Model features3 signaling compartments (caveolae, extra-caveolar membrane and cytosol) each with a local signaling cascadeAKAPs that produce local PKA and PP1 concentrations for ICaL, IKs and RyRFeedback loops for receptor desensitization, PDE stimulation and PP1 inhibition (through inhibitor protein 1)7 Substrates that are directly altered by phosphorylation: ICaL, IKs, INa, INaK, PLB, RyR and TnI

Combined with a recently published model of the canine epicardial action potential (Decker et al., Am. J. Physiol. 2009) through populations of phosphorylated and non-phosphorylated channels with distinct electrophysiological characteristics

Jordi Heijman (1,2), Paul G.A. Volders (2), Ronald L. Westra (2) and Yoram Rudy (1)

Cardiac Bioelectricity and Arrhythmia Center (CBAC)

(1) Washington University, St. Louis, MO, USA; (2) Maastricht University, Maastricht, The NetherlandsDisclosures: none (all authors)

This work has been supported by Washington University in St. Louis Cardiac Bioelectricity and Arrhythmia Center and the NIH-NHLBI Grants R37-HL33343 and RO1-HL49054. Financial support from the Hein Wellens Foundation to J.H. is gratefully acknowledged

http://cbac.wustl.eduhttp://rudylab.wustl.edu

Electrophysiological characteristicsAdjusted electrophysiological properties for phosphorylated substrates:

IKs: left-shift and increase in tail-current I-V relation, increased rate of activation, decreased rate of deactivation1,2 ICaL: increase and left-shift in peak I-V, left-shift of inactivation3

INa: increase in peak current, left-shift in activation and inactivation I-V curve4 PLB, TnI, INaK: adjustment of affinities based on experimental data5,6

1. Chen-Goodspeed et al., J Biol. Chem. 2005. 280, 1808 2. Freedman et al., J. Biol. Chem. 1996. 270, 179533. Steinberg et al., Circulation. 1995. 4. Yu et al., Circulation. 2001. 102, 25355. Hohl and Li, Circ. Res. 1991. 69, 13696. Beavo et al., PNAS. 1974. 71, 3580

β-Adrenergic signaling characteristicsComparison of the signaling model (solid lines) with experimental data (markers) shows excellent agreement

7. Kuschel et al., PNAS. 1974. 71, 3580 8. Stengl et al., Cardiovasc. Res. 2006. 72, 90 9. Nagykaldi et al., Am. J. Physiol. 2000. 279, H132910. Gao et al., J. Physiol. 1992. 449, 689 11. Sulakhe and Vo., Mol. Cell. Biochem. 1995. 149, 103 12. Kirstein et al., Eur. J. Physiol. 1996. 431, 395

1. Volders et al., Circulation 2003. 280, 1808

2. Stengl et al., J. Physiol. 2003. 551, 777

3. Antoons et al., J. Physiol. 2007. 579, 147

4. Baba et al., Cardiovasc. Res. 2004. 64, 260

5. Odermatt et al., J. Biol. Chem. 1996. 271, 14206

6. Robertson et al., J. Biol. Chem. 1982. 257, 260

Block of IKs, mimicking LQT1

AKAP / Channel disruption

IKs block has only a modest effect on APD under baseline conditions (i.e. without ISO)After ISO application APD increase under IKs block is much larger. This is most pronounced at slow rates

The IKs AKAP Yotiao provides passive regulation through local PKA and PP1 concentrations but is also critical for active phosphorylation

Mutations that disrupt IKs / Yotiao interaction prevent phosphorylation of the IKs channelThe model shows that this increases APD compared to normal ISO application, particularly in the case of complete IKs / Yotiao disruption

In many cardiac conditions, arrhythmogenic events occur during increased adrenergic tone At the cellular level stimulation of β-adrenergic receptors initiates a complex cascade, resulting in the phosphorylation of a diverse number of substratesBecause of the physiological importance of this system, many feedback loops exist to ensure precise control of this cascade. Recent experimental evidence indicates that localized signaling is critical for this specific controlOur objective was to develop a detailed compartmental computational model to analyze the (patho)physiological effects of β-adrenergic stimulation at the cellular level

NSR

JSRICa,L

Iup

PLB

Irel

INaK IKs

Troponin

TnI

INa

β1ARL βγ Gsα

AC5/6

cAMP

PDE 3/4

PP1 /2A

Gsα

C

CC

CGRK

PKI

β1ARL

Gsα

GRK

AC5/6

AC4/7Gsα

cAMP

CCC

i1PP1

PP2A

β1ARL

Gsα

AC4/7Gsα

GRK

PP1

cAMP

PDE2PDE4

CC

C

C

PKI

C

PDE2

PDE 3/4

PKI

JCav/Ecav

JCav/cyt

JEcav/cytAKA

P

PDE 2

AKA

P

AKAPInhibitionStimulation

βγ

βγ

Abbreviations:AC : adenylyl cyclase; AKAP : A-kinase anchoring protein; AP : action potential; cAMP :

cyclic AMP; CaT : calcium transient; Gs : stimulatory G-protein; ICaL : L-type calcium current; IKs : slow delayed rectifier potassium current; INa : sodium current; INaK : sodium-potassium pump; ISO : isoproterenol; PDE : phosphodiesterase; PKA : protein kinase A; PKI : protein kinase inhibitor; PP1/PP2A : protein phosphatase 1 / 2A; RyR : ryanodine receptor; TnI : troponin I;

We present a novel model that integrates a compartmental β-adrenergic signaling model with the canine ventricular action potentialThe model reproduces a wide variety of experimental data at both the subcellular signaling level and the level of the cellular electrophysiologyISO stimulation decreases APD. This effect is much more pronounced at slow rates. CaT amplitude is increased at all rates, but in particular at fast ratesIKs block has only a minor effect under baseline conditions but increases significantly under β -adrenergic stimulation, highlighting the importance of this current in cardiac (patho)physiology.Local signaling complexes (e.g. through the AKAP Yotiao) play a critical role in effective adrenergic modulation of various substrates

AP and CaT morphology Validation

Charpentier et al., Am. J. Physiol. 1996. 271, H1174

Stengl et al., J. Physiol. 2003. 551, 777

APD Rate dependenceAPD rate dependence is qualitatively similar to experimental results in canine ventricular myocytes (using different perfusate and pipette solutions):

1. Yamada and Corr, J. Cardiovasc. Electrophysiol. 1992. 3, 209

2. Waggoner et al. ,Am. J. Physiol. 2009. 296, H698

CL = 1000 ms

CL = 300 ms CL = 2000 ms

Application of a saturating dose of ISO decreases APD, particularly at slow rates and increases CaT amplitude, most prominently at fast rates.AP morphology, CaT amplitude and rate of decay are in good agreement with experimental data

CL = 300 ms CL = 1000 ms CL = 2000 ms

CL = 2000 ms CL = 1000 ms

Gs – AC relationship

ISO – AC relationship

ISO – cAMP relationship

PKA - cAMP relationship ISO and time dependence of substrate phosphorylation

IKs Tail Current I-V ICaL Peak Current I-V ICaL Inactivation I-V

INa Peak Current I-V INa In/Activation I-V Other Substrates

CL = 2000 ms