Loek van Heerebeek

Myocardial Structure and Function Differ in Systolic and Diastolic Heart Failure

Background— To support the clinical distinction between systolic heart failure (SHF) and diastolic heart failure (DHF), left ventricular (LV) myocardial structure and function were compared in LV endomyocardial biopsy samples of patients with systolic and diastolic heart failure. Methods and Results— Patients hospitalized for worsening heart failure were classified as having SHF (n=22; LV ejection fraction (EF) 34±2%) or DHF (n=22; LVEF 62±2%). No patient had coronary artery disease or biopsy evidence of infiltrative or inflammatory myocardial disease. More DHF patients had a history of arterial hypertension and were obese. Biopsy samples were analyzed with histomorphometry and electron microscopy. Single cardiomyocytes were isolated from the samples, stretched to a sarcomere length of 2.2 &mgr;m to measure passive force (Fpassive), and activated with calcium-containing solutions to measure total force. Cardiomyocyte diameter was higher in DHF (20.3±0.6 versus 15.1±0.4 &mgr;m, P<0.001), but collagen volume fraction was equally elevated. Myofibrillar density was lower in SHF (36±2% versus 46±2%, P<0.001). Cardiomyocytes of DHF patients had higher Fpassive (7.1±0.6 versus 5.3±0.3 kN/m2; P<0.01), but their total force was comparable. After administration of protein kinase A to the cardiomyocytes, the drop in Fpassive was larger (P<0.01) in DHF than in SHF. Conclusions— LV myocardial structure and function differ in SHF and DHF because of distinct cardiomyocyte abnormalities. These findings support the clinical separation of heart failure patients into SHF and DHF phenotypes.

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.

Phenotype-Specific Treatment of Heart Failure With Preserved Ejection Fraction

Heart failure (HF) with preserved ejection fraction (EF; HFpEF) accounts for 50% of HF cases, and its prevalence relative to HF with reduced EF continues to rise. In contrast to HF with reduced EF, large trials testing neurohumoral inhibition in HFpEF failed to reach a positive outcome. This failure was recently attributed to distinct systemic and myocardial signaling in HFpEF and to diversity of HFpEF phenotypes. In this review, an HFpEF treatment strategy is proposed that addresses HFpEF-specific signaling and phenotypic diversity. In HFpEF, extracardiac comorbidities such as metabolic risk, arterial hypertension, and renal insufficiency drive left ventricular remodeling and dysfunction through systemic inflammation and coronary microvascular endothelial dysfunction. The latter affects left ventricular diastolic dysfunction through macrophage infiltration, resulting in interstitial fibrosis, and through altered paracrine signaling to cardiomyocytes, which become hypertrophied and stiff because of low nitric oxide and cyclic guanosine monophosphate. Systemic inflammation also affects other organs such as lungs, skeletal muscle, and kidneys, leading, respectively, to pulmonary hypertension, muscle weakness, and sodium retention. Individual steps of these signaling cascades can be targeted by specific interventions: metabolic risk by caloric restriction, systemic inflammation by statins, pulmonary hypertension by phosphodiesterase 5 inhibitors, muscle weakness by exercise training, sodium retention by diuretics and monitoring devices, myocardial nitric oxide bioavailability by inorganic nitrate-nitrite, myocardial cyclic guanosine monophosphate content by neprilysin or phosphodiesterase 9 inhibition, and myocardial fibrosis by spironolactone. Because of phenotypic diversity in HFpEF, personalized therapeutic strategies are proposed, which are configured in a matrix with HFpEF presentations in the abscissa and HFpEF predispositions in the ordinate.

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.

Connecting heart failure with preserved ejection fraction and renal dysfunction: the role of endothelial dysfunction and inflammation

Renal dysfunction in heart failure with preserved ejection fraction (HFpEF) is common and is associated with increased mortality. Impaired renal function is also a risk factor for developing HFpEF. A new paradigm for HFpEF, proposing a sequence of events leading to myocardial remodelling and dysfunction in HFpEF, was recently introduced, involving inflammatory, microvascular, and cardiac components. The kidney might play a key role in this systemic process. Renal impairment causes metabolic and systemic derangements in circulating factors, causing an activated systemic inflammatory state and endothelial dysfunction, which may lead to cardiomyocyte stiffening, hypertrophy, and interstitial fibrosis via cross‐talk between the endothelium and cardiomyocyte compartments. Here, we review the role of endothelial dysfunction and inflammation to explain the link between renal dysfunction and HFpEF, which allows for identification of new early risk markers, prognostic factors, and unique targets for intervention.

Molecular and Cellular Basis for Diastolic Dysfunction

Heart failure with preserved ejection fraction (HFpEF) is highly prevalent and is frequently associated with metabolic risk factors. Patients with HFpEF have only a slightly lower mortality than patients with HF and reduced EF. The pathophysiology of HFpEF is currently incompletely understood, which precludes specific therapy. Both HF phenotypes demonstrate distinct cardiac remodeling processes at the macroscopic, microscopic, and ultrastructural levels. Increased diastolic left-ventricular (LV) stiffness and impaired LV relaxation are important features of HFpEF, which can be explained by changes in the extracellular matrix and the cardiomyocytes. In HFpEF, elevated intrinsic cardiomyocyte stiffness contributes to high diastolic LV stiffness. Posttranslational changes in the sarcomeric protein titin, affecting titin isoform expression and phosphorylation, contribute to elevated cardiomyocyte stiffness. Increased nitrosative/oxidative stress, impaired nitric oxide bioavailability, and down-regulation of myocardial cyclic guanosine monophosphate and protein kinase G signaling could trigger posttranslational modifications of titin, thereby augmenting cardiomyocyte and LV diastolic stiffness.

Distinct myocardial effects of beta-blocker therapy in heart failure with normal and reduced left ventricular ejection fraction

AIMS Left ventricular (LV) myocardial structure and function differ in heart failure (HF) with normal (N) and reduced (R) LV ejection fraction (EF). This difference could underlie an unequal outcome of trials with beta-blockers in heart failure with normal LVEF (HFNEF) and heart failure with reduced LVEF (HFREF) with mixed results observed in HFNEF and positive results in HFREF. To investigate whether beta-blockers have distinct myocardial effects in HFNEF and HFREF, myocardial structure, cardiomyocyte function, and myocardial protein composition were compared in HFNEF and HFREF patients without or with beta-blockers. METHODS AND RESULTS Patients, free of coronary artery disease, were divided into beta-(HFNEF) (n = 16), beta+(HFNEF) (n = 16), beta-(HFREF) (n = 17), and beta+(HFREF) (n = 22) groups. Using LV endomyocardial biopsies, we assessed collagen volume fraction (CVF) and cardiomyocyte diameter (MyD) by histomorphometry, phosphorylation of myofilamentary proteins by ProQ-Diamond phosphostained 1D-gels, and expression of beta-adrenergic signalling and calcium handling proteins by western immunoblotting. Cardiomyocytes were also isolated from the biopsies to measure active force (F(active)), resting force (F(passive)), and calcium sensitivity (pCa(50)). Myocardial effects of beta-blocker therapy were either shared by HFNEF and HFREF, unique to HFNEF or unique to HFREF. Higher F(active), higher pCa(50), lower phosphorylation of troponin I and myosin-binding protein C, and lower beta(2) adrenergic receptor expression were shared. Higher F(passive), lower CVF, lower MyD, and lower expression of stimulatory G protein were unique to HFNEF and lower expression of inhibitory G protein was unique to HFREF. CONCLUSION Myocardial effects unique to either HFNEF or HFREF could contribute to the dissimilar outcome of beta-blocker therapy in both HF phenotypes.

Transcriptional and posttranslational modifications of titin implications for diastole

See related article, pages 87–94 Myocardial diastolic stiffness has been variably attributed to extracellular matrix composition, cytoskeletal properties of cardiomyocytes, or residual diastolic crossbridge cycling because of incomplete relaxation or cytosolic calcium removal.1 Extracellular matrix and cardiomyocyte cytoskeleton are presumed to mediate chronic rises in myocardial diastolic stiffness, as occur during aging, pressure overload or heart failure, whereas residual diastolic crossbridge cycling accounts for acute changes, as observed during ischemia, exercise, or pharmacological interventions. The elegant study by Kruger et al, published in this issue of Circulation Research , challenges this conceptual framework.2 The study demonstrates that protein kinase (PK)G is capable of phosphorylating the giant cytoskeletal protein titin, as previously reported for PKA3,4 and that phosphorylation by PKG or PKA of a serine residue within the N2B fragment of titin leads to an acute fall in cardiomyofibrillar stiffness. An acute effect produced by a cytoskeletal protein invalidates the concept of distinct mediators for chronic or acute changes in myocardial diastolic stiffness. From these and other recent observations it becomes evident that the cytoskeletal protein titin can alter myocardial diastolic stiffness, both acutely and chronically, through multiple mechanisms such as isoform shifts, phosphorylation by PKG or PKA, and titin–actin interaction at the Z-disc (Figure). Figure. Titin alters cardiomyocyte stiffness through isoform shifts, phosphorylation, and titin–actin interaction. A, Sarcomeric structure with detailed view of I-band region of N2B titin isoform showing tandem immunoglobulin (Ig), N2B, and elastic PEVK segments. B through D, Shift from N2B to N2BA titin isoform (B) and phosphorylation by PKG or PKA at S469 (C) reduce stiffness of the elastic PEVK segment, whereas …