Inês Falcão-Pires

Diastolic Stiffness of the Failing Diabetic Heart Importance of Fibrosis, Advanced Glycation End Products, and Myocyte Resting Tension

Background— Excessive diastolic left ventricular stiffness is an important contributor to heart failure in patients with diabetes mellitus. Diabetes is presumed to increase stiffness through myocardial deposition of collagen and advanced glycation end products (AGEs). Cardiomyocyte resting tension also elevates stiffness, especially in heart failure with normal left ventricular ejection fraction (LVEF). The contribution to diastolic stiffness of fibrosis, AGEs, and cardiomyocyte resting tension was assessed in diabetic heart failure patients with normal or reduced LVEF. Methods and Results— Left ventricular endomyocardial biopsy samples were procured in 28 patients with normal LVEF and 36 patients with reduced LVEF, all without coronary artery disease. Sixteen patients with normal LVEF and 10 with reduced LVEF had diabetes mellitus. Biopsy samples were used for quantification of collagen and AGEs and for isolation of cardiomyocytes to measure resting tension. Diabetic heart failure patients had higher diastolic left ventricular stiffness irrespective of LVEF. Diabetes mellitus increased the myocardial collagen volume fraction only in patients with reduced LVEF (from 14.6±1.0% to 22.4±2.2%, P<0.001) and increased cardiomyocyte resting tension only in patients with normal LVEF (from 5.1±0.7 to 8.5±0.9 kN/m2, P=0.006). Diabetes increased myocardial AGE deposition in patients with reduced LVEF (from 8.8±2.5 to 24.1±3.8 score/mm2; P=0.005) and less so in patients with normal LVEF (from 8.2±2.5 to 15.7±2.7 score/mm2, P=NS). Conclusions— Mechanisms responsible for the increased diastolic stiffness of the diabetic heart differ in heart failure with reduced and normal LVEF: Fibrosis and AGEs are more important when LVEF is reduced, whereas cardiomyocyte resting tension is more important when LVEF is normal.

Hypophosphorylation of the Stiff N2B Titin Isoform Raises Cardiomyocyte Resting Tension in Failing Human Myocardium

High diastolic stiffness of failing myocardium results from interstitial fibrosis and elevated resting tension (Fpassive) of cardiomyocytes. A shift in titin isoform expression from N2BA to N2B isoform, lower overall phosphorylation of titin, and a shift in titin phosphorylation from N2B to N2BA isoform can raise Fpassive of cardiomyocytes. In left ventricular biopsies of heart failure (HF) patients, aortic stenosis (AS) patients, and controls (CON), we therefore related Fpassive of isolated cardiomyocytes to expression of titin isoforms and to phosphorylation of titin and titin isoforms. Biopsies were procured by transvascular technique (44 HF, 3 CON), perioperatively (25 AS, 4 CON), or from explanted hearts (4 HF, 8 CON). None had coronary artery disease. Isolated, permeabilized cardiomyocytes were stretched to 2.2-&mgr;m sarcomere length to measure Fpassive. Expression and phosphorylation of titin isoforms were analyzed using gel electrophoresis with ProQ Diamond and SYPRO Ruby stains and reported as ratio of titin (N2BA/N2B) or of phosphorylated titin (P-N2BA/P-N2B) isoforms. Fpassive was higher in HF (6.1±0.4 kN/m2) than in CON (2.3±0.3 kN/m2; P<0.01) or in AS (2.2±0.2 kN/m2; P<0.001). Titin isoform expression differed between HF (N2BA/N2B=0.73±0.06) and CON (N2BA/N2B=0.39±0.05; P<0.001) and was comparable in HF and AS (N2BA/N2B=0.59±0.06). Overall titin phosphorylation was also comparable in HF and AS, but relative phosphorylation of the stiff N2B titin isoform was significantly lower in HF (P-N2BA/P-N2B=0.77±0.05) than in AS (P-N2BA/P-N2B=0.54±0.05; P<0.01). Relative hypophosphorylation of the stiff N2B titin isoform is a novel mechanism responsible for raised Fpassive of human HF cardiomyocytes.

Low Myocardial Protein Kinase G Activity in Heart Failure With Preserved Ejection Fraction

Background— Prominent features of myocardial remodeling in heart failure with preserved ejection fraction (HFPEF) are high cardiomyocyte resting tension (Fpassive) and cardiomyocyte hypertrophy. In experimental models, both reacted favorably to raised protein kinase G (PKG) activity. The present study assessed myocardial PKG activity, its downstream effects on cardiomyocyte Fpassive and cardiomyocyte diameter, and its upstream control by cyclic guanosine monophosphate (cGMP), nitrosative/oxidative stress, and brain natriuretic peptide (BNP). To discern altered control of myocardial remodeling by PKG, HFPEF was compared with aortic stenosis and HF with reduced EF (HFREF). Methods and Results— Patients with HFPEF (n=36), AS (n=67), and HFREF (n=43) were free of coronary artery disease. More HFPEF patients were obese (P<0.05) or had diabetes mellitus (P<0.05). Left ventricular myocardial biopsies were procured transvascularly in HFPEF and HFREF and perioperatively in aortic stenosis. Fpassive was measured in cardiomyocytes before and after PKG administration. Myocardial homogenates were used for assessment of PKG activity, cGMP concentration, proBNP-108 expression, and nitrotyrosine expression, a measure of nitrosative/oxidative stress. Additional quantitative immunohistochemical analysis was performed for PKG activity and nitrotyrosine expression. Lower PKG activity in HFPEF than in aortic stenosis (P<0.01) or HFREF (P<0.001) was associated with higher cardiomyocyte Fpassive (P<0.001) and related to lower cGMP concentration (P<0.001) and higher nitrosative/oxidative stress (P<0.05). Higher Fpassive in HFPEF was corrected by in vitro PKG administration. Conclusions— Low myocardial PKG activity in HFPEF was associated with raised cardiomyocyte Fpassive and was related to increased myocardial nitrosative/oxidative stress. The latter was probably induced by the high prevalence in HFPEF of metabolic comorbidities. Correction of myocardial PKG activity could be a target for specific HFPEF treatment.

Diabetic cardiomyopathy: understanding the molecular and cellular basis to progress in diagnosis and treatment.

Diabetes mellitus is an important and prevalent risk factor for congestive heart failure. Diabetic cardiomyopathy has been defined as ventricular dysfunction that occurs in diabetic patients independent of a recognized cause such as coronary artery disease or hypertension. The disease course consists of a hidden subclinical period, during which cellular structural insults and abnormalities lead initially to diastolic dysfunction, later to systolic dysfunction, and eventually to heart failure. Left ventricular hypertrophy, metabolic abnormalities, extracellular matrix changes, small vessel disease, cardiac autonomic neuropathy, insulin resistance, oxidative stress, and apoptosis are the most important contributors to diabetic cardiomyopathy onset and progression. Hyperglycemia is a major etiological factor in the development of diabetic cardiomyopathy. It increases the levels of free fatty acids and growth factors and causes abnormalities in substrate supply and utilization, calcium homeostasis, and lipid metabolism. Furthermore, it promotes excessive production and release of reactive oxygen species, which induces oxidative stress leading to abnormal gene expression, faulty signal transduction, and cardiomyocytes apoptosis. Stimulation of connective tissue growth factor, fibrosis, and the formation of advanced glycation end-products increase the stiffness of the diabetic hearts. Despite all the current information on diabetic cardiomyopathy, translational research is still scarce due to limited human myocardial tissue and most of our knowledge is extrapolated from animals. This paper aims to elucidate some of the molecular and cellular pathophysiologic mechanisms, structural changes, and therapeutic strategies that may help struggle against diabetic cardiomyopathy.

Diabetes Mellitus Worsens Diastolic Left Ventricular Dysfunction in Aortic Stenosis Through Altered Myocardial Structure and Cardiomyocyte Stiffness

Background— Aortic stenosis (AS) and diabetes mellitus (DM) are frequent comorbidities in aging populations. In heart failure, DM worsens diastolic left ventricular (LV) dysfunction, thereby adversely affecting symptoms and prognosis. Effects of DM on diastolic LV function were therefore assessed in aortic stenosis, and underlying myocardial mechanisms were identified. Methods and Results— Patients referred for aortic valve replacement were subdivided into patients with AS and no DM (AS; n=46) and patients with AS and DM (AS-DM; n=16). Preoperative Doppler echocardiography and hemodynamics were implemented with perioperative LV biopsies. Histomorphometry and immunohistochemistry quantified myocardial collagen volume fraction and myocardial advanced glycation end product deposition. Isolated cardiomyocytes were stretched to 2.2-&mgr;m sarcomere length to measure resting tension (Fpassive). Expression and phosphorylation of titin isoforms were analyzed with gel electrophoresis with ProQ Diamond and SYPRO Ruby stains. Reduced LV end-diastolic distensibility in AS-DM was evident from higher LV end-diastolic pressure (21±1 mm Hg for AS versus 28±4 mm Hg for AS-DM; P=0.04) at comparable LV end-diastolic volume index and attributed to higher myocardial collagen volume fraction (AS, 12.9±1.1% versus AS-DM, 18.2±2.6%; P<0.001), more advanced glycation end product deposition in arterioles, venules, and capillaries (AS, 14.4±2.1 score per 1 mm2 versus AS-DM, 31.4±6.1 score per 1 mm2; P=0.03), and higher Fpassive (AS, 3.5±1.7 kN/m2 versus AS-DM, 5.1±0.7 kN/m2; P=0.04). Significant hypophosphorylation of the stiff N2B titin isoform in AS-DM explained the higher Fpassive and normalization of Fpassive after in vitro treatment with protein kinase A. Conclusions— Worse diastolic LV dysfunction in AS-DM predisposes to heart failure and results from more myocardial fibrosis, more intramyocardial vascular advanced glycation end product deposition, and higher cardiomyocyte Fpassive, which was related to hypophosphorylation of the N2B titin isoform.

Apelin decreases myocardial injury and improves right ventricular function in monocrotaline-induced pulmonary hypertension

We investigated the endogenous production of apelin and the cardiac and pulmonary effects of its chronic administration in monocrotaline (MCT)-induced pulmonary hypertension (PH). Male Wistar rats were injected with MCT (60 mg/kg sc) or vehicle (day 0). One week later, these animals were randomly treated during 17 days with pyroglutamylated apelin-13 (Pyr-AP13; 200 microg*kg(-1)*day(-1) ip) or a similar volume of saline, resulting in four groups: sham (n = 11), sham-AP (n = 11), MCT (n = 16), and MCT-AP (n = 13). On day 25, right ventricular (RV) and left ventricular (LV) hemodynamic and morphometric parameters were assessed. Tissue and plasma samples were collected for histological and molecular analysis. When compared with sham, the MCT group presented a significant increase of RV mass (166 +/- 38%), diameter of cardiomyocyte (40 +/- 10%), myocardial fibrosis (95 +/- 20%), peak systolic pressure (99 +/- 22%), peak rate of ventricular pressure rise (dP/dt(max); 74 +/- 24%), peak rate of ventricular pressure decline (dP/dt(min); 73 +/- 19%), and time constant tau (55 +/- 16%). In these animals, RV expression of apelin (-73 +/- 10%) and its receptor APJ (-61 +/- 20%) was downregulated, whereas mRNA expression of type B natriuretic peptide (9,606 +/- 713%), angiotensinogen (191 +/- 147%), endothelin-1 (RV, 497 +/- 156%; and LV, 799 +/- 309%), plasmatic levels of apelin (104 +/- 48%), and angiotensin 1-7 (161 +/- 151%) were increased. Chronic treatment with Pyr-AP13 significantly attenuated or normalized these changes, preventing apelin-APJ mRNA downregulation and PH-induced neurohumoral activation of several vasoconstrictors, which exacerbates apelin-APJ vasodilator effects. Therefore, apelin delayed the progression of RV hypertrophy and diastolic dysfunction. Together, these observations suggest that the apelin-APJ system may play an important role in the pathophysiology of PH, representing a potential therapeutic target since it significantly attenuates RV overload and PH-induced neurohumoral activation.

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.

Physiological, pathological and potential therapeutic roles of adipokines

Formerly regarded purely as passive energy storage, adipose tissue is now recognized as a vital endocrine organ. Adipocytes secrete diverse peptide hormones named adipokines, which act in a autocrine, paracrine or endocrine way to influence several biological functions. Adipokines comprise diverse bioactive substances, including cytokines, growth, and complement factors, which perform essential regulatory functions related to energy balance, satiety and immunity. Presently adipokines have been widely implicated in obesity, diabetes, hypertension and cardiovascular diseases. In this article we aim to present a brief description of the roles and potential therapeutic modulation of adipokines, such as leptin, resistin, adiponectin, apelin, visfatin, FABP-4, tumor necrosis factor-α (TNF-α), interleukin-6 and plasminogen activator inhibitor-1 (PAI-1).

Animal models of heart failure with preserved ejection fraction

Heart failure with preserved ejection fraction (HFpEF) constitutes a clinical syndrome in which the diagnostic criteria of heart failure are not accompanied by gross disturbances of systolic function, as assessed by ejection fraction. In turn, under most circumstances, diastolic function is impaired. Although it now represents over 50 % of all patients with heart failure, the mechanisms of HFpEF remain understood, precluding effective therapy. Understanding the pathophysiology of HFpEF has been restricted by both limited access to human myocardial biopsies and by the lack of animal models that fully mimic human pathology. Animal models are valuable research tools to clarify subcellular and molecular mechanisms under conditions where the comorbidities and other confounding factors can be precisely controlled. Although most of the heart failure animal models currently available represent heart failure with reduced ejection fraction, several HFpEF animal models have been proposed. However, few of these fulfil all the features present in human disease. In this review we will provide an overview of the currently available models to study HFpEF from rodents to large animals as well as present advantages and disadvantages of these models.

The apelinergic system: a promising therapeutic target

Importance of the field: Apelin is a bioactive peptide known as the ligand of the G-protein-coupled receptor APJ. In recent years, there has been a growing body of evidence regarding the importance of apelin and APJ in the pathophysiology of cardiovascular, metabolic and gastrointestinal diseases, brain signalling, HIV infection and tumor angiogenesis. Therefore, the apelinergic system is involved in the pathogenesis of several diseases that represent a major burden to our society. Areas covered in this review: The goal of this paper is to give an up-to-date review of existing information on apelin/APJ since the discovery of apelin in 1998, with particular focus on their involvement in the regulation of human body systems and potential therapeutic applications. What the reader will gain: An overview of the most important physiological functions of the apelinergic system and the diseases that may benefit in the future from its modulation as a therapeutic target. Take home message: Today, the established biological effects of apelin involve major cardiovascular actions, neoangiogenesis, immunologic modulation and insulinemia control as well as body fluid and glucose homeostasis. However, the physiological and pathophysiological role of endogenous apelin is still unsettled and a better and profound knowledge of this system in humans is necessary for the development of novel apelinergic-based therapeutic targets.