Alterations in cardiomyocyte function during diastolic heart failure

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1 Alterations in cardiomyocyte function during diastolic heart failure Attila Borbély VUmc UDMHSC, Division of Clinical Physiology, Institute of Cardiology, Debrecen, Hungary Laboratory for Physiology, ICaR-VU VU medical center, Amsterdam Heart failure phenotypes EF LVEDP Redfield MM. (NEJM, ; 35, ) Diastolic heart failure - Importance Diastolic heart failure ( or HFNEF) is currently diagnosed in approximately half of the heart failure patients. (Cleland JG. et al. Lancet, ; 3:131.) LV Function in Patients with First Admission for Heart Failure 37% 18% 5% ALLHAT EF<% EF -9% EF>5% N=1399 Diastolic left ventricular (LV) dysfunction occurs five times more often than systolic LV dysfunction and is an important component of diabetic cardiomyopathy. (Fischer M. et al. Eur Heart J, 3; :3., Avendano G et al., Circulation. 199; 93:139 1) 1

2 Diastolic heart failure Often presumed when symptoms and signs of heart failure occur in a patient with preserved left ventricular systolic function (normal ejection fraction / normal enddiastolic volume) at rest More common in elderly patients in whom systolic hypertension and myocardial hypertrophy contribute to cardiac dysfunction Diagnosis remains controversial Treatment is not yet well defined Diastolic heart failure the phenotype Elderly ladies Hypertension LVH Mild diabetes Mild IHD Admitted with acute pulmonary oedema ± atrial fibrillation Good response to treatment Quite well until next episode Sanderson JE Review: Heart failure with a normal ejection fraction Heart 5

3 Therapy: Epidemiological Evidence Survival improved over time in patients with heart failure and reduced LV EF (SHF) but not in patients with heart failure and preserved LV EF () (Owan TE et al. N Eng J Med: ;355:51) Treatment of Lack of prognostic benefit of current HF therapy in Overview of clinical trials in : Underuse of diagnostic guidelines in patient recruitment New perspectives: Understanding the unique pathophysiology of Diagnosis of - ESC Consensus 7 Paulus WJ et al., European Heart Journal, 7, 8;

4 Pathophysiology of Hemodynamics LVEDP, LVPSP, LVEDVI, CI, EF etc. Contractile function Measurement of Ca + - activated force AGEs Kass DA et al. (Circ Res. ; 9: ) Structure Cardiomyocyte diameter, collagen volume fraction, myofibrillar density, AGEs deposition, sarcomeric protein composition Aims of our studies 1 3 vs. vs. SHF (HFREF) and SHF patients with DM cardiomyocyte resting tension fibrosis AGEs deposition sarcomeric protein composition HF vs. Patients with aortic stenosis (AS) titin isoform phosphorylation p In vivo LV diastolic dysfunction and myocardial stiffness Methods Biopsy material endomyocardial biopsy material from transplant recipients (s), from patients with and SHF without/with diabetes mellitus, LV biopsies from explanted human donor and end-stage failing hearts Clinical parameters patient history, physical examination, medications echocardiography coronarography, invasive pressure measurements Light- and electronmicroscopy, quantitative histomorphometry cardiomyocyte diameter (MyD) determination of collagen volume fraction (CVF) (elastica von Gieson-staining) myofibrillar density, Z line thickness, AGEs deposition (CML score) Determination of myofibrillar protein composition Troponin I (TnI) phosphorylation (ELISA assay) Myofilamentary protein composition and phosphorylation (3-8% gradient SDS-PAGEs) NBA/NB titin isoform and titin isoform phosphorylation ratios (% agarose-strenghtened SDS-PAGEs, Pro-Q Diamond and SYPRO Ruby stainings, Coomassie stain )

5 Molecular determinants of cardiomyocyte contraction Mudd J.O. and Kass DA, Nature, 8; 51: Measurement of isometric contractile force generation the isolated myocyte setup Defrosting of human myocardial catheter biopsies (stored at - 8 C) Mechanical isolation and removal of membranes and associated structures with Triton X-1 Cell attachment to the set-up, sarcomere length:. μm Force measurements before and after the application of Protein Kinase A and G (PKA and PKG) Length 1 sec μm Force F total pca F passive 1 (HFNEF) Cardiomyocyte function? Extracellular matrix μm 5

6 Patient characteristics (in vivo data) Left or right ventricular endomyocardial biopsies from patients: with a history of * (n=1) with normal cardiac function (s, n=8) Hemodynamic data of control and patients HR bpm LVPSP mmhg LVEDP mmhg LVEDVI ml/m LVEF % CI l/min/m kn/m E kn/m * 8 1* *..3* *P<.5 : LV end-diastolic wall stress, E: radial myocardial stiffness modulus * All patients satisfied the European Study Group on criteria. (Borbély A. et al. Circulation 5; 111:77-781). Myocyte stiffening in F kn/m total (k ) * P<.5 * * * pca (Borbély A. et al. Circulation 5; 111:77-781). Effects of PKA F total (kn/m ) PKA 5 Before PKA After PKA pca P<.5 F total (kn/m ) 15 1 PKA 5 Before PKA After PKA pca P<.5 (Borbély A. et al. Circulation 5; 111:77-781).

7 r Effects of PKA on F passive kn/m ) F passive (k 8 P<.1 + PKA n.s. + PKA KA (kn/m ) F passive after PK P<.1 R= F passive (kn/m ) (Borbély A,, Edes I,, Paulus WJ. Circulation 5; 111:77-781). Correlation between in vitro and in vivo data LVEDP (mmhg) 3 1 P=.5 R= * * *P<.5 CVF class I. -5% II. 5-1% III. 1-15% kn/m ) ( E (kn/m ) * * P=.19 R=.58 * * P=.3 R= I II III F passive (kn/m ) CVF class : LV end-diastolic wall stress, E: radiális myocardialis stiffness modulus (Borbély A,, Edes I,, Paulus WJ. Circulation 5; 111:77-781). Troponin I - P F passive ni/ttni ratio DPT n.s. /TTnI arány deptni Kontroll P=.78. R= F passive (kn/m ) (Borbély A. et. al., Circulation. 5;111: ) 7

8 1 Summary Cardiomyocyte function - ECM 1. F passive of cardiomyocytes isolated from biopsies procured in patients with is elevated.. High F passive of cardiomyocytes is reversible as it normalizes following administration of PKA. 3. F passive and CVF combined correlated with LVEDP and LV end-diastolic p wall stress.. Troponin I phosphorylation is not involved in the high F passive, which therefore probably results from reduced phosphorylation of other sarcomeric proteins (MyBP-C and/or titin). (Borbély A. et al. Circulation 5; 111:77-781). vs. SHF Structure and function?? Similarities Differences Patient characteristics Patients with symptoms of heart failure (NYHA IV) LVEF < 5% SHF (n=) Patients LVEF 5%; LVEDP > 1 mmhg (n=) (van Heerebeek L. et al. Circulation, ;113: ) 8

9 Clinical data / hemodynamics SHF P value n Male/Female 15/7 13/9.53 Age (years) 59.1± ±.. Hypertension 3/ 1/ <.1 Diabetes Mellitus 5/ 11/.3 Obesity / 1/ <.1 HF Adm (1 st /multiple) 1/1 1/1 1. Hemodynamics Heart Rate (bpm) 8. ±.1 7. ±.3.95 LV Peak Systolic Pressure (mmhg) 11.9 ± ± 7. <.1 LV End Diastolic Pressure (mmhg) 1.9 ±.1.1 ± LV End Diastolic Volume Index (ml/m ) 119. ± ± 3.9 <.1 LV Ejection Fraction (%) 33.5 ± ±. <.1 Cardiac Index (l/min/m ).1 ±.1.7 ±.1 <.1 LV Wall Thickness (mm) 9. ± ±.3 <.1 LV Mass Index (g/m ) 13.7 ± ± LVMI/LVEDVI ratio 1.9 ± ±.1 <.1 (van Heerebeek L., Borbely A,, Paulus WJ. Circulation, ;113: ) Cardiomyocyte hypertrophy in Myocyte e diameter ( m) SHF systolic heart failure diastolic heart failure (van Heerebeek L. et al. Circulation, ;113: ) Decreased myofibrillar density in SHF SHF SHF systolic heart failure diastolic heart failure (van Heerebeek L. et al. Circulation, ;113: ) 9

10 Cardiomyocyte F passive, PKA (van Heerebeek L. et al. Circulation, ;113: ) Summary vs. SHF Myofibrill. density Ca + - sensitivity PKA-induced Ca + desensitization CM hypertrophy CM stiffness Ca + - sensitivity Myocardial contractility Myocardial stiffness (van Heerebeek L. et al., Circulation, ;113: ) 3 Diabetes mellitus (DM) Cardiomyocyte function? Extracellular matrix?? AGEs deposition 1

11 Increased AGEs deposition and fibrosis in DM + SHF (*P<.1) AGEs deposition SHF SHF CML (AGE) in DM + SHF Collagen volume fraction (%) (#P<.1) SHF SHF (van Heerebeek L. et al. Circulation, 8; 117: 3-51.) Increased F passive and Z-line thickness in DM + (*P<.1) + PKA SHF SHF SHF SHF (+1.9%, P=.5) DM + vs. DM - (+15.%, P<.1) Z-line -actinin-immunofluorescence EM (van Heerebeek L. et al. Circulation, 8; 117: 3-51.) 3 Summary DM Mechanisms responsible for the increased diastolic stiffness of the diabetic heart differ in and SHF: fibrosis and AGEs are more important in SHF, whereas cardiomyocyte resting tension is more important in. (van Heerebeek L. et al. Circulation, 8; 117: 3-51.) 11

12 What mechanisms underly elevated cardiomyocyte resting tension in heart failure? 1. changes in titin isoform composition and/or phosphorylation. altered titin-actin interaction 3. weakly-bound actin-myosin cross-bridges. oxidative stress 5.? 1. The role of titin in the myocardial stiffness Titin acts as a bidirectional spring affecting diastolic recoil and distensibility of cardiac muscle strips through isoform (NBA, NB) shifts and protein kinasemediated (PKA and PKG) phosphorylation. PKG PKA Freiburg et al. Circ Res. ; 8: Controversy between titin isoform composition and F passive CON HF AS HFREF SHF HFNEF P<.5 vs. CON NBA/NB.8... SYPRO Ruby NBA 3.3 MDa NB 3 MDa F passive (kn/m ). CON AS HF?? CON AS HF P<.5 HF vs. AS 1

13 The stiff NB titin isoform is hypophosphorylated in HF Titin MyBP-C AS HF AS HF AS HF Pro-Q Diamond AS HF Titin NBA NB P-NBA/P-NB AS * HF *P<.5 Desmin TnT *TnI MLC- Pro-Q Diamond SYPRO Ruby SYPRO Ruby NBA NB P-NBA/P-NB 1. AS HF.8... R=.8 P< NBA/NB Both PKA and PKG lower the elevated F passive in HF ive (kn/m ) F passi # ive (kn/m ) F pass 8 ## CON +PKA AS +PKA HF +PKA #P<.5 HF + PKG + PKA ## P<.5. The role of titin-actin interaction Biophysical Journal, 1997, 73: Treatment with Gelsolin FX-5 fragment 1 min 1 min 1 min 1 min Trombitas K, Granzier H., Am J Physiol., 1997, 73:C-7. 13

14 Distinct effect of gelsolin on F passive in HF and AS ve force (kn/m ) Passiv 8 HF * After gelsolin After PKA ve force (kn/m ) Passiv 8 AS * After gelsolin After PKA * P=.1 after PKA vs. after gelsolin (n=5) * P=.3 after vs. before gelsolin (n=5) 3. Weakly-bound actin-myosin cross-bridges,3-butanedione monoxime (BDM) reversible inhibitor of actin-myosin cross-bridge cycle force (kn/m ) 8 Passive HF + BDM (n=) mm BDM for 5 min Weakly-bound actin-myosin cross-bridges are unlikely to contribute to the elevated F passive in HF. The role of oxidative damage Peroxynitrite-induced -actinin nitration increases F passive of human cardiomyocytes by ~15%. (Borbély A et al., Cardiovasc Res, 7:5 33. (5)) Oxidation of myofilament protein sulfhydryl groups by,-dithiodipyridine (DTDP) raises cardiomyocyte F passive by ~8% (oxidation of myosin light chain-1 and actin). (Hertelendi Z. et al. Antioxid and Redox Signal, 7: (8)). To compare nitrotyrosine levels and localization in LV myocardial samples from: - explanted Donor hearts - end stage failing hearts of patients with ischaemic cardiomyopathy - patients with aortic stenosis - patients with - patients with SHF Data under evaluation 1

15 General conclusions Differences in LV myocardial structure and function support the clinical distinction of heart failure patients into SHF and phenotypes. The correction of the elevated F passive by PKA and PKG suggests that reduced phosphorylation of the NB titin isoform is involved in HF. To clarify the relative contribution and the physiological role of actin-titin interaction and the oxidative damage to the elevated F passive in HF requires further investigation. Clinical relevance The observed pathophysiological alterations require specific treatment strategies for patients with, in whom current therapy fails to improve prognosis. : An Inconvenient Truth! 15