Molecular Mechanisms of Contractile Dysfunction in Atrial Fibrillation

Derailment of proteostasis underlies structural and contractile dysfunction in AF

Bianca Brundel, PhD

Department of Physiology VUmc, and Clinical Pharmacy and Pharmacology UMCG

Atrial Fibrillation (AF)

• The most common cardiac arrhythmia

• Impact on patient • QoL • Mortality/morbidity • Stroke • Heart failure

• Causes of AF • Congenital heart disease (up to 30%) • Hypertension • Diabetes/Obesity

AF progression

Normal Paroxysmal Permanent

Ion-channel blockers

• Electrical remodeling: shortening APD • Changes in calcium handling Role of Calcium Handling in AF progression

Voigt, Europace 2012 AF progression

Normal Paroxysmal Permanent

Ion-channel blockers

Normal Permanent AF

Brundel, CVR 2002 Sustainable Structural damage (myolysis) Structural changes lead to contractile dysfunction and AF progression Brundel CVR 2002, De Groot and Allessie Circ 2010 Structural damage is rooted in derailment of proteostasis

Proteostasis: is homeostasis in expression, folding and function

Changes in expression

HDAC6 tubulin

Hartl, Nature 2011, Balch Science 2009 Tachypaced HL-1 mouse atrial cardiomyocytes

Control TP

Control 4 hrs TP

CaT

CS

Brundel CVR 2004, Circ Res 2006, JMCC 2006 Tachypaced Drosophila pupae

Zhang D, JMCC 2011 Den Hoed M, Nature Genetics, 2013 Zhang D, Circulation 2014 Tachypaced Drosophila pupae

Before

Before

During During

After

After

• overexpression/siRNA • Drug screen Role of Heat Shock in derailment of proteostasis and AF progression

HSP27 binds and stabilizes sarcomers expression is induced in paroxysmal AF and gets exhausted in persistent AF

Hsp27

SR PAF

CAF

% structural damage

Brundel, JMCC 2006 HSP27 represents a druggable target in AF

• HSP overexpression

Control TP

CaT

CS

HSP27

140

120 NP 100 pSP64 pHSP27-wt 80 *** *** pHSP27-uP-A 60 *** pHSP27-P-D pHSP70

% CaT amplitude CaT % 40

20 0 2 3 4 Duration pacing (hrs) TP TP HSP27

12 Brundel Circ Res 2006, JMCC 2006, 2008, PLosOne 2011 HSP27 represents a druggable target in AF

• HSP27 was overexpressed only in the heart Control TP

• HSP27 levels are reduced in persistent AF patients

HSP27 • Overexpression of HSP27 protects against contractile dysfunction

• HSP27 represents a druggable target?

TP TP HSP27 Prevent derailment of proteostasis by HDAC inhibition

Deli Zhang

Histone deacetylases (HDAC) HDACs involved in pathological cardiac remodeling

deacetylate histones

(McKinsey TA, 2007,2012; Gupta et al. 2008; Liu F et al. 2008) HDACs involved in pathological cardiac remodeling

Bind MEF2

Nuclear export

(McKinsey TA, 2007,2012; Gupta et al. 2008; Liu F et al. 2008) HDACs involved in pathological cardiac remodeling

Cytosolic

Contractile proteins

(McKinsey TA, 2007,2012; Gupta et al. 2008; Liu F et al. 2008) HDACs involved in pathological cardiac remodeling

Metabolic homeostasis

(McKinsey TA, 2007,2012; Gupta et al. 2008; Liu F et al. 2008) HDACs involved in pathological cardiac remodeling

Cardiac stress

HDACs↑ HDACi HDACI/IIa HDACIIb

Histones/nuclear export Structural & contractile proteins

Pathological Contractile dysfunction

Cardiac remodeling

(McKinsey TA, 2007,2012; Gupta et al. 2008; Liu F et al. 2008) Tubacin and Nicotinamide protect against tachypacing-induced remodeling in HL-1 atrial cardiomyocytes

HDAC inhibitors HDACs Trichostatin A (TSA) I (1,2,3,8), IIa (4,5,7,9), IIb (6,10)& IV (11)

Sodium Butyrate (SoBu) I (1,2,3,8), IIa (4,5,7,9) Nicotinamide (Nic) III (Sirtuins1-7) Tubacin HDAC6 (IIb) HL-1 atrial cardiomyocytes

NP (normal pacing)

2.5 2.0 1.5

CaT (F1/F0) 1.0

TP (tachypacing ) 2.5 # # 2.0 1.5 *

CaT (F1/F0) 1.0 Control TSA SoBu Nic Tubacin

Zhang D. Circulation 2014 Tubacin and Nicotinamide protect against tachypacing-induced remodeling in Drosophila

After TP

# #

***

Zhang D. Circulation 2014 Overview effects of HDAC inhibitors in tachypaced HL-1 cardiomyocytes and Drosophila model

Zhang D. Circulation 2014 Inhibition of Tubulin DAC (TDAC) catalytic domain of HDAC6 rescues tachypacing-induced CaT reduction

HDAC TDAC A HDAC6 wt

HDAC6 m1 DN HDAC6 m2 DN HA tag HDAC6 m1-2 DN DN GADPH HDAC6 deacetylates tubulin and promotes microtubule depolymerization

HDAC6

(Matsuyama, Shimazu et al. 2002)

. Microtubules are important regulators of calcium signaling in the heart (F.C. Howarth, et al, 1999; B.G.Kerfant, 2001).

. Taxol, a microtubule stabilizer, prevents atrial fibrillation in rabbit (Xiao, Zhang et al, 2010).

Is the protective effect of HDAC6 inhibition in AF via modulation of microtubules? Tubacin protects against tachypacing-induced depolymerization of microtubules

NP AF (4Hz)

Tubulin Ac-Tub merge Tubulin Ac-tub merge

Con

Tubacin

Zhang D. Circulation 2014 Patients with AF reveal reduced amount of (acetyl) tubulin, especially in the LAA

SR PAF PeAF

R L R L R L R L R L R L R L R L R L R L Tub A-Tub GADPH

P<0.001 P<0.001 P=0.03 P=0.02

Tub Ac-tub

Zhang D. Circulation 2014 HDAC6 is activated in AF

SR PeAF HDAC6 GADPH 1.2 150 * * 1.0

• HDAC6 is activated in AF patients0.8 100 0.6 50 0.4

• HDAC6 causes contractileHDAC6/GADPH dysfuntion 0.2 HDAC6 activity ( a. u.) ( a. activity HDAC6 0 0.0 • Is HDAC6SR aAF druggable target in AF?SR PeAF

Zhang D. Circulation 2014 Prevent derailment of proteostasis by inhbition of ER stress- induced

Marit Wiersma Autophagy

‣ Autophagy in the heart • Cell survival • Cell death • Impaired cardiomyocyte function

‣ Basal activity in every cell: • Cellular quality control • Maintenance of cellular energetic balance

Tachypacing induces autophagy in cardiomyocytes Activation of autophagy: which pathway?

mTOR

mTORC1 mTORC2

mTOR-P2448S mTOR-P2481S

S6RP-P235-236S Akt-P473S

ER stress

Autophagy ER stress signaling activates autophagy in AF

IRE1 HSPA5 HSPA5

ATF6 HSPA5 HSPA5

P eIF2α PERK HSPA5 HSPA5

ER stress sensors

ATF4 ATG12 HSPA5

autophagy Inhibition of ER stress protects against contractile dysfunction in vitro

ER autophagy inhibitors ER stress inhibitor overexpression Inhibition of ER stress protects against contractile dysfunction in Drosophila

NP TP Control Control PepA BAF 4PBA

autophagy inhibitors ER stress inhibitor

NP TP 120

100 ### ### 80 *** 60

40

Heart rate (% basal) (% of rate Heart 20

0 C C PepA BAF 4PBA Is autophagy present in human AF? Patients with persistent AF reveal autophagy and ER stress

• Activation of autophagy by upstream ER stress in patients and experimental models

• ER stress as a druggable target in AF?

Conclusions

• Tachypaced HL1 cardiomyocytes and Drosophila are useful to • uncover mechanism underlying AF progression: key role derailment of proteostasis • Identify novel druggable targets, including • HSP27 • HDAC6 • ER stress

• Findings were confirmed in patients with paroxysmal and persistent AF

• Proof-Of-Concept for druggable targets: in dog model for AF

Next presentation: Molecular Intervention in Atrial Fibrillation Department of Physiology, VUmc Amsterdam and Pharmacy and Clinical Pharmacology, UMCG Groningen

Femke Hoogstra-Berends, Marit Wiersma, 2016 Denise van Marion, Xu Hu, Deli Zhang, Robert Henning, Bianca Brundel

MHI, Canada: Stanley Nattel and XiaoYan Qi

2013T096, 2013T144, 2013T088 고맙습니다 질문?