Phosphorylation of CRYAB Induces a Condensatopathy to Worsen Post-Myocardial Infarction Left Ventricular Remodeling.

Protein aggregates are emerging therapeutic targets in rare monogenic causes of cardiomyopathy and amyloid heart disease, but their role in more prevalent heart failure syndromes remains mechanistically unexamined. We observed mis-localization of desmin and sarcomeric proteins to aggregates in human myocardium with ischemic cardiomyopathy and in mouse hearts with post-myocardial infarction ventricular remodeling, mimicking findings of autosomal-dominant cardiomyopathy induced by R120G mutation in the cognate chaperone protein, CRYAB. In both syndromes, we demonstrate increased partitioning of CRYAB phosphorylated on serine-59 to NP40-insoluble aggregate-rich biochemical fraction. While CRYAB undergoes phase separation to form condensates, the phospho-mimetic mutation of serine-59 to aspartate (S59D) in CRYAB mimics R120G-CRYAB mutants with reduced condensate fluidity, formation of protein aggregates and increased cell death. Conversely, changing serine to alanine (phosphorylation-deficient mutation) at position 59 (S59A) restored condensate fluidity, and reduced both R120G-CRYAB aggregates and cell death. In mice, S59D CRYAB knock-in was sufficient to induce desmin mis-localization and myocardial protein aggregates, while S59A CRYAB knock-in rescued left ventricular systolic dysfunction post-myocardial infarction and preserved desmin localization with reduced myocardial protein aggregates. 25-Hydroxycholesterol attenuated CRYAB serine-59 phosphorylation and rescued post-myocardial infarction adverse remodeling. Thus, targeting CRYAB phosphorylation-induced condensatopathy is an attractive strategy to counter ischemic cardiomyopathy.

Chimeric antigen receptor macrophages target and resorb amyloid plaques.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). While the precise pathophysiology of AD is incompletely understood, clinical trials of antibodies targeting aggregated forms of ß amyloid (Aß) have shown that reducing amyloid plaques can mitigate cognitive decline in patients with early-stage AD. Here, we describe what we believe to be a novel approach to target and degrade amyloid plaques by genetically engineering macrophages to express an Aß-targeting chimeric antigen receptor (CAR-Ms). When injected intrahippocampally, first-generation CAR-Ms have limited persistence and fail to significantly reduce plaque load, which led us to engineer next-generation CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. Cytokine secreting "reinforced CAR-Ms" have greater survival in the brain niche and significantly reduce plaque load locally in vivo. These findings support CAR-Ms as a platform to rationally target, resorb, and degrade pathogenic material that accumulates with age, as exemplified by targeting Aß in AD.

Identification of a leucine-mediated threshold effect governing macrophage mTOR signalling and cardiovascular risk.

High protein intake is common in western societies and is often promoted as part of a healthy lifestyle; however, amino-acid-mediated mammalian target of rapamycin (mTOR) signalling in macrophages has been implicated in the pathogenesis of ischaemic cardiovascular disease. In a series of clinical studies on male and female participants ( NCT03946774 and NCT03994367 ) that involved graded amounts of protein ingestion together with detailed plasma amino acid analysis and human monocyte/macrophage experiments, we identify leucine as the key activator of mTOR signalling in macrophages. We describe a threshold effect of high protein intake and circulating leucine on monocytes/macrophages wherein only protein in excess of ∼25 g per meal induces mTOR activation and functional effects. By designing specific diets modified in protein and leucine content representative of the intake in the general population, we confirm this threshold effect in mouse models and find ingestion of protein in excess of ∼22% of dietary energy requirements drives atherosclerosis in male mice. These data demonstrate a mechanistic basis for the adverse impact of excessive dietary protein on cardiovascular risk.

Metabolic control of mitophagy.

Mitochondrial dysfunction is a major hallmark of ageing and related chronic disorders. Controlled removal of damaged mitochondria by the autophagic machinery, a process known as mitophagy, is vital for mitochondrial homeostasis and cell survival. The central role of mitochondria in cellular metabolism places mitochondrial removal at the interface of key metabolic pathways affecting the biosynthesis or catabolism of acetyl-coenzyme A, nicotinamide adenine dinucleotide, polyamines, as well as fatty acids and amino acids. Molecular switches that integrate the metabolic status of the cell, like AMP-dependent protein kinase, protein kinase A, mechanistic target of rapamycin and sirtuins, have also emerged as important regulators of mitophagy. In this review, we discuss how metabolic regulation intersects with mitophagy. We place special emphasis on the metabolic regulatory circuits that may be therapeutically targeted to delay ageing and mitochondria-associated chronic diseases. Moreover, we identify outstanding knowledge gaps, such as the ill-defined distinction between basal and damage-induced mitophagy, which must be resolved to boost progress in this area.

Novel Roles for the Transcriptional Repressor E4BP4 in Both Cardiac Physiology and Pathophysiology.

Circadian clocks temporally orchestrate biological processes critical for cellular/organ function. For example, the cardiomyocyte circadian clock modulates cardiac metabolism, signaling, and electrophysiology over the course of the day, such that, disruption of the clock leads to age-onset cardiomyopathy (through unknown mechanisms). Here, we report that genetic disruption of the cardiomyocyte clock results in chronic induction of the transcriptional repressor E4BP4. Importantly, E4BP4 deletion prevents age-onset cardiomyopathy following clock disruption. These studies also indicate that E4BP4 regulates both cardiac metabolism (eg, fatty acid oxidation) and electrophysiology (eg, QT interval). Collectively, these studies reveal that E4BP4 is a novel regulator of both cardiac physiology and pathophysiology.

Preserving mitochondria to treat hypertrophic cardiomyopathy: From rare mitochondrial DNA mutation to heart failure therapy?

Hypertrophic cardiomyopathy and pathological cardiac hypertrophy are characterized by mitochondrial structural and functional abnormalities. In this issue of the JCI, Zhuang et al. discovered 1-deoxynojirimycin (DNJ) through a screen of mitochondrially targeted compounds. The authors described the effects of DNJ in restoring mitochondria and preventing cardiac myocyte hypertrophy in cellular models carrying a mutant mitochondrial gene, MT-RNR2, which is causally implicated in familial hypertrophic cardiomyopathy. DNJ worked via stabilization of the mitochondrial inner-membrane GTPase OPA1 and other, hitherto unknown, mechanisms to preserve mitochondrial crista and respiratory chain components. The discovery is likely to spur development of a class of therapeutics that restore mitochondrial health to prevent cardiomyopathy and heart failure.

Loss of Macrophage mTORC2 Drives Atherosclerosis via FoxO1 and IL-1ß Signaling.

BACKGROUND: The mTOR (mechanistic target of rapamycin) pathway is a complex signaling cascade that regulates cellular growth, proliferation, metabolism, and survival. Although activation of mTOR signaling has been linked to atherosclerosis, its direct role in lesion progression and in plaque macrophages remains poorly understood. We previously demonstrated that mTORC1 (mTOR complex 1) activation promotes atherogenesis through inhibition of autophagy and increased apoptosis in macrophages. METHODS: Using macrophage-specific Rictor- and mTOR-deficient mice, we now dissect the distinct functions of mTORC2 pathways in atherogenesis. RESULTS: In contrast to the atheroprotective effect seen with blockade of macrophage mTORC1, macrophage-specific mTORC2-deficient mice exhibit an atherogenic phenotype, with larger, more complex lesions and increased cell death. In cultured macrophages, we show that mTORC2 signaling inhibits the FoxO1 (forkhead box protein O1) transcription factor, leading to suppression of proinflammatory pathways, especially the inflammasome/IL (interleukin)-1ß response, a key mediator of vascular inflammation and atherosclerosis. In addition, administration of FoxO1 inhibitors efficiently rescued the proinflammatory response caused by mTORC2 deficiency both in vitro and in vivo. Interestingly, collective deletion of macrophage mTOR, which ablates mTORC1- and mTORC2-dependent pathways, leads to minimal change in plaque size or complexity, reflecting the balanced yet opposing roles of these signaling arms. CONCLUSIONS: Our data provide the first mechanistic details of macrophage mTOR signaling in atherosclerosis and suggest that therapeutic measures aimed at modulating mTOR need to account for its dichotomous functions.

Chimeric Antigen Receptor Macrophages Target and Resorb Amyloid Plaques in a Mouse Model of Alzheimer's Disease.

Substantial evidence suggests a role for immunotherapy in treating Alzheimer's disease (AD). Several monoclonal antibodies targeting aggregated forms of beta amyloid (Aß), have been shown to reduce amyloid plaques and in some cases, mitigate cognitive decline in early-stage AD patients. We sought to determine if genetically engineered macrophages could improve the targeting and degradation of amyloid plaques. Chimeric antigen receptor macrophages (CAR-Ms), which show promise as a cancer treatment, are an appealing strategy to enhance target recognition and phagocytosis of amyloid plaques in AD. We genetically engineered macrophages to express a CAR containing the anti-amyloid antibody aducanumab as the external domain and the Fc receptor signaling domain internally. CAR-Ms recognize and degrade Aß in vitro and on APP/PS1 brain slices ex vivo; however, when injected intrahippocampally, these first-generation CAR-Ms have limited persistence and fail to reduce plaque load. We overcame this limitation by creating CAR-Ms that secrete M-CSF and self-maintain without exogenous cytokines. These CAR-Ms have greater survival in the brain niche, and significantly reduce plaque load locally in vivo. These proof-of-principle studies demonstrate that CAR-Ms, previously only applied to cancer, may be utilized to target and degrade unwanted materials, such as amyloid plaques in the brains of AD mice.

Apolipoprotein M Attenuates Anthracycline Cardiotoxicity and Lysosomal Injury.

Apolipoprotein M (ApoM) binds sphingosine-1-phosphate (S1P) and is inversely associated with mortality in human heart failure (HF). Here, we show that anthracyclines such as doxorubicin (Dox) reduce circulating ApoM in mice and humans, that ApoM is inversely associated with mortality in patients with anthracycline-induced heart failure, and ApoM heterozygosity in mice increases Dox-induced mortality. In the setting of Dox stress, our studies suggest ApoM can help sustain myocardial autophagic flux in a post-transcriptional manner, attenuate Dox cardiotoxicity, and prevent lysosomal injury.

Sustained alternate-day fasting potentiates doxorubicin cardiotoxicity.

Fasting strategies are under active clinical investigation in patients receiving chemotherapy. Prior murine studies suggest that alternate-day fasting may attenuate doxorubicin cardiotoxicity and stimulate nuclear translocation of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis. In this study, human heart tissue from patients with doxorubicin-induced heart failure demonstrated increased nuclear TFEB protein. In mice treated with doxorubicin, alternate-day fasting or viral TFEB transduction increased mortality and impaired cardiac function. Mice randomized to alternate-day fasting plus doxorubicin exhibited increased TFEB nuclear translocation in the myocardium. When combined with doxorubicin, cardiomyocyte-specific TFEB overexpression provoked cardiac remodeling, while systemic TFEB overexpression increased growth differentiation factor 15 (GDF15) and caused heart failure and death. Cardiomyocyte TFEB knockout attenuated doxorubicin cardiotoxicity, while recombinant GDF15 was sufficient to cause cardiac atrophy. Our studies identify that both sustained alternate-day fasting and a TFEB/GDF15 pathway exacerbate doxorubicin cardiotoxicity.

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis.

Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1.

Association of Male Hypogonadism With Risk of Hospitalization for COVID-19.

Importance: Male sex is associated with severe COVID-19. It is not known whether the risk of hospitalization differs between men with hypogonadism, men with eugonadism, and those receiving testosterone therapy (TTh). Objective: To compare COVID-19 hospitalization rates for men with hypogonadism who were not receiving TTh, men with eugonadism, and men receiving TTh. Design, Setting, and Participants: This cohort study was conducted in 2 large academic health systems in St Louis, Missouri, among 723 men with a history of COVID-19 who had testosterone concentrations measured between January 1, 2017, and December 31, 2021. Exposures: The primary exposure was gonadal status (hypogonadism, eugonadism, and TTh). Hypogonadism was defined as a total testosterone concentration below the limit of normal provided by the laboratory (which varied from 175 to 300 ng/dL [to convert to nanomoles per liter, multiply by 0.0347]). Main Outcomes and Measures: The primary outcome was rate of hospitalization for COVID-19. Statistical adjustments were made for group differences in age, body mass index, race and ethnicity, immunosuppression, and comorbid conditions. Results: Of the 723 study participants (mean [SD] age, 55 [14] years; mean [SD] body mass index, 33.5 [7.3]), 116 men had hypogonadism, 427 had eugonadism, and 180 were receiving TTh. Men with hypogonadism were more likely than men with eugonadism to be hospitalized with COVID-19 (52 of 116 [45%] vs 53 of 427 [12%]; P < .001). After multivariable adjustment, men with hypogonadism had higher odds than men with eugonadism of being hospitalized (odds ratio, 2.4; 95% CI, 1.4-4.4; P < .003). Men receiving TTh had a similar risk of hospitalization as men with eugonadism (odds ratio, 1.3; 95% CI, 0.7-2.3; P = .35). Men receiving inadequate TTh (defined as subnormal testosterone concentrations while receiving TTh) had higher odds of hospitalization compared with men who had normal testosterone concentrations while receiving TTh (multivariable adjusted odds ratio, 3.5; 95% CI, 1.5-8.6; P = .003). Conclusions and Relevance: This study suggests that men with hypogonadism were more likely to be hospitalized after COVID-19 infection compared with those with eugonadism, independent of other known risk factors. This increased risk was not observed among men receiving adequate TTh. Screening and appropriate therapy for hypogonadism need to be evaluated as a strategy to prevent severe COVID-19 outcomes among men.

Proteasomal Degradation of TRAF2 Mediates Mitochondrial Dysfunction in Doxorubicin-Cardiomyopathy.

BACKGROUND: Cytokines such as tumor necrosis factor-α (TNFα) have been implicated in cardiac dysfunction and toxicity associated with doxorubicin (DOX). Although TNFα can elicit different cellular responses, including survival or death, the mechanisms underlying these divergent outcomes in the heart remain cryptic. The E3 ubiquitin ligase TRAF2 (TNF receptor associated factor 2) provides a critical signaling platform for K63-linked polyubiquitination of RIPK1 (receptor interacting protein 1), crucial for nuclear factor-κB (NF-κB) activation by TNFα and survival. Here, we investigate alterations in TNFα-TRAF2-NF-κB signaling in the pathogenesis of DOX cardiotoxicity. METHODS: Using a combination of in vivo (4 weekly injections of DOX 5 mg·kg-1·wk-1) in C57/BL6J mice and in vitro approaches (rat, mouse, and human inducible pluripotent stem cell-derived cardiac myocytes), we monitored TNFα levels, lactate dehydrogenase, cardiac ultrastructure and function, mitochondrial bioenergetics, and cardiac cell viability. RESULTS: In contrast to vehicle-treated mice, ultrastructural defects, including cytoplasmic swelling, mitochondrial perturbations, and elevated TNFα levels, were observed in the hearts of mice treated with DOX. While investigating the involvement of TNFα in DOX cardiotoxicity, we discovered that NF-κB was readily activated by TNFα. However, TNFα-mediated NF-κB activation was impaired in cardiac myocytes treated with DOX. This coincided with loss of K63- linked polyubiquitination of RIPK1 from the proteasomal degradation of TRAF2. Furthermore, TRAF2 protein abundance was markedly reduced in hearts of patients with cancer treated with DOX. We further established that the reciprocal actions of the ubiquitinating and deubiquitinating enzymes cellular inhibitors of apoptosis 1 and USP19 (ubiquitin-specific peptidase 19), respectively, regulated the proteasomal degradation of TRAF2 in DOX-treated cardiac myocytes. An E3-ligase mutant of cellular inhibitors of apoptosis 1 (H588A) or gain of function of USP19 prevented proteasomal degradation of TRAF2 and DOX-induced cell death. Furthermore, wild-type TRAF2, but not a RING finger mutant defective for K63-linked polyubiquitination of RIPK1, restored NF-κB signaling and suppressed DOX-induced cardiac cell death. Last, cardiomyocyte-restricted expression of TRAF2 (cardiac troponin T-adeno-associated virus 9-TRAF2) in vivo protected against mitochondrial defects and cardiac dysfunction induced by DOX. CONCLUSIONS: Our findings reveal a novel signaling axis that functionally connects the cardiotoxic effects of DOX to proteasomal degradation of TRAF2. Disruption of the critical TRAF2 survival pathway by DOX sensitizes cardiac myocytes to TNFα-mediated necrotic cell death and DOX cardiotoxicity.

Fine-Tuning Cardiac Insulin-Like Growth Factor 1 Receptor Signaling to Promote Health and Longevity.

BACKGROUND: The insulin-like growth factor 1 (IGF1) pathway is a key regulator of cellular metabolism and aging. Although its inhibition promotes longevity across species, the effect of attenuated IGF1 signaling on cardiac aging remains controversial. METHODS: We performed a lifelong study to assess cardiac health and lifespan in 2 cardiomyocyte-specific transgenic mouse models with enhanced versus reduced IGF1 receptor (IGF1R) signaling. Male mice with human IGF1R overexpression or dominant negative phosphoinositide 3-kinase mutation were examined at different life stages by echocardiography, invasive hemodynamics, and treadmill coupled to indirect calorimetry. In vitro assays included cardiac histology, mitochondrial respiration, ATP synthesis, autophagic flux, and targeted metabolome profiling, and immunoblots of key IGF1R downstream targets in mouse and human explanted failing and nonfailing hearts, as well. RESULTS: Young mice with increased IGF1R signaling exhibited superior cardiac function that progressively declined with aging in an accelerated fashion compared with wild-type animals, resulting in heart failure and a reduced lifespan. In contrast, mice with low cardiac IGF1R signaling exhibited inferior cardiac function early in life, but superior cardiac performance during aging, and increased maximum lifespan, as well. Mechanistically, the late-life detrimental effects of IGF1R activation correlated with suppressed autophagic flux and impaired oxidative phosphorylation in the heart. Low IGF1R activity consistently improved myocardial bioenergetics and function of the aging heart in an autophagy-dependent manner. In humans, failing hearts, but not those with compensated hypertrophy, displayed exaggerated IGF1R expression and signaling activity. CONCLUSIONS: Our findings indicate that the relationship between IGF1R signaling and cardiac health is not linear, but rather biphasic. Hence, pharmacological inhibitors of the IGF1 pathway, albeit unsuitable for young individuals, might be worth considering in older adults.

Peripheral monocyte-derived cells counter amyloid plaque pathogenesis in a mouse model of Alzheimer's disease.

Microglia, the parenchymal tissue macrophages in the brain, surround amyloid plaques in brains of individuals with Alzheimer's disease (AD) but are ineffective at clearing amyloid to mitigate disease progression. Recent studies in mice indicate that microglia are derived exclusively from primitive yolk sac hematopoiesis and self-renew without contribution from ontogenically distinct monocytes/macrophages of definitive adult hematopoietic origin. Using a genetic fate-mapping approach to label cells of definitive hematopoietic origin throughout life span, we discovered that circulating monocytes contribute 6% of plaque-associated macrophages in aged AD mice. Moreover, peripheral monocytes contributed to a higher fraction of macrophages in the choroid plexus, meninges, and perivascular spaces of aged AD mice versus WT control mice, indicating enrichment at potential sites for entry into the brain parenchyma. Splenectomy, which markedly reduced circulating Ly6Chi monocytes, also reduced abundance of plaque-associated macrophages of definitive hematopoietic origin, resulting in increased amyloid plaque load. Together, these results indicate that peripherally derived monocytes invade the brain parenchyma, targeting amyloid plaques to reduce plaque load.

TRAF2, an Innate Immune Sensor, Reciprocally Regulates Mitophagy and Inflammation to Maintain Cardiac Myocyte Homeostasis.

Mitochondria are essential for cardiac myocyte function, but damaged mitochondria trigger cardiac myocyte death. Although mitophagy, a lysosomal degradative pathway to remove damaged mitochondria, is robustly active in cardiac myocytes in the unstressed heart, its mechanisms and physiological role remain poorly defined. We discovered a critical role for TRAF2, an innate immunity effector protein with E3 ubiquitin ligase activity, in facilitating physiological cardiac myocyte mitophagy in the adult heart, to prevent inflammation and cell death, and maintain myocardial homeostasis.

Circadian REV-ERBs repress E4bp4 to activate NAMPT-dependent NAD+ biosynthesis and sustain cardiac function.

The heart is a highly metabolic organ that uses multiple energy sources to meet its demand for ATP production. Diurnal feeding-fasting cycles result in substrate availability fluctuations which, together with increased energetic demand during the active period, impose a need for rhythmic cardiac metabolism. The nuclear receptors REV-ERBα and ß are essential repressive components of the molecular circadian clock and major regulators of metabolism. To investigate their role in the heart, here we generated mice with cardiomyocyte (CM)-specific deletion of both Rev-erbs, which died prematurely due to dilated cardiomyopathy. Loss of Rev-erbs markedly downregulated fatty acid oxidation genes prior to overt pathology, which was mediated by induction of the transcriptional repressor E4BP4, a direct target of cardiac REV-ERBs. E4BP4 directly controls circadian expression of Nampt and its biosynthetic product NAD+ via distal cis-regulatory elements. Thus, REV-ERB-mediated E4BP4 repression is required for Nampt expression and NAD+ production by the salvage pathway. Together, these results highlight the indispensable role of circadian REV-ERBs in cardiac gene expression, metabolic homeostasis and function.