Nazha Hamdani

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.

Gigantic Business Titin Properties and Function Through Thick and Thin

The giant protein titin forms a unique filament network in cardiomyocytes, which engages in both mechanical and signaling functions of the heart. TTN, which encodes titin, is also a major human disease gene. In this review, we cover the roles of cardiac titin in normal and failing hearts, with a special emphasis on the contribution of titin to diastolic stiffness. We provide an update on disease-associated titin mutations in cardiac and skeletal muscles and summarize what is known about the impact of protein–protein interactions on titin properties and functions. We discuss the importance of titin-isoform shifts and titin phosphorylation, as well as titin modifications related to oxidative stress, in adjusting the diastolic stiffness of the healthy and the failing heart. Along the way we distinguish among titin alterations in systolic and in diastolic heart failure and ponder the evidence for titin stiffness as a potential target for pharmacological intervention in heart disease.

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.

Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Sildenafil and B-Type Natriuretic Peptide Acutely Phosphorylate Titin and Improve Diastolic Distensibility In Vivo

Background— In vitro studies suggest that phosphorylation of titin reduces myocyte/myofiber stiffness. Titin can be phosphorylated by cGMP-activated protein kinase. Intracellular cGMP production is stimulated by B-type natriuretic peptide (BNP) and degraded by phosphodiesterases, including phosphodiesterase-5A. We hypothesized that a phosphodiesterase-5A inhibitor (sildenafil) alone or in combination with BNP would increase left ventricular diastolic distensibility by phosphorylating titin. Methods and Results— Eight elderly dogs with experimental hypertension and 4 young normal dogs underwent measurement of the end-diastolic pressure-volume relationship during caval occlusion at baseline, after sildenafil, and BNP infusion. To assess diastolic distensibility independently of load/extrinsic forces, the end-diastolic volume at a common end-diastolic pressure on the sequential end-diastolic pressure-volume relationships was measured (left ventricular capacitance). In a separate group of dogs (n=7 old hypertensive and 7 young normal), serial full-thickness left ventricular biopsies were harvested from the beating heart during identical infusions to measure myofilament protein phosphorylation. Plasma cGMP increased with sildenafil and further with BNP (7.31±2.37 to 26.9±10.3 to 70.3±8.1 pmol/mL; P<0.001). Left ventricular diastolic capacitance increased with sildenafil and further with BNP (51.4±16.9 to 53.7±16.8 to 60.0±19.4 mL; P<0.001). Changes were similar in old hypertensive and young normal dogs. There were no effects on phosphorylation of troponin I, troponin T, phospholamban, or myosin light chain-1 or -2. Titin phosphorylation increased with sildenafil and BNP, whereas titin-based cardiomyocyte stiffness decreased. Conclusion— Short-term cGMP-enhancing treatment with sildenafil and BNP improves left ventricular diastolic distensibility in vivo, in part by phosphorylating titin.

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.

Terminal Differentiation, Advanced Organotypic Maturation, and Modeling of Hypertrophic Growth in Engineered Heart Tissue

Rationale: Cardiac tissue engineering should provide “realistic” in vitro heart muscle models and surrogate tissue for myocardial repair. For either application, engineered myocardium should display features of native myocardium, including terminal differentiation, organotypic maturation, and hypertrophic growth. Objective: To test the hypothesis that 3D-engineered heart tissue (EHT) culture supports (1) terminal differentiation as well as (2) organotypic assembly and maturation of immature cardiomyocytes, and (3) constitutes a methodological platform to investigate mechanisms underlying hypertrophic growth. Methods and Results: We generated EHTs from neonatal rat cardiomyocytes and compared morphological and molecular properties of EHT and native myocardium from fetal, neonatal, and adult rats. We made the following key observations: cardiomyocytes in EHT (1) gained a high level of binucleation in the absence of notable cytokinesis, (2) regained a rod-shape and anisotropic sarcomere organization, (3) demonstrated a fetal-to-adult gene expression pattern, and (4) responded to distinct hypertrophic stimuli with concentric or eccentric hypertrophy and reexpression of fetal genes. The process of terminal differentiation and maturation (culture days 7–12) was preceded by a tissue consolidation phase (culture days 0–7) with substantial cardiomyocyte apoptosis and dynamic extracellular matrix restructuring. Conclusions: This study documents the propensity of immature cardiomyocytes to terminally differentiate and mature in EHT in a remarkably organotypic manner. It moreover provides the rationale for the utility of the EHT technology as a methodological bridge between 2D cell culture and animal models.

Crucial Role for Ca2+/Calmodulin-Dependent Protein Kinase-II in Regulating Diastolic Stress of Normal and Failing Hearts via Titin Phosphorylation

Rationale: Myocardial diastolic stiffness and cardiomyocyte passive force (Fpassive) depend in part on titin isoform composition and phosphorylation. Ca2+/calmodulin-dependent protein kinase-II (CaMKII) phosphorylates ion channels, Ca2+-handling proteins, and chromatin-modifying enzymes in the heart, but has not been known to target titin. Objective: To elucidate whether CaMKII phosphorylates titin and modulates Fpassive in normal and failing myocardium. Methods and Results: Titin phosphorylation was assessed in CaMKII&dgr;/&ggr; double-knockout (DKO) mouse, transgenic CaMKII&dgr;C-overexpressing mouse, and human hearts, by Pro-Q-Diamond/Sypro-Ruby staining, autoradiography, and immunoblotting using phosphoserine-specific titin-antibodies. CaMKII-dependent site-specific titin phosphorylation was quantified in vivo by mass spectrometry using stable isotope labeling by amino acids in cell culture mouse heart mixed with wild-type (WT) or DKO heart. Fpassive of single permeabilized cardiomyocytes was recorded before and after CaMKII-administration. All-titin phosphorylation was reduced by >50% in DKO but increased by up to ≈100% in transgenic versus WT hearts. Conserved CaMKII-dependent phosphosites were identified within the PEVK-domain of titin by quantitative mass spectrometry and confirmed in recombinant human PEVK-fragments. CaMKII also phosphorylated the cardiac titin N2B-unique sequence. Phosphorylation at specific PEVK/titin N2B-unique sequence sites was decreased in DKO and amplified in transgenic versus WT hearts. Fpassive was elevated in DKO and reduced in transgenic compared with WT cardiomyocytes. CaMKII-administration lowered Fpassive of WT and DKO cardiomyocytes, an effect blunted by titin antibody pretreatment. Human end-stage failing hearts revealed higher CaMKII expression/activity and phosphorylation at PEVK/titin N2B-unique sequence sites than nonfailing donor hearts. Table. Titin Phosphosites Downregulated in CaMKII&dgr;/&ggr; DKO vs WT Mouse Hearts, Identified by Quantitative Mass Spectrometry Conclusions: CaMKII phosphorylates the titin springs at conserved serines/threonines, thereby lowering Fpassive. Deranged CaMKII-dependent titin phosphorylation occurs in heart failure and contributes to altered diastolic stress.