 min, but whereas this returned to baseline in WKY, it remained elevated in SHR for at least 4 h ( Figure 1C) . Interestingly, there were no statistically significant differences in BH 2 and biopterin in all groups although trend increases were observed ( Figures 1D and 1E ). which was restored to WKY values following short-term bolus and long-term L-phe treatment ( Figure 3A ). As anticipated, superoxide levels were higher basally in SHR compared with WKY ( Figure 3B ). Bolus dose or long-term administration of L-phe significantly reduced superoxide levels in SHR ( Figure 3B ). Superoxide dismutase, the positive control, reduced superoxide in all study groups ( Figure 3B ). Again, we observed no significant changes in BH 2 or biopterin in aortic tissue AE L-phe administration ( Figures 3C and 3D) . Unfortunately, in aortic tissues, nitrite levels fell below the limit of detection and were therefore not quantifiable. confirming that L-phe was absorbed following oral gavage ( Figure 6A , Supplemental Table 1 ). We did not detect a significant rise in the L-phe/L-tyr ratio in animals treated long-term with L-phe. This is not surprising because the long-term ad libitum L-phe dosing was at a much lower dose than the short-term bolus dose challenge. Although there were trends of decreased dopamine and increased adrenaline/ noradrenaline between SHR and WKY, these did not reach statistical significance in most tissues. The exception was the heart, where adrenaline/ noradrenaline levels were significantly higher in SHR than WKY basally, but equaled WKY levels following L-phe treatment ( Figures 6B and 6C) . aortic rings, with intact or denuded endothelium following short-term incubation with 0.5 mmol$l À1 L-phe. Data represent mean AE SEM n ¼ 6 animals (in triplicate/animal) for mesenteric arteries and aortic rings (*p < 0.05 and *** p < 0.001 for the whole curve). Table (E) shows EC 50 values (nmol$l À1 ) comparing saline control with L-phe treatment for each study. Data represent mean EC 50 values AE SEM, n ¼ 6 independent experiments (**p < 0.01 and ***p < 0.001). Ach ¼ acetylcholine; EC 50 ¼ effective concentration for 50% maximal response; other abbreviations as in Figure 1 . Heikal et al. Importantly, this is a proof-of-concept study demonstrating that GCH1-GFRP is a rational therapeutic target for vascular dysfunction. Hence, the development of L-phe mimetics that selectively bind to and enhance this protein complex may be of clinical value. Our data suggest that L-phe could itself be translated into the clinic given the minimal effects observed on catecholamines but should be advanced with caution, given L-phe's diverse biological action and potential for predictable adverse drug reactions. GCH1 binds to GFRP to form a protein complex that is receptive to allosteric regulation by both L-phe (feed forward) and BH 4 (feedback) regulation (21) . Our results have confirmed numerous previous reports that L-phe only enhances GCH1 activity when it is bound to GFRP (24, 30) . In addition to its essential cofactor role for NOS activation, BH 4 is also required by phenylalanine hydroxylase to catalyze the conversion of L-phe to L-tyr, which is further converted to dopamine, adrenaline, and noradrenaline (41). L-phe thus regulates its own metabolism via feed-forward activation of GCH1-GFRP with subsequent increases in BH 4 and hence phenylalanine hydroxylase activity. This is important because sustained elevation of L-phe can become neurotoxic (42) . Indeed, BH 4 has been successfully used as a treatment for a subset of patients with phenylketonuria (43) . Consistent with raised biopterin levels seen in patients after L-phe loading (23) and our previous observations in mice (24) To verify whether L-phe could activate the GCH1-GFRP axis functionally, a series of studies were carried out using fresh conduit (aortic) and resistance (mesenteric) blood vessels from WKY and SHR. Consistent with published reports, Ach-mediated vascular relaxation in SHR was significantly impaired in comparison to WKY rats. Short-term L-phe incubation within the organ bath significantly improved vascular relaxation in SHR vessels yet had no effect on WKY. This implies that L-phe, via local elevation of BH 4 within the vasculature, enhances NO bioavailability and endothelial function only in circumstances where the pathway is dysfunctional. This is consistent with the differential effects on plasma nitrite between SHR and WKY discussed in the preceding text. Interestingly, L-phe had a more pronounced effect on vascular relaxation in mesenteric Thus, there is still much work to be undertaken to improve the efficacy and safety of pharmacotherapies that enhance NO bioavailability, but our present study provides the first proof-of-concept data that the GCH1-GFRP complex is a rational therapeutic target to achieve BH 4 elevation and NO restoration within blood vessels. To translate these findings further, we propose 2 parallel research strategies. The first clinical devel- (4) . However, assessment of LVEF is variable and has limited ability to detect early cardiotoxicity, as many patients have histological evidence of cardiotoxicity before decrements in systolic function occur (5) . Importantly, a reduction in LVEF is often an irreversible side effect of DOX therapy, as 45% to 58% do not recover systolic function despite receiving optimal medical therapy (6). As such, early detection methods of anthracycline-induced cardiotoxicity that precede decrements in LVEF have been proposed (6-10); however, limited or inconsistent data exist regarding the efficacy of these approaches (4). The precise mechanisms of DOX-induced cardiotoxicity are not fully elucidated; although it is well established that DOX induces cardiotoxicity largely through the excessive production of reactive oxygen species (ROS) that lead to direct myocardial apoptosis, contractile abnormalities, inflammation, and vascular injury (11, 12) , and promote deleterious cardiac remodeling by increasing the activity and abundance of matrix metalloproteinases (MMPs) (13, 14) . Some studies report that elevations in circulating biomarkers of oxidative stress (myeloperoxidase) precede clinically significant systolic dysfunction in anthracycline-treated patients (8, 15 CT images were used to localize the heart and confirm the epicardial surfaces for VOI edge placement. Standard uptake values (SUVs) were then calculated for the LV myocardium, liver, and LV blood pool. Differences in blood pool and liver SUV were observed between groups (see later in the text); therefore, the ROS activity ratio was determined as the ratio between LV myocardial SUV and LV blood pool SUV to account for differences in tissue tracer clearance and bioavailability. The results of the quantitative analysis of TUNELpositive staining for all rats is shown in Figure 6E . Unlike the observed progressive histopathologic changes described in the preceding text, cellular apoptosis only trended to increase in DOX-treated rats at 8 weeks compared with time-matched controls rats (p ¼ 0.06) and was significantly increased compared with DOX-treated rats at 4 weeks (p < 0.02) ( Figure 6E ). The major finding from the present study is that 18 in response to activation of I Ca-L in cardiac myocytes isolated from treated mice (11 was used to measure mitochondrial membrane potential (J m ) as described previously (19) . Flavoprotein autofluorescence was used to measure flavoprotein oxidation based on previously described methods (20, 21) . Detailed methods are provided in the Supplemental Methods. Figure 1C ) (11, 12) . No change in J m was observed in age-matched mdx myocytes (Figures 1B and 1C) . Figure 1E ) (11) . No alteration was observed in agematched mdx myocytes ( Figures 1D and 1E) . However, myocytes isolated from 4-week-old mdx mice treated with either PMO dosing regimen exhibited a significant increase in flavoprotein oxidation in response to BayK(-) (Figures 1D and 1E ). Both treatment regimens rescued the response. BayK(þ) did not significantly alter flavoprotein oxidation in wt or mdx myocytes ( Figure 1E ). Mitochondrial electron transport chain uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone increased flavoprotein signal confirming the signal was mitochondrial in origin ( Figures 1D and 1E) . These data indicate that treatment of pre-cardiomyopathic mdx mice with a short-term, high-dose PMO treatment regimen restores metabolic activity in the mdx heart. Figures 2B and 2C) . We also examined the efficacy of a long-term, highdose PMO treatment regimen on restoring J m in isolated cardiac myocytes. Under calcium-free conditions, BayK(-) elicited a significant increase in J m in wt myocytes that was attenuated with nisoldipine ( Figure 3B) . No alteration was observed in agematched mdx myocytes (Figures 3A and 3B) . However, treatment of mdx mice with 40 mg/kg PMO 3Â per week for 19 weeks completely rescued this response ( Figures 3A and 3B) . The response was attenuated with nisoldipine ( Figure 3B ). BayK(þ) did not significantly alter J m in wt or mdx myocytes ( Figure 3B ). Oligomycin induced a significant increase in JC-1 fluorescence in mdx myocytes demonstrating weeks; other abbreviations as in Figure 1 . We also investigated the efficacy of a long-term, high-dose PMO treatment regimen on metabolic activity. BayK(-) caused a significant increase in flavoprotein oxidation in myocytes from 43-week-old wt mice that was prevented with nisoldipine ( Figure 3D) . No alteration was observed in age-matched mdx myocytes ( Figures 3C and 3D) . Figure 4B ) (11) . The response was also attenuated with mitochondrial calcium uniporter (mitochondrial calcium uptake) blocker Ru360, but not with ryanodine receptor (sarcoplasmic reticulum calcium release) blocker dantrolene ( Figure 4B) . No alteration was observed in age-matched mdx myocytes (Figures 4A and 4B) . However, myocytes isolated from 43-week-old mdx mice treated with PMO exhibited a significant increase in metabolic activity in response to BayK(-) that could be attenuated by nisoldipine or Ru360, but not dantrolene (Figures 4A and 4B) . BayK(þ) did not significantly alter metabolic activity in wt, mdx, or PMO-treated mdx myocytes ( Figures 4A and 4B ). Oligomycin induced a significant decrease in metabolic activity in all myocytes demonstrating the myocytes were metabolically active ( Figure 4B) . Figure 1 . week 49, the difference was no longer significant (27, 28) . A subsequent phase 3 double-blinded placebo-controlled study involving prolonged treatment of DMD patients with 6 mg/kg/week (48 weeks) did not improve ambulation (6-min walk test) or secondary assessments of motor function (29) . Furthermore, adverse events including kidney and liver toxicity, likely to be off-target effects of the 2 0 Omethylated phosphorothioate backbone, were reported in 46% of patients (27, 28, 30) . (Figures 3 and 4) , prevented development of the cardiomyopathy, and, importantly, is not toxic in vivo ( Figure 5) . Consistent with previous studies, we find that PMO treatment results in accumulation of as little as 5% of normal dystrophin protein expression in mdx mice ( Figure 2B ) (36) . Importantly, this is sufficient to result in functional improvement in the mdx heart ( Figures 5A and 5B ). Using the mdx mouse model of DMD, we previously For patients with life-threatening or severely debilitating disease, the wait for approval is simply too long, and can both abolish hope for those who diseases will be quickly fatal, and lead to sustained or even permanent disability for those whose diseases linger but are without effective proven therapies. It should be noted that although the terms "compassionate use" or "preapproval access" are often used informally to refer to the use of an investigational drug to treat a patient outside of a clinical trial, these terms are not defined or described in FDA regulations, which simply refer to expanded access to investigational drugs. The call for EA is not limited to individual patients. Advocacy organizations have pressed for groups of patients with rare and/or "orphan" diseases, for example, to be able to access promising new therapies prior to their approval. Indeed, social media is increasingly becoming a consumer/patient advocacy tool for implementing FDA regulatory changes and promoting access to investigational therapeutics (7). In addition, once a drug has completed phase 3 testing and is awaiting approval, patients who have Companies have also expressed concern about how data from such "compassionate use" may be applied for CMV infection (9) . Later studies also showed that ganciclovir patients were living longer (10). The FDA refused approval of ganciclovir for treatment of CMV retinitis, because they had no animal studies for that use, nor significant human placebocontrolled trials on which to base a marketing application. Many questioned whether the use of ganciclovir was wise, or safe (11, 12) . But, because of ganciclovir's known efficacy, it became paradoxically impossible to carry out human controlled trials, because such trials are only ethically justifiable if investigators are honestly uncertain about whether net positive benefits over placebo exists (13) . Furthermore, neither patients nor doctors were willing to risk assignment to placebo and further loss of eyesight after the results were published. Syntex then sought approval to study ganciclovir for treatment of CMV colitis, knowing that once marketing approval of the drug was obtained, FDA rules would allow "off-label" use for retinitis (14) . 2) the potential benefit must justify the potential risks of the treatment; and 3) providing the treatment must not compromise or interfere with the ongoing FDA drug development program, such as by critically depleting a limited supply of investigational drug that is also needed for an ongoing study or a future study that is in the planning stages (4). An "immediately life-threatening condition or disease" is defined by the FDA as "a stage of disease in which there is reasonable likelihood that death will occur within a matter of months or in which premature death is likely without early treatment." A serious disease or condition is defined as being "associated with morbidity that has substantial impact on day-to-day functioning." Furthermore, while short-lived or self-limited morbidity will usually not be a sufficient qualifying condition, the morbidity "need not be irreversible, provided it is persistent or recurrent." The FDA states that whether a condition is serious or not "is a matter of clinical judgment, based on its impact on such factors as survival, day-to-day functioning, or the likelihood that the disease, if left untreated, will progress from a less severe condition to a more serious one" (15) . and other animal species such as cats, armadillos, guinea pigs, swine and ferrets in which thalidomide had been tested, teratogenic effects had been induced only occasionally" (18) . In fact, when human birth defects began to appear in the offspring of women who had ingested thalidomide during pregnancy as a sedative and to treat nausea, researchers pointed out that thalidomide had failed to demonstrate teratogenicity in rats, and at first insisted that thalidomide could not be the culprit. In Germany, where the drug was first developed, thalidomide was held to be so safe that no prescription was required for its use, it was advertised for use in pregnant women (19) , and the drug company distributed free samples to its factory employees (18, 19) . As of now, 12 products have been approved under the Animal Rule, 7 of which were issued quickly after the guidance was published ( Table 3 There is a reasonably well-understood pathophysiological mechanism of the toxicity of the toxic substance and its prevention or substantial reduction by the product. The effect is demonstrated in more than 1 animal species expected to react with a response predictive for humans, unless the effect is demonstrated in a single animal species that represents a sufficiently well-characterized animal model for predicting the response in humans. The animal study endpoint is clearly related to the desired benefit in humans, generally the enhancement of survival or prevention of major morbidity. The data or information on the kinetics and pharmacodynamics of the product or other relevant data or information, in animals and humans, allows selection of an effective dose in humans. relationship between mortality and intensive glycemic control has not been seen in patients with heart failure whose diabetes has been managed using noninsulin-dependent therapeutic regimens. The development of heart failure is common in states of hyperinsulinemia, but not in states of glucose intolerance that is accompanied by a deficiency of insulin. This finding has been supported by the find- (20), This allows a user to access the information contained on the display "at a glance." Equivalent capabilities can be achieved using biocular displays, or the displays can provide 2 different images to provide stereoscopic image pairs. Most HMD platforms generate image pairs by using a panel display or small projector and a series of optics to enlarge the image and simulate projection at a distance to ease focusing, as illustrated for several common systems in Figure 2 . For a given projected distance and resolution, as the The perception of depth is influenced by the distance at which the eyes converge and the focal distance of the eye, referred to as vergence (left) and accommodation (right), respectively. 3-dimensional displays, which rely on stereo vision, can only influence vergence by adjusting the stereo disparity between the left and right eye, and the focal plane of the optics remains fixed. The eyes (bottom) perceive the vergence depth of the image of the sphere on the displays by the distance of the intersection of the displayed images. On the right, the accommodation distance is the distance to the focal plane at which the cube is in focus. Objects at closer and farther focal distances appear out of focus. Vergence and accommodation conflicts cause discomfort due to the disagreement in vergence and accommodation distances, particularly at close distances where accommodation cues are most influential. Silva et al. Extended reality platforms are constrained primarily by cost, size, weight, and power to achieve the highest visual quality, mobility, processing speed, and interactivity. Visual quality is dependent on resolution, brightness, focal depth, and FOV. Display technology is the most demanding aspect of extended reality, and is generally the largest design and cost constraint (23) . For 3D systems, this is compounded by requiring stereoscopic pairs of images to generate the perception of depth through vergence, or the angularity disparity between 2 displays (Figure 4 , left). The display system must compromise size and cost with providing the maximum visual quality to match the capabilities of the human visual system (HVS Depth at close distances is also perceived through accommodation ( Figure 4, right) , or the perception of depth due to disparity in focal depth, which is a challenge facing all conventional stereoscopic displays. Accommodation is required to allow a user to focus on instruments and digital objects at the same simulated distance (28) (e.g., surgical guidance overlays, within "personal space" and "action space"). Disparity between vergence depth displayed by HMD and accommodation expected by HVS is referred to the vergence and accommodation conflict (VAC) and is responsible for discomfort at these close working distances (29) . Most display systems only support a single, fixed, focal plane for all digital elements (Figure 4) , although some emerging technologies can provide multiple fixed focal planes by employing adaptive optics. Every design decision to mitigate these challenges affects applicability for use in each procedural environment. AR and MxR displays provide the best compromise between digital annotation and clear FOV. The high pixel density, large FOV devices designed for desktop or office use, require an umbilical to a high-powered workstation to support the processing required for their displays. These devices provide a larger digital display and a higherresolution display, but at the expense of complicated setup before procedures and limited These devices have the potential to provide physicians with a sterile interface that allows them to control 3D images. Early data show that this improved visualization will allow the physician to learn more quickly, interpret images more accurately, and accomplish interventions in less time. These improvements in physician performance based on better information will most likely translate into lower-cost procedures and better outcomes for patients. Peter Libby, MD, Associate Editor, JACC: Basic to Translational Science T he term "translational research" trips lightly off the tongue, but the actual practice often proves elusive. I argue here that partnerships can prove pivotal in promoting the translation of laboratory studies to advances in clinical medicine. I focus particularly on what I will define as "horizontal" and "vertical" partnerships. "Horizontal" partnerships pertain to the relationships between laboratory investigators with more patient-based or clinically-oriented participants in the biomedical enterprise. "Vertical" partnerships refer to interactions between academic investigators and those involved in biotechnology and pharmaceutical enterprises. 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Dr. Raptis has reported that he has no relationships relevant to the contents of this paper to disclose. Drs. Jennifer N.A. Silva and Southworth contributed equally to this work and are joint first authors All authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information CENTRAL ILLUSTRATION Mixed Reality Cardiac Electrophysiology Workflow Silva Although augmented reality platforms (bottom left) can show 2-dimensional (2D) data unobtrusively, mixed reality platforms (bottom center) allow for hands-free 2D and 3D visualization as well as direct sterile control of these data without otherwise obstructing the normal visual field Division of Pediatric Cardiology, Washington University School of Medicine, 1 Children's Place, CB 8116 NWT E-mail: jonsilva@wustl Advanced helmet mounted display (AHMD) Taxonomy of mixed reality visual-displays Virtual reality and pain management: current trends and future directions Affective outcomes of virtual reality exposure therapy for anxiety and specific phobias: a meta-analysis MindMaze receives FDA clearance to bring VR rehab platform to the US Pokemon Go is AR's foot in the door to our world What's new in "connected" medical devices? AMA Xperteye. The Xpert Eye solution revolutionize the modern medicine Touch Surgery brings surgery training to augmented reality First 3D television sets go on sale in UK. BBC EchoPixel True 3D Optimization of hologram computation for real-time display Computer-generated real-time digital holography: first time use in clinical medical imaging Holography for imaging in structural heart disease Children's Hospital Stanford pioneers use of VR for patient care Case Western Reserve, Cleveland Clinic collaborate with Microsoft on 'earth-shattering' mixed-reality technology for education Head tracked stereoscopic pre-surgical evaluation of major aortopulmonary collateral arteries in the newborns Abstract 15358: Improving visualization and interaction during transcatheter ablation using an augmented reality system: first-in-human experience (abstr) Intensive upper limb neurorehabilitation with virtual reality in chronic stroke: a case report Peripheral vision and pattern recognition: a review Vision and visual perception Visual Perception: Theory and Practice: Pergamon International Library of Science Perceiving layout and knowing distances: the integration, relative potency, and contextual use of different information about depth The zone of comfort: predicting visual discomfort with stereo displays KEY WORDS augmented reality, cardiology, virtual reality All authors attest they are in compliance with human studies committees and animal welfare regulations of the authors' institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information, visit Medicine, University of Washington, 2141 8th Avenue West, Seattle, Washington 98119. E-mail: lbsparrow@ yahoo.com.