Oral Hormonal Contraceptives and Cardiovascular Risks in Females.

Oral hormonal contraception (OHC) is a widely employed method in females for the prevention of unintended pregnancies, as well as for the treatment of menstrual disorders, endometriosis, and polycystic ovarian syndrome. However, it is believed that with OHCs use, some females may have higher risk of cardiovascular diseases, such as hypertension, diabetes, myocardial infarction, thrombosis, and heart failure. Although such risks are infrequently detected in healthy young females with the use of oral contraceptives, slightly elevated risks of cardiovascular diseases have been observed among reproductive-aged healthy females. However, prolonged use of OHC has also been claimed to have protective cardiac effects and may contribute to reduced risk of cardiovascular disease. In fact, the debate on whether OHC administration increases the risk of cardiovascular diseases has been ongoing with inconsistent and controversial viewpoints. Nevertheless, a great deal of work has been carried out to understand the relationship between OHC use and the occurrence of cardiovascular risk in females who use OHC for preventing the unwanted pregnancy or treatment of other disorders. Therefore, in this review we summarize the most recent available evidence regarding the association between the use of oral hormonal contraceptives and the risk for cardiovascular disease in females who are using OHC to prevent unintended pregnancy.

Sex specific considerations in cardiovascular drug therapy.

Despite major advances in cardiac research over the past three decades, cardiovascular disease (CVD) still remains the leading cause of morbidity and mortality in women and men worldwide. However, a major challenge for health care providers is that the current guidelines for cardiovascular drug therapies do not consider the impact of sex in the development of treatment plan for optimizing therapies for women. Clinical research in recent years suggests significant pharmacological and pharmacokinetic differences between females and males which have been attributed in part to differences in body composition, plasma protein binding capacity, drug metabolism and excretion. Herein, we provide a comprehensive review regarding sex- specific differences and drugs commonly used for cardiovascular diseases in women and men. Understanding how sex related differences influence drug efficacy and cardiovascular disease outcomes is crucial for not only optimizing treatment strategies for women and men but to encourage the implementation of specific guidelines that address sex difference as a consideration for treatment of cardiovascular diseases.

Agonists, inverse agonists, and antagonists as therapeutic approaches to manipulate retinoic acid-related orphan receptors.

Retinoic acid-related orphan receptors (RORs) serve as transcription factors that play a pivotal role in a myriad of physiological processes within the body. Their involvement extends to critical biological processes that confer protective effects in the heart, immune system, and nervous system, as well as contributing to the mitigation of several aggressive cancer types. These protective functions are attributed to ROR's regulation of key proteins and the management of various cellular processes, including autophagy, mitophagy, inflammation, oxidative stress, and glucose metabolism, highlighting the emerging need for pharmacological approaches to modulate ROR expression. Thus, the modulation of RORs is a rapidly growing area of research aimed not only at comprehending these receptors, but also at manipulating them to attain the desired physiological response. Despite the presence of natural ROR ligands, the development of synthetic agonists with high selectivity for these receptors holds substantial therapeutic potential. The exploration and advancement of such compounds can effectively target diseases associated with ROR dysregulation, thereby providing avenues for therapeutic interventions. Herein, we provide a comprehensive examination of the multifaceted role of ROR in diverse physiological and pathophysiological conditions, accompanied by an in-depth exploration of a spectrum of ROR agonists, inverse agonists, and antagonists.

IKKß stabilizes Mitofusin 2 and suppresses doxorubicin cardiomyopathy.

AIMS: The mitochondrial dynamics protein Mitofusin 2 (MFN2) coordinates critical cellular processes including mitochondrial bioenergetics, quality control, and cell viability. The NF-κB kinase IKKß suppresses mitochondrial injury in doxorubicin cardiomyopathy, but the underlying mechanism is undefined. METHODS AND RESULTS: Herein, we identify a novel signalling axis that functionally connects IKKß and doxorubicin cardiomyopathy to a mechanism that impinges upon the proteasomal stabilization of MFN2. In contrast to vehicle-treated cells, MFN2 was highly ubiquitinated and rapidly degraded by the proteasomal-regulated pathway in cardiac myocytes treated with doxorubicin. The loss of MFN2 activity resulted in mitochondrial perturbations, including increased reactive oxygen species (ROS) production, impaired respiration, and necrotic cell death. Interestingly, doxorubicin-induced degradation of MFN2 and mitochondrial-regulated cell death were contingent upon IKKß kinase activity. Notably, immunoprecipitation and proximity ligation assays revealed that IKKß interacted with MFN2 suggesting that MFN2 may be a phosphorylation target of IKKß. To explore this possibility, mass spectrometry analysis identified a novel MFN2 phospho-acceptor site at serine 53 that was phosphorylated by wild-type IKKß but not by a kinase-inactive mutant IKKßK-M. Based on these findings, we reasoned that IKKß-mediated phosphorylation of serine 53 may influence MFN2 protein stability. Consistent with this view, an IKKß-phosphomimetic MFN2 (MFN2S53D) was resistant to proteasomal degradation induced by doxorubicin whereas wild-type MFN2 and IKKß-phosphorylation defective MFN2 mutant (MFNS53A) were readily degraded in cardiac myocytes treated with doxorubicin. Concordantly, gain of function of IKKß or MFN2S53D suppressed doxorubicin-induced mitochondrial injury and cell death. CONCLUSIONS: The findings of this study reveal a novel survival pathway for IKKß that is mutually dependent upon and obligatory linked to the phosphorylation and stabilization of the mitochondrial dynamics protein MFN2.

Autophagy, Clock Genes, and Cardiovascular Disease.

Circadian rhythms are 24-hour cycles that regulate physical, mental, and behavioural changes of most living organisms. In the heart, circadian rhythms regulate processes such as heart rate, blood pressure, blood coagulability, and vascular tone. However, in addition to regulating physiologic processes, circadian rhythms regulate pathophysiologic processes in the heart. In this regard, circadian rhythms regulate the onset, severity, and outcome of many cardiovascular diseases (CVDs), including myocardial infarction, diabetic cardiomyopathy, doxorubicin (Dox)-induced cardiotoxicity, and heart failure. Notably, the underlying mechanism of many of these diseases is linked to impaired cellular quality control processes, such as autophagy. Autophagy is a homeostatic cellular process that regulates the removal of damaged cellular components, allowing their degradation and recycling into their basic constituents for production of cellular energy. Many studies from recent years point to a regulatory link between autophagy and circadian machinery in the control of CVDs. In this review, we highlight the recent discoveries in the field of circadian-induced autophagy in the heart and provide the molecular mechanisms and signalling pathways that underlie the crosstalk between autophagy and clock gene control in response to cardiac injury. Understanding the mechanisms that underlie circadian-induced autophagy in response to cardiac stress may prove to be beneficial in developing novel therapeutic approaches to treat cardiac disease.

Circadian regulation of genetic and hormonal risk factors of cardiovascular disease in women.

Cardiovascular disease is the leading cause of morbidity and mortality worldwide. However, sex differences can impact differently the etiology and outcome of cardiovascular disease when comparing men and women. Women have unique genetic and hormonal risk factors that can be associated with the development of cardiovascular diseases. Furthermore, certain phenotypes of cardiovascular diseases are more prevalent to women. Molecular clocks control circadian rhythms of different physiological systems in our body, including the cardiovascular system. Increased evidence in recent years points to a link between cardiovascular disease and regulation by circadian rhythms. However, the difference between circadian regulation of cardiovascular disease in women and men is poorly understood. In this review, we highlight the recent advances in circadian-regulated cardiovascular diseases with a specific focus on the pathogenesis of heart disease in women. Understanding circadian-regulated pathways and sex-specific differences between men and women may contribute to better diagnosis and development of sex-targeted interventions to better treat cardiovascular diseases.

Circadian regulated control of myocardial ischemia-reperfusion injury.

Circadian mechanisms have been associated with the pathogenesis of a variety of cardiovascular diseases, including myocardial ischemia-reperfusion injury (I-R). Myocardial ischemia resulting from impaired oxygen delivery to cardiac muscle sets into motion a cascade of cellular events that paradoxically triggers greater cardiac dysfunction upon reinstitution of coronary blood supply, a phenomenon known as I-R. I-R injury has been attributed to a number of cellular defects including increased reactive oxygen species (ROS), increased intracellular calcium and impaired mitochondrial bioenergetics that ultimately lead to cardiac cell death, ventricular remodeling and heart failure. Emerging evidence has identified a strong correlation between cellular defects that underlie I-R and the disrupted circadian. In fact, recent studies have shown that circadian dysfunction exacerbates cardiac injury following MI from impaired cellular quality control mechanisms such as autophagy, which are vital in the clearance of damaged cellular proteins and organelles such as mitochondria from the cell. The accumulation of cellular debris is posited as the central underlying cause of excessive cardiac cell death and ventricular dysfunction following MI. The complexities that govern the interplay between circadian biology and I-R injury following MI is at its infancy and understanding how circadian misalignment, such as in shift workers impacts I-R injury is of great scientific and clinical importance toward development of new therapeutic strategies using chronotherapy and circadian regulation to mitigate cardiac injury and improve cardiac outcomes after injury. In this review, we highlight recent advances in circadian biology and adaptive cellular quality control mechanisms that influence cardiac injury in response to MI injury with a specific focus on how circadian biology can be utilized to further cardiovascular medicine and patient care.

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.

Modulating mitofusins to control mitochondrial function and signaling.

Mitofusins reside on the outer mitochondrial membrane and regulate mitochondrial fusion, a physiological process that impacts diverse cellular processes. Mitofusins are activated by conformational changes and subsequently oligomerize to enable mitochondrial fusion. Here, we identify small molecules that directly increase or inhibit mitofusins activity by modulating mitofusin conformations and oligomerization. We use these small molecules to better understand the role of mitofusins activity in mitochondrial fusion, function, and signaling. We find that mitofusin activation increases, whereas mitofusin inhibition decreases mitochondrial fusion and functionality. Remarkably, mitofusin inhibition also induces minority mitochondrial outer membrane permeabilization followed by sub-lethal caspase-3/7 activation, which in turn induces DNA damage and upregulates DNA damage response genes. In this context, apoptotic death induced by a second mitochondria-derived activator of caspases (SMAC) mimetic is potentiated by mitofusin inhibition. These data provide mechanistic insights into the function and regulation of mitofusins as well as small molecules to pharmacologically target mitofusins.

NF-κB p65 Attenuates Cardiomyocyte PGC-1α Expression in Hypoxia.

Hypoxia exerts broad effects on cardiomyocyte function and viability, ranging from altered metabolism and mitochondrial physiology to apoptotic or necrotic cell death. The transcriptional coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of cardiomyocyte metabolism and mitochondrial function and is down-regulated in hypoxia; however, the underlying mechanism is incompletely resolved. Using primary rat cardiomyocytes coupled with electrophoretic mobility shift and luciferase assays, we report that hypoxia impaired mitochondrial energetics and resulted in an increase in nuclear localization of the Nuclear Factor-κB (NF-κB) p65 subunit, and the association of p65 with the PGC-1α proximal promoter. Tumor necrosis factor α (TNFα), an activator of NF-κB signaling, similarly reduced PGC-1α expression and p65 binding to the PGC-1α promoter in a dose-dependent manner, and TNFα-mediated down-regulation of PGC-1α expression could be reversed by the NF-κB inhibitor parthenolide. RNA-seq analysis revealed that cardiomyocytes isolated from p65 knockout mice exhibited alterations in genes associated with chromatin remodeling. Decreased PGC-1α promoter transactivation by p65 could be partially reversed by the histone deacetylase inhibitor trichostatin A. These results implicate NF-κB signaling, and specifically p65, as a potent inhibitor of PGC-1α expression in cardiac myocyte hypoxia.

Intersection of autophagy regulation and circadian rhythms in the heart.

Autophagy is a vital cellular mechanism that controls the removal of damaged or dysfunctional cellular components. Autophagy allows the degradation and recycling of damaged proteins and organelles into their basic constituents of amino acids and fatty acids for cellular energy production. Under basal conditions, autophagy is essential for the maintenance of cell homeostasis and function. However, during cell stress, excessive activation of autophagy can be destructive and lead to cell death. Autophagy plays a crucial role in the cardiovascular system and helps to maintain normal cardiac function. During ischemia- reperfusion, autophagy can be adaptive or maladaptive depending on the timing and extent of activation. In this review, we highlight the molecular mechanisms and signaling pathways that underlie autophagy in response to cardiac stress and therapeutic approaches to modulate autophagy by pharmacological interventions. Finally, we also discuss the intersection between autophagy and circadian regulation in the heart. Understanding the mechanisms that underlie autophagy following cardiac injury can be translated to clinical cardiology use toward improved patient treatment and outcomes.

Mitochondrial autophagy and cell survival is regulated by the circadian Clock gene in cardiac myocytes during ischemic stress.

Cardiac function is highly reliant on mitochondrial oxidative metabolism and quality control. The circadian Clock gene is critically linked to vital physiological processes including mitochondrial fission, fusion and bioenergetics; however, little is known of how the Clock gene regulates these vital processes in the heart. Herein, we identified a putative circadian CLOCK-mitochondrial interactome that gates an adaptive survival response during myocardial ischemia. We show by transcriptome and gene ontology mapping in CLOCK Δ19/Δ19 mouse that Clock transcriptionally coordinates the efficient removal of damaged mitochondria during myocardial ischemia by directly controlling transcription of genes required for mitochondrial fission, fusion and macroautophagy/autophagy. Loss of Clock gene activity impaired mitochondrial turnover resulting in the accumulation of damaged reactive oxygen species (ROS)-producing mitochondria from impaired mitophagy. This coincided with ultrastructural defects to mitochondria and impaired cardiac function. Interestingly, wild type CLOCK but not mutations of CLOCK defective for E-Box binding or interaction with its cognate partner ARNTL/BMAL-1 suppressed mitochondrial damage and cell death during acute hypoxia. Interestingly, the autophagy defect and accumulation of damaged mitochondria in CLOCK-deficient cardiac myocytes were abrogated by restoring autophagy/mitophagy. Inhibition of autophagy by ATG7 knockdown abrogated the cytoprotective effects of CLOCK. Collectively, our results demonstrate that CLOCK regulates an adaptive stress response critical for cell survival by transcriptionally coordinating mitochondrial quality control mechanisms in cardiac myocytes. Interdictions that restore CLOCK activity may prove beneficial in reducing cardiac injury in individuals with disrupted circadian CLOCK.Abbreviations: ARNTL/BMAL1: aryl hydrocarbon receptor nuclear translocator-like; ATG14: autophagy related 14; ATG7: autophagy related 7; ATP: adenosine triphosphate; BCA: bovine serum albumin; BECN1: beclin 1, autophagy related; bHLH: basic helix- loop-helix; CLOCK: circadian locomotor output cycles kaput; CMV: cytomegalovirus; COQ5: coenzyme Q5 methyltransferase; CQ: chloroquine; CRY1: cryptochrome 1 (photolyase-like); DNM1L/DRP1: dynamin 1-like; EF: ejection fraction; EM: electron microscopy; FS: fractional shortening; GFP: green fluorescent protein; HPX: hypoxia; i.p.: intraperitoneal; I-R: ischemia-reperfusion; LAD: left anterior descending; LVIDd: left ventricular internal diameter diastolic; LVIDs: left ventricular internal diameter systolic; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN2: mitofusin 2; MI: myocardial infarction; mPTP: mitochondrial permeability transition pore; NDUFA4: Ndufa4, mitochondrial complex associated; NDUFA8: NADH: ubiquinone oxidoreductase subunit A8; NMX: normoxia; OCR: oxygen consumption rate; OPA1: OPA1, mitochondrial dynamin like GTPase; OXPHOS: oxidative phosphorylation; PBS: phosphate-buffered saline; PER1: period circadian clock 1; PPARGC1A/PGC-1α: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; qPCR: quantitative real-time PCR; RAB7A: RAB7, member RAS oncogene family; ROS: reactive oxygen species; RT: room temperature; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFAM: transcription factor A, mitochondrial; TFEB: transcription factor EB; TMRM: tetra-methylrhodamine methyl ester perchlorate; WT: wild -type; ZT: zeitgeber time.

YAP/TFEB pathway promotes autophagic cell death and hypertrophic cardiomyopathy in lysosomal storage diseases.

Lysosomal storage disorders (LSD) are a group of inherited metabolic diseases characterized by lysosomal enzyme deficiency. The cardiac phenotype includes cardiomyopathy with eventual heart failure. Lysosome-mediated degradation processes, such as autophagy, maintain cellular homeostasis by discarding cellular debris and damaged organelles. Under stress, the transcription factor EB (TFEB) moves into the nucleus to activate transcription of lysosome biogenesis and autophagic proteins. In this issue of the JCI, Ikeda et al. report on their exploration of the signaling pathway involved with regulating lysosomal proteins specifically in the heart. The researchers generated a mouse model for LSD that was restricted to cardiac tissue. Unexpectedly, modulation of TFEB alone was insufficient to fully rescue the underlying clearance defect in lysosomal-associated disorders. The authors identified the Yes-associated protein (YAP)/TFEB signaling pathway as a key regulator of autophagosomes. These findings suggest that undigested autophagosomes accumulate and result in the cell death and cardiac dysfunction observed with LSD.