Distribution and function of sodium channel subtypes in human atrial myocardium.

Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary ß subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and ß subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where ß1 and ß3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with ß2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to ß3 and ß4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and ß subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation-contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".

Low-gradient aortic valve stenosis myocardial fibrosis and its influence on function and outcome.

OBJECTIVES: This prospective cohort study in patients with aortic stenosis (AS) aimed to identify surrogates of myocardial fibrosis that are easy to derive in clinical practice, allow the differentiation of low-gradient severe AS from moderate AS, and have an impact on clinical outcome. BACKGROUND: In patients with symptomatic aortic AS, a characteristic subgroup (i.e., up to one-third) exhibits severe AS with a concomitant low mean valve gradient either with preserved or reduced ejection fraction (EF). It is hypothesized that these patients tend to have an advanced stage of myocardial fibrosis and poor clinical outcome. METHODS: Eighty-six patients with moderate or severe AS were examined by echocardiography including conventional aortic valve assessment, mitral ring displacement, and strain-rate imaging. Replacement fibrosis was quantified by late-enhancement magnetic resonance imaging. Biopsy samples were taken from patients with severe AS (n = 69) at aortic valve replacement. All patients were followed for 9 months. RESULTS: Patients were divided into 4 groups according to aortic valve area (<1.0 cm(2)), mean valve gradient ≥40 mm Hg, and EF (<50%): group 1, moderate AS (n = 17); group 2, severe AS/high gradient (n = 49); group 3, severe AS/low gradient/preserved EF (n = 11); and group 4, severe AS/low gradient/decreased EF (n = 9). At baseline, a significant decrease in mitral ring displacement and systolic strain rate was detected in patients with low-gradient AS. In low-gradient groups, a higher degree of interstitial fibrosis in biopsy samples and more late-enhancement magnetic resonance imaging segments were observed. A close inverse correlation was found between interstitial fibrosis and mitral ring displacement (r = -0.79, p < 0.0001). Clinical outcome was best for patients in group 1, whereas mortality risk increased substantially in groups 2 through 4. CONCLUSIONS: In severe AS, a low gradient is associated with a higher degree of fibrosis, decreased longitudinal function, and poorer clinical outcome despite preserved EF. Mitral ring displacement differentiates between moderate AS and low-gradient/severe AS with preserved EF.

Donor antigen-specific regulatory T-cell function affects outcome in kidney transplant recipients.

Chronic transplant dysfunction, a major impediment to long-term allograft survival, is caused by several factors including an ongoing alloimmune response termed chronic rejection. To define some of these factors further, we selected 107 patients mismatched to their donors from 623 patients transplanted at a single center. Patients were categorized according to their immunosuppressive treatment and further divided into those with stable or chronic allograft dysfunction. Donor human lymphocyte antigen allopeptide-specific T-cell lines were then generated from stable patients and those with biopsy-proven chronic allograft nephropathy. Increased amounts of CD4+CD25+ regulatory T cells (Tregs) and Treg-associated gene expression profiles were found in cell lines derived from the patients with stable compared with those with chronic allograft dysfunction. Furthermore, a higher percentage of Tregs was found in patients with stable graft function on tacrolimus-based compared with cyclosporine-based immunosuppression protocols. Patients with stable graft function had a significantly higher expression of interleukin (IL)-4 and IL-10, whereas the cytokines IL-2, IL-17, and interferon-γ were significantly higher in patients with allograft dysfunction in vitro. Thus, enhancing the operational role of naturally occurring donor-specific Tregs in allograft recipients by adjusting the immunosuppression protocol may be advantageous particularly for patients with ongoing chronic rejection.

Inhibition of nuclear translocation of calcineurin suppresses T-cell activation and prevents acute rejection of donor hearts.

BACKGROUND: Inhibition of calcineurin (CnA) activity by cyclosporine A (CsA) is the mainstay in immunosuppressive therapy. CsA inhibits the phosphatase activity of the cytosolic phosphatase CnA and, therefore, prevents the dephosphorylation and subsequently nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT). However, CsA has multiple other targets within the cell and is, therefore, not specific. We developed a new approach to inhibit CnA/NFAT signaling. This synthetic peptide prevented CnA nuclear translocation in vitro. The purpose of this study was to demonstrate that this novel approach could potentially inhibit T-cell function in vitro and in vivo. METHODS: T-cell activation (Jurkat T cells, naïve rat T cells, and peripheral human T cells) was assessed by protein synthesis, interleukin (IL)-2 promoter activity, and IL-2 levels after T-cell activation. Immunohistological stainings for CnA were performed to investigate nuclear localization of CnA. The immunosuppressive effects in vivo of the synthetic peptide were investigated in rats with heterotopic transplanted hearts. RESULTS: The nuclear localization signal peptide significantly decreased alloantigen-specific T-lymphocyte proliferation, IL-2 promoter activity, and IL-2 production (338% ± 27% vs. 149% ± 11%, n=8, P<0.05) in cultured T cells by inhibition of CnA nuclear translocation. The synthetic peptide also significantly decreased the number of graft infiltrating CD8 T lymphocytes. Moreover, treatment with the synthetic inhibitory inhibited acute graft rejection (5 ± 0.6 days vs. 12 ± 2 days, n=10, P<0.05). CONCLUSIONS: Inhibition of nuclear translocation of CnA is a novel approach to inhibit the activation of the CnA/NFAT signaling cascade. Further studies have to demonstrate the long-term use of this principle in vivo.

Cardiac surgery and hematologic malignancies: a retrospective single-center analysis of 56 consecutive patients.

OBJECTIVE: Patients with a history of hematologic malignancies (HMs) are considered high-risk candidates for cardiac surgery. Increased perioperative rates of infections, thrombo-embolic complications, and bleeding disorders are reported. However, low patient numbers and lack of control groups limit all published studies. METHODS: A total of 56 patients with a history of HM underwent cardiac surgery. As many as 29 patients suffered from non-Hodgkin lymphoma, five from Hodgkin disease, and 12 from myeloproliferative disorders, one from acute lymphatic leukemia, and nine from monoclonal gammopathy. Surgery consisted of coronary artery bypass grafting, valvular surgery or combination procedures. HM patients were matched to 142 controls. Matching criteria applied consisted of sex, age, main diagnosis, and co-morbidities. RESULTS: In-hospital mortality was elevated in HM patients though not reaching significance (P = 0.7). HM patients demonstrated increased rates of vascular, pulmonary, infectious complications (P > 0.1), and transfusion requirements (P = 0.077). The long-term survival of HM patients was significantly impaired (P = 0.043). A history of irradiation or chemotherapy predisposed to postoperative respiratory insufficiency, acute renal failure, and an impaired long-term survival (P > 0.065). CONCLUSIONS: Cardiac surgery in patients with a history of a malignant hematologic disorder might achieve acceptable results. However, a higher complication and mortality rate have to be anticipated. Patients with hematologic disorders and a history of either irradiation or chemotherapy appear to be at an increased risk to develop postoperative end-organ failure.

Hemodynamic assessment of severe aortic stenosis: MRI evaluation of dynamic changes of vena contracta.

PURPOSE: Direct magnetic resonance imaging (MRI) planimetry of the maximal systolic aortic valve area does not consider temporal variations of the opening area during the ejection period. We evaluated an MRI-based methodology for the assessment of valvular dynamics in patients with severe aortic stenosis by measuring the systolic variability of the valvular blood stream, that is, the "vena contracta." MATERIALS AND METHODS: With institutional review board approval, we examined 22 patients (13 male, 9 female; mean age, 68 ±10 years) with severe aortic stenosis using 1.5 T MRI and a standardized scanning protocol consisting of gradient-echo phase-contrast velocity imaging and steady-state free precession-cine MRI before and after valve replacement therapy. Temporal changes of the aortic valve area, represented by systolic variations of the area of poststenotic turbulent flow at its smallest convergence, that is, the proximal vena contracta, were determined by MRI and quantified by a calculated parameter of temporal valve dynamics (T). T was defined as the period which the aortic valve spent over its maximal opening area (>85%) during systole. MRI was also used to determine left ventricular hypertrophy before (LVMI) and its regression (LVMR) after valve replacement. Findings were compared with transthoracic echocardiography and cardiac catheterization. RESULTS: All patients had an echocardiographic effective orifice area, EOATTE, of <1.0 cm2. The comparison of T to LVMI and LVMR revealed significant correlations (LVMI: r = -0.62; P = 0.002; LVMR: r = 0.62; P = 0.002). Further significant correlations with aortic stenosis severity were observed in the comparison with manual planimetry, invasive measurements, and echocardiographic valve areas, as well as with pressure gradients. CONCLUSIONS: MRI can measure systolic variations of the aortic valve area. Quantitative parameters of the hemodynamic relevance of valve dynamics obtained by this method correlate with established parameters of aortic stenosis severity and LVMR.

Cardiac operations in the presence of meningioma.

BACKGROUND: We investigated the effect of concomitant intracranial meningiomas on perioperative and postoperative complications after cardiac operations. Also studied was the intraoperative and perioperative management and long-term outcome of such patients. METHODS: We retrospectively evaluated 16 cardiac surgical patients with intracranial meningiomas between January 1996 and July 2007. Neurologic outcome, incidence of transient neurologic deficits, and long-term follow-up focusing on freedom from any cardiac or neurosurgical intervention were assessed. RESULTS: Five men and 11 women with a concomitant diagnosis of intracranial meningioma underwent cardiac operations using extracorporeal circulation. One patient received additional edema prophylaxis by intravenous dexamethasone. All patients were discharged home in good physical condition. Data on long-term survival were available on 14 patients, with 12 alive. Postoperatively, 2 patients died from myocardial infarction at 26.8 months and 2 from metastatic colon cancer at 57.9 months. Perioperative neurologic disorders were observed in 2 patients, comprising one stroke after intervention for aortic dissection and one thromboembolic event 2 weeks after biologic mitral valve replacement due to anticoagulation disorders. No meningioma-related adverse event was observed. CONCLUSIONS: The presence of intracranial meningioma does not appear to be a risk factor for patients undergoing cardiac operations. No meningioma-related neurologic sequelae were documented postoperatively. Neurosurgical consultation should be obtained in all patients preoperatively.

Impact of myocardial fibrosis in patients with symptomatic severe aortic stenosis.

BACKGROUND: In this prospective follow-up study, the effect of myocardial fibrosis on myocardial performance in symptomatic severe aortic stenosis was investigated, and the impact of fibrosis on clinical outcome after aortic valve replacement (AVR) was estimated. METHODS AND RESULTS: Fifty-eight consecutive patients with isolated symptomatic severe aortic stenosis underwent extensive baseline characterization before AVR. Standard and tissue Doppler echocardiography and cardiac magnetic resonance imaging (late-enhancement imaging for replacement fibrosis) were performed at baseline and 9 months after AVR. Endomyocardial biopsies were obtained intraoperatively to determine the degree of myocardial fibrosis. Patients were analyzed according to the severity of interstitial fibrosis in cardiac biopsies (severe, n=21; mild, n=15; none, n=22). The extent of histologically determined cardiac fibrosis at baseline correlated closely with New York Heart Association functional class and markers of longitudinal systolic function (all P<0.001) but not global ejection fraction or aortic valve area. Nine months after AVR, the degree of late enhancement remained unchanged, implying that AVR failed to reduce the degree of replacement fibrosis. Patients with no fibrosis experienced a marked improvement in New York Heart Association class from 2.8+/-0.4 to 1.4+/-0.5 (P<0.001). Only parameters of longitudinal systolic function predicted this functional improvement. Four patients with severe fibrosis died during follow-up, but no patient from the other groups died. CONCLUSIONS: Myocardial fibrosis is an important morphological substrate of postoperative clinical outcome in patients with severe aortic stenosis and was not reversible after AVR over the 9 months of follow-up examined in this study. Because markers of longitudinal systolic function appear to indicate sensitively both the severity of myocardial fibrosis and the clinical outcome, they may prove valuable for preoperative risk assessment in patients with aortic stenosis.

Regulatory allospecific T cell clones abrogate chronic allograft rejection.

True alloantigen-specific tolerance is the ultimate goal of solid organ transplantation, eliminating the need for long-term immunosuppression. Recent evidence suggests that Th1-derived cytokines are associated with rejection and Th2-derived cytokines with long-term allograft survival, but the roles of these subsets in rejection and tolerance are incompletely understood. Here, we analyzed the functional and regulatory capacities of T cell clones derived from tolerant and rejecting rats (Wistar rat donors, Lewis rat recipients). We generated and subcloned T cell lines from lymphocytes derived from either acutely rejecting grafts or from the grafts of CTLA4-Ig-treated tolerant rats. Pretransplantation adoptive transfer of T cell clones generated from rejected grafts (Th1 clones) accelerated acute rejection or promoted development of chronic rejection, whereas transfer of T cell clones generated from tolerized grafts (Th2 clones) protected rats from acute rejection and progressive organ dysfunction. When Th1 and Th2 clones were injected simultaneously, Th2 clones specifically regulated activation of Th1 clones. Rats that received injections of Th2 clones accepted long-term donor-specific skin grafts but acutely rejected third-party skin grafts. Tolerant rats treated with Th2 clones demonstrated an increased number of regulatory CD4+CD25+Foxp3+ cells and strong mononuclear cell staining for IL-10 but negligible IFN-gamma, IL-17, and IL-23 compared with untreated rats or those treated with Th1 clones. In summary, these results demonstrate the regulatory functions of Th2 cells in a clinically relevant allogeneic transplant model and provide new insight into the functional role of Th2 cells in preventing the process of chronic rejection.

Heterotopic rat heart transplantation (Lewis to F344): early ICAM-1 expression after 8 hours of cold ischemia.

BACKGROUND: Primary graft dysfunction is a still poorly understood complication after cardiac transplantation. Ischemia/reperfusion injury contributes to different disorders resulting in impaired graft function. METHODS: In a heterotopic rat heart transplantation model we extended graft ischemic time up to 8 hours. RESULTS: Using immunohistochemistry we detected an up to 4-fold increase in intracellular adhesion molecule-1 (ICAM-1) expression during 4 hours of reperfusion, independent of ischemic time (30-minute ischemia: 7.65 +/- 2.15 without reperfusion, 19.46 +/- 4.6 after 4-hour reperfusion; 240-minute ischemia: 5.6 +/- 1.99 and 22.3 +/- 3.77; 480-minute ischemia: 3.7 +/- 1.56 and 13.1 +/- 2.2). Eight-hour ischemic allografts had an increase in CD8-positive cells (1.37 +/- 0.5 and 2.3 +/- 0.77) and a significant increase in MHC II expression (11.48 +/- 2.1 and 18.27 +/- 1.34) during 4 hours of reperfusion. CONCLUSIONS: We hypothesize that these findings reflect an early inflammatory reaction in the allograft possibly triggered by oxidative stress. During therapeutic interventions, both of these pathways must be considered.

Cardiac allograft vasculopathy after cardiac transplantation and hormone therapy: positive effects?

BACKGROUND: There is a great deal of controversy surrounding the issue of hormone replacement therapy after transplantation. The question whether or not this therapy has effects in cardiac allograft vasculopathy (CAV), the Achilles heel of cardiac transplantation or other unique aspects of allograft function is still unknown. METHODS: We investigated the long-term effect of 17beta-estradiol as well as phytoestrogen Coumestrol, a synthetically produced phytoestrogen, on the development of CAV and the degree of fibrosis in an ovariectomized female heterotopic chronic allograft model (LEW-F344). RESULTS: We found that, 150 days after transplantation, no significant effect of estrogen application on intimal thickening of coronary arteries was observed. 17beta-estradiol and phytoestrogen Coumestrol did significantly reduce the perivascular immune reaction. However, the immune effect had no consequence on the intensity of CAV. Although neither 17beta-estradiol nor phytoestrogen Coumestrol revealed a positive effect on CAV, the group of animals treated with 17beta-estradiol showed the highest decline in heart function and the most distinct fibrosis. CONCLUSIONS: 17beta-estradiol does not affect CAV positively, but worsens cardiac allograft function and leads to increased fibrosis. This is the first study showing a negative effect of 17-beta-estradiol after heart transplantation in the long term.

Formation of 4-hydroxy-2-nonenal protein adducts in the ischemic rat heart after transplantation.

BACKGROUND: Free radicals formed during ischemia and reperfusion can lead to lipid peroxidation (LPO) and the formation of 4-hydroxy-2-nonenal (4-HNE), one of the most toxic products of LPO. Using a heterotopic rat heart transplantation model we investigated endogenous 4-HNE formation as a response to cold storage of the transplant and warm blood reperfusion in the recipient. METHODS: Lewis rat hearts were subjected to 30, 240 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution without or with transplantation and 240-minute reperfusion in F344 recipients. The amount of 4-HNE modified proteins was quantified in rat heart cryosections with an antibody recognizing cysteine-, histidine- and lysine-4-HNE Michael adducts and image analysis of immunostained tissue. RESULTS: We detected 4-HNE-modified proteins in ischemic rat hearts after transplantation and reperfusion. In hearts submitted to ischemia only, 4-HNE-protein adducts comprised 0.7 +/- 0.3% (30 minutes), 0.7 +/- 0.4% (240 minutes) and 0.2 +/- 0.1% (480 minutes) (mean +/- SEM) of the tissue area. Transplantation and reperfusion in the recipient significantly increased the amount of protein adducts to 6.8 +/- 2.6% (p = 0.041), 5.2 +/- 1.4% (p = 0.009) and 5.7 +/- 0.9% (p = 0.002) in 30-, 240- and 480-minute ischemic hearts, respectively. CONCLUSIONS: Under the conditions applied in the present study, cold ischemia for >30 minutes did not significantly alter the amount of 4-HNE protein adducts. However, because after transplantation and reperfusion, 6% of heart tissue consisted of 4-HNE-modified proteins, it can be assumed that this damage negatively affects long-term survival of the transplant.

Heterotopic rat heart transplantation: severe loss of glutathione in 8-hour ischemic hearts.

BACKGROUND: Tissue damage caused by reactive oxygen species (ROS) formed during ischemia/reperfusion seems to be an important risk factor in the failure of transplanted hearts. Although endogenous anti-oxidants protect the myocardium against free radical attack under physiologic conditions, their capacity may become limited during severe oxidative stress. Thus, we investigated the effect of 8-hour cold ischemia on the myocardial anti-oxidative defense system in a heterotopic rat heart transplantation model. METHODS: Lewis rat hearts were subjected to 30 or 480 minutes of 4 degrees C cold ischemia in Bretschneider cardioplegic solution with or without transplantation and reperfusion (30 or 240 minutes) into F344 recipients. Activity levels of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione S-transferase (GST), and concentrations of glutathione (GSH), glutathione disulfide (GSSG) and lipid hydroperoxides (LOOH) were analyzed in heart homogenates. For histology, cross-sections of the ventricles were stained with hematoxylin-eosin. RESULTS: Except for GST, enzyme activities and GSSG concentration increased and the glutathione redox ratio (GSH/GSH + 2GSSG) significantly decreased in 480-minute ischemic hearts compared with those with 30-minute ischemia. Reperfusion dramatically decreased both GSH and GSSG and increased LOOH formation but without severe histopathologic findings in the transplants. Applying a tree-structured classifier technique, GSH and LOOH were identified as significant features indicative of transplantation-induced oxidative stress. CONCLUSIONS: In the present study severe loss of glutathione and formation of LOOH indicated transplantation-induced oxidative stress in the rat heart; therefore, alterations of these parameters may hint at relevant deficits in the myocardial anti-oxidative defense and may also predict subsequent tissue damage.