key: cord-0049052-mx8ka3nj
authors: nan
title: Full Issue PDF
date: 2020-08-24
journal: JACC Basic Transl Sci
DOI: 10.1016/s2452-302x(20)30333-8
sha: 52b6b231eee8edbaae896e289a87faa72739b8e4
doc_id: 49052
cord_uid: mx8ka3nj

nan

Two novel and interacting mechanisms contributing to BHV SVD are reported: glycation and serum albumin infiltration.

Glycation product formation and serum albumin deposition were observed in 45 clinical BHV explanted due to SVD as well as BHV tissue subcutaneously implanted in rats.

In vitro exposure to glycation and serum albumin elicited collagen network misalignment similar to that seen in clinical and rat explant BHV tissue.

Glycation was sufficient to impair BHV hydrodynamic function in ISO-5840-compliant pulse duplication testing and concomitant serum albumin infiltration exacerbated these effects.

Valvular heart diseases are associated with significant cardiovascular morbidity and mortality, and often require surgical and/or percutaneous repair or replacement. Valve replacement is limited to mechanical and biological prostheses, the latter of which circumvent the need for lifelong anticoagulation but are subject to structural valve degeneration (SVD) and failure. Although calcification is heavily studied, noncalcific SVD, which represent roughly 30% of BHV failures, is relatively underinvestigated. This original work establishes 2 novel and interacting mechanisms-glycation and serum albumin incorporation-that occur in clinical valves and are sufficient to induce hallmarks of structural degeneration as well as functional deterioration. (4) and younger patients (5, 6) . BHV lifespans are limited by SVD (3, 7) to an average of between 10 and 15 years. This process is driven by cellular, biochemical, and biomechanical mechanisms arising from valve properties, patient characteristics, and the interactions between them. Calcification is observed in the majority of SVD cases; however, approximately 30% of SVD cases are not associated with significant calcification (8, 9) . Further, observation suggests that calcification might be associated with SVD without necessarily being functionally causative (8) . Several noncalcific mechanisms have been proposed to contribute to SVD, including inflammatory oxidation, tissue thickening, and collagen network degeneration (7, 10) ; however, technologies to address known mechanisms thus far have not significantly improved valve durability, suggesting that additional key mechanisms remain unidentified (6) .

Glycation is a complex constellation of nonenzymatic biochemical reactions involving the adduction of sugar-derived moieties to protein nucleophilic groups. Glycation may involve biologically significant intermediates, such as Amadori products, and culminates in the formation of biologically irreversible advanced glycation end products (AGEs) (11) (12) (13) . Glycation strongly contributes to structural and functional degeneration in various native tissues and diseases via 2 major avenues: 1) direct modification of extracellular matrix proteins via crosslinking and modification of protein interactions; and 2) modulation of cell phenotypes and instigation of inflammation via glycation product-mediated receptor signaling (14, 15) . Glycation elicits degeneration of collagenous native tissues via crosslinking and resultant disruption of collagen networks (16) (17) (18) ; yet, it has not been considered in the pathophysiology of BHV leaflets, for which collagen type I is the majority component (19, 20) . BHV lack living native cells or a functional surface barrier. Glycation in this context is 

A U G U S T 2 0 2 0 : 7 5 5 -6 6 likely to occur primarily via infiltration from the surrounding blood of: 1) glycation precursors that modify the extracellular matrix structure directly; 2) pre-glycated proteins that deposit in BHVs; and 3) nonglycated proteins that are glycated in situ by infiltrated precursors. Therefore, we hypothesized that glycation and infiltration by human serum albumin (HSA), the most abundant and glycationsusceptible circulating protein (21, 22) , synergistically contribute to BHV SVD.

PATIENT POPULATION. In order to establish clinical relevance for glycation and albumin infiltration in BHV, 45 patients with BHV aortic valve replacements requiring reoperation and BHV explantation were studied (Supplemental Table 1 Table  1 , Supplemental Methods). A transcatheter aortic valve replacement (TAVR) bioprosthesis was also obtained and analyzed.

Calcification results (Supplemental Figure 1) showed an average leaflet calcification of 126 mg of calcium/mg of leaflet mass (SD ¼ 107 mg/mg). Thirteen valves exhibited nearly no calcification (<10 mg/mg), 6 valves had intermediate calcification (between 10 and 100 mg/mg), and 26 valves had high calcification (>100 mg/mg). Unimplanted BHV were characterized with second-harmonic generation (SHG) microscopy, which demonstrated organized alignment of collagen fiber bundles ( Figures 1A and 1B) . Representative micro-computed tomography and SHG images of *p < 0.05; **p < 0.01; ***p < 0.001 for the comparisons indicated by associated brackets. Only statistically significant (p < 0.05) differences are indicated.

explanted BHVs with various degrees of calcification are shown in Figures 1C and 1D . All explanted leaflets, regardless of calcification, showed disruption of collagen alignment by SHG ( Figure 1E ) compared with unimplanted BHVs ( Figure 1B) .

Clinical explants and unimplanted BHV biomaterials (glutaraldehyde-fixed BP and porcine aortic valve) were analyzed by immunohistochemistry (IHC) for generalized AGE, the AGE receptor ligand Ncarboxymethyl-lysine (CML) (23, 24) , the AGE crosslink glucosepane (25, 26) , and HSA ( Figures 1F and 1G) .

Each of the 45 SAVR explants exhibited significant IHC staining for glycation products ( Figure 1F ) and HSA ( Figure 1G ) compared with unimplanted BHVs. and Supplemental Figures 1D and 1E ).

GLYOXAL AND HAS. To investigate the functional mechanisms of glycation and serum protein infiltration, an in vitro model using BP, 50 mmol/l glyoxal as a glycation precursor (12) , and a physiological concentration (5% w/v) of clinical-grade serum albumin was designed. IHC on BP following 24 hours of incubation demonstrated glyoxal-generated CML staining and infiltration of HSA uniformly throughout the tissue ( Figure 2A) . Coincubation with glyoxal and HSA yielded increased CML staining compared with glyoxal alone (Figure 2A ). 14 C-glyoxal was used in order to measure the glycation capacity of BP. In a 28day study, approximately 50% of the incorporated radioactivity seen at 28 days accumulated within the first 24 h ( Figure 2B ). In coincubation, 14 fication (35) (36) (37) . Calcification has also been related to deposition of calcium-binding proteins in BHV.

Although the protein deposition process generally involves endothelial barrier dysfunction and inflammatory cell-mediated events in atherosclerosis, the results reported here for albumin suggest that calcification-related protein deposition in BHV may be an atherosclerosis-unrelated, cell-free diffusive infiltration event. Nonetheless, glycation-particularly via glycated albumin-is also known to play roles in atherosclerosis-related processes via inflammatory cell activation (11, 13, 14) . It is therefore reasonable to expect that glycation contributes to established in- include the most prominent signaling AGE, CML (14, 23, 24) , and the most abundant crosslinking AGE, glucosepane (25, 26) . Thus, the mechanistic crosstalk between glycation and HSA coincubation lead to the highest degree of structural alteration by SHG assessment and electron microscopy. Additionally, the incorporation of proteins may inculcate BHV tissue with the properties of those proteins, such as calciumand lipid-binding as well as high oncotic pressure in the case of HSA.

The rat model is an established method for testing biomaterial proprieties and calcification in vivo. This model resulted in calcium phosphate deposition within the central region of BP tissue (comparable to observations in clinical explants [41] ), rather than on the surface, as noted with in vitro calcium-phosphate incubations (42) . This system also allows modeling major risk factors for BHV failure, such as patient age. (8, 43) . The lack of demonstrable glucosepane in the rat explants may be due to short (30-day) implantation times. (47, 48) . We restricted our study to a small group of representative glycation-related structures. AGE research has identified a myriad of moieties including numerous crosslinks and receptor ligands (11, 12) . The Several studies reported a strong association between type 2 diabetes mellitus and BPV SVD (2, 3) , and one of the potential causal mechanisms that has been proposed to explain this association was the high 

Patients with CAD and renal insufficiency (eGFR <60 ml/min/1.73 m 2 ) are at an increased risk of adverse outcomes.

CPC counts, an index of endogenous vascular regenerative capacity, may help stratify risk in patients with CAD and renal insufficiency.

Renal insufficiency is associated with lower CPC counts in old, but not young, patients with established CAD.

The increased risk of adverse outcomes with CAD and renal insufficiency is limited to patients with low, but not high, CPC counts. (12, 13) . This stimulation is likely a result of mobilization of progenitors from the bone marrow in response to the risk factor-mediated injury, and represents activation of the endogenous regenerative or reparative systems (5, 13, 14) . It is worth noting that continuous exposure to risk factors with age leads to depletion in CPC counts (14) . In this context, the association of renal insufficiency, a "nontraditional" CV risk factor, with CPC counts and the impact of age on this association has not been evaluated to date.

Furthermore, prior research from our group and others has shown that lower levels of CPCs are independently associated with a higher risk of adverse CV outcomes (15) (16) (17) (18) , and small studies have reported similar findings in patients with end-stage renal disease (19) (20) (21) (22) . Nonetheless, the predictive value of CPCs in patients with CAD and renal insufficiency, a high-risk group, has not been previously studied. Therefore, we sought to investigate: Figure 1 ) (27) . A subset of 20 samples increased risk of all-cause mortality (Supplemental Table 4 ). The association of renal insufficiency with Figure 3) . Thus, lower CD34þ, CD34þ/CD133þ, and CD34þ/CXCR4þ counts were associated with higher CV death or MI and all-cause mortality rates in unadjusted Cox models ( Table 3 ).

In multivariable-adjusted models, the association between hematopoietic CPC counts and CV death or MI remained significant, while only CD34þ/CD133þ Patients with coronary artery disease, renal insufficiency, and (A) CD34þ, (B) CD34þ/CD133þ, or (C) CD34þ/CXCR4þ counts below the respective median cutoffs were at a higher risk of cardiovascular (CV) death or myocardial infarction (MI). Patients with coronary artery disease and renal insufficiency but circulating progenitor cell counts above the respective median cutoffs were at a similar risk of CV death or MI as those without renal insufficiency. eGFR ¼ estimated glomerular filtration rate;

other abbreviations as in Figure 1 . Progenitor Cells, Renal Insufficiency, and CAD counts were independently associated with all-cause death ( Table 3) . No associations between CD34þ/ VEGF2Rþ counts and outcomes were observed ( Table 3 ). There were no interactions of hematopoietic CPC counts with age or with dialysis use (all p for interaction > 0.05) for both outcomes.

Age, eGFR, current smoking, heart failure history, and hemoglobin level were independent predictors of CV death or MI (Supplemental Table 5 Table 8 ).

In this large cohort study of patients with CAD, we found that the presence of renal insufficiency was associated with lower CPC counts among older par- Heart failure reduced maximum force and power by approximately 30% in the myocardium from both ventricles.

Heart failure increased the Ca 2D sensitivity of contraction, but the effect was bigger in right ventricular tissue than in left ventricular samples.

The changes in Ca 2D sensitivity may reflect ventricle-specific post-translational modifications to sarcomeric proteins. showed that heart failure increased maximum force in RV tissue but had no effect on LV samples. Belin et al. (12) found that heart failure reduced Ca 2þ sensitivity in the LV without affecting the RV,

whereas Perreault et al. (13) observed no change in LV samples but did observe an increase in the sensitivity of RV tissue.

This paper extends these studies to the human myocardium. The data showed that heart failure reduced the maximum force and power generated by multicellular preparations from both ventricles without changing contractile kinetics. Ca 2þ sensitivity was increased in failing myocardium from both ventricles, but the effect was greater for RV samples than that for LV tissue. Biochemical assays suggested that the sensitivity effects might reflect ventriclespecific modulation of the phosphorylation of cardiac troponin I (TnI). Table S1 ) and from 5 organ donors who did not have heart failure (Supplemental Table S2) were handed to a researcher in the operating room, Tension pCa curves were calculated by fitting a function of the form:

to the data, where F pas is the passive force, F act is maximum Ca 2þ -activated force, n H is the Hill coefficient, and is the free Ca 2þ concentration needed to develop half-maximum Ca 2þ -dependent force. Blair et al. Because power is the product of force and velocity, power-force curves were calculated by fitting the following function:

to the experimental data. Maximum power, P max , was interaction is a value that decreases with heart failure in samples from the LV but increases with heart failure in samples from the RV, so that the effect of heart failure depends on the ventricle that is being studied.

As previously described (15) , the linear mixed V max is believed to depend on cross bridge cycling kinetics (19) , which can be estimated by measuring the rate of force recovery (k tr ) after a quick shortening and/or re-stretch perturbation. Figure 5A shows representative records measured for a LV preparation isolated from an organ donor. Consistent with previous results from human myocardium (15) Figure 6A shows images from a representative gel that that was stained with Pro-Q Diamond to show phosphorylated proteins and then SYPRO Ruby to show total protein. Figure 6B to 6D shows data obtained from similar images Pro-Q Diamond is commonly used to study sarcomeric proteins (22À24) but the stain is not completely specific to phosphorylated proteins and Figure 1 . Abbreviations as in Figure 1 . to a given intracellular Ca 2þ transient than myocytes with low Ca 2þ sensitivities. Figure 3A confirmed previous work (7, 29) that showed that myocardium from failing hearts is more sensitive to Ca 2þ than tissue from organ donors (p < 0.005). One possibility is that the failing heart enhances Ca 2þ sensitivity to try and compensate for its reduced force-generating capacity.

Supplemental Figure S1 superposes force pCa curves measured with samples from organ donors and patients with heart failure. The curves nearly overlap at the sub-maximal activation levels where the heart normally operates.

The present data also revealed a new statistical interaction between heart failure status and cardiac ventricles. More specifically, heart failure increased the Ca 2þ sensitivity of RV myocardium more than that of LV tissue. Figures 6 and 7 show data that provide insights into the molecular mechanisms that drive these effects. Two independent measures of TnI phosphorylation (Pro-Q Diamond staining [ Figure 6B ]

and Western blotting with an antibody specific to Ser- Interestingly, Hsu et al. (11) showed that treatment with PKA equalized the Ca 2þ sensitivity of RV myocardium from organ donors and in patients with systemic sclerosis-associated pulmonary hypertension, a finding that further reinforced the importance of PKA signaling in disease. However, the present data suggested that PKA was unlikely to be the only mechanism that contributed to interventricular differences because enhanced activity of the kinase in the RV should also have increased the phosphorylation of MyBP-C. This was not observed ( Figure 6C ). (46) . Some clinical data were also unavailable.

This reduced the statistical power of tests that investigated potential relationships between cellular and organ-level function and was a particular concern for parameters related to the RV (Supplemental Table S3 ).

Sample processing was another concern. All of the hearts used were procured by the same personnel and cooled in ice cold saline within moments of being removed from the patient or donor (14) . Nevertheless, the mean time from procurement to the sample being frozen in liquid nitrogen was approximately 30 min.

As demonstrated by Walker et al. (47) , this is clearly enough time to alter the post-translational status of sarcomeric proteins, which sometimes changes on a beat-to-beat basis (48) .

Another issue was that that the patients and organ donors were treated with drugs that were intended to alter contractile properties. Catecholamine levels also rose during tissue procurement (46) 

This study was designed to determine the effect of selective optogenetic simulation of vagal efferent activity on left ventricular function in an animal (rat) model of MI-induced heart failure.

Optogenetic stimulation of dorsal brainstem vagal pre-ganglionic neurons transduced to express light-sensitive channels preserved LV function and exercise capacity in animals with MI.

The data suggest that activation of vagal efferents is critically important to deliver the therapeutic benefit of VNS in chronic heart failure. proved to be more difficult to achieve (5, 6) . Patients with heart failure on optimal medical therapy with persistent autonomic dysfunction have the worst prognosis (7). with dental acrylic ( Figure 1A) . Anesthesia was 

A U G U S T 2 0 2 0 : 7 9 9 -8 1 0 reversed with atipamezole (1 mg/kg intramuscularly).

Carprofen (5 mg/kg/day subcutaneously) was given for post-operative analgesia for 3 days, and the animals were allowed to recover for 7 days. Rats were 300 to 350 g at the time of the main experiment. Figures 1B and 1C) . were included in the analysis. There was no difference in mean infarct size between the 2 groups (p ¼ 0.14) (Figure 2A) . Table 1) . sham-operated animals ( Figure 3 ). There were no differences in heart rate (measured under urethane anesthesia) between the experimental groups ( Table 1) .

The data obtained in the present study showed that optogenetic stimulation of vagal pre-ganglionic neu- conclusion to draw in the context of a strong clinical association between low chronotropic vagal tone and the risk of death after MI or in established heart failure (40, 41) , and of the effectiveness of pharmacological interventions that lower heart rate (e.g., b-blockers and I f inhibitors) (42) (43) (44) . In most clinical trials of VNS in heart failure, the stimulating electrodes were placed on the right cervical vagus nerve, with the aim of lowering the heart rate, but it was not reliably attained (20) . Comparisons are made using 2-way analysis of variance followed by Sidak's correction for multiple comparisons. Abbreviations as in Figures 1 and 2 . Overall, the present study by Machhada et al. (6) reinforces the rationale for efferent VNS at the ventricular level in heart failure. It also draws attention to a novel additional pathway that might be involved in sympathetic-parasympathetic interaction at the cardiac level. 

This multidisciplinary work shows the feasibility of replacing the fetal pulmonary valve with a percutaneous, transcatheter, fully biodegradable tissue-engineered heart valve (TEHV), which was studied in vitro through accelerated degradation, mechanical, and hemodynamic testing and in vivo by implantation into a fetal lamb.

The TEHV exhibited only trivial stenosis and regurgitation in vitro and no stenosis in vivo by echocardiogram.

Following implantation, the fetus matured and was delivered at term. Replacing a stenotic fetal valve with a functional TEHV has the potential to interrupt the development of single-ventricle heart disease by restoring (14) were used to characterize the PCL fiber network.

Scanning electron microscopy (SEM) images of 500Â (n ¼ 7) and 1000Â (n ¼ 7) were analyzed and fibrous mesh characteristics including fiber tortuosity, alignment and density were quantified. Tortuosity was measured using ImageJ through tracing the total and end-to-end fiber lengths of a representative 12 fibers in each SEM image, with tortuosity calculated as:

For fiber alignment, the normalized orientation index (NOI) was chosen as a representative measurement and was calculated using the previously 

Pinwheeling, or localized bending of leaflet material upon closure, is known to cause increased localized bending stresses and hypothesized to correlate with decreased durability (25, 26) . Ideally, no Last, minimizing fetal blood loss through the cannula, between the time that the trocar was removed and the balloon or stent were inserted, was critical for fetal survival.

T E H V i m p l a n t a t i o n . Using what was learned in the fetal stent pilot study, TEHV implantation was attempted in 2 fetuses (#9A, #10). In fetus #9A, the TEHV was implanted in the MPA, which resulted in obstructed blood flow to the branch pulmonary arteries. Immediate bradycardia was noted, which did not respond to epinephrine. In fetus #10, the TEHV was implanted in the pulmonary annulus. Laminar flow was seen across the TEHV without regurgitation In vitro work on the valve PCL material demonstrated that the designed and created valve was Zakko et al. minimizing fetal blood loss through the introducing cannula. Guided by the new knowledge gleaned from the pilot study, subsequent implantations were more successful, but still revealed serious complications associated with this procedure including heart block, stent migration, bradycardia, and pericardial effusion. In the successful implantation, the valve demonstrated laminar flow without regurgitation but subsequently migrated to the main pulmonary artery. Still, the fetus survived to term and was alive at 18 months.

A major contribution of this paper is the development and percutaneous delivery of a fully biodegradable heart valve in the pulmonary annulus. This is the first report of transcatheter delivery of a TEHV.

Previously, researchers (4) had used nitinol stent frames for stem-cell based pulmonary valves which do not grow with the child, and require a fetal thoracotomy for delivery through the right ventricle using a delivery system that is approximately 4 times larger than that described in this work. Indeed, percutaneous delivery has many advantages including obviating the need for maternal and fetal incisions and a lower incidence of iatrogenic preterm prelabor rupture of membranes (5) .

Future work will be needed to investigate the cause of the many complications reported with this procedure, to optimize the design of the delivery system and the implant, and to fully characterize the degradation of the stent, and the rate of neotissue formation.

In summary, the work shows the first proof-of- Table 1 (9-11) .

Driven largely by budget limitations that prevented the FDA from employing sufficient personnel to provide timely processing of NDAs, FDA review times ran around 33 months in 1987 (12) . In the face of a growing AIDS crisis, the pharmaceutical industry offered funding to the FDA in the form of "user fees" in press concern that there has been a weakening of the Continued to build on previous legislation to incorporate patient perspectives into drug and device development, and to modernize clinical trials design through use of real-world evidence and clinical outcomes assessments. Provided for recruitment and retention of appropriate experts, and established 2 new expedited product development programs: 1) RMAT; and 2) breakthrough devices. Directed the FDA to create intercenter institutes to coordinate activities in major disease areas between the centers for drug, biologics and device centers, and to improve the regulation of combination products.

In addition to miscellaneous systems reporting and organizational improvements: Continued the 5-yr reauthorization cycle of human medical product user fees (PDUFA VI-see following text), and allows the FDA to use the revenue to support the marketing applications for brand-name and generic drugs, biological, and biosimilar products, and medical devices Established the renewed fees related to drugs, devices, generic drugs, and biosimilars (PDUFA) Reauthorized existing programs such as the Orphan Drug Program Included the RACE for Children Act, requiring evaluation for new molecular targets and biologics intended for adults that are specifically targeted for cancers in children, elimination of orphan exemption from pediatric studies Furthermore, the number of NDAs approved during that period that relied on at least 1 Phase III study using an active comparator rather than historic controls or placebo fell from 44% to 29%, and the proportion of NDA approvals based on nonrandomized, uncontrolled studies increased from 4% to 17%.

Despite these changes, however, the actual length of clinical trials themselves for NDAs increased from 2015 to 2017, with almost one-half (46%) including at least 1 pivotal trial of 6 months' duration or more, compared with just 26% from 1995 to 1997 (21) . DAB that treats a serious condition* that fills an unmet medical need can be approved on the basis of a surrogate endpoint: for example, lab marker, radiographic images, physical sign, or other finding that is thought to predict clinical benefit. Surrogate or intermediate clinical endpoints are allowed.

Priority Review 1992 PDUFA DAB would be a significant improvement in the safety or efficacy of the treatment, diagnosis, or prevention of serious conditions when compared to standard therapy.

Fast Track FDA Modernization Act of 1997 DABs that treat a serious condition must fulfill an unmet medical need or provide therapy that is substantially better in safety and efficacy than existing ones. Fast-Track DABs may also be eligible for Accelerated Approval and Priority Review if criteria are met, Breakthrough Therapy 2012 FDASIA DABs that are intended to treat a serious condition, and preliminary clinical evidence indicates that it may demonstrated substantial improvement over available therapy on a clinically significant endpoint. Surrogate endpoints allowed.

Regenerative Medicine Advanced Therapy 21st Century Cures Act, 2016 Regenerative medicine advanced therapy (cell therapy therapeutic tissue engineering product, human cell and tissue product, or any combination product using these) used to treat, modify, reverse, or cure a serious or life-threatening condition, and preliminary clinical evidence indicates that it has the potential to address an unmet medical need These changes decreased development times significantly (4.8 vs. 8 years) (19) . Yamashita et al. (24) found that for anticancer therapies, pursuit of any of the expedited programs was associated with reduced review times (Table 3) , by a range of 2.0 to 3.4 years.

This finding is similar to a previous study by Hwang et al. (32) . Most DAB approvals that they examined in the AA program (88%) used a noncomparative study design (24) . As more and more diseases are genetically subclassified, more drugs qualify for AA tracts.

Almost two-thirds of all INDs (64%) now qualify for such programs (14) .

The RMAT drug classification went live in March of 2017 and is too new to determine whether it has been associated with reduced development timelines (33) .

IS PATIENT SAFETY BEING COMPROMISED? Accelerated drug approval pathways shave time off of the approval process and may theoretically reduce DAB Average times in years from Investigational New Drug application to U.S. Food and Drug Administration (FDA) drug approval for anticancer drugs and biologicals achieving accelerated track designation at the FDA from 2012 to 2017 compared with overall anticancer drug approval times for the same period (median of 8.3 ys, n ¼ 115 drugs), and separated by categorization as a drug for "minor" versus "major" cancer. (24) . *Regenerative Medicine Advanced Therapy designation was created too recently to comment on review times. which announced on June 10th, 2020, that it would begin a Phase I/II study (42) . Oxford University has also indicated that trials of their vaccine will proceed along a process blending Phases I and II, and will certainly be applying for EUAs if efficacy is shown (43) . With reportedly around 100 companies and academic institutions competing to develop a COVID-19 vaccine (44), it is a virtual given that they will all try to make full use of the innovative trial designs and accelerated programs combined with early EUAs to race for approval. 

Large Animal Models of Heart Failure 

Cardiac cell death associated with aberrant heart dysfunction is the main characteristic of a myocardial infarction (MI), which can ultimately lead to HF (68, 69) . This catastrophic event occurs due to interruption of blood flow to a discrete area of the myocardium that results from partial or complete Table 2 .

The impact of TandemHeart on hemodynamics and cardiac morphology has been investigated in porcine models of acute MI or ventricular arrhythmia (151À155). For example, TandemHeart was implanted during LCx occlusion (30 min) and effectively unloaded the LV while maintaining systemic pressure, which was evident via decreased stroke volume, end-diastolic volume, and EDP (151) . Impella devices have also been investigated in swine (152, (156) (157) (158) (159) (160) (161) and ovine (162, 163) At comparable flow rates, TandemHeart decreased LV preload (end-diastolic volume), stroke volume, and contractility (dP/dt max , stroke work, pre-load recruitable stroke work) to a greater extent than the Impella.

Although the preceding studies reflect successful translational interactions and outcomes between large animal and human studies, the process is not infallible. One example is the CentriMag Circulatory ventricular assist device (Abbott Laboratories, Abbott Park, Illinois), which was recalled due to a calibrating system error linked to electromagnetic interference, which caused the device to stop (https://www.fda. gov/medical-devices/medical-device-recalls/abbottrecalls-centrimag-circulatory-support-system-motordue-pump-and-motor-issues). Difficulties with the device occurred after FDA approval, despite several preclinical studies in both sheep (164À166) and pigs (167) , 3 registered clinical trials ( Here, we review the current understanding of autophagic cell death, with a focus on autosis, and we discuss the involvement of cardiomyocyte autosis in myocardial injury and the underlying molecular mechanisms.

To explain how autophagy can kill cells under some conditions, we will first describe the physiological function of autophagy and its molecular machinery.

Autophagy is an essential catabolic process that is highly conserved in eukaryotes. The process of autophagy comprises multiple steps, including initiation, nucleation, elongation and completion, and fusion and degradation.

In the initiation step, the Unc-51-like kinase 1 (ULK1)/autophagy-related protein 1 (Atg1) complex is regulated by mammalian target of rapamycin (mTOR) inhibition in response to autophagy-inducing conditions, such as starvation. Thereafter, the ULK1/Atg1 complex activates the phosphatidylinositol 3 kinase complex to nucleate autophagosomal membranes. In the elongation step, 2 ubiquitin-like conjugation (19) . Although autophagy is involved in these 2 types of cell death, the cell death itself is mediated through other forms of cell death, including apoptosis and necrosis. It has thus been recommended to use the terms "autophagy-associated cell death" or "autophagy-mediated cell death" unless the death is prevented only by multiple autophagyinhibiting interventions but not by inhibition of other forms of cell death, such as apoptosis or necrosis.

Along these lines, it is preferable to use the term "autophagy-dependent cell death" when the aforementioned criteria have been clearly proven (20) . In phase 2, the last step of autosis, cytoplasmic organelles are drastically decreased and focal nuclear concavity and focal ballooning of the PNS are observed (21) . Cells that have died by autosis are generally more firmly attached to the culture dishes than are those that do not undergo autosis, and the increased adherence appears to be another morphological feature of autosis in vitro. To date, the best method for defining autosis is electron microscopy analysis.

Alternatively, immunofluorescence assays can also help distinguish autotic cells by detecting fragmented ER or mitochondria with nuclear concavity.

Although autosis can be inhibited by chemical inhibitors of autophagy, including 3-methyladenine, and down-regulation of the autophagic machinery, such as beclin 1 and Atg7, it cannot be inhibited by inhibitors of apoptosis, necrosis, or any other form of programmed cell death. It should be mentioned that suppression of lysosomes fails to inhibit autosis in either HeLa cells or cardiomyocytes. This suggests that autosis may be induced by excessive activation of autophagosome formation, but not by excessive degradation. We will discuss this issue in the section on the underlying mechanism of autosis.

Another important feature of autosis is its sensitivity to cardiac glycosides. A compound library screen revealed that inhibitors of Na þ ,K þ -adenosine triphosphatase (ATPase) effectively suppress autosis (6, 22) . Consistent with this finding, cardiac glycoside, a chemical inhibitor of Na þ ,K þ -ATPase, dramatically reduces Transactivator of transcription (Tat)-beclin 1induced autosis in vitro and the autosis observed in neonatal rats subjected to brain ischemia in vivo (6) .

Moreover, injection of ouabain into cardiac glycoside-sensitive mice significantly reduces myocardial I/R injury in adult mice (23) . We also noted that protein expression of Na þ ,K þ -ATPase was up-regulated in the heart in response to I/R, the time course of which coincides with that of autosis. One caveat here is that Na þ ,K þ -ATPase in rodent hearts is less sensitive to cardiac glycosides than that in human hearts is. Thus, it is difficult to test whether cardiomyocyte death can be rescued by cardiac glycosides in the murine heart. Humanized Na þ ,K þ -ATPase a1 subunit knock-in mice can be used to overcome this issue (23) . Alternatively, the a1 subunit of Na þ ,K þ -ATPase can be down-regulated with short hairpin ribonucleic acid without obvious effects on contractility.

The features of autosis compared with other forms of programmed cell death are summarized in Table 1 .

Currently, no other criteria are available to distinguish autotic cell death from other forms of cell death. We expect, however, that progress in the field During the early phase of autosis, the numbers of autophagosomes (APs), autolysosomes (ALs), and empty vacuoles (EVs) are drastically increased and separation of the inner and outer nuclear membranes is observed. The later phase is characterized by focal nuclear concavity, focal ballooning of the perinuclear space (PNS), and disappearance of subcellular organelles. ER ¼ endoplasmic reticulum; Nu ¼ nucleus.

may provide additional criteria to help identify autosis more easily and precisely.

Although autosis was initially identified in HeLa cells when very high levels of autophagy were induced by Tat-beclin 1 in vitro (6), autosis has also been observed in vivo under various conditions (Central Illustration).

Hypoxia-ischemia and reperfusion appear to be common conditions in which strong activation of autophagy and autosis are observed in multiple organs. Cerebral hypoxia-ischemia in the brain of neonatal rats promotes autosis, and cardiac glycosides, such as neriifolin, digoxin, and digitoxigenin, rescue autotic cell death in vivo (6) . Autosis is also induced in mouse kidneys subjected to I/R (22) . More recently, autosis was also observed in the heart and the cardiomyocytes therein during the late phase of I/ R. The fact that inhibition of autosis with cardiac glycosides reduces the size of infarct in all of these models suggests that autosis is widely involved in tissue injury in response to I/R (23).

ultimately kills infected cells, HIV-infected macrophages escape cell death (24) . Interestingly, autophagy-inducing peptides, including Tat-beclin 1 and Tat-viral FLICE inhibitory protein-a2, preferentially kill chronically HIV-infected human macrophages, whereas the cell death is inhibited by knockdown of ATG genes and Na þ ,K þ -ATPase a1 (25) . During the initial stage of hypoxia, epidermal growth factor receptor (EGFR) is activated and interacts with beclin 1, which activates autophagy at physiological levels and contributes to survival of cancer cells.

When hypoxia persists, however, EGFR is deactivated by autophagic degradation of caveolin 1 and dissociates from the beclin 1 complex, which in turn stimulates autophagic flux above physiological levels, thereby triggering autosis. The detailed mechanism Autosis is activated by the autophagy-inducing peptide, tyrosine aminotransferase (Tat)-beclin 1, hypoxia-ischemia in neonatal cerebral brain and ischemia-reperfusion in the heart. Autosis is regulated by the physical interaction between beclin 1 and Na þ ,K þ -adenosine triphosphatase (ATPase), which is inhibited by cardiac glycosides, by up-regulation of rubicon, which attenuates fusion of autophagosome and lysosome, and by the core autophagy machinery. Atg5 ¼ autophagy-related protein 5. 

Because I/R strongly induces autophagy in the heart, it is natural to speculate that autosis is induced, as in the case of I/R injury, in the brain and kidney. In fact, we have shown recently that autosis of cardiomyocytes is induced in the heart in vivo in response to I/R (23).

Here, we describe the key findings of this study and discuss the underlying mechanism of autosis induced by I/R. Autophagy is up-regulated by a short period of ischemia and further stimulated during reperfusion in the heart, whereas myocardial injury is attenuated 

Here, we discuss why strong activation of autophagy leads to autotic cell death ( Figure 3 ). An important consideration is the fact that induction of autosis cannot be alleviated by lysosome inhibitors. Thus, autosis is not mediated through lysosomal degradation. Increasing lines of evidence suggest that excessive accumulation of autophagosomes may mediate autotic cell death. Excessive accumulation of autophagosomes can take place when the balance between synthesis and degradation of autophagosomes is disrupted. We will discuss this issue, using the induction of autosis in response to myocardial I/R as an example.

Although autophagic flux is up-regulated during the initial hours of myocardial reperfusion, it gradually decreases thereafter (23) . We found that autosis is Currently, the sensitivity to cardiac glycosides is among the most important clues to elucidating the molecular mechanism of autosis. Na þ ,K þ -ATPase physically interacts with beclin 1 to stimulate autosis.

Cardiac glycosides disrupt the interaction between Na þ ,K þ -ATPase and beclin 1 by interacting with Na þ ,K þ -ATPase (22) . It is possible that this interaction alters ion pump activity or ion exchange-dependent effects of Na þ ,K þ -ATPase. Na þ ,K þ -ATPase modulates calcium signaling through interaction with its steroid agonist ouabain (36) . However, treatment of cardiomyocytes with a calcium chelator, 1,2-Bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetrakis(acetoxymethyl ester), did not affect autosis (23) , suggesting that calcium may not be involved in autosis. Interestingly, the interaction between Na þ ,K þ -ATPase and beclin 1 takes place at many intracellular membranes, including ER, perinuclear membranes, mitochondria, and endosomes. Thus, the Na þ ,K þ -ATPase/beclin 1 interaction may trigger autosis through its effect on membrane ion, osmolyte, and fluid homeostasis across intracellular membranes (37) . Further investigation is required to elucidate the role of the Na þ ,K þ -ATPase/beclin 1 interaction.

Another possibility is that Na þ ,K þ -ATPase alters the function of beclin 1, thereby affecting autophagic activity or vesicle trafficking. Because rubicon also physically interacts with beclin 1 (38) , modulation of beclin 1 may play a key role in mediating autosis.

However, the interaction between Na þ ,K þ -ATPase and beclin 1 alone may not affect the function of beclin 1 in such a way as to induce autophagy. Further investigation is required to clarify how the interaction between beclin 1 and either Na þ ,K þ -ATPase or rubicon affects the function and modulates the activity of autosis.

Acute myocardial infarction is among the main causes of morbidity worldwide (39 

Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis

Application of tissueengineering principles toward the development of a semilunar heart valve substitute

Association between patient age at implant and outcomes after transcatheter pulmonary valve replacement in the multicenter Melody valve trials

Small-sized conduits in the right ventricular outflow tract in young children: bicuspidalized homografts are a good alternative to standard conduits

What Is the potential of tissueengineered pulmonary valves in children?

Immediate effects and outcome of in-utero pulmonary valvuloplasty in fetuses with pulmonary atresia with intact ventricular septum or critical pulmonary stenosis

Prenatally engineered autologous amniotic fluid stem cellbased heart valves in the fetal circulation

Transcatheter (TAVR) versus surgical (AVR) aortic valve replacement: occurrence, hazard, risk factors, and consequences of neurologic events in the PARTNER trial

Metallic zinc exhibits optimal biocompatibility for bioabsorbable endovascular stents

A biodegradable synthetic graft for small arteries matches the performance of autologous vein in rat carotid arteries

A new simplified technique for making tricuspid expanded polytetrafluoroethylene valved conduit for right ventricular outflow reconstruction

A small angle light scattering device for planar connective tissue microstructural analysis

An in vitro evaluation of turbulence after transcatheter aortic valve implantation

Sinus hemodynamics variation with tilted transcatheter aortic valve deployments

Fetal transcatheter trileaflet heart valve hemodynamics: implications of scaling on valve mechanics and turbulence

Fetal cardiac ventricular volume, cardiac output, and ejection fraction determined with 4-dimensional ultrasound using spatiotemporal image correlation and virtual organ computer-aided analysis

Fetal cardiac output estimated by Doppler echocardiography during mid-and late gestation

Pulmonary and systemic arterial pressure in hyaline membrane disease

Experimental measurement of dynamic fluid shear stress on the ventricular surface of the aortic valve leaflet

A turbulence in vitro assessment of On-X and St Jude Medical prostheses

Fluid mechanics of artificial heart valves

Cardiovascular implants -Cardiac valve prostheses -Part 3: heart valve substitutes implanted by transcatheter techniques

Simulation of long-term fatigue damage in bioprosthetic heart valves: effects of leaflet and stent elastic properties

Valve-invalve outcome: design impact of a pre-existing bioprosthesis on the hydrodynamics of an Edwards Sapien XT valve

Fetal surgery for congenital heart disease

Length of gestation in ewes carrying lambs of two different breeds

Threedimensional quantitative micromorphology of preand post-implanted engineered heart valve tissues

Biomechanical properties of native and tissue engineered heart valve constructs

On the biaxial mechanical properties of the layers of the aortic valve leaflet

Stresses of natural versus prosthetic aortic valve leaflets in vivo

Birth prevalence of congenital heart disease worldwide: a systematic review and meta-analysis

Application of tissueengineering principles toward the development of a semilunar heart valve substitute

Development of tissue engineered heart valves for percutaneous transcatheter delivery in a fetal ovine model

Prenatally engineered autologous amniotic fluid stem cellbased heart valves in the fetal circulation

Preterm prelabor rupture of membranes and fetal survival after minimally invasive fetal surgery: a systematic review of the literature

How FDA Approves Drugs and Regulates Their Safety and Effectiveness

Single patient investigational new drug, and the "right to try

Food and Drug Administration

Food and Drug Administration

Food and Drug Administration. 21st Century Cures Act

The FDA Reauthorization Act of 2017

Completed PDUFA VI Deliverables

Approval Times Have Decreased in Recent Years

GAO/PEMD-96-1

Speed, safety and industry funding-from PDUFA I to PDUFA VI

FDA approval and regulation of pharmaceuticals

Prescription Drug User Fee Rates for Fiscal Year 2020

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Reform at the FDA-in need of reform

failed-certificationfaa-missed-safety-issues-in-the-737-max-systemimplicated-in-the-lion-air-crash

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Combating COVID-19 With Therapeutics

Understanding Use of Approved Drugs "Off Label

Warp Speed" operations in the COVID-19 pandemic: moving too quickly?

Trump Administration Announces Framework and Leadership for 'Operation Warp Speed

Heart disease and stroke statistics-2019 update: a report from the American Heart Association

ACCF/ AHA guideline for the management of heart failure: a report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Failure Society of America

ACCF/AHA guideline for the management of heart failure: a report of the American College of Cardiology Foundation/American Heart Association Task Force on Practice Guidelines

ESC Guidelines for the diagnosis and treatment of acute and chronic heart failure: the task force for the diagnosis and treatment of acute and chronic heart failure of the European Society of Cardiology (ESC) developed with the special contribution of the Heart Failure Association (HFA) of the ESC

Swine in cardiovascular research

Gross and microscopic anatomy of the canine intrinsic cardiac nervous system

Anatomy of the pig heart: comparisons with normal human cardiac structure

A comparative anatomic and physiologic overview of the porcine heart

Small and large animal models in cardiac contraction research: advantages and disadvantages

Large animal model of left ventricular aneurysm

Experimental animal models of coronary microvascular dysfunction

Of pigs and men and research: a review of appreciation, and the logic of the pig in human medical research

Distribution of blood flow in muscles of miniature swine during exercise

Aortic stenosis: then and now

Aortic stenosis: from pressure overload to heart failure

Myocardial remodeling with aortic stenosis and after aortic valve replacement: mechanisms and future prognostic implications

Animal models of heart failure: a scientific statement from the American Heart Association

Aortic stenosis

Coronary microvascular rarefaction and myocardial fibrosis in heart failure with preserved ejection fraction

Cardiovascular remodelling in coronary artery disease and heart failure

Oxidative capacity in failing hearts

Highenergy phosphate metabolism and creatine kinase in failing hearts: a new porcine model

Bioenergetic and functional consequences of stem cell-based VEGF delivery in pressure-overloaded swine hearts

Saxagliptin prevents increased coronary vascular stiffness in aortic-banded mini swine

Saxagliptin and tadalafil differentially alter cyclic guanosine monophosphate (cGMP) signaling and left ventricular function in aortic-banded mini-swine

Chronic low-intensity exercise attenuates cardiomyocyte contractile dysfunction and impaired adrenergic responsiveness in aorticbanded mini-swine

Chronic interval exercise training prevents BKCa channel-mediated coronary vascular dysfunction in aortic-banded miniswine

Female sex hormones and cardiac pressure overload independently contribute to the cardiogenic dementia profile in Yucatan miniature swine

The protective role of sex hormones in females and exercise prehabilitation in males on sternotomyinduced cranial hypoperfusion in aortic banded mini-swine

Loss of female sex hormones exacerbates cerebrovascular and cognitive dysfunction in aortic banded miniswine through a neuropeptide Y-Ca(2þ)-activated potassium channel-nitric oxide mediated mechanism

Carotid artery vascular mechanics serve as biomarkers of cognitive dysfunction in aortic-banded miniature swine that can be treated with an exercise intervention

Chronic exercise training prevents coronary artery stiffening in aortic-banded miniswine: role of perivascular adipose-derived advanced glycation end products

Increased stiffness is the major early abnormality in a pig model of severe aortic stenosis and predisposes to congestive heart failure in the absence of systolic dysfunction

Progressive induction of left ventricular pressure overload in a large animal model elicits myocardial remodeling and a unique matrix signature

Western diet-fed, aortic-banded Ossabaw swine: a preclinical model of cardio-metabolic heart failure

Impaired capsaicin-induced relaxation of coronary arteries in a porcine model of the metabolic syndrome

Components of metabolic syndrome and coronary artery disease in female Ossabaw swine fed excess atherogenic diet

Metabolic syndrome and coronary artery disease in Ossabaw compared with Yucatan swine

Vascular transcriptional alterations produced by juvenile obesity in Ossabaw swine

High-fat, high-fructose, high-cholesterol feeding causes severe NASH and cecal microbiota dysbiosis in juvenile Ossabaw swine

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Microvascular insulin resistance in skeletal muscle and brain occurs early in the development of juvenile obesity in pigs

Research priorities for heart failure with preserved ejection fraction: National Heart, Lung, and Blood Institute working group summary

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Upregulation of Bcl-2 proteins during the transition to pressure overload-induced heart failure

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Development of coronary collateral circulation in left circumflex Ameroidoccluded swine myocardium

Glucagon-like peptide 1 receptor activation augments cardiac output and improves cardiac efficiency in obese swine after myocardial infarction

Metformin alters the insulin signaling pathway in ischemic cardiac tissue in a swine model of metabolic syndrome

Overfed Ossabaw swine with early stage metabolic syndrome have normal coronary collateral development in response to chronic ischemia

Transplantation of autologous endothelial cells induces angiogenesis

Natriuretic peptides maintain sodium homoeostasis during chronic volume loading post-myocardial infarction in sheep

Progressive myocardial dysfunction associated with increased vascular resistance

A canine model of chronic heart failure produced by multiple sequential coronary microembolizations

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Histological remodeling in an ovine heart failure model resembles human ischemic cardiomyopathy

Selective microembolization of the circumflex coronary artery in an ovine model: dilated, ischemic cardiomyopathy and left ventricular dysfunction

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Neurohumoral changes during onset and offset of ovine heart failure: role of ANP

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The synergism between atrial fibrillation and heart failure

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Percutaneous left ventricular support devices

Intraaortic balloon support for myocardial infarction with cardiogenic shock

ESC guidelines for the diagnosis and treatment of acute and chronic heart failure 2012: the task force for the diagnosis and treatment of acute and chronic heart failure 2012 of the European Society of Cardiology. Developed in collaboration with the Heart Failure Association (HFA) of the ESC

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ACCF/AHA guideline for the management of ST-elevation myocardial infarction: a report of the American College of Cardiology Foundation/ American Heart Association Task Force on Practice Guidelines

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Management of cardiogenic shock

IABP: history-evolutionpathophysiology-indications: what we need to know

A prospective, randomized clinical trial of hemodynamic support with Impella 2.5 versus intraaortic balloon pump in patients undergoing highrisk percutaneous coronary intervention: the PROTECT II study

Experience from a randomized controlled trial with Impella 2.5 versus IABP in STEMI patients with cardiogenic pre-shock. Lessons learned from the IMPRESS in STEMI trial

Mechanically unloading the left ventricle before coronary reperfusion reduces left ventricular wall stress and myocardial infarct size

Clinical experience with the TandemHeart percutaneous ventricular assist device

Can myocardial infarct size be reduced by mechanically unloading the left ventricle?

Distinct effects of left or right atrial cannulation on left ventricular hemodynamics in a swine model of acute myocardial injury

Comparative hemodynamic effects of contemporary percutaneous mechanical circulatory support devices in a porcine model of acute myocardial infarction

Direct comparison of percutaneous circulatory support systems in specific hemodynamic conditions in a porcine model

Left ventricular assist via percutaneous transhepatic transseptal cannulation in swine

Modified TandemHeart ventricular assist device for infant and pediatric circulatory support

Speckle-tracking echocardiographic strain analysis reliably estimates degree of acute LV unloading during mechanical LV support by Impella

Support with intra-aortic balloon pump vs. Impella2.5(R) and blood flow to the heart, brain and kidneys -an experimental porcine model of ischaemic heart failure

Left ventricular unloading using an Impella CP improves coronary flow and infarct zone perfusion in ischemic heart failure

Initial experience with the pediatric Impella device: a feasibility study in a porcine model

Left ventricular unloading before reperfusion promotes functional recovery after acute myocardial infarction

Pediatric heart support with a newly developed catheter based pulsatile 12F rotary blood pump: an animal study

Short-term meimproves heart function

Pulsatile control of rotary blood pumps: Does the modulation waveform matter?

Effect of pulsatility on the mathematical modeling of rotary blood pumps

Wang-Zwische double-lumen cannula leads to total cavopulmonary support in a failing Fontan sheep model

Single double-lumen venous-venous pump-driven extracorporeal lung membrane support

Outcomes of a multicenter trial of the Levitronix CentriMag ventricular assist system for short-term circulatory support

Hybrid mouse diversity panel: a panel of inbred mouse strains suitable for analysis of complex genetic traits

Interventional Scientific Council of the American College of Cardiology. A practical approach to mechanical circulatory support in patients undergoing percutaneous coronary intervention: an interventional perspective

KEY WORDS heart failure, HFpEF, HFrEF, large animal model

Molecules and their functions in autophagy

Autophagic cell death: the story of a misnomer

Distinct roles of autophagy in the heart during ischemia and reperfusion: roles of AMP-activated protein kinase and beclin 1 in mediating autophagy

Cardiac autophagy is a maladaptive response to hemodynamic stress

Transcription factor GATA4 inhibits doxorubicin-induced autophagy and cardiomyocyte death

Autosis is a Naþ,Kþ-ATPase-regulated form of cell death triggered by autophagy-inducing peptides, starvation, and hypoxia-ischemia

Does autophagy mediate cardiac myocyte death during stress?

The mammalian ULK1 complex and autophagy initiation

The beclin 1 network regulates autophagy and apoptosis

Rubicon and PLEKHM1 negatively regulate the endocytic/autophagic pathway via a novel Rab7-binding domain

Origin of the autophagosome membrane in mammals

Molecular dissection of autophagy: two ubiquitin-like systems

The hairpin-type tail-anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes

Principles of lysosomal membrane degradation: cellular topology and biochemistry of lysosomal lipid degradation

Guidelines for evaluating myocardial cell death

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death

Autophagic programmed cell death by selective catalase degradation

Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes

Guidelines for the use and interpretation of assays for monitoring autophagy

Tyrosine kinase receptor EGFR regulates the switch in cancer cells between cell survival and cell death induced by autophagy in hypoxia

Interaction between the autophagy protein beclin 1 and Naþ, Kþ-ATPase during starvation, exercise, and ischemia

Upregulation of rubicon promotes autosis during myocardial ischemia/reperfusion injury

Understanding HIV latency: the road to an HIV cure

Induction of a Na(þ)/K(þ)-ATPasedependent form of autophagy triggers preferential cell death of human immunodeficiency virus type-1-infected macrophages

Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy

Selective cell death of latently HIV-infected CD4(þ) T cells mediated by autosis inducing nanopeptides

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages

Human immunodeficiency virus type-1 infection inhibits autophagy

EGFR inhibitors and autophagy in cancer treatment

Hypoxia-induced autophagy contributes to the chemoresistance of hepatocellular carcinoma cells

Autosis occurs in the liver of patients with severe anorexia nervosa

Molecular mechanisms of autophagy in the cardiovascular system

Induction of a massive endoplasmic reticulum and perinuclear space expansion by expression of lamin B receptor mutants and the related sterol reductases TM7SF2 and DHCR7

Disruption of the gene encoding 3beta-hydroxysterol Delta-reductase (Tm7sf2) in mice does not impair cholesterol biosynthesis

The Na-K-ATPase and calciumsignaling microdomains

Nuclear Naþ/Kþ-ATPase plays an active role in nucleoplasmic Ca2þ homeostasis

Two beclin 1-binding proteins, Atg14L and rubicon, reciprocally regulate autophagy at different stages

Global, regional, and national burden of cardiovascular diseases for 10 causes

for the CARDIOPROTECTION COST Action Investigators. Multitarget strategies to reduce myocardial ischemia/reperfusion injury: JACC Review Topic of the Week

Impaired autophagy contributes to adverse cardiac remodeling in acute myocardial infarction

Doxorubicin blocks cardiomyocyte autophagic flux by inhibiting lysosome acidification

Rag GTPases are cardioprotective by regulating lysosomal function

LncRNA CAIF inhibits autophagy and attenuates myocardial infarction by blocking p53-mediated myocardin transcription

Aldehyde dehydrogenase 2 (ALDH2) rescues myocardial ischaemia/reperfusion injury: role of autophagy paradox and toxic aldehyde

Impaired autophagosome clearance contributes to cardiomyocyte death in ischemia/reperfusion injury

Histone deacetylase (HDAC) inhibitors attenuate cardiac hypertrophy by suppressing autophagy

MiR-30-regulated autophagy mediates angiotensin IIinduced myocardial hypertrophy

Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury

Autophagy exacerbates electrical remodeling in atrial fibrillation by ubiquitin-dependent degradation

Adult mouse Beclin 1 þ/À mouse Autophagy is drastically activated during I/R in the heart Reduced myocardial infarct size with reduced autophagy in beclin 1 þ/À mice Matsui et al.Rubicon þ/À mouse Autophagic flux is increased during ischemic period but attenuated during reperfusion Myocardial infarct size is reduced by restoration of autophagic flux in rubicon þ/À mice during I/R