Brian C. Jensen

Large-scale discovery of enhancers from human heart tissue

Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of noncoding sequences highly enriched in human heart enhancers that is likely to facilitate downstream studies of the role of enhancers in development and pathological conditions of the heart.

Limited forward trafficking of connexin 43 reduces cell-cell coupling in stressed human and mouse myocardium

Gap junctions form electrical conduits between adjacent myocardial cells, permitting rapid spatial passage of the excitation current essential to each heartbeat. Arrhythmogenic decreases in gap junction coupling are a characteristic of stressed, failing, and aging myocardium, but the mechanisms of decreased coupling are poorly understood. We previously found that microtubules bearing gap junction hemichannels (connexons) can deliver their cargo directly to adherens junctions. The specificity of this delivery requires the microtubule plus-end tracking protein EB1. We performed this study to investigate the hypothesis that the oxidative stress that accompanies acute and chronic ischemic disease perturbs connexon forward trafficking. We found that EB1 was displaced in ischemic human hearts, stressed mouse hearts, and isolated cells subjected to oxidative stress. As a result, we observed limited microtubule interaction with adherens junctions at intercalated discs and reduced connexon delivery and gap junction coupling. A point mutation within the tubulin-binding domain of EB1 reproduced EB1 displacement and diminished connexon delivery, confirming that EB1 displacement can limit gap junction coupling. In zebrafish hearts, oxidative stress also reduced the membrane localization of connexin and slowed the spatial spread of excitation. We anticipate that protecting the microtubule-based forward delivery apparatus of connexons could improve cell-cell coupling and reduce ischemia-related cardiac arrhythmias.

BIN1 localizes the L-type calcium channel to cardiac T-tubules.

Cardiac tubular-like membrane invaginations contain the membrane scaffolding protein BIN1, which tethers dynamic microtubules that deliver calcium channels directly to T-tubule membrane.

Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific

Commercial antibodies are used widely to quantify and localize the α1-adrenergic receptor (AR) subtypes, α1A, α1B, and α1D. We tested ten antibodies, from abcam and Santa Cruz, using western blot with heart and brain tissue from wild-type (WT) mice and mice with systemic knockout (KO) of one or all three subtypes. We found that none of the antibodies detected a band in WT that was absent in the appropriate KO or in the KO that was null for all α1-ARs (ABDKO). We conclude that the antibodies we tested are not specific for α1-ARs. These results raise caution with prior studies using these reagents. For now, competition radioligand binding is the only reliable approach to quantify the α1-AR subtype proteins. Receptor protein localization remains a challenge.

BIN1 is reduced and Cav1.2 trafficking is impaired in human failing cardiomyocytes

BACKGROUND Heart failure is a growing epidemic, and a typical aspect of heart failure pathophysiology is altered calcium transients. Normal cardiac calcium transients are initiated by Cav1.2 channels at cardiac T tubules. Bridging integrator 1 (BIN1) is a membrane scaffolding protein that causes Cav1.2 to traffic to T tubules in healthy hearts. The mechanisms of Cav1.2 trafficking in heart failure are not known. OBJECTIVE To study BIN1 expression and its effect on Cav1.2 trafficking in failing hearts. METHODS Intact myocardium and freshly isolated cardiomyocytes from nonfailing and end-stage failing human hearts were used to study BIN1 expression and Cav1.2 localization. To confirm Cav1.2 surface expression dependence on BIN1, patch-clamp recordings were performed of Cav1.2 current in cell lines with and without trafficking-competent BIN1. Also, in adult mouse cardiomyocytes, surface Cav1.2 and calcium transients were studied after small hairpin RNA-mediated knockdown of BIN1. For a functional readout in intact heart, calcium transients and cardiac contractility were analyzed in a zebrafish model with morpholino-mediated knockdown of BIN1. RESULTS BIN1 expression is significantly decreased in failing cardiomyocytes at both mRNA (30% down) and protein (36% down) levels. Peripheral Cav1.2 is reduced to 42% by imaging, and a biochemical T-tubule fraction of Cav1.2 is reduced to 68%. The total calcium current is reduced to 41% in a cell line expressing a nontrafficking BIN1 mutant. In mouse cardiomyocytes, BIN1 knockdown decreases surface Cav1.2 and impairs calcium transients. In zebrafish hearts, BIN1 knockdown causes a 75% reduction in calcium transients and severe ventricular contractile dysfunction. CONCLUSIONS The data indicate that BIN1 is significantly reduced in human heart failure, and this reduction impairs Cav1.2 trafficking, calcium transients, and contractility.

Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure.

Epidemiology, management, and outcomes of sustained ventricular arrhythmias after continuous-flow left ventricular assist device implantation.

BACKGROUND Left ventricular assist devices (LVADs) are pivotal treatment options for patients with end-stage heart failure. Despite robust left ventricular unloading, the right ventricle remains unsupported and susceptible to hemodynamic perturbations from ventricular arrhythmias (VAs). Little is known about the epidemiology, management, resource use, and outcomes of sustained VAs in continuous-flow LVAD patients. METHODS We reviewed data from all consecutive patients receiving a continuous-flow LVAD at the University of North Carolina from January 2006 to February 2011. Patient demographics, pharmacotherapies, resource use, and outcomes were recorded. Descriptive statistics were generated, and multivariable logistic regression was used to assess the independent association of clinical variables on the development of postimplantation VAs. RESULTS Of 61 patients, 26 (43%) had sustained VAs after LVAD. Most were male (65%), had history of hypertension (65%), and had nonischemic cardiomyopathy (62%). Patients with VAs after LVAD more often had preimplant VAs (62% vs 14%, P < .01), prior implantable cardioverter-defibrillator (92% vs 71%, P = .04), and history of implantable cardioverter-defibrillator discharge (38% vs 11%, P < .01). Although length of stay was similar, those with postimplant VAs had greater rehospitalization rates, greater antiarrhythmic drug use, and frequently required external defibrillation. Using multivariable logistic regression, only history of prior VA was associated with postimplant arrhythmias (odds ratio 13.7, P < .001). CONCLUSIONS Ventricular arrhythmias in LVAD patients are common, often refractory to conservative therapy, and associated with frequent rehospitalization. Post-LVAD VAs, however, did not significantly impact survival or transplantation rates. Arrhythmia burden should be considered before LVAD placement, and future study should focus on the impact of VAs on quality of life.

Cardiac toxicity after radiotherapy for stage III non-small-cell lung cancer: Pooled analysis of dose-escalation trials delivering 70 to 90 Gy

Purpose The significance of radiotherapy (RT) -associated cardiac injury for stage III non-small-cell lung cancer (NSCLC) is unclear, but higher heart doses were associated with worse overall survival in the Radiation Therapy Oncology Group (RTOG) 0617 study. We assessed the impact of heart dose in patients treated at our institution on several prospective dose-escalation trials. Patients and Methods From 1996 to 2009, 127 patients with stage III NSCLC (Eastern Cooperative Oncology Group performance status, 0 to 1) received dose-escalated RT to 70 to 90 Gy (median, 74 Gy) in six trials. RT plans and cardiac doses were reviewed. Records were reviewed for the primary end point: symptomatic cardiac events (symptomatic pericardial effusion, acute coronary syndrome, pericarditis, significant arrhythmia, and heart failure). Cardiac risk was assessed by noting baseline coronary artery disease and calculating the WHO/International Society of Hypertension score. Competing risks analysis was used. Results In all, 112 patients were analyzed. Median follow-up for surviving patients was 8.8 years. Twenty-six patients (23%) had one or more events at a median of 26 months to first event (effusion [n = 7], myocardial infarction [n = 5], unstable angina [n = 3], pericarditis [n = 2], arrhythmia [n = 12], and heart failure [n = 1]). Heart doses (eg, heart mean dose; hazard ratio, 1.03/Gy; P = .002,), coronary artery disease ( P < .001), and WHO/International Society of Hypertension score ( P = .04) were associated with events on univariable analysis. Heart doses remained significant on multivariable analysis that accounted for baseline risk. Two-year competing risk-adjusted event rates for patients with heart mean dose < 10 Gy, 10 to 20 Gy, or ≥ 20 Gy were 4%, 7%, and 21%, respectively. Heart doses were not associated with overall survival. Conclusion Cardiac events were relatively common after high-dose thoracic RT and were independently associated with both heart dose and baseline cardiac risk. RT-associated cardiac toxicity after treatment of stage III NSCLC may occur earlier than historically understood, and heart doses should be minimized.

α1-Adrenergic Receptor Subtypes in Nonfailing and Failing Human Myocardium

Background—&agr;1-adrenergic receptors (&agr;1-ARs) play adaptive roles in the heart and protect against the development of heart failure. The 3 &agr;1-AR subtypes, &agr;1A, &agr;1B, and &agr;1D, have distinct physiological roles in mouse heart, but very little is known about &agr;1 subtypes in human heart. Here, we test the hypothesis that the &agr;1A and &agr;1B subtypes are present in human myocardium, similar to the mouse, and are not downregulated in heart failure. Methods and Results—Hearts from transplant recipients and unused donors were failing (n=12; mean ejection fraction, 24%) or nonfailing (n=9; mean ejection fraction, 59%) and similar in age (≈44 years) and sex (≈70% male). We measured the &agr;1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse-transcription polymerase chain reaction and radioligand binding. All 3 &agr;1-AR subtype mRNAs were present, and &agr;1A mRNA was most abundant (≈65% of total &agr;1-AR mRNA). However, only &agr;1A and &agr;1B binding were present, and the &agr;1B was most abundant (60% of total). In failing hearts, &agr;1A and &agr;1B binding was not downregulated, in contrast with &bgr;1-ARs. Conclusions—Our data show for the first time that the &agr;1A and &agr;1B subtypes are both present in human myocardium, but &agr;1D binding is not, and the &agr;1 subtypes are not downregulated in heart failure. Because &agr;1 subtypes in the human heart are similar to those in the mouse, where adaptive and protective effects of &agr;1 subtypes are most convincing, it might become feasible to treat heart failure with a drug targeting the &agr;1A and/or &agr;1B.

The Alpha-1D Is the Predominant Alpha-1-Adrenergic Receptor Subtype in Human Epicardial Coronary Arteries

OBJECTIVES The goal was to identify alpha-1-adrenergic receptor (AR) subtypes in human coronary arteries. BACKGROUND The alpha1-ARs regulate human coronary blood flow. The alpha1-ARs exist as 3 molecular subtypes, alpha1A, alpha1B, and alpha1D, and the alpha1D subtype mediates coronary vasoconstriction in the mouse. However, the alpha1A is thought to be the only subtype in human coronary arteries. METHODS We obtained human epicardial coronary arteries and left ventricular (LV) myocardium from 19 transplant recipients and 6 unused donors (age 19 to 70 years; 68% male; 32% with coronary artery disease). We cultured coronary rings and human coronary smooth muscle cells. We assayed alpha1- and beta-AR subtype messenger ribonucleic acid (mRNA) by quantitative real-time reverse transcription polymerase chain reaction and subtype proteins by radioligand binding and extracellular signal-regulated kinase (ERK) activation. RESULTS The alpha1D subtype was 85% of total coronary alpha1-AR mRNA and 75% of total alpha1-AR protein, and alpha1D stimulation activated ERK. In contrast, the alpha1D was low in LV myocardium. Total coronary alpha1-AR levels were one-third of beta-ARs, which were 99% the beta2 subtype. CONCLUSIONS The alpha1D subtype is predominant and functional in human epicardial coronary arteries, whereas the alpha1A and alpha1B are present at very low levels. This distribution is similar to the mouse, where myocardial alpha1A- and alpha1B-ARs mediate beneficial functional responses and coronary alpha1Ds mediate vasoconstriction. Thus, alpha1D-selective antagonists might mediate coronary vasodilation, without the negative cardiac effects of nonselective alpha1-AR antagonists in current use. Furthermore, it could be possible to selectively activate beneficial myocardial alpha1A- and/or alpha1B-AR signaling without causing coronary vasoconstriction.