Sara Leite

Myocardial Titin Hypophosphorylation Importantly Contributes to Heart Failure With Preserved Ejection Fraction in a Rat Metabolic Risk Model

Background—Obesity and diabetes mellitus are important metabolic risk factors and frequent comorbidities in heart failure with preserved ejection fraction. They contribute to myocardial diastolic dysfunction (DD) through collagen deposition or titin modification. The relative importance for myocardial DD of collagen deposition and titin modification was investigated in obese, diabetic ZSF1 rats after heart failure with preserved ejection fraction development at 20 weeks. Methods and Results—Four groups of rats (Wistar-Kyoto, n=11; lean ZSF1, n=11; obese ZSF1, n=11, and obese ZSF1 with high-fat diet, n=11) were followed up for 20 weeks with repeat metabolic, renal, and echocardiographic evaluations and hemodynamically assessed at euthanization. Myocardial collagen, collagen cross-linking, titin isoforms, and phosphorylation were also determined. Resting tension (Fpassive)–sarcomere length relations were obtained in small muscle strips before and after KCl–KI treatment, which unanchors titin and allows contributions of titin and extracellular matrix to Fpassive to be discerned. At 20 weeks, the lean ZSF1 group was hypertensive, whereas both obese ZSF1 groups were hypertensive and diabetic. Only the obese ZSF1 groups had developed heart failure with preserved ejection fraction, which was evident from increased lung weight, preserved left ventricular ejection fraction, and left ventricular DD. The underlying myocardial DD was obvious from high muscle strip stiffness, which was largely (±80%) attributable to titin hypophosphorylation. The latter occurred specifically at the S3991 site of the elastic N2Bus segment and at the S12884 site of the PEVK segment. Conclusions—Obese ZSF1 rats developed heart failure with preserved ejection fraction during a 20-week time span. Titin hypophosphorylation importantly contributed to the underlying myocardial DD.

Afterload-induced diastolic dysfunction contributes to high filling pressures in experimental heart failure with preserved ejection fraction.

Myocardial stiffness and upward-shifted end-diastolic pressure-volume (P-V) relationship (EDPVR) are the key to high filling pressures in heart failure with preserved ejection fraction (HFpEF). Nevertheless, many patients may remain asymptomatic unless hemodynamic stress is imposed on the myocardium. Whether delayed relaxation induced by pressure challenge may contribute to high end-diastolic pressure (EDP) remains unsettled. Our aim was to assess the effect of suddenly imposed isovolumic afterload on relaxation and EDP, exploiting a highly controlled P-V experimental evaluation setup in the ZSF1 obese rat (ZSF1 Ob) model of HFpEF. Twenty-week-old ZSF1 Ob (n = 12), healthy Wistar-Kyoto rats (WKY, n = 11), and hypertensive ZSF1 lean control rats (ZSF1 Ln, n = 10) underwent open-thorax left ventricular (LV) P-V hemodynamic evaluation under anesthesia with sevoflurane. EDPVR was obtained by inferior vena cava occlusions to assess LV ED chamber stiffness constant β, and single-beat isovolumic afterload acquisitions were obtained by swift occlusions of the ascending aorta. ZSF1 Ob showed increased ED stiffness, delayed relaxation, as assessed by time constant of isovolumic relaxation (τ), and elevated EDP with normal ejection fraction. Isovolumic afterload increased EDP without concomitant changes in ED volume or heart rate. In isovolumic beats, relaxation was delayed to the extent that time for complete relaxation as predicted by 3.5 × monoexponentially derived τ (τexp) exceeded effective filling time. EDP elevation correlated with reduced time available to relax, which was the only independent predictor of EDP rise in multiple linear regression. Our results suggest that delayed relaxation during pressure challenge is an important contributor to lung congestion and effort intolerance in HFpEF.

Myocardial and anti‐inflammatory effects of chronic bosentan therapy in monocrotaline‐induced pulmonary hypertension

INTRODUCTION AND OBJECTIVES Endothelin-1 antagonists are increasingly used in the treatment of pulmonary hypertension despite the lack of knowledge of their myocardial and systemic effects. We assessed the right ventricular myocardial and systemic effects of endothelin-1 antagonists in monocrotaline-induced pulmonary hypertension. METHODS Male Wistar rats (180-200 g, n=57) randomly received 60 mg/kg monocrotaline or vehicle subcutaneously. Two days later, bosentan was randomly started (300 mg/kg/day) by oral route in a subgroup of monocrotaline-injected rats, while the other monocrotaline-injected and control rats received vehicle. At 25-30 days, invasive hemodynamic assessment was performed under anesthesia, arterial blood samples were collected for gas analysis and plasma was extracted for quantification of endothelin-1, cytokines, nitrates and 6-keto-prostaglandin F1α. Right ventricular myocardium was collected for assessment of cyclooxygenase and nitric oxide synthase activity and gene expression. RESULTS The monocrotaline group developed pulmonary hypertension, low cardiac output, right ventricular hypertrophy and dilation, changes in gene expression and inflammatory activation that were attenuated in the group treated with bosentan. From a functional point of view, this group had improved right ventricular function and preserved ventriculo-vascular coupling, without deterioration in arterial gas parameters or systemic hypotension. In molecular terms, they showed reduced endothelin-1 and cytokine levels, decreased right ventricular inducible nitric oxide synthase and cyclooxygenase-2 activity and increased nitrate plasma levels compared with the non-treated group. CONCLUSIONS In this study we demonstrate that besides attenuating pulmonary hypertension, bosentan has beneficial hemodynamic, myocardial and anti-inflammatory effects.

Right ventricular end-diastolic stiffness heralds right ventricular failure in monocrotaline-induced pulmonary hypertension

Recent studies suggest right ventricular (RV) stiffness is important in pulmonary hypertension (PH) prognosis. Smaller stroke volume (SV) variation after a certain RV end-diastolic pressure (EDP) respiratory variation as assessed by spectral transfer function (STF) may identify RV stiffness. Our aim was to evaluate RV stiffness in monocrotaline (MCT)-induced PH progression and to validate STF gain between EDP and SV as marker of stiffness. Seven-week-old male Wistar rats randomly injected with 60 mg/kg MCT or vehicle were divided into three groups (n = 12 each) according to cardiac index (CI): controls (Ctrl), preserved CI (MCT pCI), and reduced CI (MCT rCI). All underwent RV pressure-volume (PV) evaluation 24-34 days after MCT, under halogenate anesthesia and constant positive-pressure ventilation. End-diastolic stiffness (βi), end-systolic elastance (Eesi), arterial elastance for indexed volumes (Eai), and preload recruitable stroke work (PRSW) were obtained and beat-to-beat fluctuations during ventilation assessed by STF. Eai was the strongest determinant of CI, alongside βi but not PRSW. MCT rCI showed impaired ventricular-vascular coupling (VVC) and higher βi, along with low end-diastolic pressure (EDP) and stroke volume index (SVi) STF gain, denoting impaired preload reserve. On multivariate analysis βi and not Eesi correlated with EDP-SVi STF gain (P < 0.001). Receiver-operating characteristics (ROC) curve analysis of EDP-SVi STF gain showed an area under curve of 0.84 for βi prediction (P = 0.002). Afterload, impaired VVC and RV stiffness are major players in RV failure. RV stiffness can be assessed by STF gain analysis of respiratory fluctuations between EDP and SVi, which may constitute a prognostic tool in PH.

Spectral transfer function analysis of respiratory hemodynamic fluctuations predicts end-diastolic stiffness in preserved ejection fraction heart failure

Preserved ejection fraction heart failure (HFpEF) diagnosis remains controversial, and invasive left ventricular (LV) hemodynamic evaluation and/or exercise testing is advocated by many. The stiffer HFpEF myocardium may show impaired stroke volume (SV) variation induced by fluctuating LV filling pressure during ventilation. Our aim was to investigate spectral transfer function (STF) gain from end-diastolic pressure (EDP) to indexed SV (SVi) in experimental HFpEF. Eighteen-week-old Wistar-Kyoto (WKY) and ZSF1 lean (ZSF1 Ln) and obese rats (ZSF1 Ob) randomly underwent LV open-chest (OC, n = 8 each group) or closed-chest hemodynamic evaluation (CC, n = 6 each group) under halogenate anesthesia and positive-pressure ventilation at constant inspiratory pressure. Beat-to-beat fluctuations in hemodynamic parameters during ventilation were assessed by STF. End-diastolic stiffness (βi) and end-systolic elastance (Eesi) for indexed volumes were obtained by inferior vena cava occlusion in OC (multibeat) or single-beat method estimates in CC. ZSF1 Ob showed higher EDP spectrum (P < 0.001), higher STF gain between end-diastolic volume and EDP, and impaired STF gain between EDP and SVi compared with both hypertensive ZSF1 Ln and normotensive WKY controls (P < 0.001). Likewise βi was only higher in ZSF1 Ob while Eesi was raised in both ZSF1 groups. On multivariate analysis βi and not Eesi correlated with impaired STF gain from EDP to SVi (P < 0.001), and receiver-operating characteristics analysis showed an area under curve of 0.89 for higher βi prediction (P < 0.001). Results support further clinical testing of STF analysis from right heart catheterization-derived EDP surrogates to noninvasively determined SV as screening/diagnostic tool to assess myocardial stiffness in HFpEF.

Chronic exercise induces pathological left ventricular hypertrophy in adrenaline-deficient mice

Adrenaline-deficient phenylethanolamine-N-methyltransferase-knockout mice (Pnmt-KO) have concentric heart remodeling and though their resting blood pressure is normal, it becomes higher during acute exercise. The aim of this study was to evaluate cardiac morphological, functional and molecular alterations after chronic exercise in adrenaline-deficient mice. Genotypes at the Pnmt locus were verified by polymerase chain reaction (PCR) of ear samples of Pnmt-KO and wild-type (WT) mice. These mice were submitted to chronic exercise training during 6weeks. Blood pressure was determined by a photoelectric pulse detector. Mice were anesthetized and cardiac morphology and function were evaluated by echocardiography and hemodynamics. IGF-1, IGF-1R, ANP and BNP mRNA were quantified by real-time PCR in left ventricle (LV) samples. Pnmt-KO mice showed increased systolic blood pressure compared with WT mice. A significant increase was found in LV mass, and LV posterior wall thickness in trained Pnmt-KO compared to trained WT mice, without significant differences in LV volumes. Acute β1-adrenergic stimulation with dobutamine increased systolic function indexes in WT mice, but not in Pnmt-KO mice. LV expression of IGF-1 and ANP was increased in trained Pnmt-KO mice when compared to trained WT mice. In conclusion, in response to chronic exercise adrenaline-deficient Pnmt-KO mice show concentric LV hypertrophy and impaired response to dobutamine, suggesting an initial stage of pathological cardiac hypertrophic remodeling. These results support the need for an efficient partial conversion of noradrenaline into adrenaline for prevention of blood pressure overshoot and thus pathological cardiac hypertrophic remodeling in chronic exercise.

Characterization of liver changes in ZSF1 rats, an animal model of metabolic syndrome

BACKGROUND The non-alcoholic fatty liver disease is the hepatic counterpart of the metabolic syndrome. ZSF1 rats are a metabolic syndrome animal model in which liver changes have not been described yet. AIM The characterization of liver histological and innate immunity changes in ZSF1 rats. METHODS Five groups of rats were included (n = 7 each group): healthy Wistar-Kyoto control rats (Ctrl), hypertensive ZSF1 lean (Ln), ZSF1 obese rats with a normal diet (Ob), ZSF1 obese rates with a high-fat diet (Ob-HFD), and ZSF1 obese rats with low-intensity exercise training (Ob-Ex). The animals were sacrificed at 20 weeks of age, their livers were collected for: a) measurements of the area of steatosis, fibrosis and inflammation (histomorphological analysis); and b) innate immunity (toll-like receptor [TLR] 2, TLR4, peroxisome proliferator-activated receptor γ [PPARγ], toll interacting protein [TOLLIP]) and inflammatory marker (tumor necrosis factor-alpha [TNFα], interleukin 1 [IL-1]) expression analysis by real-time PCR. RESULTS Ob, Ob-HFD and Ob-Ex were significantly heavier than Ln and Ctrl animals. Ob, Ob-HFD and Ob-Ex animals had impaired glucose tolerance and insulin resistance. ZSF1 Ob, Ob-HFD and Ob-Ex presented a higher degree of steatosis (3,5x; p < 0.05) than Ctrl or ZSF1 Ln rats. Steatohepatitis and fibrosis were not observed in any of the groups. No differences in expression were observed between Ctrl, Ln and Ob animals (except for the significantly higher expression of TOLLIP observed in the Ob vs Ln comparison). Ob-HFD and Ob-Ex rats showed increased expression of PPARγ and TOLLIP as compared to other groups. However, both groups also showed increased expression of TLR2 and TLR4. Nevertheless, this did not translate into a differential expression of TNFα or IL-1 in any of the groups. CONCLUSION The ZSF1 model is associated with liver steatosis but not with steatohepatitis or a significantly increased expression of innate immunity or inflammation markers.

[PP.15.22] EPINEPHRINE IS REQUIRED FOR PREVENTING BLOOD PRESSURE OVERSHOOT AND THUS CARDIAC HYPERTROPHY IN CHRONIC EXERCISE

Objective: Phenylethanolamine-N-methyltransferase-knockout (Pnmt-KO) mice are epinephrine-deficient and appear to have concentric heart remodelling. Although Pnmt-KO mice resting blood pressure is normal, it becomes higher than wild type mice during acute treadmill exercise. However, the role of epinephrine in cardiovascular response to chronic exercise remains unclear. Therefore, the aim of this study was to evaluate heart morphological, functional and molecular alterations after chronic exercise in epinephrine-deficient mice. Design and method: PCR-based genotyping was performed at the Pnmt locus of Pnmt-KO (Pnmt-/-) and wild-type (WT) mice (Pnmt+/+) (129x1/SvJ). Epinephrine and norepinephrine were quantified by RP-HPLC-ED, in adrenal glands. Animals were submitted to a 6-week chronic exercise training program performed on a motor treadmill until 20 m/min, for 55 minutes, 5 days per week. Blood pressure was determined by a photoelectric pulse detector after treadmill exercise, at rest. Mice were anaesthetized and cardiac morphology and function were evaluated by echocardiography and hemodynamics. Molecular markers of cardiac hypertrophy were evaluated by real-time PCR. Results: Systolic blood pressure was significantly increased in Pnmt-KO when compared to WT mice. A significant increase was found in left ventricular posterior wall thickness and mass in trained Pnmt-KO compared to trained WT mice, without significant differences in LV volume. Compared to basal parameters, acute &bgr;1-adrenergic stimulation with dobutamine increased cardiac index in trained WT mice, contrary to trained Pnmt-KO mice. In the left ventricle, mRNA expression of ANP and IGF-1 were significantly increased in trained Pnmt-KO mice when compared to trained WT mice. Conclusions: In conclusion, increased blood pressure overshoot in response to exercise appears to be associated with an increase in left ventricular posterior wall thickness and mass in chronic exercise, suggesting a concentric hypertrophy of the left ventricle in trained Pnmt-KO mice. In addition, acute hemodynamic stress induced by dobutamine increased systolic function index in trained WT, contrary to trained Pnmt-KO mice, suggesting a possible initial stage of pathological cardiac hypertrophy in these mice. Therefore, epinephrine appears to be essential for prevention of blood pressure overshoot and thus cardiac hypertrophy in chronic exercise.

Dose–Response Head-to-Head Comparison of Inodilators Dobutamine, Milrinone, and Levosimendan in Chronic Experimental Pulmonary Hypertension:

The choice of inodilator drug in the acute management of patients with pulmonary hypertension (PH) having right ventricular (RV) failure remains unsettled and challenging. Comprehensive experimental evaluations may provide further insight and fundamental translational research clues to support inodilator selection and clinical trial design. Our aim was to compare acute dose–response hemodynamic effects of inodilators dobutamine (DOB), milrinone (MIL), and levosimendan (LEV) in chronic experimental PH. Seven-week-old male Wistar rats were randomly injected with 60 mg·kg−1 monocrotaline (MCT) or vehicle (Ctrl, n = 7) and underwent systemic and pulmonary artery (PA) pressure and RV pressure–volume (PV) hemodynamic evaluation under halogenate anesthesia 24 to 30 days after injection. The MCT-injected animals (n = 7 each) randomly received dose–response infusions of DOB (1, 3, 6 and 12 μg·kg−1·min−1), MIL (MIL: 1, 3, 6 and 12 μg·kg−1·min−1), or LEV (0.3, 0.6, 1.2 and 2.4 μg·kg−1·min−1). Load-independent indexes were obtained by inferior vena cava occlusion at baseline and after the last dose. All inodilators increased RV ejection fraction, preload recruitable stroke work, and ventricular–vascular coupling without jeopardizing perfusion pressure. Dobutamine raised heart rate and PA pressure. Only LEV increased cardiac index and decreased PA elastance and pulmonary vascular resistance (PVR). Moreover, only LEV downward-shifted the end-diastolic PV relationship, thereby improving RV compliance. Adding sildenafil to LEV further decreased PVR. Levosimendan had beneficial acute systolic and diastolic functional effects in experimental chronic PH and RV afterload compared to DOB and MIL. It should be further tested in clinical trials enrolling patients with PH in the perioperative and critical care settings.

[OP.8A.01] EXERCISE-INDUCED HYPERTENSION AND CARDIAC HYPERTROPHY IN EPINEPHRINE-DEFICIENT MICE.

Objective: Phenylethanolamine-N-methyltransferase (Pnmt) is required for the conversion of norepinephrine to epinephrine. Bao et al (2007) described that Pnmt-knockout (Pnmt-KO) mice have an increased ratio of left ventricular (LV) posterior wall thickness to internal dimensions (LVPW/LVID) but not overall cardiac hypertrophy. Pnmt-KO mice showed normal resting blood pressure (BP) but became hypertensive during treadmill exercise. The aim of this study was to evaluate cardiac morphological and functional alterations after chronic exercise in Pnmt-KO mice. Design and method: PCR-based genotyping was performed at the Pnmt locus of 10-week-old Pnmt-KO (Pnmt-/-) and WT mice (129x1/SvJ). Epinephrine and norepinephrine were quantified by RP-HPLC-ED in adrenal glands. Animals were submitted to a 6-week exercise training program. BP was measured by a photoelectric pulse detector. Mice were anesthetized (sevoflurane, 8%) and cardiac morphology and function were evaluated by echocardiography followed by morphometric analysis. Results: Epinephrine levels were vestigial in Pnmt-KO compared with WT mice. There were no differences in systolic and diastolic BP between untrained Pnmt-KO and WT mice. However, trained Pnmt-KO mice showed a significant increase in systolic BP when compared to trained WT mice. Our findings also showed that the ratios between heart weight (HW/BW) and LV weight (LVW/BW) and body weight (BW) were significantly increased in trained compared to untrained Pnmt-KO mice. Trained Pnmt-KO mice presented higher HW/BW than trained WT mice. Untrained Pnmt-KO mice display echocardiographic results similar to those reported by Bao et al (2007) while LVPW and IVS thicknesses, LV end-diastolic internal dimension (LVIDd) and LV mass indexed for body surface area (LVMi) were significantly increased in trained Pnmt-KO mice, indicating overall LV hypertrophic changes. Indexed LV end-diastolic volume (LVEDVi), indexed stroke volume (SVi) and cardiac index (CI) were significantly higher in trained Pnmt-KO than untrained Pnmt-KO mice. Conclusions: In conclusion, the increased BP in Pnmt-KO mice in response to exercise appears to be associated with an increase in LV wall thickness and chamber volume suggesting hypertrophic remodelling of the LV in Pnmt-KO mice. Epinephrine appears to be essential for maintaining normal BP, probably through &bgr;2-adrenoceptors-induced vessel relaxation, and preventing LV hypertrophy in chronic exercise.