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 (F passive )–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 F passive 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.

Leite S, Oliveira-Pinto J, Tavares-Silva M, Abdellatif M, Fontoura D, Falcão-Pires I, Leite-Moreira AF, Lourenço AP. Echocardiography and invasive hemodynamics during stress testing for diagnosis of heart failure with preserved ejection fraction: an experimental study. Am J Physiol Heart Circ Physiol 308: H1556 -H1563, 2015. First published April 11, 2015; doi:10.1152/ajpheart.00076.2015.-Inclusion of exercise testing in diagnostic guidelines for heart failure with preserved ejection fraction (HFpEF) has been advocated, but the target population, technical challenges, and underlying pathophysiological complexity raise difficulties to implementation. Hemodynamic stress tests may be feasible alternatives. Our aim was to test Trendelenburg positioning, phenylephrine, and dobutamine in the ZSF1 obese rat model to find echocardiographic surrogates for end-diastolic pressure (EDP) elevation and HFpEF. Seventeen-week-old WistarKyoto, ZSF1 lean, and obese rats (n ϭ 7 each) randomly and sequentially underwent (crossover) Trendelenburg (30°), 5 g·Kg Ϫ1 ·min Ϫ1 dobutamine, and 7.5 g·Kg Ϫ1 ·min Ϫ1 phenylephrine with simultaneous left ventricular (LV) pressure-volume loop and echocardiography evaluation under halogenate anesthesia. Effort testing with maximum O2 consumption (V O2 max) determination was performed 1 wk later. Obese ZSF1 showed lower effort tolerance and V O2 max along with higher resting EDP. Both Trendelenburg and phenylephrine increased EDP, whereas dobutamine decreased it. Significant correlations were found between EDP and 1) peak early filling Doppler velocity of transmitral flow (E) to corresponding myocardial tissue Doppler velocity (E=) ratio, 2) E to E-wave deceleration time (E/DT) ratio, and 3) left atrial area (LAA). Diagnostic efficiency of E/DT*LAA by receiver-operating characteristic curve analysis for elevation of EDP above a cut-off of 13 mmHg during hemodynamic stress was high (area under curve, AUC ϭ 0.95) but not higher than that of E/E= (AUC ϭ 0.77, P ϭ 0.15). Results in ZSF1 obese rats suggest that noninvasive echocardiography after hemodynamic stress induced by phenylephrine or Trendelenburg can enhance diagnosis of stable HFpEF and constitute an alternative to effort testing. heart failure with preserved

Background— The combination of cardiac and renal disease driven by metabolic risk factors, referred to as cardiorenal metabolic syndrome (CRMS), is increasingly recognized as a critical pathological entity. The contribution of (micro)vascular injury to CRMS is considered to be substantial. However, mechanistic studies are hampered by lack of in vivo models that mimic the natural onset of the disease. Here, we evaluated the coronary and renal microvasculature during CRMS development in obese diabetic Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rats. Methods and Results— Echocardiographic, urine, and blood evaluations were conducted in 3 groups (Wistar-Kyoto, lean ZSF1, and obese ZSF1) at 20 and 25 weeks of age. Immunohistological evaluation of renal and cardiac tissues was conducted at both time points. At 20 and 25 weeks, obese ZSF1 rats showed higher body weight, significant left ventricular hypertrophy, and impaired diastolic function compared with all other groups. Indices of systolic function did not differ between groups. Obese ZSF1 rats developed hyperproliferative vascular foci in the subendocardium, which lacked microvascular organization and were predilection sites of inflammation and fibrosis. In the kidney, obese ZSF1 animals showed regression of the peritubular and glomerular microvasculature, accompanied by tubulointerstitial damage, glomerulosclerosis, and proteinuria. Conclusions— The obese ZSF1 rat strain is a suitable in vivo model for CRMS, sharing characteristics with the human syndrome during the earliest onset of disease. In these rats, CRMS induces microvascular fibrotic responses in heart and kidneys, associated with functional impairment of both organs.

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Falcão-Pires I, Gillebert TC, LeiteMoreira AF, Lourenço AP. Afterload-induced diastolic dysfunction contributes to high filling pressures in experimental heart failure with preserved ejection fraction. Am J Physiol Heart Circ Physiol 309: H1648 -H1654, 2015. First published September 25, 2015; doi:10.1152/ajpheart.00397.2015.-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 ϭ 12), healthy Wistar-Kyoto rats (WKY, n ϭ 11), and hypertensive ZSF1 lean control rats (ZSF1 Ln, n ϭ 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 ␤, 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 (), 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 ϫ monoexponentially derived (exp) 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.afterload; relaxation; diastolic function; heart failure with preserved ejection fraction NEW & NOTEWORTHY In a highly controlled hemodynamic evaluation setup in experimental heart failure with preserved ejection fraction, we demonstrate that delayed relaxation independently explains enddiastolic pressure elevation during suddenly imposed afterload. This is an important contribution to understanding the response to exercise or hypertensive stress in preserved ejection fraction heart failure.HEART FAILURE with preserved ejection fraction (HFpEF) remains a major unsolved health issue and a complex disease in which the contribution of various aspects is still incompletely understood (22,24). Although HFpEF pathophysiology is multifarious and HFpEF patients constitute a heterogeneous group comprising several risk factors (26), most experts would agree upon abnormalities of left ventricular (LV) relaxation and high...

Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. AbstractExtracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness‐induced changes in cardiomyocyte function could not be explained by adaptation in the microtubules. Additionally, cardiomyocytes isolated from stiff hearts of the obese ZSF1 rat model of heart failure with preserved ejection fraction show more pronounced reduction in unloaded shortening in response to matrix stiffening. Taken together, we introduce a method that allows evaluation of the influence of ECM properties on cardiomyocyte function separate from the passive inhibitory component of a stiff matrix. As such, it adds an important and physiologically relevant tool to investigate the functional consequences of cardiomyocyte–matrix interactions.

Several studies have demonstrated that administration of doxorubicin (DOXO) results in cardiotoxicity, which eventually progresses to dilated cardiomyopathy. The present work aimed to evaluate the early myocardial changes of DOXO-induced cardiotoxicity. Male New Zealand White rabbits were injected intravenously with DOXO twice weekly for 8 wk [DOXO-induced heart failure (DOXO-HF)] or with an equivolumetric dose of saline (control). Echocardiographic evaluation was performed, and myocardial samples were collected to evaluate myocardial cellular and molecular modifications. The DOXO-HF group presented cardiac hypertrophy and higher left ventricular cavity diameters, showing a dilated phenotype but preserved ejection fraction. Concerning cardiomyocyte function, the DOXO-HF group presented a trend toward increased active tension without significant differences in passive tension. The myocardial GSSG-to-GSH ratio and interstitial fibrosis were increased and Bax-to- Bcl-2 ratio presented a trend toward an increase, suggesting the activation of apoptosis signaling pathways. The macromolecule titin shifted toward the more compliant isoform (N2BA), whereas the stiffer one (N2B) was shown to be hypophosphorylated. Differential protein analysis from the aggregate-enriched fraction through gel liquid chromatography-tandem mass spectrometry revealed an increase in the histidine-rich glycoprotein fragment in DOXO-HF animals. This work describes novel and early myocardial effects of DOXO-induced cardiotoxicity. Thus, tracking these changes appears to be of extreme relevance for the early detection of cardiac damage (as soon as ventricular dilation becomes evident) before irreversible cardiac function deterioration occurs (reduced ejection fraction). Moreover, it allows for the adjustment of the therapeutic approach and thus the prevention of cardiomyopathy progression. NEW & NOTEWORTHY Identification of early myocardial effects of doxorubicin in the heart is essential to hinder the development of cardiac complications and adjust the therapeutic approach. This study describes doxorubicin-induced cellular and molecular modifications before the onset of dilated cardiomyopathy. Myocardial samples from doxorubicin-treated rabbits showed a tendency for higher cardiomyocyte active tension, titin isoform shift from N2B to N2BA, hypophosphorylation of N2B, increased apoptotic genes, left ventricular interstitial fibrosis, and increased aggregation of histidine-rich glycoprotein.

Western-type diets (WD) constitute risk factors for disease but may have distinct effects in heart failure (HF) with cardiac cachexia (CC). We evaluated hemodynamic, metabolic, and inflammatory effects of short-term WD intake in pulmonary hypertension (PH) with CC. Male Wistar rats randomly received 60 mg · kg(-1) monocrotaline (M) or vehicle (C) and consumed either a 5.4-kcal · g(-1) WD (35% animal fat, 35% simple carbohydrate, 20% protein, 0.4% Na(+)) or a 2.9-kcal · g(-1) (3% vegetable fat, 60% complex carbohydrate, 16% protein, 0.25% Na(+)) normal diet (ND) for 5 wk. Mortality, energy intake, body weight (BW), metabolism, hemodynamics, histology, apoptosis, gene expression, transcription factors, and plasma cytokines were evaluated. Compared with the C-ND group, the M-ND group had PH, HF, and mortality that were significantly attenuated in M-WD. The extent of myocardial remodeling and apoptosis was higher in M-ND than in C-ND but lower in M-WD than in M-ND, while conversely, energy intake, BW, cholesterol, and TG plasma concentrations were lower in M-ND than in C-ND but higher in M-WD than in M-ND. M-ND had increased myocardial NF-κB transcription factor activity, endothelin-1, and cytokine overexpression and higher circulating cytokine concentrations than C-ND, which were lower in M-WD than in M-ND. PPARα activity, however, was lower in M-ND, but not in M-WD, compared with the respective C groups. WD attenuated PH and CC, ameliorating survival, myocardial function, metabolism, and inflammation, through transcription factor modulation, suggesting a beneficial role in CC.