Cardiac electrophysiology and genetics:

the molecular determinants of cardiac

arrhythmias

Silvia Giuliana Priori

Dept. of Molecular Medicine Univ. Pavia
and
Molecular Cardiology Labs, IRCCS Fondazione
Maugeri
Cells Have Membranes

2
Cardiac Myocytes have “special membranes”
They can transmit and generate electricity

Myocytes are excitable cells due to the presence of a

variety of ion channels in the plasmalemma
Channels

Pore

Filter

Gate
Patch Clamping
 Patch clamp experiment set up

MICROELECTRODE

Microscope

micromanipulator

compute
Cell chamber r

oscilloscope

amplifier

Faraday cage
 Single channel recording

Amplifier

Glass micropipette (recording

electrode)  10-6 m
Seal resistance: >1 GW

Example of microscopic current

recorded from one or few channels
in a membrance patch.

Recorded currents are in the order of hundreds of nA up to 10 pA

(when multiple channels are present in the patch
 Action Potentials in the Heart
0.12-0.2 s approx. 0.44 s

PR QT
ECG Superior
vena cava Aortic artery

SA Pulmonary artery
Pulmonary
veins AV node
SA node Left atrium

Atrial muscle Mitral valve

Atria
AV Specialized
conducting Interventricular
tissue septum

Tricuspid valve Purkinje

Purkinje
Ventricluar fibers
muscle
Ventricle Inferior Descending aorta
vena cava
 Cardiac ion channels and
electrophysiological substrate
• Myocardial excitation and conducrion of the
electrical signal (action potential) depend on
the activation of voltage-gated channels.
• Voltage-dependent, means that the
conductance (ion permeability) of the
channels depends on the membrane voltage.
 Biophysical parameters

• Ion channel conductance is controlled through

distinct biophysical processes:
– Activation
– Inactivation
 Voltage – dependent activation

• Is the process in which the ion channel

opening is regulated by changes of membrane
voltage (difference of potentials between
cytosol and extracellular space)
 Voltage – dependent inactivation

• Ion channel closing is related to membrane

voltage but it has also a time-dependent
component.

• Activation and inactivation kinetics are also

regulated by several mediators (e.g.
catecholamines, ReDox status, etc)
Ventricular action
potential
 Classification of potassium channels
• Large molecular diversity
• Multiple genes and structures (alpha and beta subunits)
• We may identify groups based on the number of
transmembrane segments
– 6 TM
– 4 TM
– 2 TM
– 7TM
• In the heart the most important ion channels have six or
two TM
• K+ channels are almost invariably associated with beta
subunits.
Cardiac Potassium channels
Potassium channels Subunits
Cardiac Voltage-gated K+ Channels

Inactivating K+ channels (Ito)

“Ultra-rapid” K+ channels (IKur)

“Rapid” K+ channels (IKr)
“Slow” K+ channels (IKs)

Inward Rectifier K+ channels (Ik1)

 6TM Potassium channels
• The 6TM proteins include KCNH2 & KCNQ1 genes
the KV channels, and the Encode channels conducting
Ikr and Iks currents and they
related small-conductance have a 6TM structure
and intermediate
conductance, Ca2+-
activated K+ channels
(KCa).
• The functional channel is
formed by the tetrameric
association of these
6TM/1P subunits.
 Potassium channel kinetic
IKr IKs

The delayed rectifier IK is an important contributor to the time

course of terminal repolarization in cardiac cells.
It has two components RAPID and SLOW.
Ikr and IKs potassium currents provide the principal repolarizing
force in human ventricular myocytes.
IKr is quantitatively prominent but IKs is very important during
sympathetic activation (catecholamine-sensitive component)
 2TM potassium channels

• The 2TM K+ channels include the inward

rectifiers, the KATP channels and the G protein-
coupled channels
• The N- and C-termini of these channels are
located in the cytosol.
• The P-region between the two transmembrane
domains forms the pore, and the functional
channel is a tetramer of these 2TM/1P subunits.
Crystal structure of 2Tm K+ Channels
Kir 2.1 - Ik1
Kir 3.1+3.4 - Ik(Ach)
Kir 6.2+SUR- Ik(ATP)
 Inward Rectifier K+ channels (IK1)
IK1 stabilizes the resting membrane potential and is responsible
for shaping the initial depolarization and final repolarization of
the action potential (controls phase 4).
It is generated by the 2TM channel Kir 2.1

IK1
Figure 1. Family pedigree and ECG recordings in the SQTS family with KCNJ2 mutation.

Short QT Syndrome

Priori et al. Circ Res. 2005;96:800-807

Copyright © American Heart Association, Inc. All rights reserved.

Figure 2. A, Wild-type (top trace) and mutated (bottom trace) DNA sequences performed on
genomic DNA of individuals II-2 and III-1 (see Figure 1).

Silvia G. Priori et al. Circ Res. 2005;96:800-807

Copyright © American Heart Association, Inc. All rights reserved.

 Short QT Syndrome
type 3: KCNJ2

0.6
* * *
0.4
* 0.2
Voltage (mV)
-120 -100 -80 -60 -40 -20 0 20

K1
-0.2

Normalized I
-0.4

-0.6
WT (n=5) -0.8
Priori et al Circ Res 2005 D172N (n=7)
-1.0

-1.2
 Cardiac Na+ Channels

• Almost identical to nerve Na+ channels

(structurally and functionally)
• Expressed in all “non nodal” cardiac cells
• Responsible for initiating and propagating
the action potential.
• Mutations in the gene encoding for the
Cardiac Na+ Sodium channel’s alpha
subunit cause several inherited arrhythmias
 Voltage-Gated Sodium Channels

Physiology
Generation and propagation of action potentials
Pharmacology
Targets for local anesthetics (Class I antiarrhythmic drugs,
anticonvulsants
Genetics
Inherited disorders leading to arrhythmias and CMD
 Ion selectivity
Structure of the sodium channel

a b
+ + + +
+ + + +
+ + + +
+ + + +

Inactivation

Voltage sensing

Positive charges (K, R)

In the S4 segments
Activation and inactivation of the sodium channel
Na+ Na+ Na+

Out

activation inactivation

deactivation

In

Closed Activated Inactivated

–10 mV
Membrane voltage
–120 mV

1 A
INa

1 ms
 Molecular Cardiology
Laboratories, Fondazione
Salvatore Maugeri, IRCCS -
PAVIA, ITALY
 LQT3 mutations prolong APD by increasing Late INa

Normal Late INa Augmented Late INa

(delayed or incomplete inactivation)
0 0

Late Sodium Current (INa)

Late
nA

Peak Peak
Early after-
depolarization

Action Potential
mV
 In 1996 we hypothesized that sodium channel blockers may reverse APD
Prolongation induced by late INa
We used a toxin called Anthopleurin that increases INa to test our hypothesis

A= Resting APD
B= Anthopleurin
C= Mexiletine

A0A
Exposure to mexiletine
reverts APD prolongation
caused by Anthopleurin that
mimics LQT3

Priori et al. Circulation Research. 1996

 Sensitive to Mexiletine
Before After
QTc 480ms QTc 420ms
R1626P

QTc 570ms QTc 493ms

P1332L

QTc 455ms
P1332L QTc 506ms
• Mexiletine: does it affect arrhythmic events?

• The study population 34 LQT3 pts :

• 56% males,
• Median age 22 years (IQR 8-44),
• Mean QTc shortening 63±6 ms, p<0.0001
• Mexiletine daily dose of 8±0.5 mg/Kg.
• observation time 36 months before and after therapy
• patients with arrhythmic events from 22% to 3%,
p=0.031
• arrhythmic events/ pt ( 0.43±0.17 to 0.03±0.03,
p=0.027
• annual rate of arrhythmic events 10.3% to 0.7%,
p=0.0097.
Mazzanti et al JACC 2016

Baseline ECG First “ECG on Therapy”

Figure 4A % Patients with arrhythmias Figure 4B Arrhythmic events/patient

95% CI 7-37 0.43±0.17

p = 0.031 p = 0.027

95% CI 0-9
0.03±0.03

Figure 4C
Events per 100 person-years
95% CI 4.6-23
Mazzanti et al JACC 2016
95% CI 0.1-5.5
p = 0.0097

Mexiletine as a targeted therapy

For patients with loss of function
Mutations in the SCN5A gene.
 Cardiac Ca2+ Channels

• L-type (dihydropyridine sensitive)

• Structurally rather similar to Na+ channels
• Some functional similarity to Na+ channels
• depolarization opens Ca2+ channels
• Functionally different from Na+ channels
• slower to open
• very slow, rather incomplete inactivation
• generates much less current flow
 Role of Cardiac Ca2+ channels

• Nodal cells
– initiate and propagate action potentials- SLOW
• Non nodal cells
– controls action potential duration
– contraction
Ion channels in clinical
cardiology
 The role of ion channels
• Ion channels control cardiac excitability.
• Abnormal function of ion channels is
responsible for the onset of cardiac rhythm
disorders and it can be secondary to acquired
cardiac diseases or it may be caused by
primary disorders of ion channels (mutations)-
• This latter case is that of cardiac
“channelopathies”
 Inherited arrhythmias: an expanding field

• Long QT syndrome
• Brugada syndrome
• Catecholaminergic Polymorphic Ventricular
Tachycardia
• Short QT syndrome
• Timothy syndrome
• Andersen syndrome
• Progressive conduction defect
• Early repolarization syndrome
• …………….
 Diseases, Age and Manifestations
AGE (years of age)

0 10 20 30 40 50
CPVT

LQTS

SQTS
ARVC

BrS
 Calcium Current
Sodium current (INa): (ICa-l):
-Romano Ward syndrome - Timothy syndrome
-Brugada syndrome - Brugada syndrome
-Conduction defect - Short QT syndrome
-Atrial fibrillation SCN5A - Early repolarization
CACNA2c Extracellular
CAV3 SCN4B Intracellular
SCN1B CACNB2
SCN3B MOG1
SNTA1 ANKB
SR calcium release:
GPD1-L CACNA2D1
- Catecholaminergic
Polymorphic VT
Inward rectifier (IK1):
- Andersen syndrome
- Short QT syndrome
CASQ2 - Catecholaminergic polymorphic VT
RyR2

Slow delayed rectifier

(IKs):
- Romano Ward syndrome
-Jervell and Lange Nielsen Transient outward
syndrome current (Ito):
-Short QT syndrome - Brugada syndrome
AKAP9

KCNE1 KCNE2 KCNE5

KCNJ2
KCNQ1
KCND3
KCNH2 KCNE3
Intracellular
Fast delayed rectifier Extracellular

(IKr):
- Romano Ward syndrome
-Short QT syndrome

Napolitano C, Priori SG,Circulation, 2012

 Mechanisms and phenotypes
• Mutations of cardiac ion channels may cause a
spectrum of biophysical manifestations which can
be grossly classified as:
– Loss of function
– Gain of function
– Overlap phenotypes

• Loss of function mutations are largely prevalent

(and genetically more likely to happen given the
complexity of the molecular structures involved)
 Mechanisms and phenotypes
• The resulting clinical phenotype depend on
the effect of the mutation on the cardiac
action potential duration.
– Action potential prolongation: increase of
depolarizing currents or reduction of ripolarizing
currents
– Action potential shortening: reduction of
depolarizing currents or increase of repolarizing
currents
Action potential duration: ionic determinants
K+

- -+- +- -+-+- +
+ - - +- -+

+ -+-+- +- -+
+ -+-+- +- -+

+- -+ -+-+- +
- -+- +- -+-

Na +Ca ++

Na +
 How a Long QT is generated?
Decreased
Repolarising Currents

Increased
Depolarising Currents
 ECG in long QT syndrome
D2

V2

V5
Torsade de Pointes
Multiple genes and mechanisms for similar phenotypes  LQTS
 KEY GENES IN LQTS
Gene Locus Protein % of Disease
Long QT Syndrome (LQTS)
IKs potassium
channel alpha
KCNQ1 (LQT1) 11p15.5 subunit (Kv7.1) 30-35%
IKr potassium
channel alpha
subunit (Kv11.1 or
KCNH2 (LQT2) 7q35-q36 hERG) 25-40%
Cardiac sodium
channel alpha
SCN5A (LQT3) 3p21 subunit (NaV1.5) 5-10%

HRS / EHRA Expert Consensus Statement on the State of Genetic Testing

for the Channelopathies and Cardiomyopathies HRJ - 2011
Short QT Syndrome
 How a Short QT is generated?
Increased
Repolarising Currents

Reduced
Depolarising Currents
 Yield of genetic testing in SQTS
Variant Gene Chromosome Protein Current Effect

SQTS1 KCNH2 7q35-36 HERG IKr Gain of

function
SQTS2 KCNQ1 11p15.5 KCNQ1 IKs Gain of
function
SQTS3 KCNJ2 17q23 Kir2.1 IK1 Gain of
function
SQTS4 CACNA1c 12p13.3 CaV1.2 ICa Loss of
function
•
SQTS5
25 SQTS
CACNB2
probands
10p12
27 FM
CaVbeta2 ICa Loss of
• Mean QTc 303±13ms function

– 1 KCNQ1, 1 KCNH2, 1KCNJ2, 1 CACNA1c

• THIS YEAR (2016-2017) THE
ELECTROPHYSIOLOGY OF BRUGADA
SYNDROME WILL BE PRESENTED ON
NOVEMBER 3 AND ELECTROPHYSIOLOGY OF
CATECHOLAMINERGIC POLYMORPHIC VT WILL
BE PRESENTED ON NOVEMBER 9.
The molecular basis of the
electrocardiogram
 A novel concept:
heritability of human electrocardiogram

• The genetic background of each one of us can influence

the electrophysiological substrate (an susceptibility to
arrhythmias) in the absence of a mutation.
• Heritability represents a relevant component of the human
electrocardiogram
• In the general population, P wave, PR, QT interval duration have on
average 25-40% of their variability which is attributable to genetic
factors.
• The identification of common genetic variants responsible for this
variability would have important clinical implications.
• Single SNPs are usually associated with small effect. This implies the
evaluation of large cohorts of patients in order to detect significant
signals and differentiate them from “noise”.
 Mutations vs Polymorphisms
• Not only single genetic mutations but also
common genetic variants influence the
electrocardiogram and the shape of
repolarization.
• Single nucleotide polymorphisms (SNPS)
participate to the definition of major ECG
parameters
Pleiotropic associations
of ECG variables

Sotoodehnia N et al Nature Genetics 2010

GWAS of QRS complex duration in 40,407 healthy
subjects

Sotoodehnia N et al Nature Genetics 2010

 Most significantly
associated SNPs at 10
identified loci

ADDITIVE EFFECT
Combined effect of QT interval prolonging
alleles in unrelated individuals
 CELLULAR ELECTROPHYSIOLOGY
• The cardaci excitability Is determined by multiple ion
channels.
• Ion channels have large molecular diversity in order
to finely regulate the electrical properties and to
enable the adaptation to a changing environment
(e.g. change in heart rate, autonomic tone, etc).
• Genetic variation (mutations and SNPs) may alter the
function and cause an arrhythmogenic substrate.