Thomas Dieterle

Chronic phospholamban inhibition prevents progressive cardiac dysfunction and pathological remodeling after infarction in rats

Ablation or inhibition of phospholamban (PLN) has favorable effects in several genetic murine dilated cardiomyopathies, and we showed previously that a pseudophosphorylated form of PLN mutant (S16EPLN) successfully prevented progressive heart failure in cardiomyopathic hamsters. In this study, the effects of PLN inhibition were examined in rats with heart failure after myocardial infarction (MI), a model of acquired disease. S16EPLN was delivered into failing hearts 5 weeks after MI by transcoronary gene transfer using a recombinant adeno-associated virus (rAAV) vector. In treated (MI-S16EPLN, n = 16) and control (MI-saline, n = 18) groups, infarct sizes were closely matched and the left ventricle was similarly depressed and dilated before gene transfer. At 2 and 6 months after gene transfer, MI-S16EPLN rats showed an increase in left ventricular (LV) ejection fraction and a much smaller rise in LV end-diastolic volume, compared with progressive deterioration of LV size and function in MI-saline rats. Hemodynamic measurements at 6 months showed lower LV end-diastolic pressures, with enhanced LV function (contractility and relaxation), lowered LV mass and myocyte size, and less fibrosis in MI-S16EPLN rats. Thus, PLN inhibition by in vivo rAAV gene transfer is an effective strategy for the chronic treatment of an acquired form of established heart failure.

Myeloid Differentiation Factor-88/Interleukin-1 Signaling Controls Cardiac Fibrosis and Heart Failure Progression in Inflammatory Dilated Cardiomyopathy

Rationale: The myeloid differentiation factor (MyD)88/interleukin (IL)-1 axis activates self–antigen-presenting cells and promotes autoreactive CD4+ T-cell expansion in experimental autoimmune myocarditis, a mouse model of inflammatory heart disease. Objective: The aim of this study was to determine the role of MyD88 and IL-1 in the progression of acute myocarditis to an end-stage heart failure. Methods and Results: Using α-myosin heavy chain peptide (MyHC-α)–loaded, activated dendritic cells, we induced myocarditis in wild-type and MyD88−/− mice with similar distributions of heart-infiltrating cell subsets and comparable CD4+ T-cell responses. Injection of complete Freunds adjuvant (CFA) or MyHC-α/CFA into diseased mice promoted cardiac fibrosis, induced ventricular dilation, and impaired heart function in wild-type but not in MyD88−/− mice. Experiments with chimeric mice confirmed the bone marrow origin of the fibroblasts replacing inflammatory infiltrates and showed that MyD88 and IL-1 receptor type I signaling on bone marrow–derived cells was critical for development of cardiac fibrosis during progression to heart failure. Conclusions: Our findings indicate a critical role of MyD88/IL-1 signaling in the bone marrow compartment in postinflammatory cardiac fibrosis and heart failure and point to novel therapeutic strategies against inflammatory cardiomyopathy.

Long-term outcome of fetal cell transplantation on postinfarction ventricular remodeling and function

OBJECTIVES The purpose of this study was to determine the long-term outcome of fetal cell transplantation into myocardial infarction on left ventricular (LV) function and remodeling. BACKGROUND While neonatal cell transplantation improved function for acute myocardial infarction, long-term data on the effects of cell-transplant therapy using a more primitive cell on ventricular remodeling and function are needed.Methods. - Therefore, we injected 4 x 10(6) Fischer 344 fetal cardiac cells or medium into 1-week old infarcts in adult female Fischer rats to assess long-term outcome. RESULTS Ten months after transplantation histologic analysis showed that cell implants were readily visible within the infarct scar. Infarct wall thickness was greater in cell-treated at 0.69 +/- 0.05 mm (n = 11) vs. medium-treated hearts at 0.33 +/- 0.01 mm (n = 19; P = 0.0001). Postmortem LV volume was 0.41 +/- 0.04 ml in cell-treated vs. 0.51 +/- 0.03 ml in medium-treated hearts (P < 0.04). Ejection fraction assessed by LV angiography was 0.40 +/- 0.02 in cell-treated (n = 16) vs. 0.33 +/- 0.02 in medium-treated hearts (n = 24; P < 0.03) with trends towards smaller in vivo end-diastolic and end-systolic volumes in cell-treated vs. medium-treated hearts. Polymerase chain reaction analysis of the Sry gene of the Y chromosome was positive in four of five cell-treated and zero of five medium-treated hearts confirming viability of male cells in female donors. CONCLUSION Over the course of 10 months, fetal cardiac cell transplantation into infarcted hearts increased infarct wall thickness, reduced LV dilatation, and improved LV ejection fraction. Thus, fetal cell-transplant therapy mitigated the longer-term adverse effects of LV remodeling following a myocardial infarction.

In vivo high-efficiency transcoronary gene delivery and Cre–LoxP gene switching in the adult mouse heart

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4±14.5% (mean±s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0±5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6±5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

Incidence, clinical predictors, and prognostic impact of worsening renal function in elderly patients with chronic heart failure on intensive medical therapy

BACKGROUND Incidence, predictors, and prognostic impact of worsening renal function (WRF) in elderly patients with chronic heart failure (HF) undergoing intensive contemporary medical therapy are unknown. METHODS AND RESULTS In 566 patients (age 77 ± 8 years) included in the TIME-CHF, serum creatinine (sCr) was repeatedly measured up to 6 months. Worsening renal function was classified as increase in sCr by 0.2 to 0.3 (WRFI), 0.3 to 0.5 (WRFII), or ≥0.5 mg/dL (WRFIII) within the first 6 months. Outcome events were assessed for 18 months. RESULTS The incidence of WRF I, II, and III was 12%, 19%, and 22%, respectively. Worsening renal function III was associated with increased mortality (hazard ratio 1.98 [95% CI 1.27-3.07, P = .002] vs no WRF), whereas WRF I/II was not. History of renal failure, spironolactone treatment, higher baseline dose, and higher maximal increase in loop diuretic dose were independently associated with the occurrence of WRF III, whereas angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, and β-blocker use and allocation to N-terminal pro-B-type natriuretic peptide-guided management were not. Worsening renal function III was an independent predictor of death, death or hospitalization, and death or HF hospitalization also after adjusting for baseline characteristics. CONCLUSIONS One fifth of elderly patients with chronic HF experienced WRF III on 6-month intensive HF treatment. These patients had higher mortality, whereas patients with smaller sCr rises did not. Occurrence of WRF III was associated with high doses of loop diuretics and spironolactone use but not with other treatments.

A recombinant antibody increases cardiac contractility by mimicking phospholamban phosphorylation

Many cardiovascular disease states end in progressive heart failure. Changes in intracellular calcium handling, including a reduced activity of the sarcoplasmic reticulum calcium pump (SERCA), contribute to this contractile dysfunction. As the regulatory protein phospholamban can inhibit the calcium pump, we evaluated it as a potential target to improve cardiac function. In this study, we describe a recombinant antibody‐based protein (PLN‐Ab) that binds to the cytoplasmic domain of phospholamban. Fluorescence resonance energy transfer (FRET) studies suggest that PLN‐Ab mimics the effects of phospholamban phosphorylation. PLN‐Ab accelerated the decay of the calcium transient when expressed in neonatal rat and adult mouse ventricular cardiac myocytes. In addition, direct injection of adenovirus encoding PLN‐Ab into the diabetic mouse heart enhanced contractility when measured in vivo by echo cardiography and in ex vivo Langendorff perfused hearts. The PLN‐Ab provides a novel therapeutic approach to improving contractility through in vivo expression of an antibody inside cardiac myocytes.

Effects of Losartan Titrated to Losartan/Hydrochlorothiazide and Amlodipine on Left Ventricular Mass in Patients with Mild-to-Moderate Hypertension

To study the effects of the angiotensin II receptor antagonist Losartan and Amlodipine on left ventricular mass (LVM), we performed blood pressure measurements and transthoracic echocardiographies at baseline. After a 4-week placebo run-in period, 25 patients with mild-to-moderate essential hypertension were randomly allocated to active treatment with Losartan 50 mg titrated to Losartan 50 mg/hydrochlorothiazide (HCT) 12.5 mg (n = 11) or Amlodipine 5 mg titrated to 10 mg (n = 14) for 16 weeks. After treatment, blood pressure decreased significantly in both groups. LVM and LVM index (mean ± SD/median) in the Losartan group at baseline were 311 ± 101/288 g and 163 ± 55/150 g/m2 and decreased significantly to 252 ± 25/255 g and 133 ± 22/128 g/m2 (p = 0.003 for LVM; p = 0.01 for LVM index) after 16 weeks of active treatment. In the Amlodipine group LVM and LVM index decreased from 259 ± 47/243 g and 136 ± 25/ 131 g/m2 to 240 ± 42/234 g and 126 ± 24/123 g/m2 (n.s.). In conclusion, LVM decreased significantly as early as 16 weeks after initiation of antihypertensive treatment with the Angiotensin II antagonist Losartan.

The Value of In Vitro Diagnostic Testing in Medical Practice: A Status Report

Background In vitro diagnostic (IVD) investigations are indispensable for routine patient management. Appropriate testing allows early-stage interventions, reducing late-stage healthcare expenditure (HCE). Aim To investigate HCE on IVDs in two developed markets and to assess the perceived value of IVDs on clinical decision-making. Physician-perceived HCE on IVD was evaluated, as well as desired features of new diagnostic markers. Methods Past and current HCE on IVD was calculated for the US and Germany. A total of 79 US/German oncologists and cardiologists were interviewed to assess the number of cases where: physicians ask for IVDs; IVDs are used for initial diagnosis, treatment monitoring, or post-treatment; and decision-making is based on an IVD test result. A sample of 201 US and German oncologists and cardiologists was questioned regarding the proportion of HCE they believed to be attributable to IVD testing. After disclosing the actual IVD HCE, the physician’s perception of the appropriateness of the amount was captured. Finally, the association between physician-rated impact of IVD on decision-making and perceived contribution of IVD expenditure on overall HCE was assessed. Results IVD costs account for 2.3% and 1.4% of total HCE in the US and Germany. Most physicians (81%) believed that the actual HCE on IVDs was >5%; 19% rated the spending correctly (0–4%, p<0.001). When informed of the actual amount, 64% of physicians rated this as appropriate (p<0.0001); 66% of decision-making was based on IVD. Significantly, more physicians asked for either additional clinical or combined clinical/health economic data than for the product (test/platform) alone (p<0.0001). Conclusions Our results indicate a poor awareness of actual HCE on IVD, but a high attributable value of diagnostic procedures for patient management. New markers should deliver actionable and medically relevant information, to guide decision-making and foster improved patient outcomes.

Prominin-1+/CD133+ bone marrow-derived heart-resident cells suppress experimental autoimmune myocarditis.

AIMS Experimental autoimmune myocarditis (EAM) is a CD4(+) T cell-mediated mouse model of inflammatory heart disease. Tissue-resident bone marrow-derived cells adopt different cellular phenotypes depending on the local milieu. We expanded a specific population of bone marrow-derived prominin-1-expressing progenitor cells (PPC) from healthy heart tissue, analysed their plasticity, and evaluated their capacity to protect mice from EAM and heart failure. METHODS AND RESULTS PPC were expanded from healthy mouse hearts. Analysis of CD45.1/CD45.2 chimera mice confirmed bone marrow origin of PPC. Depending on in vitro culture conditions, PPC differentiated into macrophages, dendritic cells, or cardiomyocyte-like cells. In vivo, PPC acquired a cardiac phenotype after direct injection into healthy hearts. Intravenous injection of PPC into myosin alpha heavy chain/complete Freunds adjuvant (MyHC-alpha/CFA)-immunized BALB/c mice resulted in heart-specific homing and differentiation into the macrophage phenotype. Histology revealed reduced severity scores for PPC-treated mice compared with control animals [treated with phosphate-buffered saline (PBS) or crude bone marrow at day 21 after MyHC-alpha/CFA immunization]. Echocardiography showed preserved fractional shortening and velocity of circumferential shortening in PPC but not PBS-treated MyHC-alpha/CFA-immunized mice. In vitro and in vivo data suggested that interferon-gamma signalling on PPC was critical for nitric oxide-mediated suppression of heart-specific CD4(+) T cells. Accordingly, PPC from interferon-gamma receptor-deficient mice failed to protect MyHC-alpha/CFA-immunized mice from EAM. CONCLUSION Prominin-1-expressing, heart-resident, bone marrow-derived cells combine high plasticity, T cell-suppressing capacity, and anti-inflammatory in vivo effects.

Immediate and Sustained Blood Pressure Lowering by Urocortin 2. A Novel Approach to Antihypertensive Therapy

Recently, novel corticotropin-releasing factor-related peptides, named urocortin 1, 2, and 3, and a distinct cardiac and peripheral vascular receptor (corticotropin-releasing factor receptor 2) were described being part of a peripheral corticotropin-releasing factor system modulating cardiovascular function in response to stress. Vasorelaxation and blood pressure lowering have been reported after acute administration of these peptides. No data are available on the acute and chronic effects of urocortin 2 on blood pressure in models of arterial hypertension. To test these effects, hypertensive salt-sensitive and normotensive salt-resistant Dahl rats were randomly assigned to twice-daily applications of urocortin 2 or vehicle for 5 weeks. Blood pressure, heart rate, and left ventricular dimension and function were recorded at baseline, after initial application, and, together with cardiac and aortic expression of urocortin 2 and its receptor, after 5 weeks of treatment. Urocortin 2 significantly reduced blood pressure in hypertensive rats without affecting heart rate. Long-term urocortin 2 treatment in hypertensive rats induced sustained blood pressure reduction and diminished the development of hypertension-induced left ventricular hypertrophy and the deterioration of left ventricular contractile function. Corticotropin-releasing factor receptor 2 expression was preserved despite chronic stimulation by urocortin 2. In conclusion, our study shows that, in an animal model of arterial hypertension, urocortin 2 has immediate and sustained blood pressure–lowering effects. Beneficial effects on blood pressure, left ventricular dimension, and function, together with preserved receptor expression, suggest that corticotropin-releasing factor receptor 2 stimulation by urocortin 2 may represent a novel approach to the treatment of arterial hypertension.