Pål Aukrust

Introduction: Pulmonary arterial hypertension (PAH) is characterised by increased proliferation of smooth muscle cells in small pulmonary arteries, increased pulmonary vascular resistance and right heart failure, which may lead to death. The Notch signalling system, which consists of four Notch receptors (Notch 1–4) and five ligands (Jagged 1 and 2, Delta-like ligand 1, 3 and 4) promotes the development of PAH experimentally and has been shown to be activated in clinical PAH. Circulating Delta-like Notch ligand 1 (DLL1) is elevated in chronic heart failure, associated with disease severity and adverse outcome, but little is known about circulating DLL1 in PAH.

Purpose: Systemic inflammation in established HF is clinically relevant as many HF patients have concomitant chronic inflammatory conditions. Repetitive activation of Toll-like receptor 9 (TLR9) induces chronic inflammation. We used a pharmacological protocol to induce systemic, chronic inflammation in murine cardiomyopathy induced by cardiomyocyte specific deletion of SERCA2a. Methods: Cardiac expression of SERCA2a was deleted in 10 week old male αMHC-MerCreMer Serca2a flox/flox mice by one intraperitoneal injection of Tamoxifen, resulting in a lethal diastolic HF phenotype after 7 weeks. 4 weeks after conditional knock out (KO), mice were randomized to TLR9 agonist (CpG-B;50 mg i.p.) or PBS every 3 days. Echocardiography was performed 8 weeks after induction of HF, followed by euthanization and analyses of tissue and blood. The groups were followed for 12 weeks, registering death and morbidity (by pre-specified criteria). Non-HF control groups treated with CpG-B or PBS served as controls. Results: TLR9 stimulated mice showed alterations in levels of leukocytes, erythrocytes and platelets as measured by flow cytometry and peripheral blood count, as well as pulmonary inflammation and hypersplenism (histology). The alterations were not found in controls. Echocardiographic analyses revealed aggravation in diastolic parameters in HF mice treated with CpG B. The latter group had reduced life expectancy compared with HF controls (median 59.5 and 68.5 days respectively, p=0.003). Non-HF, CpG B treated mice did not die or display signs of disease within the observation period.

Purpose: Immune activation plays a nodal role in the pathogenesis of heart failure (HF) and Toll-like receptor 9 (TLR9) signaling significantly impacts the development of systolic HF. However, the consequence of altered TLR9 activity in diastolic HF is unknown. Methods: We engaged in a 3-generation breeding strategy using αMHC-MerCreMer Serca2a flox/flox mice crossed with TLR9-/- mice to generate several comparable mouse lines. Employing these various genetic mouse lines we addressed the importance of TLR9 on progression of diastolic HF, the latter by temporally restricted depletion of cardiac myocyte SERCA2a. Mice were allocated to two substudies; 1) a 12 week survival study registering spontaneous death or signs of severe morbidity according to pre-specified criteria (leading to euthanasia) and 2) a study examining mice at baseline, 3 weeks and 6 weeks by MRI and echocardiography. After imaging at 6 weeks mice were euthanized and tissue and blood were harvested. Results: All mice depleted of cardiomyocyte SERCA2a, but none with the SERCA2a gene intact, reached our pre-specified end-parameter within 73 days. The presence of TLR9 in this model of diastolic HF led to significant improvement in survival with a median life expectancy of 62.5 days as compared to 58 days when the TLR9 gene was deleted (p=0.007). In accordance with these findings serial imaging demonstrated an earlier onset of left ventricular restrictive filling abnormalities in the HF group depleted of TLR9. Significantly larger left atria, lower end-systolic and end-diastolic left ventricular volumes and lower stroke volumes could be detected.