Human LV Research Articles

HDAC4 controls histone methylation in response to elevated cardiac load

In patients with heart failure, reactivation of a fetal gene program, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), is a hallmark for maladaptive remodeling of the LV. The mechanisms that regulate this reactivation are incompletely understood. Histone acetylation and methylation affect the conformation of chromatin, which in turn governs the accessibility of DNA for transcription factors. Using human LV myocardium, we found that, despite nuclear export of histone deacetylase 4 (HDAC4), upregulation of ANP and BNP in failing hearts did not require increased histone acetylation in the promoter regions of these genes. In contrast, di- and trimethylation of lysine 9 of histone 3 (H3K9) and binding of heterochromatin protein 1 (HP1) in the promoter regions of these genes were substantially reduced. In isolated working murine hearts, an acute increase of cardiac preload induced HDAC4 nuclear export, H3K9 demethylation, HP1 dissociation from the promoter region, and activation of the ANP gene. These processes were reversed in hearts with myocyte-specific deletion of Hdac4. We conclude that HDAC4 plays a central role for rapid modifications of histone methylation in response to variations in cardiac load and may represent a target for pharmacological interventions to prevent maladaptive remodeling in patients with heart failure.

The Homeostatic Chemokine CCL21 Predicts Mortality and May Play a Pathogenic Role in Heart Failure

BackgroundCCL19 and CCL21, acting through CCR7, are termed homeostatic chemokines. Based on their role in concerting immunological responses and their proposed involvement in tissue remodeling, we hypothesized that these chemokines could play a pathogenic role in heart failure (HF).Methodology/Principal FindingsOur main findings were: (i) Serum levels of CCL19 and particularly CCL21 were markedly raised in patients with chronic HF (n = 150) as compared with healthy controls (n = 20). A CCL21 level above median was independently associated with all-cause mortality. (ii) In patients with HF following acute myocardial infarction (MI; n = 232), high versus low CCL21 levels 1 month post-MI were associated with cardiovascular mortality, even after adjustment for established risk factors. (iii). Explanted failing human LV tissue (n = 29) had markedly increased expression of CCL21 as compared with non-failing myocardium (n = 5). (iv) Our studies in CCR7−/− mice showed improved survival and attenuated increase in markers of myocardial dysfunction and wall stress in post-MI HF after 1 week, accompanied by increased myocardial expression of markers of regulatory T cells. (v) Six weeks post-MI, there was an increase in markers of myocardial dysfunction and wall stress in CCR7 deficient mice.Conclusions/SignificanceHigh serum levels of CCL21 are independently associated with mortality in chronic and acute post-MI HF. Our findings in CCR7 deficient mice may suggest that CCL21 is not only a marker, but also a mediator of myocardial failure. However, while short term inhibition of CCR7 may be beneficial following MI, a total lack of CCR7 during long-term follow-up could be harmful.

Lymphocyte Levels of GRK2 (βARK1) Mirror Changes in the LVAD-Supported Failing Human Heart: Lower GRK2 Associated With Improved β-Adrenergic Signaling After Mechanical Unloading

Background In human heart failure, increased expression of G protein–coupled receptor kinases (GRKs) causes the loss of β-adrenergic receptor (βAR) signaling and function. Mechanical unloading with a left ventricular assist device (LVAD) promotes reverse remodeling, which includes restoration of βAR responsiveness. We tested the hypothesis that LVAD support of the failing human heart alters the expression and activity of GRKs and we sought to determine whether changes in myocardial GRKs could be tracked in lymphocytes. Methods and Results Paired samples of human LV tissue (n = 12) and blood were obtained at the time of LVAD implantation (heart failure) and subsequent cardiac transplantation (LVAD). βAR signaling was quantified by receptor density and adenylyl cyclase activity. Immunoblotting and real-time reverse transcription polymerase chain reaction were used to measure GRK2 and GRK5 protein and mRNA levels. Rhodopsin phosphorylation was used to assess total GRK activity. Consistent with reverse remodeling, βAR density and signaling were restored to nonfailing levels after LVAD support. GRK2 protein levels were significantly reduced 55% after LVAD support and GRK2 mRNA was similarly reduced. In contrast, GRK5 protein and mRNA levels were unchanged. Total myocardial GRK activity was reduced similar to the drop in GRK2 expression. In lymphocytes, GRK2 protein levels were decreased after LVAD support and there was a significant positive correlation between myocardial and lymphocyte GRK2 levels in both heart failure and LVAD samples. Conclusion The changes in myocardial GRK2 expression and activity that are mirrored in lymphocytes provide a possible mechanism for the restoration of βAR signaling and reverse remodeling after mechanical unloading in the failing heart. Moreover, lymphocytes may provide a surrogate marker of myocardial GRK2 in these patients.

Pharmacologically regulated Fas-mediated death of adoptively transferred T cells in a nonhuman primate model

AbstractConditional suicide genes derived from pathogens have been developed to confer drug sensitivity and enhance safety of cell therapy, but this approach is limited by immune responses to the transgene product. We examined a strategy to regulate survival of transferred cells based on induction of apoptosis through oligomerization of a modified human Fas receptor by a bivalent drug (AP1903). Three macaques (Macaca nemestrina) received autologous T cells retrovirally engineered to express a Fas suicide-construct (LV’VFas). High levels of transduced cells were present in blood following cell transfer, but LV’VFas+ cells declined rapidly after AP1903 administration. A small fraction of LV’VFas+ cells resisted elimination by AP1903, in part due to insufficient levels of transgene expression in resting T cells, because reactivation of these cells in vitro enhanced sensitivity to AP1903. An immune response to the transgene product was observed, but epitope mapping indicated the response was directed to discrete components of human LV’VFas that were variant with the corresponding macaque sequences. These data demonstrate that chemically induced dimerization can be used to regulate survival of adoptively transferred T cells in vivo.

Differential coupling of m-cholinoceptors to Gi/Go-proteins in failing human myocardium

Muscarinic acetylcholine receptors (mAChRs) mediate their main cardiac effects via pertussis toxin-sensitive G-proteins. Physiological effects differ considerably between atrium and ventricle, and it is unknown to which extent these differences derive from selective receptor–G-protein coupling or further downstream events. We have characterized specific coupling between mAChRs and Gi/Go-protein isoforms in atrial and ventricular myocardium by agonist-dependent photoaffinity labeling with [ 32P]azidoanilido GTP (aaGTP) and immunoprecipitation in sarcolemmal membranes from terminally failing human hearts. The total amount of mAChRs, as determined by specific binding of [ 3H]QNB, was significantly higher in right-atrial (RA ± SEM, 959 ± 68 fmol/mg, n = 4) than in left-ventricular membranes (LV, 582 ± 53 fmol/mg, n = 6). Standardized immunoblots revealed that Giα-2 was the predominant subtype in both regions. A 40-kDa splice variant of Goα (Goα-1 and/or Goα-3) was almost exclusively detectable in RA. Levels of Giα-3 and a 39-kDa splice variant of Goα (Goα-2) were also higher in RA. Basal aaGTP binding was higher in RA than in LV for all Giα/Goα subtypes. The carbachol (10 μmol/l)-induced increase in aaGTP binding was significantly higher in RA than in LV for Goα-1/3 (336 ± 95% of LV, n = 4) and for Giα-3 (211 ± 83%), lower for Giα-2 (42 ± 5%), and was similar in both regions for Goα-2 (130 ± 62%). The differential coupling of mAChRs in human RA and LV suggests that the initiation of different physiological responses to mAChR stimulation starts with signal sorting at the receptor–G-protein level.

Expression of ten RGS proteins in human myocardium: functional characterization of an upregulation of RGS4 in heart failure.

RGS proteins (regulators of G protein signalling) negatively regulate G protein function as GTPase activating proteins. By controlling heterotrimeric G proteins they may regulate myocardial hypertrophy and contractility. We investigated the expression of RGS proteins in the human heart and whether they take part in the pathophysiological changes of heart failure. Using RNase protection assays (RPAs) RGS2, 3L, 3S, 4, 5 and 6 were identified in the myocardium from terminally failing human hearts with dilated (DCM, n=22) or ischemic (ICM, n=18) cardiomyopathy and from nonfailing donor hearts (NF, n=9). With reverse transcriptase polymerase chain reaction in addition mRNA of RGS1, 9, 12, 14 and 16 were detectable. Compared to NF in failing LV myocardium RGS4 mRNA and protein was upregulated 2-3-fold (mRNA, 10(-21) mol/microg+/-S.E.M.: NF: 22+/-5, DCM: 51+/-10*, ICM: 37+/-8; P<0.05 vs. DCM+ICM, *P<0.05 vs. NF, P<0.05 vs. DCM+ICM; protein, % of NF+/-S.E.M.: NF: 100+/-35, DCM 266+/-60*, ICM: 205+/-64, n=5, *P<0.05 vs. NF). In contrast, RGS2, 3L, 3S, 5, 6, and 16 protein and mRNA levels did not vary between failing and NF hearts. In order to investigate the impact of RGS4 on Gq/11 mediated signalling, PLC activity was measured in human LV membranes. Recombinant RGS4 blunted the endothelin-1 (ET-1) stimulated PLC activity. When overexpressed by adenoviral mediated gene transfer in rabbit ventricular myocytes RGS4 abolished the inotropic effect of ET-1. The upregulation of RGS4 in failing human myocardium diminishes Gq/11-mediated signalling and can be involved in the desensitization of Gq/11-mediated positive inotropic effects.

Mechanical unloading restores beta-adrenergic responsiveness and reverses receptor downregulation in the failing human heart.

Mechanical unloading of the failing human heart with a left ventricular assist device (LVAD) results in clinically documented reversal of chamber dilation and improvement of cardiac function. We tested the hypothesis that LVAD support normalizes the ability of cardiac muscle to respond to sympathetic nervous system stimulation by reversing the downregulation of beta-adrenergic receptors. Human LV tissue was obtained from nonfailing hearts of unmatched organ donors and failing hearts at the time of transplantation, with or without LVAD. Baseline contractile parameters and inotropic response to a beta-adrenergic agonist were measured in isolated trabecular muscles. beta-Adrenergic receptor density was quantified by radioligand binding. Results showed a significant increase in the response to beta-adrenergic stimulation after LVAD (developed tension increased by 0.76+/-0.09 g/mm(2) in nonfailing, 0.38+/-0.07 in failing, and 0.68+/-0.10 in failing+LVAD; P<0.01), accompanied by an increased density of beta-adrenergic receptors (58.7+/-9.6 fmol/mg protein in nonfailing, 26.2+/-3.8 in failing, and 63.0+/-8.3 in failing+LVAD; P<0.05). These changes were unrelated to the duration of support. Data demonstrate that mechanically supporting the failing human heart with an LVAD can reverse the downregulation of beta-adrenergic receptors and restore the ability of cardiac muscle to respond to inotropic stimulation by the sympathetic nervous system. This indicates that functional impairment of cardiac muscle in human heart failure is reversible.

Evaluation of an economical sunlamp that emits a near solar UV power spectrum for conducting photoimmunological and sunscreen immune protection studies.

Expense and inconvenience have restricted the use of the filtered xenon are lamp (solar simulator) as a UV source for conducting large-scale animal studies. Because sunscreen immunoprotective levels are significantly affected by the UV power spectrum of the source it is imperative that a solar simulating source be used for accurate measurements of sunscreen protection levels that are relevant to human LV exposures from sunlight. However, relatively inexpensive sunlamps, e.g. the UVA-340, that emit a UV power spectrum similar to that of a solar simulator are available. Unlike FS-type UVB sunlamps, which have a significant amount of effective immunosuppressive non-solar UV energy at wavelengths below 295 nm, the immunosuppression effectiveness spectrum of UVA-340 sunlamps was nearly identical to that of a solar simulator. The purpose of this study was to evaluate this sunlamp for conducting photoimmunological and sunscreen immune protection studies. Groups of C3H mice were exposed to a range of UVA-340 sunlamp doses (0.25 kJ/m2 to 20.0 kJ/m2) to establish a dose-response curve and determine the minimum immune suppression dose (MISD) for iduction of local-type suppression of contact hypersensitivity (CH). The MISD, defined as the lowest UV dose given to produce approximately 50% suppression of the CH response in mice, was determined to be 1.0 kJ/m2 for UVA-340 sunlamps. Immune protection tests on four marketed sunscreen lotions (sun protection factors [SPF] 4, 8, 15 and 30) were then conducted with UVA-340 sunlamps using MISD as the endpoint. The immune protection factors for these sunscreens were equivalent to the level of protection predicted by their labeled SPF. These results are similar to those we have previously obtained using a solar simulator. We conclude from these data that the immunosuppressive effects of UVA-340 sunlamps are similar to those of a solar simulator; however, further studies are needed to determine if UVA-340, or similar, sunlamps are a viable alternative to the solar simulator for conducting large-scale animal experiments that require a relevant UV solar spectrum.

718-1 Effect of Muscarinic Stimulation on Isovolumic Relaxation in the Normal and Falling Human Left Ventricle

Myocardial muscarinic receptor stimulation attenuates the inotropic response to Ii-adrenergic receptor stimulation in the normal and failing human LV Its effect on isovolumic LV relaxation in humans is unknown. We investigated 12 patients with congestive heart failure (CHF) (EF 33 ± 3%1 and 6 patients with normal LV function (EF 71 ± 3%). Hemodynamic responses (Millar) were measured during intracoronary infusion of dobutamine (Dob) 25 /lg/min, acetylcholine (Ach) 1 /lM, and during the combination of Dob and Ach. There were no changes in HR during drug infusions. Dob had a positive inotropic effect. increasing LV +dP/dt by 662 ± 82 mmHg/s (p &lt; 0.016) in the normal group, and by 284 ± 37 mmHg/s (p &lt; 0.016) in the CHF group. Dob also had a positive lusitropic effect. increasing isovolumic LV relaxation and reducing LVEDP in both groups. During Ach there was no change in LV +dP/dt or LV systolic pressure in either group. Ach caused a small but consistent slowing of isovolumic LV relaxation in the CHF group, which was associated with an increase in LVEDP Ach had no effect on T1/2 or T L in the normal group. The combination of Ach and Dob attenuated the positive lusitropic response to Dob in both groups Can Dab Con-2 Ach Dob/Ach NL: LVEDP 7 ± 1 6 ± 1 * 7 ± 1 7 ± 1 5 ± 1 Tau 37 ± 3 32 ± 2 * 36 ± 2 35 ± 3 35 ± 3 CHF: LVEDP 18 ± 3 15 ± 3 * 18 ± 3 21 ± 3 * 20 ± 3 Tau 54 ± 4 45 ± 3 * 52 ± 2 56 ± 3 * 53 ± 3 LVEOP in mmHg: Tau. T L in msec * p &lt; 0.016. All data mean ± SEM This study demonstrates that muscarinic receptor stimulation antagonizes the positive lusitropic effect of adrenergic stimulation in both the normal and failing human LV Furthermore, in the failing human LV, muscarinic receptor stimulation has a primary negative lusitropic effect.