Angiotensin Receptor-Neprilysin Inhibition in Patients With STEMI vs NSTEMI.

BACKGROUND: Patients who sustain an acute myocardial infarction (AMI), including ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI), remain at high risk for heart failure (HF), coronary events, and death. Angiotensin-converting enzyme inhibitors have been shown to significantly decrease the risk for cardiovascular events in both STEMI and NSTEMI patients. OBJECTIVES: The objectives were to determine whether angiotensin-receptor blockade and neprilysin inhibition with sacubitril/valsartan, compared with ramipril, has impact on reducing cardiovascular events according to the type of AMI. METHODS: The PARADISE-MI (Prospective ARNI versus ACE inhibitor trial to DetermIne Superiority in reducing heart failure Events after Myocardial Infarction) trial enrolled patients with AMI complicated by left ventricular dysfunction and/or pulmonary congestion and at least 1 risk-enhancing factor. Patients were randomized to either sacubitril/valsartan or ramipril. The primary endpoint was death from cardiovascular causes or incident HF. In this prespecified analysis, we stratified patients according to AMI type. RESULTS: Of 5,661 enrolled patients, 4,291 (75.8%) had STEMI. These patients were younger and had fewer comorbidities and cardiovascular risk factors than NSTEMI patients. After adjustment for potential confounders, the risk for the primary outcome was marginally higher in NSTEMI vs STEMI patients (adjusted HR: 1.19; 95% CI: 1.00-1.41), with borderline statistical significance (P = 0.05). The primary composite outcome occurred at similar rates in patients randomized to sacubitril/valsartan vs ramipril in STEMI (10% vs 12%; HR: 0.87; 95% CI: 0.73-1.04; P = 0.13) and NSTEMI patients (17% vs 17%; HR: 0.97; 95% CI: 0.75-1.25; P = 0.80; P interaction = 0.53). CONCLUSIONS: Compared with ramipril, sacubitril/valsartan did not significantly decrease the risk for cardiovascular death and HF in patients with AMI complicated by left ventricular dysfunction, irrespective of the type of AMI. (Prospective ARNI vs ACE Inhibitor Trial to Determine Superiority in Reducing Heart Failure Events After MI; NCT02924727).

Distinct Inflammatory Milieu in Patients With Right Heart Failure.

BACKGROUND: Right heart failure (RHF) is associated with worse clinical outcomes. In addition to hemodynamic perturbations, the syndrome of RHF involves liver congestion and dysfunction. The mechanisms that underlie heart-liver interactions are poorly understood and may involve secreted factors. As a first step to understand the cardiohepatic axis, we sought to elucidate the circulating inflammatory milieu in patients with RHF. METHODS: Blood samples were collected from the inferior vena cava and hepatic veins during right heart catheterization from 3 groups of patients: (1) controls with normal cardiac function, (2) patients with heart failure who did not meet all criteria of RHF, and (3) patients who met prespecified criteria for RHF defined by hemodynamic and echocardiographic parameters. We performed a multiplex protein assay to survey levels of several circulating markers and analyzed their association with mortality and the need for a left ventricular assist device or heart transplant. Finally, we leveraged publicly available single-cell RNA sequencing data and performed tissue imaging to evaluate the expression of these factors in the liver. RESULTS: In this study, RHF was associated with elevated levels of a subset of cytokines/chemokines/growth factors compared with controls. In particular, soluble CD163 (cluster of differentiation 163) and CXCL12 (chemokine [C-X-C motif] ligand 12) were higher in RHF and predicted left ventricular assist device/transplant-free survival in an independent validation cohort. Furthermore, single-cell RNA sequencing and immunohistochemistry of human liver biopsies suggest that these factors are expressed by Kupffer cells and may be liver derived. CONCLUSIONS: RHF is associated with a distinct circulating inflammatory profile. Soluble CD163 and CXCL12 are novel biomarkers that can prognosticate patient outcomes. Future studies to define how these molecules influence heart failure phenotypes and disease progression may lead to new approaches to the management of patients with RHF.

Necrotic cardiac myocytes skew macrophage polarization towards a classically activated phenotype.

Necrotic and dying cells release damage-associated molecular patterns (DAMPs) that can initiate sterile inflammatory responses in the heart. Although macrophages are essential for myocardial repair and regeneration, the effect of DAMPs on macrophage activation remains unclear. To address this gap in knowledge we studied the effect of necrotic cardiac myocyte extracts on primary peritoneal macrophage (PPM) cultures in vitro. We first performed unbiased transcriptomic profiling with RNA-sequencing of PPMs cultured for up to 72 hours in the presence and absence of: 1) necrotic cell extracts (NCEs) from necrotic cardiac myocytes in order to mimic the release of DAMPs; 2) lipopolysaccharide (LPS), which is known to polarize macrophages towards a classically activated phenotype and 3) Interleukin-4 (IL-4), which is known to promote polarization of macrophages towards an alternatively activated phenotype. NCEs provoke changes in differential gene expression (DEGs) that had considerable overlap with LPS-induced changes, suggesting that NCEs promote macrophage polarization towards a classically activated phenotype. Treating NCEs with proteinase-K abolished the effects of NCEs on macrophage activation, whereas NCE treatment with DNase and RNase did not affect macrophage activation. Stimulation of macrophage cultures with NCEs and LPS resulted in a significant increase in macrophage phagocytosis and interleukin-1ß secretion, whereas treatment with IL-4 had no significant effect on phagocytosis and interleukin-1ß. Taken together, our findings suggest that proteins released from necrotic cardiac myocytes are sufficient to skew the polarization of macrophages towards a classically activated phenotype.

The Effects of Angiotensin Receptor-Neprilysin Inhibition on Major Coronary Events in Patients With Acute Myocardial Infarction: Insights From the PARADISE-MI Trial.

BACKGROUND: In patients who survive an acute myocardial infarction (AMI), angiotensin-converting enzyme inhibitors decrease the risk of subsequent major cardiovascular events. Whether angiotensin-receptor blockade and neprilysin inhibition with sacubitril/valsartan reduce major coronary events more effectively than angiotensin-converting enzyme inhibitors in high-risk patients with recent AMI remains unknown. We aimed to compare the effects of sacubitril/valsartan on coronary outcomes in patients with AMI. METHODS: We conducted a prespecified analysis of the PARADISE-MI trial (Prospective ARNI vs ACE Inhibitors Trial to Determine Superiority in Reducing Heart Failure Events After MI), which compared sacubitril/valsartan (97/103 mg twice daily) with ramipril (5 mg twice daily) for reducing heart failure events after myocardial infarction in 5661 patients with AMI complicated by left ventricular systolic dysfunction, pulmonary congestion, or both. In the present analysis, the prespecified composite coronary outcome was the first occurrence of death from coronary heart disease, nonfatal myocardial infarction, hospitalization for angina, or postrandomization coronary revascularization. RESULTS: Patients were randomly assigned at a median of 4.4 [3.0-5.8] days after index AMI (ST-segment-elevation myocardial infarction 76%, non-ST-segment-elevation myocardial infarction 24%), by which time 89% of patients had undergone coronary reperfusion. Compared with ramipril, sacubitril/valsartan decreased the risk of coronary outcomes (hazard ratio, 0.86 [95% CI, 0.74-0.99], P=0.04) over a median follow-up of 22 months. Rates of the components of the composite outcomes were lower in patients on sacubitril/valsartan but were not individually significantly different. CONCLUSIONS: In survivors of an AMI with left ventricular systolic dysfunction and pulmonary congestion, sacubitril/valsartan-compared with ramipril-reduced the risk of a prespecified major coronary composite outcome. Dedicated studies are necessary to confirm this finding and elucidate its mechanism. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02924727.

Meeting the Challenges of Myocarditis: New Opportunities for Prevention, Detection, and Intervention-A Report from the 2021 National Heart, Lung, and Blood Institute Workshop.

The National Heart, Lung, and Blood Institute (NHLBI) convened a workshop of international experts to discuss new research opportunities for the prevention, detection, and intervention of myocarditis in May 2021. These experts reviewed the current state of science and identified key gaps and opportunities in basic, diagnostic, translational, and therapeutic frontiers to guide future research in myocarditis. In addition to addressing community-acquired myocarditis, the workshop also focused on emerging causes of myocarditis including immune checkpoint inhibitors and SARS-CoV-2 related myocardial injuries and considered the use of systems biology and artificial intelligence methodologies to define workflows to identify novel mechanisms of disease and new therapeutic targets. A new priority is the investigation of the relationship between social determinants of health (SDoH), including race and economic status, and inflammatory response and outcomes in myocarditis. The result is a proposal for the reclassification of myocarditis that integrates the latest knowledge of immunological pathogenesis to refine estimates of prognosis and target pathway-specific treatments.

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.

Targeting the Autophagy-Lysosome Pathway in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure.

The key biological "drivers" that are responsible for reverse left ventricle (LV) remodeling are not well understood. To gain an understanding of the role of the autophagy-lysosome pathway in reverse LV remodeling, we used a pathophysiologically relevant murine model of reversible heart failure, wherein pressure overload by transaortic constriction superimposed on acute coronary artery (myocardial infarction) ligation leads to a heart failure phenotype that is reversible by hemodynamic unloading. Here we show transaortic constriction + myocardial infarction leads to decreased flux through the autophagy-lysosome pathway with the accumulation of damaged proteins and organelles in cardiac myocytes, whereas hemodynamic unloading is associated with restoration of autophagic flux to normal levels with incomplete removal of damaged proteins and organelles in myocytes and reverse LV remodeling, suggesting that restoration of flux is insufficient to completely restore myocardial proteostasis. Enhancing autophagic flux with adeno-associated virus 9-transcription factor EB resulted in more favorable reverse LV remodeling in mice that had undergone hemodynamic unloading, whereas overexpressing transcription factor EB in mice that have not undergone hemodynamic unloading leads to increased mortality, suggesting that the therapeutic outcomes of enhancing autophagic flux will depend on the conditions in which flux is being studied.

Mechanisms and Models in Heart Failure: A Translational Approach.

Despite multiple attempts to develop a unifying hypothesis that explains the pathophysiology of heart failure with a reduced ejection fraction (HFrEF), no single conceptual model has withstood the test of time. In the present review, we discuss how the results of recent successful phase III clinical development programs in HFrEF are built upon existing conceptual models for drug development. We will also discuss where recent successes in clinical trials do not fit existing models to identify areas where further refinement of current paradigms may be needed. To provide the necessary structure for this review, we will begin with a brief overview of the pathophysiology of HFrEF, followed by an overview of the current conceptual models for HFrEF, and end with an analysis of the scientific rationale and clinical development programs for 4 new therapeutic classes of drugs that have improved clinical outcomes in HFrEF. The 4 new therapeutic classes discussed are ARNIs, SGLT2 (sodium-glucose cotransporter 2) inhibitors, soluble guanylate cyclase stimulators, and myosin activators. With the exception of SGLT2 inhibitors, each of these therapeutic advances was informed by the insights provided by existing conceptual models of heart failure. Although the quest to determine the mechanism of action of SGLT2 inhibitors is ongoing, this therapeutic class of drugs may represent the most important advance in cardiovascular therapeutics of recent decades and may lead to rethinking or expanding our current conceptual models for HFrEF.

Associations of methyl donor and methylation inhibitor levels during anti-oxidant therapy in heart failure.

Redox balance and methylation are crucial to homeostasis and are linked by the methionine-homocysteine cycle. We examined whether differences in methylation potential, measured as plasma levels of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH), occur at baseline and during anti-oxidant therapy with the xanthine oxidase inhibitor allopurinol in patients with heart failure with reduced ejection fraction. We analyzed plasma samples collected at baseline and 24 weeks in the Xanthine Oxidase Inhibition for Hyperuricemic Heart Failure Patients (EXACT-HF) study, which randomized patients with heart failure with reduced ejection fraction to allopurinol or placebo. Associations between plasma levels of SAM, SAH, SAM/SAH ratio, and outcomes, including laboratory markers and clinical events, were assessed. Despite randomization, median SAM levels were significantly lower at baseline in the allopurinol group. SAH levels at 24 weeks, and change in SAM from baseline to week 24, were significantly higher in the group of patients randomized to allopurinol compared to the placebo group. A significant correlation was observed between change in SAH levels and change in plasma uric acid (baseline to 24-week changes) in the allopurinol group. There were no significant associations between levels of SAM, SAH, and SAM/SAH ratio and clinical outcomes. Our results demonstrate significant biological variability in SAM and SAH levels at baseline and during treatment with an anti-oxidant and suggest a potential mechanism for the lack of efficacy observed in trials of anti-oxidant therapy. These data also highlight the need to explore personalized therapy for heart failure.

Improvement of ejection fraction and mortality in ischaemic heart failure.

OBJECTIVE: The frequency and predictors of improvement in left ventricular ejection fraction (LVEF) in ischaemic cardiomyopathy and its association with mortality is poorly understood. We sought to assess the predictors of LVEF improvement ≥10% and its effect on mortality. METHODS: We compared characteristics of patients enrolled in The Surgical Treatment for Ischaemic Heart Failure (STICH) trial with and without improvement of LVEF ≥10% at 24 months. A logistic regression model was constructed to determine the independent predictors of LVEF improvement. A Cox proportional hazards model was created to assess the independent association of improvement in LVEF ≥10% with mortality. RESULTS: Of the 1212 patients enrolled in STICH, 618 underwent echocardiographic assessment of LVEF at baseline and 24 months. Of the patients randomised to medical therapy plus coronary artery bypass graft surgery (CABG), 58 (19%) had an improvement in LVEF >10% compared with 51 (16%) patients assigned to medical therapy alone (p=0.30). Independent predictors of LVEF improvement >10% included prior myocardial infarction (OR 0.44, 95% CI: 0.28 to 0.71, p=0.001) and lower baseline LVEF (OR 0.94, 95% CI: 0.91 to 0.97, p<0.001). Improvement in LVEF >10% (HR 0.61, 95% CI: 0.44 to 0.84, p=0.004) and randomisation to CABG (HR 0.72, 95% CI: 0.57 to 0.90, p=0.004) were independently associated with a reduced hazard of mortality. CONCLUSIONS: Improvement of LVEF ≥10% at 24 months was uncommon in patients with ischaemic cardiomyopathy, did not differ between patients assigned to CABG and medical therapy or medical therapy alone and was independently associated with reduced mortality. TRIAL REGISTRATION NUMBER: NCT00023595.

COVID-19 Clinical Trials: A Primer for the Cardiovascular and Cardio-Oncology Communities.

The coronavirus disease-2019 (COVID-19) pandemic has resulted in a proliferation of clinical trials designed to slow the spread of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Many therapeutic agents that are being used to treat patients with COVID-19 are repurposed treatments for influenza, Ebola, or for malaria that were developed decades ago and are unlikely to be familiar to the cardiovascular and cardio-oncology communities. Here, the authors provide a foundation for cardiovascular and cardio-oncology physicians on the front line providing care to patients with COVID-19, so that they may better understand the emerging cardiovascular epidemiology and the biological rationale for the clinical trials that are ongoing for the treatment of patients with COVID-19.

COVID-19 Clinical Trials: A Primer for the Cardiovascular and Cardio-Oncology Communities.

The coronavirus disease-2019 (COVID-19) pandemic has resulted in a proliferation of clinical trials designed to slow the spread of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). Many therapeutic agents that are being used to treat patients with COVID-19 are repurposed treatments for influenza, Ebola, or for malaria that were developed decades ago and are unlikely to be familiar to the cardiovascular and cardio-oncology communities. Here, we provide a foundation for cardiovascular and cardio-oncology physicians on the front line providing care to patients with COVID-19, so that they may better understand the emerging cardiovascular epidemiology and the biological rationale for the clinical trials that are ongoing for the treatment of patients with COVID-19.

Myocardial B cells are a subset of circulating lymphocytes with delayed transit through the heart.

Current models of B lymphocyte biology posit that B cells continuously recirculate between lymphoid organs, without accumulating in peripheral healthy tissues. Nevertheless, B lymphocytes are one of the most prevalent leukocyte populations in the naive murine heart. To investigate this apparent inconsistency in the literature, we conducted a systematic analysis of myocardial B cell ontogeny, trafficking dynamics, histology, and gene expression patterns. We found that myocardial B cells represent a subpopulation of circulating B cells that make close contact with the microvascular endothelium of the heart and arrest their transit as they pass through the heart. The vast majority (>95%) of myocardial B cells remain intravascular, whereas few (<5%) myocardial B cells cross the endothelium into myocardial tissue. Analyses of mice with B cell deficiency or depletion indicated that B cells modulate the myocardial leukocyte pool composition. Analysis of B cell-deficient animals suggested that B cells modulate myocardial growth and contractility. These results transform our current understanding of B cell recirculation in the naive state and reveal a previously unknown relationship between B cells and myocardial physiology. Further work will be needed to assess the relevance of these findings to other organs.

Immunomodulatory role of non-neuronal cholinergic signaling in myocardial injury.

Whereas prior studies have demonstrated an important immunomodulatory role for the neuronal cholinergic system in the heart, the role of the non-neuronal cholinergic system is not well understood. To address the immunomodulatory role of the non-neuronal cholinergic system in the heart we used a previously validated diphtheria toxin (DT)-induced cardiomyocyte ablation model (Rosa26-DTMlc2v-Cre mice). DT-injected Rosa26-DTMlc2v-Cre mice were treated with diluent or Pyridostigmine Bromide (PYR), a reversible cholinesterase inhibitor. PYR treatment resulted in increased survival and decreased numbers of MHC-IIlowCCR2+ macrophages in DT-injected Rosa26-DTMlc2v-Cre mice compared to diluent treated Rosa26-DTMlc2v-Cre mice. Importantly, the expression of CCL2/7 mRNA and protein was reduced in the hearts of PYR-treated mice. Backcrossing Rosa26-DTMlc2v-Cre mice with a transgenic mouse line (Chat-ChR2) that constitutively overexpresses the vesicular acetylcholine transporter (VAChT) resulted in decreased expression of Ccl2/7 mRNA and decreased numbers of CD68+ cells in DT-injured Rosa26-DTMlc2v-Cre/Chat-ChR2 mouse hearts, consistent with the pharmacologic studies with PYR. In vitro studies with cultures of LPS-stimulated peritoneal macrophages revealed a concentration-dependent reduction in CCL2 secretion following stimulation with ACh, nicotine and muscarine. Viewed together, these findings reveal a previously unappreciated immunomodulatory role for the non-neuronal cholinergic system in regulating homeostatic responses in the heart following tissue injury.

Ambulatory Advanced Heart Failure in Women: A Report From the REVIVAL Registry.

OBJECTIVES: This study sought to explore clinical characteristics and outcomes in women and men with ambulatory advanced heart failure (HF). BACKGROUND: Women have been underrepresented in studies of advanced HF and have an increased mortality on the transplant waiting list and early after mechanical circulatory support (MCS). An increased understanding of the differential burden of HF between women and men is required to inform the use of mechanical circulatory support in ambulatory advanced HF patients. METHODS: REVIVAL (Registry Evaluation for Vital Information on Ventricular Assist Devices in Ambulatory Life) is a prospective, observational study of 400 outpatients with chronic systolic HF, New York Heart Association functional class II to IV, and 1 additional high-risk feature. Clinical characteristics, quality of life, and functional capacity were compared between women and men, as was a primary composite endpoint of death, durable MCS, or urgent heart transplantation at 1 year. RESULTS: REVIVAL enrolled 99 women (25% of the cohort) who had similar age, ejection fraction, INTERMACS (Interagency Registry for Mechanically Assisted Circulatory Support) profiles, medication use, and willingness to consider MCS as the men enrolled; however, women reported significantly greater limitations in quality of life with respect to physical limitation, reduced 6-min walk distance, and more frequent symptoms of depression. Nevertheless, 1-year combined risk of death, durable MCS, or urgent transplantation did not differ between women and men (24% vs. 22%; p = 0.94). CONCLUSIONS: This study represents the largest report to date of women with ambulatory advanced HF receiving contemporary therapies. Systematic elicitation of patient-reported outcome measures uncovered an added burden of HF in women and may be an appropriate target of augmented support and intervention.

Modulation of subsets of cardiac B lymphocytes improves cardiac function after acute injury.

Despite the long-standing recognition that the immune response to acute myocardial injury contributes to adverse left ventricular (LV) remodeling, it has not been possible to effectively target this clinically. Using 2 different in vivo models of acute myocardial injury, we show that pirfenidone confers beneficial effects in the murine heart through an unexpected mechanism that depends on cardiac B lymphocytes. Naive hearts contained a large population of CD19+CD11b-CD23-CD21-IgD+IgMlo lymphocytes, and 2 smaller populations of CD19+CD11b+ B1a and B1b cells. In response to tissue injury, there was an increase in neutrophils, monocytes, macrophages, as well as an increase in CD19+ CD11b- B lymphocytes. Treatment with pirfenidone had no effect on the number of neutrophils, monocytes, or macrophages, but decreased CD19+CD11b- lymphocytes. B cell depletion abrogated the beneficial effects of pirfenidone. In vitro studies demonstrated that stimulation with lipopolysaccharide and extracts from necrotic cells activated CD19+ lymphocytes through a TIRAP-dependent pathway. Treatment with pirfenidone attenuated this activation of B cells. These findings reveal a previously unappreciated complexity of myocardial B lymphocytes within the inflammatory infiltrate triggered by cardiac injury and suggest that pirfenidone exerts beneficial effects in the heart through a unique mechanism that involves modulation of cardiac B lymphocytes.

Load-Dependent Changes in Left Ventricular Structure and Function in a Pathophysiologically Relevant Murine Model of Reversible Heart Failure.

BACKGROUND: To better understand reverse left ventricular (LV) remodeling, we developed a murine model wherein mice develop LV remodeling after transverse aortic constriction (TAC) and a small apical myocardial infarct (MI) and undergo reverse LV remodeling after removal of the aortic band. METHODS AND RESULTS: Mice studied were subjected to sham (n=6) surgery or TAC+MI (n=12). Two weeks post-TAC+MI, 1 group underwent debanding (referred to as heart failure debanding [HF-DB] mice; n=6), whereas the aortic band remained in a second group (heart failure [HF] group; n=6). LV remodeling was evaluated by 2D echocardiography at 1 day, 2 weeks and 6 weeks post-TAC+MI. The hearts were analyzed by transcriptional profiling at 4 and 6 weeks and histologically at 6 weeks. Debanding normalized LV volumes, LV mass, and cardiac myocyte hypertrophy at 6 weeks in HF-DB mice, with no difference in myofibrillar collagen in the HF and HF-DB mice. LV ejection fraction and radial strain improved after debanding; however, both remained decreased in the HF-DB mice relative to sham and were not different from HF mice at 6 weeks. Hemodynamic unloading in the HF-DB mice was accompanied by a 35% normalization of the HF genes at 2 weeks and 80% of the HF genes at 4 weeks. CONCLUSIONS: Hemodynamic unloading of a pathophysiologically relevant mouse model of HF results in normalization of LV structure, incomplete recovery of LV function, and incomplete reversal of the HF transcriptional program. The HF-DB mouse model may provide novel insights into mechanisms of reverse LV remodeling.

TNF receptor-activated factor 2 mediates cardiac protection through noncanonical NF-κB signaling.

To elucidate the mechanisms responsible for cytoprotective effects of TNF receptor-activated factor 2 (TRAF2) in the heart, we employed genetic gain- and loss-of-function studies ex vivo and in vivo in mice with cardiac-restricted overexpression of TRAF2 (Myh6-TRAF2LC). Crossing Myh6-TRAF2LC mice with mice lacking canonical signaling (Myh6-TRAF2LC/Myh6-IκBαΔN) abrogated the cytoprotective effects of TRAF2 ex vivo. In contrast, inhibiting the JAK/STAT pathway did not abrogate the cytoprotective effects of TRAF2. Transcriptional profiling of WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts suggested that the noncanonical NF-κB signaling pathway was upregulated in the Myh6-TRAF2LC mouse hearts. Western blotting and ELISA for the NF-κB family proteins p50, p65, p52, and RelB on nuclear and cytoplasmic extracts from naive 12-week-old WT, Myh6-TRAF2LC, and Myh6-TRAF2LC/Myh6-IκBαΔN mouse hearts showed increased expression levels and increased DNA binding of p52 and RelB, whereas there was no increase in expression or DNA binding of the p50 and p65 subunits. Crossing Myh6-TRAF2LC mice with RelB-/+ mice (Myh6-TRAF2LC/RelB-/+) attenuated the cytoprotective effects of TRAF2 ex vivo and in vivo. Viewed together, these results suggest that crosstalk between the canonical and noncanonical NF-κB signaling pathways is required for mediating the cytoprotective effects of TRAF2.