Hypoxia-inducible factors individually facilitate inflammatory myeloid metabolism and inefficient cardiac repair.

Hypoxia-inducible factors (HIFs) are activated in parenchymal cells in response to low oxygen and as such have been proposed as therapeutic targets during hypoxic insult, including myocardial infarction (MI). HIFs are also activated within macrophages, which orchestrate the tissue repair response. Although isoform-specific therapeutics are in development for cardiac ischemic injury, surprisingly, the unique role of myeloid HIFs, and particularly HIF-2α, is unknown. Using a murine model of myocardial infarction and mice with conditional genetic loss and gain of function, we uncovered unique proinflammatory roles for myeloid cell expression of HIF-1α and HIF-2α during MI. We found that HIF-2α suppressed anti-inflammatory macrophage mitochondrial metabolism, while HIF-1α promoted cleavage of cardioprotective MerTK through glycolytic reprogramming of macrophages. Unexpectedly, combinatorial loss of both myeloid HIF-1α and HIF-2α was catastrophic and led to macrophage necroptosis, impaired fibrogenesis, and cardiac rupture. These findings support a strategy for selective inhibition of macrophage HIF isoforms and promotion of anti-inflammatory mitochondrial metabolism during ischemic tissue repair.

Macrophage AXL receptor tyrosine kinase inflames the heart after reperfused myocardial infarction.

Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.

Desmoplakin Cardiomyopathy, a Fibrotic and Inflammatory Form of Cardiomyopathy Distinct From Typical Dilated or Arrhythmogenic Right Ventricular Cardiomyopathy.

BACKGROUND: Mutations in desmoplakin (DSP), the primary force transducer between cardiac desmosomes and intermediate filaments, cause an arrhythmogenic form of cardiomyopathy that has been variably associated with arrhythmogenic right ventricular cardiomyopathy. Clinical correlates of DSP cardiomyopathy have been limited to small case series. METHODS: Clinical and genetic data were collected on 107 patients with pathogenic DSP mutations and 81 patients with pathogenic plakophilin 2 (PKP2) mutations as a comparison cohort. A composite outcome of severe ventricular arrhythmia was assessed. RESULTS: DSP and PKP2 cohorts included similar proportions of probands (41% versus 42%) and patients with truncating mutations (98% versus 100%). Left ventricular (LV) predominant cardiomyopathy was exclusively present among patients with DSP (55% versus 0% for PKP2, P<0.001), whereas right ventricular cardiomyopathy was present in only 14% of patients with DSP versus 40% for PKP2 (P<0.001). Arrhythmogenic right ventricular cardiomyopathy diagnostic criteria had poor sensitivity for DSP cardiomyopathy. LV late gadolinium enhancement was present in a primarily subepicardial distribution in 40% of patients with DSP (23/57 with magnetic resonance images). LV late gadolinium enhancement occurred with normal LV systolic function in 35% (8/23) of patients with DSP. Episodes of acute myocardial injury (chest pain with troponin elevation and normal coronary angiography) occurred in 15% of patients with DSP and were strongly associated with LV late gadolinium enhancement (90%), even in cases of acute myocardial injury with normal ventricular function (4/5, 80% with late gadolinium enhancement). In 4 DSP cases with 18F-fluorodeoxyglucose positron emission tomography scans, acute LV myocardial injury was associated with myocardial inflammation misdiagnosed initially as cardiac sarcoidosis or myocarditis. Left ventricle ejection fraction <55% was strongly associated with severe ventricular arrhythmias for DSP cases (P<0.001, sensitivity 85%, specificity 53%). Right ventricular ejection fraction <45% was associated with severe arrhythmias for PKP2 cases (P<0.001) but was poorly associated for DSP cases (P=0.8). Frequent premature ventricular contractions were common among patients with severe arrhythmias for both DSP (80%) and PKP2 (91%) groups (P=non-significant). CONCLUSIONS: DSP cardiomyopathy is a distinct form of arrhythmogenic cardiomyopathy characterized by episodic myocardial injury, left ventricular fibrosis that precedes systolic dysfunction, and a high incidence of ventricular arrhythmias. A genotype-specific approach for diagnosis and risk stratification should be used.

Sirtuin 2 regulates cellular iron homeostasis via deacetylation of transcription factor NRF2.

SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2-related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2-/- mice had decreased iron levels, while this effect was reversed in Sirt2-/- Nrf2-/- double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.

A small molecule inhibitor of PAI-1 protects against doxorubicin-induced cellular senescence.

Doxorubicin, an anthracycline antibiotic, is a commonly used anticancer drug. In spite of its widespread usage, its therapeutic effect is limited by its cardiotoxicity. On the cellular level, Doxorubicin-induced cardiotoxicity manifests as stress induced premature senescence. Previously, we demonstrated that plasminogen activator inhibitor-1 (PAI-1), a potent inhibitor of serine proteases, is an important biomarker and regulator of cellular senescence and aging. Here, we tested the hypothesis that pharmacological inhibition of cellular PAI-1 protects against stress- and aging-induced cellular senescence and delineated the molecular basis of protective action of PAI-1 inhibition. Results show that TM5441, a potent small molecule inhibitor of PAI-1, effectively prevents Doxorubicin-induced senescence in cardiomyocytes, fibroblasts and endothelial cells. TM5441 exerts its inhibitory effect on Doxorubicin-induced cellular senescence by decreasing reactive oxygen species generation, induction of antioxidants like catalase and suppression of stress-induced senescence cadre p53, p21, p16, PAI-1 and IGFBP3. Importantly, TM5441 also reduces replicative senescence of fibroblasts. Together these results for the first time demonstrate the efficacy of PAI-1 inhibitor in prevention of Doxorubicin-induced and replicative senescence in normal cells. Thus PAI-1 inhibitor may form an important adjuvant component of chemotherapy regimens, limiting not only Doxorubicin-induced cardiac senescence but also ameliorating the prothrombotic profile.

Dissecting the functions of the mammalian clock protein BMAL1 by tissue-specific rescue in mice.

The basic helix-loop-helix (bHLH)-Per-Arnt-Sim (PAS) domain transcription factor BMAL1 is an essential component of the mammalian circadian pacemaker. Bmal1-/- mice lose circadian rhythmicity but also display tendon calcification and decreased activity, body weight, and longevity. To investigate whether these diverse functions of BMAL1 are tissue-specific, we produced transgenic mice that constitutively express Bmal1 in brain or muscle and examined the effects of rescued gene expression in Bmal1-/- mice. Circadian rhythms of wheel-running activity were restored in brain-rescued Bmal1-/- mice in a conditional manner; however, activity levels and body weight were lower than those of wild-type mice. In contrast, muscle-rescued Bmal1-/- mice exhibited normal activity levels and body weight yet remained behaviorally arrhythmic. Thus, Bmal1 has distinct tissue-specific functions that regulate integrative physiology.