Restoration of Cardiac Myosin Light Chain Kinase Ameliorates Systolic Dysfunction by Reducing Superrelaxed Myosin.

BACKGROUND: Cardiac-specific myosin light chain kinase (cMLCK), encoded by MYLK3, regulates cardiac contractility through phosphorylation of ventricular myosin regulatory light chain. However, the pathophysiological and therapeutic implications of cMLCK in human heart failure remain unclear. We aimed to investigate whether cMLCK dysregulation causes cardiac dysfunction and whether the restoration of cMLCK could be a novel myotropic therapy for systolic heart failure. METHODS: We generated the knock-in mice (Mylk3+/fs and Mylk3fs/fs) with a familial dilated cardiomyopathy-associated MYLK3 frameshift mutation (MYLK3+/fs) that had been identified previously by us (c.1951-1G>T; p.P639Vfs*15) and the human induced pluripotent stem cell-derived cardiomyocytes from the carrier of the mutation. We also developed a new small-molecule activator of cMLCK (LEUO-1154). RESULTS: Both mice (Mylk3+/fs and Mylk3fs/fs) showed reduced cMLCK expression due to nonsense-mediated messenger RNA decay, reduced MLC2v (ventricular myosin regulatory light chain) phosphorylation in the myocardium, and systolic dysfunction in a cMLCK dose-dependent manner. Consistent with this result, myocardium from the mutant mice showed an increased ratio of cardiac superrelaxation/disordered relaxation states that may contribute to impaired cardiac contractility. The phenotypes observed in the knock-in mice were rescued by cMLCK replenishment through the AAV9_MYLK3 vector. Human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation reduced cMLCK expression by 50% and contractile dysfunction, accompanied by an increased superrelaxation/disordered relaxation ratio. CRISPR-mediated gene correction, or cMLCK replenishment by AAV9_MYLK3 vector, successfully recovered cMLCK expression, the superrelaxation/disordered relaxation ratio, and contractile dysfunction. LEUO-1154 increased human cMLCK activity ≈2-fold in the Vmax for ventricular myosin regulatory light chain phosphorylation without affecting the Km. LEUO-1154 treatment of human induced pluripotent stem cell-derived cardiomyocytes with MYLK3+/fs mutation restored the ventricular myosin regulatory light chain phosphorylation level and superrelaxation/disordered relaxation ratio and improved cardiac contractility without affecting calcium transients, indicating that the cMLCK activator acts as a myotrope. Finally, human myocardium from advanced heart failure with a wide variety of causes had a significantly lower MYLK3/PPP1R12B messenger RNA expression ratio than control hearts, suggesting an altered balance between myosin regulatory light chain kinase and phosphatase in the failing myocardium, irrespective of the causes. CONCLUSIONS: cMLCK dysregulation contributes to the development of cardiac systolic dysfunction in humans. Our strategy to restore cMLCK activity could form the basis of a novel myotropic therapy for advanced systolic heart failure.

AMPK regulates cell shape of cardiomyocytes by modulating turnover of microtubules through CLIP-170.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

Evaluation of the efficacy of creatine chemical exchange saturation transfer imaging in assessing testicular maturity.

Purpose: Microscopic testicular sperm extraction is the most effective treatment for NOA, but the sperm retrieval rate is low and depends on testicular maturity. However, there are limited useful tests to assess testicular maturity. Chemical exchange saturation transfer (CEST) imaging is a new magnetic resonance imaging (MRI) technique that can image the distribution of trace substances in vivo. We focused on the potential role of creatine (Cr) in testes and hypothesized that Cr-CEST could indicate intratesticular spermatogenesis. Methods: We performed Cr-CEST by using 7T MRI on wild-type C57B6/J mice and several types of male infertility models such as Sertoli-cell only (SCO) (Kitw/Kitwv), maturation arrest (MA) (Zfp541 knockout mouse and Kctd19 knockout mouse), and teratozoospermia (Tbc1d21 knockout mouse). After performing Cr-CEST, histological analysis was performed. Results: The SCO and MA models showed decreased CEST signal intensity (p < 0.05), while no reduction was observed in the teratozoospermia model (p = 1.0). CEST signal intensity increased as the spermatogenesis stage progressed from the SCO model to the MA and teratozoospermia models. Furthermore, CEST signal intensity was reduced in 4-week-old wild-type mice with immature testes (p < 0.05). Conclusions: This study suggests that Cr-CEST evaluates intratesticular spermatogenesis noninvasively and provides a new therapeutic strategy for treating male infertility.

Aberrant accumulation of TMEM43 accompanied by perturbed transmural gene expression in arrhythmogenic cardiomyopathy.

Arrhythmogenic cardiomyopathy (ACM) caused by TMEM43 p.S358L is a fully penetrant heart disease that results in impaired cardiac function or fatal arrhythmia. However, the molecular mechanism of ACM caused by the TMEM43 variant has not yet been fully elucidated. In this study, we generated knock-in (KI) rats harboring a Tmem43 p.S358L mutation and established induced pluripotent stem cells (iPSCs) from patients based on the identification of TMEM43 p.S358L variant from a family with ACM. The Tmem43-S358L KI rats exhibited ventricular arrhythmia and fibrotic myocardial replacement in the subepicardium, which recapitulated the human ACM phenotype. The four-transmembrane protein TMEM43 with the p.S358L variant (TMEM43S358L ) was found to be modified by N-linked glycosylation in both KI rat cardiomyocytes and patient-specific iPSC-derived cardiomyocytes. TMEM43S358L glycosylation increased under the conditions of enhanced endoplasmic reticulum (ER) stress caused by pharmacological stimulation or age-dependent decline of the ER function. Intriguingly, the specific glycosylation of TMEM43S358L resulted from the altered membrane topology of TMEM43. Moreover, unlike TMEM43WT , which is mainly localized to the ER, TMEM43S358L accumulated at the nuclear envelope of cardiomyocytes with the increase in glycosylation. Finally, our comprehensive transcriptomic analysis demonstrated that the regional differences in gene expression patterns between the inner and outer layers observed in the wild type myocardium were partially diminished in the KI myocardium prior to exhibiting histological changes indicative of ACM. Altogether, these findings suggest that the aberrant accumulation of TMEM43S358L underlies the pathogenesis of ACM caused by TMEM43 p.S358L variant by affecting the transmural gene expression within the myocardium.

The CR9 element is a novel mechanical load-responsive enhancer that regulates natriuretic peptide genes expression.

Enhancers regulate gene expressions in a tissue- and pathology-specific manner by altering its activities. Plasma levels of atrial and brain natriuretic peptides, encoded by the Nppa and Nppb, respectively, and synthesized predominantly in cardiomyocytes, vary depending on the severity of heart failure. We previously identified the noncoding conserved region 9 (CR9) element as a putative Nppb enhancer at 22-kb upstream from the Nppb gene. However, its regulatory mechanism remains unknown. Here, we therefore investigated the mechanism of CR9 activation in cardiomyocytes using different kinds of drugs that induce either cardiac hypertrophy or cardiac failure accompanied by natriuretic peptides upregulation. Chronic treatment of mice with either catecholamines or doxorubicin increased CR9 activity during the progression of cardiac hypertrophy to failure, which is accompanied by proportional increases in Nppb expression. Conversely, for cultured cardiomyocytes, doxorubicin decreased CR9 activity and Nppb expression, while catecholamines increased both. However, exposing cultured cardiomyocytes to mechanical loads, such as mechanical stretch or hydrostatic pressure, upregulate CR9 activity and Nppb expression even in the presence of doxorubicin. Furthermore, the enhancement of CR9 activity and Nppa and Nppb expressions by either catecholamines or mechanical loads can be blunted by suppressing mechanosensing and mechanotransduction pathways, such as muscle LIM protein (MLP) or myosin tension. Finally, the CR9 element showed a more robust and cell-specific response to mechanical loads than the -520-bp BNP promoter. We concluded that the CR9 element is a novel enhancer that responds to mechanical loads by upregulating natriuretic peptides expression in cardiomyocytes.

Accurate Estimation of the Duration of Testicular Ischemia Using Creatine Chemical Exchange Saturation Transfer (CrCEST) Imaging.

BACKGROUND: In the management of testicular torsion, estimating the duration of testicular ischemia is essential for deciding on an appropriate surgical treatment, but there are currently limited evaluation methods. PURPOSE: To perform testicular creatine chemical exchange saturation transfer (CrCEST) imaging and to evaluate its ability to accurately estimate the duration of testicular ischemia. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 control mice (n = 6) and testicular ischemia models induced by clamping the spermatic cord (n = 14). Eight of testicular ischemia models were serially imaged at two or three timepoints and a total of 26 images of ischemic testis were obtained. The ischemic duration ranged from 6-42 hours. FIELD STRENGTH/SEQUENCE: 11.7T vertical-bore MRI/segment fast low-angle shot acquisition for CEST. ASSESSMENT: CrCEST imaging was performed and the magnetization transfer ratio for the CrCEST effect (MTRCr** ) was calculated in control mice and testicular ischemia models. Correlation analysis between the duration of testicular ischemia and MTRCr** decline was performed. STATISTICAL TESTS: Paired t-test, and Pearson's correlation analysis. RESULTS: In control mice, the CrCEST effect in testes was significantly more than five times higher than that in skeletal muscle. MTRCr** did not differ significantly between the right and left testes (8.6 ± 0.8 vs. 8.3 ± 0.6, P = 0.96). In testicular ischemia models, MTRCr** of ischemic testes was significantly lower than that of controls (4 ± 2 vs. 8.9 ± 0.6, P < 0.001). Correlation analysis revealed a strong linear correlation between MTRCr** decline and the duration of ischemia (r = 0.96, P < 0.001). DATA CONCLUSION: A decreased CrCEST effect in ischemic testes correlated well with ischemic duration. Testicular CrCEST imaging was useful for accurately estimating the duration of testicular ischemia. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Visualization of Spatial Distribution of Spermatogenesis in Mouse Testes Using Creatine Chemical Exchange Saturation Transfer Imaging.

BACKGROUND: When determining treatment strategies for male infertility, it is important to evaluate spermatogenesis and its spatial distribution in the testes. PURPOSE: To investigate the usefulness of creatine chemical exchange saturation transfer (CrCEST) imaging for evaluating spermatogenesis and its spatial distribution. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 control mice (n = 5) and model mice of male infertility induced by whole testis X-ray irradiation (n = 11) or localized X-ray irradiation to lower regions of testes (n = 3). FIELD STRENGTH/SEQUENCE: A 11.7-T vertical-bore magnetic resonance imaging (MRI)/segmented fast low-angle shot acquisition for CEST. ASSESSMENT: The magnetization transfer ratio for the CrCEST effect (MTRCr* ) was calculated in each testis of the control mice and X-ray irradiation model mice at 10, 15, 20, and 30 days after irradiation. Correlation analysis was performed between MTRCr* and Johnsen's score, a histological score for spermatogenesis. In the localized X-ray irradiation model, regional MTRCr* and Johnsen's score were calculated for correlation analysis. STATISTICAL TESTS: Unpaired t-test, one-way analysis of variance with Tukey's HSD test and Pearson's correlation analysis. A P value < 0.05 was considered statistically significant. RESULTS: In the irradiation model, CrCEST imaging revealed a significant linear decrease of MTRCr* after irradiation (control, 8.7 ± 0.6; 10 days, 7.9 ± 0.8; 15 days, 6.5 ± 0.6; 20 days, 5.4 ± 1.0; 30 days, 4.4 ± 0.8). A significant linear correlation was found between MTRCr* and Johnsen's score (Pearson's correlation coefficient (r) = 0.79). In the localized irradiation model, CrCEST imaging visualized a significant regional decrease of MTRCr* in the unshielded region (shielded, 6.9 ± 0.7; unshielded, 4.9 ± 1.0), and a significant linear correlation was found between regional MTRCr* and Johnsen's score (r = 0.78). DATA CONCLUSION: Testicular CrCEST effects correlated well with spermatogenesis. CrCEST imaging was useful for evaluating spermatogenesis and its spatial distribution. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

In vivo real-time ATP imaging in zebrafish hearts reveals G0s2 induces ischemic tolerance.

Most eukaryotic cells generate adenosine triphosphate (ATP) through the oxidative phosphorylation system (OXPHOS) to support cellular activities. In cultured cell-based experiments, we recently identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS, and showed that G0s2 protects cultured cardiomyocytes from hypoxia. In this study, we examined the in vivo protective role of G0s2 against hypoxia by generating both loss-of-function and gain-of-function models of g0s2 in zebrafish. Zebrafish harboring transcription activator-like effector nuclease (TALEN)-mediated knockout of g0s2 lost hypoxic tolerance. Conversely, cardiomyocyte-specific transgenic zebrafish hearts exhibited strong tolerance against hypoxia. To clarify the mechanism by which G0s2 protects cardiac function under hypoxia, we introduced a mitochondrially targeted FRET-based ATP biosensor into zebrafish heart to visualize ATP dynamics in in vivo beating hearts. In addition, we employed a mosaic overexpression model of g0s2 to compare the contraction and ATP dynamics between g0s2-expressing and non-expressing cardiomyocytes, side-by-side within the same heart. These techniques revealed that g0s2-expressing cardiomyocyte populations exhibited preserved contractility coupled with maintained intra-mitochondrial ATP concentrations even under hypoxic condition. Collectively, these results demonstrate that G0s2 provides ischemic tolerance in vivo by maintaining ATP production, and therefore represents a promising therapeutic target for hypoxia-related diseases.

Higd1a improves respiratory function in the models of mitochondrial disorder.

The respiratory chain (RC) transports electrons to form a proton motive force that is required for ATP synthesis in the mitochondria. RC disorders cause mitochondrial diseases that have few effective treatments; therefore, novel therapeutic strategies are critically needed. We previously identified Higd1a as a positive regulator of cytochrome c oxidase (CcO) in the RC. Here, we test that Higd1a has a beneficial effect by increasing CcO activity in the models of mitochondrial dysfunction. We first demonstrated the tissue-protective effects of Higd1a via in situ measurement of mitochondrial ATP concentrations ([ATP]mito) in a zebrafish hypoxia model. Heart-specific Higd1a overexpression mitigated the decline in [ATP]mito under hypoxia and preserved cardiac function in zebrafish. Based on the in vivo results, we examined the effects of exogenous HIGD1A on three cellular models of mitochondrial disease; notably, HIGD1A improved respiratory function that was coupled with increased ATP synthesis and demonstrated cellular protection in all three models. Finally, enzyme kinetic analysis revealed that Higd1a significantly increased the maximal velocity of the reaction between CcO and cytochrome c without changing the affinity between them, indicating that Higd1a is a positive modulator of CcO. These results corroborate that Higd1a, or its mimic, provides therapeutic options for the treatment of mitochondrial diseases.

Cardiac Fibroblasts Play Pathogenic Roles in Idiopathic Restrictive Cardiomyopathy.

BACKGROUND: Restrictive cardiomyopathy (RCM) is characterized by impaired ventricular relaxation. Although several mutations were reported in some patients, no mutations were identified in cardiomyocyte expressing genes of other patients, indicating that pathological mechanisms underlying RCM could not be determined by cardiomyocytes only. Cardiac fibroblasts (CFs) are a major cell population in the heart; however, the pathological roles of CFs in cardiomyopathy are not fully understood.Methods and Results:This study established 4 primary culture lines of CFs from RCM patients and analyzed their cellular physiology, the effects on the contraction and relaxation ability of healthy cardiomyocytes under co-culture with CFs, and RNA sequencing. Three of four patients hadTNNI3mutations. There were no significant alterations in cell proliferation, apoptosis, migration, activation, and attachment. However, when CFs from RCM patients were co-cultured with healthy cardiomyocytes, the relaxation velocity of cardiomyocytes was significantly impaired both under direct and indirect co-culture conditions. RNA sequencing revealed that gene expression profiles of CFs in RCM were clearly distinct from healthy CFs. The differential expression gene analysis identified that several extracellular matrix components and cytokine expressions were dysregulated in CFs from RCM patients. CONCLUSIONS: The comprehensive gene expression patterns were altered in RCM-derived CFs, which deteriorated the relaxation ability of cardiomyocytes. The specific changes in extracellular matrix composition and cytokine secretion from CFs might affect pathological behavior of cardiomyocytes in RCM.

A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2.

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G0/G1 switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FOF1-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Mutant KCNJ3 and KCNJ5 Potassium Channels as Novel Molecular Targets in Bradyarrhythmias and Atrial Fibrillation.

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.

Mouse skeletal muscle creatine chemical exchange saturation transfer (CrCEST) imaging at 11.7T MRI.

BACKGROUND: Creatine chemical exchange saturation transfer (CrCEST) imaging is expected to be a novel evaluation method of muscular energy metabolism. PURPOSE: To develop CrCEST imaging of mouse skeletal muscle and to validate this technique by measuring changes in Cr concentration of ischemic hindlimbs. STUDY TYPE: Prospective. ANIMAL MODEL: C57BL/6 mice (n = 6), mild hindlimb ischemic mice (n = 6), and severe hindlimb ischemic mice (n = 6). FIELD STRENGTH/SEQUENCE: Magnetic resonance angiography (MRA), CrCEST imaging, and phosphorus magnetic resonance spectroscopy (31 P MRS) obtained at 11.7T. ASSESSMENT: MRA and 31 P MRS were performed to confirm the presence of ischemia following the compression by rubber tourniquet. CrCEST imaging was performed and magnetization transfer ratio asymmetry (MTRasym ), which reflects Cr concentration, and was calculated in severe ischemia models, mild ischemia models, and control mice. Follow-up CrCEST imaging was performed after the release of ischemia in the mild ischemia models. STATISTICAL TESTS: Mean ± SD, one-way analysis of variance (ANOVA) with Tukey's HSD test, unpaired or paired t-test. RESULTS: MRA revealed the loss of blood flow of the femoral artery in the ischemic hindlimb. 31 P MRS revealed different degrees of PCr decrease in severe and mild ischemic hindlimb (n = 3 per group, normal hindlimb: 1.0 ± 0, mild ischemic hindlimb: 0.77 ± 0.13, severe ischemic hindlimb: 0 ± 0). CrCEST imaging inversely revealed a significant stepwise increase in the MTRasym ratio of ischemic hindlimbs compared with controls (control, mild ischemia, and severe ischemia; 0.99 ± 0.04, 1.36 ± 0.08, and 1.59 ± 0.23, respectively, P < 0.0001). In addition, follow-up CrCEST imaging after the release of ischemia revealed normalization of the MTRasym ratios (recovered hindlimb: 1.01 ± 0.05). DATA CONCLUSION: We demonstrated an increase in the MTRasym of ischemic hindlimbs, along with a decrease of PCr. We demonstrated the normalization of MTRasym after the release of ischemia and developed CrCEST imaging of mouse skeletal muscle. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2020;51:563-570.

Non-Ischemic Heart Failure With Reduced Ejection Fraction Is Associated With Altered Intestinal Microbiota.

BACKGROUND: Research suggests that heart failure with reduced ejection fraction (HFrEF) is a state of systemic inflammation that may be triggered by microbial products passing into the bloodstream through a compromised intestinal barrier. However, whether the intestinal microbiota exhibits dysbiosis in HFrEF patients is largely unknown.Methods and Results:Twenty eight non-ischemic HFrEF patients and 19 healthy controls were assessed by 16S rRNA analysis of bacterial DNA extracted from stool samples. After processing of sequencing data, bacteria were taxonomically classified, diversity indices were used to examine microbial ecology, and relative abundances of common core genera were compared between groups. Furthermore, we predicted gene carriage for bacterial metabolic pathways and inferred microbial interaction networks on multiple taxonomic levels.Bacterial communities of both groups were dominated by the Firmicutes and Bacteroidetes phyla. The most abundant genus in both groups wasBacteroides. Although α diversity did not differ between groups, ordination by ß diversity metrics revealed a separation of the groups across components of variation.StreptococcusandVeillonellawere enriched in the common core microbiota of patients, whileSMB53was depleted. Gene families in amino acid, carbohydrate, vitamin, and xenobiotic metabolism showed significant differences between groups. Interaction networks revealed a higher degree of correlations between bacteria in patients. CONCLUSIONS: Non-ischemic HFrEF patients exhibited multidimensional differences in intestinal microbial communities compared with healthy subjects.

Rationale and Design of the Multicenter Trial on Japan Working Group on the Effects of Angiotensin Receptor Blockers Selection (Azilsartan vs. Candesartan) on Diastolic Function in the Patients Suffering from Heart Failure with Preserved Ejection Fraction: J-TASTE Trial.

BACKGROUND: Previous studies suggest that the pathophysiology of heart failure with preserved ejection fraction (HFpEF) is characterized not only by high ventricular stiffness, but also by vascular stiffness. Azilsartan has higher vascular affinity compared with other angiotensin II receptor blockers (ARBs), which were proven to have no beneficial effects on clinical outcomes in patients with HFpEF in earlier clinical trials. We aimed to test the hypothesis that azilsartan may improve left ventricular diastolic function in HFpEF patients with hypertension in this trial. METHODS: The Effects of Angiotensin Receptor Blockers on Diastolic Function in Patients Suffering from Heart Failure with Preserved Ejection Fraction: J-TASTE trial is a multicenter, randomized, open-labeled, and assessor(s)-blinded, active controlled using candesartan, parallel-group clinical trial, to compare changes in left ventricular (LV) diastolic dysfunction between HFpEF patients with hypertension who have received candesartan or azilsartan for 48 weeks. The primary endpoint is the change in early diastolic wave height/early diastolic mitral annulus velocity (E/e') assessed by echocardiography from the baseline to the end of the study (48 weeks). A total of 190 patients will be recruited into the study. CONCLUSIONS: The design of the J-TASTE trial will provide data on whether differences between the effects of the two tested drugs on LV diastolic function exist in HFpEF patients with hypertension and will improve understanding of the pathophysiological role of vascular stiffness on diastolic function.

Higd1a is a positive regulator of cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the only enzyme that uses oxygen to produce a proton gradient for ATP production during mitochondrial oxidative phosphorylation. Although CcO activity increases in response to hypoxia, the underlying regulatory mechanism remains elusive. By screening for hypoxia-inducible genes in cardiomyocytes, we identified hypoxia inducible domain family, member 1A (Higd1a) as a positive regulator of CcO. Recombinant Higd1a directly integrated into highly purified CcO and increased its activity. Resonance Raman analysis revealed that Higd1a caused structural changes around heme a, the active center that drives the proton pump. Using a mitochondria-targeted ATP biosensor, we showed that knockdown of endogenous Higd1a reduced oxygen consumption and subsequent mitochondrial ATP synthesis, leading to increased cell death in response to hypoxia; all of these phenotypes were rescued by exogenous Higd1a. These results suggest that Higd1a is a previously unidentified regulatory component of CcO, and represents a therapeutic target for diseases associated with reduced CcO activity.

Clinical Significance of Pulmonary Arterial Capacitance Calculated by Echocardiography in Patients With Advanced Heart Failure.

BACKGROUND: Advanced left heart failure (HF) often accompanies post-capillary pulmonary hypertension related to RV afterload. Although pulmonary arterial capacitance (PAC), a measure of pulmonary artery compliance, reflects right ventricular (RV) afterload, the clinical utility of PAC obtained by echocardiography (echo-PAC) is not well established in advanced HF.Methods and Results:We performed right heart catheterization in advanced HF patients (n=30), calculating echo-PAC as stroke volume/(tricuspid regurgitation pressure gradient-pulmonary regurgitation pressure gradient). The difference between the echo-PAC and catheter-measured PAC values was insignificant (0.21±0.17 mL/mmHg, P=0.23). Echo-PAC values predicted both pulmonary arterial wedge pressure (PAWP) ≥18 mmHg and pulmonary vascular resistance ≥3 Wood units (P=0.02, area under the curve: 0.88, cutoff value: 1.94 mL/mmHg). Next, we conducted an outcome study with advanced HF patients (n=72). Patients with echo-PAC <1.94 mL/mmHg had more advanced New York Heart Association functional class, higher B-type natriuretic peptide plasma levels, larger RV and lower RV fractional area change than those with echo-PAC ≥1.94 mL/mmHg. They also had a significantly higher rate of ventricular assist device implantation or death, even after adjustment for indices related to HF severity or RV function during a 1-year follow-up period (P<0.01). CONCLUSIONS: Decreased PAC as measured by echocardiography, indicating elevated PAWP and RV dysfunction, predicted poorer outcomes in patients with advanced HF.

Evaluation of intramitochondrial ATP levels identifies G0/G1 switch gene 2 as a positive regulator of oxidative phosphorylation.

The oxidative phosphorylation (OXPHOS) system generates most of the ATP in respiring cells. ATP-depleting conditions, such as hypoxia, trigger responses that promote ATP production. However, how OXPHOS is regulated during hypoxia has yet to be elucidated. In this study, selective measurement of intramitochondrial ATP levels identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of OXPHOS. A mitochondria-targeted, FRET-based ATP biosensor enabled us to assess OXPHOS activity in living cells. Mitochondria-targeted, FRET-based ATP biosensor and ATP production assay in a semiintact cell system revealed that G0s2 increases mitochondrial ATP production. The expression of G0s2 was rapidly and transiently induced by hypoxic stimuli, and G0s2 interacts with OXPHOS complex V (FoF1-ATP synthase). Furthermore, physiological enhancement of G0s2 expression prevented cells from ATP depletion and induced a cellular tolerance for hypoxic stress. These results show that G0s2 positively regulates OXPHOS activity by interacting with FoF1-ATP synthase, which causes an increase in ATP production in response to hypoxic stress and protects cells from a critical energy crisis. These findings contribute to the understanding of a unique stress response to energy depletion. Additionally, this study shows the importance of assessing intramitochondrial ATP levels to evaluate OXPHOS activity in living cells.