Accepted Manuscript

Will cardiac optogenetics find the way through the obscure angles of heart
physiology?

Nicola Pianca, Tania Zaglia, Marco Mongillo

PII: S0006-291X(16)31978-7
DOI: 10.1016/j.bbrc.2016.11.104
Reference: YBBRC 36808

To appear in: Biochemical and Biophysical Research Communications

Received Date: 2 November 2016

Accepted Date: 17 November 2016

Please cite this article as: N. Pianca, T. Zaglia, M. Mongillo, Will cardiac optogenetics find the
way through the obscure angles of heart physiology?, Biochemical and Biophysical Research
Communications (2016), doi: 10.1016/j.bbrc.2016.11.104.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 ACCEPTED MANUSCRIPT

Will cardiac optogenetics find the way through the

obscure angles of heart physiology?

PT
Nicola Pianca1,2*, Tania Zaglia1,2*, Marco Mongillo1,2,3

RI
1
University of Padova, Department of Biomedical Sciences, via Ugo Bassi 58/B, 35131 Padova,
Italy; 2, Venetian Institute of Molecular Medicine, via G. Orus, 2, 35129, Padova, Italy; 3, CNR

SC
institute of Neurosciences, viale Colombo 3, 35133 Padova, Italy

U
AN
M
D
TE
C EP
AC

Correspondence to:
Marco Mongillo, MD, PhD
email: marco.mongillo@unipd.it
phone: +39 0497923229
Fax: +39 0497923250

1
 ACCEPTED MANUSCRIPT
ABSTRACT

Optogenetics is a technique exploded in the last 10 years, which revolutionized several areas of

biological research. The brightest side of this technology is the use of light to modulate non-

invasively, with high spatial resolution and millisecond time scale, excitable cells genetically

modified to express light-sensitive microbial ion channels (opsins). Neuroscience has first benefited

PT
from such fascinating strategy, in intact organisms. By shining light to specific neuronal

RI
subpopulations, optogenetics allowed unearth the mechanisms involved in cell-to-cell

communication within the context of intact organs, such as the brain, formed by complex neuronal

SC
circuits. More recently, scientists looked at optogenetics as a tool to answer some of the questions,

remained in the dark, of cardiovascular physiology. In this review, we focus on the application of

U
optogenetics in the study of the heart, a complex multicellular organ, homing different populations
AN
of excitable cells, spatially and functionally interconnected. Moving from the first proof-of-
M

principle works, published in 2010, to the present time, we discuss the in vitro and in vivo

applications of optogenetics for the study of electrophysiology of the different cardiac cell types,
D

and for the dissection of cellular mechanisms underlying arrhythmias. We also present how
TE

molecular biology and technology foster the evolution of cardiac optogenetics, with the aim to

further our understanding of fundamental questions in cardiac physiology and pathology. Finally,
EP

we confer about the therapeutic potential of such biotechnological strategy for the treatment of heart

rhythm disturbances (e.g. cardiac pacing, cardioversion).

C
AC

Keywords:
cardiac optogenetics;
channelrhodopsin-2;
cardiac arrhythmias;
heart physiology;

2
 ACCEPTED MANUSCRIPT
1. Introduction
The discovery that the biological activity of a large class of cells relies on changes in the

distribution of charges between the inner and outer side of the plasma membrane, regulating the so-

called membrane potential, has led to the gross classification of cell types into excitable and non-

excitable. Unsurprisingly, the functional study of physiology and pathophysiology of excitable

PT
cells, which notably include neurons and muscles, has made intense use of electricity, either applied

locally to single cells in vitro (i.e. with a patch clamp pipette) or delivered to an intact organ (i.e. via

RI
extracellular electrodes). Whilst these approaches uncovered the fundamentals of excitable cell

SC
biology, they have been suffering from invasiveness and lack of selectivity, which limit their utility

in the understanding of the function of specific cells in their complex physiologic environment,

U
often including several interconnected excitable cell types. The most visionary scientists in the
AN
field, had "been interested for some time in potential methods by which mammalian neurons

might be transfected with a gene whose product would permit light-triggering of depolarizations
M

and action potentials" (RY Tsien, in [1]), in other words, they were seeking strategies for non-

invasive control of excitable cell function. Once again, Nature itself brought us the solution,
D

represented by ion-channels sensitive to light, whose function is at the basis of the phototaxic
TE

behavior of unicellular organisms, mostly algae [2]. Not many could envisage that such simple
EP

organisms would offer the instruments to uncovering physiology of the most complicated

mammalians, and above all, humans. These tools are represented by the class of proteins,
C

collectively named opsins, which, combined to the progress in molecular biology, opened the
AC

revolutionary road of optogenetics.

2. Fundamentals of optogenetics
The prototypical opsin, and the most used so far, is Channelrhodopsin-2 (ChR2), a seven

transmembrane domain, light-gated cation channel, which undergoes a rapid conformational change

upon illumination with light at a relatively narrow range of excitation wavelengths, around 470nm.

When ChR2 is expressed on cell membranes, absorption of blue light opens the channel, triggering

3
 ACCEPTED MANUSCRIPT
an inward current, which results in membrane depolarization, rapidly reversible with the channel

closure in the dark [3-5]. These properties were exemplified by the initial works using ChR2 in

cultured neurons, demonstrating that the delivery of short flashes of blue light was able to elicit

action potentials and modulate neuronal activity, with elevated temporal resolution [6, 7]. Such

biotechnology was soon combined with transgenesis to transfer the approach to various model

PT
organisms ranging from C. elegans, to D. melanogaster, D. rerio, and rodents, demonstrating that

RI
cellular activity could be non-invasively modulated also in the complexity of intact, living organs

(in vivo optogenetics) [8-10]. The availability of different strategies to restrict expression of ChR2

SC
to a given cell lineage was exploited to selectively interrogate the physiology of specific circuits of

excitable cells, with high temporal and spatial precision [11, 12]. This profoundly innovative

U
biotechnology was initially applied to both neurophysiology studies and to address disease
AN
mechanisms [13, 14], and prompted scientists to the research of rhodopsins with enhanced
M

properties, which were later discovered or genetically engineered [15, 16]. As a result of these

studies, the optogenetics toolbox now includes: i) opsins which can be targeted, thanks to specific
D

promoters, to a variety of cell types; ii) opsin variants which, depending on their ionic selectivity,
TE

can be used to depolarize or hyperpolarize cell membranes (e.g. Na+ vs. Cl-); iii) spectral variants,

activated by light at different wavelengths (i.e. absorbing red light) and iv) fast-kinetics variants,
EP

allowing high photostimulation rates [17] (see Appendix).

In summary, the necessary ingredients of an in vivo optogenetics experiment are:

C
AC

i) the model organism, which can be selected among different species, and, in small rodents among

a wide variety of strains with cell type restricted opsin targeting (see Appendix).

ii) the light source. This can be as simple as a manually held light guide, suited for acute

photoactivation of superficial cells, or more complex, like stably implanted optrodes or light-

emitting cuffs for deep brain or peripheral neuron stimulation, respectively, in freely moving

animals (see Appendix).

4
 ACCEPTED MANUSCRIPT
iii) a suitable readout method which, depending on the experimental setting, ranges from

physiology assessments (EEG, behaviour, gait, muscle contraction force) to morphology and

biochemistry (cell signaling, differentiation and engraftment).

3. Optogenetics in cardiac physiology

PT
The central nervous system has been the initial and preferred target of the in vivo optogenetic

investigations in laboratory animals. This is due to the obvious interest in defining the

RI
neurobiological basis of brain function, with a method surpassing the intrinsic limitations of the

SC
existing experimental techniques. It is well appreciated that different neuron types establish specific

interactions to generate complex neural circuits, which underlie the neuro-anatomical basis of brain

U
function. The development and use of optogenetics, by enabling temporally precise control of the
AN
activity of neurons in living systems, has allowed understand the function and regulation of specific

circuits [11, 13]. The distinctive advantages of optogenetics have been applied to address, with
M

unprecedented specificity and accuracy, both basic neurophysiology, ranging from the proof-of-

principle control of voluntary movement [18, 19] to more complex phenomena involving behavior
D

and emotions [20, 21] and neuropathology, from neurodegenerative disease [14, 22], to the study of
TE

the mechanisms of addiction [23]. Similar to the complex functional architecture of the Central
EP

Nervous System, heart function also relies on the co-existence of different types of excitable cells,

namely the conducting and working cardiomyocytes (CMs), which distribute in the organ with a
C

precise topology, display peculiar electrophysiological properties, and are interconnected to ensure
AC

the correct cardiac performance. In addition to the intrinsic mechanisms regulating its function, the

adaptation of heart activity to the perfusional demand of the organism, is also regulated by extrinsic

factors, especially the autonomic nervous system neurons, which interact and modulate the

properties of their myocardial cellular targets. The dysfunction of the intrinsic and extrinsic

myocardial cells, as well as of their regulated interactions, is implicated in several cardiovascular

diseases, including arrhythmias and heart failure [24-26]. Most molecular cardiology research has

5
 ACCEPTED MANUSCRIPT
meticulously studied isolated heart cells in vitro, or has focused on global cardiac morphology, but

the behaviour of the integrated regulatory cellular network of the heart remained largely

unexplored, mainly due to technical difficulties. In fact, the dissection of the selective role of the

individual myocardial cell types in vivo, using conventional electrical stimulation, is limited by lack

of specificity, and the complex heart cytoarchitecture, in which differently connected excitable cells

PT
often co-exist within the same region. With this in mind, cardiac optogenetics extends beyond an

RI
alternative to electrical pacing and, thanks to its cell type specificity, the high spatial flexibility, and

its minimal invasiveness, represents an invaluable tool to answer unresolved questions in cardiac

SC
physiology and disease mechanisms (Figure 1).

U
3.1 Physiology of the intrinsic heart regulation
AN
Optogenetics was brought to light in cardiology research by two proof-of-principle studies, which

were published simultaneously in 2010, in mouse and zebrafish models, respectively [27, 28].
M

These reports have demonstrated that optogenetics could be used to control cardiac rhythm,

generate ectopic sources of heart activation, or interfere with the cardiac conduction system,
D

causing heart slowing or block. By showing, almost magically, that the heart rate could be
TE

modulated with a touch of light, these seminal studies set the basis for subsequent works, which
EP

exploited in full the properties of optogenetics to illuminate researchers on the function of the

specific cellular systems of the heart, and on their intercellular communication. Soon after these
C

demonstrations, a large number of investigators followed the light path of optogenetics in cultured
AC

or isolated heart cells in vitro, with scopes ranging from the identification of strategies to improve

the cardiac differentiation of embryonic/induced pluripotent stem cells [29, 30], to the establishment

of light controlled cardiac cell monolayers, suited to the study of the mechanisms of intercellular

conduction of heart activation waves [31-34]. Furthermore, co-cultures between two different

myocardial cell populations, i.e. excitable cardiomyocytes and non-excitable cardiac fibroblasts,

were used to study their coupling in a partial replicate of the cardiac cell network on a dish [35, 36].

6
 ACCEPTED MANUSCRIPT
Only later the properties of optogenetics were exploited in intact animals, a task made simpler by

the improvements in the techniques of transgenesis, and the development of double-floxed ChR2

mice, suitable for cell specific expression upon breeding with one of the many CRE-recombinase

expressing strains (see Appendix) (Figure 1).

By breeding double-floxed-ChR2 mice with strains expressing CRE recombinase under either a

PT
wide cardiomyocyte promoter (-MyHC-CRE) or one restricted to conduction system cells (Cx40-

RI
CRE), we set out to compare the effect of light-activation in specific regions of the common

contractile myocardium (Figure 2a) vs. that of the sole conduction system cells (Figure 2b).

SC
Targeting of the excitation light beam to different regions of heart surface, allowed the stimulation,

in vivo, of the conduction system at different levels, from the His-bundle, to the septal and the distal

U
intraventricular Purkinje fibers (PFs), which evoked heartbeats, each with the corresponding ECG
AN
morphology. Previously, conducting cardiomyocytes were studied in vitro and in silico, but the
M

direct assessment of their function in vivo, with conventional approaches (i.e. electrical stimulation),

was hampered by their anatomical location (i.e. subendocardial region) and proximity to other cell
D

types. These results demonstrated that cell specific optogenetics, combined with spatially controlled
TE

light delivery, allowed gather information on the electrophysiology of such hard-to-study cellular

system, in vivo. As an example, by adopting protocols derived from cardiac electrophysiological

EP

studies (e.g. the so-called extrastimulus protocol), we determined electrophysiological parameters

C

of Purkinje fibers, such as the effective refractory period (ERP), clinically relevant in
AC

arrhythmogenesis [37]. Altogether, our results further demonstrate that optogenetics is a potent tool

to probe the functional anatomy (from structure to function) of cellular subsystems in complex

intact organs. In perspective, the reverse approach (from function to structure) could be applied, to

identify disease mechanisms, by optically scanning a complex organ and correlating abnormal

responses to photoactivation with the underlying tissue architecture.

7
 ACCEPTED MANUSCRIPT
3.2 Physiology of the extrinsic heart regulation
In addition to the autoregulation dependent on the intrinsic properties of the myocardium, heart

function is regulated by a large number of extrinsic factors, including neurogenic mechanisms

depending on the autonomic nervous system. Autonomic neurons, of both the sympathetic and

parasympathetic branches, innervate the myocardium, and operate fine control of heart rate and

PT
contractility in both basal conditions and during stresses. The cardiac efferent neuronal network

originates from central nuclei (paraventricular nucleus) for the parasympathetic axis, and from the

RI
brainstem for the sympathetic, and enters the heart walls, where postganglionic neurons take contact

SC
with their effector cells. Understanding the hierarchy of the brain-heart connection network, and the

neuro-effector coupling between post-ganglionic cardiac neurons (sympathetic and parasympathetic

U
efferents) and heart cells, is fundamental, as alterations in autonomic input to the heart may have the
AN
catastrophic effects of triggering life-threatening arrhythmias [24].

Optogenetic stimulation of cardiovascular responses has initially been achieved by photoactivation

M

of central nuclei, or selected neuronal circuits, of the cardiac autonomic network. Selective
D

optogenetic stimulation of cerebellar Purkinje cells, demonstrated the role played by these cells in
TE

blood pressure adaptation to postural changes [38]. In subsequent studies, optogenetics was used for

investigation of the brainstem dorsal motor nucleus of the vagus nerve, in the modulation of the
EP

phenomenon of preconditioning in ischemic cardiomyopathy [39]. These studies sign somewhat the

transitional phase between neuronal and cardiac optogenetics, and they also show that the method
C

can probe the function of high-level elements of the brain-heart regulatory axis with elevated
AC

accuracy. We expect that the use of different experimental protocols (e.g. photoactivation of

cervical ganglia, intrinsic cardiac ganglia), with sympathetic neuron-specific ChR2 expression, can

increase the details on the functional anatomy of the neuro-cardiac regulatory system. Given its

limited invasiveness and the high spatial resolution, optogenetics is also poised to the study of the

cardiac neuronal interface, enabling local control of neuronal subsets depending on illumination

patterns. The method has recently been applied to study of interactions between neurons and heart

8
 ACCEPTED MANUSCRIPT
in ex vivo preparations of perfused heart from sympathetic neuron-expressing ChR2 mice. Neuronal

photoactivation resulted, as expected, in increased beating rate and contraction force, showing the

correct function of such optogenetic assay. Moreover, this study demonstrated that over-activation

of sympathetic neurons induced cardiac arrhythmias, showing the causal link between the two

events [40]. The cardiac-neural interface has also been studied with optogenetics in vitro, in co-

PT
cultures between sympathetic neuron-differentiated human pluripotent stem cells expressing ChR2

RI
and cardiomyocytes, showing that the methodology can be used to assess the functional intercellular

coupling [41].

SC
In summary, the value of optogenetics as a tool to study cardiovascular physiology is emerging in

the recent years. While the more conventional approaches have already been validated, we look

U
forward to seeing other sophisticated applications of optogenetics to address unresolved issues in
AN
the physiology of heart regulation
M

4. Optogenetics in cardiac arrhythmias

D

4.1 Tissue determinants of extrasystolic heart beats and arrhythmia triggers

TE

Arrhythmias are complex phenomena, involving multiple myocardial cell types, with a wide variety

of consequences ranging from benign palpitations, to contractile failure and sudden cardiac death.
EP

Arrhythmogenic conditions include both genetically-determined (e.g. Catecholaminergic

Polymorphic Ventricular Tachycardia, Brugada syndrome, long QT syndrome , Arrhythmogenic

C
AC

CardioMyopathy) and acquired diseases (e.g. myocardial hypertrophy, ischemia, fibrosis). Although

the specific arrhythmia mechanisms may differ for each case, the essential requirements are: i)

altered cellular electrophysiology, which creates the permissive myocardial environment (substrate)

and ii) an arrhythmia trigger, most likely represented by abnormal depolarizations of confined

groups of cardiac cells. Arrhythmia research has used transgenesis to generate disease models in

mice, as well as interventions or drug treatments to replicate the conditions which determine the

arrhythmogenic myocardial substrate. This has clearly provided fundamental tools to investigate the

9
 ACCEPTED MANUSCRIPT
pathophysiology of arrhythmias. On the contrary, information on the factors which define efficient

focal triggers, relied on numerical modeling and were not addressed directly. Stochastic

depolarization of cardiomyocytes, independent from the cardiac activation wave, occurs at low

frequency in normal hearts, but is silent most of the times, as it is not able to activate a conducted

beat. This is due to the protection ensured by the electrotonic coupling between cardiomyocytes,

PT
allowing the current generated by the spontaneous depolarization in sparse cells to be 'sunk' by the

RI
surrounding polarized myocardium. A minimal critical mass of cardiomyocytes, depolarizing

simultaneously, is therefore needed to overcome the source-sink mismatch and result in a premature

SC
ventricular contraction (PVC) which, in the presence of a permissive myocardial substrate, may

evolve into chaotic and self-sustained ventricular arrhythmias. Conventional electrical stimulation is

U
not suited to determine the characteristics of the critical arrhyhmogenic mass, because the electrical
AN
pulses generate a rather inhomogeneous tissue depolarization [42], and the size and topology of the
M

stimulated area cannot be spatially controlled [43]. In addition, prolonged stimulation are associated

with production of toxic byproducts (H2, Cl2) and changes in pH [44], all of which are confounding
D

factors in the experiments. To define the tissue determinants of a focal source of heart activation in
TE

vivo, we exploited the spatial selectivity, the temporal resolution and the possibility of precisely

shaping the illuminated volume, all of which are intrinsic properties of optogenetics (Figures 1 and
EP

3a). The liminal tissue volume to generate a conducted beat was probed in cardiac-expressing ChR2

mice, by gradually increasing the fiber optic size and light intensity, which reflected on the
C
AC

photoactivated surface area, and light penetration within the heart walls, respectively (Figure 3a,b).

Light-matter interaction models were subsequently used to extrapolate the number of

cardiomyocytes enclosed in the minimal photoactivated volume required to elicit ectopies in a

normal mouse heart (critical mass), which corresponded to about 1300-1800 cardiomyocytes [37].

Notably, the results obtained with such direct optogenetic approach were different by two orders of

magnitude from the previous estimates (around 800.000, in rabbit heart), based on simulation

models [45].

10
 ACCEPTED MANUSCRIPT

4.2 Role of Purkinje fibers in arrhythmogenesis

Several reports in the literature have suggested that the PF network is a frequent triggering site of

arrhythmias [26, 46]. This has been attributed to the intrinsic properties of such cell type, which,

shows in vitro peculiar electrophysiologic characteristics and increased susceptibility to alterations

PT
in intracellular Ca2+ handling linked to cell depolarizations [47]. Although these properties explain

why PFs may depolarize abnormally (out of time, out of space), for activation of PFs to generate

RI
ectopic beats, a minimal critical cell mass must overcome the source-sink mismatch, as described

SC
above for the working myocardium. The coupling resistance between PFs and the surrounding

myocardium, and the peculiar topology of the PF system, which approximates a cable-like structure,

U
are both factors expected to decrease the critical number of cells required to generate ectopies, when
AN
compared to well-connected working cardiomyocytes. Due to the technical difficulties in addressing

these properties in vivo, these concepts have been modeled [45] and only recently proven directly
M

using optogenetics [37]. The results of the study demonstrate that around 100 PFs are sufficient to

elicit ectopic ventricular beats, in the normal heart. With respect to the numerical modeling, the
D

flexibility of optogenetic activation of different heart sectors, combined to high resolution imaging
TE

of the PF network distribution, allowed increase the understanding of the functional anatomy of the
EP

cardiac conduction system. Data on the role of PFs in cardiac arrhythmogenesis have been obtained

using chemicals (e.g. Lugol's solution) to ablate this cellular component. This approach is allegedly
C

specific, but implies the disruption of the first subendocardial layers where PFs are localized. Based
AC

on the evidence that endocardial Lugol's administration modified the arrhythmic phenotype in a

mouse model of CPVT, Cerrone et al. concluded that PFs are necessary and sufficient to trigger and

sustain ventricular arrhythmias in such genetic background [48]. By using the non-invasive and

conservative optogenetic approach, we aimed to investigate the causal role of PFs in

arrhythmogenesis, in a transgenic mouse expressing ChR2 under control of selective PF promoter in

the RyRR2474S background (unpublished). Surprisingly, photoactivated after-depolarizations in

11
 ACCEPTED MANUSCRIPT
PFs failed to trigger sustained arrhythmias, suggesting that ectopies originating from the PF

network are not sufficient to trigger and sustain the ventricular tachycardia typical of the disease.

While these discrepancies may be due to the different mouse strain and experimental methodology,

we have obtained results, in a model of acquired arrhythmias, represented by acute myocardial

ischemia, supporting that the synchronous depolarization of both PFs and the surrounding

PT
cardiomyocytes is required to trigger and sustain ventricular arrhythmias (Figure 3c,d) [37]. While

RI
further research will be needed to elucidate these phenomena at more mechanistic level, our results

demonstrate that optogenetics, applied to genetic or acquired animal models of arrhythmias, may

SC
provide important advancements on the pathogenesis of cardiac diseases.

5. Research and development of new light sources

U
AN
The use of light emission in cells, tissues, or intact animals has dominated experimental biology,

over the last few decades, and this has brought to the wide offer of thoroughly tested molecular
M

tools and instrumental devices which is now available. The explosion in the number of experiments

using optogenetics in different, and often unexplored tissue contexts, in vivo, has similarly
D

stimulated the rapidly evolving research of new suitable technologies. One of the most obvious
TE

technological developments of cardiac optogenetics, has been the combination of light as actuator
EP

(optogenetics) with light detection as a readout (optical mapping). Such approach uses voltage

sensitive dyes in opsin expressing hearts to record at high-speed the dynamics of cardiac activation
C

waves. This method has been applied as proof-of-concept in ex vivo preparations [37, 40, 49, 50]
AC

(Figure 4), but given that both optogenetics and optical mapping have independently been used in

vivo, it is foreseeable that the combination of the two will soon result in the "all optical" approach

for arrhythmia mechanism studies.

The development of novel devices to control light delivery in space and time, is an expanding

interdisciplinary area in optogenetic research, which unites biologist and biotechnologists,

physicists and engineers at least. From the simple optics of the first pioneering studies, investigators

12
 ACCEPTED MANUSCRIPT
have designed innovative strategies, which include stereotactic placement of light-emitting optrodes

in the brain, cuffs for photoactivation of peripheral nerves, and multisite optical interfaces, as

exhaustively reviewed by Deisseroth [12].

In cardiac optogenetics, new hardware being developed, is finalized, for example, to the

investigation of arrhythmic events characterized by complex spatial and temporal cardiac activation

PT
dynamics (e.g. polymorphic ventricular tachycardia). Research will soon use flexible meshes of

RI
micron-scale LED, to achieve complex spatial-temporal illumination patterns on the heart surface

[51-53]. This will allow e.g. to phenocopy the behavior of arrhythmic waves (e.g. spiral waves,

SC
reentry mechanisms), or to obtain information on the role of cell types so far neglected (e.g. cardiac

neurons), in the regulation of cardiac physiology and pathology. Compared to the experimental

U
setting used in fluorescence microscopy experiments, which are prone to artifacts and variability
AN
due to the experimental conditions (e.g. ex vivo preparations, anesthesia), the newer devices being
M

developed for cardiac optogenetics are finalized to interrogate biological processes, in the natural

context of the intact organism, in the freely moving animal. For example, in analogy to the
D

implantation of programmable light sources on the animal skull, commonly used for neuro-
TE

optogenetics, micron-scale LED suitable for fixed positioning in the thoracic cavity, would allow

cardiac optogenetics to be performed in conscious animals. The tendency is to move from the
EP

minimal-invasiveness to the distinctive non-invasiveness of optogenetics.

Furthermore, such approach could serve as the basis to develop a platform for the in vivo screening
C
AC

of drugs (e.g. anti-arrhythmics), extending an assay recently established in vitro. In this respect it

was described in literature an automated system where optical sensing and actuation allow to test

the effects of theoretically more than 600 drugs per hour on cardiomyocyte electrophysiology [54].

6. Therapeutic potential of cardiac optogenetics

At first glance, it may seem that the translational perspectives of cardiac optogenetics are far-

fetched, since the unconditional requirement of this method is the expression of an algal-derived

13
 ACCEPTED MANUSCRIPT
protein in men. However, recent development of safe and efficient gene delivery strategies, which

counts several clinical trials approved by the FDA, brings closer the possible clinical application of

optogenetics for disease therapy. The first example is represented by a recently approved clinical

trial testing the use of opsins in ganglionic retinal neurons for the treatment of a degenerative retinal

disease, called retinitis pigmentosa [55, 56]. In cardiology, the most obvious use of optogenetics

PT
would possibly be directed to the treatment of arrhythmias, which is now based on the use of well-

RI
established electronic devices (pacemaker, Implanted Cardioverter Defibrillator), to correct heart

activation sequence and prevent sudden cardiac death [57]. These approaches have some

SC
limitations, including the tissue damage due to electrical currents or physical contacts with the

pacemaker leads [58-60]. For all the outstanding properties described above, cardiac optogenetics

U
has the potential to overcome this drawbacks, given its distinctive contact-less feature and the
AN
virtually damage free use of light as an actuator [61-63]. One of the potential clinical application of
M

cardiac optogenetics, is the so-called 'optical defibrillation' of the arrhythmic heart. As a first step,

different computational models have been used to simulate how opsin channel kinetics, delivery
D

mode and cardiac distribution can be modulated to obtain optical heart defibrillation [64-66].
TE

Moreover, the possibility to obtain light-driven termination of arrhythmias was tested in vitro, using

murine isolated neonatal cardiomyocytes or human cardiomyocyte-derived cell lines expressing

EP

light sensitive opsins [31, 34, 67]. These studies brought, recently, to proof-of-principle research in

which the feasibility of optogenetic control of heart rhythm disturbances was tested in vivo.
C
AC

Interestingly, these studies demonstrated the possibility of multiple site pacing, which is particularly

important for an envisioned application in cardiac resynchronization therapy [49] and the

termination of ventricular fibrillation [68]. Moreover, an ex vivo study demonstrated that patterned

illumination of the heart at low light intensity was able to terminate ventricular tachycardia [50]. In

addition to these applications, cell-type selectivity of optogenetics could be exploited to achieve

heart activation at selective levels of the conduction system, such as the His-bundle or the PFs. This

could greatly improve the current therapeutic approaches based on the non-specific electrical

14
 ACCEPTED MANUSCRIPT
stimulation of relatively large regions of the myocardium. For the reasons detailed above, and

predicted by computational modeling [61, 66], selective stimulation of PFs could represent a more

efficient and controlled way to obtain ventricular pacing. Along the same lines, optogenetic pacing

of the His-bundle is potentially an improved strategy to obtain the so-called 'ventricular

resynchronization'. The term is referred to the correction of the differences in the timing of

PT
activation of the right and left ventricles, which causes negative effects on pumping efficiency of

RI
the heart, and is currently achieved with implantation of dual chamber pacemakers. The use of His-

bundle pacing, compared to standard biventricular pacing, would refine the approach allowing more

SC
efficient and physiologic ventricular activation [69, 70]. Optogenetic pacing would allow high

definition control of the photoactivated area, and cellular selectivity [61], resulting in selective His-

U
bundle stimulation as previously shown by us as a proof-of-concept using Cx40-ChR2 mice [37]
AN
(Figure 2b). All these examples of possible therapeutic uses of optogenetics indicate that the
M

methodology has a certain appeal for specific cardiac disturbances, mainly related to heart

electrophysiology. The major obstacle to the sight of clinical applications of cardiac optogenetics
D

remains how to express ChR2 in the human heart at uniform level in all regions, and stably in time.
TE

To solve this problem, two main ways are currently pursued: i) cell- and ii) viral-mediated delivery.

In the first case, the idea is based on the engraftment of non-excitable cells, manipulated in vitro to
EP

express ChR2, which are capable of forming electrical coupling with cardiomyocytes, rendering

them the ability to be light-stimulated. The tandem cell unit approach has, at the time being, only
C
AC

been validated in vitro [32, 36, 71], and its possible success in vivo depends on both the efficiency

of long-term myocardial cell engraftment, which is a highly debated issue in regenerative medicine,

and the reliability of electrical coupling with existing cardiomyocytes, which may dramatically

insist on arrhythmic risk [61, 72]. Viral-mediated expression of ChR2 in the heart has been

achieved, in rodents, using lentiviral, adenoviral, or adeno-associated viral vectors [31, 34, 35, 73-

75]. In these preclinical models, the stability in opsin expression may not represent a major

problem, as functional and optically activatable ChR2 was still present in cardiomyocytes one year

15
 ACCEPTED MANUSCRIPT
after the virus injection [49, 68, 76]. Here, important concerns are represented by other limiting

factors, including the presence of pre-existing neutralizing antibodies to the ChR2-carrying virus

type, the non-uniform expression of ChR2, that could result in heterogeneity of depolarization of

myocardium, potentially acting as a substrate for arrhythmias, and the ectopic expression of the

transgene in other cell types infected by the virus [61, 77]. In summary, optogenetics is, at the time

PT
being, far from the translation to cardiovascular therapy, and there are still major limitations to

RI
overcome, but the field is moving forward at fast pace, and the distinguished features of the method

could offer important advantages - as delineated above - in the treatment of some specific

SC
conditions.

7. Concluding remarks
U
AN
Cardiac optogenetics has introduced itself to the world of neurosciences as a revolutionary and long

sought-after method to modulate the function of specific neuronal circuits in living and conscious
M

animals. The applications of such biotechnology for cardiac studies have lagged behind, while the
D

appropriate tools, including both the animal models and experimental protocols were developed.
TE

We are now at the stage when a significant optogenetics toolkit is validated and available, and

described in a series of research papers. What we are looking forward to is new exciting questions,
EP

to fire the explosion of cardiac optogenetics, and shed light on the obscure angles of heart

physiology.
C
AC

Appendix
Sources of information and tools for optogenetics experiments:
Animal models: https://www.jax.org
Constructs: http://web.stanford.edu/group/dlab/optogenetics/
https://www.addgene.org
Hardware: http://www.scientifica.uk.com
https://www.thorlabs.com
http://www.noldus.com

16
 ACCEPTED MANUSCRIPT
http://web.stanford.edu/group/dlab/optogenetics/hardware.html

SOURCES OF FUNDING
The work was supported by Telethon Foundation, Grant n. GGP11224 to Marco Mongillo.

CONFLICT OF INTEREST

PT
Nothing to declare.

RI
REFERENCES

SC
[1] A. Adamantidis, S. Arber, J.S. Bains, et al., Optogenetics: 10 years after ChR2 in neurons--
views from the community, Nat Neurosci, 18 (2015) 1202-1212.
[2] M.A. Lawson, D.N. Zacks, F. Derguini, et al., Retinal analog restoration of photophobic
responses in a blind Chlamydomonas reinhardtii mutant. Evidence for an archaebacterial like

U
chromophore in a eukaryotic rhodopsin, Biophys J, 60 (1991) 1490-1498.
[3] G. Nagel, T. Szellas, W. Huhn, et al., Channelrhodopsin-2, a directly light-gated cation-selective
AN
membrane channel, Proc Natl Acad Sci U S A, 100 (2003) 13940-13945.
[4] T. Suzuki, K. Yamasaki, S. Fujita, et al., Archaeal-type rhodopsins in Chlamydomonas: model
structure and intracellular localization, Biochem Biophys Res Commun, 301 (2003) 711-717.
M

[5] O.A. Sineshchekov, K.H. Jung, J.L. Spudich, Two rhodopsins mediate phototaxis to low- and
high-intensity light in Chlamydomonas reinhardtii, Proc Natl Acad Sci U S A, 99 (2002) 8689-
8694.
[6] E.S. Boyden, F. Zhang, E. Bamberg, et al., Millisecond-timescale, genetically targeted optical
D

control of neural activity, Nat Neurosci, 8 (2005) 1263-1268.

[7] X. Li, D.V. Gutierrez, M.G. Hanson, et al., Fast noninvasive activation and inhibition of neural
TE

and network activity by vertebrate rhodopsin and green algae channelrhodopsin, Proc Natl Acad Sci
U S A, 102 (2005) 17816-17821.
[8] G. Nagel, M. Brauner, J.F. Liewald, et al., Light activation of channelrhodopsin-2 in excitable
EP

cells of Caenorhabditis elegans triggers rapid behavioral responses, Curr Biol, 15 (2005) 2279-
2284.
[9] C. Wyart, F. Del Bene, E. Warp, et al., Optogenetic dissection of a behavioural module in the
vertebrate spinal cord, Nature, 461 (2009) 407-410.
C

[10] D. Bellmann, A. Richardt, R. Freyberger, et al., Optogenetically Induced Olfactory Stimulation

AC

in Drosophila Larvae Reveals the Neuronal Basis of Odor-Aversion behavior, Front Behav
Neurosci, 4 (2010) 27.
[11] K. Deisseroth, Circuit dynamics of adaptive and maladaptive behaviour, Nature, 505 (2014)
309-317.
[12] K.L. Montgomery, S.M. Iyer, A.J. Christensen, et al., Beyond the brain: Optogenetic control in
the spinal cord and peripheral nervous system, Sci Transl Med, 8 (2016) 337rv335.
[13] A.M. Aravanis, L.P. Wang, F. Zhang, et al., An optical neural interface: in vivo control of
rodent motor cortex with integrated fiberoptic and optogenetic technology, J Neural Eng, 4 (2007)
S143-156.
[14] T. Bordia, X.A. Perez, J.E. Heiss, et al., Optogenetic activation of striatal cholinergic
interneurons regulates L-dopa-induced dyskinesias, Neurobiol Dis, 91 (2016) 47-58.
[15] F. Zhang, J. Vierock, O. Yizhar, et al., The microbial opsin family of optogenetic tools, Cell,
147 (2011) 1446-1457.

17
 ACCEPTED MANUSCRIPT
[16] A. Berndt, K. Deisseroth, OPTOGENETICS. Expanding the optogenetics toolkit, Science, 349
(2015) 590-591.
[17] K. Deisseroth, Stanford Optogenetics Resource Center.
[18] E. Azim, J. Jiang, B. Alstermark, et al., Skilled reaching relies on a V2a propriospinal internal
copy circuit, Nature, 508 (2014) 357-363.
[19] R.D. Proville, M. Spolidoro, N. Guyon, et al., Cerebellum involvement in cortical
sensorimotor circuits for the control of voluntary movements, Nat Neurosci, 17 (2014) 1233-1239.
[20] J. Courtin, F. Chaudun, R.R. Rozeske, et al., Prefrontal parvalbumin interneurons shape
neuronal activity to drive fear expression, Nature, 505 (2014) 92-96.

PT
[21] S.B. Wolff, J. Grundemann, P. Tovote, et al., Amygdala interneuron subtypes control fear
learning through disinhibition, Nature, 509 (2014) 453-458.
[22] D.S. Roy, A. Arons, T.I. Mitchell, et al., Memory retrieval by activating engram cells in mouse

RI
models of early Alzheimer's disease, Nature, 531 (2016) 508-512.
[23] A. Luthi, C. Luscher, Pathological circuit function underlying addiction and anxiety disorders,
Nat Neurosci, 17 (2014) 1635-1643.

SC
[24] K. Fukuda, H. Kanazawa, Y. Aizawa, et al., Cardiac innervation and sudden cardiac death,
Circ Res, 116 (2015) 2005-2019.
[25] C. Antzelevitch, Heterogeneity and cardiac arrhythmias: an overview, Heart Rhythm, 4 (2007)
964-972.

U
[26] P.A. Boyden, W. Dun, R.B. Robinson, Cardiac Purkinje fibers and Arrhythmias; The GK Moe
award Lecture 2015, Heart Rhythm, (2016).
AN
[27] A.B. Arrenberg, D.Y. Stainier, H. Baier, et al., Optogenetic control of cardiac function,
Science, 330 (2010) 971-974.
[28] T. Bruegmann, D. Malan, M. Hesse, et al., Optogenetic control of heart muscle in vitro and in
M

vivo, Nat Methods, 7 (2010) 897-900.

[29] O.J. Abilez, Cardiac optogenetics, Conf Proc IEEE Eng Med Biol Soc, 2012 (2012) 1386-
1389.
[30] Y. Zhuge, B. Patlolla, C. Ramakrishnan, et al., Human pluripotent stem cell tools for cardiac
D

optogenetics, Conf Proc IEEE Eng Med Biol Soc, 2014 (2014) 6171-6174.
[31] B.O. Bingen, M.C. Engels, M.J. Schalij, et al., Light-induced termination of spiral wave
TE

arrhythmias by optogenetic engineering of atrial cardiomyocytes, Cardiovasc Res, 104 (2014) 194-
205.
[32] Z. Jia, V. Valiunas, Z. Lu, et al., Stimulating cardiac muscle by light: cardiac optogenetics by
EP

cell delivery, Circ Arrhythm Electrophysiol, 4 (2011) 753-760.

[33] C.M. Ambrosi, P.M. Boyle, K. Chen, et al., Optogenetics-enabled assessment of viral gene and
cell therapy for restoration of cardiac excitability, Sci Rep, 5 (2015) 17350.
[34] R.A. Burton, A. Klimas, C.M. Ambrosi, et al., Optical control of excitation waves in cardiac
C

tissue, Nat Photonics, 9 (2015) 813-816.

AC

[35] J. Yu, E. Entcheva, Inscribing Optical Excitability to Non-Excitable Cardiac Cells: Viral
Delivery of Optogenetic Tools in Primary Cardiac Fibroblasts, Methods Mol Biol, 1408 (2016)
303-317.
[36] U. Nussinovitch, R. Shinnawi, L. Gepstein, Modulation of cardiac tissue electrophysiological
properties with light-sensitive proteins, Cardiovasc Res, 102 (2014) 176-187.
[37] T. Zaglia, N. Pianca, G. Borile, et al., Optogenetic determination of the myocardial
requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2, Proc Natl
Acad Sci U S A, 112 (2015) E4495-4504.
[38] T. Tsubota, Y. Ohashi, K. Tamura, et al., Optogenetic inhibition of Purkinje cell activity
reveals cerebellar control of blood pressure during postural alterations in anesthetized rats,
Neuroscience, 210 (2012) 137-144.

18
 ACCEPTED MANUSCRIPT
[39] S. Mastitskaya, N. Marina, A. Gourine, et al., Cardioprotection evoked by remote ischaemic
preconditioning is critically dependent on the activity of vagal pre-ganglionic neurones, Cardiovasc
Res, 95 (2012) 487-494.
[40] A.M. Wengrowski, X. Wang, S. Tapa, et al., Optogenetic release of norepinephrine from
cardiac sympathetic neurons alters mechanical and electrical function, Cardiovasc Res, 105 (2015)
143-150.
[41] Y. Oh, G.S. Cho, Z. Li, et al., Functional Coupling with Cardiac Muscle Promotes Maturation
of hPSC-Derived Sympathetic Neurons, Cell Stem Cell, 19 (2016) 95-106.
[42] A.M. Gillis, V.G. Fast, S. Rohr, et al., Spatial changes in transmembrane potential during

PT
extracellular electrical shocks in cultured monolayers of neonatal rat ventricular myocytes, Circ
Res, 79 (1996) 676-690.
[43] S.B. Knisley, N. Trayanova, F. Aguel, Roles of electric field and fiber structure in cardiac

RI
electric stimulation, Biophys J, 77 (1999) 1404-1417.
[44] D.R. Merrill, M. Bikson, J.G. Jefferys, Electrical stimulation of excitable tissue: design of
efficacious and safe protocols, J Neurosci Methods, 141 (2005) 171-198.

SC
[45] Y. Xie, D. Sato, A. Garfinkel, et al., So little source, so much sink: requirements for
afterdepolarizations to propagate in tissue, Biophys J, 99 (2010) 1408-1415.
[46] M.M. Scheinman, Role of the His-Purkinje system in the genesis of cardiac arrhythmia, Heart
Rhythm, 6 (2009) 1050-1058.

U
[47] T.J. Herron, M.L. Milstein, J. Anumonwo, et al., Purkinje cell calcium dysregulation is the
cellular mechanism that underlies catecholaminergic polymorphic ventricular tachycardia, Heart
AN
Rhythm, 7 (2010) 1122-1128.
[48] M. Cerrone, S.F. Noujaim, E.G. Tolkacheva, et al., Arrhythmogenic mechanisms in a mouse
model of catecholaminergic polymorphic ventricular tachycardia, Circ Res, 101 (2007) 1039-1048.
M

[49] U. Nussinovitch, L. Gepstein, Optogenetics for in vivo cardiac pacing and resynchronization
therapies, Nat Biotechnol, 33 (2015) 750-754.
[50] C. Crocini, C. Ferrantini, R. Coppini, et al., Optogenetics design of mechanistically-based
stimulation patterns for cardiac defibrillation, Sci Rep, 6 (2016) 35628.
D

[51] L. Xu, S.R. Gutbrod, A.P. Bonifas, et al., 3D multifunctional integumentary membranes for
spatiotemporal cardiac measurements and stimulation across the entire epicardium, Nat Commun, 5
TE

(2014) 3329.
[52] T.I. Kim, J.G. McCall, Y.H. Jung, et al., Injectable, cellular-scale optoelectronics with
applications for wireless optogenetics, Science, 340 (2013) 211-216.
EP

[53] A. Klimas, E. Entcheva, Toward microendoscopy-inspired cardiac optogenetics in vivo:

technical overview and perspective, J Biomed Opt, 19 (2014) 080701.
[54] A. Klimas, C.M. Ambrosi, J. Yu, et al., OptoDyCE as an automated system for high-
throughput all-optical dynamic cardiac electrophysiology, Nat Commun, 7 (2016) 11542.
C

[55] V. Busskamp, S. Picaud, J.A. Sahel, et al., Optogenetic therapy for retinitis pigmentosa, Gene
AC

Ther, 19 (2012) 169-175.

[56] B.S. Henriksen, R.E. Marc, P.S. Bernstein, Optogenetics for retinal disorders, J Ophthalmic
Vis Res, 9 (2014) 374-382.
[57] J. Tomzik, K.C. Koltermann, M. Zabel, et al., Quality of Life in Patients with an Implantable
Cardioverter Defibrillator: A Systematic Review, Front Cardiovasc Med, 2 (2015) 34.
[58] J.P. Daubert, W. Zareba, D.S. Cannom, et al., Inappropriate implantable cardioverter-
defibrillator shocks in MADIT II: frequency, mechanisms, predictors, and survival impact, J Am
Coll Cardiol, 51 (2008) 1357-1365.
[59] B.D. Powell, L.A. Saxon, J.P. Boehmer, et al., Survival after shock therapy in implantable
cardioverter-defibrillator and cardiac resynchronization therapy-defibrillator recipients according to
rhythm shocked. The ALTITUDE survival by rhythm study, J Am Coll Cardiol, 62 (2013) 1674-
1679.

19
 ACCEPTED MANUSCRIPT
[60] A.C. Ruwald, C. Schuger, A.J. Moss, et al., Mortality reduction in relation to implantable
cardioverter defibrillator programming in the Multicenter Automatic Defibrillator Implantation
Trial-Reduce Inappropriate Therapy (MADIT-RIT), Circ Arrhythm Electrophysiol, 7 (2014) 785-
792.
[61] P.M. Boyle, T.V. Karathanos, N.A. Trayanova, "Beauty is a light in the heart": the
transformative potential of optogenetics for clinical applications in cardiovascular medicine, Trends
Cardiovasc Med, 25 (2015) 73-81.
[62] C.M. Ambrosi, E. Entcheva, Optogenetics' promise: pacing and cardioversion by light?, Future
Cardiol, 10 (2014) 1-4.

PT
[63] P.M. Boyle, E. Entcheva, N.A. Trayanova, See the light: can optogenetics restore healthy
heartbeats? And, if it can, is it really worth the effort?, Expert Rev Cardiovasc Ther, 12 (2014) 17-
20.

RI
[64] T.V. Karathanos, J.D. Bayer, D. Wang, et al., Opsin spectral sensitivity determines the
effectiveness of optogenetic termination of ventricular fibrillation in the human heart: A simulation
study, J Physiol, (2016).

SC
[65] T.V. Karathanos, P.M. Boyle, N.A. Trayanova, Optogenetics-enabled dynamic modulation of
action potential duration in atrial tissue: feasibility of a novel therapeutic approach, Europace, 16
Suppl 4 (2014) iv69-iv76.
[66] P.M. Boyle, J.C. Williams, C.M. Ambrosi, et al., A comprehensive multiscale framework for

U
simulating optogenetics in the heart, Nat Commun, 4 (2013) 2370.
[67] B. Hofmann, V. Maybeck, S. Eick, et al., Light induced stimulation and delay of cardiac
AN
activity, Lab Chip, 10 (2010) 2588-2596.
[68] T. Bruegmann, P.M. Boyle, C.C. Vogt, et al., Optogenetic defibrillation terminates ventricular
arrhythmia in mouse hearts and human simulations, J Clin Invest, (2016).
M

[69] D.L. Lustgarten, E.M. Crespo, I. Arkhipova-Jenkins, et al., His-bundle pacing versus
biventricular pacing in cardiac resynchronization therapy patients: A crossover design comparison,
Heart Rhythm, 12 (2015) 1548-1557.
[70] B.J. Pang, S. Kumar, M.A. Tacey, et al., Capturing the His-Purkinje system is not possible
D

from conventional right ventricular apical and nonapical pacing sites, Pacing Clin Electrophysiol,
37 (2014) 724-730.
TE

[71] U. Nussinovitch, L. Gepstein, Optogenetics for suppression of cardiac electrical activity in

human and rat cardiomyocyte cultures, Neurophotonics, 2 (2015) 031204.
[72] N.W. Smit, R. Coronel, Stem cells can form gap junctions with cardiac myocytes and exert
EP

pro-arrhythmic effects, Front Physiol, 5 (2014) 419.

[73] O.J. Abilez, J. Wong, R. Prakash, et al., Multiscale computational models for optogenetic
control of cardiac function, Biophys J, 101 (2011) 1326-1334.
[74] C.M. Ambrosi, E. Entcheva, Optogenetic control of cardiomyocytes via viral delivery,
C

Methods Mol Biol, 1181 (2014) 215-228.

AC

[75] A. Mayra, H. Tomimitsu, T. Kubodera, et al., Intraperitoneal AAV9-shRNA inhibits target

expression in neonatal skeletal and cardiac muscles, Biochem Biophys Res Commun, 405 (2011)
204-209.
[76] C.C. Vogt, T. Bruegmann, D. Malan, et al., Systemic gene transfer enables optogenetic pacing
of mouse hearts, Cardiovasc Res, 106 (2015) 338-343.
[77] A.K. Zaiss, D.A. Muruve, Immunity to adeno-associated virus vectors in animals and humans:
a continued challenge, Gene Ther, 15 (2008) 808-816.

20
 ACCEPTED MANUSCRIPT
FIGURE LEGENDS

Figure 1. Advantages of cardiac optogenetic compared to standard electrical methods to study

heart electrophysiology.
Electrical stimulation of the heart is a conventional technique in clinical and experimental settings.

The application of electrical pulses to the heart surface results in the depolarization of all

PT
myocardial cells within a tissue volume, which depends on the current intensity and duration, but

RI
also from tissue factors (e.g. intercellular resistance), causing difficult control on the activated

myocardial volume. In optogenetics, each ChR2 expressing cell within the illuminated volume is

SC
directly and simultaneously depolarized, and in combination with control on the irradiated tissue

volume, and genetic targeting of opsin expression, these properties allow precise selective control

U
of specific heart structures. Genetic targeting of the light sensitive opsin to the cell type of interest
AN
can be easily achieved with Cre-Lox technology. The figure illustrates examples of promoters used
M

to drive cell specific expression of ChR2 in the different myocardial cells: alpha Myosin Heavy

Chain (MyHC) for cardiomyocytes, Connexin 40 (Cx40) for Purkinje fibers and Tyrosine
D

Hydroxilase (TOH) for sympathetic neurons. Red-bordered cells identify opsin expressing cells.
TE

Yellow cells identify depolarized cells.

EP

Figure 2. Selective electrophysiological investigation of the different myocardial cells using

cardiac optogenetic in vivo.
C

The cell selectivity of optogenetics is exemplified by the results of experiments using two animal
AC

models, in which ChR2 is expressed: in all cardiomyocytes (a) or only in conductive

cardiomyocytes (b). ECG traces show the result of heart photostimulation, performed in an

anesthetized open-chest mouse model, by light delivered via fiber optic positioned on the heart

surface. Photostimulation of different heart regions in the first model (a), as well as different

myocardial cells in the second model (b), evoke arrhythmic beats with diverse and characteristic

ECG morphology. (a, left panel) Fluorescence image of a mouse heart expressing ChR2, fused to a

red fluorescent protein, in all cardiomyocyte under the control of -MyHC promoter. On the right
21
 ACCEPTED MANUSCRIPT
panel, representative ECG traces showing ectopic beat generation upon epicardial illumination of

the left and right ventricles. Stars indicate the sites of illumination. (b, left panel) Fluorescence

image of a mouse heart expressing ChR2, fused to a red fluorescent protein, only in the atria and

ventricular conduction system, under the control of Cx40 promoter. On the right panel,

representative ECG traces showing the induction of arrhythmic beats arising from His-bundle cells

PT
(1) and Purkinje fiber cells (2) obtained through epicardial illumination at the sites indicated in the

RI
picture. Blue arrowheads indicate light pulses (Figure modified from [37]).

SC
Figure 3. Optogenetics as a valuable tool for the study of arrhythmia mechanisms.
The high spatial and temporal resolution combined with cell type specificity of cardiac

U
optogenetics, can be used to test myocardial physiology with new protocols and technical
AN
capabilities. Optogenetics was used to measure the minimal number of simultaneously depolarized

cells needed to generate an extrasystole in the normal heart (a, b) and to define the role of PF
M

selective activation in the generation of arrhythmias, in a pathologic context (i.e. myocardial

ischemia) (c, d). The cartoon in (a) shows the fundamental steps of the optogenetic assay of tissue
D

vulnerability to premature ventricular contractions (i.e. extrasystoles). The assay consists in the
TE

gradual decrease of illuminated myocardial volume, by acting on optical fiber diameter (d) and light
EP

intensity (h), to define the liminal conditions required for PVC activation. In the left panel an

example of subthreshold stimulation is shown, unable to generate a conducted beat. In the right
C

panel, the volume illuminated is large enough to evoke a globally spreading depolarizing wave, as
AC

shown in the corresponding ECG trace. Blue arrows indicate light pulses. The graph in (b) shows

the percentage of induced beats with the different conditions. (c) Photograph of a mouse heart

stimulated in vivo with an optical fiber. The black dot highlights the site of left anterior descending

coronary artery ligature and the dashed line indicates the ischemic area. (d) Representative ECG

traces showing the photostimulation of mouse hearts with global ChR2 expression (MyHC-ChR2)

and Purkinje fiber specific expression of ChR2 (Cx40-ChR2), during acute ischemia. Stimulation of

22
 ACCEPTED MANUSCRIPT
MyHC-ChR2 hearts, evoked sustained arrhythmias in 8 out of 10 mice, top panel. The same

photostimulation protocol failed to trigger sustained arrhythmias in Cx40-ChR2 mice (n = 8 mice)

(Bottom panel). Blue bars indicate the light pulses. (Figure modified from [37]).

Figure 4. All optical stimulation and sensing in ex vivo mouse hearts.

PT
Epicardial optical mapping is an imaging methodology using high speed acquisitions in hearts

loaded with fluorescent membrane potential dyes, the combination of this technique with

RI
optogenetics, results in an all-optical system using light both as actuator and as readout. Here a

SC
proof-of-principle of this approach is shown in isolated-perfused heart. The heart, expressing ChR2

in cardiomyocytes, was loaded with red-emitting voltage sensitive dye RH1691, and focally paced

U
through an optical fiber carrying blue light. (a) Activation map of an ex vivo photostimulated heart
AN
(Scale bar, 0.5 cm; color bar in ms). (b) Representative monophasic potential (MAP) trace during

photostimulation. (c) Optical mapping (Vm) of one light pulse. Images are snapshots taken every 2
M

ms (Scale bar, 0.5 cm; color bar in a.u.). The asterisks show the MAP electrode position. The
D

dashed lines indicate the optical fiber position. (Figure modified from [37]).
TE
C EP
AC

23
 ACCEPTED MANUSCRIPT
Electrical stimulation Optical stimulation

Optical fiber
Electrode

Cell: Cardiomyocyte
Promoter: MyHC
[37]

PT
Cell: Purkinje fiber
Promoter: Cx40
[37]

RI
Cardiomyocyte Purkinje fiber Neuron process

Cell: Sympathetic neuron

SC
Promoter: TOH
Opsin expressing cells Depolarized cells [40]

U
AN
M
D
TE
C EP
AC

Figure 1
a ACCEPTED MANUSCRIPT
b
-MyHC-td-Tomato-ChR2 Cx40-td-Tomato-ChR2
- RV Cardiomyocytes 1 - Purkinje fibers
RA RA
LA
2
LA
RV 1
100ms 100ms
RV
IVS
LV - LV Cardiomyocytes 2 - His bundle
LV

PT
1.5 mm ChR2 1 mm ChR2

RI
U SC
AN
M
D
TE
C EP
AC

Figure 2
a b
ACCEPTED MANUSCRIPT

h 1 h 2
d d

optical
fiber
2 1

PT
25 ms

c d

RI
-MyHC-ChR2

SC
optical
fiber LAD coronary
ligation 250 ms
LV
5s 5s

U
RV
ischemic area
Cx40-ChR2
AN
M
D
TE
C EP
AC

Figure 3
a b MANUSCRIPT
ACCEPTED

ms

PT
c

RI
U SC
AN
M
D
TE
EP
C
AC

Figure 4
 ACCEPTED MANUSCRIPT

HIGHLIGHTS

PT
- Optogenetics enables non-invasive control of specific excitable cells in vivo

- Cardiac optogenetics is useful to study the physiology of myocardial cell subsystems

RI
- Cardiac optogenetics has been applied in preclinical research of arrhythmogenesis

SC
- Gene manipulation and technical advancements nears cardiac optogenetics to therapy

U
AN
M
D
TE
C EP
AC