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Background The Life Extension Medical Decision law enacted on February 4, 2018 in South Korea was the first to consider the suspension of futile life-sustaining treatment, and its enactment caused a big controversy in Korean society. However, no study has evaluated whether the actual implementation of life-sustaining treatment has decreased after the enforcement of this law. This study aimed to compare the provision of patient consent before and after the enforcement of this law among cancer patients who visited a tertiary university hospital's emergency room to understand the effects of this law on the clinical care of cancer patients. Methods This retrospective single cohort study included advanced cancer patients aged over 19 years who visited the emergency room of a tertiary university hospital. The two study periods were as follows: from February 2017 to January 2018 (before) and from May 2018 to April 2019 (after). The primary outcome was the length of hospital stay. The consent rates to perform cardiopulmonary resuscitation (CPR), intubation, continuous renal replacement therapy (CRRT), and intensive care unit (ICU) admission were the secondary outcomes. Results The length of hospital stay decreased after the law was enforced from 4 to 2 days (p = 0.001). The rates of direct transfers to secondary hospitals and nursing hospitals increased from 8.2 to 21.2% (p = 0.001) and from 1.0 to 9.7%, respectively (p p = 0.032). For CPR and CRRT, the consent rates decreased from 1.0 to 0.0% and from 13.9 to 8.8%, respectively, but the differences were not significant (p = 0.226 and p = 0.109, respectively). Conclusion After the enforcement of the Life Extension Medical Decision law, the length of stay in the tertiary university hospital decreased in patients who established their life-sustaining treatment plans in the emergency room. Moreover, the rate of consent for ICU admission decreased.

Aims Emipagliflozin (EMPA) is a potent inhibitor of the renal sodium-glucose co-transporter 2 (SGLT2) and an effective treatment for type-2 diabetes. In patients with diabetes and heart failure, EMPA has cardioprotective effects independent of improved glycaemic control, despite SGLT2 not being expressed in the heart. A number of non-canonical mechanisms have been proposed to explain these cardiac effects, most notably an inhibitory action on cardiac Na+/H+ exchanger 1 (NHE1), causing a reduction in intracellular [Na+] ([Na+]i). However, at resting intracellular pH (pHi), NHE1 activity is very low and its pharmacological inhibition is not expected to meaningfully alter steady-state [Na+]i. We re-evaluate this putative EMPA target by measuring cardiac NHE1 activity. Methods and results The effect of EMPA on NHE1 activity was tested in isolated rat ventricular cardiomyocytes from measurements of pHi recovery following an ammonium pre-pulse manoeuvre, using cSNARF1 fluorescence imaging. Whereas 10 µM cariporide produced near-complete inhibition, there was no evidence for NHE1 inhibition with EMPA treatment (1, 3, 10, or 30 µM). Intracellular acidification by acetate-superfusion evoked NHE1 activity and raised [Na+]i, reported by sodium binding benzofuran isophthalate (SBFI) fluorescence, but EMPA did not ablate this rise. EMPA (10 µM) also had no significant effect on the rate of cytoplasmic [Na+]i rise upon superfusion of Na+-depleted cells with Na+-containing buffers. In Langendorff-perfused mouse, rat and guinea pig hearts, EMPA did not affect [Na+]i at baseline nor pHi recovery following acute acidosis, as measured by 23Na triple quantum filtered NMR and 31P NMR, respectively. Conclusions Our findings indicate that cardiac NHE1 activity is not inhibited by EMPA (or other SGLT2i’s) and EMPA has no effect on [Na+]i over a wide range of concentrations, including the therapeutic dose. Thus, the beneficial effects of SGLT2i’s in failing hearts should not be interpreted in terms of actions on myocardial NHE1 or intracellular [Na+]., Graphical Abstract

Cardiac diastolic dysfunction is an independent predictor of mortality, regardless of LV systolic function. However, the current guidelines that define cardiac diastolic dysfunction may underrate the clinical implications of those with indeterminate diastolic function.We sought to evaluate the prognostic implications of indeterminate diastolic function on echocardiography and its association with coronary microvascular dysfunction (CMD).A total of 330 patients without LV systolic dysfunction and significant epicardial coronary stenosis (fractional flow reserve0.80) were analyzed from a prospective registry. Cardiac diastolic dysfunction was defined according to two algorithms depending on the presence of myocardial disease. First, the presence of myocardial disease and evidence of elevated LV filling pressure indicated diastolic dysfunction. Second, diastolic function in those without myocardial disease was defined using echocardiographic parameters (E/e', e' velocity, tricuspid regurgitation velocity, and left atrial volume index). Patients who did not meet half of the available criteria were classified as having indeterminate diastolic function. CMD was defined as coronary flow reserve2.0 and index of microcirculatory resistance≥25U. The primary outcome was cardiovascular death or admission for heart failure at 5 years.Coronary flow reserve was lower in patients with indeterminate diastolic function compared with those with no diastolic dysfunction (3.5±1.6 vs. 3.2±1.6, P=0.002). The prevalence of CMD was also higher in patients with indeterminate diastolic function than those with no diastolic dysfunction (10.6% vs. 4.9%, P0.034). Patients with indeterminate diastolic function showed significantly higher risk of cardiovascular death or admission for heart failure than those without, but not greater than those with definite diastolic dysfunction (cumulative incidence: 12.6%, 27.2%, and 32.7%, respectively, log-rank P0.001). Presence of CMD and elevated LV filling pressure (E/e'14) were independent predictors for cardiovascular death or admission for heart failure in patients with indeterminate diastolic function.Patients with indeterminate diastolic function on echocardiogram showed higher risk of cardiovascular death or admission for heart failure than those with no diastolic dysfunction. Presence of CMD and elevated LV filling pressure were independent predictors for cardiovascular death or admission for heart failure among patients with indeterminate diastolic function.

No abstract available.

Introduction: Mechanical circulatory support such as the left ventricular assist device (LVAD) has become widely implemented in the treatment of end-stage heart failure, whether as bridge-to-transplant or as destination therapy. The hemodynamic effects of arrhythmia on LVADs and its management are significant in determining the long-term outcome of these patients. Both atrial arrhythmia and ventricular arrhythmia are commonly seen after implantation of the device. There are no strict guidelines, however, on the need for intensive management of arrhythmias in LVAD. In this case report, we present a patient with new onset atrial fibrillation after LVAD implantation which leads to acute decompensating heart failure. The patient was treated with catheter ablation. The intervention demonstrated positive outcomes for this patient. Patient concerns: The patient was a Korean male, who presented with dyspnea, fatigue and generalized edema after persistent atrial fibrillation precipitated by implantation of the left ventricular assist device. Diagnosis: The patient was diagnosed with acute decompensating heart failure that was aggravated by recurrent atrial arrhythmia. Intervention: We attempted to relieve symptoms of right ventricular dysfunction by method of strict rhythm control in this patient. The patient underwent radiofrequency catheter ablation for recurrent atrial fibrillation. Outcome: The patient showed improved clinical symptoms, BNP levels, and echocardiogram parameters immediately after the procedure as well as during long term outpatient follow up. Conclusion: In this case report, we present the first successful case in Korea of atrial fibrillation in LVAD treated with catheter ablation. This case suggests setting catheter ablation as a routine first-line treatment for atrial arrhythmia in LVAD patients, especially when the arrhythmia predisposes the patient at risk for decompensating heart failure.

Iron deficiency is present in ~50% of heart failure (HF) patients. Large multicenter trials have shown that treatment of iron deficiency with i.v. iron benefits HF patients, but the underlying mechanisms are not known. To investigate the actions of iron deficiency on the heart, mice were fed an iron-depleted diet, and some received i.v. ferric carboxymaltose (FCM), an iron supplementation used clinically. Iron-deficient animals became anemic and had reduced ventricular ejection fraction measured by magnetic resonance imaging. Ca2+ signaling, a pathway linked to the contractile deficit in failing hearts, was also significantly affected. Ventricular myocytes isolated from iron-deficient animals produced smaller Ca2+ transients from an elevated diastolic baseline but had unchanged sarcoplasmic reticulum (SR) Ca2+ load, trigger L-type Ca2+ current, or cytoplasmic Ca2+ buffering. Reduced fractional release from the SR was due to downregulated RyR2 channels, detected at protein and message levels. The constancy of diastolic SR Ca2+ load is explained by reduced RyR2 permeability in combination with right-shifted SERCA activity due to dephosphorylation of its regulator phospholamban. Supplementing iron levels with FCM restored normal Ca2+ signaling and ejection fraction. Thus, 2 Ca2+-handling proteins previously implicated in HF become functionally impaired in iron-deficiency anemia, but their activity is rescued by i.v. iron supplementation., Iron deficiency, a common comorbidity in heart failure, reduces the strength of cardiac contraction through effects on Ca2+-handling proteins crucial for normal cardiac function but typically deranged failing hearts.

Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain. DOI: http://dx.doi.org/10.7554/eLife.19804.001, eLife digest Many proteins inside cells require iron to work properly, and so this mineral is an essential part of the diets of most mammals. However, because too much iron in the body is also bad for health, mammals possess several proteins whose role is to maintain the balance of iron. Two proteins in particular, called hepcidin and ferroportin, are thought to be important in this process. Some ferroportin is found in the cells that line the gut (where iron is absorbed into the body) and is required to release this iron into the bloodstream. It is also found in the spleen, which is where iron is removed from old red blood cells so that it can be recycled. The liver produces hepcidin to control when ferroportin is active in the gut and spleen. Both hepcidin and ferroportin are also found in heart cells. In 2015, a study reported that that heart ferroportin plays an important role in heart activity. However, it was not clear what role hepcidin plays in this organ. Now, Lakhal-Littleton et al. – including many of the researchers from the previous work – have genetically engineered mice such that they specifically lacked heart hepcidin, or had a version of ferroportin in their heart that does not respond to hepcidin. The experiments show that these changes caused fatal heart failure in the mice because ferroportin releases iron from heart cells in an uncontrolled manner. Lakhal-Littleton et al. were able to prevent heart failure by injecting the animals with iron directly into the bloodstream. These findings show that hepcidin produced outside the liver has a role in controlling the levels of iron in the body’s organs. Other organs such as the brain, kidney and placenta all have their own forms of hepcidin and ferroportin; further work could investigate the roles of these proteins. Finally, another challenge for the future will be to test whether new drugs that are being developed to block or mimic hepcidin from the liver have the potential to treat heart conditions in humans. DOI: http://dx.doi.org/10.7554/eLife.19804.002

Prolyl hydroxylase domain protein 2 (PHD2) (also known as EGLN1) is a key oxygen sensor in mammals that posttranslationally modifies hypoxia-inducible factor α (HIF-α) and targets it for degradation. In addition to its catalytic domain, PHD2 contains an evolutionarily conserved zinc finger domain, which we have previously proposed recruits PHD2 to the HSP90 pathway to promote HIF-α hydroxylation. Here, we provide evidence that this recruitment is critical both in vitro and in vivo. We show that in vitro, the zinc finger can function as an autonomous recruitment domain to facilitate interaction with HIF-α. In vivo, ablation of zinc finger function by a C36S/C42S Egln1 knock-in mutation results in upregulation of the erythropoietin gene, erythrocytosis, and augmented hypoxic ventilatory response, all hallmarks of Egln1 loss of function and HIF stabilization. Hence, the zinc finger ordinarily performs a critical positive regulatory function. Intriguingly, the function of this zinc finger is impaired in high-altitude-adapted Tibetans, suggesting that their adaptation to high altitude may, in part, be due to a loss-of-function EGLN1 allele. Thus, these findings have important implications for understanding both the molecular mechanism of the hypoxic response and human adaptation to high altitude.

The three principal proteins of the oxygen-sensing pathway regulating the EPO gene are PHD2, HIF-2α, and VHL [1]. PHD2 is a prolyl hydroxylase that site-specifically modifies HIF-2α in an oxygen-dependent manner[2]. The primary site of hydroxylation is Pro-531 of HIF-2α, and this posttranslational modification allows recognition by VHL, a component of an E3 ubiquitin ligase complex [3–5]. VHL recognizes hydroxylated, but not unmodified, HIF. Under normoxic conditions, VHL targets HIF-2α for constitutive degradation. Under hypoxic conditions, this modification is attenuated, allowing stabilization of HIF-2α. HIF-2α then transactivates genes that promote adaptation to hypoxic conditions. A key gene is that encoding for EPO, the central regulator of red cell mass, and the transcriptional upregulation of the EPO results in increased circulating levels of EPO, increased red cell mass, and hence increased oxygen delivery to tissues[6, 7]. Recent studies have identified erythrocytosis-associated mutations in the genes that encode for these three proteins of the oxygen-sensing pathway[1, 8–11]. These include heterozygous mutations in the PHD2 gene, heterozygous mutations in the HIF2A gene, and either homozygous or compound heterozygous mutations in the VHL gene[12–14]. Current evidence indicates that the PHD2 and VHL mutations lead to loss of function of the respective proteins, while the HIF2A mutations lead to a gain of function of HIF-2α [1]. Intriguingly, haplotypes in the HIF2A and PHD2 genes have also been associated with adaptation to high altitudes in Tibetans, highlighting a central role for these genes in hypoxic adaptation[15–17]. All of these issues make the documentation of human mutations in this pathway of considerable interest. In the present report, we identify two new HIF2A mutations associated with erythrocytosis. Patient A, a 27 year old female, presented with dizzy episodes, and her routine blood picture showed a hemoglobin (Hb) of 17.4 g/dl, a hematocrit (Hct) of 0.51 with a white cell count of 5.8 × 109/l and normal platelet counts. The oxygen dissociation curve and abdominal ultrasound were both normal. She was a smoker. There is no history of thrombosis or pulmonary hypertension and no family history of erythrocytosis. No family members were available for screening. No splenomegaly was detected. No mutations of JAK2 V617F, JAK2 exon 12, VHL, or PHD2 were detected. Repeat Hb level was 17.6 g/dl and at this time her serum EPO was 6.3 mU/ml (reference range 5.0–25.0 mU/ml). She remains asymptomatic with a Hb at this level. Patient B, an asymptomatic 49-year-old Brazilian male, presented with an increased Hb of 21.0 g/dl, Hct of 0.65, white cell count of 7.3 × 109/l, and platelet count of 236 × 109/l during routine blood tests. There was no history of either thromboembolic events or pulmonary hypertension, nor any family history of erythrocytosis. His father and grandfather both died of acute ischemic cerebral vascular events. He did not smoke, nor did he use any medications. Arterial blood gas analysis showed normal oxygen saturation and p50 values. EPO level was 38.2 mU/mL (reference range 5.0–25.0 mU/ml). Abdominal ultrasound was normal, as were ferritin and C-reactive protein levels. No mutations of JAK2 V617F, JAK2 exon 12, EPOR exon 8, VHL, or PHD2 were detected. The patient has been treated with phlebotomies and acetylsalicylic acid. Patient C presented at age 35 with a raised Hb of 18.2 g/dl and Hct of 0.52. His white cell count was 3.5 × 109/l and platelet count 200 × 109/l. He is a smoker. There was no splenomegaly and no evidence of renal disease or pulmonary hypertension. His serum EPO level was 7.8 mU/ml (reference range 5.0–25.0 mU/ml). Sequencing both VHL and PHD2 did not detect any mutations. He remains asymptomatic. Sequencing of exon 12 of HIF2A in these three individuals revealed two novel mutations (Figure 1A). Patient A was heterozygous for a c.1604 T>C mutation (middle panel), which exchanges Met for Thr at amino acid 535 (p.Met535Thr; M535T). Patient B had an identical heterozygous mutation, and this mutation was not present in his only son, who had a normal Hb level (data not shown). Patient C possessed a C to G change at c.1620 (c.1620C>G), resulting in a p.Phe540Leu (F540L) mutation (lower panel). In the case of patient C, a family history of erythrocytosis was confirmed, but no family members were available for screening. The serum EPO levels for patients A and C were within the reference range, while the EPO level for patient B was elevated. It may be noted that in many of the described cases with HIF2A mutations, it is well above the reference range [18–20]. Figure 1 Genetic testing for HIF2A mutations. (A)Detection of the c.1604 T>C and c.1620C>G mutations by PCR-direct sequencing. PCR-direct sequencing was performed on total peripheral blood DNA using specific primers to amplify exon 12. Sequencing ... Both mutations share two key features with previously described HIF2A mutations[1]. First, they are heterozygous. Second, they affect residues that are C-terminal and in close proximity to the primary site of prolyl hydroxylation in HIF-2α, Pro-531(Figure 1B). In fact, the M535T mutation is now the third mutation that has been reported to affect Met-535, the first two being M535I and M535V[18, 19]. The M535V mutation has previously been shown to impair interaction of HIF-2α with PHD2 [21]. Furthermore, HIF-1α Met-568, which corresponds to HIF-2α Met-535, is a contact residue for PHD2 in the cocrystal structure of HIF-1α (556–574):PHD2[22], therefore making it likely that Met-535 of HIF-2α is one as well. The F540L affects a residue that has, thus far, not been a mutational target in the context of erythrocytosis. To examine the functional consequences of the F540L mutation, we proceeded as follows. To assess its effect on the interaction between HIF-2α and VHL, we performed a competition assay in which we incubated immobilized (biotinylated) and hydroxylated (Hyp-564) HIF-1α (556–574) peptide with VHL in the absence or presence of wild type or F540L Hyp-531 HIF-2α (527–542) peptide. In the absence of HIF-2α peptide, VHL binds to immobilized Hyp HIF-1α, as expected (Figure 2A, lane 3). In the presence of Hyp HIF-2α peptide, this binding is diminished, and we find that the mutant HIF-2α is a substantially weaker inhibitor than wild type (compare lanes 5 and 4), implying that this mutation weakens the interaction with VHL. Figure 2 Binding assays. (A) Recombinant FlagVHL was incubated without (lane 2) or with(lanes 3 to 5) biotinylated Hyp-564 HIF-1α (556–574) peptide prebound to streptavidin-agarose, in the absence(lane 3)or presence of 5 nM wild type (WT) or F540L ... To assess the effect of the F540L mutation on the interaction of HIF-2α and PHD2, we performed a direct binding assay in which we examined the binding of PHD2 to immobilized glutathione S-transferase fused to either wild type or F540L HIF-2α (516–549). We find substantially less binding to the latter than the former (compare lanes 4 and 3), indicating that this mutation also weakens the interaction with PHD2. We therefore conclude that the F540L mutation impairs the interaction of HIF-2α with both PHD2 and VHL. Taken together, these observations support the assignment of the M535T and F540L HIF2A mutations as new causes of erythrocytosis. The F540L mutation, in addition, is noteworthy for several reasons. First, the F540L mutation affects a residue that is the most distant from Pro-531 of any of the erythrocytosis-associated mutations that have been reported thus far. Second, like many, though not all, HIF-2α mutations, it impairs interaction with both its upstream modifying enzyme, PHD2, and its downstream ubiquitin ligase, VHL. Third, it is a seemingly conservative substitution of a bulky hydrophobic amino acid with another bulky hydrophobic residue, yet it produces significant functional defects. It may be noted in this regard that Phe-540 is conserved in HIF-2α proteins from mammalian species, human HIF-1α and HIF-3α, as well as HIF-2α proteins from chicken, frog, and zebrafish (Figure 1B). The importance of this residue is further highlighted by x-ray crystallographic studies, which show that the corresponding residue in HIF-1α, Phe-572, is a contact residue for both PHD2 and for VHL [22–24].

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated “striated muscle contraction” as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.